Compositions and methods for immunotherapy

Abstract

The present disclosure relates to methods and compositions for conferring and / or augmenting immune responses mediated by cellular immunotherapy, for example, by adoptive transfer of tumor-specific genetically modified lymphocyte subsets. The present disclosure provides compositions comprising genetically modified lymphocytes expressing at least two transgenes capable of modulating the immune system and the innate and adaptive immune responses.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 935,308, filed November 14, 2019. The foregoing application is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION The present invention relates generally to compositions and methods for treating cancer or tumors in a subject, and more particularly to compositions and methods for treating cancer or tumors in a subject by modulating the subject's immune system. [Background technology]

[0003] Background of the Invention Adoptive cell transfer or adoptive cell therapy (ACT) has shown promise as a treatment for cancer patients, but it faces two major obstacles: the short-term survival of transplanted cells in cancer patients and the hostile, immunosuppressive tumor microenvironment.

[0004] To overcome these limitations, several options have been proposed. For example, one clinical trial tested the administration of interleukin 2 (IL-2) simultaneously with ACT. IL-2 is a potent immunostimulant; therefore, it promotes immune responses and increases the survival rate of transplanted cells. However, this approach was unsuccessful due to the toxicity associated with IL-2. U.S. Patent No. 7,381,405 describes a method for preparing IL-2-transduced lymphocytes for ACT that secrete IL-2. This approach is based on the hypothesis that lymphocytes secrete their own growth factors (such as IL-2) and therefore are less dependent on other exogenous factors for in vivo survival. Despite successful results in an in vitro setting, this approach was deemed ineffective in clinical trials. IL-2-transduced lymphocytes were less effective in treating cancer than non-transduced lymphocytes (Heemskerk et al., Human Gene Therapy, 2008).

[0005] The emergence of chimeric antigen receptor (CAR) T cells has provided a useful tool for improving ACT. TRUCK (International Publication No. WO 2017 / 108805 (Patent Document 2)) and Armored CAR (U.S. Patent No. 10,124,023 (Patent Document 3)) are representative examples of CAR T cells further genetically modified to secrete recombinant interleukin-12 (IL-12) and CD40L, respectively. However, these strategies have drawbacks. For example, in TRUCK, high transgenic IL-12 production restricted T cell proliferation and increased apoptosis, resulting in limited therapeutic efficacy. Furthermore, the clinical application of armored CAR T cells has so far been limited to liquid tumors.

[0006] Solid tumors and their microenvironments have presented a series of challenges to the success of ACT therapy. These challenges include efficient tumor trafficking and invasion and overcoming tumor-mediated immunosuppression. Despite numerous efforts, state-of-the-art ACT therapies have not provided functional durability for long-term efficacy within the immunosuppressive solid tumor microenvironment.

[0007] Therefore, there is a pressing need to identify novel ACT therapies that can confer functional persistence to cells and / or alter the cytokine milieu to overcome the immunosuppressive tumor microenvironment. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent No. 7,381,405 [Patent Document 2] International Publication No. WO 2017 / 108805 [Patent Document 3] U.S. Patent No. 10,124,023 [Non-patent literature]

[0009] [Non-Patent Document 1] Heemskerk et al., Human Gene Therapy, 2008 Summary of the Invention

[0010] The present disclosure addresses the above-mentioned needs in many aspects. In one aspect, the present disclosure provides a composition comprising a plurality of genetically modified lymphocytes that express at least two transgenes (e.g., therapeutic transgenes) for modulating the immune system of a subject.

[0011] In some embodiments, the transgene is selected from the group consisting of an antibody, an antibody fragment, a receptor, a decoy, a checkpoint blockade modulator, a cytokine, a chemokine, a hormone, a cell exclusion tag, and combinations thereof.

[0012] In some embodiments, the decoy is selected from the group consisting of PD1, CTLA4, LAG3, VEGFR1, TIM3, TIGIT, and SIRPα decoy. In some embodiments, the decoy is a PD1 decoy. In some embodiments, the PD-1 decoy is a PD-1.IgG4 (e.g., PD-1.IgG4Fc) decoy.

[0013] In some embodiments, the cytokine is selected from the group consisting of LIGHT or a variant / fragment thereof, IL-33 or a variant / fragment thereof, IL-2 or a variant / fragment thereof, IL-15 or a variant / fragment thereof, IL-12 or a variant / fragment thereof, and CD40L or a variant / fragment thereof. In some embodiments, the cytokine is a mutant cytokine.

[0014] In some embodiments, the cell exclusion tag is selected from the group consisting of tEGFR, Her2, CD20, and CD19.

[0015] In some embodiments, the at least two transgenes comprise two or more of PD-1 decoy or a variant / fragment thereof, IL-2 variant / fragment, LIGHT or a variant / fragment thereof, IL-33 or a variant / fragment thereof, and CD40L or a variant / fragment thereof. In some embodiments, the at least two transgenes further comprise a truncated EGFR (tEGFR) or a variant / fragment thereof, a truncated HER2 (tHER2) or a variant / fragment thereof, or CD20 or a variant / fragment thereof. In some embodiments, the PD-1 decoy or a variant / fragment thereof and tEGFR or a variant / fragment thereof (or tHER2 or a variant / fragment thereof, CD20 or a variant / fragment thereof, CD19 or a variant / fragment thereof) are carried on the same vector.

[0016] In some embodiments, the at least two transgenes are (a) a PD-1 decoy or a variant thereof and a tEGFR or a variant thereof; (b) a PD-1 decoy or a variant thereof and an IL-2 variant; (c) a PD-1 decoy or a variant thereof and LIGHT or a variant thereof; (d) a PD-1 decoy or a variant thereof and an IL-33 or a variant thereof; (e) a PD-1 decoy or a variant thereof and CD40L or a variant thereof; (f) a PD-1 decoy or a variant thereof, an IL-2 variant, and an IL-33 or a variant thereof; (g) a PD-1 decoy or a variant thereof, a tEGFR or a variant thereof, and an IL-2 variant; (h) a PD-1 decoy or a variant thereof, a tEGFR or a variant thereof, and an IL-2 variant; or a variant thereof, and LIGHT or a variant thereof; (i) PD-1 decoy or a variant thereof, tEGFR or a variant thereof, and IL-33 or a variant thereof; (j) PD-1 decoy or a variant thereof, tEGFR or a variant thereof, and CD40L or a variant thereof; (k) PD-1 decoy or a variant thereof, tEGFR or a variant thereof, IL-2 variant, and IL-33 or a variant thereof; (l) PD-1 decoy or a variant thereof, tEGFR or a variant thereof, IL-2 variant, and CD40L or a variant thereof; or (m) PD-1 decoy or a variant thereof, tEGFR or a variant thereof, IL-33 variant, and CD40L or a variant thereof.

[0017] In some embodiments, the PD-1 decoy comprises the amino acid sequence of any one of SEQ ID NOs: 1-4, 6-17, 42, 44, 47-48, and 51-52, or an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 1-4, 6-17, 42, 44, 47-48, and 51-52.

[0018] In some embodiments, the IL-2 variant comprises the amino acid sequence of any one of SEQ ID NOs:21-23, or an amino acid sequence having at least 80% identity to any one of SEQ ID NOs:21-23.

[0019] In some embodiments, the IL-33 comprises the amino acid sequence of any one of SEQ ID NOs:25 and 27, or an amino acid sequence having at least 80% identity to any one of SEQ ID NOs:25 and 27.

[0020] In some embodiments, LIGHT comprises the amino acid sequence of any one of SEQ ID NOs:28-29 and 31, or an amino acid sequence having at least 80% identity to any one of SEQ ID NOs:28-29 and 31.

[0021] In some embodiments, CD40L comprises the amino acid sequence of any one of SEQ ID NOs: 32-34, 36, and 38, or an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 32-34, 36, and 38.

[0022] In some embodiments, tEGFR comprises an amino acid sequence having at least 80% identity to, or the amino acid sequence of SEQ ID NO: 40. In some embodiments, HER2 comprises an amino acid sequence having at least 80% identity to, or the amino acid sequence of SEQ ID NO: 45. In some embodiments, CD20 comprises an amino acid sequence having at least 80% identity to, or the amino acid sequence of SEQ ID NO: 49.

[0023] In some embodiments, the transgene comprises an antibody or antibody fragment selected from the group consisting of VEGF, TGF-B, 4-1BB, CD28, CD27, NKG2D, PD1, PDL1, and CTLA4 antibodies. In certain embodiments, the antibody is a PD1 antibody.

[0024] In some embodiments, the plurality of lymphocytes comprises at least two lymphocyte subsets. In certain embodiments, the plurality of lymphocytes consists of two lymphocyte subsets. In some embodiments, each subset of the plurality of lymphocytes expresses at least one transgene. In some embodiments, the at least two transgenes are different from one another.

[0025] In some embodiments, the plurality of lymphocytes comprises (i) a first subset expressing at least two transgenes; and (ii) a second subset expressing at least two transgenes, wherein at least one of the transgenes of the first subset is different from a transgene of the second subset, or at least one of the transgenes of the first subset is common to a transgene of the second subset.

[0026] In some embodiments, (i) a first subset expresses at least a PD-1 decoy or a variant thereof and an IL-2 variant, and a second subset expresses at least a PD-1 decoy or a variant thereof and LIGHT or a variant thereof; (ii) a first subset expresses at least a PD-1 decoy or a variant thereof and an IL-2 variant, and a second subset expresses at least a PD-1 decoy or a variant thereof and IL-33 or a variant thereof; (iii) a first subset expresses at least a PD-1 decoy or a variant thereof and an IL-2 variant, and a second subset expresses at least a PD-1 decoy or a variant thereof and CD40L or a variant thereof; (iv) (v) the first subset expresses at least PD-1 decoy or a variant thereof and LIGHT or a variant thereof, and the second subset expresses at least PD-1 decoy or a variant thereof and IL-33 or a variant thereof; or (vi) the first subset expresses at least PD-1 decoy or a variant thereof and LIGHT or a variant thereof, and the second subset expresses at least PD-1 decoy or a variant thereof and CD40L or a variant thereof.

[0027] In some embodiments, the first subset or the second subset further expresses tEGFR or a variant thereof, truncated HER2 (tHER2) or a variant thereof, CD20 or a variant thereof, or CD19 or a variant thereof.

[0028] In some embodiments, (i) a first subset expresses at least a PD-1 decoy or a variant thereof, tEGFR or a variant thereof, and an IL-2 variant, and a second subset expresses at least a PD-1 decoy or a variant thereof, tEGFR or a variant thereof, and LIGHT or a variant thereof; (ii) a first subset expresses at least a PD-1 decoy or a variant thereof, tEGFR or a variant thereof, and an IL-2 variant, and a second subset expresses at least a PD-1 decoy or a variant thereof, tEGFR or a variant thereof, and IL-33 or a variant thereof; (iii) a first subset expresses at least a PD-1 decoy or a variant thereof, tEGFR or a variant thereof, and an IL-2 variant, and a second subset expresses at least a PD-1 decoy or a variant thereof, tEGFR or a variant thereof, and CD40L or a variant thereof; (iv) (v) the first subset expresses at least PD-1 decoy or a variant thereof, tEGFR or a variant thereof, and LIGHT or a variant thereof, and the second subset expresses at least PD-1 decoy or a variant thereof, tEGFR or a variant thereof, and LIGHT or a variant thereof, and the second subset expresses at least PD-1 decoy or a variant thereof, tEGFR or a variant thereof, and CD40L or a variant thereof; or (vi) the first subset expresses at least PD-1 decoy or a variant thereof, tEGFR or a variant thereof, and IL-33 or a variant thereof, and the second subset expresses at least PD-1 decoy or a variant thereof, tEGFR or a variant thereof, and CD40L or a variant thereof.

[0029] In some embodiments, the first subset or the second subset further expresses tEGFR or a variant thereof, tHER2 or a variant thereof, or CD20 or a variant thereof.

[0030] In some embodiments, the two subsets are combined in a ratio of about 1:1 to about 1:100. In some embodiments, the two subsets are combined in a ratio of about 1:1.

[0031] In some embodiments, the lymphocytes are autologous. In some embodiments, the lymphocytes are tumor-infiltrating lymphocytes. In some embodiments, the lymphocytes express a chimeric antigen receptor (CAR). In some embodiments, the lymphocytes express a recombinant T cell receptor (TCR). In some embodiments, the recombinant T cell receptor (TCR) exhibits reactivity to NY-ESO1, MAGE-A1, MAGE-A3, MAGE A-10, MAGE-C2, SSX2, MAGE-A12, or a combination thereof.

[0032] Also included within the scope of this disclosure is a pharmaceutical composition comprising an effective amount of the above-described composition and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent.

[0033] Further provided in this disclosure are kits containing effective amounts of the above compositions.

[0034] In another aspect, the present disclosure provides a method for preparing the composition, comprising the steps of: (a) providing a plurality of lymphocytes; (b) introducing nucleic acid molecules encoding at least two transgenes into the plurality of lymphocytes to obtain a plurality of genetically modified lymphocytes; and (c) expanding the plurality of genetically modified lymphocytes in a cell culture medium.

[0035] Alternatively, the method includes the steps of: (a) providing a plurality of lymphocytes; (b) introducing two or more nucleic acid molecules into the plurality of lymphocytes, thereby obtaining a plurality of genetically modified lymphocytes, wherein each of the two or more nucleic acid molecules encodes at least one transgene; and (c) expanding the plurality of genetically modified lymphocytes in a cell culture medium.

[0036] In some embodiments, the at least two transgenes comprise two or more of PD-1 decoy, IL-2 variant / fragment, LIGHT or variant / fragment thereof, IL-33 or variant / fragment thereof, and CD40L or variant / fragment thereof. In some embodiments, the at least two transgenes further comprise tEGFR or variant / fragment thereof. In some embodiments, the PD-1 decoy or variant / fragment thereof and tEGFR or variant / fragment thereof (or tHER2 or variant / fragment thereof, CD20 or variant / fragment thereof, or CD19 or variant / fragment thereof) are carried on the same vector.

[0037] In some embodiments, the method includes: (a) introducing a first nucleic acid molecule encoding at least two transgenes into a first plurality of lymphocytes to obtain the first plurality of genetically modified lymphocytes; and (b) introducing a second nucleic acid molecule encoding at least two transgenes into a second plurality of lymphocytes to obtain the second plurality of genetically modified lymphocytes.

[0038] In some embodiments, the method further comprises expanding the first plurality of lymphocytes in cell culture medium after the step of introducing the first nucleic acid, or expanding the second plurality of lymphocytes in cell culture medium after the step of introducing the second nucleic acid.

[0039] In some embodiments, the method further includes combining the first plurality of genetically modified lymphocytes with the second plurality of genetically modified lymphocytes at a predetermined ratio of about 1:1 to about 1:100 (e.g., 1:1).

[0040] In some aspects, the cell culture medium is a defined cell culture medium. In some aspects, the cell culture medium comprises a neoantigen peptide.

[0041] In yet another aspect, the present disclosure further provides a method of treating cancer / tumor or chronic infection in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a composition or pharmaceutical composition as described above.

[0042] In some embodiments, the cancer is selected from the group consisting of melanoma, sarcoma, ovarian cancer, prostate cancer, lung cancer, bladder cancer, MSI-high tumors, head and neck tumors, kidney cancer, and breast cancer.

[0043] In some embodiments, the composition is administered by intravenous infusion. In some embodiments, the method further comprises administering a second therapeutic agent to the subject. In some embodiments, the second therapeutic agent is an anti-cancer or anti-tumor agent. In some embodiments, the composition or pharmaceutical composition is administered to the subject before, after, or simultaneously with the second therapeutic agent.

