Ex VIVO generated antigen-specific CD4+ t cells with enhanced self-renewal and cytotoxic activity for immunotherapy of cancer

Abstract

A method for generating antigen-specific CD4+ cytotoxic T cells comprising providing a plurality of CD4+ T cells; stimulating the CD4+ T cells with at least one tumor associated-antigen (TAA) peptide and / or at least one viral peptide; and expanding the CD4+ T cells in the presence of at least one pro-inflammatory cytokine. Also provided are enriched populations and compositions comprising the generated antigen-specific CD4+ cytotoxic T cells, and methods of treating, ameliorating, or preventing cancers and disease by administering the cells to subjects.

Description

[0001] Docket: 93597 / 7387 (92420-A-PCT)

[0002] EX VIVO GENERATED ANTIGEN-SPECIFIC CD4+ T CELLS WITH ENHANCED SELF-RENEWAL AND CYTOTOXIC ACTIVITY FOR IMMUNOTHERAPY OF CANCER AND VIRAL DISEASES

[0003] [1] Throughout this application, various publications are referenced, including referenced in parenthesis. The disclosures of all publications mentioned in this application in their entireties are hereby incorporated by reference into this application in order to provide additional description of the art to which this invention pertains and of the features in the art which can be employed with this invention.

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] [2] This application claims the benefit of U.S. Provisional Application No. 63 / 712,703, filed October 28, 2024, the contents of which are hereby incorporated by reference.

[0006] BACKGROUND OF THE INVENTION

[0007] [3] CD4+ T cells are often present in tumor infiltrating lymphocytes (TILs) and TIL-like products, displaying antigen-specific activity but have not been formally studied despite their central role in orchestrating the immune response. Using realistic animal models of adoptive immunotherapy, antigen-specific CD4+ T cells have been suggested to have a capacity to completely eradicate advanced tumors. However, generation of autologous or allogeneic TIL-like multi-tumor associated antigen (TAA)-specific CD4+ T cells has not been demonstrated, and prior studies have not studied the feasibility of such an approach.

[0008] BRIEF SUMMARY OF THE INVENTION

[0009] [4] A method for generating a population of antigen-specific CD4+ cytotoxic T cells comprising: providing a plurality of CD4+ T cells; stimulating the CD4+ T cells with at least one tumor associated-antigen (TAA) peptide, and / or at least one viral peptide, preferably at least one peptide derived from a viral oncoprotein; and expanding the CD4+ T cells in the presence of at least one pro-inflammatory cytokine, so as to produce a population of antigen-specific CD4+ cytotoxic T cells.

[0010] 1

[0011] 4916-0122-4820v.8 [5] A pharmaceutical composition comprising an antigen-specific CD4+ cytotoxic T cell generated by a method described herein.

[0012] [6] A population of cells comprising the antigen-specific CD4+ cytotoxic T cells generated by a method described herein.

[0013] [7] A method of treating, ameliorating, or reducing development of a cancer or disease in a subject comprising administering a pharmaceutical composition or population of cells described herein to the subj ect.

[0014] [8] A method of treating, ameliorating, or reducing development a disease in an immunosuppressed subject or subject on active chemotherapy comprising administering a pharmaceutical composition or population of cells described herein to the subject.

[0015] [9] An adoptive T-cell therapy for treating a cancer comprising administering a pharmaceutical composition or population of cells (e.g., antigen-specific CD4+ cytotoxic T cells) described herein to a subject having the cancer, so as to treat the cancer.

[0016]

[0010] An adoptive T cell therapy for treating a viral infection or virus-associated disease comprising administering a pharmaceutical composition or population of cells (e.g., antigenspecific CD4+ cytotoxic T cells) described herein to a subject having a viral infection or virus- associated disease, so as to treat the viral infection or virus-associated disease.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018]

[0011] Fig. 1: Schematic of a method for generating antigen specific Thl.l7-CTLs. Purified Total T, CD4+T, or naive CD4+ T cells were stimulated with either PBMCs or monocyte-derived dendritic cells loaded with overlapping peptide libraries of either viral antigens, tumor associated antigens, or neoantigens and expanded in the presence of pro-inflammatory cytokines for 12-14 days to produce antigen specific Thl.l7-CTLs. Standard Thl-CTLs were produced in neutral conditions. In both conditions, the purified cells were cultured without IL-2 for 72 hours to allow proper stimulation of antigen reactive cells, followed by replenishing media with IL-2 every 2-3 days until the end of the culture.

[0019]

[0012] Figs. 2A-2C: Purified EBV-specific Thl. l7-CD4+CTLs generated under pro- inflammatory conditions display superior reactivity as compared to Thl- CTLs (Fig. 2A), and

[0020] 2

[0021] 4916-0122-4820v.8 unfractionated EBV-CTLs produced by the expansion of Total (non-purified) T cells cultured in neutral (Thl) or pro-inflammatory conditions (Fig. 2B) against all EBV antigens (Fig. 2C).

