TIL expansion process using specific cytokine combinations and / or AKTi treatment

Abstract

Provided herein are methods of producing TILs via (i) REP pre-stimulation with a combination of interferon gamma (IFNγ) and anti-PD-1 antibody, with or without a CD40 agonist, and with or without an anti-CTLA-4 antibody; (ii) various concentrations of IL-15 and IL-21, with or without an AKT inhibitor (AKTi), with or without low concentrations of IL-2, during REP expansion and / or REP pre-expansion; (iii) low concentrations of IL-2 and AKTi during REP expansion and / or REP pre-expansion; (iv) a combination of (i) and (ii); or (v) a combination of (i) and (iii).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 375,209, filed September 9, 2022, and U.S. Provisional Application No. 63 / 331,757, filed April 15, 2022, all of which are incorporated by reference in their entireties. [Background technology]

[0002] The treatment of large refractory cancers using adoptive autologous transfer of tumor-infiltrating lymphocytes (TILs) represents a powerful approach to treating 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 to 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 research is ongoing and additional treatment options are needed (Kverneland et al., Oncotarget, 2020, 11(22), 2092-2105).

[0003] Furthermore, current TIL production and treatment processes are limited by length, cost, sterility concerns, and other factors described herein, severely limiting the ability to treat patients who are refractory to other checkpoint inhibitor therapies. There is an urgent need to provide TIL production processes and therapies based on such processes that are suitable for use in treating patients who have few or no remaining viable treatment options. The present invention fills this need by providing a production process for use in generating more robust TILs.

[0004] The present invention provides improved processes and methods for preparing TILs with specific combinations of cytokines and other cell culture media components to prepare therapeutic TIL populations with increased therapeutic efficacy, reduced effector differentiation, improved memory and stem-like attributes, and increased functionality. Summary of the Invention

[0005] (i) REP pre-stimulation with a combination of interferon gamma (IFNγ), anti-PD-1, CD40 agonism, and / or CTLA-4 agonism; (ii) various combinations of IL-21, IL-15, low concentration IL-2, and AKT inhibitors (AKTi) during REP expansion and / or REP pre-expansion; (iii) low concentration IL-2 and / or AKTi during REP expansion and / or REP pre-expansion; (iv) a combination of (i) and (ii); or (v) provided herein are methods of producing a TIL population via a combination of (i) and (iii).

[0006] The present invention provides a method for generating a tumor infiltrating lymphocyte (TIL) population, the method comprising: (a) resecting a tumor from a cancer patient, the tumor comprising a first population of TILs; (b) processing the tumor into a plurality of tumor fragments; (c) adding a plurality of tumor fragments to the closed system; (d) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (c) to step (d) occurs without opening the system, and optionally the cell culture medium is replaced on D3, D4, D5, D6 or D7 to produce a second TIL population; (e) performing a second expansion by culturing the second TIL population in cell culture medium having 3000 IU / mL or less of IL-2, and / or a protein kinase B (AKT) inhibitor, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is performed in a sealed container that provides a second gas-permeable surface area, and the transition from step (d) to step (e) occurs without opening the system, and optionally the cell culture medium is replaced on D3, D4, D5, D6, or D7 to produce a third TIL population; (f) harvesting the third population of TILs obtained from step (f), wherein the transition from step (e) to step (f) occurs without opening the system; (g) transferring the harvested third population of TILs from step (g) to an infusion bag, wherein the transition from step (f) to step (g) occurs without opening the system; (h) optionally, cryopreserving the infusion bag containing the harvested third population of TILs from step (g) using a cryopreservation process.

[0007] The present invention provides a method for generating a tumor infiltrating lymphocyte (TIL) population, the method comprising: (a) obtaining a tumor from a cancer patient, the tumor comprising a first population of TILs; (b) processing the tumor into a tumor digest; (c) adding the tumor digest to the closed system; (d) performing a first expansion by culturing the first TIL population in cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (c) to step (d) occurs without opening the system, and optionally the cell culture medium is replaced on day 3 of the first expansion, day 4 of the first expansion, day 5 of the first expansion, day 6 of the first expansion, or day 7 of the first expansion to produce the second TIL population; (e) performing a second expansion by culturing the second TIL population in cell culture medium having IL-2 at a concentration of 3000 IU / mL or less, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain a third TIL population, wherein the third TIL population comprises a therapeutic TIL population, and wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and the transition from step (d) to step (e) occurs without opening the closed system, and optionally the cell culture medium is removed on day 3 of the second expansion, day 4 of the second expansion, day 5 of the second expansion, day 6 of the second expansion, producing a third TIL population that is replaced on day 7 of the second expansion, or day 8 of the second expansion; (f) harvesting the third population of TILs obtained from step (f), wherein the transition from step (e) to step (f) occurs without opening the closed system; (g) transferring the harvested third population of TILs from step (g) to an infusion bag, wherein the transition from step (f) to (g) occurs without opening the closed system.

[0008] The present invention provides a method for generating a tumor infiltrating lymphocyte (TIL) population, the method comprising: (a) obtaining a tumor from a cancer patient, the tumor comprising a first population of TILs; (b) processing the tumor into tumor fragments or tumor digests; (c) adding tumor fragments or tumor digests to the closed system; (d) performing a first expansion by culturing the first TIL population in cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (c) to step (d) occurs without opening the system, and optionally the cell culture medium is replaced on day 3 of the first expansion, day 4 of the first expansion, day 5 of the first expansion, day 6 of the first expansion, or day 7 of the first expansion to produce the second TIL population; (e) performing a second expansion by culturing the second TIL population in cell culture medium having IL-2 at a concentration of 3000 IU / mL or less, OKT-3, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain a third TIL population, wherein the third TIL population comprises a therapeutic TIL population, and wherein the second expansion is performed in a sealed container that provides a second gas permeable surface area, and the transition from step (d) to step (e) occurs without opening the closed system, and optionally the cell culture medium is replaced on day 3 of the second expansion, day 4 of the second expansion, day 5 of the second expansion, day 6 of the second expansion, or day 7 of the second expansion; (f) harvesting the third population of TILs obtained from step (f), wherein the transition from step (e) to step (f) occurs without opening the closed system; (g) transferring the harvested third population of TILs from step (g) to an infusion bag, wherein the transition from step (f) to (g) occurs without opening the closed system.

[0009] The present invention provides a method for generating a tumor infiltrating lymphocyte (TIL) population, the method comprising: (a) obtaining a tumor from a cancer patient, the tumor comprising a first population of TILs; (b) processing the tumor into a tumor digest and cryopreserving the tumor digest; (c) thawing the cryopreserved tumor digest and adding the tumor digest to the closed system; (d) performing a first expansion by culturing the first TIL population in cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (c) to step (d) occurs without opening the system, and optionally the cell culture medium is replaced on day 3 of the first expansion, day 4 of the first expansion, day 5 of the first expansion, day 6 of the first expansion, or day 7 of the first expansion to produce the second TIL population; (e) treating the second TIL population with IL-2, OKT-3, and and performing a second expansion by culturing the TILs in cell culture medium with antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain a third TIL population, the third TIL population comprising a therapeutic TIL population, the second expansion being performed in a closed container that provides a second gas permeable surface area, and the transition from step (d) to step (e) occurs without opening the closed system, and optionally the cell culture medium is replaced on day 3 of the second expansion, day 4 of the second expansion, day 5 of the second expansion, day 6 of the second expansion, or day 7 of the second expansion. (f) harvesting the third population of TILs obtained from step (f), wherein the transition from step (e) to step (f) occurs without opening the closed system; (g) transferring the harvested third population of TILs from step (g) to an infusion bag, wherein the transition from step (f) to (g) occurs without opening the system.

[0010] The present invention provides a method for generating a tumor infiltrating lymphocyte (TIL) population, the method comprising: (a) performing a first expansion by culturing tumor fragments obtained from a tumor obtained from a cancer patient to generate a first TIL population in cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (c) to step (d) occurs without opening the system, and optionally the cell culture medium is replaced on day 3 of the first expansion, day 4 of the first expansion, day 5 of the first expansion, day 6 of the first expansion, or day 7 of the first expansion to produce the second TIL population; (b) performing a second expansion by culturing the second TIL population in cell culture medium having IL-2 at a concentration of 3000 IU / mL or less, OKT-3, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain a third TIL population, wherein the third TIL population comprises a therapeutic TIL population, and wherein the second expansion is performed in a sealed container providing a second gas permeable surface area, and the transition from step (a) to step (b) occurs without opening the closed system, and optionally the cell culture medium is replaced on day 3 of the second expansion, day 4 of the second expansion, day 5 of the second expansion, day 6 of the second expansion, or day 7 of the second expansion to produce a third TIL population; (c) harvesting the third population of TILs obtained from step (b), wherein the transition from step (e) to step (f) occurs without opening the closed system; (d) transferring the harvested third population of TILs from step (g) to an infusion bag, wherein the transition from step (f) to (g) optionally occurs without opening the closed system.

[0011] The present invention provides a method for generating a tumor infiltrating lymphocyte (TIL) population, the method comprising: (a) performing a first expansion by culturing a tumor digest obtained from a tumor obtained from a cancer patient to generate a first TIL population in cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (c) to step (d) occurs without opening the system, and optionally the cell culture medium is replaced on day 3 of the first expansion, day 4 of the first expansion, day 5 of the first expansion, day 6 of the first expansion, or day 7 of the first expansion to produce the second TIL population; (b) The second TIL population is treated with IL-2, OKT-3, and and performing a second expansion by culturing the TILs in cell culture medium with antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain a third TIL population, the third TIL population comprising a therapeutic TIL population, the second expansion being performed in a sealed container that provides a second gas permeable surface area, and the transition from step (a) to step (b) occurs without opening the chain system, and optionally the cell culture medium is replaced on day 3 of the second expansion, day 4 of the second expansion, day 5 of the second expansion, day 6 of the second expansion, or day 7 of the second expansion to produce a third TIL population; (c) harvesting the third population of TILs obtained from step (b), wherein the transition from step (e) to step (f) occurs without opening the closed system; (d) transferring the harvested third population of TILs from step (g) to an infusion bag, wherein the transition from step (f) to (g) optionally occurs without opening the closed system.

[0012] The present invention provides a method for generating a tumor infiltrating lymphocyte (TIL) population, the method comprising: (a) performing a first expansion by culturing tumor fragments or tumor digests obtained from a tumor obtained from a cancer patient to generate a first TIL population in cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (c) to step (d) occurs without opening the system, and optionally the cell culture medium is replaced on day 3 of the first expansion, day 4 of the first expansion, day 5 of the first expansion, day 6 of the first expansion, or day 7 of the first expansion to produce the second TIL population; (b) performing a second expansion by culturing the second TIL population in cell culture medium having IL-2 at a concentration of 3000 IU / mL or less, OKT-3, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain a third TIL population, wherein the third TIL population comprises a therapeutic TIL population, and wherein the second expansion is performed in a sealed container providing a second gas permeable surface area, and the transition from step (a) to step (b) occurs without opening the chain system, and optionally the cell culture medium is replaced on day 3 of the second expansion, day 4 of the second expansion, day 5 of the second expansion, day 6 of the second expansion, or day 7 of the second expansion to produce a third TIL population; (c) harvesting the third population of TILs obtained from step (b), wherein the transition from step (e) to step (f) occurs without opening the closed system; (d) transferring the harvested third population of TILs from step (g) to an infusion bag, wherein the transition from step (f) to (g) optionally occurs without opening the closed system.

[0013] The present invention provides a method for generating a tumor infiltrating lymphocyte (TIL) population, the method comprising: (a) thawing a cryopreserved tumor digest obtained from a tumor obtained from a cancer patient, and performing a first expansion by culturing the tumor digest to generate a first TIL population in cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (c) to step (d) occurs without opening the system, and optionally the cell culture medium is replaced on day 3 of the first expansion, day 4 of the first expansion, day 5 of the first expansion, day 6 of the first expansion, or day 7 of the first expansion to produce the second TIL population; (b) The second TIL population is treated with IL-2, OKT-3, and and performing a second expansion by culturing the TILs in cell culture medium with antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain a third TIL population, the third TIL population comprising a therapeutic TIL population, the second expansion being performed in a sealed container that provides a second gas permeable surface area, and the transition from step (a) to step (b) occurs without opening the chain system, and optionally the cell culture medium is replaced on day 3 of the second expansion, day 4 of the second expansion, day 5 of the second expansion, day 6 of the second expansion, or day 7 of the second expansion to produce a third TIL population; (c) harvesting the third population of TILs obtained from step (b), wherein the transition from step (e) to step (f) occurs without opening the closed system; (d) transferring the harvested third population of TILs from step (g) to an infusion bag, wherein the transition from step (f) to (g) optionally occurs without opening the closed system.

[0014] 9. The method of claim 1, further comprising the step of cryopreserving the infusion bag containing the harvested third population of TILs using a cryopreservation process.

[0015] The present invention provides a method for generating a tumor infiltrating lymphocyte (TIL) population, the method comprising: (a) obtaining a tumor from a cancer patient, the tumor comprising a first population of TILs; (b) processing the tumor into a plurality of tumor fragments; (c) adding a plurality of tumor fragments to the closed system; (d) performing a first expansion by culturing the first TIL population in cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (c) to step (d) occurs without opening the system, and optionally the cell culture medium is replaced on day 3 of the first expansion, day 4 of the first expansion, day 5 of the first expansion, day 6 of the first expansion, or day 7 of the first expansion to produce the second TIL population; (e) performing a second expansion by culturing the second TIL population in cell culture medium having IL-2 at a concentration of 3000 IU / mL or less, OKT-3, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain a third TIL population, wherein the third TIL population comprises a therapeutic TIL population, wherein the second expansion is performed in a sealed container that provides a second gas permeable surface area, and wherein the transition from step (d) to step (e) occurs without opening the closed system, and optionally the cell culture medium is replaced on day 3 of the second expansion, day 4 of the second expansion, day 5 of the second expansion, day 6 of the second expansion, or day 7 of the second expansion.

[0016] The present invention provides a method for generating a tumor infiltrating lymphocyte (TIL) population, the method comprising: (a) obtaining a tumor from a cancer patient, the tumor comprising a first population of TILs; (b) processing the tumor into a tumor digest; (c) adding the tumor digest to the closed system; (d) performing a first expansion by culturing the first TIL population in cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (c) to step (d) occurs without opening the system, and optionally the cell culture medium is removed on day 3 of the first expansion, day 4 of the first expansion, or on day 6 of the first expansion. producing a second population of TILs that is replaced on day 5 of the first expansion, day 6 of the first expansion, or day 7 of the first expansion; (e) performing a second expansion by culturing the second TIL population in cell culture medium having IL-2 at a concentration of 3000 IU / mL or less, OKT-3, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain a third TIL population, wherein the third TIL population comprises a therapeutic TIL population, wherein the second expansion is performed in a sealed container that provides a second gas permeable surface area, and wherein the transition from step (d) to step (e) occurs without opening the closed system, and optionally the cell culture medium is replaced on day 3 of the second expansion, day 4 of the second expansion, day 5 of the second expansion, day 6 of the second expansion, or day 7 of the second expansion.

[0017] The present invention provides a method for generating a tumor infiltrating lymphocyte (TIL) population, the method comprising: (a) obtaining a tumor from a cancer patient, the tumor comprising a first population of TILs; (b) processing the tumor into tumor fragments or tumor digests; (c) adding tumor fragments or tumor digests to the closed system; (d) performing a first expansion by culturing the first TIL population in cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (c) to step (d) occurs without opening the system, and optionally the cell culture medium is replaced on day 3 of the first expansion, day 4 of the first expansion, day 5 of the first expansion, day 6 of the first expansion, or day 7 of the first expansion to produce the second TIL population; (e) performing a second expansion by culturing the second TIL population in cell culture medium having IL-2 at a concentration of 3000 IU / mL or less, OKT-3, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain a third TIL population, wherein the third TIL population comprises a therapeutic TIL population, wherein the second expansion is performed in a sealed container that provides a second gas permeable surface area, and wherein the transition from step (d) to step (e) occurs without opening the closed system, and optionally the cell culture medium is replaced on day 3 of the second expansion, day 4 of the second expansion, day 5 of the second expansion, day 6 of the second expansion, or day 7 of the second expansion.

[0018] The present invention provides a method for generating a tumor infiltrating lymphocyte (TIL) population, the method comprising: (a) obtaining a tumor from a cancer patient, the tumor comprising a first population of TILs; (b) processing the tumor into a tumor digest and cryopreserving the tumor digest; (c) thawing the cryopreserved tumor digest and adding the tumor digest to the closed system; (d) performing a first expansion by culturing the first TIL population in cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (c) to step (d) occurs without opening the system, and optionally the cell culture medium is replaced on day 3 of the first expansion, day 4 of the first expansion, day 5 of the first expansion, day 6 of the first expansion, or day 7 of the first expansion to produce the second TIL population; (e) performing a second expansion by culturing the second TIL population in a cell culture medium having IL-2 at a concentration of 3000 IU / mL or less, OKT-3, and antigen-presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain a third TIL population, the third TIL population comprising a therapeutic TIL population; producing a third TIL population, wherein the second expansion is performed in a closed container that provides a second gas permeable surface area, and the transition from step (d) to step (e) occurs without opening the closed system, and optionally the cell culture medium is replaced on day 3 of the second expansion, day 4 of the second expansion, day 5 of the second expansion, day 6 of the second expansion, or day 7 of the second expansion; (f) harvesting the third population of TILs obtained from step (f), wherein the transition from step (e) to step (f) occurs without opening the closed system; (g) transferring the harvested third population of TILs from step (g) to an infusion bag, wherein the transition from step (f) to (g) occurs without opening the closed system.

[0019] The present invention provides a method for generating a tumor infiltrating lymphocyte (TIL) population, the method comprising: (a) performing a first expansion by culturing tumor fragments obtained from a tumor obtained from a cancer patient to generate a first TIL population in cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (c) to step (d) occurs without opening the system, and optionally the cell culture medium is replaced on day 3 of the first expansion, day 4 of the first expansion, day 5 of the first expansion, day 6 of the first expansion, or day 7 of the first expansion to produce the second TIL population; (b) performing a second expansion by culturing the second TIL population in cell culture medium having IL-2 at a concentration of 3000 IU / mL or less, OKT-3, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain a third TIL population, wherein the third TIL population comprises a therapeutic TIL population, wherein the second expansion is performed in a sealed container providing a second gas permeable surface area, and wherein the transition from step (a) to step (b) occurs without opening the closed system, and optionally the cell culture medium is replaced on day 3 of the second expansion, day 4 of the second expansion, day 5 of the second expansion, day 6 of the second expansion, or day 7 of the second expansion.

[0020] The present invention provides a method for generating a tumor infiltrating lymphocyte (TIL) population, the method comprising: (a) performing a first expansion by culturing a tumor digest obtained from a tumor obtained from a cancer patient to generate a first TIL population in cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (c) to step (d) occurs without opening the system, and optionally the cell culture medium is replaced on day 3 of the first expansion, day 4 of the first expansion, day 5 of the first expansion, day 6 of the first expansion, or day 7 of the first expansion to produce the second TIL population; (b) performing a second expansion by culturing the second TIL population in cell culture medium having IL-2 at a concentration of 3000 IU / mL or less, OKT-3, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain a third TIL population, wherein the third TIL population comprises a therapeutic TIL population, wherein the second expansion is performed in a sealed container providing a second gas permeable surface area, and wherein the transition from step (a) to step (b) occurs without opening the closed system, and optionally the cell culture medium is replaced on day 3 of the second expansion, day 4 of the second expansion, day 5 of the second expansion, day 6 of the second expansion, or day 7 of the second expansion.

[0021] The present invention provides a method for generating a tumor infiltrating lymphocyte (TIL) population, the method comprising: (a) performing a first expansion by culturing tumor fragments or tumor digests obtained from a tumor obtained from a cancer patient to generate a first TIL population in cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (c) to step (d) occurs without opening the system, and optionally the cell culture medium is replaced on day 3 of the first expansion, day 4 of the first expansion, day 5 of the first expansion, day 6 of the first expansion, or day 7 of the first expansion to produce the second TIL population; (b) performing a second expansion by culturing the second TIL population in cell culture medium having IL-2 at a concentration of 3000 IU / mL or less, OKT-3, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain a third TIL population, wherein the third TIL population comprises a therapeutic TIL population, wherein the second expansion is performed in a sealed container providing a second gas permeable surface area, and wherein the transition from step (a) to step (b) occurs without opening the closed system, and optionally the cell culture medium is replaced on day 3 of the second expansion, day 4 of the second expansion, day 5 of the second expansion, day 6 of the second expansion, or day 7 of the second expansion.

[0022] The present invention provides a method for generating a tumor infiltrating lymphocyte (TIL) population, the method comprising: (a) thawing a cryopreserved tumor digest obtained from a tumor obtained from a cancer patient, and performing a first expansion by culturing the tumor digest to generate a first TIL population in cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (c) to step (d) occurs without opening the system, and optionally the cell culture medium is replaced on day 3 of the first expansion, day 4 of the first expansion, day 5 of the first expansion, day 6 of the first expansion, or day 7 of the first expansion to produce the second TIL population; (b) performing a second expansion by culturing the second TIL population in cell culture medium having IL-2 at a concentration of 3000 IU / mL or less, OKT-3, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain a third TIL population, wherein the third TIL population comprises a therapeutic TIL population, wherein the second expansion is performed in a sealed container providing a second gas permeable surface area, and wherein the transition from step (a) to step (b) occurs without opening the closed system, and optionally the cell culture medium is replaced on day 3 of the second expansion, day 4 of the second expansion, day 5 of the second expansion, day 6 of the second expansion, or day 7 of the second expansion.

[0023] In some embodiments, the IL-2 is a lower dose of IL-2.

[0024] In some embodiments, the culture medium in step (d) comprises no more than 3000 IU / mL of IL-2, and / or a protein kinase B (AKT) inhibitor.

[0025] In some embodiments, the culture medium in step (e) comprises a protein kinase B (AKT) inhibitor.

[0026] In some embodiments, the culture medium in step (d) and / or step (e) comprises IL-15 and / or IL-21.

[0027] In some embodiments, the method comprises, during D0, D1 or D2 of step (d), The method further comprises adding IFNγ and an anti-PD-1 antibody at a concentration of 0 ng / ml.

[0028] In some embodiments, the method further comprises adding a CD40 agonist and / or an inhibitor of CTLA-4 during DO, D1 or D2 of step (d).

[0029] In some embodiments, after step (a) and before step (b), the method comprises enzymatically digesting the plurality of tumor fragments to obtain the first population of TILs.

[0030] In some embodiments, the first TIL population is obtained 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 the cancer.

[0031] In some embodiments, the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY1125976, perifosine, oridonin, herbacetin, tehranolide, isoliquirigenin, scutellarin, and honokiol.

[0032] The present invention provides a method for generating a tumor infiltrating lymphocyte (TIL) population, the method comprising: (a) resecting a tumor from a cancer patient, the tumor comprising a first population of TILs; (b) processing the tumor into a plurality of tumor fragments; (c) adding a plurality of tumor fragments to the closed system; (d) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container that provides a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and the transition from step (c) to step (d) occurs without opening the system, and optionally the cell culture medium is replaced on D3, D4, D5, D6 or D7 to produce a second TIL population; (e) performing a second expansion by culturing the second TIL population in cell culture medium comprising IL-15 and / or IL-21, OKT-3, and antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain a third TIL population, the third TIL population comprising a therapeutic TIL population, and wherein the second expansion is performed in a sealed container providing a second gas permeable surface area, and the transition from step (d) to step (e) occurs without opening the system, and optionally the cell culture medium is replaced on D3, D4, D5, D6, or D7 to produce a third TIL population; (f) harvesting the third population of TILs obtained from step (f), wherein the transition from step (e) to step (f) occurs without opening the system; (g) transferring the harvested third population of TILs from step (g) to an infusion bag, wherein the transition from step (f) to (g) occurs without opening the system; (h) optionally, cryopreserving the infusion bag containing the harvested third population of TILs from step (g) using a cryopreservation process.

[0033] In some embodiments, the culture medium in step (d) comprises IL-15 and / or IL-21.

[0034] In some embodiments, the IL-2 is a lower dose of IL-2.

[0035] In some embodiments, the culture medium in step (d) and / or step (e) comprises 3000 IU / mL or less of IL-2, and / or a protein kinase B (AKT) inhibitor.

[0036] In some embodiments, the method further comprises adding IFNγ and an anti-PD-1 antibody at a concentration of 200 ng / ml during DO, D1 or D2 in step (d).

[0037] In some embodiments, the method further comprises adding a CD40 agonist and / or an inhibitor of CTLA-4 during D0, D1 or D2 in step (d).

[0038] In some embodiments, after step (a) and before step (b), the method comprises enzymatically digesting the plurality of tumor fragments to obtain the first population of TILs.

[0039] In some embodiments, the first TIL population is obtained 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 the cancer.

[0040] In some embodiments, the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY1125976, perifosine, oridonin, herbacetin, teheranolide, isoliquirigenin, scutellarin, and honokiol.

[0041] The present invention provides a method for generating a tumor infiltrating lymphocyte (TIL) population, the method comprising: (a) resecting a tumor from a cancer patient, the tumor comprising a first population of TILs; (b) processing the tumor into a plurality of tumor fragments; (c) adding a plurality of tumor fragments to the closed system; (d) performing a first expansion by culturing the first TIL population in a cell culture medium containing IL-2 to produce a second TIL population, wherein the first expansion is performed in a sealed container providing a first gas permeable surface area, and the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and an inhibitor of IFNγ and / or PD-1 is added during D0, D1 or D2 of step (d), and the transition from step (c) to step (d) occurs without opening the system, and optionally the cell culture medium is replaced on D3, D4, D5, D6 or D7 to produce a second TIL population; (e) performing a second expansion by culturing the second TIL population in cell culture medium with OKT-3 and antigen presenting cells (APCs) to produce a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain a third TIL population, the third TIL population being a therapeutic TIL population, and wherein the second expansion is performed in a sealed container that provides a second gas permeable surface area, and the transition from step (d) to step (e) occurs without opening the system, and optionally the cell culture medium is replaced on D3, D4, D5, D6, or D7 to produce a third TIL population; (f) harvesting the third population of TILs obtained from step (f), wherein the transition from step (e) to step (f) occurs without opening the system; (g) transferring the harvested third population of TILs from step (g) to an infusion bag, wherein the transition from step (f) to step (g) occurs without opening the system; (h) optionally, using a cryopreservation process to preserve the harvested third fraction from step (g); and cryopreserving the infusion bag containing the TIL population.