[0044] [The present invention 1001] A composition comprising a plurality of genetically modified lymphocytes expressing at least two transgenes for modulating the immune system of a subject. [The present invention 1002] 1001. The composition of claim 1001, wherein the transgene is selected from the group consisting of an antibody, an antibody fragment, a receptor, a decoy, a checkpoint blockade modulator, a cytokine, a chemokine, a hormone, a cell exclusion tag, and combinations thereof. [The present invention 1003] The composition of claim 1002, wherein the decoy is selected from the group consisting of PD1, CTLA4, LAG3, VEGFR1, TIM3, TIGIT, and SIRPα decoy. [The present invention 1004] The composition of the present invention 1003, wherein the decoy is a PD1 decoy. [The present invention 1005] 1004. The composition of the present invention, wherein the PD-1 decoy is a PD-1.IgG4 decoy. [The present invention 1006] 1002. The composition of claim 10, wherein the cytokine is selected from the group consisting of LIGHT or a variant thereof, IL-33 or a variant thereof, IL-2 or a variant thereof, IL-15 or a variant thereof, IL-12 or a variant thereof, and CD40L or a variant thereof. [The present invention 1007] The composition of claim 1006, wherein the cytokine is a mutant cytokine. [The present invention 1008] 1002. The composition of claim 1002, wherein the cell exclusion tag is selected from the group consisting of truncated EGFR (tEGFR), HER2, CD20, and CD19. [The present invention 1009] Any of the aforementioned compositions of the present invention, wherein the at least two transgenes comprise two or more of PD-1 decoy or a variant thereof, IL-2 variant, LIGHT or a variant thereof, IL-33 or a variant thereof, and CD40L or a variant thereof. [The present invention 1010] 1009. The composition of claim 10, wherein the at least two transgenes further comprise tEGFR or a variant thereof, truncated HER2 (tHER2) or a variant thereof, CD20 or a variant thereof, or CD19 or a variant thereof. [The present invention 1011] At least two transgenes (a) PD-1 decoy or its variant and tEGFR or its variant; (b) PD-1 decoy or its variants and IL-2 variants; (c) PD-1 decoy or a variant thereof and LIGHT or a variant thereof; (d) PD-1 decoy or a variant thereof and IL-33 or a variant thereof; (e) PD-1 decoy or a variant thereof and CD40L or a variant thereof; (f) PD-1 decoy or a variant thereof, an IL-2 variant, and an IL-33 or a variant thereof; (g) PD-1 decoy or its variant, tEGFR or its variant, and IL-2 variant; (h) PD-1 decoy or a variant thereof, tEGFR or a variant thereof, and LIGHT or a variant thereof; (i) PD-1 decoy or a variant thereof, tEGFR or a variant thereof, and IL-33 or a variant thereof; (j) PD-1 decoy or a variant thereof, tEGFR or a variant thereof, and CD40L or a variant thereof; (k) PD-1 decoy or a variant thereof, tEGFR or a variant thereof, IL-2 variant, and IL-33 or a variant thereof; (l) a PD-1 decoy or a variant thereof, a tEGFR or a variant thereof, an IL-2 variant, and a CD40L or a variant thereof; or (m) PD-1 decoy or a variant thereof, tEGFR or a variant thereof, IL-33 variant, and CD40L or a variant thereof. The composition of claim 1009 or 1010, comprising: [The present invention 1012] The composition of the present invention 1010, wherein the PD-1 decoy or variant thereof is carried on the same vector as tEGFR or a variant thereof, tHER2 or a variant thereof, CD20 or a variant thereof, or CD19 or a variant thereof. [The present invention 1013] Any of the compositions of claims 1004 to 1005 and 1009 to 1011, wherein the PD-1 decoy comprises an amino acid sequence of any one of SEQ ID NOs: 1 to 4, 6 to 17, 42, 44, 47 to 48, and 51 to 52, or an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 1 to 4, 6 to 17, 42, 44, 47 to 48, and 51 to 52. [The present invention 1014] Any of the compositions of claims 1006 to 1013, wherein the IL-2 variant comprises the amino acid sequence of any one of SEQ ID NOs:21 to 23, or an amino acid sequence having at least 80% identity to any one of SEQ ID NOs:21 to 23. [The present invention 1015] Any of the compositions of claims 1006 to 1014, wherein IL-33 comprises the amino acid sequence of any one of SEQ ID NOs: 25 and 27, or an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 25 and 27. [The present invention 1016] Any of the compositions of claims 1006 to 1015, wherein LIGHT comprises the amino acid sequence of any one of SEQ ID NOs: 28 to 29 and 31, or an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 28 to 29 and 31. [The present invention 1017] Any of the compositions of claims 1006 to 1016, wherein CD40L comprises the amino acid sequence of any one of SEQ ID NOs: 32 to 34, 36, and 38, or an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 32 to 34, 36, and 38. [The present invention 1018] Any of the compositions of claims 1002 and 1010 to 1017, wherein tEGFR comprises an amino acid sequence having at least 80% identity to SEQ ID NO:40 or the amino acid sequence of SEQ ID NO:40; HER2 comprises an amino acid sequence having at least 80% identity to SEQ ID NO:45 or the amino acid sequence of SEQ ID NO:45; and CD20 comprises an amino acid sequence having at least 80% identity to SEQ ID NO:49 or the amino acid sequence of SEQ ID NO:49. [The present invention 1019] 1002. The composition of claim 1002, wherein the antibody or antibody fragment is selected from the group consisting of VEGF, TGF-B, 4-1BB, CD28, CD27, NKG2D, PD1, PDL1, and CTLA4 antibodies. [The present invention 1020] The composition of the present invention 1019, wherein the antibody is a PD1 antibody. [The present invention 1021] Any of the aforementioned compositions of the present invention, wherein the plurality of lymphocytes comprises at least two lymphocyte subsets. [The present invention 1022] Any of the compositions of the present invention, wherein the plurality of lymphocytes consists of two lymphocyte subsets. [The present invention 1023] The composition of claim 1021 or 1022, wherein each subset of the plurality of lymphocytes expresses at least one transgene. [The present invention 1024] The composition of any of claims 1021 to 1023, wherein at least two transgenes are different from each other. [The present invention 1025] Several lymphocytes (i) a first subset expressing at least two transgenes; and (ii) a second subset expressing at least two transgenes; and wherein at least one of the transgenes of the first subset is different from the transgenes of the second subset, or at least one of the transgenes of the first subset is in common with the transgenes of the second subset. [The present invention 1026] (i) a first subset expresses at least a PD-1 decoy or a variant thereof and an IL-2 variant, and a second subset expresses at least a PD-1 decoy or a variant thereof and LIGHT or a variant thereof; (ii) the first subset expresses at least a PD-1 decoy or a variant thereof and an IL-2 variant, and the second subset expresses at least a PD-1 decoy or a variant thereof and an IL-33 or a variant thereof; (iii) the first subset expresses at least a PD-1 decoy or a variant thereof and an IL-2 variant, and the second subset expresses at least a PD-1 decoy or a variant thereof and CD40L or a variant thereof; (iv) the first subset expresses at least PD-1 decoy or a variant thereof and LIGHT or a variant thereof, and the second subset expresses at least PD-1 decoy or a variant thereof and IL-33 or a variant thereof; (v) a first subset expresses at least PD-1 decoy or a variant thereof and LIGHT or a variant thereof, and a second subset expresses at least PD-1 decoy or a variant thereof and CD40L or a variant thereof; or (vi) the first subset expresses at least PD-1 decoy or a variant thereof and IL-33 or a variant thereof, and the second subset expresses at least PD-1 decoy or a variant thereof and CD40L or a variant thereof; Composition of the present invention 1025. [The present invention 1027] 1027. The composition of claim 1026, wherein the first subset or the second subset further expresses tEGFR or a variant thereof, tHER2 or a variant thereof, CD20 or a variant thereof, or CD19 or a variant thereof. [The present invention 1028] (i) a first subset expresses at least a PD-1 decoy or a variant thereof, a tEGFR or a variant thereof, and an IL-2 variant, and a second subset expresses at least a PD-1 decoy or a variant thereof, a tEGFR or a variant thereof, and LIGHT or a variant thereof; (ii) the first subset expresses at least a PD-1 decoy or a variant thereof, a tEGFR or a variant thereof, and an IL-2 variant, and the second subset expresses at least a PD-1 decoy or a variant thereof, a tEGFR or a variant thereof, and an IL-33 or a variant thereof; (iii) the first subset expresses at least PD-1 decoy or a variant thereof, tEGFR or a variant thereof, and an IL-2 variant, and the second subset expresses at least PD-1 decoy or a variant thereof, tEGFR or a variant thereof, and CD40L or a variant thereof; (iv) the first subset expresses at least PD-1 decoy or a variant thereof, tEGFR or a variant thereof, and LIGHT or a variant thereof, and the second subset expresses at least PD-1 decoy or a variant thereof, tEGFR or a variant thereof, and IL-33 or a variant thereof; (v) the first subset expresses at least PD-1 decoy or a variant thereof, tEGFR or a variant thereof, and LIGHT or a variant thereof, and the second subset expresses at least PD-1 decoy or a variant thereof, tEGFR or a variant thereof, and CD40L or a variant thereof; or (vi) the first subset expresses at least PD-1 decoy or a variant thereof, tEGFR or a variant thereof, and IL-33 or a variant thereof, and the second subset expresses at least PD-1 decoy or a variant thereof, tEGFR or a variant thereof, and CD40L or a variant thereof; The composition of the present invention 1027. [The present invention 1029] 1029. Any of the compositions of claims 1021 to 1028, wherein the two subsets are combined in a ratio of about 1:1 to about 1:100. [The present invention 1030] 1029. The composition of claim 10, wherein the two subsets are combined in a ratio of about 1:1. [The present invention 1031] Any of the preceding compositions of the present invention, wherein the lymphocytes are autologous. [The present invention 1032] Any of the compositions of the present invention, wherein the lymphocytes are tumor-infiltrating lymphocytes. [The present invention 1033] Any of the aforementioned compositions of the present invention, wherein the lymphocytes express a chimeric antigen receptor (CAR). [The present invention 1034] Any of the preceding compositions of the present invention, wherein the lymphocytes express a recombinant T cell receptor (TCR). [This invention 1035] The composition of lymphocytes of the present invention 1034, wherein the recombinant T cell receptor (TCR) exhibits reactivity to NY-ESO1, MAGE-A1, MAGE-A3, MAGE A-10, MAGE-C2, SSX2, MAGE-A12, or a combination thereof. [The present invention 1036] A pharmaceutical composition comprising an effective amount of any of the compositions of the present invention and a pharmaceutically acceptable carrier. [This invention 1037] The pharmaceutical composition of claim 1032, further comprising a second therapeutic agent. [The present invention 1038] A kit comprising an effective amount of any of the compositions of the present inventions 1001 to 1035 or an effective amount of any of the pharmaceutical compositions of the present inventions 1036 to 1037. [This invention 1039] providing a plurality of lymphocytes; introducing nucleic acid molecules encoding at least two transgenes into said plurality of lymphocytes to obtain a plurality of genetically modified lymphocytes; and expanding the plurality of genetically modified lymphocytes in a cell culture medium. A method for preparing any one of the compositions of inventions 1001 to 1035, comprising: [The present invention 1040] providing a plurality of lymphocytes; introducing two or more nucleic acid molecules into said plurality of lymphocytes, thereby obtaining a plurality of genetically modified lymphocytes, wherein each of said two or more nucleic acid molecules encodes at least one transgene; and expanding the plurality of genetically modified lymphocytes in a cell culture medium. A method for preparing any one of the compositions of inventions 1001 to 1035, comprising: [The present invention 1041] The method of any one of claims 1039 to 1040, wherein the at least two transgenes comprise two or more of PD-1 decoy, IL-2 variant, LIGHT or a variant thereof, IL-33 or a variant thereof, and CD40L or a variant thereof. [The present invention 1042] 1042. The method of claim 1041, wherein the at least two transgenes further comprise tEGFR or a variant thereof, tHER2 or a variant thereof, CD20 or a variant thereof, or CD19 or a variant thereof. [This invention 1043] At least two transgenes (a) PD-1 decoy or its variant and tEGFR or its variant; (b) PD-1 decoy or its variants and IL-2 variants; (c) PD-1 decoy or a variant thereof and LIGHT or a variant thereof; (d) PD-1 decoy or a variant thereof and IL-33 or a variant thereof; (e) PD-1 decoy or a variant thereof and CD40L or a variant thereof; (f) PD-1 decoy or a variant thereof, an IL-2 variant, and an IL-33 or a variant thereof; (g) PD-1 decoy or its variant, tEGFR or its variant, and IL-2 variant; (h) PD-1 decoy or a variant thereof, tEGFR or a variant thereof, and LIGHT or a variant thereof; (i) PD-1 decoy or a variant thereof, tEGFR or a variant thereof, and IL-33 or a variant thereof; (j) PD-1 decoy or a variant thereof, tEGFR or a variant thereof, and CD40L or a variant thereof; (k) PD-1 decoy or a variant thereof, tEGFR or a variant thereof, IL-2 variant, and IL-33 or a variant thereof; (l) a PD-1 decoy or a variant thereof, a tEGFR or a variant thereof, an IL-2 variant, and a CD40L or a variant thereof; or (m) PD-1 decoy or a variant thereof, tEGFR or a variant thereof, IL-33 variant, and CD40L or a variant thereof. The method of the present invention 1041 or 1042, comprising: [This invention 1044] The method of claim 1042, wherein the PD-1 decoy is carried on the same vector as the tEGFR or variant thereof, tHER2 or variant thereof, CD20 or variant thereof, or CD19 or variant thereof. [This invention 1045] introducing into a first plurality of lymphocytes a first nucleic acid molecule encoding at least two transgenes to obtain a first plurality of genetically modified lymphocytes; and introducing a second nucleic acid molecule encoding at least two transgenes into a second plurality of lymphocytes to obtain a second plurality of genetically modified lymphocytes. A method for preparing any one of the compositions of inventions 1021 to 1035, comprising: [The present invention 1046] The method of the present invention 1045, comprising, after the step of introducing the first nucleic acid, expanding the first plurality of lymphocytes in cell culture medium, or, after the step of introducing the second nucleic acid, expanding the second plurality of lymphocytes in cell culture medium. [This invention 1047] The method of any one of claims 1045 to 1046, further comprising combining the first plurality of genetically modified lymphocytes with the first plurality of genetically modified lymphocytes at a predetermined ratio of about 1:1 to about 1:100. [This invention 1048] The method of any of claims 1039 to 1040 and 1046, wherein the cell culture medium is a defined cell culture medium. [This invention 1049] The method of claim 1048, wherein the cell culture medium comprises a neoantigen peptide. [The present invention 1050] A method for treating cancer / tumor or chronic infection in a subject, comprising the step of administering a therapeutically effective amount of any of the compositions of present inventions 1001 to 1035 or any of the pharmaceutical compositions of present inventions 1036 to 1037 to a subject in need thereof. [This invention 1051] The method of claim 1050, wherein the cancer is selected from the group consisting of melanoma, sarcoma, ovarian cancer, prostate cancer, lung cancer, bladder cancer, MSI-high tumors, head and neck tumors, kidney cancer, and breast cancer. [This invention 1052] 1052. The method of any one of claims 1050 to 1051, wherein said composition is administered by intravenous infusion. [This invention 1053] The method of any of claims 1050 to 1052, further comprising the step of administering a second therapeutic agent to the subject. [This invention 1054] The method of claim 1053 and the pharmaceutical composition of claim 1037, wherein the second therapeutic agent is an anti-cancer or anti-tumor agent. [This invention 1055] The method of any one of claims 1053 to 1054, wherein said composition or said pharmaceutical composition is administered to the subject before, after or simultaneously with a second therapeutic agent. The foregoing summary is not intended to define every aspect of the present disclosure; additional aspects are described in other sections, such as the following detailed description. It should be understood that this entire document is intended to be related as a unified disclosure, and that all combinations of features described herein are contemplated, even if those combinations are not found together in the same sentence, paragraph, or section of this document. Other features and advantages of the present invention will become apparent from the detailed description that follows. However, it should be understood that the detailed description and specific examples, while indicating particular embodiments of the present disclosure, are given by way of example only, as various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0045] [Figure 1-1]Figure 1A shows that OT-1 CD8+ T cells were genetically modified to secrete PD1.IgG4 decoy in combination with either IL-2 variant (IL-2V), LIGHT, IL-33, or CD40L. Figure 1A shows that OT-1 CD8+ T cells were genetically modified to secrete both PD1.IgG4 and mutant IL2. Transduction efficiency was determined by FACS (Figure 1A; left panel), and secretion was assessed by ELISA (Figure 1A; center and right panels). Figure 1B shows that OT-1 CD8+ T cells were genetically modified to secrete both PD1.IgG4 and LIGHT. Transduction efficiency was determined by FACS (Figure 1B; left and middle left panels), and secretion was assessed by ELISA (Figure 1B; middle right and right panels). Figure 1C shows that OT-1 CD8+ T cells were genetically modified to secrete both PD1.IgG4 and IL-33. Transduction efficiency was determined by FACS (Figure 1C; left and middle left panels), and secretion was assessed by ELISA (Figure 1C; middle right and right panels). Figure 1D shows that OT-1 CD8+ T cells were genetically modified to secrete both PD1.IgG4 and CD40L. Transduction efficiency was determined by FACS (Figure 1D; left and middle left panels), and secretion was assessed by ELISA (Figure 1C; middle right and right panels). [Figure 1-2] See description of Figure 1-1. [Figure 2-1]This is a series of figures showing that adoptive transfer of OT-1 CD8+ T cells genetically modified to secrete a combination of three immunomodulatory factors significantly improved tumor control of established, large B16-OVA tumors in the absence of preconditioning. Figures 2A and 2B show the tumor growth curves (Figure 2A) and overall survival curves (Figure 2B) of mice administered OT-1 CD8+ T cells secreting PD-1.IgG4, IL-2V, and LIGHT. Figures 2C and 2D show the tumor growth curves (Figure 2C) and overall survival curves (Figure 2D) of mice administered OT-1 CD8+ T cells secreting PD-1.IgG4, IL-33, and LIGHT. Figures 2E and 2F show the tumor growth curves (Figure 2E) and overall survival curves (Figure 2F) of mice administered OT-1 CD8+ T cells secreting PD-1.IgG4, IL-33, and IL-2V. Experiments were performed blindly with six animals per group. Figures 2G and 2H show tumor growth curves (Figure 2G) and overall survival curves (Figure 2H) for mice administered OT-1 CD8+ T cells secreting PD-1, IgG4, IL-2V, and CD40L. Survival analysis was performed using a log-rank Mantel-Cox model. Tumor growth at day 27 was compared using the Kruskal-Wallis test, comparing each group to mice administered UT OT-1 CD8+ T cells. Correction for multiple comparisons was performed using the Dunn test; *p<0.05, **p<0.001, ****p<0.0001. [Figure 2-2] See description of Figure 2-1. [Figure 3-1]This is a series of figures showing that orthogonal T-cell engineering improves ACT efficacy in immunocompetent hosts through the expansion of adoptively transferred CD8+ T cells and the mobilization of endogenous antitumor immunity. Figure 3A shows the experimental design. Figure 3B shows a waterfall plot showing the change in tumor volume from day 17. The best response (smallest tumor volume) observed in each animal at least 12 days after the first ACT was used for calculation (* day 24 after tumor inoculation, ** day 31 after tumor inoculation). Objective response rates (ORR) include complete responses (CR; 100% reduction in tumor volume) and partial responses (PR; ≤-30% tumor change). Figures 3C, 3D, 3E, and 3F show that B16-OVA tumor-bearing mice were treated with either transfected or non-transfected OT1 cells as indicated; tumors were then harvested on days 17 and 24, and cell quantification was performed by flow cytometry. Data are from three independent experiments (n ≥ 5 mice / group). Figure 3C shows the total number of CD8+ TILs on day 24. Figure 3D shows the total number of CD45.1+ OT1 TILs on days 17 and 24. Figure 3E shows the total number of endogenous CD45.1neg CD8 TILs on days 17 and 24. Figure 3F shows the total number of endogenous and exogenous TCF1+ CD8+ TILs on day 24. Figure 3G shows representative immunofluorescence micrographs of tumor sections from each experimental group on day 24, showing OT1 and endogenous TCF1+ CD8+ TILs. Filled triangles: TCF1+ OT1; open triangles: TCF1negOT1; open arrows: TCF1+ endogenous CD8+ TILs. Figure 3H shows that PD1d / 2V / 33+ OT-1 cells were administered as previously indicated to B16-OVA-tumor-bearing CD8KO mice or wtC57BL6 mice that were also treated with the drug FTY720 at 100 μg / mouse (administered i.p. every 3 days starting 2 days before the first cell transfer). Figure 3I shows that B16-OVA tumor-bearing mice were treated as indicated, and tumors were then harvested on day 24. Tregs were quantified by flow cytometry. Data are from three independent experiments (n ≥ 5 mice / group).Bar graphs of CD8+ / Treg ratios are shown. Figures 3J and 3K show tumor growth control over time in B16-OVA tumor-bearing mice treated with PD1d / 2V / 33+ OT-1 cells in the presence or absence of depleting antibodies specific for the indicated surface markers, 250 μg / mouse, administered i.p. starting 1 day before the first cell transfer and maintained every 3 days: CD4 (maintained until day 55 after tumor inoculation) (Figure 3J) and Ly6G (Figure 3K). A representative experiment out of two independent experiments (n = 6 mice / group) is shown (Figures 3H, 3J, and 3K). Brown-Forsythe and Welch ANOVA tests combined with Tukey's test to correct for multiple comparisons were used to compare different groups (Figures 3C, 3D, 3E, and 3F) and tumor volumes (Figures 3H, 3J, and 3K). Comparisons between days 17 and 24 (D and F) used two-tailed Student's t-test with Welch's correction: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. [Figure 3-2] See description of Figure 3-1. [Figure 3-3] See description of Figure 3-1. [Figure 4-1]This series of figures demonstrates that orthogonal engineering induces novel subsets of effector-like CD8 T cells distinct from terminally exhausted and transiently CX3CR1+ effector-like cells. Figure 4A shows the experimental design. B16-OVA tumor-bearing mice were treated as indicated; tumors were then harvested on days 17 and 24, and CD45+ cell suspensions enriched for CD8+ TILs were obtained by FACS sorting and single-cell sequencing using 10X Genomics. Figure 4B shows a UMAP plot demonstrating a low-dimensional representation of cellular heterogeneity and the results of unsupervised clustering, with contour plots depicting areas of high cell density for each treatment. Figure 4C shows the CD8 TIL state predicted by TILPRED (top) and a volcano plot (bottom) showing significantly differentially expressed genes between GzmC+ C5 cells and GzmCneg terminally exhausted cells. Figure 4D shows the projection of PD-1d / 2V / 33 (day 24) TILs onto a reference TIL map using ProjecTILs. On the right, radar plots show the expression levels of key T cell markers for the projected T cell state and the reference exhausted T cell state. Figure 4E shows dot plots showing cluster-specific markers. Figure 4F shows gene signature enrichment analysis (GSEA) of CD8 TIL Tox knockout cells in Gzmc+ C5 cells and Gzmcneg C4 cells. Figure 4G shows B16-OVA tumor-bearing mice treated with either transfected or untransfected OT1 cells on days 12 and 15 after tumor cell inoculation. Tumors or spleens (PD1d / 2V / 33 OT1) were harvested on day 24, and intracellular expression of granzyme C was analyzed by flow cytometry. Summary of two independent experiments is shown (n≧5 animals / group) (UT: non-transduced). One-way ANOVA test was used in combination with Dunnett's test to correct for multiple comparisons. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. [Figure 4-2] See description of Figure 4-1. [Figure 4-3] See description of Figure 4-1. [Figure 5] This is a series of figures showing that orthogonal engineering uncouples TOX expression from co-inhibitory receptor expression in GzmC+ TCF1neg CD8+ TILs. Figure 5A shows the analysis of exogenous and endogenous CD8+ T cell compartments based on granzyme C and TCF1 expression at day 24. OT1 TILs were not harvested from tumors after PD1d / 33 ACT (UT: untransduced). Figure 5B shows the gating strategy for evaluating TOX and phenotypic markers. PD1d / 2V were not included in the statistical analysis because CD8 TILs were mostly TCF1+. Figure 5C shows surface expression of PD-1 on TCF1neg CD8+ TIL cells. Figure 5D shows surface expression of TIM-3 on PD-1+ TCF1neg CD8+ TILs. For these two surface markers, data from four independent experiments (PD1d / 2V / 33) or two experiments (other groups) are shown (n = 4 or 5 mice per experiment, 24 days after tumor inoculation). Figure 5E shows TOX expression in PD-1+ TCF1neg CD8+ TILs. Data from two independent experiments (PD1d / 2V / 33, PD1d / 33) or one experiment (other groups) are shown (n = 4 or 5 mice per experiment, 24 days after tumor inoculation). Figure 5F shows KLRG1 surface expression in TCF1neg CD8 TILs. A representative experiment from two independent experiments (n = 5 mice per group) is shown. One-way ANOVA was used in combination with Dunnett's test to correct for multiple comparisons; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Naive OT-I T cells isolated from the spleens of non-tumor-bearing mice were used as an internal negative control for FACS staining. [Figure 6-1]Figure 6 shows that GzmC+ TCF1neg CD8+ TILs are polyfunctional effector cells with negligible expression of co-inhibitory receptors. OT1 and endogenous CD8 TILs from animals treated with transgenic OT1 cells or untransduced (UT) OT1 cells were analyzed on day 24 for quantification of effector molecules in PD-1+ TCF1neg CD8+ TILs. OT1 TILs were not recovered from tumors after PD1d / 33 ACT or UT. PD1d / 2V TILs were not included in the statistical analysis because most CD8 TILs were TCF1+. Figure 6A shows surface expression of CD69. Figure 6B shows intracellular expression of Ki-67. Figure 6C shows intracellular expression of granzyme B. Figure 6D shows the normalized MFI of granzyme B expression relative to naive OT-1 T cells isolated from non-tumor-bearing mice. Figure 6E shows the coexpression of granzyme B, and Figure 6F shows the intracellular expression of TNFα and IFNγ after 4 hours of ex vivo stimulation with aCD3 and aCD28 antibodies. Data shown in Figures 6A and 6B were obtained from two independent experiments (PD1d / 2V / 33, PD1d / 33) or one experiment (other groups) (n = 4–6 mice per group). Data shown in Figures 6C, 6D, 6E, and 6F were obtained from two independent experiments (PD1d / 2V / 33) or one experiment (other groups) (n = 4–6 mice per group). One-way ANOVA was used in combination with Dunnett's test to correct for multiple comparisons; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Naive OT-1 T cells isolated from the spleens of non-tumor-bearing mice were used as an internal negative control for FACS staining. Figures 6G and 6H show tumor growth control over time in B16-OVA tumor-bearing mice treated with PD1d / 2V / 33+ OT-1 cells in the presence or absence of 250 μg / mouse of antibodies specific for the indicated surface markers; αPD-L1 (Figure 6G) and αPD-L1+αTIM3 (Figure 6H), administered i.p. starting 1 day before the first cell transfer and maintained every 3 days for up to 6 doses.Figure 6I shows tumor growth control over time in B16-OVA tumor-bearing mice treated with PD1d / 2V / 33+ OT1 cells or OT1 T cells genetically engineered to secrete IL-2V and IL-33 (without the PD-1 ectodomain). Similar to PD1d / 2V / 33, this arm contained a 1:1 mixture of IgG4 / IL-33-expressing OT-1 cells (without PD-1 decoy) and IgG4 / IL-2V-expressing OT-1 cells. Expression of both IgG4 Fc and IL-33 was confirmed by FACS and ELISA and was not significantly different from PD1d / 33 (n = 8 mice / group). Figures 6G, 6H, and 6I show a representative experiment of two independent experiments (n = 6 mice / group). Naive OT-1 T cells isolated from the spleens of non-tumor-bearing mice were used as an internal negative control for FACS staining. [Figure 6-2] See description of Figure 6-1. [Figure 7]Figure 7A shows the analysis of PD-1 expression in TCF1+ CD8+ TILs collected on day 24. Figure 7B shows the gating strategy for evaluating TOX expression in PD-1+ TCF1+ cells. Figure 7C shows the analysis of TOX expression in GzmC+PD-1+TCF1+ CD8 TIL cells and GzmCnegPD-1+TCF1+ CD8 TIL cells from PD1d / 2V. Data shown in Figures 7A, 7B, and 7C were obtained from two independent experiments (n = 4–6 mice / group). One-way ANOVA was used in combination with Dunnett's test for correction of multiple comparisons; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Figure 7D shows a comparative analysis of granzyme C expression (MFI normalized to naive OT1) in TCF1+ CD8+ TILs harvested on day 24 from mice treated with PD1d / 2V / 33+ OT1, relative to endogenous TCF1+ CD8 TILs harvested on day 12 after tumor inoculation (baseline, n = 6 mice) and TCF1+ transgenic OT1 cells after in vitro expansion (before ACT, n = 14 mice) (three independent experiments, n > 4 mice / group). Naive OT-1 T cells isolated from the spleens of non-tumor-bearing mice were used as an internal negative control for FACS staining. [Figure 8-1]This series of figures demonstrates that the novel TCF1negCD8+ TIL effector state induced by orthogonal engineering is dynamically associated with tumor response. Figure 8A shows the experimental design. B16-OVA tumor-bearing mice were treated as indicated on days 12 and 15 after tumor cell inoculation. Tumors were harvested on days 17, 24, and 38, and CD45+ cell suspensions enriched for CD8+ TILs were obtained by FACS sorting and subjected to single-cell sequencing using 10X Genomics. Figure 8B shows a UMAP plot depicting the cellular heterogeneity of only the PD1d / 2V / 33 sample across different time points and a low-dimensional representation of the results of unsupervised clustering, where contour plots delineate areas of high cell density for each treatment. Dot plots (bottom) indicate cluster-specific markers. Figure 8C shows the projection of clusters C5 and C6 onto the reference TIL map using ProjecTILs. The independent component IC26 also significantly separates the unique cluster C5 observed during tumor control from TILs obtained during escape. Bottom right: Volcano plot showing significantly differentially expressed genes between clusters C6 and C5. Figure 8D shows the analysis of granzyme C expression in total CD8+ TIL cells collected during tumor control (day 24) and escape (day 38). CD8+ T cells present in the spleens of Triple_Combo-treated mice were included as controls, as were CD8+ TILs from untreated or UT OT1-treated mice. Data from two independent experiments (PD1d / 2V / 33) are shown. Figure 8E shows the analysis of OT1 (CD45.1+) intratumoral persistence in total CD8+ TILs collected during tumor control (day 24) and escape (day 38). Figure 8F shows analysis of exogenous and endogenous CD8+ TILs harvested during tumor control (day 24) and escape (day 38) based on PD-1 and TCF1 expression.Analysis of intracellular expression of granzyme B (Figure 8G), TNFα, and INFγ (Figure 8H) in PD-1+ TCF1neg CD8 TILs collected during tumor control (day 24) or escape (day 38) after 4 hours of ex vivo stimulation with anti-CD3 and anti-CD28 antibodies. Data from two independent experiments (tumor control) or one experiment (escape) are shown (n = 4–6 mice per group). A two-tailed Student's t-test was used to compare two groups. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Naive OT-1 T cells isolated from the spleens of non-tumor-bearing mice were used as an internal negative control for FACS staining. [Figure 8-2] See description of Figure 8-1. [Figure 9A] Figure 9A shows the characterization of hCD8+ T cells transduced with PD-1 decoy variants and PD-1 decoy variants plus tEGFR. Figure 9A shows a titration ELISA of soluble monomeric PD1 decoy variants (bacterially produced) on plates coated with human PDL1 protein. Bound PD1 decoy molecules were detected with an anti-His tag antibody. Compared to WT PD1 decoy, the PD-1 decoy variant 4XMUT_M70 and variant 6XDM exhibited 10-fold and approximately 7.5-fold enhanced PD-L1 binding, respectively. [Figure 9B] Figure 9B shows the detection of tEGFR and intracellular PD1 decoy in retrovirally transduced CD8+ T cells. [Figure 9C]This series of figures shows the characterization of hCD8+ T cells transduced with PD-1 decoy variants and PD-1 decoy variants plus tEGFR. Figure 9C shows IFNγ production by NY-TCR(I53F)-transduced CD8+ T cells coexpressing PD-1 decoy (variant) and tEGFR. These transduced T cells were cocultured with different PD-L1+ target tumor cells (100,000 cells of each cell type) at a 1:1 ratio for 48 hours. NA8 is HLA / A2+ NY-ESO-1-, while SAOS2 and A375 are HLA / A2+ NY-ESO-1+. Supernatants were collected after 48 hours, diluted 1 / 25, and assayed for the presence of IFNγ using a commercially available ELISA kit from Thermo. Data from a representative T cell donor are shown. In all assays, the variants performed better than the WT PD-1 decoy. [Figure 9D] Figure 9C shows a series of figures characterizing hCD8+ T cells transduced with PD-1 decoy variants and PD-1 decoy variants plus tEGFR. Figure 9D shows the results of an ADCC assay of human T cells transduced to express PD1 decoy (4XMUT_M70E) and tEGFR. CD8 T cells transduced with the PD1 decoy_tEGFR retrovirus were labeled with chromium. These transduced T cells were co-cultured with anti-EGFR Ab and with PBMCs from the same donor at different ratios. The negative control was NT (non-transduced) T cells, and killing was assessed at 4 hours. As a positive control, T cells were treated with HCl. [Figure 10A] This is a series of figures showing the results of an antibody-dependent cellular cytotoxicity (ADCC) assay when T cells were genetically modified to express tEGFR. Figure 10A shows tEGFR-transduced CD8+ T cells loaded with chromium and cultured with PBMCs at different ratios for 4-5 hours with decreasing concentrations of the anti-EGFR antibody cetuximab. As a negative control, tCD30-transduced T cells were used in the assay with the highest concentration of cetuximab (100 μg / ml). Released chromium is used as an indicator of T cell lysis. [Figure 10B]Figure 10B and Figure 10C show the results of an antibody-dependent cellular cytotoxicity (ADCC) assay when T cells were genetically modified to express tEGFR. Figure 10B and Figure 10C show the results of an ADCC assay on tHER2-transduced CD8+ T cells (left: Herceptin (Figure 10B); right: Kadcyla (Figure 10C)). [Figure 10C] Figure 10B and Figure 10C show the results of an antibody-dependent cellular cytotoxicity (ADCC) assay when T cells were genetically modified to express tEGFR. Figure 10B and Figure 10C show the results of an ADCC assay on tHER2-transduced CD8+ T cells (left: Herceptin (Figure 10B); right: Kadcyla (Figure 10C)). [Figure 10D] Figure 10D shows the results of an ADCC assay on CD20-transduced CD8+ T cells, where T cells were genetically modified to express tEGFR. [Figure 11A] Figure 11 shows a series of diagrams depicting representative constructs carrying transgenes used for lymphocyte transduction, including PD-1 decoy and tEGFR (Figure 11A), a CD40L variant (Figure 11B), and an IL-2 variant (also called IL-2V) (Figure 11C), respectively. [Figure 11B] See legend to Figure 11A. [Figure 11C] See legend to Figure 11A. DETAILED DESCRIPTION OF THE INVENTION

[0046] Detailed Description of the Invention The present disclosure relates to methods and compositions for conferring and / or augmenting immune responses mediated by cellular immunotherapy, for example, by adoptive transfer of tumor-specific genetically modified subsets of lymphocytes. The present disclosure provides compositions comprising genetically modified lymphocytes expressing at least two transgenes capable of modulating the immune system and innate and adaptive immune responses. The disclosed methods and compositions are illustrative of a platform technology called Genetic Engineering for the Enhanced Performance of T-cells (GEEP-T™). GEEP-T™ aims to provide genetically modified lymphocytes with enhanced anti-tumor function and methods for developing such lymphocytes.

[0047] A. Compositions and Kits In one aspect, the present disclosure provides a composition comprising a plurality of genetically modified lymphocytes that express at least two transgenes (e.g., therapeutic transgenes) for modulating the immune system of a subject.

[0048] In some embodiments, the lymphocytes are peripheral blood lymphocytes (PBLs). In some embodiments, the lymphocytes are tumor-infiltrating lymphocytes (TILs). Lymphocytes include T cells, B cells, NK cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and basophils. In some embodiments, the lymphocytes are derived from CD34 hematopoietic stem cells, embryonic stem cells, or induced pluripotent stem cells. The lymphocytes can be autologous, allogeneic, syngeneic, or xenogeneic. In some embodiments, the lymphocytes are autologous. In some embodiments, the lymphocytes are human lymphocytes.