[0022]

[0013] Fig. 3: EBV-specific Thl.l7-CD4+ CTLs recognize natural tumor targets, display superior proliferation, persistence, and anti -turn or activity in vitro and in vivo.

[0023]

[0014] Fig. 4: Thl. l7-CTLs show resistance to immunosuppression. Thl.l7-CTLs overexpress MDR1 compared to Thl-CTLs, and a superior MDRl-specific efflux capacity, and retain superior reactivity in presence of tacrolimus-induced immunosuppression.

[0024]

[0015] Fig. 5: Tumor associated antigen (PRAME) specific Thl and Thl.17 CD4+ CTLs generated using autologous naive CD4 T cells and MoDCs pulsed with peptide library of PRAME antigen. Data shows superior antigen specific reactivity, polyfunctionality, and higher avidity of Thl.17 cells compared to conventional Thl CTLs.

[0025]

[0016] Fig. 6: Data showing superior polyfunctionality of PRAME- specific Thl.17 CTLs compared to Thl . CTLs.

[0026]

[0017] Fig. 7: Thl.17 CTLs retain ability to produce IL-2 - a marker of in vivo persistence — despite prolonged ex vivo expansion and repeated stimulation. CFSE assays further demonstrate their superior proliferative capacity compared to non-proliferating Thl CTLs.

[0027]

[0018] Fig. 8: Thl .17 cells are less differentiated compared to Thl CTLs, exhibit tissue resident phenotype, and show reduced expression of exhaustion markers.

[0028]

[0019] Fig. 9: Tumor associated antigen-specific Thl .17-CTLs recognize naturally processed antigens in vitro (either full-length tumor protein or leukemic blasts) and eradicate leukemic blasts in vivo upon adoptive transfer into tumor-bearing animals.

[0029]

[0020] Fig. 10: Successful generation of highly reactive, polyfunctional, and multi-tumor associated antigen-specific Thl .17 CD4+ CTLs from subjects with Myelodysplastic syndrome and Merkel cell carcinoma with robust reactivity, higher expansion, and better phenotype compared to Thl CTLs.

[0030]

[0021] Fig. 11: Thl .17-CTLs have superior bioenergetics profile as compared to standard Thl

[0031] CD4+ CTLs.

[0032] 3

[0033] 4916-0122-4820v.8

[0022] Fig. 12: Successful generation of neoantigen specific Th 1.17 CTLs targeting common KRAS hotspot mutations.

[0034]

[0023] Fig. 13: Generation Thl.17 TILs from head and neck tumor resection samples demonstrating improved expansion, enhanced viability and favorable phenotypic features.

[0035] DETAILED DESCRIPTION OF THE INVENTION

[0036]

[0024] A method for generating a population of antigen-specific CD4+ cytotoxic T cells comprising: providing a plurality of CD4+ T cells; stimulating the CD4+ T cells with at least one tumor associated-antigen (TAA) peptide, and / or at least one viral peptide, preferably at least one peptide derived from a viral oncoprotein; and expanding the CD4+ T cells in the presence of at least one pro-inflammatory cytokine, so as to produce a population of antigen-specific CD4+ cytotoxic T cells.

[0037]

[0025] In some embodiments, the plurality of CD4+ T cells are purified. In some embodiments, the plurality of CD4+ T cells are purified from a mixture of cells isolated from a sample, e.g., a blood sample or a whole blood sample, isolated from a subject. As a non-limiting example, the plurality of CD4+ T cells are purified from peripheral blood mononuclear cells (PBMCs) isolated from a subject.

[0038]

[0026] In some embodiments, the at least one viral peptide is an immunodominant viral antigen from a virus, or a peptide derived therefrom.

[0039]

[0027] In some embodiments, the at least one pro-inflammatory cytokine is IL-1 [3, IL-6, IL-7, IL-15, IL-21, IL-23, and / or TGF-0.

[0040]

[0028] In some embodiments, expanding the CD4+ T cells in the presence of at least one pro- inflammatory cytokine comprises expanding the cells in the presence of a combination of IL-ip, IL-6, IL-7, IL-15, IL-21, IL-23, and / or TGF-0.

[0041]

[0029] In some embodiments, the at least one pro-inflammatory cytokine includes a combination of cytokines comprising IL-10, IL-6, IL-7, IL-15, IL-21, IL-23, and / or TGF-0.

[0042] 4

[0043] 4916-0122-4820v.8

[0030] In some embodiments, the at least one pro-inflammatory cytokine is at a concentration of 1000 - 0.1 ng / ml.

[0044]

[0031] In some embodiments the at least one pro-inflammatory cytokine is a combination of IL-ip (preferably at 1000-0.1 ng / ml), IL-6 (preferably at 1000-0.1 ng / ml), IL-7 (preferably at 1000-0.1 ng / ml), IL-15 (preferably at 1000-0.1 ng / ml), IL-21 (preferably at 1000-0.1 ng / ml), IL- 23 (preferably at 1000-0.1 ng / ml), TGF-0 (preferably at 1000-0.1 ng / ml). In some embodiments the at least one pro-inflammatory cytokines have human sequences.