[0042] In some embodiments, the method further comprises adding a CD40 agonist and / or an inhibitor of CTLA-4 during D0, D1 or D2 of step (d).

[0043] In some embodiments, the IL-2 is a lower dose of IL-2.

[0044] In some embodiments, the culture medium in step (d) and / or step (e) comprises no more than 3000 IU / mL of IL-2, and / or a protein kinase B (AKT) inhibitor.

[0045] In some embodiments, the culture medium in step (d) and / or step (e) comprises IL-15 and / or IL-21.

[0046] In some embodiments, after step (a) and before step (b), the method comprises enzymatically digesting the plurality of tumor fragments to obtain the first population of TILs.

[0047] In some embodiments, the first TIL population is obtained 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 the cancer.

[0048] In some embodiments, the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY1125976, perifosine, oridonin, herbacetin, teheranolide, isoliquirigenin, scutellarin, and honokiol.

[0049] The present invention provides a method for generating a tumor infiltrating lymphocyte (TIL) population, the method comprising: (a) resecting a tumor from a cancer patient, the tumor comprising a first population of TILs; (b) performing an initial expansion (or first expansion by priming) by culturing the first TIL population in cell culture medium comprising IL-2, optionally OKT-3 (an anti-CD3 antibody), and optionally IL-15 and / or IL-21, wherein the first expansion by priming is optionally performed in a sealed container providing a first gas permeable surface area, and the first expansion by priming is performed for about 1 to 8 days to obtain a second TIL population, and the transition from step (a) to step (b) optionally occurs without opening the system, and optionally the cell culture medium is replaced on D3, D4, D5, D6 or D7; (c) performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain a third TIL population, wherein the second cell culture medium comprises 3000 IU / mL or less of IL-2, OKT-3 (anti-CD3 antibody), IL-15 and / or IL-21, APC, and / or a protein kinase B (AKT) inhibitor, and the rapid expansion is performed over a period of 14 days or less, and optionally the rapid second expansion can proceed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days after initiation of the rapid second expansion, and optionally the cell culture medium is replaced on D3, D4, D5, D6, or D7 to obtain a third TIL population; (d) harvesting a third population of TILs; (e) transferring the harvested third population of TILs from step (d) to an infusion bag, wherein the transition from step (d) to (e) occurs without opening the system; (f) cryopreserving the infusion bag containing the harvested third population of TILs from step (e) using a cryopreservation process.

[0050] In some embodiments, the IL-2 is a lower dose of IL-2.

[0051] In some embodiments, the culture medium in step (b) comprises no more than 3000 IU / mL of IL-2, and / or a protein kinase B (AKT) inhibitor.

[0052] In some embodiments, the culture medium in step (b) and / or step (c) comprises IL-15 and / or IL-21.

[0053] In some embodiments, the method further comprises adding an inhibitor of IFNγ and PD-1 at a concentration of 200 ng / ml during DO, D1 or D2 of step (b).

[0054] In some embodiments, the method further comprises adding a CD40 agonist and / or an inhibitor of CTLA-4 during D0, D1 or D2 of step (b).

[0055] In some embodiments, the first TIL population is obtained 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 the cancer.

[0056] In some embodiments, the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY1125976, perifosine, oridonin, herbacetin, teheranolide, isoliquirigenin, scutellarin, and honokiol.

[0057] The present invention provides a method for generating a tumor infiltrating lymphocyte (TIL) population, the method comprising: (a) resecting a tumor from a cancer patient, the tumor comprising a first population of TILs; (b) performing an initial expansion (or first expansion by priming) by culturing the first TIL population in cell culture medium comprising IL-2, optionally OKT-3 (an anti-CD3 antibody), and optionally IL-15 and / or IL-21, wherein the first expansion by priming is optionally performed in a sealed container providing a first gas permeable surface area, and the first expansion by priming is performed for about 1 to 8 days to obtain a second TIL population, and the transition from step (a) to step (b) optionally occurs without opening the system, and optionally the cell culture medium is replaced on D3, D4, D5, D6 or D7; (c) performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain a third TIL population, wherein the second cell culture medium comprises IL-2, OKT-3 (anti-CD3 antibody), IL-15 and / or IL-21, APC, and the rapid expansion is performed over a period of 14 days or less, and optionally the rapid second expansion can proceed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days after the initiation of the rapid second expansion, and optionally the cell culture medium is replaced on D3, D4, D5, D6, or D7 to obtain a third TIL population; (d) harvesting a third population of TILs; (e) transferring the harvested third population of TILs from step (d) to an infusion bag, wherein the transition from step (d) to (e) occurs without opening the system; (f) The third collection of harvested TILs from step (e) using a cryopreservation process. and cryopreserving the infusion bag containing the group.

[0058] In some embodiments, the IL-2 is a lower dose of IL-2.

[0059] In some embodiments, the culture medium in step (b) and / or step (c) comprises 3000 IU / mL or less of IL-2, and / or a protein kinase B (AKT) inhibitor.

[0060] In some embodiments, the culture medium in step (b) and / or step (c) comprises IL-15 and / or IL-21.

[0061] In some embodiments, the method further comprises adding an inhibitor of IFNg and PD-1 at a concentration of 200 ng / ml during DO, D1 or D2 of step (b).

[0062] In some embodiments, the method further comprises adding a CD40 agonist and / or an inhibitor of CTLA-4 during DO, D1 or D2 of step (b).

[0063] In some embodiments, the first TIL population is obtained 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 the cancer.

[0064] In some embodiments, the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY1125976, perifosine, oridonin, herbacetin, teheranolide, isoliquirigenin, scutellarin, and honokiol.

[0065] The present invention provides a method for generating a tumor infiltrating lymphocyte (TIL) population, the method comprising: (a) resecting a tumor from a cancer patient, the tumor comprising a first population of TILs; (b) performing initial expansion (or first expansion by priming) by culturing the first TIL population in cell culture medium comprising IL-2, optionally OKT-3 (anti-CD3 antibody), and optionally IL-15 or IL-21, wherein the first expansion by priming is optionally performed in a sealed container providing a first gas permeable surface area, and the first expansion by priming is performed for about 1 to 8 days to obtain a second TIL population, and an inhibitor of IFNγ and / or PD-1 is added during D0, D1 or D2 of step (b), and the transition from step (a) to step (b) optionally occurs without opening the system, and optionally the cell culture medium is replaced on D3, D4, D5, D6 or D7; (c) performing a rapid second expansion of the second TIL population in a second cell culture medium to obtain a third TIL population, wherein the second cell culture medium comprises IL-2, OKT-3 (an anti-CD3 antibody), and optionally IL-15 and / or IL-21, APC, and the rapid expansion is performed over a period of 14 days or less, and optionally the rapid second expansion can proceed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days after the initiation of the rapid second expansion, and optionally the cell culture medium is replaced on D3, D4, D5, D6, or D7 to obtain a third TIL population; (d) harvesting a third population of TILs; (e) transferring the harvested third population of TILs from step (g) to an infusion bag, wherein the transition from step (f) to step (g) occurs without opening the system. Steps (h) cryopreserving the infusion bag containing the harvested third population of TILs from step (g) using a cryopreservation process.

[0066] In some embodiments, the IL-2 is a lower dose of IL-2.

[0067] In some embodiments, the culture medium in step (b) and / or step (c) comprises 3000 IU / mL or less of IL-2, and / or a protein kinase B (AKT) inhibitor.

[0068] In some embodiments, the culture medium in step (b) and / or step (c) comprises IL-15 and / or IL-21.

[0069] In some embodiments, the method further comprises adding an inhibitor of IFNγ and PD-1 at a concentration of 200 ng / ml during DO, D1 or D2 of step (d).

[0070] In some embodiments, the method further comprises adding a CD40 agonist and / or an inhibitor of CTLA-4 during D0, D1 or D2 in step (d).

[0071] In some embodiments, after step (a) and before step (b), the method comprises enzymatically digesting the plurality of tumor fragments to obtain the first population of TILs.

[0072] In some embodiments, the first TIL population is obtained 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 the cancer.

[0073] In some embodiments, the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY1125976, perifosine, oridonin, herbacetin, teheranolide, isoliquirigenin, scutellarin, and honokiol.

[0074] In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), renal cancer, and renal cell carcinoma.

[0075] In some embodiments, the first TIL population is stimulated with IFNg (interferon gamma), an inhibitor of PD-1 (e.g., an anti-PD-1 antibody), a CD40 agonist (e.g., CD40L, an anti-CD40 agonist antibody), and / or a CTLA-4 agonist (e.g., an anti-CTLA-4 agonist antibody) for up to about 48 hours prior to the first expansion, and optionally for 24 hours or 48 hours prior to the first expansion.

[0076] In some embodiments, in the first expansion step by priming, the cell culture medium further comprises antigen-presenting cells (APCs), and the number of APCs in the culture medium in the rapid second expansion step is greater than the number of APCs in the culture medium in the first expansion step by priming.

[0077] In some embodiments, antigen presenting cells (APCs) are added to the first TIL population, the second TIL population, or both.

[0078] In some embodiments, CD40 or CD40L is present in the cell culture medium at an initial concentration of about 30 ng / mL during stimulation.

[0079] In some embodiments, IFNg is present in the cell culture medium during stimulation at an initial concentration of about 200 ng / mL.

[0080] 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.

[0081] In some embodiments, IL-2 is present in the cell culture medium in the second expansion at an initial concentration of 1000 IU / mL to 6000 IU / mL.

[0082] In some embodiments, the low dose of IL-2 comprises 1000 IU / mL or less.

[0083] 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.

[0084] 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.

[0085] In some embodiments, the stimulation cell culture medium further comprises a cytokine selected from the group consisting of IL-4, IL-7, IL-15, IL-21, and combinations thereof.

[0086] In some embodiments, the first expansion is carried out over a period of about 11 days.

[0087] In some embodiments, the first expansion is performed using a gas-permeable container.

[0088] In some embodiments, the second expansion is performed using a gas-permeable container.

[0089] In some embodiments, the stimulation is performed using a gas-permeable container.

[0090] The present invention also provides populations of TILs produced using the methods described herein.

[0091] In some embodiments, the TIL population is about 2.3 x 10 10 ~Approx. 13.7×10 10 Including TIL.

[0092] The present invention also provides pharmaceutical compositions comprising the TIL populations described herein.

[0093] The present invention also provides a method of treating a cancer patient, comprising administering a population of TILs or a pharmaceutical composition thereof described herein.

[0094] In some embodiments, the method further comprises treating the patient with a non-myeloablative lymphodepletion regimen prior to administering the third population of TILs to the patient.

[0095] In some embodiments, the non-myeloablative lymphodepletion regimen includes cyclophosphamide at 60 mg / m 2 / day for 2 days, followed by fludarabine at 25 mg / m 2 / day for three days.

[0096] In some embodiments, the non-myeloablative lymphodepletion regimen includes cyclophosphamide at 60 mg / m 2 / day and fludarabine at a dose of 25 mg / m 2 / day for 2 days, followed by fludarabine at 25 mg / m 2 / day for 3 days.

[0097] In some embodiments, the non-myeloablative lymphodepletion regimen includes cyclophosphamide at 60 mg / m 2 / day and fludarabine at a dose of 25 mg / m 2 / day for 2 days, followed by fludarabine at 25 mg / m 2 / day for 1 day.

[0098] In some embodiments, cyclophosphamide is administered with mesna.

[0099] In some embodiments, the method further comprises treating the patient with an IL-2 regimen starting the day after administering the TILs to the patient.

[0100] In some embodiments, the method further comprises treating the patient with an IL-2 regimen starting on the same day that the TILs are administered to the patient.

[0101] In some embodiments, the IL-2 regimen is a high-dose IL-2 regimen comprising 600,000 or 720,000 IU / kg aldesleukin, or a biosimilar or variant thereof, administered as a 15-minute bolus intravenous infusion every 8 hours until tolerated.

[0102] In some embodiments, a therapeutically effective population of TILs is administered, about 2.3 x 10 10 ~Approx. 13.7×10 10 Including TIL.

[0103] In some embodiments, the TILs comprise genetically modified TILs, and optionally, the first TIL population and / or the second TIL population are genetically modified to reduce expression of CD39 and CD69.

[0104] In some embodiments, the TILs express CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, TET2, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, The method further comprises a genetic modification that reduces expression of one or more immune checkpoint genes selected from the group including FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, and TOX.

[0105] In some embodiments, the one or more immune checkpoint genes are selected from the group including PD-1, CBL-B, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TIGIT, TET2, TGFβ, and PKA.

[0106] In some embodiments, the genetically modified TILs comprise a genetic modification that enhances expression of one or more immune checkpoint genes in at least a portion of the therapeutic TIL population. Further comprising, wherein the immune checkpoint gene(s) is / are selected from the group comprising CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, NOTCH 1 / 2 intracellular domain (ICD), and / or NOTCH ligand mDLL1.

[0107] In some embodiments, the genetic modification step is carried out using a programmable nuclease that mediates the generation of a double- or single-strand break in the one or more immune checkpoint genes.

[0108] In some embodiments, the genetic modification is performed using one or more methods selected from CRISPR, TALE, zinc finger, and combinations thereof.

[0109] In some embodiments, the method comprises CRISPR technology.

[0110] In some embodiments, the CRISPR method is a CRISPR / Cas9 method.

[0111] In some embodiments, the genetic modification comprises a TALE method.

[0112] In some embodiments, the genetic modification comprises zinc finger technology.

[0113] In some embodiments, processing a tumor sample obtained from a subject into a tumor digest comprises incubating the tumor sample in an enzyme medium.

[0114] In some embodiments, processing a tumor sample obtained from a subject into a tumor digest further comprises mechanically disrupting the tumor sample to dissociate the tumor sample.

[0115] In some embodiments, processing a tumor sample obtained from a subject into a tumor digest further comprises purifying the dissociated tumor sample using density gradient separation.

[0116] In some embodiments, the enzyme medium comprises DNase.

[0117] In some embodiments, the enzyme medium contains 30 units / mL of DNase.

[0118] In some embodiments, the enzyme medium comprises collagenase.

[0119] In some embodiments, the enzyme medium comprises 1.0 mg / mL collagenase.

[0120] In some embodiments, the harvested therapeutic TIL population comprises sufficient TILs for use in administering a therapeutically effective dose to a subject.

[0121] In some embodiments, the therapeutically effective dose is about 1×10 9 ~Approx. 9×10 10 Including TIL.

[0122] In some embodiments, the APCs comprise peripheral blood mononuclear cells (PBMCs).

[0123] In some embodiments, the therapeutic TIL population harvested in step (e) exhibits an increased subpopulation of CD4+ cells compared to the first and / or second TIL populations.

[0124] In some embodiments, the PBMCs are supplemented at a TIL:PBMC ratio of about 1:25.

[0125] In some embodiments, the first expansion step occurs in about 3 to 11 days.

[0126] In some embodiments, the second expansion step is performed in about 7-11 days.

[0127] In some embodiments, the first expansion and the second expansion are each performed separately within a period of 11 days.

[0128] In some embodiments, the first expansion in step and the second expansion in step are each performed separately within a period of 11 to 12 days.

[0129] In some embodiments, steps (a) through (e), (f), (g), or (h) are carried out in about 10 days to about 24 days.

[0130] In some embodiments, steps (a)-(e), (f), (g), and (h) are carried out in about 15 days to about 24 days.

[0131] In some embodiments, steps (a) through (e), (f), (g), or (h) are carried out in about 20 days to about 24 days.

[0132] In some embodiments, steps (a)-(e), (f), (g), and (h) are carried out in about 20 to about 22 days.

[0133] In some embodiments, the expansion process (steps (a) through (f)) is carried out within about 22 days.

[0134] In some embodiments, the expansion process (steps (a) through (f)) is carried out within about 24 days.

[0135] In some embodiments, the expansion process (steps (a) through (f)) is carried out within about 26 days.

[0136] In some embodiments, the priming first expansion and the rapid second expansion occur over a period of 21 days or less.

[0137] In some embodiments, the priming first expansion and the rapid second expansion occur over a period of no more than 16 or 17 days.

[0138] In some embodiments, the first expansion by priming is carried out over a period of no more than 7 or 8 days.

[0139] In some embodiments, the rapid second expansion occurs over a period of 11 days or less.

[0140] In some embodiments, the second TIL population is at least 50-fold more numerous than the first TIL population.

[0141] In some embodiments, the low dose of IL-2 is between 100 and 3000 IU / mL, optionally less than 1000 IU / mL.

[0142] In some embodiments, the concentration of IL-15 is about 10 ng / mL. In some embodiments, IL-21 is added at about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, about 55 ng / mL, or about 60 ng / mL, optionally at about 10 ng / mL.

[0143] In some embodiments, the concentration of IL-21 is about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, about 55 ng / mL, or about 60 ng / mL, optionally about 10 ng / mL.

[0144] In some embodiments, the concentration of the AKT inhibitor is about 1 uM, about 2 uM, about 3 uM, about 4 uM, about 5 uM, about 10 uM, about 20 uM, about 30 uM, about 40 uM, about 50 uM, or about 100 uM, optionally about 5 uM.

[0145] In some embodiments, the concentration of IFNg (interferon gamma) for stimulation is 200 ng / ml.

[0146] In some embodiments, the inhibitor of PD-1 is an anti-PD-1 antibody.

[0147] In some embodiments, the anti-PD-1 antibody is pembrolizumab or nivolumab.

[0148] In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody.

[0149] In some embodiments, the CTLA-4 agonist is a CTLA-4 antibody.

[0150] In some embodiments, the CTLA-4 antibody is ipilimumab, tremelimumab, or zalifrelimab.

[0151] The present invention also provides a population of TILs according to any of the methods described herein.

[0152] The present invention also provides a population of TILs cryopreserved by any of the methods described herein.

[0153] In some embodiments, IL-2 is at 1000 IU / ml, IL-21 is at 10 ng / ml, and AKTi is at 5 uM.

[0154] The present invention provides a method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) performing a first priming expansion by culturing a first TIL population obtained from a tumor from a cancer patient in a cell culture medium comprising IL-2, optionally OKT-3, and antigen-presenting cells (APCs) to produce a second TIL population, wherein the first priming expansion is performed in a container comprising a first gas permeable surface area, and the first priming expansion is performed for a first period of about 1 to 7 / 8 days to obtain a second TIL population, wherein the second TIL population is greater in number than the first TIL population, and optionally the cell culture medium is replaced on day 3 of the first expansion, day 4 of the first expansion, day 5 of the first expansion, day 6 of the first expansion, or day 7 of the first expansion; (b) performing a second rapid expansion by supplementing the 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 in the second rapid expansion is at least twice the number of APCs added in step (b), and the second rapid expansion is performed for a second period of about 1 to 11 days to obtain the third TIL population, the third TIL population being a therapeutic TIL population, and the second rapid expansion is performed in a container comprising a second gas permeable surface area, and optionally, the cell culture medium is replaced on day 3 of the second expansion, day 4 of the second expansion, day 5 of the second expansion, day 6 of the second expansion, or day 7 of the second expansion; (c) harvesting the therapeutic TIL population obtained from step (b).

[0155] In some embodiments, the method comprises: (d) transferring the harvested third population of TILs from step (c) to an infusion bag; (e) cryopreserving the infusion bag containing the harvested third population of TILs from step (d) using a cryopreservation process.

[0156] In some embodiments, the culture medium in step (a) comprises no more than 3000 IU / mL of IL-2, and / or a protein kinase B (AKT) inhibitor.

[0157] In some embodiments, the culture medium in step (a) or step (b) comprises a protein kinase B (AKT) inhibitor.

[0158] In some embodiments, the culture medium in step (a) or step (b) comprises an AKT inhibitor at a concentration of about 0.1 μM to about 10 μM.

[0159] In some embodiments, the culture medium in step (a) or step (b) comprises an AKT inhibitor at a concentration of about 1 μM.

[0160] In some embodiments, the culture medium in step (a) and / or step (b) comprises IL-15 and / or IL-21.

[0161] In some embodiments, the culture medium in step (a) and / or step (b) comprises IL-15 and / or IL-21 at a concentration of about 1 ng / mL to about 100 ng / mL.

[0162] In some embodiments, the culture medium in step (a) and / or step (b) comprises IL-15 and / or IL-21 at a concentration of about 10 ng / mL.

[0163] In some embodiments, the method further comprises adding IFNγ and an anti-PD-1 antibody at a concentration of 200 ng / ml during DO, D1 or D2 of step (a) and / or step (b).

[0164] In some embodiments, the method further comprises adding a CD40 agonist and / or an inhibitor of CTLA-4 during DO, D1 or D2 of step (a) and / or step (b).

[0165] In some embodiments, the AKT inhibitor is selected from the group consisting of ipatasertib, GSK690693, GSK2141795, GSK2110183, AZD5363, GDC-0068, AT7867, CCT128930, MK-2206, BAY1125976, perifosine, oridonin, herbacetin, teheranolide, isoliquirigenin, scutellarin, and phosphatidylcholine. Nokiol is selected from the group consisting of:

[0166] In some embodiments, the AKT inhibitor is AZD5363.

[0167] In some embodiments, the method includes enzymatically digesting a plurality of tumor fragments to obtain a tumor digest comprising a first population of TILs.

[0168] In some embodiments, the first TIL population is obtained 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 the cancer.

[0169] In some embodiments, during the second expansion, the second TIL population is split into one or more cultures and supplemented with additional cell culture medium containing IL-2, OKT-3, and APC.

[0170] 120. The method of claim 119, wherein the second TIL population is divided into 2, 3, 4, 5, 6, 7, 8, 9, or 10 cultures.

[0171] In some embodiments, the second TIL population is split on day 2 of the second expansion, day 3 of the second expansion, day 4 of the second expansion, or day 5 of the second expansion.

[0172] 10. The method of any one of the preceding claims, wherein in some embodiments, a closed system is optional.

[0173] 10. The method of any one of the preceding claims, in some embodiments, wherein the system is not closed.

[0174] Some embodiments of the present disclosure provide therapeutic TIL populations produced using the methods disclosed herein.

[0175] In some embodiments, the therapeutic TIL population is about 2.3 x 10 10 ~Approx. 13.7×10 10 Including TIL.

[0176] In some embodiments, the therapeutic TIL population is about 1 x 10 9 ~Approx. 1×10 11 Including TIL.

[0177] In some embodiments, the therapeutic TIL population exhibits enhanced polyfunctionality compared to a TIL population produced using a reference TIL production process.

[0178] In some embodiments, the therapeutic TIL population exhibits a more stem-like phenotype compared to a TIL population produced using a reference TIL production process.

[0179] In some embodiments, the therapeutic TIL population exhibits an increased frequency of less activated and / or differentiated TILs compared to a TIL population produced using a reference TIL production process.

[0180] In some embodiments, the therapeutic TIL populations exhibit improved tumor cell killing in an allogeneic setting compared to TIL populations produced using a reference TIL production process.

[0181] In some embodiments, the therapeutic TIL population exhibits reduced expression of CD27, CD28, CD62L, and IL-7R compared to a TIL population produced using a reference TIL production process. The present invention shows increased expression of memory-associated markers selected from the group consisting of:

[0182] In some embodiments, the therapeutic TIL population exhibits reduced expression of activation markers selected from the group consisting of CD38, CD39, and CD69 compared to a TIL population produced using a reference TIL production process.

[0183] In some embodiments, the therapeutic TIL population exhibits reduced expression of an inhibition / depletion-associated marker selected from the group consisting of LAG3, TIM3, TIGIT, and TOX compared to a TIL population produced using a reference TIL production process.