[0049] In some embodiments, the lymphocytes may be tumor-infiltrating lymphocytes (TILs). In some embodiments, the lymphocytes may express a chimeric antigen receptor (CAR). In some embodiments, the lymphocytes may express a recombinant T cell receptor (TCR). The CAR or TCR can bind to a cancer antigen. In some embodiments, the CAR or TCR may exhibit reactivity to NY-ESO1, MAGE-A1, MAGE-A3, MAGE A-10, MAGE-C2, SSX2, MAGE-A12, or a combination thereof.

[0050] In some embodiments, the transgene encodes a molecule selected from the group consisting of a soluble receptor, a decoy, a dominant negative, a microenvironment modulator, an enzyme, an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, a translocase, a kinase, a transporter, a modifier, a molecular chaperone, an ion channel, an antibody, a cytokine, a chemokine, a hormone, DNA, a ribozyme, a biosensor, an epigenetic modifier, a transcription factor, a coding RNA, a non-coding RNA, a small RNA, a long RNA, an IRES element, or an exosome shuttle RNA.

[0051] In some embodiments, the transgene encodes at least two molecules selected from the group consisting of a soluble receptor, a decoy, a dominant negative, a microenvironment modulator, an enzyme, an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, a translocase, a kinase, a transporter, a modifier, a molecular chaperone, an ion channel, an antibody, a cytokine, a chemokine, a hormone, DNA, a ribozyme, a biosensor, an epigenetic modifier, a transcription factor, a coding RNA, a non-coding RNA, a small RNA, a long RNA, an IRES element, or an exosome shuttle RNA.

[0052] In some embodiments, two or more molecules encoded by the transgenes are linked by a self-cleaving peptide sequence. In some embodiments, expression of the transgene is controlled by a constitutively activated promoter. In some embodiments, expression of the transgene is controlled by an inducible promoter. In some embodiments, expression of the transgene is induced by the activation state of the lymphocyte. In some embodiments, the transgene is introduced into the lymphocyte via integration-competent gammaretrovirus or lentivirus, DNA transfer, etc.

[0053] In some embodiments, the transgene is selected from the group consisting of an antibody, an antibody fragment, a receptor, a decoy, a checkpoint blockade modulator, a cytokine, a chemokine, a hormone, a cell exclusion tag, and combinations thereof.

[0054] In some embodiments, the antibody or antibody fragment can be a VEGF, TGF-B, 4-1BB, CD28, CD27, NKG2D, PD1, PDL1, or CTLA4 antibody. In some embodiments, the antibody is a PD1 antibody. In some embodiments, the decoy can be a PD1, CTLA4, LAG3, VEGFR1, TIM3, TIGIT, or SIRPα decoy. In some embodiments, the decoy is a PD1 decoy, e.g., a PD-1.IgG4 decoy.

[0055] In some embodiments, the cytokine is selected from the group consisting of LIGHT or a variant / fragment thereof, IL-33 or a variant / fragment thereof, IL-2 or a variant / fragment thereof, IL-15 or a variant / fragment thereof, IL-12 or a variant / fragment thereof, and CD40L or a variant / fragment thereof. In some embodiments, the cytokine is a mutant cytokine.

[0056] In some embodiments, the cell exclusion tag is selected from the group consisting of tEGFR, Her2, CD20, and CD19.

[0057] In some embodiments, the transgene comprises two or more of PD-1 decoy or a variant / fragment thereof, IL-2 variant / fragment, LIGHT or a variant / fragment thereof, IL-33 or a variant / fragment thereof, and CD40L or a variant / fragment thereof. In some embodiments, the transgene further comprises tEGFR or a variant / fragment thereof, tHER2 or a variant / fragment thereof, CD20 or a variant / fragment thereof, or CD19 or a variant / fragment thereof.

[0058] In some embodiments, the PD-1 decoy or variant / fragment thereof is carried on the same vector as a cellular exclusion tag (CET), such as tEGFR or variant / fragment thereof, tHER2 or variant / fragment thereof, CD20 or variant / fragment thereof, or CD19 or variant / fragment thereof.

[0059] In some embodiments, the at least two transgenes are (a) a PD-1 decoy or a variant / fragment thereof and a tEGFR or a variant / fragment thereof; (b) a PD-1 decoy or a variant / fragment thereof and an IL-2 variant / fragment; (c) a PD-1 decoy or a variant / fragment thereof and LIGHT or a variant / fragment thereof; (d) a PD-1 decoy or a variant / fragment thereof and an IL-33 or a variant / fragment thereof; (e) a PD-1 decoy or a variant / fragment thereof and CD40L or a variant / fragment thereof; (f) a PD-1 decoy or a variant / fragment thereof, an IL-2 variant / fragment, and an IL-33 or a variant / fragment thereof; (g) a PD-1 decoy or a variant / fragment thereof, a tEGFR or a variant / fragment thereof, and an IL-2 variant / fragment; (h) a PD-1 decoy or a variant / fragment thereof, a tEGFR or a variant / fragment thereof, and an IL-2 variant / fragment thereof. (i) a PD-1 decoy or a variant / fragment thereof, tEGFR or a variant / fragment thereof, and IL-33 or a variant / fragment thereof; (j) a PD-1 decoy or a variant / fragment thereof, tEGFR or a variant / fragment thereof, and CD40L or a variant / fragment thereof; (k) a PD-1 decoy or a variant / fragment thereof, tEGFR or a variant / fragment thereof, IL-2 variant / fragment thereof, and IL-33 or a variant / fragment thereof; (l) a PD-1 decoy or a variant / fragment thereof, tEGFR or a variant / fragment thereof, IL-2 variant / fragment thereof, and CD40L or a variant / fragment thereof; or (m) a PD-1 decoy or a variant / fragment thereof, tEGFR or a variant / fragment thereof, IL-33 variant / fragment, and CD40L or a variant / fragment thereof.

[0060] In some embodiments, the PD-1 decoy comprises the amino acid sequence of any one of SEQ ID NOs: 1-4, 6-17, 42, 44, 47-48, and 51-52, or an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) identity to any one of SEQ ID NOs: 1-4, 6-17, 42, 44, 47-48, and 51-52.

[0061] In some embodiments, the IL-2 variant comprises the amino acid sequence of any one of SEQ ID NOs:21-23, or an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) identity to any one of SEQ ID NOs:21-23.

[0062] In some embodiments, the IL-33 comprises the amino acid sequence of any one of SEQ ID NOs:25 and 27, or an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) identity to any one of SEQ ID NOs:25 and 27.

[0063] In some embodiments, LIGHT comprises the amino acid sequence of any one of SEQ ID NOs:28-29 and 31, or an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) identity to any one of SEQ ID NOs:28-29 and 31.

[0064] In some embodiments, CD40L comprises the amino acid sequence of any one of SEQ ID NOs: 32-34, 36, and 38, or an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) identity to any one of SEQ ID NOs: 32-34, 36, and 38.

[0065] In some embodiments, the tEGFR comprises an amino acid sequence having at least 80% identity to or the amino acid sequence of SEQ ID NO:40.

[0066] In some embodiments, HER2 comprises an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) identity to or the amino acid sequence of SEQ ID NO:45.

[0067] In some embodiments, CD20 comprises an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) identity to or the amino acid sequence of SEQ ID NO:49.

[0068] Also included within the scope of the present disclosure are novel PD-1 decoy variants having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) identical to any one of SEQ ID NOs: 6-17, 42, 44, 47-48, and 51-52, or comprising the amino acid sequence of any one of SEQ ID NOs: 6-17, 42, 44, 47-48, and 51-52.

[0069] In some embodiments, the composition comprises at least two lymphocyte subsets. For example, the composition may comprise two, three, four, five, or more genetically modified lymphocyte subsets. Each subset of genetically modified lymphocytes can express at least one transgene. For example, each subset of genetically modified lymphocytes can express two, three, four, five, or more transgenes.

[0070] In some embodiments, the composition comprises two genetically modified leukocyte subsets, each subset expressing at least one transgene. In some embodiments, the composition comprises two genetically modified leukocyte subsets, each subset expressing two transgenes. In some embodiments, the composition comprises three genetically modified leukocyte subsets, each subset expressing at least one transgene. In some embodiments, the composition comprises four genetically modified leukocyte subsets, each subset expressing at least one transgene. In some embodiments, the composition comprises five or more genetically modified leukocyte subsets, each subset expressing at least one transgene.

[0071] In some embodiments, the composition comprises at least two genetically modified leukocyte subsets, wherein each subset expresses at least two transgenes and each subset shares one transgene, hi some embodiments, the composition comprises at least two genetically modified leukocyte subsets, wherein each subset expresses at least two transgenes and each subset expresses a different transgene.

[0072] In some embodiments, the plurality of lymphocytes can include (i) a first subset expressing at least two transgenes; and (ii) a second subset expressing at least two transgenes, wherein at least one of the transgenes of the first subset is different from or at least one of the transgenes of the first subset is in common with a transgene of the second subset. In some embodiments, the composition of lymphocytes can express three transgenes after combining the first and second subsets.

[0073] In some embodiments, (i) a first subset expresses at least a PD-1 decoy or a variant / fragment thereof and an IL-2 variant / fragment, and a second subset expresses at least a PD-1 decoy or a variant / fragment thereof and LIGHT or a variant / fragment thereof; (ii) a first subset expresses at least a PD-1 decoy or a variant / fragment thereof and an IL-2 variant / fragment, and a second subset expresses at least a PD-1 decoy or a variant / fragment thereof and IL-33 or a variant / fragment thereof; (iii) a first subset expresses at least a PD-1 decoy or a variant / fragment thereof and an IL-2 variant / fragment, and a second subset expresses at least a PD-1 decoy or a variant / fragment thereof and CD40L or a variant / fragment thereof; (iv) (v) the first subset expresses at least PD-1 decoy or a variant / fragment thereof and LIGHT or a variant / fragment thereof, and the second subset expresses at least PD-1 decoy or a variant / fragment thereof and IL-33 or a variant / fragment thereof; (v) the first subset expresses at least PD-1 decoy or a variant / fragment thereof and LIGHT or a variant / fragment thereof, and the second subset expresses at least PD-1 decoy or a variant / fragment thereof and CD40L or a variant / fragment thereof; or (vi) the first subset expresses at least PD-1 decoy or a variant / fragment thereof and IL-33 or a variant / fragment thereof, and the second subset expresses at least PD-1 decoy or a variant / fragment thereof and CD40L or a variant / fragment thereof.

[0074] In some embodiments, the first subset or the second subset further expresses tEGFR or a variant thereof, tHER2 or a variant thereof, CD20 or a variant thereof, or CD19 or a variant thereof.

[0075] In some embodiments, (i) a first subset expresses at least a PD-1 decoy or a variant / fragment thereof, tEGFR or a variant / fragment thereof, and an IL-2 variant / fragment, and a second subset expresses at least a PD-1 decoy or a variant / fragment thereof, tEGFR or a variant / fragment thereof, and LIGHT or a variant / fragment thereof; (ii) a first subset expresses at least a PD-1 decoy or a variant / fragment thereof, tEGFR or a variant / fragment thereof, and an IL-2 variant / fragment, and a second subset expresses at least a PD-1 decoy or a variant / fragment thereof, tEGFR or a variant / fragment thereof, and IL-33 or a variant / fragment thereof; or (iii) a first subset expresses at least a PD-1 decoy or a variant / fragment thereof, tEGFR or a variant / fragment thereof, and IL-2 variant / fragment thereof, and a (iv) the first subset expresses at least PD-1 decoy or a variant / fragment thereof, tEGFR or a variant / fragment thereof, and LIGHT or a variant / fragment thereof, and the second subset expresses at least PD-1 decoy or a variant / fragment thereof, tEGFR or a variant / fragment thereof, and IL-33 or a variant / fragment thereof; (v) the first subset expresses at least PD-1 decoy or a variant / fragment thereof, tEGFR or a variant / fragment thereof, and LIGHT or a variant / fragment thereof, and the second subset expresses at least PD-1 decoy or a variant / fragment thereof, tEGFR or a variant / fragment thereof, and CD40L or a variant / fragment thereof;or (vi) the first subset expresses at least PD-1 decoy or a variant / fragment thereof, tEGFR or a variant / fragment thereof, and IL-33 or a variant / fragment thereof, and the second subset expresses at least PD-1 decoy or a variant / fragment thereof, tEGFR or a variant / fragment thereof, and CD40L or a variant / fragment thereof;

[0076] As used herein, the term "variant" refers to a first molecule related to a second molecule (also called a "parent" molecule). A variant molecule can be derived from, isolated from, based on, or homologous to the parent molecule. As used herein, a "functional variant" of a protein refers to a variant of the protein that retains at least partial activity of the protein. Functional variants include variants, including polymorphs, which may be insertion, deletion, or substitution variants. Functional variants also include fusion products of such proteins with another, usually unrelated, nucleic acid, protein, polypeptide, or peptide. Functional variants can be naturally occurring or artificially created.

[0077] In some embodiments, variants of a transgene can include one or more conservative modifications. Transgene variants with one or more conservative modifications can retain desired functional properties, which can be tested using functional assays known in the art.

[0078] As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or change the binding properties of a protein containing that amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine); acidic side chains (e.g., aspartic acid, glutamic acid); uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan); nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine); beta-branched side chains (e.g., threonine, valine, isoleucine); and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine), and contain one or more conservative modifications. Cas proteins with one or more conservative modifications can retain desired functional properties, which can be tested using functional assays known in the art.

[0079] As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between two sequences is a function of the number of identical positions shared by these sequences (i.e., % homology = number of identical positions / total number of positions x 100), taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. The comparison of sequences and the determination of the percent identity between two sequences can be achieved using a mathematical algorithm, as described in the non-limiting examples below.

[0080] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) incorporated into the GAP program of the GCG software package (available at www.gcg.com), using either a Blossum62 matrix or a PAM250 matrix, gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0081] The term "homolog" or "homologous" when used in reference to a polypeptide refers to a high degree of sequence identity between two polypeptides, or a high degree of similarity between their three-dimensional structures, or a high degree of similarity between their active sites and mechanisms of action. In some embodiments, a homolog has greater than 60% sequence identity with a reference sequence, more preferably greater than 75% sequence identity, and even more preferably greater than 90% sequence identity. The term "substantial identity" as applied to a polypeptide means that two peptide sequences share at least 75% sequence identity when optimally aligned, for example, by the GAP or BESTFIT programs using default gap weights.

[0082] As used herein, a "fragment" of a peptide or polypeptide refers to a peptide, polypeptide, or protein that is less than full-length. For example, a peptide or polypeptide fragment can have a length of at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, or at least about 40 amino acids, or a single unit length thereof. For example, a fragment can be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or more amino acids in length. There is no upper limit to the size of a peptide fragment. However, in some embodiments, a peptide fragment can be less than about 500 amino acids, less than about 400 amino acids, less than about 300 amino acids, or less than about 250 amino acids in length.

[0083] Also included within the scope of the present disclosure are variants, mutants, and homologs that share substantial identity to the transgenes. For example, such variants and homologs may have a sequence that shares at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequences of the transgenes described herein.

[0084] In some embodiments, the described transgene variants are fusion polypeptides comprising the transgene sequence fused (e.g., fused at the N-terminus or C-terminus) to a fusion partner. In some embodiments, the fusion partner comprises a fragment of a human immunoglobulin polypeptide sequence (e.g., a CH3 domain; or part or all of an Fc region, such as IgG4Fc). For example, PD-1 or a variant / fragment thereof, IL-2 or a variant / fragment thereof, IL-33 or a variant / fragment thereof, CD40L or a variant / fragment thereof, or LIGHT or a variant / fragment thereof can be fused at the N-terminus or C-terminus, linked to a fusion partner, such as IgG4Fc or a variant / fragment thereof, either directly or indirectly via a linker.

[0085] The term "fusion polypeptide" or "fusion protein" refers to a protein created by linking two or more polypeptide sequences together. Fusion polypeptides encompassed by the present invention include the translation product of a chimeric gene construct that joins a nucleic acid sequence encoding a first polypeptide with a nucleic acid sequence encoding a second polypeptide to form a single open reading frame. In other words, a "fusion polypeptide" or "fusion protein" is a recombinant protein of two or more proteins linked by a peptide bond or through several peptides. Fusion proteins may also include a peptide linker between the two domains.

[0086] Immunosuppressive polypeptides known to bind to and suppress or reduce immune responses include CD47, PD-1, CTLA-4, and their corresponding ligands, such as SIRPα, PD-L1, PD-L2, B7-1, and B7-2. Such polypeptides are present in the tumor microenvironment and inhibit immune responses against neoplastic cells. In various embodiments, inhibiting, blocking, or antagonizing the interaction of immunosuppressive polypeptides and / or their ligands via a transgene enhances the immune response of immunoresponsive cells. In one aspect, the transgene can function as a genetic knockdown of inhibitory / checkpoint molecules, including, but not limited to, PD-1, CTLA-4, LAG-3, TIGIT, VISTA, TIM-3, and CBL-B.

[0087] Costimulatory polypeptides known to stimulate or enhance immune responses upon binding include CD28, OX-40, 4-1BB, CD27, NKG2D, and their corresponding ligands, e.g., B7-1, B7-2, OX-40L, 4-1BBL, CD70, and NKG2D ligands. Such polypeptides are present in the tumor microenvironment and activate immune responses against neoplastic cells. In various embodiments, promoting, stimulating, or agonizing inflammatory polypeptides and / or their ligands via transgenes enhances the immune response of immunocompetent cells.

[0088] In some embodiments, the transgene is a cytokine or growth factor. The terms "growth factor" and "cytokine" refer to signaling molecules that regulate cellular activity in an autocrine, paracrine, or endocrine manner. They exert their biological functions by binding to specific receptors and activating associated downstream signaling pathways, which then regulate gene transcription in the nucleus and ultimately stimulate a biological response (Nicola N. Oxford; New York: Oxford University Press; 1994). Growth factors and cytokines affect various physiological processes in adults, including cell proliferation, differentiation, apoptosis, immune or hematopoietic responses, morphogenesis, angiogenesis, metabolism, wound healing, and tissue homeostasis. Historically, growth factors were considered biological components that positively influence cell growth and proliferation, while cytokines were generally considered to indicate immune or hematopoietic responses. However, the convergence of different lines of research has revealed that "cytokine" and "growth factor" may have similar functions; therefore, these terms are used interchangeably herein.

[0089] TGF-β Superfamily: The TGF-β superfamily includes TGF-β proteins, bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), glial-derived neurotrophic factors (GDNFs), activins, inhibins, Nodal, Lefty, and Müllerian inhibitory substance (MIS). Members of the TGF-β superfamily are multifunctional regulators of diverse biological processes, including morphogenesis, embryonic development, adult stem cell differentiation, immune regulation, wound healing, inflammation, and cancer. (1) BMP-like family: BMPs (i.e., BMP1-10, BMP-15), GDFs (i.e., GDF1-15), AMH (2) GDNF family: GDNF, Artemin, Neuturin, and Persephone (3) TGF-β-like family: TGF-βs (i.e., TGF-β-1, TGF-β-2, TGF-β-3), activins (i.e., activin A / AB / B, inhibin A / B), Nodal

[0090] Epidermal Growth Factor (EGF): Members of the EGF family include EGF, TGF-α, neuregulin, amphiregulin, and betacellulin. EGF family members are best known for their ability to stimulate cell proliferation, differentiation, and survival. Dysregulation of members of this family and their receptors is closely associated with tumorigenesis (Herbst RS. International Journal of Radiation Oncology, Biology, Physics 2004, 59(2 Suppl):21-26).

[0091] Platelet-Derived Growth Factor (PDGF): Platelet-derived growth factor (PDGF) is a potent mitogenic and chemotactic protein. Currently, four PDGF proteins (PDGFA, PDGFB, PDGFC, and PDGFD) are known, encoded by four genes. PDGF is secreted as disulfide-linked homodimers or heterodimers containing PDGF-AA, PDGF-BB, PDGF-CC, PDGF-DD, and PDGF-AB. Two PDGF receptors, PDGFRα and PDGFRβ, are known to possess intrinsic tyrosine kinase activity, both of which can form heterodimers and homodimers. Ligand binding promotes receptor dimerization and autophosphorylation, resulting in the activation of multiple downstream intracellular signaling cascades. Signaling through PDGFRα is essential for the development of the facial skeleton, hair follicles, spermatogenesis, oligodendrocytes, and astrocytes, as well as the development of lungs and intestinal villi, while signaling through PDGFRβ is crucial for the development of blood vessels, kidneys, and white adipocytes (Heldin CH. Cell Commun Signal 2013, 11:97).

[0092] Fibroblast growth factor (FGF) family: In humans, 22 members of the FGF family have been identified, all of which are heparin-binding proteins. High-affinity interaction with cell surface heparan sulfate proteoglycans is essential for FGF signaling mediated by receptor tyrosine kinases (Ornitz DM, Itoh N. Genome Biology 2001, 2(3): REVIEWS3005). FGFs are multipotent proteins that are primarily mitogenic, but also have regulatory, morphogenetic, and endocrine functions. FGFs are involved in embryonic development (Heldin CH: Targeting the PDGF signaling pathway in tumor treatment. Cell Commun Signal 2013, 11:97), vascularization of mature tissues / systems (Kim BS, et al. Biochemical and biophysical research communications 2014, 450(4):1333-1338), keratinocyte organization (Tsuboi R, et al. The Journal of Investigative Dermatology 1993, 101(1):49-53), and wound healing (Lee JG, Kay EP. Investigative Ophthalmology & Visual Science 2006, 47(4):1376-1386).

[0093] Insulin-like growth factors (IGFs): Insulin-like growth factors (IGFs) are proteins with high sequence similarity to insulin. IGF receptors are disulfide-linked heterotetrameric transmembrane proteins with a cytoplasmic tyrosine kinase domain. There are two types of IGF receptors: IGF-I-R and IGF-II-R. IGF availability can be regulated by IGF-binding proteins 1-6 (Griffeth RJ, et al. Basic and Clinical Andrology 2014, 24:12). The primary effect of IGFs is cell growth. In fact, most of the effects of pituitary growth hormone are mediated by IGFs, primarily IGF-1. Growth hormone stimulates many tissues, especially the liver, to synthesize and secrete IGF-1, which then stimulates both hypertrophy (an increase in cell size) and hyperplasia (an increase in cell number) in most tissues, including bone. IGF can also induce neuronal survival, protect chondrocytes, and activate bone cells (Brahmkhatri VP, et al. BioMed research international 2015, 2015:538019).

[0094] Vascular endothelial growth factor (VEGF): VEGF is a homodimeric glycoprotein growth factor that acts specifically on endothelial cells (Ferrara N, Gerber HP, LeCouter. Nature Medicine 2003, 9(6):669-676). They regulate angiogenesis and vascular permeability, particularly during embryonic development, skeletal growth, and reproductive function. They also play an important role in hematopoiesis. VEGF signals primarily through the tyrosine kinases VEGFR1 and VEGFR2, stimulating cell survival, proliferation, migration, and / or adhesion (Ferrara N. Endocrine Reviews 2004, 25(4):581-611). Deregulation of VEGF is associated with tumors, intraocular neovascularization, and other diseases (Ferrara N, et al. Nature Medicine 2003, 9(6):669-676). Members of the VEGF gene family include VEGF / VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and placental growth factor (PlGF) (Holmes DI, Zachary I. Genome Biology 2005, 6(2):209).

[0095] Hepatocyte growth factor (HGF): HGF is secreted by mesenchymal cells and acts as a multifunctional cytokine primarily on cells of epithelial and endothelial origin. It regulates cell growth, cell motility, and morphogenesis by activating tyrosine kinase signaling cascades via HGFR (Okada M, et al. Pediatric Research 2004, 56(3):336-344). HGF has been shown to play a major role in embryonic organ development, adult organ regeneration, and wound healing. Furthermore, its ability to stimulate mitogenesis, cell motility, and matrix invasion gives it a central role in angiogenesis and tumorigenesis (Sharma NS, et al. FASEB 2010, 24(7):2364-2374).

[0096] Tumor necrosis factor (TNF): Cytokines known to be involved in tumor cell apoptosis were initially classified under the tumor necrosis factor (TNF) family. All TNF family members share a conserved C-terminal domain of the trimer, called the "TNF homology domain" or THD. Responsible for receptor binding, the THD shares approximately 20-30% sequence identity among family members. Most ligands are synthesized as membrane-bound proteins, but soluble forms can be generated by limited proteolysis (Bodmer JL, et al. Trends in Biochemical Sciences 2002, 27(1):19-26). The first two members of this family identified were TNFα and TNFβ. To date, 19 TNF superfamily ligands have been identified, along with 32 TNF superfamily receptors. While many TNF superfamily members promote or inhibit apoptosis, they also regulate important functions of both the innate and adaptive immune systems, including natural killer cell activation, T cell costimulation, and B cell homeostasis and activation (Croft M. Nature Reviews Immunology 2009, 9(4):271-285). LIGHT (lymphotoxin homologous, inducible, and competes with HSV glycoprotein D for herpesvirus entry mediator, a receptor expressed by T lymphocytes) is a type II transmembrane glycoprotein of the TNF ligand superfamily. LIGHT is expressed on immature DCs and activated T cells and binds to three distinct receptors: herpesvirus entry mediator (HVEM), lymphotoxin β receptor (LTβR), and decoy receptor 3 / TR6. Upon binding to HVEM, LIGHT costimulates T cells, promoting proliferation and cytokine production. Another example is CD154, also known as CD40 ligand or CD40L. It is a protein expressed primarily on activated T cells and is a member of the TNF superfamily of molecules. It binds to CD40 on antigen-presenting cells and exerts various effects depending on the type of target cell.Yet another example is the Fas ligand (FasL or CD95L or CD178). Fas ligand / receptor interactions play an important role in the regulation of the immune system and in cancer progression.