[0045]

[0032] In some embodiments, the stimulating comprises exposing the CD4+ T cells to an overlapping peptide library. In some embodiments, the stimulating comprises exposing the CD4+ T cells to a full-length antigen and / or to a full-length protein encoded by a virus or tumor cell. In some embodiments, the T cells are total T cells or naive CD4+ cells.

[0046]

[0033] In some embodiments, the at least one tumor associated antigen (TAA) peptide is at least one of a Cancer-Testis Antigen (CTA) peptide, Preferentially Expressed Antigen in Melanoma (PRAME) peptide, New York esophageal squamous cell carcinoma (NYESO1) peptide, Cancer / Testis Antigen Family 45 Member Al (CT45A1) peptide, Melanoma-associated antigen 3 (MAGE-A3) peptide, MAGE-A1, MAGE-A4, and Wilms’ tumor 1 (WT-1) peptide, and / or a ACRBP, ZNF165, ZNF645, XAGE5, XAGE3, XAGE2, XAGE1B, WT1, VENTXP1, TULP2, TTK, TSSK6, TSPY3, TSPY2, TSGA10, TPTE, TPPP2, TMPRSS12, TMEM108, TMEFF2, TMEFF1, THEG, TFDP3, TEX15, TEX14, TEX101, TEKT5, TDRD6, TDRD1, TAF7L, SYCP1, SYCE1, SSX9, SSX7, SSX6, SSX5, SSX4B, SSX4, SSX3, SSX2, SSX1, SPO11, SPEF2, SPATA19, SPANXN5, SPANXN4, SPANXN3, SPANXN2, SPANXN1, SPANXD, SPANXC, SPANXB1, SPANXA2, SPANXA1, SPAG9, SPAG8, SPAG6, SPAG4, SPAG17, SPAG1, SPACA3, SPA17, SLCO6A1, SEMG1, SAGE1, ROPN1, RGS22, RBM46, PRSS55, PRSS54, PRM2, PRM1, PRAME, POTEH, POTEG, POTEE, POTED, POTEC, POTEB, POTEA, PLAC1, PIWIL2, PBK, PASD1, PAGE5, PAGE4, PAGE3, PAGE2B, PAGE2, PAGE1, OTOA, OIP5, ODF4, ODF3, ODF2, ODF1, NXF2B, NXF2, NR6A1, NOL4, NLRP4, MORC1, MAGEC3, MAGEC2, MAGECI, MAGEB6, MAGEB5, MAGEB4, MAGEB3, MAGEB2, MAGEB1, MAGEA9B, MAGEA9, MAGEA8, MAGEA6, MAGEA5, MAGEA4, MAGEA3, MAGEA2B, MAGEA2, MAGEA12, MAGEA11, MAGEA10, MAGEA1, MAEL, LY6K, LUZP4, LIPI, LEMD1, LDHC, KIAA0100, IL13RA2, IGSF11, HSPB9, HORMAD2,

[0047] 5

[0048] 4916-0122-4820v.8 H0RMAD1, GPATCH2, GPAT2, GAGE2A, GAGE13, GAGE12J, GAGE12H, GAGE12G, GAGE12F, GAGE12E, GAGE12D, GAGE12C, GAGE12B, GAGE1, FTHL17, FMR1NB, FBXO39, FATE1, FAM46D, FAM133A, ELOVL4, DSCR8, DPPA2, DNAJB8, DMRT1, DKKL1, DDX53, DDX43, DCAF12, CTNNA2, CTCFL, CTAGE5, CTAGE1, CTAG2, CTAG1B, CTAG1A, CT83, CT47B1, CT47A9, CT47A8, CT47A7, CT47A6, CT47A5, CT47A4, CT47A3, CT47A2, CT47A11, CT47A10, CT47A1, CT45A6, CT45A5, CT45A3, CT45A2, CT45A1, CSAG2, CSAG1, CRISP2, CPXCR1, COX6B2, CEP55, CEP290, CCDC83, CCDC62, CCDC36, CCDC33, CCDC110, CALR3, CAGE1, CABYR, BRDT, BIRC5, BAGE2, ATAD2, ARX, ARMC3, ANKRD45, AKAP4, AKAP3, ADAM29, ADAM2, ADAM I 2. uPAR, ACTL8, or a Long interspersed nuclear element- 1 (LINE-1) peptide.