[0184] In some embodiments, the therapeutic TIL population exhibits increased expression of functional markers selected from the group consisting of GZMB, CXCR3, IFNg, TNFa, and IL-2 compared to a TIL population produced using a reference TIL production process. [Brief explanation of the drawings]

[0185] [Figure 1] An exemplary Gen2 (Process 2A) chart providing an overview of steps A-F. [Figure 2A] 1 is a process flow diagram of an embodiment of Gen2 (Process 2A) for TIL fabrication. [Figure 2B] 1 is a process flow diagram of an embodiment of Gen2 (Process 2A) for TIL fabrication. [Figure 2C] 1 is a process flow diagram of an embodiment of Gen2 (Process 2A) for TIL fabrication. [Figure 3] FIG. 1 shows a diagram of an embodiment of an exemplary manufacturing process (approximately 22 days) for cryopreserved TILs. [Figure 4] FIG. 1 shows a diagram of an embodiment of Gen2 (Process 2A), a 22-day process for TIL fabrication. [Figure 5] 1 is a comparison table of steps A-F from exemplary embodiments of Process 1C and Gen2 (Process 2A) for TIL fabrication. [Figure 6] Detailed comparison of Process 1C and Gen2 (Process 2A) embodiments for TIL fabrication. [Figure 7] 1. Exemplary Gen3 TIL fabrication process. [Figure 8A] A comparison of embodiments of the 2A process (an approximately 22 day process) and the Gen3 process (an approximately 14-16 day process) for TIL fabrication is shown. [Figure 8B] An exemplary Process Gen3 chart providing an overview of steps A-F (approximately a 14-16 day process). [Figure 8C] A chart providing three exemplary Gen3 processes along with an overview of steps A-F (approximately 14- to 16-day processes) for each of the three process variations. [Figure 8D] An exemplary modified Gen2-like process (approximately a 22-day process) providing an overview of steps A-F. [Figure 8E] A comparison of the 2A process (approximately a 22 day process) and an embodiment of the Gen3 process (approximately a 14-22 day process) for TIL fabrication is shown. [Figure 8F] Exemplary process (a) CD39 / CD69 double negative, (b) CD39 / CD69LO / LO, or combined (i) and (ii) TIL expansion method Gen3 chart providing an overview of steps A-F (approximately a 14- to 22-day process). [Figure 8G] Exemplary embodiments of (a) CD39 / CD69 double negative, (b) CD39 / CD69LO / LO, or a combination of (i) and (ii) TIL expansion methods using pre-selection as described herein. [Figure 8H] An exemplary TS-TIL process (approximately a 22-day process) providing an overview of steps A-F. [Figure 8I] An exemplary modified I-TIL process (approximately a 22-day process) providing an overview of steps A-F. [Figure 8J] An exemplary modified TS-TIL process (approximately a 22-day process) providing an overview of steps A-F. [Figure 9] 1 provides an experimental flow chart for the comparison between Gen2 (Process 2A) and Gen3 processes. [Figure 10] 1 shows a comparison of various Gen2 (Process 2A) and Gen3.1 process embodiments. [Figure 11] 1 is a table illustrating various features of embodiments of the Gen2, Gen2.1, and Gen3.0 processes. [Figure 12] Summary of media conditions for an embodiment of the Gen3 process, designated Gen3.1. [Figure 13] 1 is a table illustrating various features of embodiments of the Gen2, Gen2.1, and Gen3.0 processes. [Figure 14] 1 is a table comparing various features of embodiments of the Gen2 and Gen3.0 processes. [Figure 15] 1 is a table providing media use in various embodiments of the described expansion process. [Figure 16] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 17]Schematic of an exemplary embodiment of a method for expanding T cells from hematopoietic malignancies using the Gen3 expansion platform. [Figure 18] Structures IA and IB are provided. The cylinders refer to individual polypeptide binding domains. Structures IA and IB comprise three linearly linked TNFRSF-binding domains derived from antibodies that bind, for example, to 4-1BBL or 4-1BB, that fold to form a trivalent protein, which is then linked to a second trivalent protein via IgG1-Fc (comprising the CH3 and CH2 domains), which is then used to link two of the trivalent proteins together via disulfide bonds (small oblong ellipses), stabilizing the structure and providing an agonist that can bring together the six receptor and intracellular signaling domains of the signaling protein to form a signaling complex. The TNFRSF-binding domains shown as cylinders can be, for example, scFv domains comprising VH and VL chains connected by a linker that may contain hydrophilic residues and Gly and Ser sequences for flexibility, and Glu and Lys for solubility. [Figure 19] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 20] 1 provides a process overview of an exemplary embodiment of the Gen3.1 process (16-day process). [Figure 21] Schematic of an exemplary embodiment of the Gen3.1 testing process (16-17 day process). [Figure 22] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 23A] 1 is a comparison table of an exemplary Gen2 process and an exemplary Gen3 process. [Figure 23B] 1 is a comparison table of an exemplary Gen2 process and an exemplary Gen3 process. [Figure 24] Schematic of an exemplary embodiment of the preparation timeline for the Gen3 process (16 / 17 day process). [Figure 25] Schematic of an exemplary embodiment of the Gen3 process (14-16 day process). [Figure 26A] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 26B] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 27] Schematic of an exemplary embodiment of the Gen3 process (16-day process). [Figure 28] Comparison of Gen2, Gen2.1, and Gen3 process (16-day process) embodiments. [Figure 29] Comparison of Gen2, Gen2.1, and Gen3 process (16-day process) embodiments. [Figure 30] Components of a Gen3 embodiment. [Figure 31] Flowchart comparison of Gen3 embodiments (Gen3.0, Gen3.1 control, Gen3.1 test). [Figure 32] Components of an exemplary embodiment of the Gen3 process (16-17 day process) are shown. [Figure 33] Approval criteria table [Figure 34] Schematic diagram of the workflow of Example 15. [Figure 35] Schematic diagram of the workflow of Example 17. [Figure 36A] TIL expansion (A) and viability (B) under standard or modified REP conditions, including different concentrations of IL-2 or IL-15 given once or twice during the REP expansion process. [Figure 36B] TIL expansion (A) and viability (B) under standard or modified REP conditions, including different concentrations of IL-2 or IL-15 given once or twice during the REP expansion process. [Figure 37-1] Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+), and live TILs following standard or modified REP conditions containing different concentrations of IL-2 or IL-15 given once or twice during the REP expansion process. [Figure 37-2]Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+), and live TILs following standard or modified REP conditions containing different concentrations of IL-2 or IL-15 given once or twice during the REP expansion process. [Figure 38A] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions, including different concentrations of IL-2 or IL-15 given once or twice during the REP expansion process. TILs were thawed and stained to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, and (E) TIM3 by flow cytometry. [Figure 38B] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions, including different concentrations of IL-2 or IL-15 given once or twice during the REP expansion process. TILs were thawed and stained to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, and (E) TIM3 by flow cytometry. [Figure 38C] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions, including different concentrations of IL-2 or IL-15 given once or twice during the REP expansion process. TILs were thawed and stained to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, and (E) TIM3 by flow cytometry. [Figure 38D] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions, including different concentrations of IL-2 or IL-15 given once or twice during the REP expansion process. TILs were thawed and stained to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, and (E) TIM3 by flow cytometry. [Figure 38E]Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions, including different concentrations of IL-2 or IL-15 given once or twice during the REP expansion process. TILs were thawed and stained to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, and (E) TIM3 by flow cytometry. [Figure 39A] Frequency of PD-1 and TIM3 subsets in CD8+ and CD4+ TILs following standard or modified REP conditions with different concentrations of IL-2 or IL-15 given once or twice during the REP expansion process. (A) Frequency of PD-1+TIM3+ TILs and (B) PD-1-TIM3- TILs. [Figure 39B] Frequency of PD-1 and TIM3 subsets in CD8+ and CD4+ TILs following standard or modified REP conditions with different concentrations of IL-2 or IL-15 given once or twice during the REP expansion process. (A) Frequency of PD-1+TIM3+ TILs and (B) PD-1-TIM3- TILs. [Figure 40A] TIL expansion (A) and viability (B) following standard or modified REP conditions containing 1 uM GDC-0068 or IL-15 alone or in combination with IL-21, with or without IL-21, when added once or twice during the expansion process. [Figure 40B] TIL expansion (A) and viability (B) following standard or modified REP conditions containing 1 uM GDC-0068 or IL-15 alone or in combination with IL-21, with or without IL-21, when added once or twice during the expansion process. [Figure 41] Frequencies of NK, NKT cells and viable cells following standard or modified REP conditions, including 1 uM GDC-0068 or IL-15 alone or in combination with IL-21, with or without IL-21, when added once or twice during the expansion process. [Figure 42A]Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions, including 1 uM GDC-0068 or IL-15 alone or in combination with IL-21, or different concentrations of IL-2 in combination with IL-21, when added once or twice during the expansion process. TILs were thawed and stained to measure expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, and (E) TIM3 by flow cytometry. [Figure 42B] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions, including 1 uM GDC-0068 or IL-15 alone or in combination with IL-21, or different concentrations of IL-2 in combination with IL-21, when added once or twice during the expansion process. TILs were thawed and stained to measure expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, and (E) TIM3 by flow cytometry. [Figure 42C] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions, including 1 uM GDC-0068 or IL-15 alone or in combination with IL-21, or different concentrations of IL-2 in combination with IL-21, when added once or twice during the expansion process. TILs were thawed and stained to measure expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, and (E) TIM3 by flow cytometry. [Figure 42D] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions, including 1 uM GDC-0068 or IL-15 alone or in combination with IL-21, or different concentrations of IL-2 in combination with IL-21, when added once or twice during the expansion process. TILs were thawed and stained to measure expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, and (E) TIM3 by flow cytometry. [Figure 42E]Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions, including 1 uM GDC-0068 or IL-15 alone or in combination with IL-21, or different concentrations of IL-2 in combination with IL-21, when added once or twice during the expansion process. TILs were thawed and stained to measure expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, and (E) TIM3 by flow cytometry. [Figure 43A] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions, including 1 uM GDC-0068 or IL-15 alone or in combination with IL-21, or different concentrations of IL-2 in combination with IL-21, when added once or twice during the expansion process. TILs were thawed and stained to measure expression of (A) CD25 (B) CD38, and (C) NKG2D by flow cytometry. [Figure 43B] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions, including 1 uM GDC-0068 or IL-15 alone or in combination with IL-21, or different concentrations of IL-2 in combination with IL-21, when added once or twice during the expansion process. TILs were thawed and stained to measure expression of (A) CD25 (B) CD38, and (C) NKG2D by flow cytometry. [Figure 43C] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions, including 1 uM GDC-0068 or IL-15 alone or in combination with IL-21, or different concentrations of IL-2 in combination with IL-21, when added once or twice during the expansion process. TILs were thawed and stained to measure expression of (A) CD25 (B) CD38, and (C) NKG2D by flow cytometry. [Figure 44A]Expression of (A) CD69+CD39+ and (B) CD69-CD39-CD8+ TILs following standard or modified REP conditions, including 1 uM GDC-0068 or IL-15 alone or in combination with IL-21, with or without IL-21, when added once or twice during the expansion process. [Figure 44B] Expression of (A) CD69+CD39+ and (B) CD69-CD39-CD8+ TILs following standard or modified REP conditions, including 1 uM GDC-0068 or IL-15 alone or in combination with IL-21, with or without IL-21, when added once or twice during the expansion process. [Figure 45A] Frequency of PD-1 and TIM3 subsets in CD8+ and CD4+ TILs following standard or modified REP conditions, including 1 uM GDC-0068 or IL-15 alone or at different concentrations of IL-2 in combination with IL-21, with or without combination with IL-21, when added once or twice during the expansion process. (A) Frequency of PD-1+TIM3+ TILs and (B) PD-1-TIM3- TILs. [Figure 45B] Frequency of PD-1 and TIM3 subsets in CD8+ and CD4+ TILs following standard or modified REP conditions, including 1 uM GDC-0068 or IL-15 alone or at different concentrations of IL-2 in combination with IL-21, with or without combination with IL-21, when added once or twice during the expansion process. (A) Frequency of PD-1+TIM3+ TILs and (B) PD-1-TIM3- TILs. [Figure 46A] TIL expansion (A) and viability (B) under standard or modified REP conditions containing IL-2 or IL-15 alone or in combination with different concentrations of IL-21 added twice during the expansion process. [Figure 46B] TIL expansion (A) and viability (B) under standard or modified REP conditions containing IL-2 or IL-15 alone or in combination with different concentrations of IL-21 added twice during the expansion process. [Figure 47A] Marker expression on CD8+ and CD4+ TILs under standard or modified REP conditions, including IL-2 or IL-15 alone or in combination with different concentrations of IL-21 added twice during the expansion process. TILs were thawed and stained to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, and (E) TIM3 by flow cytometry. [Figure 47B] Marker expression on CD8+ and CD4+ TILs under standard or modified REP conditions, including IL-2 or IL-15 alone or in combination with different concentrations of IL-21 added twice during the expansion process. TILs were thawed and stained to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, and (E) TIM3 by flow cytometry. [Figure 47C] Marker expression on CD8+ and CD4+ TILs under standard or modified REP conditions, including IL-2 or IL-15 alone or in combination with different concentrations of IL-21 added twice during the expansion process. TILs were thawed and stained to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, and (E) TIM3 by flow cytometry. [Figure 47D] Marker expression on CD8+ and CD4+ TILs under standard or modified REP conditions, including IL-2 or IL-15 alone or in combination with different concentrations of IL-21 added twice during the expansion process. TILs were thawed and stained to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, and (E) TIM3 by flow cytometry. [Figure 47E] Marker expression on CD8+ and CD4+ TILs under standard or modified REP conditions, including IL-2 or IL-15 alone or in combination with different concentrations of IL-21 added twice during the expansion process. TILs were thawed and stained to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, and (E) TIM3 by flow cytometry. [Figure 48A]Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions with IL-2 or IL-15 alone or in combination with different concentrations of IL-21 added twice during the expansion process. TILs were thawed and stained to measure expression of (A) CD25 and (B) CD38 by flow cytometry. [Figure 48B] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions with IL-2 or IL-15 alone or in combination with different concentrations of IL-21 added twice during the expansion process. TILs were thawed and stained to measure expression of (A) CD25 and (B) CD38 by flow cytometry. [Figure 49A] Expression of (A) CD69+CD39+ and (B) CD69-CD39-CD8+ TILs following standard or modified REP conditions containing IL-2 or IL-15 alone or in combination with different concentrations of IL-21 added twice during the expansion process. [Figure 49B] Expression of (A) CD69+CD39+ and (B) CD69-CD39-CD8+ TILs following standard or modified REP conditions containing IL-2 or IL-15 alone or in combination with different concentrations of IL-21 added twice during the expansion process. [Figure 50A] Frequency of CD40L expression on (A) CD8+ (B) CD4+ TILs after overnight stimulation with TransAct and 4 h of culture the following day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions containing IL-2 or IL-15 alone or in combination with different concentrations of IL-21, which were added twice during the expansion process. [Figure 50B] Frequency of CD40L expression on (A) CD8+ (B) CD4+ TILs after overnight stimulation with TransAct and 4 h of culture the following day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions containing IL-2 or IL-15 alone or in combination with different concentrations of IL-21, which were added twice during the expansion process. [Figure 51A]TIL expansion (A) and viability (B) under standard or modified REP conditions containing IL-2 or IL-15 alone or in combination with different concentrations of IL-21 with or without 5uM AKTi (GDC-0068) added twice during the expansion process. [Figure 51B] TIL expansion (A) and viability (B) under standard or modified REP conditions containing IL-2 or IL-15 alone or in combination with different concentrations of IL-21 with or without 5uM AKTi (GDC-0068) added twice during the expansion process. [Figure 52A] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions, including IL-2 or IL-15 alone or in combination with different concentrations of IL-21 with or without 5uM AKTi (GDC-0068) added twice during the expansion process. TILs were thawed and stained to measure expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, and (E) TIM3 by flow cytometry. [Figure 52B] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions, including IL-2 or IL-15 alone or in combination with different concentrations of IL-21 with or without 5uM AKTi (GDC-0068) added twice during the expansion process. TILs were thawed and stained to measure expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, and (E) TIM3 by flow cytometry. [Figure 52C] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions, including IL-2 or IL-15 alone or in combination with different concentrations of IL-21 with or without 5uM AKTi (GDC-0068) added twice during the expansion process. TILs were thawed and stained to measure expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, and (E) TIM3 by flow cytometry. [Figure 52D]Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions, including IL-2 or IL-15 alone or in combination with different concentrations of IL-21 with or without 5uM AKTi (GDC-0068) added twice during the expansion process. TILs were thawed and stained to measure expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, and (E) TIM3 by flow cytometry. [Figure 52E] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions, including IL-2 or IL-15 alone or in combination with different concentrations of IL-21 with or without 5uM AKTi (GDC-0068) added twice during the expansion process. TILs were thawed and stained to measure expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, and (E) TIM3 by flow cytometry. [Figure 53A] Expression of (A) CD69+CD39+ and (B) CD69-CD39-CD8+ TILs following standard or modified REP conditions containing IL-2 or IL-15 alone or in combination with different concentrations of IL-21 with or without 5uM AKTi (GDC-0068) added twice during the expansion process. [Figure 53B] Expression of (A) CD69+CD39+ and (B) CD69-CD39-CD8+ TILs following standard or modified REP conditions containing IL-2 or IL-15 alone or in combination with different concentrations of IL-21 with or without 5uM AKTi (GDC-0068) added twice during the expansion process. [Figure 54] Summary of media conditions for an embodiment of the I-TIL process. [Figure 55] Summary of media conditions for an embodiment of the I-TIL process. [Figure 56A] TIL expansion (A) and survival (B) under standard or modified REP conditions containing IL-21 (30 ng / ml) alone or in combination with different doses of IL-2 or IL-15 with or without Panobinostat. [Figure 56B]TIL expansion (A) and survival (B) under standard or modified REP conditions containing IL-21 (30 ng / ml) alone or in combination with different doses of IL-2 or IL-15 with or without Panobinostat. [Figure 57] Frequencies of CD8+, CD4+ and live TILs under standard or modified REP conditions including IL-21 (30 ng / ml) alone or in combination with different doses of IL-2 or IL-15 with or without Panobinostat. [Figure 58A] Marker expression on CD8+ and CD4+ TILs under standard or modified REP conditions containing IL-21 (30 ng / ml) alone or in combination with different doses of IL-2 or IL-15 with or without panobinostat. TILs were thawed and stained to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, and (E) TIM3 by flow cytometry. [Figure 58B] Marker expression on CD8+ and CD4+ TILs under standard or modified REP conditions containing IL-21 (30 ng / ml) alone or in combination with different doses of IL-2 or IL-15 with or without panobinostat. TILs were thawed and stained to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, and (E) TIM3 by flow cytometry. [Figure 58C] Marker expression on CD8+ and CD4+ TILs under standard or modified REP conditions containing IL-21 (30 ng / ml) alone or in combination with different doses of IL-2 or IL-15 with or without panobinostat. TILs were thawed and stained to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, and (E) TIM3 by flow cytometry. [Figure 58D]Marker expression on CD8+ and CD4+ TILs under standard or modified REP conditions containing IL-21 (30 ng / ml) alone or in combination with different doses of IL-2 or IL-15 with or without panobinostat. TILs were thawed and stained to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, and (E) TIM3 by flow cytometry. [Figure 58E] Marker expression on CD8+ and CD4+ TILs under standard or modified REP conditions containing IL-21 (30 ng / ml) alone or in combination with different doses of IL-2 or IL-15 with or without panobinostat. TILs were thawed and stained to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, and (E) TIM3 by flow cytometry. [Figure 59A] Frequency of PD-1 and TIM3 subsets in CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (30 ng / ml) alone or in combination with different doses of IL-2 or IL-15 with or without panobinostat. (A) Frequency of PD-1+TIM3+ TILs and (B) PD-1-TIM3- TILs. [Figure 59B] Frequency of PD-1 and TIM3 subsets in CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (30 ng / ml) alone or in combination with different doses of IL-2 or IL-15 with or without panobinostat. (A) Frequency of PD-1+TIM3+ TILs and (B) PD-1-TIM3- TILs. [Figure 60A] TIL expansion (A) and viability (B) under standard or modified REP conditions containing different concentrations of IL-2 in combination with IL-21 or 2-HC. [Figure 60B] TIL expansion (A) and viability (B) under standard or modified REP conditions containing different concentrations of IL-2 in combination with IL-21 or 2-HC. [Figure 61-1]Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+) and live TILs following standard or modified REP conditions containing different concentrations of IL-2 in combination with IL-21 or 2-HC. [Figure 61-2] Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+) and live TILs following standard or modified REP conditions containing different concentrations of IL-2 in combination with IL-21 or 2-HC. [Figure 62A] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing different concentrations of IL-2 in combination with IL-21 or 2-HC. TILs were thawed and stained to measure expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, and (E) TIM3 by flow cytometry. [Figure 62B] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing different concentrations of IL-2 in combination with IL-21 or 2-HC. TILs were thawed and stained to measure expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, and (E) TIM3 by flow cytometry. [Figure 62C] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing different concentrations of IL-2 in combination with IL-21 or 2-HC. TILs were thawed and stained to measure expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, and (E) TIM3 by flow cytometry. [Figure 62D] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing different concentrations of IL-2 in combination with IL-21 or 2-HC. TILs were thawed and stained to measure expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, and (E) TIM3 by flow cytometry. [Figure 62E]Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing different concentrations of IL-2 in combination with IL-21 or 2-HC. TILs were thawed and stained to measure expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, and (E) TIM3 by flow cytometry. [Figure 63A] TIL expansion (A) and viability (B) under standard or modified REP conditions with 1 uM GDC-0068 or IL-15 alone or with or without combination with IL-21, and different concentrations of IL-2 in combination with IL-21, when added once or twice during the expansion process. [Figure 63B] TIL expansion (A) and viability (B) under standard or modified REP conditions with 1 uM GDC-0068 or IL-15 alone or with or without combination with IL-21, and different concentrations of IL-2 in combination with IL-21, when added once or twice during the expansion process. [Figure 64-1] Frequency of PD-1 and TIM3 subsets in CD8+ and CD4+ TILs following standard or modified REP conditions containing different concentrations of IL-2 in combination with IL-21 or 2-HC. (A) Frequency of PD-1+TIM3+ TILs and (B) PD-1-TIM3- TILs. [Figure 64-2] Frequency of PD-1 and TIM3 subsets in CD8+ and CD4+ TILs following standard or modified REP conditions containing different concentrations of IL-2 in combination with IL-21 or 2-HC. (A) Frequency of PD-1+TIM3+ TILs and (B) PD-1-TIM3- TILs. [Figure 64-3] Frequency of PD-1 and TIM3 subsets in CD8+ and CD4+ TILs following standard or modified REP conditions containing different concentrations of IL-2 in combination with IL-21 or 2-HC. (A) Frequency of PD-1+TIM3+ TILs and (B) PD-1-TIM3- TILs. [Figure 65A]Frequency of (A) IFNg (B) TNFa (C) IFNg + TNFa + (D) Ki-67 (E) PD-1 and (F) TOX expression on CD8+ TILs after overnight stimulation with TransAct and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions with 1 uM GDC-0068 or IL-15 alone, or different concentrations of IL-2 in combination with IL-21, with or without combination with IL-21, when added once or twice during the expansion process. [Figure 65B] Frequency of (A) IFNg (B) TNFa (C) IFNg + TNFa + (D) Ki-67 (E) PD-1 and (F) TOX expression on CD8+ TILs after overnight stimulation with TransAct and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions with 1 uM GDC-0068 or IL-15 alone, or different concentrations of IL-2 in combination with IL-21, with or without combination with IL-21, when added once or twice during the expansion process. [Figure 65C] Frequency of (A) IFNg (B) TNFa (C) IFNg + TNFa + (D) Ki-67 (E) PD-1 and (F) TOX expression on CD8+ TILs after overnight stimulation with TransAct and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions with 1 uM GDC-0068 or IL-15 alone, or different concentrations of IL-2 in combination with IL-21, with or without combination with IL-21, when added once or twice during the expansion process. [Figure 65D]Frequency of (A) IFNg (B) TNFa (C) IFNg + TNFa + (D) Ki-67 (E) PD-1 and (F) TOX expression on CD8+ TILs after overnight stimulation with TransAct and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions with 1 uM GDC-0068 or IL-15 alone, or different concentrations of IL-2 in combination with IL-21, with or without combination with IL-21, when added once or twice during the expansion process. [Figure 65E] Frequency of (A) IFNg (B) TNFa (C) IFNg + TNFa + (D) Ki-67 (E) PD-1 and (F) TOX expression on CD8+ TILs after overnight stimulation with TransAct and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions with 1 uM GDC-0068 or IL-15 alone, or different concentrations of IL-2 in combination with IL-21, with or without combination with IL-21, when added once or twice during the expansion process. [Figure 65F] Frequency of (A) IFNg (B) TNFa (C) IFNg + TNFa + (D) Ki-67 (E) PD-1 and (F) TOX expression on CD8+ TILs after overnight stimulation with TransAct and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions with 1 uM GDC-0068 or IL-15 alone, or different concentrations of IL-2 in combination with IL-21, with or without combination with IL-21, when added once or twice during the expansion process. [Figure 66-1] Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+), NK, NKT cells and viable cells following standard or modified REP conditions containing IL-2 or IL-15 alone or in combination with different concentrations of IL-21 added twice during the expansion process. [Figure 66-2]Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+), NK, NKT cells and viable cells following standard or modified REP conditions containing IL-2 or IL-15 alone or in combination with different concentrations of IL-21 added twice during the expansion process. [Figure 66-3] Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+), NK, NKT cells and viable cells following standard or modified REP conditions containing IL-2 or IL-15 alone or in combination with different concentrations of IL-21 added twice during the expansion process. [Figure 66-4] Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+), NK, NKT cells and viable cells following standard or modified REP conditions containing IL-2 or IL-15 alone or in combination with different concentrations of IL-21 added twice during the expansion process. [Figure 66-5] Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+), NK, NKT cells and viable cells following standard or modified REP conditions containing IL-2 or IL-15 alone or in combination with different concentrations of IL-21 added twice during the expansion process. [Figure 66-6] Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+), NK, NKT cells and viable cells following standard or modified REP conditions containing IL-2 or IL-15 alone or in combination with different concentrations of IL-21 added twice during the expansion process. [Figure 67A] Frequency of PD-1 and TIM3 subsets in CD8+ and CD4+ TILs following standard or modified REP conditions, including IL-2 or IL-15 alone or in combination with different concentrations of IL-21 added twice during the expansion process. (A) Frequency of PD-1+TIM3+ TILs and (B) PD-1-TIM3- TILs. [Figure 67B]Frequency of PD-1 and TIM3 subsets in CD8+ and CD4+ TILs following standard or modified REP conditions, including IL-2 or IL-15 alone or in combination with different concentrations of IL-21 added twice during the expansion process. (A) Frequency of PD-1+TIM3+ TILs and (B) PD-1-TIM3- TILs. [Figure 68A] Frequency of (A) IFNg, (B) TNFa, (C) IFNg + TNFa + (D) Ki-67, (E) PD-1, and (F) TOX expression on CD8+ TILs after overnight stimulation with TransAct and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions containing IL-2 or IL-15 alone or in combination with different concentrations of IL-21, which were added twice during the expansion process. [Figure 68B] Frequency of (A) IFNg, (B) TNFa, (C) IFNg + TNFa + (D) Ki-67, (E) PD-1, and (F) TOX expression on CD8+ TILs after overnight stimulation with TransAct and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions containing IL-2 or IL-15 alone or in combination with different concentrations of IL-21, which were added twice during the expansion process. [Figure 68C] Frequency of (A) IFNg, (B) TNFa, (C) IFNg + TNFa + (D) Ki-67, (E) PD-1, and (F) TOX expression on CD8+ TILs after overnight stimulation with TransAct and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions containing IL-2 or IL-15 alone or in combination with different concentrations of IL-21, which were added twice during the expansion process. [Figure 68D] Frequency of (A) IFNg, (B) TNFa, (C) IFNg + TNFa + (D) Ki-67, (E) PD-1, and (F) TOX expression on CD8+ TILs after overnight stimulation with TransAct and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions containing IL-2 or IL-15 alone or in combination with different concentrations of IL-21, which were added twice during the expansion process. [Figure 68E] Frequency of (A) IFNg, (B) TNFa, (C) IFNg + TNFa + (D) Ki-67, (E) PD-1, and (F) TOX expression on CD8+ TILs after overnight stimulation with TransAct and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions containing IL-2 or IL-15 alone or in combination with different concentrations of IL-21, which were added twice during the expansion process. [Figure 68F] Frequency of (A) IFNg, (B) TNFa, (C) IFNg + TNFa + (D) Ki-67, (E) PD-1, and (F) TOX expression on CD8+ TILs after overnight stimulation with TransAct and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions containing IL-2 or IL-15 alone or in combination with different concentrations of IL-21, which were added twice during the expansion process. [Figure 69A] Frequency of (A) Ki-67 (B) PD-1, and (C) TOX expression on CD4+ TILs after overnight stimulation with TransAct and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions, including IL-2 or IL-15 alone or in combination with different concentrations of IL-21, which were added twice during the expansion process. [Figure 69B] Frequency of (A) Ki-67 (B) PD-1, and (C) TOX expression on CD4+ TILs after overnight stimulation with TransAct and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions, including IL-2 or IL-15 alone or in combination with different concentrations of IL-21, which were added twice during the expansion process. [Figure 69C] Frequency of (A) Ki-67 (B) PD-1, and (C) TOX expression on CD4+ TILs after overnight stimulation with TransAct and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions, including IL-2 or IL-15 alone or in combination with different concentrations of IL-21, which were added twice during the expansion process. [Figure 70-1] Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+) and viable cells following standard or modified REP conditions containing IL-2 or IL-15 alone or in combination with different concentrations of IL-21 with or without 5uM AKTi (GDC-0068) added twice during the expansion process. [Figure 70-2] Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+) and viable cells following standard or modified REP conditions containing IL-2 or IL-15 alone or in combination with different concentrations of IL-21 with or without 5uM AKTi (GDC-0068) added twice during the expansion process. [Figure 71A] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions, including IL-2 or IL-15 alone or in combination with different concentrations of IL-21 with or without 5uM AKTi (GDC-0068) added twice during the expansion process. TILs were thawed and stained to measure (A) CD25 and (B) CD38 expression by flow cytometry. [Figure 71B] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions, including IL-2 or IL-15 alone or in combination with different concentrations of IL-21 with or without 5uM AKTi (GDC-0068) added twice during the expansion process. TILs were thawed and stained to measure (A) CD25 and (B) CD38 expression by flow cytometry. [Figure 72A] Frequency of PD-1 and TIM3 subsets in CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 or IL-15 alone or in combination with different concentrations of IL-21 with or without 5uM AKTi (GDC-0068) added twice during the expansion process. (A) Frequency of PD-1+TIM3+ TILs and (B) PD-1-TIM3- TILs. [Figure 72B]Frequency of PD-1 and TIM3 subsets in CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 or IL-15 alone or in combination with different concentrations of IL-21 with or without 5uM AKTi (GDC-0068) added twice during the expansion process. (A) Frequency of PD-1+TIM3+ TILs and (B) PD-1-TIM3- TILs. [Figure 73A] Frequency of (A) IFNg (B) TNFa (C) IFNg+TNFa+ (D) Ki-67 (E) PD-1 and (F) TOX expression on CD8+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions, including IL-2 or IL-15 alone or in combination with different concentrations of IL-21 with or without 5uM AKTi (GDC-0068) added twice during the expansion process. [Figure 73B] Frequency of (A) IFNg (B) TNFa (C) IFNg+TNFa+ (D) Ki-67 (E) PD-1 and (F) TOX expression on CD8+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions, including IL-2 or IL-15 alone or in combination with different concentrations of IL-21 with or without 5uM AKTi (GDC-0068) added twice during the expansion process. [Figure 73C] Frequency of (A) IFNg (B) TNFa (C) IFNg+TNFa+ (D) Ki-67 (E) PD-1 and (F) TOX expression on CD8+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions, including IL-2 or IL-15 alone or in combination with different concentrations of IL-21 with or without 5uM AKTi (GDC-0068) added twice during the expansion process. [Figure 73D]Frequency of (A) IFNg (B) TNFa (C) IFNg+TNFa+ (D) Ki-67 (E) PD-1 and (F) TOX expression on CD8+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions, including IL-2 or IL-15 alone or in combination with different concentrations of IL-21 with or without 5uM AKTi (GDC-0068) added twice during the expansion process. [Figure 73E] Frequency of (A) IFNg (B) TNFa (C) IFNg+TNFa+ (D) Ki-67 (E) PD-1 and (F) TOX expression on