[0097] Interleukins (IL): Interleukins are a large group of immunoregulatory proteins that control the growth, differentiation, and activation of cells of the immune or hematopoietic system during immune responses. Based on distinguishing structural features, known ILs are broadly divided into four groups: IL1-like cytokines, class I helical cytokines (IL4-like, gamma chain, and IL-6 / 12-like), class II helical cytokines (IL-10-like and IL-28-like), and IL-17-like cytokines (Table 1). TIFF0007819923000001.tif83166

[0098] Interferons (IFNs): IFNs are a group of signaling proteins produced and released by host cells in response to the presence of pathogens such as viruses, bacteria, parasites, or tumor cells. Interferons also have immunoregulatory functions; they suppress B cell activation, enhance T cell activity, and enhance the cell-killing ability of natural killer cells. More than 20 different IFN genes and proteins have been identified in animals, including humans. They are generally divided into two classes: type I IFNs and type II IFNs. Type I IFNs, also known as viral IFNs, include IFN-α, IFN-β, and IFN-ω. Type II IFNs are also known as immune IFNs (IFN-γ). Viral IFNs are induced by viral infection, whereas type II IFNs are induced by mitogenic or antigenic stimulation. Most types of virus-infected cells can synthesize type I IFNs in cell culture. In contrast, IFN-γ is synthesized only by specific cells of the immune system, such as natural killer cells, CD4 Th1 cells, and CD8 cytotoxic suppressor cells (Samuel CE. Clinical Microbiology Reviews 2001, 14(4):778-809, Table of Contents).

[0099] In some embodiments, the transgene is a decoy receptor. By "decoy receptor" is meant a receptor that can effectively recognize and bind to a particular growth factor or cytokine, but is structurally incapable of signaling or activating the intended receptor complex. It acts as an inhibitor, binding to a ligand and preventing the ligand from binding to the canonical receptor.

[0100] In some embodiments, the transgene is a soluble decoy. "Soluble decoy" refers to a polypeptide that is expressed and secreted from a cell and binds to a specific receptor on another cell, thereby inhibiting the binding of a natural ligand to the receptor. Non-limiting examples of soluble decoys include PD1 decoy, CTLA-4 decoy, LAG3 decoy, VEGFR1 decoy, TIM3 decoy, TIGIT decoy, and SIRPα decoy. In one embodiment, the PD-1 decoy is expressed and secreted by lymphoid cells, and such a PD-1 decoy occupies the binding site of PD-L1 on antigen-presenting cells (APCs), thereby inhibiting the binding of natural PD-1 on T cells to PDL-1 on APCs, thereby inhibiting the immunosuppressive signaling of T cells and thereby enhancing the immune response of T cells.

[0101] PD-1 decoy: PD-1 is a potent negative regulator of T lymphocytes in the tumor microenvironment. In one embodiment, T cells expressing a dominant-negative deletion mutant of PD-1 (a non-limiting example of a PD-1 decoy) were generated by retroviral transduction. This PD-1 decoy increased IFN-γ secretion by antigen-specific T cells in response to tumor cells expressing the cognate antigen. In another embodiment, a soluble fragment of the PD-1 ectodomain (a non-limiting example of a PD-1 decoy) with higher binding affinity to PDL-1 is administered as a competitive antagonist of PDL-1. Non-limiting examples of soluble PD-1 ectodomain variants are disclosed in Maute et al. PNAS 2015 Nov 24; 112(47): E6506-E6514. In yet another embodiment, a PD-1 decoy molecule comprising the ectodomain of PD1 fused to the Fc region of human IgG4 (PD-1.IgG4) can be used in vivo for enhanced tumor control. In another embodiment, such a PD-1 decoy can be expressed and secreted by TILs.

[0102] PD-1 decoys as described in this disclosure can also be created by computer-based rational design to develop variants with enhanced binding and / or solubility to the PD-1 ectodomain. For example, single and multiple amino acid substitutions predicted to enhance the binding affinity of PD-1 to PD-L1 are evaluated in recombinant soluble proteins produced in a bacterial expression system. These variants can be evaluated for binding to plate-captured PD-L1 in a direct titration ELISA; variants of interest are then cloned into retroviral vectors to assess secretion by T cells. PD-1 decoys that exhibited poor solubility when produced in bacteria were discarded, as poor solubility generally corresponds to no or low production by T cells.

[0103] The PD-1 decoy produced by genetically modified human T cells also contained an Fc portion (e.g., IgG4 Fc) to enhance protein binding and stability. The PD1-Fc decoy produced by primary human T cells can be evaluated by ELISA. To assess functionality, a coculture assay was established in which primary human T cells were co-modified to express the A2 / NY-ESO-1 T cell receptor (TCR) (via lentiviral transduction) to enable tumor cell recognition, as well as the PD1-Fc decoy and cell surface tEGFR (encoded by a bicistronic retroviral vector). These co-transduced T cells (or control T cells containing only the TCR or only the PD1 decoy) were cultured in a 200-well platelet-free (PDL1)-treated control cell culture. POS The PD1-Fc decoy variants were co-cultured with target tumor cells. The IFNγ levels present in the co-culture supernatant were evaluated to determine the optimal PD-1 decoy variant (i.e., the higher the IFNγ level, the better the PD1 decoy is at blocking PD-L1 on the surface of target tumor cells). 4XMUT_M70 and 6XDM are among the PD1-Fc decoy variants that exhibit high binding affinity to PD-L1 and high solubility (Figure 9A-D).

[0104] Cellular Elimination Tag (CET): Transgenes such as PD1-Fc decoy can be constitutively expressed from bicistronic retroviral vectors that also encode a CET, such as tEGFR, tHER2, CD20, or CD19 (Figure 10A-D). The purpose of the CET is fourfold. First, it serves as a means of assessing transduction efficiency. Second, it can be used to enrich for genetically modified cells (on anti-EGFR-coated beads) if necessary. Third, it can be used to track genetically modified T cells in patients after engraftment (by FACS from blood samples or tumor biopsies). Finally, it can be used as an exclusion tag via ADCC in the event of toxicity in cetuximab-treated patients. A truncated human EGFR polypeptide (huEGFRt) lacking the extracellular N-terminal ligand-binding domain and intracellular receptor tyrosine kinase activity but retaining the native amino acid sequence, type I transmembrane cell surface localization, and a conformationally intact binding epitope of the pharmaceutical-grade anti-EGFR monoclonal antibody cetuximab (Erbitux) has been described by Want et al. (Wang X, et al. Blood. 2011 Aug 4;118(5):1255-63. Epub 2011 Jun 7). Other examples of CET ADCC include tHER2 (using Herceptin or Kadcyla), CD20 (using rituximab), and CD19. CD20 as a CET has been described by Griffioen et al. (Griffioen M, et al. Haematologica. 2009 Sep;94(9):1316-20). CD19 as a CET has been described by Budde et al. (Budde, et al. Blood 2013; 122 (21): 1660) and Annesley et al. (Annesley et al., Blood 2019; 134 (Supplement_1): 223).

[0105] LIGHT: LIGHT is a type II transmembrane glycoprotein of the TNF ligand superfamily (Mauri et al. Immunity 1998 Jan; 8(1):21-30). It is expressed on immature dendritic cells and activated T cells (Tamada K et al. J Immunol. 2000 Apr 15; 164(8):4105-10) and binds to three distinct receptors: herpesvirus entry mediator (HVEM), lymphotoxin beta receptor (LTβR), and decoy receptor 3 / TR6. Upon binding to HVEM, LIGHT costimulates T cells, promoting proliferation and cytokine production (Tamada et al. Nat Med. 2000 Mar; 6(3):283-9). In one embodiment, the LIGHT protein can be genetically modified to be expressed and secreted by TILs.

[0106] IL-33: Cytokines are central intercellular mediators in the inflammatory tumor microenvironment, and interleukin-33 (IL-33) is considered an alarmin released after cell injury. IL-33 was discovered as a member of the IL-1 family of cytokines. The IL-1 gene family contains 11 members (IL-1α, IL-1β, IL-1RA, IL-18, IL-36RA, IL-36α, IL-37, IL-36β, IL-36γ, IL-38, and IL-33) that regulate and initiate inflammatory responses by inducing a complex network of proinflammatory cytokines and through the expression of integrins on leukocytes and endothelial cells (Interleukin-1 in the pathogenesis and treatment of inflammatory diseases. (Dinarello CA., Blood. 2011 Apr 7; 117(14):3720-32). The process of tumorigenesis can trigger antitumor immune responses. Type 1 immune responses are key components of cellular immunity, including tumor-inducing IFN-γ-producing Th1 cells, cytotoxic T lymphocytes, NK T cells, and γδ T cells, and limit tumor growth and metastasis (Galon J et al. Science. 2006 Sep 29; 313(5795):1960-4). Because inflammation is another important component of malignant tumors, IL-33 may play a role in improving cancer surveillance and immunity against tumors. In one embodiment of the present invention, IL-33 can be genetically modified to be expressed and secreted by TILs.

[0107] IL-2: Interleukin-2 (IL-2) was one of the first cytokines discovered and molecularly characterized. It was shown to primarily support the growth and proliferation of T cells and NK cells. IL-2 was approved for clinical use in 1992, but the precise biology of its receptor is still under investigation. Systemic high-dose (HD) IL-2 treatment has produced durable responses in patients with melanoma and renal cancer, but only in a relatively small number of patients. Furthermore, systemic HD IL-2 treatment induces significant toxicity, further limiting its clinical relevance. IL-2 promotes the activation and proliferation of T cells and NK cells in vitro. In one embodiment, IL-2 or its functional variants can be genetically modified to be expressed and secreted by TILs. Such TILs can be further engineered to secrete additional transgenes.

[0108] CD40L: As immune costimulatory molecules, CD40 and its ligand, CD40L, can complement each other. Previous studies have shown that CD40 and CD40L play crucial roles in humoral and cellular immunity, and that CD40 and CD40L expression is closely related to the onset and progression of various diseases (Elgueta et al. Immunol Rev 2009; 229:152-172). CD40 has been found to be highly expressed in bladder, breast, ovarian, and other tumors (Hussain et al. Br J Cancer 2011; 88:586). CD40L, as the major ligand for CD40, is primarily expressed on the surface of activated CD4+ T cells. When CD40 binds to CD40L, CD40L can activate T lymphocytes and activate the Fas-mediated apoptosis pathway in tumor cells.

[0109] In another aspect, the genetically modified lymphocytes can be incorporated into a pharmaceutical composition suitable for administration. Pharmaceutical compositions generally comprise substantially isolated / purified lymphocytes in a form suitable for administration to a subject and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are determined in part not only by the specific composition to be administered, but also by the specific method used to administer the composition. Pharmaceutical compositions are generally formulated in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0110] The terms "pharmaceutically acceptable" and "physiologically acceptable" when referring to compositions, carriers, diluents, and reagents are used interchangeably and include materials that can be administered to a subject without producing undesirable physiological effects to an extent that would prohibit administration of the composition. For example, a "pharmaceutically acceptable excipient" includes excipients that are generally safe, non-toxic, and useful in preparing a desired pharmaceutical composition, and includes excipients that are acceptable for veterinary use as well as human pharmaceutical use.

[0111] Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin.The use of such media and compounds for pharmaceutically active substances is well known in the art.Unless conventional media or compounds are incompatible with the disclosed compositions, their use in the compositions is contemplated.In some embodiments, a second therapeutic agent, such as an anti-cancer agent or an anti-tumor agent, can also be incorporated into the pharmaceutical composition.

[0112] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0113] In some embodiments, the composition comprises genetically modified lymphocytes as described above, and optionally a cryoprotectant (e.g., glycerol, DMSO, PEG).

[0114] The compositions or pharmaceutical compositions described herein can be provided in a kit. In one embodiment, the kit includes (a) a container containing the composition and, optionally, (b) informational material. The informational material can be instructional, instructional, marketing, or other material related to the methods described herein and / or the use of the agent for therapeutic benefit. For example, the kit can include instructions for manufacture, the therapeutic regimen to be used, and the duration of administration. In certain embodiments, the kit also includes an additional therapeutic agent (e.g., a checkpoint modulator). The kit can include one or more containers, each containing a different reagent. For example, the kit includes a first container containing the composition and a second container for the additional therapeutic agent.

[0115] The container can contain a unit dosage of the pharmaceutical composition. In addition to the composition, the kit can include other ingredients, such as a solvent or buffer, an adjuvant, a stabilizer, or a preservative.

[0116] The kit optionally includes a device suitable for administering the composition, such as a syringe or other suitable delivery device. The device can be provided pre-loaded with one or both agents, or the device can be empty but suitable for loading.

[0117] B. Methods for preparing compositions In another aspect, the present disclosure further provides a method for preparing the composition, comprising: (a) providing a plurality of lymphocytes; (b) introducing nucleic acid molecules encoding at least two transgenes into the plurality of lymphocytes to obtain a plurality of genetically modified lymphocytes; and (c) expanding the plurality of genetically modified lymphocytes in a cell culture medium.

[0118] In some embodiments, the method includes: (a) providing a plurality of lymphocytes; (b) introducing two or more nucleic acid molecules into the plurality of lymphocytes, thereby obtaining a plurality of genetically modified lymphocytes, wherein each of the two or more nucleic acid molecules encodes at least one transgene; and (c) expanding the plurality of genetically modified lymphocytes in a cell culture medium.

[0119] In some embodiments, the transgene comprises two or more of PD-1 decoy, IL-2 variant / fragment, LIGHT or variant / fragment thereof, IL-33 or variant / fragment thereof, and CD40L or variant / fragment thereof. In some embodiments, the transgene further comprises tEGFR or variant / fragment thereof. In some embodiments, the PD-1 decoy or variant / fragment thereof and tEGFR or variant / fragment thereof (or tHER2 or variant / fragment thereof, CD20 or variant / fragment thereof, or CD19 or variant / fragment thereof) are carried on the same vector.

[0120] In some embodiments, the at least two transgenes are (a) a PD-1 decoy or a variant thereof and a tEGFR or a variant thereof; (b) a PD-1 decoy or a variant thereof and an IL-2 variant; (c) a PD-1 decoy or a variant thereof and LIGHT or a variant thereof; (d) a PD-1 decoy or a variant thereof and an IL-33 or a variant thereof; (e) a PD-1 decoy or a variant thereof and CD40L or a variant thereof; (f) a PD-1 decoy or a variant thereof, an IL-2 variant, and an IL-33 or a variant thereof; (g) a PD-1 decoy or a variant thereof, a tEGFR or a variant thereof, and an IL-2 variant; (h) a PD-1 decoy or a variant thereof, a tEGFR or a variant thereof, and an IL-2 variant; or a variant thereof, and LIGHT or a variant thereof; (i) PD-1 decoy or a variant thereof, tEGFR or a variant thereof, and IL-33 or a variant thereof; (j) PD-1 decoy or a variant thereof, tEGFR or a variant thereof, and CD40L or a variant thereof; (k) PD-1 decoy or a variant thereof, tEGFR or a variant thereof, IL-2 variant, and IL-33 or a variant thereof; (l) PD-1 decoy or a variant thereof, tEGFR or a variant thereof, IL-2 variant, and CD40L or a variant thereof; or (m) PD-1 decoy or a variant thereof, tEGFR or a variant thereof, IL-33 variant, and CD40L or a variant thereof.

[0121] In some embodiments, the method can include (a) introducing a first nucleic acid molecule encoding at least two transgenes into a first plurality of lymphocytes to obtain the first plurality of genetically modified lymphocytes; and (b) introducing a second nucleic acid molecule encoding at least two transgenes into a second plurality of lymphocytes to obtain the second plurality of genetically modified lymphocytes. In some embodiments, the method can further include combining the first plurality of genetically modified lymphocytes with the second plurality of genetically modified lymphocytes at a predetermined ratio of about 1:1 to about 1:100 (e.g., 1:1, 1:2, 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100).

[0122] In some embodiments, the method comprises: a) introducing transgenes into different lymphocyte subsets, wherein each subset expresses at least one transgene; and b) combining at least two subsets of lymphocytes. In some embodiments, each subset expresses at least two transgenes according to the above embodiments. In some embodiments, the composition of lymphocytes expresses at least three different transgenes.

[0123] In some embodiments, the method for obtaining the tumor-specific genetically modified leukocyte subset composition can be performed in vitro or ex vivo. In a more particular form, the method can be as disclosed in PCT / EP2018 / 080343, the contents of which are incorporated herein by reference in their entirety.

[0124] In some embodiments, the method can additionally include expanding the first plurality of lymphocytes in cell culture medium after the step of introducing the first nucleic acid, or expanding the second plurality of lymphocytes in cell culture medium after the step of introducing the second nucleic acid.

[0125] The terms "culturing" or "expanding" refer to maintaining or culturing cells under conditions that allow the cells to proliferate and avoid senescence. For example, cells can be cultured in a medium that optionally contains one or more growth factors, i.e., a growth factor cocktail. In some embodiments, the cell culture medium is a defined cell culture medium. The cell culture medium may also contain neoantigen peptides. Stable cell lines may be established to allow for continuous cell growth.

[0126] a. Lymphocytes Prior to the lymphocyte expansion and genetic modification described herein, a source of lymphocytes is obtained from a subject. Lymphocytes can be obtained from several sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. As described herein, various lymphocyte lines available in the art can be used. Lymphocytes can be obtained from a unit of blood drawn from a subject using various techniques known to those skilled in the art, such as Ficoll™ separation. An individual's circulating blood cells are obtained by apheresis. Apheresis products typically contain lymphocytes, such as T lymphocytes, monocytes, granulocytes, B lymphocytes, other nucleated white blood cells, red blood cells, and platelets. Cells collected by apheresis can be washed to remove the plasma fraction and placed in an appropriate buffer or medium for subsequent processing steps. Cells may be washed with phosphate-buffered saline (PBS). Alternatively, the wash solution may lack calcium, magnesium, or many, if not all, divalent cations. As those skilled in the art will readily appreciate, the wash step can be accomplished by methods known to those skilled in the art, such as using a semi-automated continuous-flow centrifuge (e.g., a Cobe 2991 cell processor, a Baxter CytoMate, or an e1Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as Ca2+-free, Mg2+-free PBS, PlasmaLyte A, or other saline solutions with or without buffers. Alternatively, undesirable components of the apheresis sample can be removed and the cells resuspended directly in culture medium.

[0127] As described herein, lymphocytes can be isolated from peripheral blood by lysis of red blood cells and depletion of monocytes, for example, by centrifugation through a PERCOLL™ gradient or counterflow centrifugal elutriation. If desired, specific lymphocyte subpopulations, such as T lymphocytes (i.e., CD3+, CD28+, CD4+, CD8+, CD45RA+, or CD45RO+ T lymphocytes), can be further isolated by positive or negative selection techniques. For example, T lymphocytes can be isolated by incubating with anti-CD3 / anti-CD28 conjugated beads (i.e., 3x28), such as DYNABEADS® M-450 CD3 / CD28 T, for a sufficient time (i.e., 30 minutes to 24 hours) to positively select the desired T lymphocytes. When isolating T lymphocytes from leukemia patients, longer incubation times, such as 24 hours, can be used to improve cell performance. Longer incubation times can be used to isolate T lymphocytes in situations where T lymphocytes are scarce compared to other cell types, such as isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or immunocompromised individuals. Those skilled in the art will recognize that multiple rounds of selection may be used. It may be desirable to perform a selection procedure and use "unselected" cells in the activation and expansion process. "Unselected" cells can also be subjected to a new round of selection.

[0128] Enrichment of lymphocyte (e.g., T lymphocyte) populations by negative selection can be achieved using a combination of antibodies against surface markers specific to the cells being negatively selected. One method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the cells being negatively selected. For example, to enrich CD4+ cells by negative selection, monoclonal antibodies typically include antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Alternatively, regulatory T lymphocytes are depleted using anti-C25 conjugated beads or other similar selection methods.

[0129] Stimulatory lymphocytes can also be frozen after a washing step. Without being bound by theory, the freezing and subsequent thawing steps result in a more homogenous product by eliminating granulocytes and, to some extent, monocytes from the cell population. After a washing step that removes plasma and platelets, the cells can be suspended in a freezing solution. While many solutions and freezing parameters are known in the art and may be useful in this regard, one method is to use PBS containing 20% ​​DMSO and 8% human serum albumin, or a medium containing 10% dextran 40, 5% glucose, human albumin, and 7.5% DMSO, or 31.25% Plasmalyte A, 31.25% glucose 5%, 0.45% NaCl, 10% dextran 40, 5% glucose, 20% human serum albumin, and 7.5% DMSO, or other suitable cell freezing media containing, for example, Hespan and PlasmaLyte A. The cells can then be frozen to -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other controlled freezing methods are also available, including immediate uncontrolled freezing at -20°C or in liquid nitrogen.

[0130] Cryopreserved cells can be thawed and washed as described herein, left at room temperature for 1 hour, and then activated using the methods of the present invention. Lymphocytes can be expanded and frozen as described herein and then used. As described herein, samples can be collected from patients immediately after diagnosis of a particular disease described herein, but before any treatment. Cells can be isolated from a subject's blood sample or apheresis prior to various related therapies, including, but not limited to, treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolic acid, and FK506, antibodies or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxane, fludarabine, cyclosporidine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and radiation. These drugs inhibit calcium-dependent calcineurin phosphatase (e.g., cyclosporine and FK506) or inhibit p70S6 kinase (rapamycin), which is important in growth factor-induced signal transduction (Liu et al., Cell 66: 807-815, 1991; Henderson et al., Immun 73: 316-321, 1991; Bierer et al., Curr. Opin. Immun., 5: 763-773, 1993). Cells can be isolated from patients and frozen for later use in conjunction with (e.g., before, concurrently with, or after) bone marrow or stem cell transplantation, T-lymphocyte ablation with chemotherapeutic agents such as fludarabine, external beam radiotherapy (XRT), cyclophosphamide, or therapy with antibodies such as OKT3 or CAMPATH. As described herein, cells can be isolated prior to therapy involving ablation of B lymphocytes, such as with an agent reactive with CD20, e.g., Rituxan, and frozen for use after the therapy.

[0131] Either before or after genetically modifying lymphocytes (e.g., T lymphocytes) to express a desired transgene, the lymphocytes can generally be activated and expanded using methods such as those described in, for example, U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005.

[0132] b. Vector Transgenes can be introduced into lymphoid cells using a variety of methods, including, but not limited to, transduction of cells with integration-competent gammaretroviruses or lentiviruses, and DNA transfer.

[0133] A wide variety of vectors can be used to express transgenes. The ability of certain viruses to infect cells or enter cells through receptor-mediated endocytosis, and their ability to integrate into the genome of host cells and stably and efficiently express viral genes, makes them attractive candidates for introducing foreign nucleic acids into cells. Therefore, in certain embodiments, viral vectors are used to introduce nucleotide sequences encoding one or more transgenes or fragments thereof into host cells for expression. Viral vectors can contain nucleotide sequences encoding one or more transgenes or fragments thereof, operably linked to one or more control sequences, such as promoters. Alternatively, viral vectors may not contain control sequences, and instead rely on control sequences in host cells to drive the expression of transgenes or fragments thereof. Non-limiting examples of viral vectors that can be used to deliver nucleic acids include adenoviral vectors, AAV vectors, and retroviral vectors.

[0134] For example, adeno-associated virus (AAV) can be used to introduce the nucleotide sequence encoding one or more transgenes or fragments thereof into host cells for expression.AAV system has been previously described and is generally well known in the art (Kelleher and Vos, Biotechniques, 17(6):1110-7, 1994; Cotten et al., Proc Natl Acad Sci USA, 89(13):6094-6098, 1992; Curiel, Nat Immun, 13(2-3):141-64, 1994; Muzyczka, Curr Top Microbiol Immunol, 158:97-129, 1992).Details about the construction and use of rAAV vectors are described in, for example, U.S. Patent No. 5,139,941 and U.S. Patent No. 4,797,368, each of which is incorporated herein by reference in its entirety for all purposes.

[0135] In some embodiments, retroviral expression vectors can be used to introduce nucleotide sequences encoding one or more transgenes or fragments thereof into host cells for expression. These systems have been previously described and are generally well known in the art (Nicolas and Rubinstein, in Rodriguez and Denhardt, eds., Stoneham: Butterworth, pp. 494-513, 1988; Temin, in Gene Transfer, Kucherlapati (ed.), New York: Plenum Press, pp. 149-188, 1986). Examples of vectors for eukaryotic expression in mammalian cells include AD5, pSVL, pCMV, pRc / RSV, pcDNA3, pBPV, and the like, as well as vectors derived from viral systems such as vaccinia virus, adeno-associated virus, herpes virus, and retroviruses, using promoters such as CMV, SV40, EF-1, UbC, RSV, ADV, BPV, and β-actin.

[0136] Retroviruses can also be combined with appropriate packaging cell lines, in which case the capsid protein functions to infect target cells. Typically, cells and virus are incubated in culture for at least about 24 hours. Cells are then grown in culture for a shorter time interval, e.g., 24-73 hours, or at least two weeks, depending on the application. They may also be grown for five weeks or longer before analysis. Commonly used retroviral vectors are "defective," meaning they are unable to produce the viral proteins necessary for productive infection. Vector replication requires propagation in a packaging cell line. Retrovirus host cell specificity is determined by the envelope protein, env (pl20), which is provided by the packaging cell line. There are at least three types of envelope proteins: ecotropic, amphotropic, and xenotropic. Retroviruses packaged with ecotropic envelope proteins, such as MMLV, are capable of infecting most mouse and rat cell types. Ecotropic packaging cell lines include BOSC23. Retroviruses with amphotropic envelope proteins, such as 4070A, are capable of infecting most mammalian cell types, including humans, dogs, and mice. Amphotropic packaging cell lines include PA12 and PA317. Retroviruses packaged with xenotropic envelope proteins, such as AKR env, are capable of infecting most mammalian cell types, except for mouse cells. The vectors can contain genes that must subsequently be removed, for example, using a recombinase system such as Cre / Lox, or cells expressing them can be destroyed by including genes that allow for selective toxicity, for example, herpesvirus TK, BCL-xs, etc. An appropriate inducible promoter is activated in the desired target cell type, either the transfected cell or its progeny.

[0137] Non-limiting examples of vectors useful in the present invention include the retroviral vector SFG.MCS and helper plasmids RD114 and Peg-Pam3 (Arber et al. J Clin Invest 2015 Jan 2; 125(1): 157-168), the lentiviral vector pRRL and helper plasmids R8.74 and pMD2G (e.g., Addgene Plasmid #12259). In some embodiments, the Sleeping Beauty transposon system can be used (Deniger et al. 2016 Mol Ther. Jun; 24(6): 1078-1089). In some embodiments, a transgene can be introduced into cells by, for example, electroporation (Xiaojun et al. 2017 Protein Cell, 8(7): 514-526) or the Cell Squeeze® method, which deforms cells as they pass through a small opening, disrupting the cell membrane and allowing the material to enter the cell. Such electroporation of transgene-encoding RNA allows for transient expression of the transgene in cells and can limit toxicity and other undesirable effects of genetically modified cells (Barrett et al. 2011 Hum Gene Ther. Dec; 22 (12): 1575-1586).