[0049]

[0034] In some embodiments, the stimulating comprises exposing the CD4+ T cells to an overlapping peptide library spanning any one of PRAME, WT-1, NYESO-1, MAGE-A3, ACRBP, ZNF165, ZNF645, XAGE5, XAGE3, XAGE2, XAGE1B, WT1, VENTXP1, TULP2, TTK, TSSK6, TSPY3, TSPY2, TSGA10, TPTE, TPPP2, TMPRSS12, TMEM108, TMEFF2, TMEFF1, THEG, TFDP3, TEX15, TEX14, TEX101, TEKT5, TDRD6, TDRD1, TAF7L, SYCP1, SYCE1, SSX9, SSX7, SSX6, SSX5, SSX4B, SSX4, SSX3, SSX2, SSX1, SPO11, SPEF2, SPATA19, SPANXN5, SPANXN4, SPANXN3, SPANXN2, SPANXN1, SPANXD, SPANXC, SPANXB1, SPANXA2, SPANXA1, SPAG9, SPAG8, SPAG6, SPAG4, SPAG17, SPAG1, SPACA3, SPA17, SLCO6A1, SEMG1, SAGE1, ROPN1, RGS22, RBM46, PRSS55, PRSS54, PRM2, PRM1, PRAME, POTEH, POTEG, POTEE, POTED, POTEC, POTEB, POTEA, PLAC1, PIWIL2, PBK, PASD1, PAGE5, PAGE4, PAGE3, PAGE2B, PAGE2, PAGE1, OTOA, OIP5, ODF4, ODF3, ODF2, ODF1, NXF2B, NXF2, NR6A1, NOL4, NLRP4, M0RC1, MAGEC3, MAGEC2, MAGECI, MAGEB6, MAGEB5, MAGEB4, MAGEB3, MAGEB2, MAGEB1, MAGEA9B, MAGEA9, MAGEA8, MAGEA6, MAGEA5, MAGEA4, MAGE A3, MAGEA2B, MAGEA2, MAGEA12, MAGEA11, MAGEA10, MAGEA1, MAEL, LY6K, LUZP4, LIPI, LEMD1, LDHC, K1AA0100, 1L13RA2, 1GSF11, HSPB9, H0RMAD2, H0RMAD1, GPATCH2, GPAT2, GAGE2A, GAGE13, GAGE 12 J, GAGE12H, GAGE12G, GAGE12F, GAGE12E, GAGE 12D, GAGE12C, GAGE12B, GAGE1, FTHL17, FMR1NB, FBXO39, FATE1, FAM46D, FAM133A, ELOVL4, DSCR8, DPPA2, DNAJB8, DMRT1, DKKL1, DDX53, DDX43, DCAF12, CTNNA2, CTCFL, CTAGE5, CTAGE1, CTAG2, CTAG1B, CTAG1A, CT83, CT47B1, CT47A9, CT47A8, CT47A7, CT47A6, CT47A5, CT47A4, CT47A3, CT47A2, CT47A11, CT47A10, CT47A1,

[0050] 6

[0051] 4916-0122-4820v.8 CT45A6, CT45A5, CT45A3, CT45A2, CT45A1, CSAG2, CSAG1, CRISP2, CPXCR1, COX6B2, CEP55, CEP290, CCDC83, CCDC62, CCDC36, CCDC33, CCDC110, CALR3, CAGE1, CAB YR, BRDT, BIRC5, BAGE2, ATAD2, ARX, ARMC3, ANKRD45, AKAP4, AKAP3, ADAM29, ADAM2, ADAM12, uPAR, ACTL8, and / or a Long interspersed nuclear element-1 (LINE-1).

[0052]

[0035] In some embodiments, the stimulating comprises exposing the CD4+ T cells to overlapping peptide libraries spanning PRAME, WT-1, NYESO-1 and MAGE-A3.

[0053]

[0036] In some embodiments, the at least one tumor associated antigen (TAA) peptide is a neoantigen and / or antigen product of a KRAS, NRAS, BRAF, TET2, JAK2, or p53 gene, preferably having a cancer-associated mutation. In some embodiments the TAA is a product of a fusion gene, preferably a BCR-AB fusion, EML4-ALK fusion, TMPRSS2-ERG fusion, FGFR3-TACC3 fusion, a NTRK fusion, or a PAX3-F0X01 fusion. In some embodiments is an antigenic product of a mutation identified in an individual cancer.

[0054]

[0037] In some embodiments, the viral oncoprotein is an Ebstein-Barr Virus oncoprotein, preferably at least one of Epstein-Barr nuclear antigen 1 (EBNA1), Latent Membrane Protein 1 (LMP1), and Latent Membrane Protein 2A (LMP2A), a papilloma virus (HPV) E6 antigen, an HPV E7 antigens, hepatitis B virus (HBV) Hepatitis B X protein (HBx), a PreS / S protein, a hepatitis C virus (HCV) core protein, NS3 / NS4A, NS5A, Merkel Cell Polyomavirus (MCpyV) Large T (LT) antigen, MCpyV small T (ST) antigen, Human Herpes Virus 8 (HHV8) Latency- Associated Nuclear Antigen (LANA), a vCyclin antigen, a vFLIP antigen, vGPCR antigen, Human T-lymphotropic virus 1 (HTLV-1) Tax antigen, a Human T-lymphotropic virus 2 (HTLV-2) Tax antigen, Simian Virus 40 (SV40) Large T (T-ag) antigen, and SV40 Small T antigen (t-ag).

[0055]

[0038] In some embodiments, the viral oncoprotein is a Merkel Cell Polyomavirus Large T (LT) antigen.