CD8+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions, including IL-2 or IL-15 alone or in combination with different concentrations of IL-21 with or without 5uM AKTi (GDC-0068) added twice during the expansion process. [Figure 73F] Frequency of (A) IFNg (B) TNFa (C) IFNg+TNFa+ (D) Ki-67 (E) PD-1 and (F) TOX expression on CD8+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions, including IL-2 or IL-15 alone or in combination with different concentrations of IL-21 with or without 5uM AKTi (GDC-0068) added twice during the expansion process. [Figure 74A] Frequency of (A) Ki-67 (B) PD-1, and (C) TOX expression on CD4+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions, including IL-2 or IL-15 alone or in combination with different concentrations of IL-21 with or without 5uM AKTi (GDC-0068), which was added twice during the expansion process. [Figure 74B]Frequency of (A) Ki-67 (B) PD-1, and (C) TOX expression on CD4+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions, including IL-2 or IL-15 alone or in combination with different concentrations of IL-21 with or without 5uM AKTi (GDC-0068), which was added twice during the expansion process. [Figure 74C] Frequency of (A) Ki-67 (B) PD-1, and (C) TOX expression on CD4+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions, including IL-2 or IL-15 alone or in combination with different concentrations of IL-21 with or without 5uM AKTi (GDC-0068), which was added twice during the expansion process. [Figure 75A] Frequency of CD40L expression on (A) CD8+ (B) CD4+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 hours of culture the following day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions, including IL-2 or IL-15 alone or in combination with different concentrations of IL-21 with or without 5uM AKTi (GDC-0068), added twice during the expansion process. [Figure 75B] Frequency of CD40L expression on (A) CD8+ (B) CD4+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 hours of culture the following day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions, including IL-2 or IL-15 alone or in combination with different concentrations of IL-21 with or without 5uM AKTi (GDC-0068), added twice during the expansion process. [Figure 76A] Fold expansion (A) and viability (B) under modified pre-REP conditions containing different concentrations of IL-2 alone or in combination with different concentrations of GDC-0068 or IL-21 (10 ng / ml) added twice during the pre-REP expansion process. [Figure 76B]Fold expansion (A) and viability (B) under modified pre-REP conditions containing different concentrations of IL-2 alone or in combination with different concentrations of GDC-0068 or IL-21 (10 ng / ml) added twice during the pre-REP expansion process. [Figure 77A] Frequency of (A) CD127 and (B) CD62L on CD8 TILs under modified pre-REP conditions, including different concentrations of IL-2 alone or in combination with different concentrations of GDC-0068 or IL-21 (10 ng / ml) added twice during the pre-REP expansion process. [Figure 77B] Frequency of (A) CD127 and (B) CD62L on CD8 TILs under modified pre-REP conditions, including different concentrations of IL-2 alone or in combination with different concentrations of GDC-0068 or IL-21 (10 ng / ml) added twice during the pre-REP expansion process. [Figure 78A] Frequency of (A) CD69-CD39- and (B) CD69+CD39+CD8 TILs on CD8 TILs under modified pre-REP conditions, including different concentrations of IL-2 alone or in combination with different concentrations of GDC-0068 or IL-21 (10 ng / ml) added twice during the pre-REP expansion process. [Figure 78B] Frequency of (A) CD69-CD39- and (B) CD69+CD39+CD8 TILs on CD8 TILs under modified pre-REP conditions, including different concentrations of IL-2 alone or in combination with different concentrations of GDC-0068 or IL-21 (10 ng / ml) added twice during the pre-REP expansion process. [Figure 79] Frequency of Tcm-like CD8 TILs under modified pre-REP conditions, including different concentrations of IL-2 alone or in combination with different concentrations of GDC-0068 or IL-21 (10 ng / ml) added twice during the pre-REP expansion process. [Figure 80A] TIL expansion (A) and viability (B) under standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) alone or in combination with different previously titrated concentrations of AKT inhibitors added twice during the expansion process. [Figure 80B] TIL expansion (A) and viability (B) under standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) alone or in combination with different previously titrated concentrations of AKT inhibitors added twice during the expansion process. [Figure 81A] Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+) and viable cells following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) alone or in combination with different previously titrated concentrations of AKT inhibitors added twice during the expansion process. [Figure 81B] Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+) and viable cells following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) alone or in combination with different previously titrated concentrations of AKT inhibitors added twice during the expansion process. [Figure 81C] Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+) and viable cells following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) alone or in combination with different previously titrated concentrations of AKT inhibitors added twice during the expansion process. [Figure 81D] Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+) and viable cells following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) alone or in combination with different previously titrated concentrations of AKT inhibitors added twice during the expansion process. [Figure 82A] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) alone or in combination with different previously titrated concentrations of AKT inhibitors added twice during the expansion process. TILs were thawed and stained to measure expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT by flow cytometry. [Figure 82B]Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) alone or in combination with different previously titrated concentrations of AKT inhibitors added twice during the expansion process. TILs were thawed and stained to measure expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT by flow cytometry. [Figure 82C] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) alone or in combination with different previously titrated concentrations of AKT inhibitors added twice during the expansion process. TILs were thawed and stained to measure expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT by flow cytometry. [Figure 82D] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) alone or in combination with different previously titrated concentrations of AKT inhibitors added twice during the expansion process. TILs were thawed and stained to measure expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT by flow cytometry. [Figure 82E] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) alone or in combination with different previously titrated concentrations of AKT inhibitors added twice during the expansion process. TILs were thawed and stained to measure expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT by flow cytometry. [Figure 82F]Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) alone or in combination with different previously titrated concentrations of AKT inhibitors added twice during the expansion process. TILs were thawed and stained to measure expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT by flow cytometry. [Figure 83A] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) alone or in combination with different previously titrated concentrations of AKT inhibitors added twice during the expansion process. TILs were thawed and stained to measure (A) CD25 and (B) CD38 expression by flow cytometry. [Figure 83B] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) alone or in combination with different previously titrated concentrations of AKT inhibitors added twice during the expansion process. TILs were thawed and stained to measure (A) CD25 and (B) CD38 expression by flow cytometry. [Figure 84A] Expression of (A) CD69+CD39+ and (B) CD69-CD39-CD8+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) alone or in combination with different previously titrated concentrations of AKT inhibitors added twice during the expansion process. [Figure 84B] Expression of (A) CD69+CD39+ and (B) CD69-CD39-CD8+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) alone or in combination with different previously titrated concentrations of AKT inhibitors added twice during the expansion process. [Figure 85A]Frequency of PD-1 and TIM3 subsets in CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) alone or in combination with different AKT inhibitors at previously titrated concentrations added twice during the expansion process. (A) Frequency of PD-1+TIM3+ TILs and (B) PD-1-TIM3- TILs. [Figure 85B] Frequency of PD-1 and TIM3 subsets in CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) alone or in combination with different AKT inhibitors at previously titrated concentrations added twice during the expansion process. (A) Frequency of PD-1+TIM3+ TILs and (B) PD-1-TIM3- TILs. [Figure 86A] Frequency of (A) IFNg, (B) TNFa, (C) IFNg + TNFa + (D) Ki-67, (E) PD-1, and (F) TOX expression on CD8+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions, including IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) alone or in combination with different previously titrated concentrations of AKT inhibitors, which were added twice during the expansion process. [Figure 86B] Frequency of (A) IFNg, (B) TNFa, (C) IFNg + TNFa + (D) Ki-67, (E) PD-1, and (F) TOX expression on CD8+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions, including IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) alone or in combination with different previously titrated concentrations of AKT inhibitors, which were added twice during the expansion process. [Figure 86C]Frequency of (A) IFNg, (B) TNFa, (C) IFNg + TNFa + (D) Ki-67, (E) PD-1, and (F) TOX expression on CD8+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions, including IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) alone or in combination with different previously titrated concentrations of AKT inhibitors, which were added twice during the expansion process. [Figure 86D] Frequency of (A) IFNg, (B) TNFa, (C) IFNg + TNFa + (D) Ki-67, (E) PD-1, and (F) TOX expression on CD8+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions, including IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) alone or in combination with different previously titrated concentrations of AKT inhibitors, which were added twice during the expansion process. [Figure 86E] Frequency of (A) IFNg, (B) TNFa, (C) IFNg + TNFa + (D) Ki-67, (E) PD-1, and (F) TOX expression on CD8+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions, including IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) alone or in combination with different previously titrated concentrations of AKT inhibitors, which were added twice during the expansion process. [Figure 86F] Frequency of (A) IFNg, (B) TNFa, (C) IFNg + TNFa + (D) Ki-67, (E) PD-1, and (F) TOX expression on CD8+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions, including IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) alone or in combination with different previously titrated concentrations of AKT inhibitors, which were added twice during the expansion process. [Figure 87A]Frequency of (A) Ki-67 (B) PD-1, and (C) TOX expression on CD4+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions, including IL-2 (1000 IU / ml) plus IL-21 (10 ng / ml) alone or in combination with different previously titrated concentrations of AKT inhibitors, which were added twice during the expansion process. [Figure 87B] Frequency of (A) Ki-67 (B) PD-1, and (C) TOX expression on CD4+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions, including IL-2 (1000 IU / ml) plus IL-21 (10 ng / ml) alone or in combination with different previously titrated concentrations of AKT inhibitors, which were added twice during the expansion process. [Figure 87C] Frequency of (A) Ki-67 (B) PD-1, and (C) TOX expression on CD4+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions, including IL-2 (1000 IU / ml) plus IL-21 (10 ng / ml) alone or in combination with different previously titrated concentrations of AKT inhibitors, which were added twice during the expansion process. [Figure 88A] Frequency of CD40L expression on (A) CD8+ (B) CD4+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 h of culture the following day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions, including IL-2 (1000 IU / ml) plus IL-21 (10 ng / ml) alone or in combination with different, previously titrated concentrations of AKT inhibitors, which were added twice during the expansion process. [Figure 88B]Frequency of CD40L expression on (A) CD8+ (B) CD4+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 h of culture the following day in the presence of Brefeldin A. TILs were expanded under standard or modified REP conditions, including IL-2 (1000 IU / ml) plus IL-21 (10 ng / ml) alone or in combination with different, previously titrated concentrations of AKT inhibitors, which were added twice during the expansion process. [Figure 89A] TIL expansion (A) and survival (B) under standard or modified pre-REP conditions, including lower doses of IL-2, with or without various doses of IL-21 or the AKT inhibitor MK-2206. [Figure 89B] TIL expansion (A) and survival (B) under standard or modified pre-REP conditions, including lower doses of IL-2, with or without various doses of IL-21 or the AKT inhibitor MK-2206. [Figure 90A] Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+) and viable cells following standard or modified pre-REP conditions containing lower doses of IL-2 with or without various doses of IL-21 or the AKT inhibitor MK-2206. [Figure 90B] Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+) and viable cells following standard or modified pre-REP conditions containing lower doses of IL-2 with or without various doses of IL-21 or the AKT inhibitor MK-2206. [Figure 90C] Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+) and viable cells following standard or modified pre-REP conditions containing lower doses of IL-2 with or without various doses of IL-21 or the AKT inhibitor MK-2206. [Figure 90D] Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+) and viable cells following standard or modified pre-REP conditions containing lower doses of IL-2 with or without various doses of IL-21 or the AKT inhibitor MK-2206. [Figure 91A]Marker expression on CD8+ and CD4+ TILs following standard or modified pre-REP conditions, including lower doses of IL-2 with or without various doses of IL-21 or the AKT inhibitor MK-2206. TILs were stained by flow cytometry to measure expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT. [Figure 91B] Marker expression on CD8+ and CD4+ TILs following standard or modified pre-REP conditions, including lower doses of IL-2 with or without various doses of IL-21 or the AKT inhibitor MK-2206. TILs were stained by flow cytometry to measure expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT. [Figure 91C] Marker expression on CD8+ and CD4+ TILs following standard or modified pre-REP conditions, including lower doses of IL-2 with or without various doses of IL-21 or the AKT inhibitor MK-2206. TILs were stained by flow cytometry to measure expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT. [Figure 91D] Marker expression on CD8+ and CD4+ TILs following standard or modified pre-REP conditions, including lower doses of IL-2 with or without various doses of IL-21 or the AKT inhibitor MK-2206. TILs were stained by flow cytometry to measure expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT. [Figure 91E] Marker expression on CD8+ and CD4+ TILs following standard or modified pre-REP conditions, including lower doses of IL-2 with or without various doses of IL-21 or the AKT inhibitor MK-2206. TILs were stained by flow cytometry to measure expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT. [Figure 91F]Marker expression on CD8+ and CD4+ TILs following standard or modified pre-REP conditions, including lower doses of IL-2 with or without various doses of IL-21 or the AKT inhibitor MK-2206. TILs were stained by flow cytometry to measure expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT. [Figure 92A] Marker expression on CD8+ and CD4+ TILs following standard or modified pre-REP conditions, including lower doses of IL-2, with or without various doses of IL-21 or the AKT inhibitor MK-2206. TILs were stained to measure expression of (A) CD25 and (B) CD38 by flow cytometry. [Figure 92B] Marker expression on CD8+ and CD4+ TILs following standard or modified pre-REP conditions, including lower doses of IL-2, with or without various doses of IL-21 or the AKT inhibitor MK-2206. TILs were stained to measure expression of (A) CD25 and (B) CD38 by flow cytometry. [Figure 93A] Expression of (A) CD69+CD39+ and (B) CD69-CD39-CD8+ TILs following standard or modified pre-REP conditions, including lower doses of IL-2 with or without various doses of IL-21 or the AKT inhibitor MK-2206. [Figure 93B] Expression of (A) CD69+CD39+ and (B) CD69-CD39-CD8+ TILs following standard or modified pre-REP conditions, including lower doses of IL-2 with or without various doses of IL-21 or the AKT inhibitor MK-2206. [Figure 94A] Frequency of PD-1 and TIM3 subsets in CD8+ and CD4+ TILs following standard or modified pre-REP conditions, including lower doses of IL-2, with or without various doses of IL-21 or the AKT inhibitor MK-2206. (A) Frequency of PD-1+TIM3+ TILs and (B) PD-1-TIM3- TILs. [Figure 94B]Frequency of PD-1 and TIM3 subsets in CD8+ and CD4+ TILs following standard or modified pre-REP conditions, including lower doses of IL-2, with or without various doses of IL-21 or the AKT inhibitor MK-2206. (A) Frequency of PD-1+TIM3+ TILs and (B) PD-1-TIM3- TILs. [Figure 95A] Frequency of (A) IFNg (B) TNFa (C) IFNg+TNFa+ (D) Ki-67 (E) PD-1, and (F) TOX expression on CD8+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified pre-REP conditions, including lower doses of IL-2, with or without various doses of IL-21 or the AKT inhibitor MK-2206. [Figure 95B] Frequency of (A) IFNg (B) TNFa (C) IFNg+TNFa+ (D) Ki-67 (E) PD-1, and (F) TOX expression on CD8+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified pre-REP conditions, including lower doses of IL-2, with or without various doses of IL-21 or the AKT inhibitor MK-2206. [Figure 95C] Frequency of (A) IFNg (B) TNFa (C) IFNg+TNFa+ (D) Ki-67 (E) PD-1, and (F) TOX expression on CD8+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified pre-REP conditions, including lower doses of IL-2, with or without various doses of IL-21 or the AKT inhibitor MK-2206. [Figure 95D]Frequency of (A) IFNg (B) TNFa (C) IFNg+TNFa+ (D) Ki-67 (E) PD-1, and (F) TOX expression on CD8+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified pre-REP conditions, including lower doses of IL-2, with or without various doses of IL-21 or the AKT inhibitor MK-2206. [Figure 95E] Frequency of (A) IFNg (B) TNFa (C) IFNg+TNFa+ (D) Ki-67 (E) PD-1, and (F) TOX expression on CD8+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified pre-REP conditions, including lower doses of IL-2, with or without various doses of IL-21 or the AKT inhibitor MK-2206. [Figure 95F] Frequency of (A) IFNg (B) TNFa (C) IFNg+TNFa+ (D) Ki-67 (E) PD-1, and (F) TOX expression on CD8+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 hours of culture the next day in the presence of Brefeldin A. TILs were expanded under standard or modified pre-REP conditions, including lower doses of IL-2, with or without various doses of IL-21 or the AKT inhibitor MK-2206. [Figure 96A] Frequency of (A) Ki-67 (B) PD-1, and (C) TOX expression on CD4+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 hours of culture the following day in the presence of Brefeldin A. TILs were expanded under standard or modified pre-REP conditions, including lower doses of IL-2, with or without various doses of IL-21 or the AKT inhibitor MK-2206. [Figure 96B] Frequency of (A) Ki-67 (B) PD-1, and (C) TOX expression on CD4+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 hours of culture the following day in the presence of Brefeldin A. TILs were expanded under standard or modified pre-REP conditions, including lower doses of IL-2, with or without various doses of IL-21 or the AKT inhibitor MK-2206. [Figure 96C] Frequency of (A) Ki-67 (B) PD-1, and (C) TOX expression on CD4+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 hours of culture the following day in the presence of Brefeldin A. TILs were expanded under standard or modified pre-REP conditions, including lower doses of IL-2, with or without various doses of IL-21 or the AKT inhibitor MK-2206. [Figure 97A] Frequency of CD40L expression on (A) CD8+ (B) CD4+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 h of culture the following day in the presence of Brefeldin A. TILs were expanded under standard or modified pre-REP conditions, including lower doses of IL-2, with or without various doses of IL-21 or the AKT inhibitor MK-2206. [Figure 97B] Frequency of CD40L expression on (A) CD8+ (B) CD4+ TILs after overnight stimulation with aCD3 / CD28 beads and 4 h of culture the following day in the presence of Brefeldin A. TILs were expanded under standard or modified pre-REP conditions, including lower doses of IL-2, with or without various doses of IL-21 or the AKT inhibitor MK-2206. [Figure 98A] TIL expansion (A) and survival (B) under standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different AKT inhibitors. [Figure 98B] TIL expansion (A) and survival (B) under standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different AKT inhibitors. [Figure 99A] Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+) and viable cells under standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different AKT inhibitors. [Figure 99B]Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+) and viable cells under standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different AKT inhibitors. [Figure 99C] Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+) and viable cells under standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different AKT inhibitors. [Figure 99D] Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+) and viable cells under standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different AKT inhibitors. [Figure 100A] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different AKT inhibitors. TILs were stained to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT by flow cytometry. [Figure 100B] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different AKT inhibitors. TILs were stained to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT by flow cytometry. [Figure 100C] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different AKT inhibitors. TILs were stained to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT by flow cytometry. [Figure 100D]Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different AKT inhibitors. TILs were stained to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT by flow cytometry. [Figure 100E] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different AKT inhibitors. TILs were stained to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT by flow cytometry. [Figure 100F] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different AKT inhibitors. TILs were stained to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT by flow cytometry. [Figure 101A] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different AKT inhibitors. TILs were stained to measure (A) CD25 and (B) CD38 expression by flow cytometry. [Figure 101B] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different AKT inhibitors. TILs were stained to measure (A) CD25 and (B) CD38 expression by flow cytometry. [Figure 102A] Expression of (A) CD69+CD39+ and (B) CD69-CD39-CD8+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different AKT inhibitors. [Figure 102B]Expression of (A) CD69+CD39+ and (B) CD69-CD39-CD8+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different AKT inhibitors. [Figure 103A] Frequency of PD-1 and TIM3 subsets in CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different AKT inhibitors. (A) Frequency of PD-1+TIM3+ TILs and (B) PD-1-TIM3- TILs. [Figure 103B] Frequency of PD-1 and TIM3 subsets in CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different AKT inhibitors. (A) Frequency of PD-1+TIM3+ TILs and (B) PD-1-TIM3- TILs. [Figure 104A] Frequency of (A) IFNg (B) TNFa, and (C) IL-2 expressing CD8+ TILs after 6 h of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were expanded under standard or modified REP conditions, including IL-2 (1000 IU / ml) plus IL-21 (10 ng / ml) with different AKT inhibitors. [Figure 104B] Frequency of (A) IFNg (B) TNFa, and (C) IL-2 expressing CD8+ TILs after 6 h of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were expanded under standard or modified REP conditions, including IL-2 (1000 IU / ml) plus IL-21 (10 ng / ml) with different AKT inhibitors. [Figure 104C] Frequency of (A) IFNg (B) TNFa, and (C) IL-2 expressing CD8+ TILs after 6 h of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were expanded under standard or modified REP conditions, including IL-2 (1000 IU / ml) plus IL-21 (10 ng / ml) with different AKT inhibitors. [Figure 105A]Frequency of (A) IFNg (B) TNFa, and (C) IL-2 expressing CD4+ TILs after 6 h of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were expanded under standard or modified REP conditions, including IL-2 (1000 IU / ml) plus IL-21 (10 ng / ml) with different AKT inhibitors. [Figure 105B] Frequency of (A) IFNg (B) TNFa, and (C) IL-2 expressing CD4+ TILs after 6 h of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were expanded under standard or modified REP conditions, including IL-2 (1000 IU / ml) plus IL-21 (10 ng / ml) with different AKT inhibitors. [Figure 105C] Frequency of (A) IFNg (B) TNFa, and (C) IL-2 expressing CD4+ TILs after 6 h of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were expanded under standard or modified REP conditions, including IL-2 (1000 IU / ml) plus IL-21 (10 ng / ml) with different AKT inhibitors. [Figure 106A] Frequency of CXCR3 expression on (A) CD8+ and (B) CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different AKT inhibitors. [Figure 106B] Frequency of CXCR3 expression on (A) CD8+ and (B) CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different AKT inhibitors. [Figure 107A] TIL expansion (A) and viability (B) under standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different concentrations of AZD5363. [Figure 107B] TIL expansion (A) and viability (B) under standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different concentrations of AZD5363. [Figure 108A]Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+) and viable cells under standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different concentrations of AZD5363. [Figure 108B] Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+) and viable cells under standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different concentrations of AZD5363. [Figure 108C] Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+) and viable cells under standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different concentrations of AZD5363. [Figure 108D] Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+) and viable cells under standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different concentrations of AZD5363. [Figure 109A] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different concentrations of AZD5363. TILs were stained to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT by flow cytometry. [Figure 109B] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different concentrations of AZD5363. TILs were stained to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT by flow cytometry. [Figure 109C]Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different concentrations of AZD5363. TILs were stained to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT by flow cytometry. [Figure 109D] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different concentrations of AZD5363. TILs were stained to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT by flow cytometry. [Figure 109E] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different concentrations of AZD5363. TILs were stained to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT by flow cytometry. [Figure 109F] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different concentrations of AZD5363. TILs were stained to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT by flow cytometry. [Figure 110A] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different concentrations of AZD5363. TILs were stained to measure the expression of (A) CD25 and (B) CD38 by flow cytometry. [Figure 110B]Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different concentrations of AZD5363. TILs were stained to measure the expression of (A) CD25 and (B) CD38 by flow cytometry. [Figure 111A] Expression of (A) CD69+CD39+ and (B) CD69-CD39-CD8+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different concentrations of AZD5363. [Figure 111B] Expression of (A) CD69+CD39+ and (B) CD69-CD39-CD8+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different concentrations of AZD5363. [Figure 112A] Frequency of PD-1 and TIM3 subsets in CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different concentrations of AZD5363. (A) Frequency of PD-1+TIM3+ TILs and (B) PD-1-TIM3- TILs. [Figure 112B] Frequency of PD-1 and TIM3 subsets in CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different concentrations of AZD5363. (A) Frequency of PD-1+TIM3+ TILs and (B) PD-1-TIM3- TILs. [Figure 113A] Frequency of IFNg+TNFa+ expressing (A) IFNg (B) TNFa and (C) CD8+ TILs after 6 hours of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were expanded under standard or modified REP conditions, including IL-2 (1000 IU / ml) and IL-21 (10 ng / ml) with different concentrations of AZD5363. [Figure 113B]Frequency of IFNg+TNFa+ expressing (A) IFNg (B) TNFa and (C) CD8+ TILs after 6 hours of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were expanded under standard or modified REP conditions, including IL-2 (1000 IU / ml) and IL-21 (10 ng / ml) with different concentrations of AZD5363. [Figure 113C] Frequency of IFNg+TNFa+ expressing (A) IFNg (B) TNFa and (C) CD8+ TILs after 6 hours of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were expanded under standard or modified REP conditions, including IL-2 (1000 IU / ml) and IL-21 (10 ng / ml) with different concentrations of AZD5363. [Figure 114A] Frequency of IFNg+TNFa+ expressing (A) IFNg (B) TNFa and (C) CD4+ TILs after 6 hours of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were expanded under standard or modified REP conditions, including IL-2 (1000 IU / ml) and IL-21 (10 ng / ml) with different concentrations of AZD5363. [Figure 114B] Frequency of IFNg+TNFa+ expressing (A) IFNg (B) TNFa and (C) CD4+ TILs after 6 hours of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were expanded under standard or modified REP conditions, including IL-2 (1000 IU / ml) and IL-21 (10 ng / ml) with different concentrations of AZD5363. [Figure 114C] Frequency of IFNg+TNFa+ expressing (A) IFNg (B) TNFa and (C) CD4+ TILs after 6 hours of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were expanded under standard or modified REP conditions, including IL-2 (1000 IU / ml) and IL-21 (10 ng / ml) with different concentrations of AZD5363. [Figure 115A]Frequency of GZMB expression on (A) CD8+ and (B) CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different concentrations of AZD5363. [Figure 115B] Frequency of GZMB expression on (A) CD8+ and (B) CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different concentrations of AZD5363. [Figure 116A] Frequency of CXCR3 expression on (A) CD8+ and (B) CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different concentrations of AZD5363. [Figure 116B] Frequency of CXCR3 expression on (A) CD8+ and (B) CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different concentrations of AZD5363. [Figure 117A] Frequency of TOX expression on (A) CD8+ and (B) CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different concentrations of AZD5363. [Figure 117B] Frequency of TOX expression on (A) CD8+ and (B) CD4+ TILs following standard or modified REP conditions containing IL-2 (1000 IU / ml) + IL-21 (10 ng / ml) with different concentrations of AZD5363. [Figure 118A] TIL expansion (A) and viability (B) under standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of different concentrations of IL-2 or IL-15. [Figure 118B] TIL expansion (A) and viability (B) under standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of different concentrations of IL-2 or IL-15. [Figure 119A]Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+) and viable cells under standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of different concentrations of IL-2 or IL-15. [Figure 119B] Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+) and viable cells under standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of different concentrations of IL-2 or IL-15. [Figure 119C] Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+) and viable cells under standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of different concentrations of IL-2 or IL-15. [Figure 119D] Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+) and viable cells under standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of different concentrations of IL-2 or IL-15. [Figure 120A] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of different concentrations of IL-2 or IL-15. TILs were stained by flow cytometry to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT. [Figure 120B] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of different concentrations of IL-2 or IL-15. TILs were stained by flow cytometry to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT. [Figure 120C]Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of different concentrations of IL-2 or IL-15. TILs were stained by flow cytometry to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT. [Figure 120D] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of different concentrations of IL-2 or IL-15. TILs were stained by flow cytometry to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT. [Figure 120E] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of different concentrations of IL-2 or IL-15. TILs were stained by flow cytometry to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT. [Figure 120F] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of different concentrations of IL-2 or IL-15. TILs were stained by flow cytometry to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT. [Figure 121A] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of different concentrations of IL-2 or IL-15. TILs were stained to measure the expression of (A) CD25 and (B) CD38 by flow cytometry. [Figure 121B]Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of different concentrations of IL-2 or IL-15. TILs were stained to measure the expression of (A) CD25 and (B) CD38 by flow cytometry. [Figure 122A] Expression of (A) CD69+CD39+ and (B) CD69-CD39-CD8+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of different concentrations of IL-2 or IL-15. [Figure 122B] Expression of (A) CD69+CD39+ and (B) CD69-CD39-CD8+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of different concentrations of IL-2 or IL-15. [Figure 123A] Frequency of PD-1 and TIM3 subsets in CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of different concentrations of IL-2 or IL-15. (A) Frequency of PD-1+TIM3+ TILs and (B) PD-1-TIM3- TILs. [Figure 123B] Frequency of PD-1 and TIM3 subsets in CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of different concentrations of IL-2 or IL-15. (A) Frequency of PD-1+TIM3+ TILs and (B) PD-1-TIM3- TILs. [Figure 124A] Frequency of IL-2 expressing (A) IFNg (B) TNFa and (C) IFNg + TNFa + (D) CD8 + TILs after 6 h of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were expanded under standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of different concentrations of IL-2 or IL-15. [Figure 124B]Frequency of IL-2 expressing (A) IFNg (B) TNFa and (C) IFNg + TNFa + (D) CD8 + TILs after 6 h of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were expanded under standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of different concentrations of IL-2 or IL-15. [Figure 124C] Frequency of IL-2 expressing (A) IFNg (B) TNFa and (C) IFNg + TNFa + (D) CD8 + TILs after 6 h of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were expanded under standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of different concentrations of IL-2 or IL-15. [Figure 124D] Frequency of IL-2 expressing (A) IFNg (B) TNFa and (C) IFNg + TNFa + (D) CD8 + TILs after 6 h of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were expanded under standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of different concentrations of IL-2 or IL-15. [Figure 125A] Frequency of (A) IFNg (B) TNFa, and (C) IFNg + TNFa (D) IL-2 expressing CD4+ TILs after 6 h of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were expanded under standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of different concentrations of IL-2 or IL-15. [Figure 125B] Frequency of (A) IFNg (B) TNFa, and (C) IFNg + TNFa (D) IL-2 expressing CD4+ TILs after 6 h of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were expanded under standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of different concentrations of IL-2 or IL-15. [Figure 125C]Frequency of (A) IFNg (B) TNFa, and (C) IFNg + TNFa (D) IL-2 expressing CD4+ TILs after 6 h of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were expanded under standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of different concentrations of IL-2 or IL-15. [Figure 125D] Frequency of (A) IFNg (B) TNFa, and (C) IFNg + TNFa (D) IL-2 expressing CD4+ TILs after 6 h of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were expanded under standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of different concentrations of IL-2 or IL-15. [Figure 126A] Frequency of GZMB expression on (A) CD8+ and (B) CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of different concentrations of IL-2 or IL-15. [Figure 126B] Frequency of GZMB expression on (A) CD8+ and (B) CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of different concentrations of IL-2 or IL-15. [Figure 127A] Frequency of CXCR3 expression on (A) CD8+ and (B) CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of different concentrations of IL-2 or IL-15. [Figure 127B] Frequency of CXCR3 expression on (A) CD8+ and (B) CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of different concentrations of IL-2 or IL-15. [Figure 128A] TIL expansion (A) and viability (B) under standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 128B]TIL expansion (A) and viability (B) under standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 129A] Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+) and viable cells under standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 129B] Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+) and viable cells under standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 129C] Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+) and viable cells under standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 129D] Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+) and viable cells under standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 130A] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). TILs were stained by flow cytometry to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT. [Figure 130B]Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). TILs were stained by flow cytometry to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT. [Figure 130C] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). TILs were stained by flow cytometry to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT. [Figure 130D] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). TILs were stained by flow cytometry to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT. [Figure 130E] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). TILs were stained by flow cytometry to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT. [Figure 130F] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). TILs were stained by flow cytometry to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, and (F) TIGIT. [Figure 131A] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). TILs were stained to measure the expression of (A) CD25 and (B) CD38 by flow cytometry. [Figure 131B] Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). TILs were stained to measure the expression of (A) CD25 and (B) CD38 by flow cytometry. [Figure 132A] Expression of (A) CD69+CD39+ and (B) CD69-CD39-CD8+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 132B] Expression of (A) CD69+CD39+ and (B) CD69-CD39-CD8+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 133A] Frequency of PD-1 and TIM3 subsets in CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). (A) Frequency of PD-1+TIM3+ TILs and (B) PD-1-TIM3- TILs. [Figure 133B] Frequency of PD-1 and TIM3 subsets in CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). (A) Frequency of PD-1+TIM3+ TILs and (B) PD-1-TIM3- TILs. [Figure 134A] Frequency of IL-2 expressing (A) IFNg (B) TNFa and (C) IFNg+TNFa+ (D) CD8+ TILs after 6 hours of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were in standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 134B] Frequency of IL-2 expressing (A) IFNg (B) TNFa and (C) IFNg+TNFa+ (D) CD8+ TILs after 6 hours of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were in standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 134C] Frequency of IL-2 expressing (A) IFNg (B) TNFa and (C) IFNg+TNFa+ (D) CD8+ TILs after 6 hours of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were in standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 134D] Frequency of IL-2 expressing (A) IFNg (B) TNFa and (C) IFNg+TNFa+ (D) CD8+ TILs after 6 hours of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were in standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 135A]Frequency of (A) IFNg, (B) TNFa, and (C) IFNg + TNFa, (D) IL-2 expressing CD4+ TILs after 6 h of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were expanded under standard or modified REP conditions with IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 135B] Frequency of (A) IFNg, (B) TNFa, and (C) IFNg + TNFa, (D) IL-2 expressing CD4+ TILs after 6 h of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were expanded under standard or modified REP conditions with IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 135C] Frequency of (A) IFNg, (B) TNFa, and (C) IFNg + TNFa, (D) IL-2 expressing CD4+ TILs after 6 h of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were expanded under standard or modified REP conditions with IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 135D] Frequency of (A) IFNg, (B) TNFa, and (C) IFNg + TNFa, (D) IL-2 expressing CD4+ TILs after 6 h of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were expanded under standard or modified REP conditions with IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 136A] Frequency of GZMB expression on (A) CD8+ and (B) CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 136B]Frequency of GZMB expression on (A) CD8+ and (B) CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 137A] Frequency of CXCR3 expression on (A) CD8+ and (B) CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 137B] Frequency of CXCR3 expression on (A) CD8+ and (B) CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 138A] The Invigo-T expansion process increases TIL yield and viability. TIL expansion (A) and viability (B) under standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 138B] The Invigo-T expansion process increases TIL yield and viability. TIL expansion (A) and viability (B) under standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 139A] The Invigo-T expansion process increases the number of CD4+ (Foxp3-) cells while decreasing the number of Foxp3+ cells. Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+), and viable cells under standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 139B]The Invigo-T expansion process increases the number of CD4+ (Foxp3-) cells while decreasing the number of Foxp3+ cells. Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+), and viable cells under standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 139C] The Invigo-T expansion process increases the number of CD4+ (Foxp3-) cells while decreasing the number of Foxp3+ cells. Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+), and viable cells under standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 139D] The Invigo-T expansion process increases the number of CD4+ (Foxp3-) cells while decreasing the number of Foxp3+ cells. Frequencies of CD8, CD4(Foxp3-), CD4(Foxp3+), and viable cells under standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 140A] The Invigo-T expansion process increases the expression of memory-associated markers and decreases the expression of inhibitory receptors. Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). TILs were stained by flow cytometry to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, (F) TIGIT, (G) CD27, (H) CD62L, (I) IL-7R, and (J) TOX. [Figure 140B]The Invigo-T expansion process increases the expression of memory-associated markers and decreases the expression of inhibitory receptors. Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). TILs were stained by flow cytometry to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, (F) TIGIT, (G) CD27, (H) CD62L, (I) IL-7R, and (J) TOX. [Figure 140C] The Invigo-T expansion process increases the expression of memory-associated markers and decreases the expression of inhibitory receptors. Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). TILs were stained by flow cytometry to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, (F) TIGIT, (G) CD27, (H) CD62L, (I) IL-7R, and (J) TOX. [Figure 140D] The Invigo-T expansion process increases the expression of memory-associated markers and decreases the expression of inhibitory receptors. Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). TILs were stained by flow cytometry to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, (F) TIGIT, (G) CD27, (H) CD62L, (I) IL-7R, and (J) TOX. [Figure 140E]The Invigo-T expansion process increases the expression of memory-associated markers and decreases the expression of inhibitory receptors. Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). TILs were stained by flow cytometry to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, (F) TIGIT, (G) CD27, (H) CD62L, (I) IL-7R, and (J) TOX. [Figure 140F] The Invigo-T expansion process increases the expression of memory-associated markers and decreases the expression of inhibitory receptors. Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). TILs were stained by flow cytometry to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, (F) TIGIT, (G) CD27, (H) CD62L, (I) IL-7R, and (J) TOX. [Figure 140G] The Invigo-T expansion process increases the expression of memory-associated markers and decreases the expression of inhibitory receptors. Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). TILs were stained by flow cytometry to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, (F) TIGIT, (G) CD27, (H) CD62L, (I) IL-7R, and (J) TOX. [Figure 140H]The Invigo-T expansion process increases the expression of memory-associated markers and decreases the expression of inhibitory receptors. Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). TILs were stained by flow cytometry to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, (F) TIGIT, (G) CD27, (H) CD62L, (I) IL-7R, and (J) TOX. [Figure 140I] The Invigo-T expansion process increases the expression of memory-associated markers and decreases the expression of inhibitory receptors. Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). TILs were stained by flow cytometry to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, (F) TIGIT, (G) CD27, (H) CD62L, (I) IL-7R, and (J) TOX. [Figure 140J] The Invigo-T expansion process increases the expression of memory-associated markers and decreases the expression of inhibitory receptors. Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). TILs were stained by flow cytometry to measure the expression of (A) CD28, (B) CD127, (C) PD-1, (D) LAG3, (E) TIM3, (F) TIGIT, (G) CD27, (H) CD62L, (I) IL-7R, and (J) TOX. [Figure 141A]The Invigo-T expansion process reduces the activation state of TILs. Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). TILs were stained to measure the expression of (A) CD25, (B) CD38, (C) CD39, and (D) CD69 by flow cytometry. [Figure 141B] The Invigo-T expansion process reduces the activation state of TILs. Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). TILs were stained to measure the expression of (A) CD25, (B) CD38, (C) CD39, and (D) CD69 by flow cytometry. [Figure 141C] The Invigo-T expansion process reduces the activation state of TILs. Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). TILs were stained to measure the expression of (A) CD25, (B) CD38, (C) CD39, and (D) CD69 by flow cytometry. [Figure 141D] The Invigo-T expansion process reduces the activation state of TILs. Marker expression on CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). TILs were stained to measure the expression of (A) CD25, (B) CD38, (C) CD39, and (D) CD69 by flow cytometry. [Figure 142A]The Invigo-T expansion process maintains a reduced activation state in TILs after stimulation. Expression of (A) CD69+CD39+ and (B) CD69-CD39-CD8+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). Expression of CD38 (C) and CD39 (D) was assessed by FACS after REP following OKT3 stimulation at 1 μg / ml for 6 hours in the presence of Brefeldin A. [Figure 142B] The Invigo-T expansion process maintains a reduced activation state in TILs after stimulation. Expression of (A) CD69+CD39+ and (B) CD69-CD39-CD8+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). Expression of CD38 (C) and CD39 (D) was assessed by FACS after REP following OKT3 stimulation at 1 μg / ml for 6 hours in the presence of Brefeldin A. [Figure 142C] The Invigo-T expansion process maintains a reduced activation state in TILs after stimulation. Expression of (A) CD69+CD39+ and (B) CD69-CD39-CD8+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). Expression of CD38 (C) and CD39 (D) was assessed by FACS after REP following OKT3 stimulation at 1 μg / ml for 6 hours in the presence of Brefeldin A. [Figure 142D] The Invigo-T expansion process maintains a reduced activation state in TILs after stimulation. Expression of (A) CD69+CD39+ and (B) CD69-CD39-CD8+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). Expression of CD38 (C) and CD39 (D) was assessed by FACS after REP following OKT3 stimulation at 1 μg / ml for 6 hours in the presence of Brefeldin A. [Figure 143A] Frequency of PD-1 and TIM3 subsets in CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). (A) Frequency of PD-1+TIM3+ TILs and (B) PD-1-TIM3- TILs. [Figure 143B] Frequency of PD-1 and TIM3 subsets in CD8+ and CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). (A) Frequency of PD-1+TIM3+ TILs and (B) PD-1-TIM3- TILs. [Figure 144A] Frequency of IFNg+TNFa+IL-2+ expressing (A) IFNg, (B) TNFa, and (C) IFNg+TNFa+ (D) IL-2 and (E) CD8+ TILs after 6 hours of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were grown in standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 144B] Frequency of IFNg+TNFa+IL-2+ expressing (A) IFNg, (B) TNFa, and (C) IFNg+TNFa+ (D) IL-2 and (E) CD8+ TILs after 6 hours of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were grown in standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 144C]Frequency of IFNg+TNFa+IL-2+ expressing (A) IFNg, (B) TNFa, and (C) IFNg+TNFa+ (D) IL-2 and (E) CD8+ TILs after 6 hours of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were grown in standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 144D] Frequency of IFNg+TNFa+IL-2+ expressing (A) IFNg, (B) TNFa, and (C) IFNg+TNFa+ (D) IL-2 and (E) CD8+ TILs after 6 hours of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were grown in standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 144E] Frequency of IFNg+TNFa+IL-2+ expressing (A) IFNg, (B) TNFa, and (C) IFNg+TNFa+ (D) IL-2 and (E) CD8+ TILs after 6 hours of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were grown in standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 145A] Frequency of IFNg+TNFa+IL-2+ expressing (A) IFNg, (B) TNFa, and (C) IFNg+TNFa, (D) IL-2, and (E) CD4+ TILs after 6 hours of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were grown in standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 145B]Frequency of IFNg+TNFa+IL-2+ expressing (A) IFNg, (B) TNFa, and (C) IFNg+TNFa, (D) IL-2, and (E) CD4+ TILs after 6 hours of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were grown in standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 145C] Frequency of IFNg+TNFa+IL-2+ expressing (A) IFNg, (B) TNFa, and (C) IFNg+TNFa, (D) IL-2, and (E) CD4+ TILs after 6 hours of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were grown in standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 145D] Frequency of IFNg+TNFa+IL-2+ expressing (A) IFNg, (B) TNFa, and (C) IFNg+TNFa, (D) IL-2, and (E) CD4+ TILs after 6 hours of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were grown in standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 145E] Frequency of IFNg+TNFa+IL-2+ expressing (A) IFNg, (B) TNFa, and (C) IFNg+TNFa, (D) IL-2, and (E) CD4+ TILs after 6 hours of stimulation with plate-bound OKT3 in the presence of Brefeldin A and Monensin. TILs were grown in standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 146A]Frequency of GZMB expression on (A) CD8+ and (B) CD4+ and (C) CD107a+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 146B] Frequency of GZMB expression on (A) CD8+ and (B) CD4+ and (C) CD107a+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 146C] Frequency of GZMB expression on (A) CD8+ and (B) CD4+ and (C) CD107a+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 147A] Frequency of CXCR3 expression on (A) CD8+ and (B) CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 147B] Frequency of CXCR3 expression on (A) CD8+ and (B) CD4+ TILs following standard or modified REP conditions containing IL-21 (10 ng / ml) and AZD5363 in the presence of IL-2 (1000 IU / ml) or IL-15 (10 ng / ml). [Figure 148A] The Invigo-T expansion process increases the polyfunctionality of TILs. CD8+ TILs expanded by each indicated process were assessed for cytokine expression by FACS after REP following OKT3 stimulation at 1 μg / ml for 6 hours in the presence of Brefeldin A. Similar results were observed for CD4+ TILs (not shown). [Figure 148B]The Invigo-T expansion process increases the polyfunctionality of TILs. CD8+ TILs expanded by each indicated process were assessed for cytokine expression by FACS after REP following OKT3 stimulation at 1 μg / ml for 6 hours in the presence of Brefeldin A. Similar results were observed for CD4+ TILs (not shown). [Figure 148C] The Invigo-T expansion process increases the polyfunctionality of TILs. CD8+ TILs expanded by each indicated process were assessed for cytokine expression by FACS after REP following OKT3 stimulation at 1 μg / ml for 6 hours in the presence of Brefeldin A. Similar results were observed for CD4+ TILs (not shown). [Figure 149A] The Invigo-T expansion process increases TIL cytotoxicity in an allogeneic setting. TILs generated by the indicated processes (A) were cocultured with KILR® THP-1 cells at an effector-to-target cell ratio of 10:1 for 24 hours to measure cytotoxicity in an allogeneic setting, and (B, C) were stimulated with TransAct every 3 days, then cocultured with KILR® THP-1 cells for 24 hours and stained by flow cytometry. Control and Invigo-T-treated TILs were cocultured with autologous tumor cells for 24 hours. IFNg levels were measured by ELISA after 24 hours (D, E). [Figure 149B] The Invigo-T expansion process increases TIL cytotoxicity in an allogeneic setting. TILs generated by the indicated processes (A) were cocultured with KILR® THP-1 cells at an effector-to-target cell ratio of 10:1 for 24 hours to measure cytotoxicity in an allogeneic setting, and (B, C) were stimulated with TransAct every 3 days, then cocultured with KILR® THP-1 cells for 24 hours and stained by flow cytometry. Control and Invigo-T-treated TILs were cocultured with autologous tumor cells for 24 hours. IFNg levels were measured by ELISA after 24 hours (D, E). [Figure 149C]The Invigo-T expansion process increases TIL cytotoxicity in an allogeneic setting. TILs generated by the indicated processes (A) were cocultured with KILR® THP-1 cells at an effector-to-target cell ratio of 10:1 for 24 hours to measure cytotoxicity in an allogeneic setting, and (B, C) were stimulated with TransAct every 3 days, then cocultured with KILR® THP-1 cells for 24 hours and stained by flow cytometry. Control and Invigo-T-treated TILs were cocultured with autologous tumor cells for 24 hours. IFNg levels were measured by ELISA after 24 hours (D, E). [Figure 149D] The Invigo-T expansion process increases TIL cytotoxicity in an allogeneic setting. TILs generated by the indicated processes (A) were cocultured with KILR® THP-1 cells at an effector-to-target cell ratio of 10:1 for 24 hours to measure cytotoxicity in an allogeneic setting, and (B, C) were stimulated with TransAct every 3 days, then cocultured with KILR® THP-1 cells for 24 hours and stained by flow cytometry. Control and Invigo-T-treated TILs were cocultured with autologous tumor cells for 24 hours. IFNg levels were measured by ELISA after 24 hours (D, E). [Figure 149E] The Invigo-T expansion process increases TIL cytotoxicity in an allogeneic setting. TILs generated by the indicated processes (A) were cocultured with KILR® THP-1 cells at an effector-to-target cell ratio of 10:1 for 24 hours to measure cytotoxicity in an allogeneic setting, and (B, C) were stimulated with TransAct every 3 days, then cocultured with KILR® THP-1 cells for 24 hours and stained by flow cytometry. Control and Invigo-T-treated TILs were cocultured with autologous tumor cells for 24 hours. IFNg levels were measured by ELISA after 24 hours (D, E). [Figure 150A] Overall phenotypic changes observed with Invigo-T on (A) CD8+ TILs and (B) CD4+ TILs. [Figure 150B]Overall phenotypic changes observed with Invigo-T on (A) CD8+ TILs and (B) CD4+ TILs. [Figure 151A] Results from bulk TCRseq of control or Invigo-T expanded TILs. Bulk TCRb sequencing was performed on control or Invigo-T treated TILs. Shown are (A) Shannon diversity index (B) and Simpson clonality index (C) of control vs. Invigo-T. Data are presented as mean ± SD and n=5 per group. [Figure 151B] Results from bulk TCRseq of control or Invigo-T expanded TILs. Bulk TCRb sequencing was performed on control or Invigo-T treated TILs. Shown are (A) Shannon diversity index (B) and Simpson clonality index (C) of control vs. Invigo-T. Data are presented as mean ± SD and n=5 per group. [Figure 152A] Different predicted cell type distributions between CTRL and Invigo-T scRNAseq samples. Single-cell RNA sequencing (scRNAseq) was performed on control and Invigo-T treated TILs. (A) UMAP dimensionality reduction visualization labeled based on automated reference atlas predicted cell type annotations. (B) UMAP visualization of reference mapping results for control (left) vs. Invigo-T (right). (C) Bar plot visualization of mean cluster frequencies for control vs. Invigo-T groups. [Figure 152B] Different predicted cell type distributions between CTRL and Invigo-T scRNAseq samples. Single-cell RNA sequencing (scRNAseq) was performed on control and Invigo-T treated TILs. (A) UMAP dimensionality reduction visualization labeled based on automated reference atlas predicted cell type annotations. (B) UMAP visualization of reference mapping results for control (left) vs. Invigo-T (right). (C) Bar plot visualization of mean cluster frequencies for control vs. Invigo-T groups. [Figure 152C]Different predicted cell type distributions between CTRL and Invigo-T scRNAseq samples. Single-cell RNA sequencing (scRNAseq) was performed on control and Invigo-T treated TILs. (A) UMAP dimensionality reduction visualization labeled based on automated reference atlas predicted cell type annotations. (B) UMAP visualization of reference mapping results for control (left) vs. Invigo-T (right). (C) Bar plot visualization of mean cluster frequencies for control vs. Invigo-T groups. [Figure 153] Distinct CD8 T cell cluster distribution between CTRLs and Invigo-T expanded TILs. CD8 T cell UMAP dimensionality reduction visualization colored based on groups (left) and Seurat-defined clusters (right). [Figure 154A] Invigo-T enriches genes associated with stem-like cells while reducing exhaustion-associated genes on CD8+ TILs. (A) Heatmap visualization of the top 20 differentially expressed genes in CD8 T cell subsets comparing control and Invigo-T groups. (B) CD8 T cell subset heatmap visualization of specific genes of interest related to stemness (left), exhaustion (middle), or metabolism (right) comparing control and Invigo-T groups. (C) CD8 T cell subset gene set heatmap visualization of stem or exhaustion gene signatures showing the raw z-score of the UCell score average for each sample grouped by control vs. Invigo-T. [Figure 154B-1] Invigo-T enriches genes associated with stem-like cells while reducing exhaustion-associated genes on CD8+ TILs. (A) Heatmap visualization of the top 20 differentially expressed genes in CD8 T cell subsets comparing control and Invigo-T groups. (B) CD8 T cell subset heatmap visualization of specific genes of interest related to stemness (left), exhaustion (middle), or metabolism (right) comparing control and Invigo-T groups. (C) CD8 T cell subset gene set heatmap visualization of stem or exhaustion gene signatures showing the raw z-score of the UCell score average for each sample grouped by control vs. Invigo-T. [Figure 154B-2]Invigo-T enriches genes associated with stem-like cells while reducing exhaustion-associated genes on CD8+ TILs. (A) Heatmap visualization of the top 20 differentially expressed genes in CD8 T cell subsets comparing control and Invigo-T groups. (B) CD8 T cell subset heatmap visualization of specific genes of interest related to stemness (left), exhaustion (middle), or metabolism (right) comparing control and Invigo-T groups. (C) CD8 T cell subset gene set heatmap visualization of stem or exhaustion gene signatures showing the raw z-score of the UCell score average for each sample grouped by control vs. Invigo-T. [Figure 154B-3] Invigo-T enriches genes associated with stem-like cells while reducing exhaustion-associated genes on CD8+ TILs. (A) Heatmap visualization of the top 20 differentially expressed genes in CD8 T cell subsets comparing control and Invigo-T groups. (B) CD8 T cell subset heatmap visualization of specific genes of interest related to stemness (left), exhaustion (middle), or metabolism (right) comparing control and Invigo-T groups. (C) CD8 T cell subset gene set heatmap visualization of stem or exhaustion gene signatures showing the raw z-score of the UCell score average for each sample grouped by control vs. Invigo-T. [Figure 154C] Invigo-T enriches genes associated with stem-like cells while reducing exhaustion-associated genes on CD8+ TILs. (A) Heatmap visualization of the top 20 differentially expressed genes in CD8 T cell subsets comparing control and Invigo-T groups. (B) CD8 T cell subset heatmap visualization of specific genes of interest related to stemness (left), exhaustion (middle), or metabolism (right) comparing control and Invigo-T groups. (C) CD8 T cell subset gene set heatmap visualization of stem or exhaustion gene signatures showing the raw z-score of the UCell score average for each sample grouped by control vs. Invigo-T. [Figure 155A]Invigo-T enriches for the response-associated CD39-CD69- gene cluster. Violin plot of CD8 T cell subset UCell scores for (A) CD39-CD69- and (B) CD39+CD69+ gene signatures from Krishna et al., Science 2020 Dec 11;370(6522):1328-1334, comparing control with Invigo-T. (C) Violin plot of CD8 T cell subset UCell scores for other gene signatures of interest, comparing control with Invigo-T. [Figure 155B] Invigo-T enriches for the response-associated CD39-CD69- gene cluster. Violin plot of CD8 T cell subset UCell scores for (A) CD39-CD69- and (B) CD39+CD69+ gene signatures from Krishna et al., Science 2020 Dec 11;370(6522):1328-1334, comparing control with Invigo-T. (C) Violin plot of CD8 T cell subset UCell scores for other gene signatures of interest, comparing control with Invigo-T. [Figure 155C-1] Invigo-T enriches for the response-associated CD39-CD69- gene cluster. Violin plot of CD8 T cell subset UCell scores for (A) CD39-CD69- and (B) CD39+CD69+ gene signatures from Krishna et al., Science 2020 Dec 11;370(6522):1328-1334, comparing control with Invigo-T. (C) Violin plot of CD8 T cell subset UCell scores for other gene signatures of interest, comparing control with Invigo-T. [Figure 155C-2]Invigo-T enriches for the response-associated CD39-CD69- gene cluster. Violin plot of CD8 T cell subset UCell scores for (A) CD39-CD69- and (B) CD39+CD69+ gene signatures from Krishna et al., Science 2020 Dec 11;370(6522):1328-1334, comparing control with Invigo-T. (C) Violin plot of CD8 T cell subset UCell scores for other gene signatures of interest, comparing control with Invigo-T. [Figure 155C-3] Invigo-T enriches for the response-associated CD39-CD69- gene cluster. Violin plot of CD8 T cell subset UCell scores for (A) CD39-CD69- and (B) CD39+CD69+ gene signatures from Krishna et al., Science 2020 Dec 11;370(6522):1328-1334, comparing control with Invigo-T. (C) Violin plot of CD8 T cell subset UCell scores for other gene signatures of interest, comparing control with Invigo-T. [Figure 155C-4] Invigo-T enriches for the response-associated CD39-CD69- gene cluster. Violin plot of CD8 T cell subset UCell scores for (A) CD39-CD69- and (B) CD39+CD69+ gene signatures from Krishna et al., Science 2020 Dec 11;370(6522):1328-1334, comparing control with Invigo-T. (C) Violin plot of CD8 T cell subset UCell scores for other gene signatures of interest, comparing control with Invigo-T. [Figure 155C-5]Invigo-T enriches for the response-associated CD39-CD69- gene cluster. Violin plot of CD8 T cell subset UCell scores for (A) CD39-CD69- and (B) CD39+CD69+ gene signatures from Krishna et al., Science 2020 Dec 11;370(6522):1328-1334, comparing control with Invigo-T. (C) Violin plot of CD8 T cell subset UCell scores for other gene signatures of interest, comparing control with Invigo-T. [Figure 156-1] Invigo-T treated CD8 TILs show increased expression of memory-associated genes. Violin plot of normalized expression of CD8 T cell subsets of specific genes of interest comparing control and Invigo-T. [Figure 156-2] Invigo-T treated CD8 TILs show increased expression of memory-associated genes. Violin plot of normalized expression of CD8 T cell subsets of specific genes of interest comparing control and Invigo-T. [Figure 156-3] Invigo-T treated CD8 TILs show increased expression of memory-associated genes. Violin plot of normalized expression of CD8 T cell subsets of specific genes of interest comparing control and Invigo-T. [Figure 157A]Pseudotime trajectory analysis shows the majority of Invigo-T-treated CD8+ TILs in a less differentiated state, with a subset of more differentiated cells. Invigo-T shows an improved phenotype when comparing cluster 3 vs. 2 on CD8+ TILs. (A) CD8 T cell UMAP dimensionality reduction visualization colored based on group (left) and Monocle3-defined pseudotime (right). (B) Violin plot of CD8 T cell subset Monocle3 pseudotime comparing control vs. Invigo-T. (C) CD8 T cell UMAP dimensionality reduction visualization colored based on group (left) and Seurat-defined cluster (right), with two of the most differentiated clusters (2 and 3) highlighted via red boxes. (D) Heatmap visualization of the top 20 differentially expressed genes in the highlighted clusters comparing control (cluster 2) vs. Invigo-T (cluster 3). Data are presented as distributions of all values ​​unless otherwise stated, n=5 per group, and p-values ​​are based on the Mann-Whitney U test. [Figure 157B] Pseudotime trajectory analysis shows the majority of Invigo-T-treated CD8+ TILs in a less differentiated state, with a subset of more differentiated cells. Invigo-T shows an improved phenotype when comparing cluster 3 vs. 2 on CD8+ TILs. (A) CD8 T cell UMAP dimensionality reduction visualization colored based on group (left) and Monocle3-defined pseudotime (right). (B) Violin plot of CD8 T cell subset Monocle3 pseudotime comparing control vs. Invigo-T. (C) CD8 T cell UMAP dimensionality reduction visualization colored based on group (left) and Seurat-defined cluster (right), with two of the most differentiated clusters (2 and 3) highlighted via red boxes. (D) Heatmap visualization of the top 20 differentially expressed genes in the highlighted clusters comparing control (cluster 2) vs. Invigo-T (cluster 3). Data are presented as distributions of all values ​​unless otherwise stated, n=5 per group, and p-values ​​are based on the Mann-Whitney U test. [Figure 157C]Pseudotime trajectory analysis shows the majority of Invigo-T-treated CD8+ TILs in a less differentiated state, with a subset of more differentiated cells. Invigo-T shows an improved phenotype when comparing cluster 3 vs. 2 on CD8+ TILs. (A) CD8 T cell UMAP dimensionality reduction visualization colored based on group (left) and Monocle3-defined pseudotime (right). (B) Violin plot of CD8 T cell subset Monocle3 pseudotime comparing control vs. Invigo-T. (C) CD8 T cell UMAP dimensionality reduction visualization colored based on group (left) and Seurat-defined cluster (right), with two of the most differentiated clusters (2 and 3) highlighted via red boxes. (D) Heatmap visualization of the top 20 differentially expressed genes in the highlighted clusters comparing control (cluster 2) vs. Invigo-T (cluster 3). Data are presented as distributions of all values ​​unless otherwise stated, n=5 per group, and p-values ​​are based on the Mann-Whitney U test. [Figure 157D] Pseudotime trajectory analysis shows the majority of Invigo-T-treated CD8+ TILs in a less differentiated state, with a subset of more differentiated cells. Invigo-T shows an improved phenotype when comparing cluster 3 vs. 2 on CD8+ TILs. (A) CD8 T cell UMAP dimensionality reduction visualization colored based on group (left) and Monocle3-defined pseudotime (right). (B) Violin plot of CD8 T cell subset Monocle3 pseudotime comparing control vs. Invigo-T. (C) CD8 T cell UMAP dimensionality reduction visualization colored based on group (left) and Seurat-defined cluster (right), with two of the most differentiated clusters (2 and 3) highlighted via red boxes. (D) Heatmap visualization of the top 20 differentially expressed genes in the highlighted clusters comparing control (cluster 2) vs. Invigo-T (cluster 3). Data are presented as distributions of all values ​​unless otherwise stated, n=5 per group, and p-values ​​are based on the Mann-Whitney U test. [Figure 158]Differential gene expression analysis on CD4 TILs. Heatmap visualization of the top 20 differentially expressed genes in CD4 T cell subsets comparing control and Invigo-T groups. [Figure 159A] Pseudotime trajectory analysis shows a majority of Invigo-T treated CD4 TILs in a less differentiated state compared to control TILs. (A) CD4 T cell UMAP dimensionality reduction visualization colored based on group (left) and Monocle3-defined pseudotime (right). (B) Violin plot of CD4 T cell subset Monocle3 pseudotime comparing control and Invigo-T. [Figure 159B] Pseudotime trajectory analysis shows a majority of Invigo-T treated CD4 TILs in a less differentiated state compared to control TILs. (A) CD4 T cell UMAP dimensionality reduction visualization colored based on group (left) and Monocle3-defined pseudotime (right). (B) Violin plot of CD4 T cell subset Monocle3 pseudotime comparing control and Invigo-T. [Figure 160A] Invigo-T-treated CD4 TILs show increased expression of memory-related genes and decreased expression of exhaustion-related gene signatures compared to control-treated TILs. (A) CD4 T cell subset gene set heatmap visualization of stem or exhaustion gene signatures showing the raw z-score of the UCell score average for each sample grouped by control vs. Invigo-T. (B) Violin plot of normalized expression of CD4 T cell subsets of specific genes of interest comparing control vs. Invigo-T. Data are shown as distributions of all values ​​unless otherwise noted, n=5 per group. [Figure 160B-1]Invigo-T-treated CD4 TILs show increased expression of memory-related genes and decreased expression of exhaustion-related gene signatures compared to control-treated TILs. (A) CD4 T cell subset gene set heatmap visualization of stem or exhaustion gene signatures showing the raw z-score of the UCell score average for each sample grouped by control vs. Invigo-T. (B) Violin plot of normalized expression of CD4 T cell subsets of specific genes of interest comparing control vs. Invigo-T. Data are shown as distributions of all values ​​unless otherwise noted, n=5 per group. [Figure 160B-2] Invigo-T-treated CD4 TILs show increased expression of memory-related genes and decreased expression of exhaustion-related gene signatures compared to control-treated TILs. (A) CD4 T cell subset gene set heatmap visualization of stem or exhaustion gene signatures showing the raw z-score of the UCell score average for each sample grouped by control vs. Invigo-T. (B) Violin plot of normalized expression of CD4 T cell subsets of specific genes of interest comparing control vs. Invigo-T. Data are shown as distributions of all values ​​unless otherwise noted, n=5 per group. [Figure 160B-3] Invigo-T-treated CD4 TILs show increased expression of memory-related genes and decreased expression of exhaustion-related gene signatures compared to control-treated TILs. (A) CD4 T cell subset gene set heatmap visualization of stem or exhaustion gene signatures showing the raw z-score of the UCell score average for each sample grouped by control vs. Invigo-T. (B) Violin plot of normalized expression of CD4 T cell subsets of specific genes of interest comparing control vs. Invigo-T. Data are shown as distributions of all values ​​unless otherwise noted, n=5 per group. [Figure 160B-4]Invigo-T-treated CD4 TILs show increased expression of memory-related genes and decreased expression of exhaustion-related gene signatures compared to control-treated TILs. (A) CD4 T cell subset gene set heatmap visualization of stem or exhaustion gene signatures showing the raw z-score of the UCell score average for each sample grouped by control vs. Invigo-T. (B) Violin plot of normalized expression of CD4 T cell subsets of specific genes of interest comparing control vs. Invigo-T. Data are shown as distributions of all values ​​unless otherwise noted, n=5 per group. [Figure 160B-5] Invigo-T-treated CD4 TILs show increased expression of memory-related genes and decreased expression of exhaustion-related gene signatures compared to control-treated TILs. (A) CD4 T cell subset gene set heatmap visualization of stem or exhaustion gene signatures showing the raw z-score of the UCell score average for each sample grouped by control vs. Invigo-T. (B) Violin plot of normalized expression of CD4 T cell subsets of specific genes of interest comparing control vs. Invigo-T. Data are shown as distributions of all values ​​unless otherwise noted, n=5 per group.