[0138] In some embodiments, genome editing technologies, such as the CRISPR / Cas9 system, designer zinc fingers, transcription activator-like effectors (TALEs), or homing meganucleases, can be used to induce transgene expression in immune cells. Generally, the term "CRISPR / Cas9 system" refers collectively to the transcripts and other elements involved in inducing the expression or activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (transactivating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr-mate sequences (including "direct repeats" and partial direct repeats processed by tracrRNA in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), or other sequences and transcripts from CRISPR loci. One or more elements of the CRISPR system can be derived from type I, type II, or type III CRISPR systems. Alternatively, one or more elements of a CRISPR system may be derived from a particular organism that contains an endogenous CRISPR system, such as Streptococcus pyogenes. Generally, CRISPR systems are characterized by an element (also called a protospacer in the context of endogenous CRISPR systems) that facilitates the formation of a CRISPR complex at the site of the target sequence.

[0139] In some embodiments, the genetic modification is introduced by transfecting lymphocyte cells with a vector (e.g., a lentiviral vector) encoding one or more transgenes or functional fragments thereof and CA9 or a functional fragment thereof. In some embodiments, one or more transgenes or functional fragments thereof and CA9 or a functional fragment thereof can be introduced into immune cells using one, two, or more vectors.

[0140] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing exogenous vectors and / or nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0141] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems, such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro and in vivo release vehicle is a liposome (e.g., artificial membrane vesicle).

[0142] When a non-viral delivery system is used, an exemplary delivery vehicle is a liposome. The use of lipid formulations for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo) is contemplated. In another aspect, the nucleic acid may be associated with a lipid. The lipid-associated nucleic acid may be encapsulated in the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, bound to the liposome via a linking molecule attached to both the liposome and the oligonucleotide, enclosed in the liposome, complexed with the liposome, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained in a suspension in lipids, the contents of or complexed with micelles, or otherwise associated with lipids. Lipid, lipid / DNA, or lipid / expression vector-related compositions are not limited to any particular structure in solution. For example, they can exist in a bilayer structure, as micelles, or in a "collapsed" structure. They may also simply be dispersed in the solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances and can be natural or synthetic. For example, lipids include the naturally occurring lipid droplets in the cytoplasm, as well as the class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0143] Suitable lipids for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma (St. Louis, MO); dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; and dimyristyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids (Birmingham, AL). Lipid stock solutions in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a generic term that encompasses a variety of unique multilamellar lipid vesicles formed by the formation of bilayers or closed lipid aggregates. Liposomes can be characterized as vesicular structures with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in excess aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure, trapping dissolved water and solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, some compositions exhibit structures in solution that differ from the typical vesicle structure. For example, lipids may form micelles or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also considered.

[0144] Regardless of the method used to introduce exogenous nucleic acid into a host cell, the presence of the recombinant DNA sequence in the host cell can be confirmed by a series of tests. Such assays include, for example, "molecular biological" assays well known to those skilled in the art, such as Southern and Northern blots, RT-PCR, and PCR; biochemical assays, such as detecting the presence or absence of specific peptides by immunological means (ELISA and Western blot) or by the assays described herein to identify agents within the scope of the present invention.

[0145] C. Treatment method The present disclosure further provides a method of treating cancer or tumors, comprising administering to a subject in need thereof a therapeutically effective amount of a composition or pharmaceutical composition, as described above.

[0146] As used herein, the terms "subject" and "patient" are used interchangeably, regardless of whether the subject has received or is currently receiving any treatment. As used herein, the term "subject" can refer to any vertebrate, including, but not limited to, mammals (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats and mice, non-human primates (e.g., monkeys such as cynomolgus monkeys and chimpanzees), and humans). The subject can be human or non-human. In a more exemplary aspect, the mammal is a human.

[0147] In some embodiments, the subject is a human. In some embodiments, the subject has cancer. In some embodiments, the subject is immune-depleted.

[0148] As used to describe the present invention, "cancer," "tumor," and "malignant tumor" all equally relate to hyperplasia of a tissue or organ. When the tissue is part of the lymphatic or immune system, the malignant cells may include non-solid tumors of circulating cells. Malignant tumors of other tissues or organs may give rise to solid tumors. The methods of the present invention can be used to treat lymphocytes, circulating immune cells, and solid tumors.

[0149] Treatable cancers include not only vascularized tumors, but also non-vascularized or substantially non-vascularized tumors.Cancers may be non-solid tumors (e.g., hematological tumors such as leukemia and lymphoma) or solid tumors.The types of cancers that can be treated with the compositions of the present invention include, but are not limited to, carcinomas, blastomas, and sarcomas, as well as certain leukemias or malignant lymphoid tumors, benign tumors, and malignant tumors, such as sarcomas, carcinomas, and melanomas.Also included are adult tumors / cancers and pediatric tumors / cancers.

[0150] Hematologic cancer is a cancer of the blood or bone marrow. Examples of hematologic (or blood-derived) cancers include leukemias, such as acute leukemia (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloid leukemia, promyelocytic, myelomonocytic, monocytic, and erythroid leukemia), chronic leukemia (such as chronic myelocytic (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and aggressive), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0151] A solid tumor is an abnormal mass of tissue that usually does not contain cysts or fluid. Solid tumors can be benign or malignant. Various types of solid tumors are named for the type of cells that form them (sarcoma, carcinoma, lymphoma, etc.). Examples of solid tumors, such as sarcomas and carcinomas, include: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovial, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colorectal cancer, lymphoid malignancies, pancreatic cancer, breast cancer, gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, endometrial cancer, cervical cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, and papillary adenocarcinoma. , medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocarcinoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder cancer, melanoma, and CNS tumors (such as gliomas) (brain stem glioma, mixed glioma, etc.), glioblastoma (including astrocytoma, CNS lymphoma, germinoma, medulloblastoma, schwannoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastases).

[0152] In some embodiments, the cancer is selected from the group consisting of melanoma, sarcoma, ovarian cancer, prostate cancer, lung cancer, bladder cancer, MSI-high tumors, head and neck tumors, kidney cancer, and breast cancer.

[0153] The pharmaceutical compositions described above can be administered in a manner appropriate for the disease to be treated (or prevented). The dosage and frequency of administration are determined by factors such as the patient's condition and the type and severity of the patient's disease, but the appropriate dosage can be determined by clinical trials.

[0154] When an "immunologically effective amount," "antitumor effective amount," "tumor suppression 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 age, weight, tumor size, degree of infection or metastasis, and condition of the patient (subject). Generally, pharmaceutical compositions containing lymphocytes described herein are administered in an amount of 10 4 ~10 9cells / kg body weight, e.g. 10 5 ~10 6 It can be said that the lymphocyte composition can be administered at a dose of 1000 cells / kg body weight (including all integer values ​​within these ranges). The lymphocyte composition can also be administered at these doses several times. The cells can be administered using injection techniques well known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dose and treatment regimen for a particular patient can be easily determined by those skilled in the medical field by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0155] The composition can be administered by any convenient method, including infusion or injection (i.e., intravenously, intrathecally, intramuscularly, intraluminally, intratracheally, intraperitoneally, or subcutaneously), transdermally, or other methods known in the art. Administration can be once every two weeks, once a week, or more frequently, although the frequency may be reduced during the maintenance phase of the disease or disorder. In some embodiments, the composition is administered by intravenous infusion.

[0156] In certain cases, cells activated and expanded using the methods described herein, or other methods known in the art in which lymphocytes are expanded to therapeutic levels, are administered to patients in conjunction with (e.g., before, simultaneously with, or after) a number of related therapies. Also, as described herein, lymphocytes can be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolic acid, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-cancer antibodies, CD3 or other antibody therapy, cytoxan, fludarabine, cyclosporidine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and radiation.

[0157] The compositions of the present invention can also be administered to patients in conjunction with (e.g., before, simultaneously with, or after) bone marrow transplantation, therapy involving T lymphocyte ablation with chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. As described herein, the compositions can also be administered after B lymphocyte ablative therapy, such as with an agent that reacts with CD20, e.g., Rituxan. For example, a subject can undergo standard treatment with high-dose chemotherapy followed by a transplant of peripheral blood stem cells. In certain cases, after transplantation, the subject receives an infusion of expanded lymphocytes, or expanded lymphocytes are administered before or after surgery.

[0158] In some embodiments, the method may further comprise administering a second therapeutic agent to the subject. The second therapeutic agent is an anti-cancer or anti-tumor agent. In some embodiments, the composition is administered to the subject before, after, or simultaneously with the second therapeutic agent, such as a chemotherapeutic agent or an immunotherapeutic agent.

[0159] In some embodiments, the method further comprises administering a therapeutically effective amount of an immune checkpoint modulator. Examples of immune checkpoint modulators include PD1, PDL1, CTLA4, TIM3, LAG3, and TRAIL. The checkpoint modulator can be administered simultaneously, separately, or in parallel with the composition of the present invention.

[0160] A "chemotherapeutic agent" refers to a compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXANTM); alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, methyldopa, and uredopa; ethylenimines and methylamelamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptofen phycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including the synthetic analogs KW-2189 and CBI-TMI); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, e.g., chlorambucil, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, e.g., enediyne antibiotics (e.g., calicheamicin, e.g., Agnew Chem. Intl. Ed. Engl. 33:183-186 (1994); dynemicins, such as dynemicin A; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomycins, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin). including epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfilomycin, puromycin, quelamycin, rhodrubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as fulvastatin, fluvastatin, fluvastatin, fluvastatin; Folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; epothilon; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids, such as maytansine, and ansamitocins; mitoguazone; mitoxantrone;Mopidamol; Nitracrine; Pentostatin; Fenamet; Pirarubicin; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK®; Razoxane; Rhizoxin; Schizofuran; Spirogermanium; Tenuazonic acid; Triaziquone; 2,2',2''-Trichlorotriethylamine; Trichothecenes (especially T-2 toxin, verrucarin A, roridin A, anguidine); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxoids, such as paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronic acid; CPT-11; the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. Also included within this definition are antihormonal agents that act to regulate or inhibit hormone action on tumors, such as antiestrogens, e.g., tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and antiandrogens, e.g., flutamide, nilutamide, bicalutamide, leuprolide, xeloda, gemcitabine, KRAS mutation covalent inhibitors, and goserelin;and pharmaceutically acceptable salts, acids, or derivatives of any of the above. Additional examples include irinotecan, oxaliplatin, and other standard colon cancer regimens.

[0161] "Immunotherapeutic agents" may include biologic agents useful in the treatment of cancer. In some embodiments, immunotherapeutic agents include immune checkpoint inhibitors (e.g., inhibitors of PD-1, PD-L1, TIM-3, LAG-3, VISTA, DKG-α, B7-H3, B7-H4, TIGIT, CTLA-4, BTLA, CD160, TIM1, IDO, LAIR1, IL-12, or combinations thereof). Examples of immunotherapeutic agents include atezolizumab, avelumab, blinatumomab, daratumumab, cemiplimab, durvalumab, elotuzumab, laherparepvec, ipilimumab, nivolumab, obinutuzumab, ofatumumab, pembrolizumab, cetuximab, and talimogene.

[0162] D. definition To aid in understanding the detailed description of the compositions and methods according to the present disclosure, several clear definitions are provided to facilitate clear disclosure of the various aspects of the present disclosure. 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 disclosure belongs.

[0163] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this invention belongs.The following references provide those skilled in the art with the general definitions of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd Edition 1994); The Cambridge Dictionary of Science and Technology (Walker, ed., 1988); The Glossary of Genetics, 5th Edition, R. Rieger et al. (ed.), Springer Verlag (1991); and Hale & Marham, The HarperCollins Dictionary of Biology (1991).As used herein, the following terms have the meanings ascribed to them below, unless otherwise specified.

[0164] As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcript) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. Transcription products and encoded polypeptides can be collectively referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell.

[0165] As used herein, the term "recombinant" refers to a cell, microorganism, nucleic acid molecule, or vector that has been modified by the introduction of an exogenous nucleic acid molecule or in which the expression of an endogenous nucleic acid molecule or gene has been regulated. It can be deregulated or modified to be constitutively modified, and such modifications or alterations can be introduced by genetic engineering. Genetic modifications include, for example, the introduction of nucleic acid molecules (which may include expression control elements such as promoters) encoding one or more proteins or enzymes, or the addition, deletion, substitution, or other functional disruption or functional addition of another nucleic acid molecule to the genetic material of a cell. Exemplary modifications include modifications in the coding region of a heterologous or homologous polypeptide derived from a reference or parent molecule or functional fragment thereof.

[0166] By "transgene" or "therapeutic transgene" is meant a molecule selected from the group consisting of a soluble receptor, a decoy, a decoy receptor, a dominant negative, a microenvironment modulator, an enzyme, an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, a translocase, a kinase, a transporter, a modifier, a molecular chaperone, an ion channel, an antibody, a cytokine, a growth factor, a chemokine, a hormone, DNA, a ribozyme, a biosensor, an epigenetic modifier, a transcription factor, a coding RNA, a non-coding RNA, a small RNA, a long RNA, an IRES element, or an exosome shuttle RNA.

[0167] As used herein, the term "functional variant" refers to an altered transgene that has substantial or significant sequence identity or similarity to a wild-type transgene, and such a functional variant retains the biological activity of the wild-type transgene of that variant. In some embodiments, a functional variant of a transgene is used.

[0168] As used herein, the term "antigen-recognizing receptor" refers to a receptor that can activate immune cells (e.g., T cells) in response to antigen binding. Exemplary antigen-recognizing receptors can be natural or genetically modified TCRs, or genetically modified TCR-like mAbs (Hoydahl et al. Antibodies 2019 8:32), or CARs in which a tumor antigen-binding domain is fused to an intracellular signaling domain that can activate immune cells (e.g., T cells).T cell clones expressing natural TCRs against specific cancer antigens have been previously described (Traversari et al., J Exp Med, 1992 176:1453-7; Ottaviani et al., Cancer Immunol Immunother, 2005 54:1214-20; Chaux et al., J Immunol, 1999 163:2928-36; Luiten and van der Bruggen, Tissue Antigens, 2000 55:149-52; van der Bruggen et al., Eur J Immunol, 1994 24:3038-43; Huang et al., J Immunol, 1999 162:6849-54; Ma et al., Int J Cancer, 2004 109:698-702; Ebert et al., Cancer Res, 2009 69:1046-54; Ayyoub et al. J Immunol 2002 168:1717-22; Chaux et al., European Journal of Immunology, 2001 31:1910-16; Wang et al., Cancer Immunol Immunother, 2007 56:807-18; Schultz et al., Cancer Research, 2000 60:6272-75; Cesson et al., Cancer Immunol Immunother, 2010 60:23-25; Zhang et al., Journal of Immunology, 2003 171:219-25; Gnjatic et al., PNAS, 2003 100:8862-67; Chen et al., PNAS, 2004). In one embodiment, such TCRs can be sequenced and genetically engineered into TILs for use in adoptive cell therapy.In certain aspects, TCRs that recognize MAGE-A1 antigen, MAGE-A3 antigen, MAGE A-10 antigen, MAGE-C2 antigen, NY-ESO-1 antigen, SSX2 antigen, and MAGE-A12 antigen can be genetically modified into TILs for use in adoptive cell therapy.In still other embodiments, the genetically modified TILs with TCRs are further modified to secrete transgenes.In still other embodiments, CARs are used.In other embodiments, CARs are further modified to secrete transgenes.

[0169] As used herein, the term "antibody" refers not only to intact antibody molecules but also to fragments of antibody molecules that retain immunogen-binding ability. Such fragments are also well known in the art and are routinely used both in vitro and in vivo. Thus, as used herein, the term "antibody" refers not only to intact immunoglobulin molecules but also to the well-known active fragments f(ab')2 and fab. f(ab')2 and fab fragments, which lack the Fe fragment of intact antibodies, may be cleared from the circulation more quickly and exhibit less nonspecific tissue binding than intact antibodies (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Antibodies of the present invention include whole native antibodies, bispecific antibodies; chimeric antibodies; fab, fab', single-chain V-region fragments (scFv), fusion polypeptides, and unconventional antibodies.

[0170] As used herein, the term "single-chain variable fragment" or "scFv" refers to a fusion protein in which the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin are covalently linked to form a VH:VL heterodimer. The heavy (VH) and light (VL) chains are either directly linked or linked by a peptide-encoding linker (e.g., 10, 15, 20, or 25 amino acids) that connects the N-terminus of the VH to the C-terminus of the VL or the C-terminus of the VH to the N-terminus of the VL. The linker is typically rich in glycine for flexibility and serine or threonine for solubility. Despite the removal of the constant regions and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. Single-chain Fv polypeptide antibodies can be expressed from nucleic acids containing the VH and VL coding sequences described in Huston et al. (Proc. Nat. Acad. Sci., 85:5879-5883, 1988). See also U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778, and U.S. Patent Publication Nos. 20050196754 and 20050196754. Antagonist scFvs with inhibitory activity have been described (see, e.g., Zhao et al., Hybridoma (Larchmont) 2008 27(6):455-51; Peter et al., J cachexia sarcopenia muscle 2012 Aug. 12; Shieh et al., J Immunol 2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife et al., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 1995 2(10:31-40)).Agonistic scFvs with stimulatory activity have been described (see, e.g., Peter et al., J Bioi Chern 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev Immunol 1997 17(5-6):427-55; Ho et al., Biochim Biophys Acta 2003 1638(3):257-66).

[0171] As used herein, "treating" or "treatment" refers to the administration of a compound or agent to a subject having a disease for the purpose of curing, alleviating, mitigating, relieving, delaying the onset of, preventing, or ameliorating the disease, symptoms of the disease, conditions secondary to the disease, or predisposition to the disease.

[0172] The terms "elicit" or "enhance" in the context of an immune response refer to eliciting or increasing an immune response, such as increasing the ability of immune cells to target and / or kill cancer cells or to target and / or kill pathogens and pathogen-infected cells (e.g., EBV-positive cancer cells).

[0173] As used herein, the term "immune response" refers to any type of immune response, including, but not limited to, an innate immune response (e.g., activation of the Toll receptor signaling cascade), a cellular immune response (e.g., a response mediated by T cells (e.g., antigen-specific T cells) and non-specific cells of the immune system), and a humoral immune response (e.g., a response mediated by B cells (e.g., via the production and secretion of antibodies into plasma, lymph, and / or tissue fluids)). The term "immune response" encompasses all aspects of the ability of a subject's immune system to respond to antigens and / or immunogens (e.g., both the initial response to an immunogen (e.g., a pathogen) and the acquired (e.g., memory) response that is a result of the adaptive immune response).

[0174] As used herein, the term "in vitro" refers to events that take place in an artificial environment, e.g., in a test tube or reaction vessel, cell culture, etc., rather than within a multicellular organism.

[0175] As used herein, the term "in vivo" refers to events that take place within a multicellular organism, such as a non-human animal.

[0176] As used herein, the term "disease" is generally intended to be synonymous with, and used interchangeably with, the terms "disorder" and "condition" (as in medical condition), in that it reflects an abnormal condition of the human or animal body or part thereof that impairs normal function, is usually manifested by characteristic signs and symptoms, and results in a reduction in the length or quality of life of a human or animal.

[0177] The terms "reduce," "decrease," "reduction," "reduction," or "inhibition" are all used herein to generally mean a statistically significant reduction. However, for the avoidance of doubt, "reduced," "reduction," "reduction," or "inhibition" refers to a reduction of at least 10% compared to a reference level, e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or up to 100% (e.g., a loss of level compared to a reference sample), or any reduction between 10% and 100% compared to a reference level.

[0178] As used herein, the term "modulate" is meant to refer to any change in a biological state, ie, an increase, a decrease, and the like.

[0179] The terms "increased," "increase," "enhance," or "activate" are all used herein to generally mean an increase by a statistically significant amount; for the avoidance of doubt, the terms "increased," "increase," "enhance," or "activate" mean an increase of at least 10% compared to a base level, for example, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or up to a 100% increase, or any increase between 10-100% compared to a base level, or at least about a 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold increase, or any increase between 2-fold and 10-fold or more compared to a base level.

[0180] The terms "effective amount," "effective dose," or "effective dosage" are defined as an amount sufficient to achieve, or at least partially achieve, a desired effect. A "therapeutically effective amount" or "therapeutically effective dosage" of a drug or therapeutic agent is the amount of the drug that, when used alone or in combination with other therapeutic agents, promotes disease regression as evidenced by a decrease in the severity of symptoms, an increase in the frequency and duration of symptom-free periods, or prevention of functional impairment or disability due to disease affliction. A "prophylactically effective amount" or "prophylactically effective dosage" of a drug is the amount of the drug that, when administered alone or in combination with other therapeutic agents to a subject at risk of developing a disease or suffering a disease recurrence, inhibits the onset or recurrence of the disease. The ability of a therapeutic or prophylactic agent to promote disease regression or inhibit the occurrence or recurrence of a disease can be assessed using various methods known to those skilled in the art, for example, by measuring the activity of the agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0181] Doses are often expressed in relation to body weight, so a dose expressed as [g, mg, or other unit] / kg (or g, mg, etc.) usually refers to [g, mg, or other unit] "per kg (or g, mg, etc.) body weight," even if the term "body weight" is not explicitly stated.

[0182] The term "agent" is used herein to refer to a compound, a mixture of compounds, a biopolymer (such as a nucleic acid, an antibody, a protein or portion thereof, e.g., a peptide), or an extract made from biological material such as a bacterial, plant, fungal, or animal (e.g., mammalian) cell or tissue. The activity of such an agent may make it suitable as a "therapeutic agent," which is a biologically, physiologically, or pharmacologically active substance that acts locally or systemically in a subject.

[0183] The terms "therapeutic agent," "therapeutic agent," or "therapeutic agent" are used interchangeably and refer to a molecule or compound that has some beneficial effect when administered to a subject. Beneficial effects include enabling a diagnostic determination; ameliorating a disease, symptom, disorder, or pathological condition; inhibiting or preventing the onset of a disease, symptom, disorder, or condition; and generally resolving a disease, symptom, disorder, or pathological condition.

[0184] As used herein, "combination" therapy, unless otherwise clear from the context, refers to the administration of two or more therapeutic agents in a coordinated manner, including, but not limited to, simultaneous administration. Specifically, combination therapy encompasses both coadministration (e.g., administration of a co-formulation or simultaneous administration of separate therapeutic compositions) and sequential or sequential administration, as long as the administration of one therapeutic agent is somehow conditioned on the administration of another therapeutic agent. For example, one therapeutic agent can be administered only after another therapeutic agent has been administered and allowed to act for the indicated period of time. See, for example, Kohrt et al. (2011) Blood 117:2423.

[0185] The terms "sample," "test sample," and "patient sample" may be used interchangeably herein. A sample may be serum, urine, plasma, amniotic fluid, cerebrospinal fluid, cell (e.g., antibody-producing cells), or tissue sample. Such samples may be used directly as obtained from a patient, or may be pretreated by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, etc., to alter the properties of the sample in some way as described herein or known in the art. As used herein, the terms "sample" and "biological sample" generally refer to biological material that is tested for and / or suspected of containing an analyte of interest, such as an antibody. A sample may be any tissue sample from a subject. A sample may also contain proteins from a subject.

[0186] As used herein, the terms "inhibit" and "antagonize" mean to measurably decrease or completely prevent the expression, stability, function, or activity of a molecule, reaction, interaction, gene, mRNA, and / or protein. Inhibitors are compounds, e.g., antagonists, that bind to proteins, genes, and mRNAs and partially or totally block stimulation, reducing, preventing, delaying activation, inactivating, desensitizing, or downregulating their stability, expression, function, and activity.

[0187] "Parenteral" administration of the compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection or infusion techniques.

[0188] As used herein, the term "pharmaceutical composition" refers to a mixture of at least one compound useful in the present invention with other chemical components, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, and / or excipients. A pharmaceutical composition facilitates administration of the compound to an organism.

[0189] As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, that does not destroy the biological activity or properties of the composition and that is relatively non-toxic; that is, such a material can be administered to an individual without producing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0190] The term "pharmaceutically acceptable carrier" includes pharmaceutically acceptable salts, pharmaceutically acceptable materials, compositions, or carriers, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials, that are involved in carrying or transporting a compound of the present invention in or to a subject so that the compound performs its intended function. Typically, such compounds are carried or transported from one organ or part of the body to another. Each salt or carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject. Some examples of materials that can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository wax; fats and oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol. Glycols; polyols such as glycerin, sorbitol, mannitol, polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free distilled water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer; diluents; granulating agents; lubricants; binders; disintegrating agents; wetting agents; emulsifiers; coloring agents; release agents; coating agents; sweeteners; flavoring agents; fragrances; preservatives; antioxidants; plasticizers; gelling agents; thickeners; hardeners; setting agents; suspending agents; surfactants; humectants; carriers; stabilizers; and other non-toxic, compatible substances used in pharmaceutical formulations, or any combination thereof. As used herein, "pharmaceutically acceptable carrier" also includes any and all coating agents, antibacterial and antifungal agents, and absorption delaying agents that are compatible with the activity of the compound and physiologically acceptable to the subject. Supplementary active compounds can also be incorporated into the compositions.

[0191] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0192] The terms "including," "comprising," "containing," or "having," and variations thereof, are meant to encompass the items listed thereafter and equivalents thereof, as well as additional subject matter, unless expressly stated otherwise.

[0193] The phrases "in one embodiment," "in various embodiments," "in some embodiments," etc. are used repeatedly. Such phrases do not necessarily refer to the same embodiment, although they may unless the context dictates otherwise.

[0194] The term "and / or" or " / " means any one of the items, any combination of the items, or all of the items with which this term is associated.

[0195] The term "substantially" does not exclude "completely", for example, a composition that is "substantially free" of Y may be completely free of Y. Where appropriate, the term "substantially" may be omitted from the definition of the present invention.

[0196] As used herein, the term "approximately" or "about," when applied to one or more values ​​of interest, refers to a value similar to the stated reference value. In some embodiments, unless otherwise specified or clear from the context, the term "approximately" or "about" refers to a range of numbers that is within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or lesser) of the stated reference value (unless the number exceeds 100% of the possible values). Unless otherwise indicated herein, the term "about" is intended to include values, e.g., weight percent, that approximate the stated range and are equivalent in terms of the functionality of the individual components, compositions, or embodiments.