[0056]

[0039] In some embodiments, the viral oncoprotein is encoded by any one of Human T- lymphotropic virus 1 (HTLV1), Human Papillomavirus (HPV), Hepatitis B Virus, Hepatitis C virus, Human Herpesvirus-8 (HHV8), and / or simian vacuolating virus 40 (SV40).

[0057]

[0040] In some embodiments, stimulating the CD4+ T cells further comprises co-culturing the CD4+ T cells with antigen presenting cells (APCs), antigen presenting cells (APCs), peripheral

[0058] 7

[0059] 4916-0122-4820v.8 blood mononuclear cells (PBMCs), monocyte-derived dendritic cells (MoDCs), wherein said APCs, PBMCs, and / or MoDCs have been exposed to the at least one tumor associated-antigen (TAA) peptide, and / or at least one viral peptide. In some embodiments, the APCs, PBMCs, and / or MoDCs have been exposed to a CT A, a neoantigen, or a fusion gene product, or at a protein from which the TAA is derived.

[0060]

[0041] In some embodiments, the MoDCs have been genetically engineered to express the at least one TAA or viral peptide. In some embodiments, the MoDCs have been genetically engineered to express a full-length protein encoded by a cancer cell, a fragment thereof, or single epitope thereof. In some embodiments, the MoDCs have been genetically engineered to express a viral antigen and / or a single viral epitope.

[0061]

[0042] In some embodiments, the MoDCs have been exposed to a full-length synthetic protein representing a cancer antigen, tumor antigen, or viral antigen of interest or have been transduced with a polynucleotide encoding the antigen of interest.

[0062]

[0043] In some embodiments, the MoDCs have been exposed to tumor lysate or apoptotic tumor cells derived from a subject’s cancer or leukemia.

[0063]

[0044] In some embodiments, the provided CD4+ T cells and / or APCs, PBMCs, and / or MoDCs are autologous cells isolated from a single subject (e.g., a subject suffering from cancer).

[0064]

[0045] In some embodiments, the provided CD4+ T cells and / or APCs, PBMCs, and / or MoDCs are allogeneic cells isolated from a single subject (e.g., a healthy donor).

[0065]

[0046] In some embodiments the CD4+ T cells and / or APCs and / or MoDCs are purified from peripheral blood mononuclear cells (PBMCs) isolated from a subject.

[0066]

[0047] In some embodiments, the provided CD4+ T cells and / or APCs, PBMCs, and / or MoDCs are purified from bone marrow (BM) isolated from a subject.

[0067]

[0048] In some embodiments, the provided CD4+ T cells and / or APCs, PBMCs, and / or MoDCs are purified from a tumor sample resected from a subject.

[0068]

[0049] In some embodiments, the at least one pro-inflammatory cytokine is a type 1 / 17 cytokine.

[0069] 8

[0070] 4916-0122-4820v.8

[0050] In some embodiments, the at least one pro-inflammatory cytokine is at least one of IL- 1P, IL-6, IL-7, IL-15, IL-21, IL-23, and / or TGF- .

[0071]

[0051] In some embodiments, the CD4+ T cells are exposed to the at least one proinflammatory cytokine about 72 hours after the stimulating.

[0072]

[0052] In some embodiments, the provided plurality of CD4+ T cells are naive.

[0073]

[0053] In some embodiments, the expanding comprises culturing the stimulated CD4+ T cells with the at least one proinflammatory cytokine for about 7-35 days, about 10-31 days, about 10- 14 days, about 10-12 days, or about 24-28 days.

[0074]

[0054] In some embodiments, the method further comprises repeating the stimulating step at least once, preferably within 7-14 days of first performing the stimulating step, more preferably at about 10 days after first performing the stimulating step.

[0075]

[0055] In some embodiments, the CD4+ T cells are exposed to the at least one pro- inflammatory cytokine within about 72 hours of each stimulation.

[0076]

[0056] In some embodiments, the population of antigen-specific CD4+ cytotoxic T cells produced by the methods described herein are tumor infiltrating leukocytes, tumor infiltrating leukocyte (TIL)-like cells, and / or multi-tumor associated-antigen (TAA)-specific CD4+ cytotoxic T cells; or wherein the population of antigen-specific CD4+ cytotoxic T cells produced by the method are EBV-specific cytotoxic T cells (EBV-CTLs).In some embodiments, the antigenspecific CD4+ cytotoxic T cells produced by the methods described herein are Merkel Cell Polyomavirus-specific T cells, or HTLVl, HPV, HBV, or HHV8-specific T cells

[0077]

[0057] In some embodiments, the method further comprises purifying the population of antigen-specific CD4+ cytotoxic T cells so as to increase the percentage of cells that are antigenspecific CD4+ cytotoxic T cells.

[0078]

[0058] In some embodiments, the provided CD4+ T cells are isolated from a subject suffering from, or at risk of suffering from, a myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML), post-transplant lymphoproliferative disease (PTLD), a B-cell or T cell lymphoma, a Latency 1 associated disease, a Latency 2 associated disease, a Latency 3 associated disease, EBV reactivation or infection, a virus-associated cancer or disease, a urothelial carcinoma, an epithelial cancer, a sarcoma, a glioma, and / or a solid tumor.