[0186] Brief Description of Sequence Listing SEQ ID NO: 1 is the amino acid sequence of the heavy chain of muromonab.

[0187] SEQ ID NO: 2 is the amino acid sequence of the light chain of muromonab.

[0188] SEQ ID NO: 3 is the amino acid sequence of recombinant human IL-2 protein.

[0189] SEQ ID NO: 4 is the amino acid sequence of aldesleukin.

[0190] SEQ ID NO: 5 is an IL-2 form.

[0191] SEQ ID NO: 6 is the amino acid sequence of nemvaleukin alpha.

[0192] SEQ ID NO: 7 is an IL-2 form.

[0193] SEQ ID NO: 8 is a mucin domain polypeptide.

[0194] SEQ ID NO: 9 is the amino acid sequence of recombinant human IL-4 protein.

[0195] SEQ ID NO: 10 is the amino acid sequence of recombinant human IL-7 protein.

[0196] SEQ ID NO: 11 is the amino acid sequence of recombinant human IL-15 protein.

[0197] SEQ ID NO: 12 is the amino acid sequence of recombinant human IL-21 protein.

[0198] SEQ ID NO: 13 is the IL-2 sequence.

[0199] SEQ ID NO: 14 is the IL-2 mutein sequence.

[0200] SEQ ID NO: 15 is the sequence of an IL-2 mutein.

[0201] SEQ ID NO: 16 is HCDR1_IL-2 of IgG.IL2R67A.H1.

[0202] SEQ ID NO: 17 is HCDR2 of IgG.IL2R67A.H1.

[0203] SEQ ID NO: 18 is the HCDR3 of IgG.IL2R67A.H1.

[0204] SEQ ID NO: 19 is HCDR1_IL-2 Kabat of IgG.IL2R67A.H1.

[0205] SEQ ID NO: 20 is the HCDR2 Kabat of IgG.IL2R67A.H1.

[0206] SEQ ID NO: 21 is the HCDR3 Kabat of IgG.IL2R67A.H1.

[0207] SEQ ID NO: 22 is the HCDR1_IL-2 clotia of IgG.IL2R67A.H1.

[0208] SEQ ID NO: 23 is the HCDR2 clone of IgG.IL2R67A.H1.

[0209] SEQ ID NO: 24 is the HCDR3 clone of IgG.IL2R67A.H1.

[0210] SEQ ID NO: 25 is HCDR1_IL-2 IMGT of IgG.IL2R67A.H1.

[0211] SEQ ID NO: 26 is HCDR2 IMGT of IgG.IL2R67A.H1.

[0212] SEQ ID NO: 27 is the HCDR3 IMGT of IgG.IL2R67A.H1.

[0213] SEQ ID NO: 28 is the V H It's a chain.

[0214] SEQ ID NO: 29 is the heavy chain of IgG.IL2R67A.H1.

[0215] SEQ ID NO: 30 is the LCDR1 Kabat of IgG.IL2R67A.H1.

[0216] SEQ ID NO: 31 is the LCDR2 Kabat of IgG.IL2R67A.H1.

[0217] SEQ ID NO: 32 is the LCDR3 Kabat of IgG.IL2R67A.H1.

[0218] SEQ ID NO: 33 is the LCDR1 chothia of IgG.IL2R67A.H1.

[0219] SEQ ID NO: 34 is the LCDR2 chothia of IgG.IL2R67A.H1.

[0220] SEQ ID NO: 35 is the LCDR3 chothia of IgG.IL2R67A.H1.

[0221] SEQ ID NO: 36 is V L It's a chain.

[0222] SEQ ID NO: 37 is the light chain.

[0223] SEQ ID NO: 38 is the light chain.

[0224] SEQ ID NO: 39 is the light chain.

[0225] SEQ ID NO: 40 is the amino acid sequence of human 4-1BB.

[0226] SEQ ID NO: 41 is the amino acid sequence of mouse 4-1BB.

[0227] SEQ ID NO: 42 is the heavy chain of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0228] SEQ ID NO: 43 is the light chain of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0229] SEQ ID NO: 44 represents the heavy chain variable region (V) of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566). H )

[0230] SEQ ID NO: 45 is the light chain variable region (V) of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566). L )

[0231] SEQ ID NO: 46 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0232] SEQ ID NO: 47 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0233] SEQ ID NO: 48 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0234] SEQ ID NO: 49 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0235] SEQ ID NO: 50 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0236] SEQ ID NO: 51 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody utomilumab (PF-05082566).

[0237] SEQ ID NO: 52 is the heavy chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0238] SEQ ID NO: 53 is the light chain of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0239] SEQ ID NO: 54 is the heavy chain variable region (VH) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0240] SEQ ID NO: 55 is the light chain variable region (VL) of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0241] SEQ ID NO: 56 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0242] SEQ ID NO: 57 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0243] SEQ ID NO: 58 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0244] SEQ ID NO: 59 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0245] SEQ ID NO: 60 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0246] SEQ ID NO: 61 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody urelumab (BMS-663513).

[0247] SEQ ID NO: 62 is the Fc domain of the TNFRSF agonist fusion protein.

[0248] SEQ ID NO: 63 is the linker of the TNFRSF agonist fusion protein.

[0249] SEQ ID NO: 64 is the linker of the TNFRSF agonist fusion protein.

[0250] SEQ ID NO: 65 is the linker of the TNFRSF agonist fusion protein.

[0251] SEQ ID NO: 66 is the linker of the TNFRSF agonist fusion protein.

[0252] SEQ ID NO: 67 is the linker for the TNFRSF agonist fusion protein.

[0253] SEQ ID NO: 68 is the linker for the TNFRSF agonist fusion protein.

[0254] SEQ ID NO: 69 is the linker for the TNFRSF agonist fusion protein.

[0255] SEQ ID NO: 70 is the linker of the TNFRSF agonist fusion protein.

[0256] SEQ ID NO: 71 is the linker for the TNFRSF agonist fusion protein.

[0257] SEQ ID NO: 72 is the linker for the TNFRSF agonist fusion protein.

[0258] SEQ ID NO: 73 is the Fc domain of the TNFRSF agonist fusion protein.

[0259] SEQ ID NO: 74 is the linker for the TNFRSF agonist fusion protein.

[0260] SEQ ID NO: 75 is the linker for the TNFRSF agonist fusion protein.

[0261] SEQ ID NO: 76 is the linker for the TNFRSF agonist fusion protein.

[0262] SEQ ID NO: 77 is the 4-1BB ligand (4-1BBL) amino acid sequence.

[0263] SEQ ID NO: 78 is the soluble portion of the 4-1BBL polypeptide.

[0264] SEQ ID NO: 79 is the heavy chain variable region (V) of 4-1BB agonist antibody 4B4-1-1 version 1 H )

[0265] SEQ ID NO: 80 represents the light chain variable region (V) of 4-1BB agonist antibody 4B4-1-1 version 1 L )

[0266] SEQ ID NO: 81 is the heavy chain variable region (V H )

[0267] SEQ ID NO: 82 is the light chain variable region (V) of 4-1BB agonist antibody 4B4-1-1 version 2 L )

[0268] SEQ ID NO: 83 identifies the heavy chain variable region (V H )

[0269] SEQ ID NO: 84 identifies the light chain variable region (V L )

[0270] SEQ ID NO: 85 is the amino acid sequence of human OX40.

[0271] SEQ ID NO: 86 is the amino acid sequence of mouse OX40.

[0272] SEQ ID NO: 87 is the heavy chain of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0273] SEQ ID NO: 88 is the light chain of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0274] SEQ ID NO: 89 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562). H )

[0275] SEQ ID NO: 90 is the OX40 agonist monoclonal antibody tabolixizumab (MEDI The light chain variable region (V L )

[0276] SEQ ID NO: 91 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0277] SEQ ID NO: 92 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0278] SEQ ID NO: 93 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0279] SEQ ID NO: 94 is the light chain CDR1 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0280] SEQ ID NO: 95 is the light chain CDR2 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0281] SEQ ID NO: 96 is the light chain CDR3 of the OX40 agonist monoclonal antibody tabolixizumab (MEDI-0562).

[0282] SEQ ID NO: 97 is the heavy chain of the OX40 agonist monoclonal antibody 11D4.

[0283] SEQ ID NO: 98 is the light chain of the OX40 agonist monoclonal antibody 11D4.

[0284] SEQ ID NO: 99 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 11D4. H )

[0285] SEQ ID NO: 100 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 11D4. L )

[0286] SEQ ID NO: 101 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0287] SEQ ID NO: 102 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0288] SEQ ID NO: 103 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0289] SEQ ID NO: 104 is the light chain CDR1 of the OX40 agonist monoclonal antibody 11D4.

[0290] SEQ ID NO: 105 is the light chain CDR2 of the OX40 agonist monoclonal antibody 11D4.

[0291] SEQ ID NO: 106 is the light chain CDR3 of the OX40 agonist monoclonal antibody 11D4.

[0292] SEQ ID NO: 107 is the heavy chain of the OX40 agonist monoclonal antibody 18D8.

[0293] SEQ ID NO: 108 is the light chain of the OX40 agonist monoclonal antibody 18D8.

[0294] SEQ ID NO: 109 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 18D8. H )

[0295] SEQ ID NO: 110 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 18D8. L )

[0296] SEQ ID NO: 111 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0297] SEQ ID NO: 112 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0298] SEQ ID NO: 113 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0299] SEQ ID NO: 114 is the light chain CDR1 of the OX40 agonist monoclonal antibody 18D8.

[0300] SEQ ID NO: 115 is the light chain CDR2 of the OX40 agonist monoclonal antibody 18D8.

[0301] SEQ ID NO: 116 is the light chain CDR3 of the OX40 agonist monoclonal antibody 18D8.

[0302] SEQ ID NO: 117 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody Hu119-122. H )

[0303] SEQ ID NO: 118 is the light chain variable region (V) of the OX40 agonist monoclonal antibody Hu119-122. L )

[0304] SEQ ID NO: 119 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0305] SEQ ID NO: 120 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0306] SEQ ID NO: 121 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0307] SEQ ID NO: 122 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu119-122.

[0308] SEQ ID NO: 123 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu119-122.

[0309] SEQ ID NO: 124 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu119-122.

[0310] SEQ ID NO: 125 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody Hu106-222. H )

[0311] SEQ ID NO: 126 is the light chain variable region (V) of the OX40 agonist monoclonal antibody Hu106-222. L )

[0312] SEQ ID NO: 127 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0313] SEQ ID NO: 128 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0314] SEQ ID NO: 129 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0315] SEQ ID NO: 130 is the light chain CDR1 of the OX40 agonist monoclonal antibody Hu106-222.

[0316] SEQ ID NO: 131 is the light chain CDR2 of the OX40 agonist monoclonal antibody Hu106-222.

[0317] SEQ ID NO: 132 is the light chain CDR3 of the OX40 agonist monoclonal antibody Hu106-222.

[0318] SEQ ID NO: 133 is the OX40 ligand (OX40L) amino acid sequence.

[0319] SEQ ID NO: 134 is the soluble portion of the OX40L polypeptide.

[0320] SEQ ID NO: 135 is an alternative soluble portion of the OX40L polypeptide.