[0197] When values ​​and ranges are provided herein, it is understood that all values ​​and ranges encompassed within those values ​​and ranges are meant to be encompassed within the scope of the present invention. Moreover, all values ​​within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the application.

[0198] As used herein, the term "each," when used in reference to a collection of items, is intended to identify each individual item in the set, but does not necessarily refer to every item in the set. Exceptions may occur where express disclosure or context clearly dictates otherwise.

[0199] Any and all examples provided herein, or the use of exemplary language (e.g., "etc.") are intended merely to facilitate a better understanding of the invention and do not limit the scope of the invention unless specifically claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0200] All methods described herein may be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. For any provided methods, the method steps may be performed simultaneously or sequentially. When method steps are performed sequentially, those steps may be performed in any order unless otherwise specified.

[0201] Where a method includes a combination of steps, any and all combinations or subcombinations of the steps are encompassed within the scope of the present disclosure unless otherwise stated herein.

[0202] Each publication, patent application, patent, and other reference cited herein is incorporated herein by reference in its entirety to the extent that it does not contradict this disclosure. The publications disclosed herein are provided solely for their disclosure prior to the filing date of the present invention. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0203] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art and are to be included within the spirit and scope of this application and the appended claims. [Example]

[0204] E. Example Example 1 This example describes the materials and methods used in the subsequent examples.

[0205] Mice and cell lines Six-week-old female C57BL / 6 mice were purchased from Harlan (Harlan, The Netherlands) and maintained in accordance with the animal facility guidelines at the University of Lausanne (UNIL, Epalinges, Switzerland). C57BL / 6 OT-1 CD45.1+ and C57BL / 6 CD8a- / - mice were described in Hogquist KA et al. (Hogquist KA et al. Cell 76(1):17-27 PubMed: 8287475 MGI: J:92867) and Fung-Leung WP et al. (Fung-Leung WP et al. Cell 65(3):443-9 PubMed: 1673361 MGI: J:68956). All in vivo experiments were performed with the approval of the Service of Consumer and Veterinary Affairs (SCAV) of the canton of Vaud, Switzerland.

[0206] The ovalbumin-expressing B16 melanoma cell line (B16-OVA) was previously generated by retroviral transduction of the B16.F10 cell line purchased from ATCC and cultured as a monolayer in DMEM supplemented with 10% fetal calf serum (FCS), 100 U / ml penicillin, and 100 μg / ml streptomycin sulfate. Cells were passaged twice weekly to maintain them under exponential growth conditions and routinely tested for mycoplasma contamination. The Phoenix Eco retroviral ecotropic packaging cell line, derived from immortalized normal human embryonic kidney (HEK) cells, was maintained in RPMI 1640-Glutamax medium supplemented with 10% heat-inactivated FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin sulfate.

[0207] Human embryonic kidney (HEK) 293T cells were purchased from ATCC (CRL-3216) and cultured in RPMI 1640 Glutamax medium (Invitrogen), 10% FBS (heat-inactivated at 56°C for 30 minutes; Gibco), and 1% penicillin / streptomycin (ThermoFisher Scientific). HEK 293T cells were used to generate retroviral and lentiviral particles. HLA-A2.1 pos / NY-ESO pos Melanoma cell lines Me275 and A375, and HLA-A2.1 pos / NY-ESO neg The cell line NA8 (obtained from the UNIL Department of Oncology) was cultured in IMDM supplemented with 10% FBS and 1% penicillin / streptomycin.

[0208] Design of bicistronic expression cassettes The retroviral vector pMSGV1 (a murine stem cell virus (MSCV)-based splice gag vector) containing the MSCV long terminal repeat (LTR) was used as the backbone for all constructs. The expression cassette typically encoded the signal peptide of mouse IgGκ chain region V-III MOPC 321 (e.g., Uniprot ID: P01650) (SEQ ID NO: 20), followed by the N-terminal ectodomain of mouse PD-1 (e.g., Uniprot ID: Q02242, residues S21-Q167, C83S) (SEQ ID NO: 1) (herein referred to as PD-1.IgG4 decoy) fused to human IgG4_Fc (e.g., Uniprot ID: P01861.1, residues P104-K327) (SEQ ID NO: 19). Restriction sites AgeI and EcoRI were located at the 5' and 3' ends of this first portion, respectively. The second part consisted of a T2A sequence followed by the signal peptide of mouse IFN-β (e.g., Uniprot ID: P01575.1) (SEQ ID NO: 39), followed by a gene string encoding one of the following molecules: mouse IL-33 (e.g., Uniprot ID: Q8BVZ5.1, residues S109 to I266) (SEQ ID NO: 27), mouse LIGHT (e.g., Uniprot ID: Q9QYH9.1, residues D72 to V239) (SEQ ID NO: 31), mouse CD40L (e.g., Uniprot ID: P27548, residues M112 to L260) (SEQ ID NO: 33), and no alpha mutant IL-2 (e.g., Uniprot ID: P27548, residues M112 to L260) (SEQ ID NO: 33). ID: P60568.1, residues A21 to T153, mutations: R58A, F62A, Y65A, E82A, and C145S) (SEQ ID NO: 23). Restriction sites MluI and SalI were located at the 5' and 3' ends of this second part, respectively. As a result, after each cloning, the following constructs were obtained: PD-1.IgG4_T2A_IL-2 V, PD-1.IgG4_T2A_IL-33, PD-1.IgG4_T2A_LIGHT, and PD-1.IgG4_T2A_CD40L. All gene strings were synthesized at GeneArt AG with mouse codon optimization, and all constructs were fully sequenced at Microsynth AG after cloning into the MSGV vector.

[0209] As shown in Figures 11A-C, codon-optimized gene strings encoding PD1 decoys, truncated EGFR, CD40 ligand decoys, and IL-2 variants, separated by picornavirus-derived 2A sequences, were ordered from GeneArt (ThermoFisher Scientific) and cloned into the retroviral vectors pSFG (for constitutive expression) or pSFG-SIN (for self-inactivation) for activation-based gene expression under the NFAT promoter. The vectors were amplified in Stellar competent cells (Escherichia coli HST08, #636763, Takara Biosciences) and purified using a plasmid mini / maxi-prep kit (Genomed) after sequence confirmation (Microsynth AG).

[0210] A gene string encoding the HLA / A2:NY-ESO-1 peptide-based T cell receptor (TCR), including TCRα23 and TCRβ13.1, was ordered from GeneArt (ThermoFisher Scientific). The TCRα and TCRβ chains were codon-optimized and separated by a picornavirus-derived 2A sequence. This gene string was inserted into the lentiviral vector pRRL, which deleted most of the U3 region of the 3' long terminal repeat, resulting in a self-inactivating 3' long terminal repeat (SIN).

[0211] Lentivirus production 10×10 6HEK 293T cells were seeded in a T150 flask containing RPMI complete medium (RPMI 1640 Glutamax medium (Invitrogen), 10% FBS (Gibco), 1% penicillin / streptomycin). Approximately 24 hours later (at 70-80% confluency), the cells were transfected with 7 μg of pVSV-G (VSV glycoprotein expression plasmid), 18 μg of R874 (Rev and Gag / Pol expression plasmid), and 15 μg of pRRL transgene plasmid using a mixture of 107 μl of Turbofect and 2 ml of Optimem medium (product number 51985026, Invitrogen). After a 30-minute incubation at room temperature, the DNA mixture was added on top of the cells, and the volume was adjusted to a total of 30 ml. After 24 hours, the medium was refreshed, and viral supernatant was harvested 48 hours after transfection. Viral particles were concentrated by ultracentrifugation and resuspended in 400 μl of RPMI complete medium. Virus aliquots of 100–200 μl per Eppendorf tube were prepared and stored at −80°C.

[0212] Retrovirus production 1 x 10 Phoenix Eco cells per T-150 tissue culture flask in 25 ml of medium 7Cells were seeded at 1000 x 1000 cells per well and, 24 hours later, transfected with 14.4 μg of pCL-Eco retroviral packaging vector and 21.4 μg of pMSGV transfer plasmid using Turbofect (Thermo Fisher Scientific). All plasmids were purified using the JETSTAR 2.0 Plasmid Maxiprep Kit (Genomed). The transfection mixture was prepared in 2 ml of Optimem at a 3:1 ratio of Turbofect:plasmid and incubated at room temperature for 30 minutes. The medium was then removed from a T-150 flask containing 80-90% confluent Phoenix Eco cells, and the transfection mixture was added and incubated for 1 minute, after which 25 ml of fresh medium was added. 48 hours after transfection, the viral supernatant was harvested, followed by the addition of 25 ml of fresh medium. A second harvest was performed 24 hours later. The viral particles in both SNs were concentrated by ultracentrifugation at 24,000 g for 2 hours at 4°C using a Beckman JS-24 rotor (Beckman Coulter), suspended in 0.5 ml of mouse T cell medium, and the viral titer was measured. Finally, the retrovirus was aliquoted, frozen on dry ice, and stored at -80°C.

[0213] Another example is 10 x 10 6HEK 293T cells were seeded overnight at 37°C in 17 ml of RPMI, 10% fetal bovine serum (FBS, Gibco), and 1% penicillin / streptomycin (ThermoFisher Scientific) in a T150 flask. The next day (at 85-95% confluency of 293T cells), a mixture of 120 μl of Turbofect (Life Technologies) and 3 ml of OptiMem was prepared per transfection (per T150 flask), which was then combined with retroviral plasmids: 22 μg of PamPeg, 7 μg of RDF-RD114, and 18 μg of SFG or SFG-SIN encoding the gene of interest. The medium was gently removed from the 293T cells, and the retroviral-plasmid mixture was pipetted onto the 293T cells. After resting for 5 minutes, an additional 16 ml of medium was gently added. The cells were then incubated overnight at 37°C. The next day, the medium was refreshed, and the following day (at 48 h), virus was harvested from the filtered supernatant by ultracentrifugation (24,000 x g for 2 h). For a second virus harvest at 72 h, fresh medium was added to 293T cells. Virus aliquots of 100–200 μl per Eppendorf tube were prepared from both days and stored at -80°C.

[0214] Transduction of mouse T cells Primary mouse OT-1 cells were isolated from single-cell suspensions of dissociated spleens from 6-10-week-old CD45.1+ congenic OT-1 C57BL / 6 mice using the Mouse Pan T cell Isolation Kit II (Miltenyi Biotec, catalog no. 130-095-130) and cultured in RPMI 1640-Glutamax medium (T cell medium) supplemented with 10% heat-inactivated FBS, 100 U / ml penicillin, 100 μg / ml streptomycin sulfate, 1 mM pyruvate, 50 μM BME, and 10 mM non-essential amino acids.

[0215] The cultures were maintained at a cell density of 0.5–1 × 10 cells / ml and replenished with fresh T cell medium every other day until day 15. (The medium was supplemented with 10 IU / ml human alpha-free IL-2 alone until day 3, and then with 10 ng / ml hIL-7 and IL-15.) On day 7, cellular expression of the molecules was assessed by intracellular flow cytometry analysis, and their presence in the supernatant was assessed by ELISA. Finally, the genetically modified OT-1 T cells were adjusted according to the transduction efficiency of the PD-1.IgG4 decoy before cell transplantation. Recombinant human IL-7 and human IL-15 were obtained from Miltenyi Biotec.

[0216] Isolated naive OT-1 T cells were plated at 1 x 10 in a 24-well plate. 6 T cells were seeded at 100 cells / ml in T cell culture medium and stimulated with αCD-3 / αCD-28 Ab-coated beads (Invitrogen) and 10 IU / ml human α-free IL-2. Twenty-four hours after activation, T cells were first transduced with retrovirus at a multiplicity of infection (MOI) of 10. Transduction was performed in non-tissue culture grade 24-well plates (Becton Dickinson Labware) pre-coated with 20 mg / ml recombinant RetroNectin (RetroNectin; Takara) overnight at 4°C, washed, and blocked with 2% bovine serum albumin (BSA) in PBS for 30 minutes at room temperature, followed by a final wash. After adding retrovirus (250 μl), the plates were centrifuged at 2000 x g for 1.5 hours at 32°C. 125 μl of supernatant was aspirated, and 1 × 10 cells were transduced. 6 Activated T cells were transferred to each coated well. The plates were centrifuged at 1200 rpm for 10 minutes and incubated overnight at 37°C, 5% CO2. The second transduction was performed 48 hours after activation according to the protocol described above. On day 7, cellular expression of the molecules was assessed by intracellular flow cytometry analysis, and their presence in the supernatant was assessed by ELISA. Finally, genetically modified OT-1 T cells were conditioned based on their expression of PD-1.IgG4 decoy before cell transfer.

[0217] Cultures were grown at 0.5–1 × 10 6 Cells were maintained at a cell density of 10 cells / ml and replenished with fresh T cell medium every other day until day 15, following an in vitro expansion protocol optimized to generate CD44+CD62L+TCF1+ central memory CD8 T cells. T cell medium was supplemented with 10 IU / ml human alpha-free IL-2 alone until day 3, and then with 10 ng / ml hIL-7 / IL-15 until the end of culture. Recombinant human IL-7 and human IL-15 were obtained from Miltenyi Biotec.

[0218] Purification and activation of human T cells Apheresis and buffy coat samples from healthy donors were purchased from Transfusion Interregionale CRS SA (Epalinges, Switzerland) with written consent under a protocol approved by the University Institutional Review Board. Peripheral blood mononuclear cells (PBMCs) were prepared by Lymphoprep (StemCell Technologies) density gradient centrifugation and depleted using CD8 or CD4 magnetic microbeads (Miltenyi) according to the manufacturer's protocol. + or CD4 + T cells were negatively isolated. + and CD4 + T cells were stimulated with anti-CD3 / CD28 beads (Invitrogen) at a bead:T cell ratio of 2:1 in the presence of human IL-2 (GlaxoSmithKline).

[0219] Lentiviral and retroviral transduction of human T cells Lentiviral transduction of T cells was performed 24 hours after activation by direct addition of viral particles (MOI 20) to the culture medium and enhanced by simultaneous addition of Lentiboost (Sirion Biotech). Retroviral transduction of T cells was performed 48 hours after activation. T cells were transferred to retronectin-coated plates where retroviral particles had been spun at 2000 x g for 1.5 hours. The following day, T cells were removed from the retronectin-coated plates. Anti-CD3 / anti-CD28 beads (Thermo Fisher Scientific) were removed 5 days after activation, and thereafter T cells were cultured at 0.5–1 × 10 in RPMI 1640-Glutamax (Thermo Fisher) supplemented with 10% heat-inactivated FBS (Gibco), 1% penicillin / streptomycin, 10 ng / ml human IL-7 (Miltenyi), and 10 ng / ml IL-15 (Miltenyi). 6 T cells / ml.

[0220] Co-transduction of human T cells with lentivirus and retrovirus For co-transduction, human T cells were purified and incubated for 18-22 hours with bead activation (0.5 x 10 cells per 48 wells). 6 T cells +1×10 6 After incubation with anti-CD3 / anti-CD28 beads (50 IU / ml IL-2), concentrated lentivirus (100 μl) was added. Optionally, 1 μl of Lentiboost (Sirion Biotech) was also added to enhance transduction efficiency. The following day, transduced T cells were transferred to retronectin-coated plates pre-spun with retroviral particles at 2000 x g for 1.5 hours. The following day, T cells were transferred to tissue culture plates. On day 5, the beads were removed, and T cells were transferred to larger wells and fed with fresh medium supplemented with 10 ng / ml IL-15 and 10 ng / ml IL-7. Fresh medium and cytokines were fed every 2–3 days. From days 7–10, cotransduction efficiency could be measured by flow cytometry.

[0221] Retroviral transduction of tumor-infiltrating lymphocytes (TILs) Thawed TILs were previously propagated from dissociated patient tumor fragments. 0.5 × 10 6 TILs were stimulated in a 48-well plate with 500 μl RPMI, 10% FBS, 25 μl GMP-grade TransAct (1:20, Miltenyi Biotech) and 6000 IU / ml IL-2. Non-tissue culture plates were coated overnight at 4°C with Retronectin (Takara Bio, 1 mg / ml diluted 50x, 250 μl per 48-well plate). The next day, Retronectin was removed and the plates were blocked with 500 μl RPMI, 10% FBS (Gibco), and 1% penicillin / streptomycin for 30 minutes at 37°C. The medium was then removed, and 50–100 μl of concentrated retrovirus was added to 50 μl of medium and spun at 2000 g for 1 hour at 25°C. The supernatant was then removed, and TILs were added and spun at 1000 g for 10 minutes at 25°C. After overnight incubation at 37°C, TILs were transferred to 48-well tissue culture plates containing fresh medium. On day 5, TILs were transferred to larger well plates and supplemented with fresh medium (RPMI, 10% FBS (Gibco), 1% penicillin / streptomycin, 6000 IU / ml IL-2). Transduction efficiency was assessed on days 7–10. From day 5 onward, fresh medium was supplied every 2–3 days.

[0222] Flow cytometry analysis All FACS data were acquired on an LCRII flow cytometer (BD) and analyzed using FlowJo software. Dead cell exclusion was performed using fixable aqua dead dyes L34965 or L34975 (Invitrogen) according to the manufacturer's instructions. The following antibodies were used for T cell staining: anti-Vb13.1:PE (IM2292, BD Biosciences), anti-IFNγ:PeCy7 (502527, Biolegend), and tetramer (A2 / NY-ESO-1 157-165 TCR transduction efficiency was assessed using a ELISA (in-house produced) stain.

[0223] Flow cytometry analysis to assess the expression of immunoregulatory factors by gene-transduced T cells One-week-old transduced OT-1 T cells were incubated with 50 μl of Live / Dead Fixable aqua dead in PBS for 30 minutes at room temperature, washed, and then incubated again with 50 μl of FCR blocking reagent (clone 2.4G2, BD Pharmingen) for 30 minutes at 4°C. The cells were washed again and incubated with surface marker antibodies against CD3 (145-2C11, Invitrogen), CD8α (53-6.7, BioLegend), and CD45.1 (A20, BioLegend) for an additional 30 minutes at 4°C. The following antibodies were used for intracellular staining: anti-human hIgG4-Fc (Abcam, clone HP6025) to detect PD-1.IgG4 decoy, and anti-mouse IL-33 (eBioscience, clone 396118). After surface staining, transfected OT-1 cells were washed twice and fixed / permeabilized using FoxP3 transcription factor staining buffer set (Invitrogen) according to the manufacturer's recommendations. For detection of each molecule, cells were further washed and incubated with the respective antibody for 30 minutes at room temperature. Cells were washed, resuspended in PBS supplemented with 2% BSA and 0.01% azide (FACS buffer), acquired on a BD flow cytometer, LSRII cytometer, and analyzed using FlowJo software v11 (Tree Star).

[0224] Flow cytometry analysis to assess the production of intracellular cytokines or PD1-Fc decoy or CD40L decoy To assess intracellular cytokine production or PD1 decoy or CD40L decoy production by FACS, 50,000 live T cells per well in a round-bottom 96-well plate were activated for 7 hours with a combination of plate-coated anti-CD3 (5 μg / ml) and soluble anti-CD28 (2 μg / ml) antibodies (or anti-CD3 / anti-CD28 beads). To prevent protein secretion, Golgi stop (BD Biosciences) was added to the wells at a dilution of 1:400 1.5 hours after the start of the assay. T cells were fixed and permeabilized using a standard fixation / permeabilization kit (BD Biosciences) according to the manufacturer's instructions, and then assessed for their transduction efficiency or ability to produce molecules of interest. Anti-Fc antibodies were used to detect the decoys. For cytokines of interest (IL-2, IFN-γ), specific antibodies were used.

[0225] ELISA to assess secretion of immunomodulatory factors by transfected T cells One week after activation and transduction, 10 transfected OT-1 T cells were seeded in 1 ml of serum-free RPMI medium for 72 h. SNs were then harvested and tested for each molecule. For PD1.IgG4, a modified ELISA was used with the following setup: plates were coated with anti-mouse PD1 Ab (R&D, AF1021, 2 μg / ml), the plates were incubated with SNs, and PD1.IgG4 was detected with anti-hIgG4-HRP Ab (Abcam, ab99817, diluted 1:1000).

[0226] IL-2 V For the IL-2 antibody, a modified ELISA was used with the following setup: plates were coated with anti-human IL-2 Ab (R&D, AF-202-NA, 3 μg / ml), and the plates were incubated with the supernatants to measure IL-2. VThe fusion molecules TIM-3.IgG4 or IL-2 were detected with biotinylated polyclonal anti-human IL-2 Ab (Invitrogen, 13-7028-81, diluted 1:500) followed by streptavidin-HRP (BioLegend, diluted 1:1000). V SN from OT-1 T cells transduced to express either LIGHT or TNFSF14 was used as a negative control. Three commercially available ELISA kits were used to detect LIGHT, IL-33, and CD40L: the Mouse LIGHT / TNFSF14 DuoSet ELISA (DY1794-05) developed by R&D, the LEGEND MAX™ Mouse IL-33 ELISA Kit (436407) developed by BioLegend, and the Mouse CD40Ligand / TNFSF5 ELISA Kit (NBP1-92662) developed by Novus Biological.

[0227] Adoptive cell transfer in tumor-bearing mice B16-OVA tumor cells were harvested with 0.05% trypsin, washed, and resuspended in PBS for injection. 5 Tumor cells were injected subcutaneously into the right flank of 7-week-old C57BL / 6 mice. On day 11 (mean tumor volume 100-200 mm), 3 ), mice were regrouped to have comparable mean tumor volumes between experimental arms (n ≥ 5 mice / group). On days 12 and 15, 5 × 10 6 Mice were treated with intravenous transfer of transgenic CD44+ CD62L+ TCF1+ OT-1 T cells or control non-transgenic OT-1. Mice were monitored three times weekly, and tumor length (L; maximum longitudinal measurement) and width (W; maximum transverse measurement) were measured with calipers by an independent blinded investigator. Tumor volume (V) was calculated using the following formula: V = (L × W) 2 ) / 2. The mean tumor volume per group was plotted ±SD. 3 Mice were sacrificed when they reached 0.05°C, or if they became distressed or moribund according to the regulations.

[0228] ELISA to assess secretion of immunomodulatory factors by transfected T cells 1 x 10 cells 1 week after transduction 6 Transfected OT-1 T cells were seeded in 1 ml of serum-free RPMI medium in a 24-well plate for 72 hours. Then, SNs were collected and tested for each molecule by ELISA. PD1.IgG4 homemade ELISA: Coating Ab: anti-mouse PD-1 (R&D, AF1021, 2 μg / ml), Detection Ab: anti-hIgG4-HRP (Abcam, ab99817, diluted 1:1000). IL-2 V Homemade ELISA: Coating Ab: anti-human IL-2 (R&D, AF-202-NA, 3 μg / ml), secondary Ab: biotinylated polyclonal anti-human IL-2 (Invitrogen, 13-7028-81, diluted 1:500), streptavidin-HRP (BioLegend, diluted 1:1000). Fusion molecules: TIM-3, IgG4, or IL-2. V SN from OT-1 T cells transduced to express either IL-33 or IL-34 was used as a negative control. IL-33 was detected using the commercially available LEGEND MAX™ Mouse IL-33 ELISA Kit (436407) developed by BioLegend.

[0229] ADCC measured by chromium release assay Thaw autologous PBMCs, 1 x 10 6 The cells were added to a 6-well plate at a concentration of 0.5 × 10 cells / ml in the presence of 10 ng / ml GM-CSF at 3 ml per well. 6 EGFR +T cells were loaded with 50 μCi of chromium-51, resuspended, and placed in a 37°C water bath for approximately 1 hour. The T cells were then washed twice, suspended at a concentration of 400,000 cells / ml, and 50 μl (=2,000 cells) of T cells were transferred per well. Cetuximab (anti-EGFR antibody) was prepared at 300 μg / ml or 30 μg / ml. 50 μl of cetuximab was added to the T cells and incubated at 37°C for 30 minutes. PBMC (effector cells) were cultured at 1.2 × 10 in RPMI, 10% FBS (Gibco), and 1% penicillin / streptomycin. 6 cells / ml. A 1 / 3 dilution of effector PBMCs (1.2 × 10 6 , 0.4×10 6 , 1.33×10 6 , and 0.42 × 10 6 PBMC / ml) and 50 μl of tEGFR + The anti-EGFR antibody was added to wells containing T cells. Different effector:target cell ratios (30:1, 10:1, 3:1, 1:1, in triplicate) were set up. As a positive control, 1x TritonX was added to the T cells (=maximum chromium release). All negative controls (medium only, T cells without PBMCs, T cells + PBMCs but no antibody, etc.) were set up. The plates were spun at 1500 rpm and placed at 37°C for 4-5 hours. 50 μl of the supernatant was transferred to a lumaplate well and allowed to dry overnight. The next day, chromium levels were assessed using a topCounter.

[0230] Co-culture assays and ELISAs to measure cytokine production TCR-T cells co-modified to express PD1 decoy and truncated EGFR (and all control T cell conditions) were added to 1 × 10 6 TCR + T cells / ml, and tumor cells were added at a concentration of 1 × 10 6The T cells and tumor cells were prepared at 100 μl / ml. 100 μl of each were combined in a 96-well round-bottom plate. The plate was spun at 1500 rpm for 1 minute and incubated at 37°C for 48–72 hours. IFN-γ levels in the supernatant were assessed by ELISA (Invitrogen) according to the manufacturer's recommendations.

[0231] Co-culture assay and ELISA to assess the secretion of PD1 and CD40L decoys 1×10 6 Primary UTD-cotransduced T cells (engineered to express NY TCR and secrete PD1 decoy and tEGFR) were plated at 1 x 10 per well in a 96-well round-bottom plate. 6 The cells were co-cultured in duplicate with target cells in a final volume of 200 μL of complete RPMI medium. The plates were spun at 1500 rpm for 1 minute and incubated at 37°C. After 24 hours, the co-culture supernatants were collected and tested for the presence of PD1-Fc fusion decoy molecules by capturing them with plate-bound anti-PD1 antibody or plate-bound human PD-L1 protein. Bound PD1-Fc decoy molecules were detected with anti-IgG-Fc Ab. The same conditions were used to assess CD40L decoy secreted into the supernatant, except that a commercially available ELISA kit (Invitrogen) was used.