[0079] 9

[0080] 4916-0122-4820v.8

[0059] In some embodiments, the provided CD4+T cells are isolated from a subject who has undergone, or is expected to undergo, a solid organ transplantation and / or immunosuppression therapy or a subject who is immunodeficient. or from a subject who has undergone, or is expected to undergo, a solid organ transplantation and / or immunosuppression therapy and / or chemotherapy

[0081]

[0060] In some embodiments of the antigen-specific CD4+ cytotoxic T cells, the provided CD4+ T cells are human.

[0082]

[0061] A pharmaceutical composition comprising an antigen-specific CD4+ cytotoxic T cell generated by a method described herein.

[0083]

[0062] A population of cells comprising the antigen-specific CD4+ cytotoxic T cells generated by a method described herein.

[0084]

[0063] In some embodiments, the population is enriched in antigen-specific CD4+ cytotoxic T cells over the percentage of antigen-specific CD4+ cytotoxic T cells that is obtainable as an untreated biological sample from a human subject.

[0085]

[0064] In some embodiments, the population of antigen-specific CD4+ cytotoxic T cells is more proliferative than a population of antigen-specific CD4+ cytotoxic T cells cultured in the presence of neutral cytokines and not in the presence of proinflammatory cytokines. In some embodiments, the population is more than twice as proliferative. In some embodiments, the population is more than three times as proliferative. In some embodiments, the population is more than four times as proliferative.

[0086]

[0065] In some embodiments, the population of antigen-specific CD4+ cytotoxic T cells is enriched in antigen-specific CD4+ T cells displaying tissue resident markers (CD103, CD39, CD69). In some embodiments, the population is enriched with antigen-specific CD4+ cytotoxic T cells concurrently producing multiple cytokines (IFN-gamma, TNF-alpha and IL-2) as compared to the antigen-specific CD4+ T cells cultured in the presence of neutral cytokines and not in the presence of proinflammatory cytokines. In some embodiments, the population is enriched in antigen-specific CD4+ T cells displaying increased resistance to immunosuppression and / or chemotherapy mediated by expression of multi-drug resistance 1 (MDR1) efflux pump and / or aldehyde dehydrogenase (ALDH) enzymes. In some embodiments, the population overexpresses

[0087] 10

[0088] 4916-0122-4820v.8 Multi-Drug Resistance Protein 1 (MDR1) as compared to an otherwise identical standard Thl population of CTLs. In some embodiments, the population has increased resistance to tacrolimus- mediated immunosuppression as compared to an otherwise identical standard Thl population of CTLs.

[0089]

[0066] A method of treating, ameliorating, or reducing development of a cancer or disease in a subject comprising administering a pharmaceutical composition or population of cells described herein to the subject.

[0090]

[0067] In some embodiments, a somatic mutation to a KRAS, NRAS, p53, BRAF, TET2, JAK 1, JAK2, and / or JAK3 gene is present in a cancer cell isolated, identified, or derived from the subject. In some embodiments, the cancer cell isolated, identified, or derived from the subject comprises a fusion gene product and other cancer-related mutation.

[0091]

[0068] In some embodiments, the cancer or disease is myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML), post-transplant lymphoproliferative disease (PTLD), a B-cell lymphoma, a Latency 1 associated disease, Latency 2 associated disease, Latency 3 associated disease, a head and neck cancer, or an epithelial cancer, or a solid tumor.

[0092]

[0069] A method of treating, ameliorating, or reducing development a disease in an immunosuppressed subject or subject on active chemotherapy comprising administering a pharmaceutical composition or population of cells described herein to the subject.

[0093]

[0070] In some embodiments, the administering comprises adoptive transfer of EBV-specific cytotoxic T cells (EBV-CTLs).

[0094]

[0071] In some embodiments, the subject has post-transplant lymphoproliferative disease (PTLD) or an EBV-associated lymphoma.

[0095]

[0072] In some embodiments, the subject has had a transplant, or receives active immunosuppressive medications (e g., calcineurin inhibitors, tacrolimus, and / or steroids) or cytotoxic chemotherapy.

[0096]

[0073] In some embodiments, the subject has a head and neck cancer, urothelial cancer, epithelial cancer, esophageal cancer, colon cancer, skin cancer, lung cancer, prostate cancer, a melanoma, sarcoma, glioma, glioblastoma, or a malignant tumor.

[0097] 11

[0098] 4916-0122-4820v.8

[0074] In some embodiments, the subject has MDS or AML.

[0099]

[0075] In some embodiments, the subject has a solid or hematological tumor.

[0100]

[0076] In some embodiments, the subject has a KRAS, NRAS, p53, BRAF, TET2, JAK 1, JAK2, and / or JAK3 gene mutation.