[0321] SEQ ID NO: 136 is the heavy chain variable region (V H )

[0322] SEQ ID NO: 137 is the light chain variable region (V) of OX40 agonist monoclonal antibody 008. L )

[0323] SEQ ID NO: 138 is the heavy chain variable region (V) of OX40 agonist monoclonal antibody 011. H )

[0324] SEQ ID NO: 139 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 011. L )

[0325] SEQ ID NO: 140 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 021. H )

[0326] SEQ ID NO: 141 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 021. L )

[0327] SEQ ID NO: 142 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody 023. H )

[0328] SEQ ID NO: 143 is the light chain variable region (V) of the OX40 agonist monoclonal antibody 023. L )

[0329] SEQ ID NO: 144 is the heavy chain variable region (V H )

[0330] SEQ ID NO: 145 is the light chain variable region (V) of the OX40 agonist monoclonal antibody L )

[0331] SEQ ID NO: 146 is the heavy chain variable region (V H )

[0332] SEQ ID NO: 147 is the light chain variable region (V) of the OX40 agonist monoclonal antibody L )

[0333] SEQ ID NO: 148 is the heavy chain variable region (V H )

[0334] SEQ ID NO: 149 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody. H )

[0335] SEQ ID NO: 150 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0336] SEQ ID NO: 151 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0337] SEQ ID NO: 152 is the heavy chain variable region (V H )

[0338] SEQ ID NO: 153 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody. H )

[0339] SEQ ID NO: 154 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0340] SEQ ID NO: 155 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody. L )

[0341] SEQ ID NO: 156 is the heavy chain variable region (V H )

[0342] SEQ ID NO: 157 is the light chain variable region (V) of the OX40 agonist monoclonal antibody L )

[0343] SEQ ID NO: 158 is the heavy chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0344] SEQ ID NO: 159 is the light chain amino acid sequence of the PD-1 inhibitor nivolumab.

[0345] SEQ ID NO: 160 represents the heavy chain variable region (V) of the PD-1 inhibitor nivolumab H ) amino acid sequence.

[0346] SEQ ID NO: 161 represents the light chain variable region (V) of the PD-1 inhibitor nivolumab L ) amino acid sequence.

[0347] SEQ ID NO: 162 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0348] SEQ ID NO: 163 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0349] SEQ ID NO: 164 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0350] SEQ ID NO: 165 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor nivolumab.

[0351] SEQ ID NO: 166 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor nivolumab.

[0352] SEQ ID NO: 167 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor nivolumab.

[0353] SEQ ID NO: 168 is the heavy chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0354] SEQ ID NO: 169 is the light chain amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0355] SEQ ID NO: 170 represents the heavy chain variable region (V) of the PD-1 inhibitor pembrolizumab H ) amino acid sequence.

[0356] SEQ ID NO: 171 is the light chain variable region (V) of the PD-1 inhibitor pembrolizumab L ) amino acid sequence.

[0357] SEQ ID NO: 172 is the heavy chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0358] SEQ ID NO: 173 is the heavy chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0359] SEQ ID NO: 174 is the heavy chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0360] SEQ ID NO: 175 is the light chain CDR1 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0361] SEQ ID NO: 176 is the light chain CDR2 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0362] SEQ ID NO: 177 is the light chain CDR3 amino acid sequence of the PD-1 inhibitor pembrolizumab.

[0363] SEQ ID NO: 178 is the heavy chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0364] SEQ ID NO: 179 is the light chain amino acid sequence of the PD-L1 inhibitor durvalumab.

[0365] SEQ ID NO: 180 represents the heavy chain variable region (V) of the PD-L1 inhibitor durvalumab H ) amino acid sequence.

[0366] SEQ ID NO: 181 represents the light chain variable region (V) of the PD-L1 inhibitor durvalumab L ) amino acid sequence.

[0367] SEQ ID NO: 182 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0368] SEQ ID NO: 183 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0369] SEQ ID NO: 184 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0370] SEQ ID NO: 185 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0371] SEQ ID NO: 186 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0372] SEQ ID NO: 187 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor durvalumab.

[0373] SEQ ID NO: 188 is the heavy chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0374] SEQ ID NO: 189 is the light chain amino acid sequence of the PD-L1 inhibitor avelumab.

[0375] SEQ ID NO: 190 represents the heavy chain variable region (V) of the PD-L1 inhibitor avelumab H ) amino acid sequence.

[0376] SEQ ID NO: 191 is the light chain variable region (V) of the PD-L1 inhibitor avelumab L ) amino acid sequence.

[0377] SEQ ID NO: 192 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0378] SEQ ID NO: 193 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0379] SEQ ID NO: 194 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0380] SEQ ID NO: 195 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor avelumab.

[0381] SEQ ID NO: 196 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor avelumab.

[0382] SEQ ID NO: 197 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor avelumab.

[0383] SEQ ID NO: 198 is the heavy chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0384] SEQ ID NO: 199 is the light chain amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0385] SEQ ID NO: 200 represents the heavy chain variable region (V) of the PD-L1 inhibitor atezolizumab H ) amino acid sequence.

[0386] SEQ ID NO: 201 is the light chain variable region (V) of the PD-L1 inhibitor atezolizumab L ) amino acid sequence.

[0387] SEQ ID NO: 202 is the heavy chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0388] SEQ ID NO: 203 is the heavy chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0389] SEQ ID NO: 204 is the heavy chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0390] SEQ ID NO: 205 is the light chain CDR1 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0391] SEQ ID NO: 206 is the light chain CDR2 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0392] SEQ ID NO: 207 is the light chain CDR3 amino acid sequence of the PD-L1 inhibitor atezolizumab.

[0393] SEQ ID NO: 208 is the heavy chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0394] SEQ ID NO: 209 is the light chain amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0395] SEQ ID NO: 210 represents the heavy chain variable region (V) of the CTLA-4 inhibitor ipilimumab H ) amino acid sequence.

[0396] SEQ ID NO: 211 represents the light chain variable region (V) of the CTLA-4 inhibitor ipilimumab L ) amino acid sequence.

[0397] SEQ ID NO: 212 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0398] SEQ ID NO: 213 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0399] SEQ ID NO: 214 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0400] SEQ ID NO: 215 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0401] SEQ ID NO: 216 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor ipilimumab.

[0402] SEQ ID NO: 217 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor ipilimumab do.

[0403] SEQ ID NO: 218 is the heavy chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0404] SEQ ID NO: 219 is the light chain amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0405] SEQ ID NO: 220 represents the heavy chain variable region (V) of the CTLA-4 inhibitor tremelimumab H ) amino acid sequence.

[0406] SEQ ID NO: 221 represents the light chain variable region (V) of the CTLA-4 inhibitor tremelimumab L ) amino acid sequence.

[0407] SEQ ID NO: 222 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0408] SEQ ID NO: 223 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0409] SEQ ID NO: 224 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0410] SEQ ID NO: 225 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0411] SEQ ID NO: 226 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0412] SEQ ID NO: 227 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor tremelimumab.

[0413] SEQ ID NO: 228 is the heavy chain amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0414] SEQ ID NO: 229 is the light chain amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0415] SEQ ID NO: 230 is the heavy chain variable region (V) of the CTLA-4 inhibitor zalifrelimab H ) amino acid sequence.

[0416] SEQ ID NO: 231 is the light chain variable region (V) of the CTLA-4 inhibitor zalifrelimab L ) amino acid sequence.

[0417] SEQ ID NO: 232 is the heavy chain CDR1 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0418] SEQ ID NO: 233 is the heavy chain CDR2 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0419] SEQ ID NO: 234 is the heavy chain CDR3 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0420] SEQ ID NO: 235 is the light chain CDR1 amino acid sequence of the CTLA-4 inhibitor zalifrelimab be.

[0421] SEQ ID NO: 236 is the light chain CDR2 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0422] SEQ ID NO: 237 is the light chain CDR3 amino acid sequence of the CTLA-4 inhibitor zalifrelimab.

[0423] SEQ ID NO: 238 is the CD40 agonist CD40L. DETAILED DESCRIPTION OF THE INVENTION

[0424] I. Introduction Provided herein are methods of producing TILs by (i) REP pre-stimulation with a combination of interferon gamma (IFN-γ) and anti-PD-1, with or without CD40 agonism and with or without CTLA-4 agonism, (ii) adding various cytokine combinations, such as IL-15, IL-21, low concentration IL-2, with or without AKT inhibition (AKTi), during REP expansion and / or REP pre-expansion, and / or (iii) adding low concentration IL-2 and AKTi during REP expansion and / or REP pre-expansion. Also provided herein are methods of treatment using such TILs.

[0425] 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.

[0426] 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 some 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.

[0427] The term "in vivo" refers to events that take place inside a subject's body.

[0428] 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.

[0429] The term "ex vivo" refers to events involving the administration of a therapy or treatment to cells, tissues, and / or organs that have been removed from a subject's body. Suitably, the cells, tissues, and / or organs may be returned to the subject's body in a surgical or therapeutic manner.

[0430] The term "rapid expansion" refers to an increase in the number of antigen-specific TILs by at least about 3-fold (or 4, 5, 6, 7, 8, or 9-fold) over a one-week period, more preferably by at least about 10-fold (or 20, 30, 40, 50, 60, 70, 80, or 90-fold) over a one-week period, or most preferably by at least about 100-fold over a one-week period. Several rapid expansion protocols are described herein.

[0431] As used herein, "tumor infiltrating lymphocytes" or "TILs" refers to a population of cells that are initially acquired as leukocytes that have left the bloodstream of a subject and migrated to a tumor. TILs include CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + These include, but are not limited to, T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from a patient tissue sample as outlined herein (sometimes referred to as "freshly harvested"), and "secondary TILs" are any expanded or propagated TIL cell populations discussed herein, including, but not limited to, bulk TILs and expanded TILs ("REP TILs" or "post-REP TILs"). TIL cell populations may include genetically modified TILs.

[0432] TILs can generally be defined either biochemically using cell surface markers or functionally by their ability to infiltrate tumors and achieve therapy. TILs can generally be classified by the expression of one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally or alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into patients. TILs can be further characterized by potency; for example, TILs can be considered potent if their interferon (IFN) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL. For example, TILs can be considered potent if interferon (IFNγ) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL, greater than about 300 pg / mL, greater than about 400 pg / mL, greater than about 500 pg / mL, greater than about 600 pg / mL, greater than about 700 pg / mL, greater than about 800 pg / mL, greater than about 900 pg / mL, or greater than about 1000 pg / mL.

[0433] "CD39 / CD69 double negative and / or CD39 LO / CD69 LO TILs” or “CD39 / CD69 double negative and / or CD39 LO / CD69 LO "TIL population" or grammatical variations of any of the foregoing means a TIL or TIL population that exhibits, on average, undetectable, low, or reduced levels of the cell surface proteins CD39 and CD69 compared to any TIL / TIL population from which the referenced TIL or TIL population is obtained.

[0434] "CD39 / CD69 double negative and / or CD39 LO / CD69 LO Enriched TILs" or "CD39 / CD69 double negative and / or CD39 LO / CD69 LO"Enriched TIL population" or grammatical variations of any of the foregoing means a TIL or TIL population enriched for TILs that have, on average, undetectable, low, or reduced levels of the cell surface proteins CD39 and CD69 compared to any TIL / TIL population from which the referenced TIL or TIL population is obtained. Any enrichment means may be used to enrich for CD39 / CD69 double negative and / or CD39 LO / CD69 LO Enriched TILs can be obtained, CD39 / CD69 double negative and / or CD39 LO / CD69 LO This involves sorting or selection of TILs.

[0435] As used herein, a "population of cells" (including TILs) refers to several cells that share a common trait. Generally, a population is roughly 1 x 10 6 ~1×10 10 The number of TILs ranges from approximately 1 x 10 to 1 x 10, with different TIL populations containing different numbers. For example, the initial growth of primary TILs in the presence of IL-2 is approximately 1 x 10 8 REP expansion typically results in a bulk TIL population of 1.5 x 10 cells for injection. 9 ~1.5×10 10 This is done to provide a population of cells.

[0436] "Cryopreserved TILs" as used herein refer to primary, bulk, or expanded TILs. Cryopreservation refers to the processing and storage of TILs (i.e., TILs) at temperatures ranging from approximately -150°C to -60°C. General methods for cryopreservation are described elsewhere herein, including in the Examples. For clarity, "cryopreserved TILs" can be distinguished from frozen tissue samples that may be used as a source of primary TILs.

[0437] By "thawed cryopreserved TILs" herein is meant a population of TILs that have previously been cryopreserved and then processed to return to room temperature or above, including but not limited to, cell culture temperature or the temperature at which the TILs can be administered to a patient.

[0438] TILs can generally be defined either biochemically using cell surface markers or functionally by their ability to infiltrate tumors and achieve therapy. TILs can generally be classified by expression of one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into patients.

[0439] The term "cryopreservation media" or "cryopreservation medium" refers to any medium that can be used for cryopreserving cells. Such media can include media containing 7% to 10% DMSO. Exemplary media include CryoStor CS10, Hyperthermasol, and combinations thereof. The term "CS10" refers to cryopreservation media obtained from Stemcell Technologies or Biolife Solutions. CS10 medium may be referred to by the trade name "CryoStor® CS10." CS10 medium is a serum-free, animal-component-free medium that contains DMSO.

[0440] The term "central memory T cells" refers to cells that are CD45R0+ and CCR7 (CCR7 高 ) and CD62L (CD62 高 ) is a subset of T cells that constitutively express the CD4 receptor. The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors of central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. After TCR triggering, central memory T cells primarily secrete IL-2 and CD40L as effector molecules. Central memory T cells predominate in the CD4 compartment in the blood and are proportionally enriched in lymph nodes and tonsils in humans.

[0441] The term "effector memory T cells" refers to T cells that are CD45R0+ like central memory T cells, but have lost constitutive expression of CCR7 (CCR7 低 ), heterogeneous or low CD62L expression (CD62L 低 ), refers to a subset of human or mammalian T cells. The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors of central memory T cells include BLIMP1. Effector memory T cells rapidly secrete high levels of inflammatory cytokines after antigen stimulation, including interferon gamma, IL-4, and IL-5. Effector memory T cells predominate in the CD8 compartment in the blood and are proportionally enriched in the lung, liver, and intestine in humans. CD8+ effector memory T cells carry large amounts of perforin.

[0442] The term "closed system" refers to a system that is closed to the external environment. Any closed system suitable for cell culture methods can be used in the methods of the present invention. Closed systems include, but are not limited to, sealed G containers. Once tumor segments are added to the closed system, TI The system is not opened to the external environment until L is ready to be administered to the patient.

[0443] The terms "fragmenting," "fragments," and "fragmented" as used herein to describe processes for destroying tumors include mechanical fragmentation methods such as crushing, slicing, dividing, and mincing tumor tissue, as well as any other method for disrupting the physical structure of tumor tissue.

[0444] The terms "peripheral blood mononuclear cells" and "PBMCs" refer to peripheral blood cells with round nuclei, including lymphocytes (T cells, B cells, NK cells) and monocytes. When used as antigen-presenting cells (PBMCs are a type of antigen-presenting cell), the peripheral blood mononuclear cells are preferably irradiated allogeneic peripheral blood mononuclear cells.

[0445] 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.

[0446] The term "anti-CD3 antibody" refers to an antibody or variant thereof, e.g., a monoclonal antibody, including a human, humanized, chimeric, or murine antibody, directed against the CD3 receptor in the T cell antigen receptor of mature T cells. Anti-CD3 antibodies include OKT-3, also known as muromonab. Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab.

[0447] The term "OKT-3" (also referred to herein as "OKT3") refers to a monoclonal antibody, including a human, humanized, chimeric, or murine antibody against the CD3 receptor in the T cell antigen receptor of mature T cells, or a biosimilar or variant thereof, including commercially available forms such as OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab, or variants, conservative amino acid substitutions, glycoforms, or biosimilars thereof. The amino acid sequences of the heavy and light chains of muromonab are shown in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2). A hybridoma capable of producing OKT-3 has been deposited with the American Type Culture Collection and assigned ATCC accession number CRL8001. A hybridoma capable of producing OKT-3 has also been deposited with the European Collection of Authenticated Cell Cultures (ECACC) and assigned catalog number 86022706.

[0448] [Table 1]

[0449] The term "IL-2" (also referred to herein as "IL2") refers to the T cell growth factor known as interleukin-2 and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants. IL-2 is described, for example, in Nelson, J. Immunol. 2004, 172, 3983-88 and Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which are incorporated herein by reference. The amino acid sequence of recombinant human IL-2 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 3). For example, the term IL-2 encompasses human recombinant IL-2 forms such as aldesleukin (PROLEUKIN, commercially available from multiple suppliers at 22 million IU per single-use vial), as well as the recombinant IL-2 form (catalog number CYT-209-b) marketed by CellGenix, Inc., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA, and other commercial equivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125 human IL-2) is a non-glycosylated human recombinant IL-2 form with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 4). The term IL-2 also refers to the pegylated IL-2 prodrug bempegaldesleukin (NKTR-214, an IL-2 prodrug in which an average of six lysine residues are replaced with [(2,7-bis{[methylpoly(oxyethylene)]carbamoyl}-9H-fluoren-9-yl)methoxy]carbonyl). 6The present invention also encompasses pegylated forms of IL-2 described herein, including pegylated human recombinant IL-2 such as SEQ ID NO: 4, which is available from Nektar Therapeutics (South San Francisco, CA, USA) or can be prepared by methods known in the art, such as the method described in Example 19 of International Patent Application Publication No. WO2018 / 132496 A1 or Example 1 of U.S. Patent Application Publication No. 2019 / 0275133 A1, the disclosures of which are incorporated herein by reference. Benpegaldesleukin (NKTR-214) and other pegylated IL-2 molecules suitable for use in the present invention are described in U.S. Patent Application Publication No. 2014 / 0328791 A1 and International Patent Application Publication No. WO2012 / 065086 A1, the disclosures of which are incorporated herein by reference. Alternative forms of conjugated IL-2 suitable for use in the present invention are described in U.S. Patent Nos. 4,766,106, 5,206,344, 5,089,261, and 4,902,502, the disclosures of which are incorporated herein by reference. Formulations of IL-2 suitable for use in the present invention are described in U.S. Patent No. 6,706,289, the disclosure of which is incorporated herein by reference.

[0450] In some embodiments, a suitable IL-2 form for use in the present invention is THOR-707, available from Synthorx, Inc. The preparation and properties of THOR-707 and additional alternative forms of IL-2 suitable for use in the present invention are described in U.S. Patent Application Publication Nos. 2020 / 0181220 A1 and 2020 / 0330601 A1, the disclosures of which are incorporated herein by reference. In some embodiments, a suitable IL-2 form for use in the present invention is an interleukin-2 (IL-2) complex comprising an isolated and purified IL-2 polypeptide and a conjugation moiety that binds to the isolated and purified IL-2 polypeptide at an amino acid position selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107, 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, 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 an unnatural amino acid. In some embodiments, the unnatural amino acid is N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-dopa, fluorinated phenylalanine, isopropyl and selenocysteine, or selenocysteine. In some embodiments, the IL-2 complex has a decreased affinity for the IL-2 receptor alpha (IL-2Rα) subunit compared to a wild-type IL-2 polypeptide, hi 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 compared to a wild-type IL-2 polypeptide. In some embodiments, the conjugated moiety is IL-2. and IL-2Rα. In some embodiments, the conjugated moiety comprises a water-soluble polymer. In some embodiments, the additional conjugated moiety comprises a water-soluble polymer. In some embodiments, each of the water-soluble polymers independently comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharide), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), or a combination thereof. In some embodiments, each of the water-soluble polymers independently comprises PEG. In some embodiments, the PEG is linear PEG or branched PEG. In some embodiments, each of the water-soluble polymers independently comprises a polysaccharide. In some embodiments, the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, each of the water-soluble polymers independently comprises a glycan. In some embodiments, each of the water-soluble polymers independently comprises a polyamine. In some embodiments, the conjugation moiety comprises a protein. In some embodiments, the additional conjugation moieties comprise proteins. In some embodiments, each of the proteins independently comprises albumin, transferrin, or transthyretin. In some embodiments, each of the proteins independently comprises an Fc portion. In some embodiments, each of the proteins independently comprises an Fc portion of an IgG. In some embodiments, the conjugation moiety comprises a polypeptide. In some embodiments, the additional conjugation moieties comprise polypeptides.In some embodiments, each of the proteins independently comprises an XTEN peptide, a glycine-rich homoamino acid polymer (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), 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 conjugation moiety is directly attached to the isolated and purified IL-2 polypeptide. In some embodiments, the conjugation moiety is indirectly attached to the isolated and purified IL-2 polypeptide via a linker. In some embodiments, the linker comprises a homobifunctional linker.In some embodiments, the homobifunctional linker is selected from the group consisting of the Romant reagents dithiobis(succinimidyl propionate) DSP, 3′3′-dithiobis(sulfosuccinimidyl propionate) (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), ethylene glycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N′-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3′-dithiobispropionimidate (DTBP), 1,4-di-(3′-(2′-pyridyldithio)bis ... (e) propionamido) butane (DPDPB), bismaleimidohexane (BMH), halogenated aryl-containing compounds (DFDNB), such as 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl] disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid, N,N'-ethylene-bis(iodoacetamide), or N,N'-hexamethylenebis(iodoacetamide). In some embodiments, the linker comprises a heterobifunctional linker. In some embodiments, the heterobifunctional linker is: , N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamido]hexanoate (sulfo-LC-sMPT), succinimidyl-4-( N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MB), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MB), N-succinimidyl (4-iodoacetyl)aminobenzoate (sIAB), sulfosuccinimidyl (4-iodoacetyl)aminobenzoate (sulfo-sIAB), Succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ-maleimidobutyryloxy)succinimide ester (GMB), N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMB), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (slAXX ... succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6-(((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive crosslinkers such as 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M2C2H),3-(2-pyridyldithio)propionyl hydrazide (PDPH), N-hydroxysuccinimidyl-4-azidosalicylate (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylate (sulfo-NHs-AsA), sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (H sAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NO), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3'-dithiopropionate ( sAND), N-succinimidyl-4(4-azidophenyl)-1,3'-dithiopropionate (sADP), N-sulfosuccinimidyl (4-azidophenyl)-1,3'-dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(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 (s 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 comprises a cleavable linker, optionally including a dipeptide linker. In some embodiments, the dipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys. In some embodiments, the linker comprises a non-cleavable linker. In some embodiments, the linker optionally comprises a maleimido group, including maleimidocaproyl (mc), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC). In some embodiments, the linker further comprises a spacer. In some embodiments, the spacer comprises p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), a derivative, or an analog thereof. In some embodiments, the conjugated moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the additional conjugate 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, the IL-2 form suitable for use in the present invention is pegylated as disclosed in U.S. Patent Application Publication Nos. 2020 / 0181220 A1 and 2020 / 0330601 A1. In some embodiments, an IL-2 form suitable for use in the present invention is an IL-2 conjugate comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:5, and wherein AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 relative to amino acid position in SEQ ID NO:5. In some embodiments, the IL-2 polypeptide comprises an N-terminal deletion of one residue relative to SEQ ID NO:5.In some embodiments, forms of IL-2 suitable for use in the present invention lack IL-2R alpha chain association but retain normal binding to the intermediate affinity IL-2R beta-gamma signaling complex.

[0451] In some embodiments, a suitable IL-2 form for use in the present invention is an IL-2 conjugate comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:5, and wherein AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 relative to amino acid position in SEQ ID NO:5. In some embodiments, a suitable IL-2 form for use in the present invention is an IL-2 conjugate comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:5, and wherein AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 relative to amino acid position in SEQ ID NO:5. In some embodiments, a suitable IL-2 form for use in the present invention is an IL-2 conjugate comprising an IL-2 polypeptide comprising N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugation moiety comprising polyethylene glycol (PEG), wherein the IL-2 polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:5, and wherein AzK substitutes an amino acid at position K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 relative to amino acid position in SEQ ID NO:5.

[0452] In some embodiments, a form of IL-2 suitable for use in the present invention is nemvaleukin alpha, also known as ALKS-4230 (SEQ ID NO: 6), available from Alkermes, Nembareukin alfa is available from Nembareukin alfa, which is a compound having a peptidyl linker ( 60 GG 61 ) and fused to human interleukin-2 fragment (62-132) via a peptidyl linker ( 133 GSGGGS 138 Human interleukin-2 receptor α-chain fragment (139-303) fused via a nucleotide sequence (Cys), produced in Chinese hamster ovary (CHO) cells, and glycosylated. 125 >Ser 51 ; human interleukin-2 (IL-2) (4-74)-peptide (62-132) fused via a G2 peptide linker (60-61) and human interleukin-2 receptor alpha chain (IL2R subunit alpha, IL2Rα, IL2RA) (1-165)-peptide (139-303) fused via a GSG3S peptide linker (133-138), produced in Chinese hamster ovary (CHO) cells, and alpha-glycosylated human interleukin-2 (IL-2) (75-133)-peptide [Cys 125(51)>Ser]-mutant (1-59). The amino acid sequence of nemvaleukin alfa is set forth in SEQ ID NO: 6. In some embodiments, nemvaleukin alfa exhibits the following post-translational modifications: disulfide bridges at the following positions: 31-116, 141-285, 184-242, 269-301, 166-197, or 166-199, 168-199, or 168-197 (using the numbering of SEQ ID NO: 6), and glycosylation sites at the following positions: N187, N206, T212, using the numbering of SEQ ID NO: 6. The preparation and properties of nemvaleukin alfa, as well as additional alternative forms of IL-2 suitable for use in the present invention, are described in U.S. Patent Application Publication No. 2021 / 0038684 A1 and U.S. Patent No. 10,183,979, the disclosures of which are incorporated herein by reference. In some embodiments, an IL-2 form suitable for use in the present invention is a protein having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity to SEQ ID NO:6. In some embodiments, an IL-2 form suitable for use in the present invention has the amino acid sequence set forth in SEQ ID NO:6 or conservative amino acid substitutions thereof. In some embodiments, an IL-2 form suitable for use in the present invention is a fusion protein comprising amino acids 24-452 of SEQ ID NO:7, or a variant, fragment, or derivative thereof. In some embodiments, an IL-2 form suitable for use in the present invention is a fusion protein comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity to amino acids 24-452 of SEQ ID NO:7, or a variant, fragment, or derivative thereof. Other IL-2 forms suitable for use in the present invention are described in U.S. Pat. No. 10,183,979, the disclosure of which is incorporated herein by reference.Optionally, in some embodiments, a form of IL-2 suitable for use in the present invention is a fusion protein comprising a first fusion partner linked to a second fusion partner by a mucin domain polypeptide linker, wherein the first fusion partner is IL-1Rα or a protein having at least 98% amino acid sequence identity to IL-1Rα and having receptor antagonist activity for IL-Rα, the second fusion partner comprises all or a portion of an immunoglobulin comprising an Fc region, and the mucin domain polypeptide linker comprises SEQ ID NO:8 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:8, and wherein the half-life of the fusion protein is improved compared to the fusion of the first fusion partner with the second fusion partner in the absence of the mucin domain polypeptide linker.