[0232] Immune subset depletion, checkpoint blockade and FTY720 treatment Specific cell subsets were depleted by administering 250 μg / dose of depleting antibodies i.p. every 3 days starting 1 day before treatment: CD4 T cells with α-mouse CD4 (clone GK1.5, BioXcell), NK cells with α-mouse NK1.1 (clone PK136, BioXcell), and neutrophils with α-mouse Ly6G (clone 1A8, BioXcell). For checkpoint blockade, mice received 250 μg / dose of α-mouse PD-L1 (BioXcell, 10F.962) and α-mouse TIM-3 (BioXcell, RMT3-23) i.p. injections every 3 days. To block lymphocyte leakage from secondary lymphoid organs, a stock solution of FTY720 (obtained from SIGMA) (10 mg / ml in DMSO) was prepared and diluted to 1 mg / ml with water prior to administration. Finally, 100 μg of the drug was administered i.p. every 3 days, starting 2 days before treatment. Both immune cell depletion and sequestration (FTY720) were confirmed by flow cytometry of PBMCs.

[0233] Preparation of tumor single-cell suspensions, antibodies for flow cytometry, and ex vivo restimulation for cytokine production Five and 12 days after the initial adoptive cell transfer, tumors were excised and dissociated into single-cell suspensions using a commercially available mouse tumor dissociation kit (Miltenyi Biotec, 130-096-730) by combining mechanical dissociation and enzymatic degradation of the extracellular matrix. Following single-cell suspension, 2.5 × 10 6Viable cells were seeded into a 96-well plate and incubated with 50 μl of Live / Dead Fixable aqua dead in PBS for 30 minutes at room temperature. Fc receptors were then blocked by incubation with 50 μl of purified anti-CD16 / CD32 mAb (clone 2.4G2, BD Pharmingen) for 30 minutes at 4°C. Cells were then stained with fluorochrome-conjugated mAbs of interest in 50 μl of FACS buffer for 30 minutes at 4°C. Cells were then washed twice and fixed / permeabilized with FoxP3 Transcription Factor Staining Buffer Set (Invitrogen) for intracellular staining. Analysis of stained cells was performed using an LSRII cytometer and FlowJo software.

[0234] The following antibodies were used: CD45.1 (clone A20, BioLegend), CD3 (clone 145-2C11, Invitrogen), CD4 (clone GK1.5, BioLegend), CD8 (clone 53.6.7, BioLegend), FOXP3 (clone FJK-16S, Invitrogen), NK1.1 (clone PK136, BioLegend), CD44 (clone IM7, BioLegend), PD-1 (clone 29F.1A12, BioLegend), LY6C (clone HK1.4, BioLegend), Granzyme C (clone SFC1D8, BioLegend), TCF1 (clone C63D9, Cell Signaling Technology), anti-rabbit IgG (H+L), F(ab')2 fragment AF488 or PE conjugate (Cell Signaling Technology). Proteins included: granzyme B (clone GB11, Novul Biologicals), CD69 (clone H1.2F3, BioLegend), TIM-3 (clone RMT3-23, BioLegend), CD137 / 4-1BB (clone 17B5, Invitrogen), KLRG1 (clone 2F1 / KLRG1, BioLegend), KI67 (clone 16A8, BioLegend), IFNg (clone XM61.L, Invitrogen), TNFa (clone MP6-XT22, BioLegend), TOX (clone TXRX10, Invitrogen), and CD45 (clone 30-F11, BioLegend).

[0235] Fluorescence minus one (FMO) controls were stained in parallel using an antibody panel in which one antibody was sequentially omitted. FMO staining was performed as a control for the following antibodies: TCF1, Ki67, 4-1BB, Granzyme B, TNFα, IFNg, PD-1, and TIM-3. An isotype control was used for Granzyme C staining (clone HTK888, BioLegend). Absolute cell counts were obtained during acquisition on the flow cytometer using Precision Count Beads™ (BioLegend).

[0236] For detection of cytokine production, tumor single cell suspension (2.5 × 10 6 Viable cells (number of cells) were restimulated in vitro in 24-well plates with 1 μg / ml well-coated anti-mouse CD3 (clone 17A2, Invitrogen) and 2 μg / ml soluble anti-mouse CD28 (clone 37.51, Invitrogen) in the presence of Brefeldin A (5 μg / ml) for 4 hours. Cells were surface stained before fixation and permeabilization, followed by intracellular staining, as described above.

[0237] Immunofluorescence labeling and microscopy For immunohistochemical analysis, tumor tissues were isolated, fixed overnight in 1% PFA in PBS, infiltrated overnight in 30% sucrose, embedded in OCT compound, and frozen. Cryostat sections were collected onto Superfrost Plus slides (Fisher Scientific), air-dried, and preincubated with a blocking solution containing BSA, normal mouse serum, normal donkey serum (Sigma), and 0.1% Triton. They were then labeled overnight at 4°C with primary antibodies diluted in 0.1% Triton-containing PBS. After washing with 0.1% Triton-containing PBS, secondary reagents were applied at room temperature for 45 minutes. Finally, after further washing with PBS and 0.1% Triton, nuclei were stained with DAPI (Sigma), followed by PBS washes and mounting in DABCO (in-house). Images were acquired with a Zeiss AxioImager Z1 microscope and an AxioCam MRC5 camera. Images were processed using Fiji (NIH) or Adobe Photoshop. Exposure and image processing were identical for groups of mice to allow direct comparison.

[0238] Antibodies (ab) used for CD8 / CD45.1 / CD105 labeling: 1°ab: rat-a-mouse CD8a (clone 53-6.7), rabbit-a-mouse CD105 (clone MJ7 / 18), mouse-a-mouse CD45.1 biotin (clone A20.1). 2°reagents: donkey-a-rat Alexa 488 (Invitrogen, #A21208), donkey-a-rabbit Cy3 (Jackson ImmunoResearch, #711-165-152), streptavidin APC (Biolegend, #405207).

[0239] Antibodies (ab) used for CD8-CD45.1-TCF1 labeling were: 1°ab: rat-a-mouse CD8a (53-6.7), rabbit-a-mouse TCF-1 (Cellsignalling, clone C63D9, #2203), mouse-a-mouse CD45.1 biotin (clone A20.1). 2°ab: donkey-a-rat Alexa 488 (Invitrogen, #A21208), donkey-a-rabbit Cy3 (Jackson ImmunoResearch, #711-165-152), streptavidin APC (Biolegend, #405207).

[0240] Single-cell RNA sequencing analysis To select high-quality CD8 TIL transcriptomes, we filtered the aggregated UMI count matrix generated by CellRanger. First, we retained cells with 500–5,000 detected genes, UMI counts between 2,000–30,000, mitochondrial content less than 5%, and ribosomal protein content less than 50%. Next, we filtered CD8 T cells for those expressing Cd2, Cd8a, and CD8b1 (≥1 UMI) but not Cd4 (0 UMI). We further removed cells expressing Cd14, Csf1r, Cd19, Spi1, Foxp3, H2-Aa, and H2-Ab1, resulting in 1,788 high-quality CD8 TIL transcriptomes.

[0241] For dimensionality reduction, we first identified highly variable genes (HVGs) using the Seurat 3.1.1 vst method with default parameters (Stuart et al., Cell, vol. 177, issue 7, p1888-1902.e21, June 13, 2019). Next, mitochondrial, ribosomal protein-coding, and cell cycle genes (those with Gene Ontology term GO:0007049) were removed from the set of HVGs, and the remaining HVGs (1649) were scaled to have mean = 0 and variance = 1. The standardized HVGs were used for the first step of dimensionality reduction using PCA and the second set using UMAP (implemented in Seurat v3.1.1) on the first 10 principal components (with other parameters by default). Clustering was performed using the shared nearest neighbor method in Seurat with default parameters: FindNeighbors and FindClusters with a resolution of 0.2. For supervised classification of CD8 TIL status, TILPRED (https: / / github.com / carmonalab / TILPRED; Santiago J. Carmona, et al., OncoImmunology, 9:1 (2020)) was used with default parameters. Differentially expressed genes between clusters were identified using FindAllMarkers and MAST v1.10 (Finak, G., et al., Genome Biol 16, 278 (2015)) with parameters min.pct=0.25 and logfc.threshold=0.25. To compare the Gzmc cluster with the traditional exhausted cluster, the original "exhausted" cluster was sub-clustered by increasing the "resolution" parameter to 0.3.Differential expression analysis between the refined exhaustion cluster and the Gzmc cluster was assessed using FindAllMarkers and MAST v1.10 with parameters min.pct = 0.1 and logfc.threshold = 0.25. Gene set enrichment analysis of these clusters against the TOX-KO signature (Scott, AC, et al. Nature 571, 270-274 (2019)) was calculated using the GSEA function from the clusterProfiler package v3.12 (Guangchuang Yu, et al., OMICS: A Journal of Integrative Biology. May 2012, 284-287) with default parameters, using the top 200 differentially expressed cluster genes with p-values ​​< 0.01, ordered by decreasing fold change.

[0242] statistical analysis Normal distribution of data was assessed using the Shapiro-Will normality test. To compare two groups, we used a two-tailed Student's t-test (if normal distribution and homogeneous variances were present) or a Welch-corrected t-test (if normal distribution but unequal variances were present). If data were not normally distributed, we used the nonparametric Mann-Whitney test. A similar strategy was followed when comparing three or more groups. If data were not normally distributed, we used the Kruskal-Wallis test. If data were normally distributed and homogeneous variances were present, we used one-way ANOVA. If data were normally distributed but unequal variances were present, we used the Brown-Forsythe and Welch ANOVA tests. Corrections for multiple comparisons were performed using the Dunn's test (for the Kruskal-Wallis test), the Dunnett's test (for the one-way ANOVA), and the Tukey's test (for the Brown-Forsythe test). Survival analysis was performed using the log-rank Mantel-Cox model. The Pearson correlation test was used to calculate the correlation between the number of TCF1+ OT-1 intratumoral CD8 T cells and the total number of tumor-infiltrating OT-1 cells. All statistical analyses were performed using GraphPad Prism 8.0; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0243] Statistical analysis of tumor control was performed using the percent change in tumor volume relative to day 17 after tumor inoculation. The best response (smallest tumor volume) observed in each animal at least 12 days after the first ACT was used for calculation. Objective response rates and clinical benefit rates by treatment group were calculated based on the total number of mice per group. (1) Objective response included complete response (CR; 100% reduction in tumor volume) and partial response (PR; ≤-30% tumor change), and (2) Clinical benefit included CR, PR, and stable disease (-30% < tumor change ≤ +20%).

[0244] Predicted probabilities for the variables "objective response" and "clinical benefit" were calculated using exact logistic regression. The values ​​of tumor transformation as a continuous variable were further analyzed using linear regression. A p-value of less than 0.05 was considered statistically significant.

[0245] Example 2 PD1 decoy molecule was injected into IL-2 V The sequences of PD1.IgG4, LIGHT, or interleukin-33 (IL-33) were cloned into retroviral constructs; each construct was codon-optimized to encode only two molecules separated by the self-cleaving peptide T2A. Therefore, at least four different constructs were available: i) PD1.IgG4_T2A_IL-2 V ,PD1 decoy and IL-2 V ii) PD1.IgG4_T2A_LIGHT, expressing PD1 decoy and LIGHT; iii) PD1.IgG4_T2A_IL-33, expressing PD1 decoy and IL-33; and iv) PD1.IgG4_T2A_CD40L, expressing PD1 decoy and CD40L.

[0246] Figure 1 shows the efficiency of transfection and transduction of OT-1 T cells with the designed constructs. For each composition tested, the cells exhibited high transduction efficiency and high secretion levels for each of the expressed and secreted proteins.

[0247] Using specific ELISA, genetically modified OT-1 T cells were shown to inhibit the immune regulatory factors PD1, IgG4, T2A, and IL-2 V It was also confirmed that these cells were able to secrete specific combinations of PD1.IgG4_T2A_LIGHT, PD1.IgG4_T2A_IL-33, and PD1.IgG4_T2A_CD40L.

[0248] These results demonstrate that T cells can be successfully genetically engineered to express two distinct exogenous secreted proteins, and that these cells display evolved properties by continuing to express and secrete large amounts of each exogenous protein.

[0249] Example 3 As shown in Figure 2A and Figure 2B, PD1.IgG4, LIGHT, and IL-2 V ACT with OT-1 T cells secreting PD1.IgG4 significantly improved the control of established large B16-OVA tumors. This combination strategy induced tumor regression after ACT and improved overall survival compared with responses to untransduced OT-1 T cells. As shown in Figure 2C and Figure 2D, ACT with OT-1 T cells secreting PD1.IgG4, LIGHT, and IL-33 also significantly improved the control of established large B16-OVA tumors. This combination strategy demonstrated superior antitumor activity compared with the combination of PD1.IgG4 and LIGHT. The combination of PD1.IgG4, LIGHT, and IL-33 improved overall survival compared with responses to untransduced OT-1 T cells.

[0250] PD1.IgG4, IL-2 V Adoptive transfer of PD1.IgG4, IL-2, and IL-33-secreting OT-1 T cells nearly doubled overall survival compared with administration of non-transduced OT-1 T cells, and extended overall survival by more than 10 days compared with other combination strategies (Figure 2F). As shown in Figure 2G and Figure 2H, the PD1.IgG4, IL-2, and IL-33-secreting OT-1 T cells significantly increased overall survival compared with non-transduced OT-1 T cells. V ACT with OT-1 T cells secreting CD40L significantly improved the control of large, established B16-OVA tumors. This combined strategy induced tumor regression after ACT and improved overall survival compared with responses to untransduced OT-1 T cells.

[0251] In summary, T cells can be genetically engineered to secrete combinations of immunomodulatory factors to control advanced tumors. The above examples also highlight the therapeutic feasibility of combining populations of T cells with the same antigen specificity but different secretory properties to obtain higher-order combinations of immunomodulatory factors. A key advantage of this approach is the enhanced safety, as the molecules are primarily secreted in the tumor microenvironment (i.e., not administered systemically).

[0252] Example 4 Adoptive immunotherapy offers the opportunity to reprogram T cells and the tumor microenvironment. As demonstrated in this example, orthogonal engineering of adoptively transferred T cells containing an IL-2Rβγ-binding IL-2 variant, PD1 decoy, and IL-33 enhances the ability of transferred CD8 T cells to differentiate. + cells and endogenous CD8 + This reprogramming of both T cells resulted in cell-autonomous T cell expansion, engraftment, and tumor control in immune-competent hosts. Tumor-infiltrating lymphocytes (TILs) adopted a novel effector state characterized by TOX suppression, granzyme C abundance, and effector molecules, survival, and progenitor markers, distinct from canonical TOX-driven exhaustion. Dynamically driven by the interaction of IL-2 variants with IL-33, TILs in this state uncoupled persistence from TOX-driven exhaustion and successfully controlled tumors. Thus, rational T cell engineering without host lymphodepletion not only enables optimal reprogramming of adoptively transferred T cells but also mobilizes endogenous immunity into a novel state compatible with tumor control.

[0253] It was hypothesized that T cells possess unique properties that allow them to autonomously reach the required proliferative state in the absence of lymphocyte depletion, as well as the desired functional state corresponding to moderately immunogenic tumor engraftment and tumor rejection. In this disclosure, we aimed to modify T cells through orthogonal combinatorial engineering, i.e., by introducing genes whose products result in desired perturbation (reprogramming T cells and enabling them to reprogram adaptive and innate immunity in the TME). The PD-1 / PD-L1 inhibitory pathway was targeted by secreted PD-1 decoy (PD1d), a fusion molecule containing the ectodomain of mouse PD-1 bound to the Fc region of human IgG4. To support T cell proliferation, a human IL-2 variant (IL-2Rα chain, CD25, which does not engage with the high-affinity IL-2Rα chain) was introduced. V ) was used (T. Carmenate et al., Journal of Immunology 190, 6230-6238 (2013); G. Rojas et al., Scientific Reports 9, 800 (2019)). Compared to wild-type IL-2, the key advantages of this molecule are its low toxicity and reduced blockade by regulatory T cells (Tregs). Also, unlike wild-type IL-2, which promotes terminal effector differentiation, IL-2 V CD8 is a favorable characteristic of ACT + It was hypothesized that IL-33 promotes T cell stemness (JG Crompton, et al. Immunol Rev 257, 264-276 (2014)). Finally, IL-33 was used to generate tumor-favorable inflammatory signals. Two retroviral vectors were constructed; one carrying soluble PD1d and IL-2. V (PD1d / 2 V One encodes soluble PD1d and murine IL-33 (PD1d / 33 module), and the other encodes soluble PD1d and murine IL-33 (PD1d / 33 module). +T cells were transduced separately with retroviruses carrying one or the other module, and then pooled in a 1:1 ratio to express the triple combination (PD1d / 2 V We created an ACT cocktail with OT1 T cells (T1 / 33). OT1 T cells were also used to treat advanced B16-OVA melanoma tumors in immunocompetent recipient mice. As shown below, this example demonstrates how these interventions can enhance the CD8 + What might lead to a T cell state and the desired CD8 T cell status to achieve T cell engraftment and tumor regression in the absence of lymphodepletion preconditioning or exogenous cytokine support? + We will consider what the state of T cells is.

[0254] Orthogonal T cell engineering improves the efficacy of ACT in a cell-autonomous manner As the first engineering module, we first evaluated the antitumor potential of PD-1 decoy. This molecule was fully expressed and secreted by genetically modified OT1 cells and bound to plate-immobilized PD-L1 in vitro (which was competitively eliminated by saturating PD-L1 neutralizing antibodies). ACT using PD1d-transfected OT1 cells showed significant antitumor activity in vivo in lymphocyte-depleted (irradiated) mice. Next, we transfected OT1 cells with PD1d / 2. V The PD1d / 33 module or the PD1d / 33 module were efficiently transduced to express PD1d and IL-2, respectively. V We tested whether PD1d and IL-33 could be secreted simultaneously. PD1d expression was assessed, resulting in transduction efficiencies of over 75%, and simultaneous secretion of all molecules was confirmed by ELISA.

[0255] Next, orthogonal engineered ACT was performed without lymphocyte depletion preconditioning (Figure 3A). In the absence of lymphocyte depletion or exogenous cytokines, palpable (100 m 3 ) administered to tumor-bearing mice, approximately 70% of T CM and about 30% T EM5 × 10 cells with the (effector memory) phenotype 6 Two injections of untransduced OT1 cells failed to control tumor growth (Figure 3B). V The same dose of OT1 cells transfected with either PD1d / 2 or PD2d / 2 had a slight (but not significant) effect on tumor growth (Figure 3B; Tables 2 and 3), while the double gene-modified PD1d / 2 V -OT1 cells did not prove more effective under the same conditions. Systemic administration of anti-(α)PD-L1 antibodies with non-transduced OT1 cells produced results comparable to those of PD1d-OT1 cells. Similarly, IL-33-transduced OT1 cells were minimally effective, as were PD1d / 33-OT1 cells. Surprisingly, PD1d / 2 V / 33-OT1 cells (i.e., 1:1 mixed PD1d / 2 V Treatment with PD1d / 2 cells and PD1d / 33 cells was statistically significantly superior to any other treatment (Figure 3B; Tables 2 and 3). The objective response rate (ORR) of this treatment approach was 85.7%, with a predicted probability of occurrence of 83.3% (Tables 2 and 3), compared with 0-9% for all other cases. Finally, PD1d / 2 cells in the absence of lymphodepletion and exogenous cytokine support were statistically significantly superior to any other treatment (Figure 3B; Tables 2 and 3). V Confirming the efficacy of / 33-OT1 cells, early treatment of mice (starting on day 6) resulted in complete tumor eradication and cure.

[0256] Orthogonal engineering in immunocompetent hosts demonstrates the efficacy of adoptively transferred CD8 + This results in cell-autonomous proliferation of tumor cells and the contribution of endogenous antitumor immunity. Next, double gene-modified PD1d / 2 V Or, the antitumor effect of PD1d / 33 was enhanced by triple gene-modified PD1d / 2 V At baseline (day 12), treatment-naïve B16-OVA tumors expressed CD44 + CD8 exhibits antigen-experienced cell phenotype + showed moderate spontaneous infiltration of T cells. Adoptively transferred CD8 +T cells began to accumulate in tumors within 4 days, and by day 5 (day 17) after ACT, tumors in mice treated with triple-modified cells already had significantly higher levels of CD8 + T cell engraftment was demonstrated. One week later (day 24), PD1d / 2 V Tumors in mice treated with / 33-OT1 cells were significantly higher in CD8+ cells than in mice treated with the double-transformed cells. + The double-transduced cells produced significantly more TILs, as well as CD8+ T cells, than mice treated with untransduced OT1 cells. + The number of TILs was significantly higher in the OT1 (CD45.1) group (Figure 3C). + ) Focusing on TIL, tumors 2 weeks after ACT showed PD1d / 2 V Significant proliferation of PD1d / 2 cells was observed (Fig. 3D). V PD1d / 33-OT1 and PD1d / 33-OT1 cells showed moderate and minimal engraftment, respectively. Thus, the efficacy of the triple gene-modified combination ACT was associated with the unique intratumoral expansion of adoptively transferred T cells and tumor regression.

[0257] Given that ACT was performed in fully immunocompetent hosts, we investigated whether endogenous immune effector cells contributed to its efficacy. Surprisingly, the total CD8 + Approximately 50% or more of TILs are endogenous (CD45.1 neg CD45.2 + ) (Figure 3E). V Although some proliferation of endogenous TILs was observed in tumors treated with -OT1 or PD1d / 33-OT1 cells, these were particularly prominent in tumors treated with the triple gene-modified cells.

[0258] CD8 +The presence of a pool of stem-like cells expressing the TCF1 transcription factor within the T cell compartment has previously been associated with their ability to mobilize immunity against tumors (and chronic viral infections) upon PD-1 blockade, and the use of such progenitor cells in ACT may improve efficacy. However, conditions in the TME lack TCF1 expression. + CD8 + Notably, TCF1 does not promote the presence or persistence of TILs. + OT1 + Significant proliferation of TILs was observed with triple gene-modified PD1d / 2 V / 33 or double gene-modified PD1d / 2 V This was particularly observed after transfection with OT1 cells (Fig. 3F and Fig. 3G), and IL-2 V These two approaches, which share the same trait, were the only ones shown to expand the stem-like compartment. Importantly, the stem-like TCF1 + CD8 + The expansion of PD1d / 2 TILs also extended to endogenous TILs (Figures 3F and 3G). V After / 33-ACT, 10-20% of OT1 and endogenous CD8 + 30–50% of TILs express TCF1, and TCF1 + A strong direct correlation was observed between the presence of OT1 cells and the total number of OT1 TILs. V Genetic modification approaches including these have achieved conditions that promote stemness and, consequently, the persistence of transferred T cells as well as endogenous T cells.

[0259] Importantly, effective tumor control by transfected OT1 cells was due to the TCF1 + CD8 + Not only increased presence of TILs but also a large number of TCF1 neg Effector-like CD8 + TIL(PD1d / 2 V OT1 TILs and endogenous CD8 TILs were also associated with cytotoxicity (a condition that was only met after 33-ACT) (Figure 3G). Indeed, in these tumors, 80–90% of OT1 TILs and endogenous CD8 TILs were + 50-70% of TILs are TCF1 negImportantly, the high frequency of TCF1 + CD8 + TILs are PD1d / 2 V -ACT, but these tumors showed TCF1 neg CD8 + Much less TIL and IL-2 V TCF1 + CD8 + However, in the absence of IL-33 co-expression, these cells were unable to express TCF1 neg These results confirm that Tcf1 suppression is associated with effector differentiation. In contrast, PD1d / 33-ACT cells exhibited low TCF1 + CD8 + and high TCF1 neg CD8 + was associated with poor TIL frequency and overall poor TIL proliferation (Figures 3F and 3G).

[0260] To understand the contribution of endogenous T cells in tumor control after ACT, CD8 knockout tumor-bearing mice were treated with PD1d / 2 T cells under the same conditions. V Treatment with 1 / 33-OT1 cells. Endogenous CD8 + We observed that the antitumor effect of ACT was lost in the absence of T cells (Figure 3H). + T cell engagement was crucial for effective tumor control. Surprisingly, tumor control did not depend on the recruitment of endogenous T cells from lymph nodes, as evidenced by coadministration of FTY720, a drug that impairs lymphocyte egress from lymph nodes (Figure 3H). Thus, tumor control by triple gene-modified ACT required the recruitment of systemic CD8 T cells. + Rather than recruiting T cells to the tumor, in situ expansion of pre-existing endogenous TILs was utilized and required.

[0261] Next, we evaluated the interaction of ACT with tumor Tregs. V secretion is more pronounced in CD8 than in Tregs + preferentially expand CD8+ Consistent with maximal cell proliferation, the CD8 / Treg ratio was PD1d / 2 V The highest level was observed after / 33-ACT (Figure 3I). + TILs are CD8 + Less overall proliferation than TILs, PD1d / 2 V / 33-CD4 before ACT + Antibody-mediated depletion of T cells did not impair tumor control and mouse survival, but rather significantly improved them (Figure 3J).

[0262] Finally, we investigated whether triple gene-modified ACT mobilizes innate immunity. V After ACT, especially PD1d / 2 V With / 33-ACT, tumor NK cells were increased but not activated, and they were not necessary. Importantly, however, tumor control was co-dependent on neutrophil recruitment and activation (Figure 3K). Thus, orthogonal engineering achieves tumor regression in immunocompetent hosts through the recruitment of both adaptive and innate immunity. While limited examples of ACT-mediated tumor control exist in lymphocyte-depleted mice bearing hematologic tumors, this is the first demonstration of successful ACT in an advanced, poorly immunogenic solid tumor in the absence of adjuvant therapy (SK Vodnala et al., Science 363, eaau0135 (2019)).