[0101]

[0077] A method for generating a population of antigen-specific CD4+ cytotoxic T cells comprising: providing a plurality of CD4+ T cells; stimulating the CD4+ T cells with at least one viral, bacterial, parasitical or bacterial antigen peptide; and expanding the CD4+ T cells in the presence of at least one pro-inflammatory cytokine, so as to produce a population of antigen-specific CD4+ cytotoxic T cells.

[0102]

[0078] In some embodiments of the methods herein, the disease is a viral infection or is caused by a viral infection. In some embodiments, the disease is a virus-associated malignancy caused by Merkel Cell Polyomavirus, Human Papillomavirus (HPV), Hepatitis B Virus, Hepatitis C Virus, Human Herpesvirus-8 (HHV8), Human T-lymphotropic virus 1 (HTLV1), or simian vacuolating virus 40 (SV40).

[0103]

[0079] In some embodiments, the disease is a parasitical infection or is caused by a parasitical infection. In some embodiments, the disease is a bacterial infection or is caused by bacterial infection. In some embodiments, the disease is a fungal infection or is caused by a fungal infection.

[0104]

[0080] In some embodiments, the antigens or antigenic targets as discussed in relation to the methods disclosed herein are viral antigens. Non-limiting examples include from: Adeno- Associated Virus (AAV) Antigens, Adenovirus Antigens, ASFV Antigens, Chikungunya Virus Antigens, Coronavirus Antigens, Coxsackie Virus Antigens, Crimean-Congo Hemorrhagic Fever Virus Antigens, Cytomegalovirus Antigens, Dengue Virus Antigens, Eastern Equine Encephalitis Virus (EEEV) Antigens, Ebola Virus Antigens, Echovirus Antigens, Enterovirus Antigens, Epstein-Barr Virus Antigens, HAV Antigens, HBV Antigens, HCMV Antigens, HCV Antigens, HDV Antigens, HEV Antigens, HIV Antigens, HPV Antigens, HSV Antigens, HTLV Antigens, Influenza A Virus Antigens, Influenza B Virus Antigens, Japanese Encephalitis Antigens,

[0105] 12

[0106] 4916-0122-4820v.8 Leukemia Virus Antigens, Marburg Virus Antigens, Measles Virus Antigens, Metapneumovirus Antigens, Molluscum Contagiosum Antigens, Mumps Virus Antigens, Nipah Virus Antigens, Norovirus Antigens, Orf Virus Antigens, Parainfluenza Virus Antigens, Parvovirus Antigens, Poliovirus Antigens, Rabies Virus Antigens, Respiratory Syncytial Virus Antigens, Rhinovirus Antigens, Rift Valley Fever Virus Antigens, Rotavirus Antigens, Rubella Virus Antigens, Simian (Macaque) Immunodeficiency Virus Antigens, TBEV Antigens, Tobacco Etch Virus Antigens, Varicella Zoster Virus Antigens, Variola Antigens, Venezuelan Equine Encephalitis Virus Antigens, West Nile Virus Antigens, Yellow Fever Virus Antigens, Zika Virus Antigens, or other viral antigens.

[0107]

[0081] In some embodiments, the antigens or antigenic targets as discussed in relation to the methods disclosed herein are bacterial antigens. Non-limiting examples of bacterial antigens include: Burkholderia pseudomallei Antigens, Legionella Pneumophila Antigens, Neisseria gonorrhoeae Antigens, Salmonella Antigens, Staphylococcus Antigens, Ureaplasma urealyticum Antigens, Vibrio cholerae Antigens, Aeromonas Antigens, Arthrobacter Globiformis Antigens, Bacillus Antigens, Bordetella Pertussis Antigens, Borrelia Antigens, Brucella Abortus Antigens, Campylobacter Jejuni Antigens, Candida Albicans Antigens, Chlamydia Trachomatis Antigens, Chlamydophila Pneumoniae Antigens, Clostridium tetani Antigens, Corynebacterium Diphtheriae Antigens, E.coli Antigens, Haemophilus influenza Antigens, Helicobacter pylori Antigens, Leptospira biflexa Antigens, Listeria Monocytogenes Antigens, Mycobacterium Tuberculosis Antigens, Mycoplasma Pneumoniae Antigens, Other Bacterial Antigens, Salmonella typhimurium Antigens, Streptococcus Pneumoniae Antigens, Streptomyces Avidinii Antigens, Treponema Pallidum Antigens, and Yersinia Enterocolitica Antigens.

[0108]

[0082] In some embodiments, the antigens or antigenic targets as discussed in relation to the methods disclosed herein are fungal antigens. Fungal antigens include, for example, Aspergillus and Saccharomyces antigens.

[0109]

[0083] In some embodiments, the antigens or antigenic targets as discussed in relation to the methods disclosed herein are parasite antigens. Non-limiting examples of parasitic antigens include: Leishmania Antigens, Plasmodium Falciparum Antigens, Plasmodium Vivax Antigens, Plasmodium Antigens, Toxoplasma Gondii Antigens, Acanthamoeba Antigens, Caenorhabditis

[0110] 13

[0111] 4916-0122-4820v.8 Elegans Antigens, Echinococcus Granulosus Antigens, Malaria Antigens, Schistosoma Japoni cum Antigens, Trichomonas Vaginalis Antigens, and Trypanosoma cruzi (T. cruzi) Antigens.