[0453] [Table 2]

[0454] In some embodiments, IL-2 forms suitable for use in the present invention comprise a heavy chain variable region (V) comprising complementarity determining regions HCDR1, HCDR2, and HCDR3. H ) and a light chain variable region (V L ) and V H or V L and an IL-2 molecule or a fragment thereof grafted onto the CDR of a heavy chain variable region (V) comprising complementarity determining regions HCDR1, HCDR2, and HCDR3. H ) and a light chain variable region (V L ) and V H or V Land an IL-2 molecule or fragment thereof grafted onto the CDRs of, wherein the IL-2 molecule is a mutein, and the antibody cytokine grafted protein preferentially expands T effector cells over regulatory T cells. In some embodiments, the IL-2 regimen comprises administration of an antibody described in U.S. Patent Application Publication No. 2020 / 0270334 A1, the disclosure of which is incorporated herein by reference. In some embodiments, the antibody cytokine grafted protein comprises a heavy chain variable region (VH) comprising complementarity determining regions HCDR1, HCDR2, and HCDR3, a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3, and a VL comprising complementarity determining regions HCDR1, HCDR2, and LCDR3. H or V L CD and an IL-2 molecule or a fragment thereof grafted into R, 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 heavy chain and an IgG light chain selected from the group consisting of an IgG light chain comprising SEQ ID NO: 39 and an IgG heavy chain comprising SEQ ID NO: 38, an IgG light chain comprising SEQ ID NO: 37 and an IgG heavy chain comprising SEQ ID NO: 29, an IgG light chain comprising SEQ ID NO: 39 and an IgG heavy chain comprising SEQ ID NO: 29, an IgG light chain comprising SEQ ID NO: 37 and an IgG heavy chain comprising SEQ ID NO: 38.

[0455] In some embodiments, the IL-2 molecule or fragment thereof is V H In some embodiments, the IL-2 molecule or fragment thereof is a mutein. H In some embodiments, the IL-2 molecule or fragment thereof is a mutein. H In some embodiments, the IL-2 molecule or fragment thereof is a mutein. L In some embodiments, the IL-2 molecule or a fragment thereof is grafted onto LCDR1 of V LIn some embodiments, the IL-2 molecule or fragment thereof is grafted onto LCDR2 of V L The LCDR3 of the IL-2 molecule is grafted onto the LCDR3 of the IL-2 molecule, and the IL-2 molecule is a mutein.

[0456] The insertion of the IL-2 molecule can be at or near the N-terminal region of the CDR, the middle region of the CDR, or at or near the C-terminal region of the CDR. In some embodiments, the antibody cytokine graft protein comprises an IL-2 molecule incorporated into the CDR, where the IL2 sequence does not frameshift the CDR sequence. In some embodiments, the antibody cytokine graft protein comprises an IL-2 molecule incorporated into the CDR, where the IL-2 sequence replaces all or part of the CDR sequence. The replacement with the IL-2 molecule can be at or near the N-terminal region of the CDR, the middle region of the CDR, or the C-terminal region of the CDR. The replacement with the IL-2 molecule can be as little as one or two amino acids of the CDR sequence, or the entire CDR sequence.

[0457] In some embodiments, the IL-2 molecule is directly grafted onto the CDR without a peptide linker and without additional amino acids between the CDR sequence and the IL-2 sequence. In some embodiments, the IL-2 molecule is indirectly grafted onto the CDR using a peptide linker with one or more additional amino acids between the CDR sequence and the IL-2 sequence.

[0458] In some embodiments, the IL-2 molecule described herein is an IL-2 mutein. In some cases, the IL-2 mutein comprises an R67A substitution. In some embodiments, the IL-2 mutein comprises the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the IL-2 mutein comprises the amino acid sequence of Table 1 of U.S. Patent Application Publication No. 2020 / 0270334 A1, the disclosure of which is incorporated herein by reference.

[0459] In some embodiments, the antibody cytokine transplant protein comprises an HCDR1 selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 25. In some embodiments, the antibody cytokine transplant protein comprises an HCDR1 selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 16. In some embodiments, the antibody cytokine transplant protein comprises an HCDR1 selected from the group consisting of an HCDR2 selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, and SEQ ID NO: 26. In some embodiments, the antibody cytokine transplant protein comprises an HCDR3 selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, and SEQ ID NO: 27. In some embodiments, the antibody cytokine transplant protein comprises a V comprising the amino acid sequence of SEQ ID NO: 28. H In some embodiments, the antibody sequence 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 chain comprising the amino acid sequence of SEQ ID NO: 36. L In some embodiments, the antibody cytokine transplant protein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, the antibody cytokine transplant protein comprises a V region comprising the amino acid sequence of SEQ ID NO: 28. H V comprising the region and the amino acid sequence of SEQ ID NO: 36 LIn some embodiments, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:29 and a light chain region comprising the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:29 and a light chain region comprising the amino acid sequence of SEQ ID NO:39. In some embodiments, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:38 and a light chain region comprising the amino acid sequence of SEQ ID NO:37. In some embodiments, the antibody cytokine transplant protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO:38 and a light chain region comprising the amino acid sequence of SEQ ID NO:39. In some embodiments, the antibody cytokine transplant protein comprises IgG.IL2F71A.H1 or IgG.IL2R67A.H1 of U.S. Patent Application Publication No. 2020 / 0270334 A1, or a variant, derivative, or fragment thereof, or conservative amino acid substitutions thereof, or a protein having at least 80%, at least 90%, at least 95%, or at least 98% sequence identity thereto. In some embodiments, the antibody component of the antibody cytokine transplant proteins described herein comprises immunoglobulin sequences, framework sequences, or CDR sequences of palivizumab. In some embodiments, the antibody cytokine transplant proteins described herein have a longer serum half-life than a wild-type IL-2 molecule, such as, but not limited to, aldesleukin or an equivalent molecule. In some embodiments, the antibody cytokine transplant proteins described herein have a sequence set forth in Table 3.

[0460] [Table 3-1] [Table 3-2]

[0461] The term "IL-4" (also referred to herein as "IL4") refers to the cytokine known as interleukin 4, which is produced by Th2 T cells, as well as eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naive helper T cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. Upon activation by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and class II MHC expression, inducing class switching from B cells to IgE and IgG1 expression. Recombinant human IL-4 suitable for use in the present invention is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-211) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 9).

[0462] 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 It is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-254) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number Gibco PHC0071). The amino acid sequence of recombinant human IL-7 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 10).

[0463] The term "IL-15" (also referred to herein as "IL15") refers to the T-cell growth factor known as interleukin-15 and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants. IL-15 is described, for example, in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the disclosure of which is incorporated herein by reference. IL-15 shares β and γ signaling receptor subunits with IL-2. Recombinant human IL-15 is a single, non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular weight of 12.8 kDa. Recombinant human IL-15 is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (catalog number CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, MA, USA (human IL-15 recombinant protein, catalog number 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the present invention is shown in Table 2 (SEQ ID NO: 11).

[0464] The term "IL-21" (also referred to herein as "IL21") refers to the pleiotropic cytokine protein known as interleukin-21, and includes all forms of IL-21, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants. IL-21 is described, for example, in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014, 13, 379-95, the disclosure of which is incorporated herein by reference. IL-21 primarily stimulates natural killer T cells and activated human CD4 + Recombinant human IL-21 is produced by T cells. It is a single, non-glycosylated polypeptide chain containing 132 amino acids with a molecular weight of 15.4 kDa. Recombinant human IL-21 is available from ProSpec-Tany. It is commercially available from several suppliers, including 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).

[0465] When an "antitumor effective amount," "tumor inhibiting effective amount," or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in the age, weight, tumor size, extent of infection or metastasis, and health status of the patient (subject). Generally, the tumor-infiltrating lymphocytes (e.g., secondary TILs or genetically modified cytotoxic lymphocytes) described herein are administered at a dose of 10 per kg of body weight. 4 ~10 11 cells (e.g., 10 per kg body weight) 5 ~10 6 , 10 5 ~10 10 , 10 5 ~10 11 , 10 6 ~10 10 , 10 6 ~10 11 , 10 7 ~10 11 , 10 7 ~10 10 , 10 8 ~10 11 , 10 8 ~10 10 , 10 9 ~10 11, or 10 9 ~10 10 The TIL (optionally including genetically modified cytotoxic lymphocytes) compositions may also be administered multiple times at these dosages. Tumor TIL (optionally including genetically engineered TIL) can be administered by using injection techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 1988, 319, 1676). Optimal dosages and treatment regimes for a particular patient can be readily determined by one skilled in the medical arts by monitoring the patient for signs of disease and adjusting treatment accordingly.

[0466] The terms "hematological malignancies," "blood system malignancies," or terms of related meaning, refer to cancers and tumors of mammalian hematopoietic and lymphatic tissues, including, but not limited to, blood, bone marrow, lymph nodes, and lymphatic tissues. Hematological malignancies are also referred to as "liquid tumors." Hematological malignancies may include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), multiple myeloma, acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphoma. The term "B-cell hematological malignancies" refers to hematological malignancies affecting B cells.

[0467] 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.

[0468] The term "microenvironment" as used herein may refer to the solid or hematological tumor microenvironment as a whole, or to individual subsets of cells within the microenvironment. As used herein, the tumor microenvironment refers to a complex mixture of "cells, soluble factors, signaling molecules, extracellular matrix, and mechanical cues that promote neoplastic transformation, support tumor growth and invasion, protect tumors from host immunity, foster therapeutic resistance, and provide a niche for successful and dominant metastasis," as described in Swartz, et al., Cancer Res., 2012, 72, 2473. Tumors express antigens that are recognized by T cells, but tumor clearance by the immune system is rare due to immunosuppression by the microenvironment.

[0469] In some embodiments, the invention includes methods of treating cancer with a TIL population, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of TILs according to the invention. In some embodiments, a TIL population may be provided, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of TILs according to the invention. In some embodiments, the non-myeloablative chemotherapy is cyclophosphamide 60 mg / kg / day for 2 days (27 and 26 days before TIL infusion) and fludarabine 25 mg / m2 / day for 5 days (27-23 days before TIL infusion). In some embodiments, after non-myeloablative chemotherapy and TIL infusion according to the invention (day 0), the patient receives an intravenous infusion of IL-2 at 720,000 IU / kg every 8 hours to physiological tolerance.

[0470] Experimental findings indicate that lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes plays an important role in enhancing therapeutic efficacy by eliminating regulatory T cells and competing elements of the immune system ("cytokine sinks"). Accordingly, some embodiments of the present invention utilize a lymphodepletion step (sometimes referred to as "immunosuppressive conditioning") on patients prior to introducing the TILs of the present invention.

[0471] 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. A therapeutically effective amount 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 refers to a compound or combination of compounds that induces a specific response (e.g., a specific antibody or antibody combination) in target cells. The term also applies to a dose that induces a specific effect (e.g., a reduction in 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 by which the compound is delivered.

[0472] The terms "treatment," "treating," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect can be preventative, in terms of completely or partially preventing the disease or condition, and / or therapeutic, in terms of partially or completely curing the disease and / or side effects caused by the disease. "Treatment," as used herein, encompasses any treatment of disease in a mammal, particularly a human, and includes (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with it; (b) suppressing the disease, i.e., arresting its onset or progression; and (c) palliating the disease, i.e., causing regression of the disease and / or alleviating one or more disease symptoms. "Treatment" is also intended to encompass the delivery of an agent to provide a pharmacological effect even in the absence of a disease or condition. For example, "treatment" encompasses the delivery of a composition capable of eliciting an immune response or conferring immunity in the absence of a pathology, e.g., in the case of a vaccine.

[0473] The term "heterologous," when used with reference to portions of a nucleic acid or protein, indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids are typically produced recombinantly, with two or more sequences from unrelated genes arranged to create a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source, or coding regions from different sources. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0474] The terms "sequence identity," "percent identity," and "percent sequence identity" (or their synonyms, e.g., "99% identical") in the context of two or more nucleic acids or polypeptides refer to two or more sequences or subsequences that are the same or have a specified percentage of the same nucleotide or amino acid residues when compared and aligned for maximum correspondence (introducing gaps, if necessary), without considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignment of amino acid or nucleotide sequences are known in the art. Suitable programs for determining percent sequence identity include, for example, the BLAST suite of programs available from the BLAST website of the U.S. government's National Center for Biotechnology Information. Comparison between two sequences can be performed using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, and BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or MegAlign, available from DNASTAR, are additional publicly available software programs that can be used to align sequences. Those skilled in the art can determine the appropriate parameters for maximum alignment depending on the particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0475] As used herein, the term "variant" encompasses antibodies or fusion proteins that contain 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. The variant may include, but is not limited to, 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. The variant retains the ability of the reference antibody to specifically bind to the antigen. The term variant also includes pegylated antibodies or proteins.

[0476] As used herein, "tumor infiltrating lymphocytes" or "TILs" refers to a population of cells that are initially acquired as leukocytes that have left the bloodstream of a subject and migrated to a tumor. TILs include CD8 + Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4 + These include, but are not limited to, T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. "Primary TILs" are those obtained from a patient tissue sample as outlined herein (sometimes referred to as "freshly harvested"). "Secondary TILs" are any TIL cell populations that have been expanded or propagated as discussed herein, including, but not limited to, bulk TILs, expanded TILs ("REP TILs"), and "reREP TILs" as discussed herein. reREP TILs can include, for example, second expanded TILs or second additional expanded TILs (e.g., those described in step D of FIG. 8, including TILs designated as reREP TILs).

[0477] TILs can generally be defined either biochemically using cell surface markers or functionally by their ability to infiltrate tumors and achieve therapy. TILs can generally be classified by the expression of one or more of the following biomarkers: CD4, CD8, TCRαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally or alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into patients. TILs can be further characterized by potency; for example, TILs can be considered potent if their interferon (IFN) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL. For example, TILs can be considered potent if interferon (IFNγ) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL, greater than about 300 pg / mL, greater than about 400 pg / mL, greater than about 500 pg / mL, greater than about 600 pg / mL, greater than about 700 pg / mL, greater than about 800 pg / mL, greater than about 900 pg / mL, or greater than about 1000 pg / mL.

[0478] 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.

[0479] The term "RNA" defines a molecule containing at least one ribonucleotide residue. The term "ribonucleotide" defines a nucleotide having a hydroxyl group at the 2' position of a bD-ribofuranose moiety. The term RNA includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. The nucleotides of the RNA molecules described herein can also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNA.

[0480] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is used herein to refer to a compound or a mixture thereof. and any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inactive ingredients. The use of such pharmaceutically acceptable carriers or excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or 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.

[0481] The terms "about" and "approximately" mean within a statistically significant range of values. Such a range may be within one order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5%. The acceptable deviation encompassed by the term "about" or "approximately" depends on the particular system under study and can be readily understood by one of ordinary skill in the art. Furthermore, as used herein, the terms "about" and "approximately" mean that dimensions, sizes, formulations, parameters, shapes, and other quantities and characteristics are not, and need not be, exact, but may be approximate and / or larger or smaller, as appropriate, reflecting tolerances, conversion factors, rounding, measurement errors, etc., and other factors known to those of ordinary skill in the art. In general, a dimension, size, formulation, parameter, shape, or other quantity or characteristic is "about" or "approximately" whether or not expressly stated as such. It should be noted that embodiments of widely different sizes, shapes, and dimensions may employ the described configurations.

[0482] When used in the appended claims, the transitional terms "comprising," "consisting essentially of," and "consisting of" define the claim in its original and amended form, in terms of whether additional, unrecited claim elements or steps, if any, are excluded from the scope of the claim(s). The term "comprising" is intended to be inclusive or open-ended and does not exclude any additional, unrecited elements, methods, steps, or materials. The term "consisting of" excludes any element, step, or material other than those specified in the claim, and in the latter case, also excludes impurities normally associated with the specified material(s). The term "consisting essentially of" limits the claim to the specified element, step, or material(s) and does not materially affect the basic and novel feature(s) of the claimed invention. All compositions, methods, and kits described herein embodying the present invention may, in alternative embodiments, be more specifically defined by any of the transitional terms "comprising," "essentially consisting of," and "consisting of."

[0483] The terms "antibody" and its plural "antibodies" refer to whole immunoglobulins and any antigen-binding fragment ("antigen-binding portion") or single chains thereof. "Antibody" also refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding portions thereof. Each heavy chain contains a heavy chain variable region (referred to herein as V H Each light chain is composed of a light chain variable region (abbreviated herein as V) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. L The light chain constant region consists of one domain, C L The V of the antibody H and V L The regions can be further subdivided into regions of hypervariability, termed complementarity-determining regions (CDRs) or hypervariable regions (HVRs), which may be interspersed with more conserved regions called framework regions (FRs). H and VL is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with one or more antigen epitopes. The constant region of the antibody can 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. do.

[0484] The term "antigen" refers to a substance that induces an immune response. In some embodiments, an antigen is a molecule that can be bound by an antibody or TCR when presented by a major histocompatibility complex (MHC) molecule. As used herein, the term "antigen" also encompasses T cell epitopes. An antigen can additionally be recognized by the immune system. In some embodiments, an antigen can induce a humoral or cellular immune response, leading to the activation of B and / or T lymphocytes. In some cases, this may require that the antigen contain or be bound by a Th cell epitope. An antigen may also have one or more epitopes (e.g., B- and T-epitopes). In some embodiments, an antigen preferably reacts with a corresponding antibody or TCR, typically in a highly specific and selective manner, and not with many other antibodies or TCRs that may be induced by other antigens.

[0485] The terms "monoclonal antibody," "mAb," "monoclonal antibody composition," or their plurals, refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Monoclonal antibodies specific for a particular receptor can be produced using knowledge and techniques in the art by injecting a test subject with an appropriate antigen and then isolating hybridomas expressing antibodies with the desired sequence or functional characteristics. DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector and then transfected into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulin protein to obtain the synthesis of the monoclonal antibody in the recombinant host cells. Recombinant production of antibodies is described in more detail below.

[0486] As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) V L , V H , C L (ii) a Fab fragment, which is a monovalent fragment consisting of two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V H and an Fd fragment consisting of the CH1 domain, (iv) a V of a single arm of an antibody L and V H Fv fragment consisting of domains, (v) V H or VL (v) domain antibody (dAb) fragments, which may consist of two domains, V and VD (Ward, et al., Nature, 1989, 341, 544-546), and (vi) isolated complementarity-determining regions (CDRs). L and V H are encoded by separate genes, which can be synthesized using recombinant methods. L and V H The domains may be joined by a synthetic linker that allows them to pair as a single protein chain to form a monovalent molecule known as a single-chain Fv (scFv) (see, e.g., Bird, et al., Science 1988, 242, 423-426, and Huston, et al., Proc. Natl. Acad. Sci. USA 1988, 85, 5879-5883). Such scFv antibodies are also intended to be encompassed by the term "antigen-binding portion" or "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. In some embodiments, the scFv protein domain comprises a V H Part and V L The scFv molecule contains a V L Do If the main component is the N-terminal portion of the scFv molecule, V L -LV H , or V H If the domain is the N-terminal portion of the scFv molecule, V H -LV LMethods for producing scFv molecules and designing suitable peptide linkers are described in U.S. Pat. Nos. 4,704,692, 4,946,778, R. Raag and M. Whitlow, "Single Chain Fvs," FASEB Vol. 9:73-80 (1995), and RE Bird and BW Walker, Single Chain Antibody Variable Regions, TIBTECH, Vol. 9:132-137 (1991), the disclosures of which are incorporated herein by reference.

[0487] As used herein, the term "human antibody" is intended to ...

Claims

1. A method for producing a tumor-infiltrating lymphocyte (TIL) population, wherein the method is (a) A step of producing a second TIL population by performing a first expansion by culturing tumor fragments or tumor digests obtained from a tumor obtained from a cancer patient, thereby generating a first TIL population in a cell culture medium containing IL-2, wherein the first expansion is performed in a sealed container providing a first gas-permeable surface area, and the first expansion is performed for about 3 to 14 days, (b) The method comprising the step of producing a third TIL population by performing a second expansion on the second TIL population by culturing it in a cell culture medium having IL-2, OKT-3, and antigen-presenting cells (APCs) at a concentration of 3000 IU / mL or less, wherein the second expansion is performed for about 7 to 14 days to obtain the third TIL population, the third TIL population includes a therapeutic TIL population, the second expansion is performed in a sealed container providing a second gas-permeable surface area, and the transition from step (a) to step (b) occurs without opening the closed system.

2. (c) A step of collecting the third TIL population obtained from step (b), wherein the transition from step (b) to step (c) occurs without opening the closed system, (d) A step of transferring the third TIL population collected from step (c) to an injection bag, wherein the transition from step (c) to (d) occurs without opening the closed system, The method according to claim 1, further comprising:

3. A method for producing a tumor-infiltrating lymphocyte (TIL) population, wherein the method is (a) A step of producing a second TIL population by performing a first expansion on a first TIL population obtained from a tumor of a cancer patient in a cell culture medium containing IL-2, wherein the first expansion is performed for about 3 to 14 days to obtain the second TIL population, and (b) A step of performing a second expansion on the second TIL population by culturing it in a cell culture medium having IL-2, OKT-3, and antigen-presenting cells (APCs) at a concentration of 3000 IU / mL or less to produce a third TIL population, wherein the second expansion is performed for about 7 to 14 days to obtain the third TIL population, and the third TIL population produces a third TIL population that includes a therapeutic TIL population. (c) A step of collecting the third TIL population obtained from step (b), The method comprising (d) transferring the third TIL population collected from step (c) to an injection bag.

4. The method according to any one of claims 1 to 3, wherein the cell culture medium is replaced on the third, fourth, fifth, sixth, or seventh day of the first expansion and / or the second expansion.

5. (e) The method of claim 2 or 3, further comprising the step of freeze-drying the injection bag containing the collected third TIL population using a freeze-drying process.

6. The method according to any one of claims 1 to 3, wherein the culture medium for the first expansion comprises IL-2 and / or a protein kinase B (AKT) inhibitor at a concentration of 3000 IU / mL or less.

7. The method according to any one of claims 1 to 3, wherein the culture medium for the second expansion does not contain added IL-2.

8. The method according to any one of claims 1 to 3, wherein the culture medium for the second expansion comprises a protein kinase B (AKT) inhibitor.

9. The method according to any one of claims 1 to 3, wherein the culture medium for the first expansion and / or the second expansion comprises IL-15 and / or IL-21.

10. In D0, D1, or D2, in the culture medium for the first expansion, i) IFNγ and anti-PD-1 antibodies at a concentration of 200 ng / ml; and / or ii) CD40 agonists and / or CTLA-4 inhibitors, The method according to any one of claims 1 to 3, further comprising adding

11. The method according to any one of claims 1 to 3, wherein, prior to the first expansion, the method comprises the step of enzymatically digesting a plurality of tumor fragments to obtain a tumor digest product comprising the first TIL population.

12. The method according to any one of claims 1 to 3, wherein the first TIL population is obtained by surgical excision, needle biopsy, core biopsy, microbiopsy, or other means for obtaining a sample containing a mixture of tumor cells and TIL cells from the cancer.

13. The method according to any one of claims 1 to 3, wherein at least a portion of the first TIL population, the second TIL population, and / or the third TIL population are gene-edited to enhance or reduce gene expression.

14. The method according to claim 13, wherein the gene encodes a protein selected from the group consisting of IL-12, IL-15, IL-2, IL-4, IL-7, IL-10, IL-18, and IL-21.

15. The method according to any one of claims 1 to 3, wherein the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, triple-negative breast cancer, cancer caused by human papillomavirus, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), kidney cancer, and renal cell carcinoma.

16. The method according to any one of claims 1 to 3, wherein the first TIL population is stimulated for up to about 48 hours prior to the first expansion with IFNg (interferon gamma), a PD-1 inhibitor (e.g., an anti-PD-1 antibody), a CD40 agonist (e.g., CD40L, an anti-CD40 agonist antibody), and / or a CTLA-4 agonist (e.g., an anti-CTLA-4 agonist antibody).

17. The first extension and the second extension are, i) over a period of about 11 days; or ii) Within the period of the 5th, 6th, 7th, 8th, or 9th, The method according to any one of claims 1 to 3, performed individually.

18. A therapeutic TIL population manufactured using the method described in any one of claims 1 to 3, wherein the therapeutic TIL population is superior to a TIL population manufactured using a reference TIL manufacturing process. Exhibiting enhanced polyfunctionality, Showing a more trunk-like phenotype, This indicates an increased frequency of TILs with less activation and / or differentiation. It shows improved tumor cell killing in an allogeneic, allogeneic setting. The increased expression of memory-related markers selected from the group consisting of CD27, CD28, CD62L, and IL-7R is observed. This shows a decrease in the expression of an activation marker selected from the group consisting of CD38, CD39, and CD69. Decreased expression of an inhibition / depletion-related marker selected from the group consisting of LAG3, TIM3, TIGIT, and TOX, and / or It shows increased expression of a functional marker selected from the group consisting of GZMB, CXCR3, IFNg, TNFa, and IL-2. The aforementioned therapeutic TIL group.

19. A therapeutic TIL population according to claim 13, for use in the treatment of cancer patients.

20. The aforementioned cancer patient, i) Before using the therapeutic TIL population, a non-myeloablative lymphocyte depletion regimen, and / or ii) A therapeutic TIL population for use according to claim 19, further provided to an IL-2 regime initiated on the same day or the following day as the use of the therapeutic TIL population.