[0263] Intratumoral GzmC induced by orthogonal engineering + TCF1 neg Effector CD8 + A novel subset of T cells persists independent of TOX To further understand the molecular status of TILs associated with tumor control by triple-modified ACT and the influence of individual double-modified modules, we analyzed TILs by single-cell (sc) RNA-seq (Figure 4A). +Unsupervised clustering analysis of TILs revealed five distinct transcriptome states (clusters C1–C5) (Figure 4B). To interpret these results, we used the machine learning tool TILPRED, which assigns cells to previously identified molecular TIL states in untreated mouse tumors (SJ Carmona, et al. OncoImmunology 9, 1737369 (2020)). TILs from tumors treated with untransfected OT1 cells exhibited similar characteristics to TILs from untreated tumors, with a predominance of progenitor and terminal exhausted cell pools (C4), and fewer circulating (C3), effector memory (C2), and naive cells (C1) (Figure 4B and Figure 4C). Thus, without genetic modification and host conditioning, ACT did not affect TIL state. PD1d / 2 V After -ACT, TILs were treated with TCF1 + Consistent with the significant proliferation of cells, TILs showed a predominant naive-like pool (C1), with some addition of effector memory (C2), circulating (C3), and progenitor and terminal exhausted cells (C4). Conversely, TILs after PD1 / 33-ACT showed a predominant effector memory state (C2) and contained some circulating cells (C3). Thus, IL-2 VLocal expression of IL-14 alone or IL-33 alone redirected TILs toward a naive-like state and an effector memory state, respectively. Furthermore, the combination of these two cytokines resulted in an entirely novel state (C5) associated with tumor control, distinct from any state supported by each cytokine individually. C5 was observed exclusively in triple-altered ACT TILs during the response phase and not in any other tumor condition (Figure 4B). The novelty of this state was further supported by ProjecTILs, a tool that projects (sc)RNAseq data onto a reference TIL atlas; this revealed that while cells in clusters C1–C4 matched previously described reference states, C5 emerged as a novel state never before described, characterized by the upregulation of a unique effector-like transcriptional program (Figure 4D).

[0264] C5, the majority of TILs identified by both TILPRED and ProjecTILs, was composed of "terminal-exhausted" CD8 TILs, just like TILs in cluster C4. +They were broadly classified as terminally exhausted TILs. Indeed, cells in both clusters shared relatively high expression of co-inhibitory receptor genes, such as Pdcd1, Lag3, Tigit, Havcr2 / TIM3, and Entpd1 / CD39, as well as the costimulatory receptor and activation marker Tnfsfr9 / 4-1BB (Figure 4E). Given their separation by UMAP and the upregulation of the effector-like transcriptional program in ProjecTILs, we used differential expression analysis to identify distinct molecular features of C5 and C4 terminally exhausted TILs. Compared with canonical C4 terminally exhausted TILs, C5 TILs displayed a unique effector signature, accompanied by a marked downregulation of the exhaustion-related transcription factors Tox, bhlhe40, and Batf, as well as multiple inhibitory receptors. Notably, they also downregulated Cx3cr1, a characteristic marker of transient effector-like exhausted cells (Figure 4C, bottom; Table 3). Furthermore, C5 TILs express multiple granzymes, most notably Gzmc, which constitutes a C5-specific marker (Figure 4E), the anti-apoptotic gene Bcl2, and precursor CD8 + Ly6c2 (CX3CR1), a T cell-associated marker + C5 cells significantly upregulated effector cell markers, including α-tocopherol (α-tocopherol), which are absent in transient effector-like exhausted cells. Consistently, C5 cells were enriched for the signature of Tox knockout CD8 TILs compared with C4 (Figure 4F). Correspondingly, OT1 (approximately 80%) and endogenous (approximately 70%) CD8 TILs during the responder phase were significantly upregulated. + FACS confirmed that the majority of TILs expressed granzyme C (Figure 4G). + CD8 + No GzmC cells were detected in the spleen, indicating that a local cue in the reprogrammed TME specifically drove T cells to this state in tumors. Importantly, only a small frequency of GzmC + CD8 + T cells were endogenous CD8 +This was found not only in TILs or OT1 cells after in vitro expansion, but also in the remaining experimental groups (Fig. 4G); V / 33-ACT leads to a high degree of intratumoral reprogramming of TILs, including endogenous TILs, resulting in CD8 + Generated a novel phenotype of T cell effector, apparently involving IL-2 V These results indicate that local interaction of IL-33 with IL-1 is required.

[0265] TOX neg / low GzmC + TCF1 neg Effector CD8 + TILs are multipotent cells that exhibit insignificant expression of co-inhibitory receptors PD1d / 2 V GzmC is a major contributor to tumor rejection after / 33-ACT + Effector CD8 + The status of TILs was further characterized. A gating strategy was used to identify TCF1 in the OT1 and endogenous compartments. neg Effector CD8 + The cells were identified as PD1 / 2. V The majority of OT1 and endogenous GzmC after / 33-ACT + CD8 + Two-thirds of TILs are actually TCF1 neg These cells were then cultured with TCF1 cells from other groups, when available. neg Effector CD8 + Compared with TILs (Figure 5B) (analyzed from PD1d / 33-OT1, non-transduced OT1, and non-transduced OT1 ACT+αPD-L1); importantly, all of these cells expressed GzmC neg (Fig. 5A). V GzmC from / 33-ACT + TCF1 neg CD8 + TILs, as well as GzmC from other groups neg TCF1 neg CD8 +The majority of effector OT1 and endogenous TILs express PD-1 + (Fig. 5C), and a significant proportion of these cells also expressed TIM3 + Surprisingly, consistent with the (sc)RNA-seq data, OT1 cells expressed almost no TOX and only endogenous PD-1. + GzmC + TCF1 neg Only about 40% of the cells expressed TOX (Fig. 5E). + ) and C4(GzmC neg ) Consistent with being one of the most differentially expressed genes between exhausted cells, PD1 + GzmC + TCF1 neg The majority of OT1 and endogenous TILs are terminally exhausted CD8 + They showed high expression of LY6C, a marker not present in TILs. + TCF1 neg OT1 or endogenous TILs also showed low or absent expression of KLRG1, a marker of short-lived effector cells (Figure 5F). V TOX from / 33-ACT low / neg GzmC + TCF1 neg CD8 + Demonstrating that TILs are not canonical terminally exhausted cells, we found that the majority of OT1 and approximately half of endogenous cells express CD69, suggesting recent TCR-induced activation (Figure 6A). Furthermore, TOX from both the OT1 and endogenous compartments was significantly increased. neg / low GzmC + PD-1 + TCF1 neg CD8 + T cells expressed more Ki-67 than TILs from the other groups (Figure 6B). V Most OT1 and endogenous CD8 from / 33-ACT + TILs (but not from other groups) expressed GrzmB according to (sc)RNAseq analysis. + These PD-1 +Some OT1 cells have double GzmC high and GzmB high A significant fraction of PD1d / 2 was identified (Figure 6E), which was not detected in endogenous cells. V PD-1 recruited during tumor regression from / 33-ACT + CD8 + TILs were examined for cytokine responses to ex vivo CD3 / CD28 stimulation. Approximately half of the OT1 and endogenous TILs produced TNFα upon stimulation, and some cells produced both TNFα and IFNγ (Figure 6F), demonstrating multifunctional effector properties. Thus, triple-gene-modified ACTs induce potent tumor-rejecting CD8 T cells without acquiring the TOX program. + They give rise to a unique phenotype known as effector TILs.

[0266] To verify whether co-inhibitory receptors such as PD-1 or TIM-3 are uncoupled from the TOX exhaustion program under the achieved unique TIL state and whether their suppressive function is insignificant, we performed a PD1d / 2 V We combined αPD-L1 / 33-ACT with αPD-L1 antibody or αPD-L1 / αTIM3 dual antibody. However, no improvement in tumor control was observed (Figures 6G and 6H). Therefore, PD-1 blockade suppresses IL-2 V It was inferred that this was completely unnecessary in the context of combined IL-33 / IL-33 gene-modified ACT. In fact, removing the PD-1 ectodomain from the PD-1_IgG4 decoy did not result in any significant improvement in activity. V Tumor control by ACTs carrying only the / 33 module was not affected (Figure 6I). Collectively, these data indicate that orthogonal engineering of T cells with βγ-binding IL-2 and IL-33 in immunocompetent hosts enabled the generation of a novel effector TIL state (in which TOX remained repressed and co-inhibitory receptors were expressed but negligible) with the ability to control tumors.

[0267] Orthogonal Engineering is TOX neg / low GzmC + Promotes differentiation of progenitor cells As with chronic viral infections, CD8 + T cell-mediated antitumor responses, even after PD-1 blockade, are driven by intratumoral TCF1 cells with stem-like properties. + PD-1 + Pre-exhaustion CD8 + They may be maintained by T cells, which express the transcription factor TOX, which is essential for their generation and persistence. neg IL-2 under ACT conditions inhibits TOX in cells V Considering the role of IL-2 V TCF1 + We investigated whether PD1d / IL-2 also suppresses the TOX program in progenitor cells. V During the response phase after / 33-ACT, significant numbers of TCF1 + CD8 + TILs were detected (Figures 3F and 3G). All of these cells expressed GzmC (Figure 5A), and most expressed PD-1. + (Fig. 7A), these (both OT1 and endogenous) were mostly TOX. neg (Figures 7B and 7C). Thus, orthogonal engineering of ACT resulted in tumor-responsive TCF1 + Progenitor cells already deactivate the TOX program and upregulate GzmC (Figure 7D). Importantly, PD1d / 2 V -TCF1 after ACT + PD-1 + CD8 + Downregulation of TOX in TILs was also observed, whereas significant expression in the same TIL subset after PD1 / 33-ACT was higher than that of IL-2. V TCF1 + CD8 + This indicates that TOX is suppressed at the progenitor cell level.

[0268] PD1d / IL-2 V -After ACT, PD-1 neg TCF1 + Progenitor cells (both OT1 and endogenous) were identified at significant frequencies (Fig. 7A). negHowever, antigen-experienced T CM or T EM Thus, IL-2 V In the presence of tumor-specific TCF1 + TILs express PD-1 + However, in the presence of IL-33 alone, endogenous TCF1 + PD-1 neg CD8 + 40% of TIL is TOX + And most of them are T EM Thus, orthogonal engineering was performed on TCF1 cells. + We conclude that IL-2 reprograms the stem-like compartment toward a transcriptional program upregulated by the repression of the TOX program and the expression of GzmC. Importantly, these two programs appear to be independent. V was a crucial factor supporting stemness and persistence in a TOX-independent manner, whereas pro-CD8 + GzmC in TILs + Combination with IL-33 was required to also trigger activation of the differentiation program.

[0269] GzmC + Dynamic evolution of effector states Finally, GzmC after triple gene modification ACT + The dynamic evolution of TIL status was evaluated. CD8 TILs collected 5 days after ACT (day 17) were + (sc)RNA-seq data from TILs at tumor response (day 24) and PD1d / 2 VWe compared (sc)RNA-seq data from tumors that escaped (day 38) after an initial response to / 33-ACT (Figure 8A). Adding an escape timepoint to all previous TILs did not alter the previously described cellular annotations and cluster distribution. Thus, early after ACT, TILs were primarily distributed among proliferative (C3), effector memory (C2), and exhausted pools (C4) (Figure 8B). By day 12 after ACT, cells transitioned to a new C5 state, associated with tumor regression. Interestingly, subsequent progression was associated with TIL transition from C5 to a new C6 state (Figure 8B). This new adaptive state upon tumor escape is associated with the rejection-associated C5 GzmC state. + The effector state was certainly distinct (Figures 8B and 8C). As expected from the inability of PD-1 (or TIM-3) blockade to control tumors (Figures 6G, 6H, and 6I), the escaping cells were far removed from the exhausted, terminally differentiated cells of C4 and expressed significantly lower levels of TOX than these cells (Figure 8B). V Clustering of the / 33 sample alone provided improved resolution, revealing that C6 cells, close to but distinct from the canonical effector memory C2 state observed early after ACT or during the response, exhibit lower Fosl2, Bcl2, Gzma, Gzmb, and Tnfrsf9 (CD137) (Figure 8B). Consistently, ProjecTILs analysis revealed that the C6 state during escape deviates from the reference map effector memory state, downregulating a Fosl2-driven effector gene program. Thus, escape is not mediated by canonical exhaustion, and cells are, at least in large part, in the PD1d / 2 phase. V It retained the TOX repression program characteristic of / 33-ACT but lost expression of GzmC (and GzmB).

[0270] The above observations were verified by flow cytometry. After triple gene modification ACT, the characteristic GzmC expression in cluster 5 was observed. + CD8 +The TIL population significantly expanded within the tumor from days 5 to 12, consistent with tumor regression (Figure 8D), but these cells were lost upon tumor escape. Furthermore, a significant decrease in OT1 cells was observed upon tumor progression (Figure 8E), reflecting the TCF1 expression of both OT1 and endogenous TILs. neg This was associated with a shrinkage of the CD8+ / CD9+ population (Figure 8F). Consistent with the C6 status, residual CD8+ / CD9 ... + TILs are PD-1 neg / lo T EM Dear CD8 + T exhibiting the phenotype and propagating in vitro EM Dear CD8 + Finally, TCF1 cells collected during escape showed a significant downregulation of granzyme B compared to T cells. neg PD-1 + CD8 + T cells were collected during tumor control and were TCF1 neg PD-1 + CD8 + Compared to T cells, TILs exhibited a loss of both granzyme B expression and polyfunctionality (Figures 8G and 8H). Thus, we conclude that the optimal TIL effector state is dynamically linked to tumor response.

[0271] Consideration This example demonstrates that orthogonal combinatorial T cell engineering in the context of solid tumor ACT can successfully overcome homeostatic barriers in the host, resulting in profound reprogramming of TILs and the tumor microenvironment and tumor regression in the absence of lymphodepletion or exogenous cytokine support. Performing ACT in an immunocompetent host not only dramatically reduces the toxicity and cost of current ACT, but also offers the unique advantage of fully leveraging the host's immune system. This example further demonstrates that, under these circumstances, endogenous CD8 + cells (especially existing CD8 + Both TILs and neutrophils are recruited to tumors and are essential for achieving tumor regression.

[0272] Recent studies using high-dimensional computational analysis have demonstrated the expression of multiple types of CD8 T cells in human and mouse tumors, including naive-like, effector memory, cytotoxic, and exhausted CD8 T cells. + We have revealed the existence of a cytotoxic TIL state (SJ Carmona, et al. OncoImmunology 9, 1737369 (2020)). While the cytotoxic TIL state observed primarily in human samples is mostly enriched in bystander cells, there is substantial evidence that the exhausted compartment is enriched in tumor-specific CD8 TILs (AM van der Leun, et al. Nature Reviews Cancer 20, 218-232 (2020)). Notably, this compartment is highly heterogeneous, consisting of either precursor or terminal exhausted CD8 TILs. + T cells are composed of a series of cellular states hierarchically organized along the differentiation axis. To date, immune checkpoint blockade (ICB) has achieved significant clinical responses, but its effectiveness is primarily based on inducing a change in the exhausted CD8 T cell state already present before treatment, rather than inducing a novel, non-exhausted, effector-like state (J.-C. Beltra et al., Immunity 52, 825-841.e828 (2020)). Therefore, pharmacological reprogramming of CD8 TILs toward such a "desired" effector state represents an effective strategy for improving clinical responses to current immunotherapies.

[0273] T cell engineering offers limitless opportunities to rationally reprogram TILs and, in a paracrine manner, the TME. This example demonstrates the use of IL-2 variants that engage only the βγ chain receptors in CD8 T cells. + We demonstrate orthogonal engineering of PD-1 blockade to stimulate T cells, together with IL-33, a potent innate immune activator, to reprogram the TME. This combination suppresses the unique expression of multiple granzymes (most notably granzyme C) and exhausted CD8+ cells during chronic viral infection and in cancer. +This led to the adoption by both exogenous and endogenous TILs of a novel effector state distinguished by the suppression of TOX, a transcription factor important for the generation and maintenance of T cell populations (AC Scott et al., Nature 571, 270-274 (2019)). This state was characterized by significant local CD8 expression in the TME. + This was reproducibly associated with T cell proliferation, potent effector function, and effective tumor control. PD-1 and other co-inhibitory receptors remained expressed under this novel program; this indicates that their upregulation under persistent antigen stimulation is not strictly TOX-dependent. Furthermore, pharmacological blockade of the PD-1 and TIM3 pathways confirmed that their expression is functionally insignificant.

[0274] CD8 + The T cell exhaustion program is regulated by the expression of TCF1 + Stably implemented in the progenitor compartment. However, increased TCF1 expression through orthogonal engineering + CD8+ cells remained negative for TOX and upregulated granzyme C, a marker never detected in the canonical progenitor-exhausted cell compartment. Therefore, the treatment-induced progenitor-like cell state is consistent with the TCF1 expression profile consistently reported in chronic viral infections and cancer. + TOX + Diverging from a progenitor state. Similarly, multifunctional GzmC was amplified by orthogonal engineering. + TCF1 neg PD-1 + TOX low / neg The effector-like state is a canonical terminal exhausted cell state (TOX). + PD-1 + CX3CR1 neg GzmC neg ) as well as from a transient effector-like exhausted cell state (CX3CR1 + TIM3 + PD-1 +) is also diverged from the canonical GzmC. Indeed, TOX is downregulated in this state. neg TCF1 + These cells arise from progenitor exhausted cells and do not significantly upregulate Gzmc (J.-C. Beltra et al., Immunity 52, 825-841.e828 (2020)). Furthermore, it is possible to upregulate Gzmc by orthogonal engineering. + They express Cx3cr1 and Klrg1, which are significantly downregulated in the effector state. + T help cells express CX3CR1 + These cells are required for the formation of an effector state. However, these cells are harmful in the context of this approach. Therefore, TCF1 + and TCF1 neg GzmC + CD8 + TILs are novel TOX-independent CD8 + They were hypothesized to represent the progenitor and effector states, respectively, of the T cell differentiation program.

[0275] Therapeutic manipulation of TOX has emerged as a promising strategy to abolish T cell exhaustion in the context of cancer. Recently, it has been demonstrated that TOX knockdown or deletion of TOX2 improves CAR-T cell function, and heterozygous deletion of TOX enhances antitumor T cell responses (H. Seo et al., Proceedings of the National Academy of Sciences 116, 12410-12415 (2019); O. Khan et al., Nature 571, 211-218 (2019)). This example demonstrates the ability of non-exhausted, highly functional CD8 + This represents an alternative and novel approach to therapeutically targeting TOX to induce an effector state. Indeed, it is possible to induce not only transferred T cells but also endogenous CD8 + IL-2 also inhibits T cell proliferation in the tumor compartment. VIt was shown that IL-33 promoted both CD8 T cell stemness and suppressed TOX. On the other hand, IL-33 promoted CD8 T cell stemness and suppressed Tcf1 expression, possibly indirectly through TME reprogramming, by upregulating Gzmc and suppressing Tcf1 expression. + This optimal TIL state is lost upon tumor escape, and the resulting EM-like state promotes the differentiation of T cells into multifunctional effector cells. + This was not only distinct from the normal effector memory CD8 TIL state, but also from the canonical effector memory CD8 TIL state. Thus, tumor escape in response to orthogonal T cell engineering was not mediated by reactivation of an exhaustion program by TOX, but rather by optimal GzmC activation. + Effector CD8 + Intratumoral CD8 status + This was due to a lack of T cell differentiation, which was due to exhausted CD8 T cells that were reactivated by PD-1 blockade. + This is a crucial difference as T cells reacquire their exhausted phenotype during tumor escape.

[0276] In summary, this example demonstrates the use of CD8 + We demonstrate that orthogonal combinatorial engineering of T cells, specifically those secreting a CD25-unengaged variant of IL-2 and the alarmin IL-33, can control advanced melanoma tumors in the absence of preconditioning, cytokine therapy, or other support (e.g., vaccination). While combinatorial T cell therapy was non-curative, CD4 depletion enabled long-term survival; this indicates that Tregs are involved in disease progression, thus providing opportunities for further combinatorial therapeutic intervention. Thus, the present disclosure provides a novel approach to develop highly functional CD8 T cells with the ability to reprogram the TME and control advanced, poorly immunogenic solid tumors. + This demonstrates the feasibility of clinical translation of combinatorial gene-modified T cells to induce the condition.

[0277] Table 2. Observed response and predicted probability of objective response and clinical benefit for each treatment group TIFF0007819923000002.tif82166Note: Objective response includes complete response (CR; 100% tumor volume reduction) and partial response (PR; ≤-30% tumor change). Clinical benefit includes CR, PR, and stable disease (-30% < tumor change ≤ +20%). Probability of occurrence was calculated using exact logistic regression.

[0278] Table 3. Predicted tumor size change from baseline in each group using linear regression TIFF0007819923000003.tif93166*: -100% is the minimum plausible value for tumor size change. Note: p-values ​​compare each treatment effect to the triple combination (PD1d+IL-2V+IL-33). Adjusted R of the model 2 :43.8%.

[0279] Example 5 CD40L decoy and IL-2 variants and combined GEEP therapy We constructed a SIN retroviral vector encoding a trimeric CD40L decoy and a variant of IL-2 that does not bind to CD25. Because these molecules are expressed under the control of NFAT, they are produced only by activated T cells; this should occur only in the tumor microenvironment. In preclinical models, the IL-2 variant promoted a less differentiated phenotype and supported engraftment (i.e., T cell survival) in vivo. Preclinical studies also demonstrated that CD40L promotes tumor control. It can act on antigen-presenting cells, such as dendritic cells, to activate them and thereby provide better T cell support. Therefore, the CD40L decoy is a reprogrammer of the tumor microenvironment.

[0280] T cells are first genetically modified with PD1 decoy-tEGFR and then transduced with CD40L decoy and IL2 by co-transduction or by mixing with a different genetically modified T cell population.V The tEGFR (also called cell exclusion tag (CET)) is used as a means to assess transduction efficiency and, if necessary, can be used to enrich for genetically modified cells (on anti-EGFR-coated beads). It can also be used as a means to track genetically modified T cells in patients after engraftment (by FACS from collected blood samples or tumor biopsies). Furthermore, it can be used as an exclusion tag via ADCC if toxicity occurs in patients receiving cetuximab.

[0281] Table 4. Representative sequences of example transgenes TIFF0007819923000004.tif226166TIFF0007819923000005.tif226166TIFF0007819923000006.tif221166TIFF0007819923000007.t if226166TIFF0007819923000008.tif221166TIFF0007819923000009.tif226166TIFF0007819923000010.tif225166TIFF0007819923 000011.tif166166TIFF0007819923000012.tif225166TIFF0007819923000013.tif228166TIFF0007819923000014.tif226166TIFF00 07819923000015.tif225166TIFF0007819923000016.tif176166TIFF0007819923000017.tif196166TIFF0007819923000018.tif84166

Claims

1. 1. A composition comprising a plurality of genetically modified lymphocytes expressing at least two transgenes for modulating the immune system of a subject, The composition, wherein the transgene comprises a PD-1-Fc fusion decoy in combination with CD40L having an amino acid sequence of any one of SEQ ID NOs: 32-34, 36, and 38.

2. The composition of claim 1, wherein the PD-1-Fc fusion decoy is a PD-1.IgG4 decoy.

3. 3. The composition of claim 1 or 2, wherein the transgene further comprises a truncated EGFR (tEGFR), HER2, truncated HER2 (tHER2), CD20, or CD19.

4. 3. The composition of claim 1, wherein the transgene comprises a PD-1-Fc fusion decoy, a CD40L having the amino acid sequence of any one of SEQ ID NOs: 32-34, 36, and 38, and an IL-2 variant having the amino acid sequence of any one of SEQ ID NOs: 21-23.

5. The composition of any one of claims 1 to 4, wherein the PD-1-Fc fusion decoy comprises an amino acid sequence of any one of SEQ ID NOs: 1-4, 6-17, 42, 44, 47-48, and 51-52.

6. The composition of claim 1 , wherein the plurality of lymphocytes comprises at least two lymphocyte subsets.

7. The composition of claim 6, wherein the plurality of lymphocytes consists of two lymphocyte subsets.

8. Several lymphocytes (i) a first subset expressing at least two transgenes; and (ii) a second subset expressing at least two transgenes; 7. The composition of claim 6, wherein at least one of the transgenes of the first subset is different from the transgenes of the second subset or at least one of the transgenes of the first subset is in common with the transgenes of the second subset.

9. (i) the first subset or the second subset expresses a transgene comprising a PD-1-Fc fusion decoy in combination with CD40L having the amino acid sequence of any one of SEQ ID NOs: 32-34, 36, and 38; or (ii) the first subset or the second subset expresses a transgene comprising a PD-1-Fc fusion decoy, a CD40L having an amino acid sequence of any one of SEQ ID NOs: 32-34, 36, and 38, and an IL-2 variant having an amino acid sequence of any one of SEQ ID NOs: 21-23; 7. The composition of claim 6.

10. 7. The composition of claim 6, wherein the first subset or the second subset further expresses tEGFR, HER2, truncated HER2 (tHER2), CD20, or CD19.

11. The composition according to any one of claims 6 to 10, wherein the two subsets are combined in a ratio of 1:1 to 1:

100.

12. 12. The composition of claim 11, wherein the two subsets are combined in a 1:1 ratio.

13. The composition of any one of claims 1 to 12, wherein the lymphocytes are autologous.

14. The composition according to any one of claims 1 to 13, wherein the lymphocytes are tumor-infiltrating lymphocytes.

15. The composition of any one of claims 1 to 14, wherein the lymphocytes express a chimeric antigen receptor (CAR) or a recombinant T cell receptor (TCR).

16. The composition of claim 15, wherein the TCR exhibits reactivity to NY-ESO1, MAGE-A1, MAGE-A3, MAGE A-10, MAGE-C2, SSX2, MAGE-A12, or a combination thereof.

17. A pharmaceutical composition or kit comprising an effective amount of the composition of any one of claims 1 to 16 and a pharmaceutically acceptable carrier.

18. The pharmaceutical composition or kit of claim 17, wherein the pharmaceutical composition or kit further comprises a second therapeutic agent.

19. 17. The composition of any one of claims 1 to 16 for use in treating cancer / tumor or chronic infection in a subject, wherein the cancer is selected from the group consisting of melanoma, sarcoma, ovarian cancer, prostate cancer, lung cancer, bladder cancer, MSI-high tumors, head and neck tumors, kidney cancer, and breast cancer.

20. 20. The composition of claim 19, wherein the composition is administered by intravenous infusion.

Images