[0112]

[0084] An adoptive T-cell therapy for treating a cancer comprising administering a pharmaceutical composition or population of cells described herein to a subject having the cancer, so as to treat the cancer.

[0113]

[0085] In some embodiments of the methods herein relating to a cancer, the cancer is a solid tumor. In some embodiments, the cancer is a hematological cancer. In some embodiments, the cancer expresses CD19, B7H3 (CD276), BCMA (CD269), ALPPL2, Claudin 18.2, CD123, CD171, CD179a, CD20, CD213A2, CD22, CD24, CD246, CD272, CD30, CD33, CD38, CD44v6, CD46, CD71, CD97, CEA, Claudin 6(CLDN6), CLECL1, CS-1, DLL-3, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, Ephrin B2, FAP, FLT3, GCC, GD2, GD3, GM3, GPRC5D, HER2 (ERBB2 / neu), IGLL1, IL-l lRa, KIT (CD117), KLK2, LY6G6D, MUC1, NCAM, p53R175H, PAP, PDGFR-P, PRAME, PRSS21, PSCA, PSMA, R0R1, SIRPa, SSEA-4, TAG72, TEM1 / CD248, TEM7R, TSHR, VEGFR2, ALPI, citrullinated vimentin, cMet, and / or Axl. In some embodiments, the cancer expresses DLL3, LY6G6D, Claudin 6, GCC, p53R175H, and / or WT-1, PRAME, ACRBP, ZNF165, ZNF645, XAGE5, XAGE3, XAGE2, XAGE1B, WT1, VENTXP1, TULP2, TTK, TSSK6, TSPY3, TSPY2, TSGA10, TPTE, TPPP2, TMPRSS12, TMEM108, TMEFF2, TMEFF1, THEG, TFDP3, TEX15, TEX14, TEX101, TEKT5, TDRD6, TDRD1, TAF7L, SYCP1, SYCE1, SSX9, SSX7, SSX6, SSX5, SSX4B, SSX4, SSX3, SSX2, SSX1, SPO11, SPEF2, SPATA19, SPANXN5, SPANXN4, SPANXN3, SPANXN2, SPANXN1, SPANXD, SPANXC, SPANXB1, SPANXA2, SPANXA1, SPAG9, SPAG8, SPAG6, SPAG4, SPAG17, SPAG1, SPACA3, SPA17, SLCO6A1, SEMG1, SAGE1, R0PN1, RGS22, RBM46, PRSS55, PRSS54, PRM2, PRM1, PRAME, POTEH, POTEG, POTEE, POTED, POTEC, POTEB, POTEA, PLAC1, PIWIL2, PBK, PASD1, PAGE5, PAGE4, PAGE3, PAGE2B, PAGE2, PAGE1, OTOA, OIP5, ODF4, ODF3, ODF2, ODF1, NXF2B, NXF2, NR6A1, N0L4, NLRP4, M0RC1, MAGEC3, MAGEC2, MAGECI, MAGEB6, MAGEB5, MAGEB4, MAGEB3, MAGEB2, MAGEB1, MAGEA9B, MAGEA9, MAGEA8, MAGEA6, MAGEA5, MAGEA4, MAGE A3, MAGEA2B, MAGEA2, MAGEA12, MAGEA11, MAGEA10, MAGEA1, MAEL, LY6K, LUZP4, LIPI, LEMD1, LDHC, KIAA0100, IL13RA2, IGSF11, HSPB9, H0RMAD2, H0RMAD1, GPATCH2, GPAT2, GAGE2A, GAGE13, GAGE12J, GAGE12H, GAGE12G, GAGE12F, GAGE12E, GAGE12D, GAGE12C, GAGE12B, GAGE1, FTHL17, FMR1NB,

[0114] 14

[0115] 4916-0122-4820v.8 FBXO39, FATE1, FAM46D, FAM133A, EL0VL4, DSCR8, DPPA2, DNAIB8, DMRT1 , DKKL1, DDX53, DDX43, DCAF12, CTNNA2, CTCFL, CTAGE5, CTAGE1, CTAG2, CTAG1B, CTAG1A, CT83, CT47B1, CT47A9, CT47A8, CT47A7, CT47A6, CT47A5, CT47A4, CT47A3, CT47A2, CT47A11, CT47A10, CT47A1, CT45A6, CT45A5, CT45A3, CT45A2, CT45A1, CSAG2, CSAG1, CRISP2, CPXCR1, COX6B2, CEP55, CEP290, CCDC83, CCDC62, CCDC36, CCDC33, CCDC110, CALR3, CAGE1, CABYR, BRDT, BIRC5, BAGE2, ATAD2, ARX, ARMC3, ANKRD45, AKAP4, AKAP3, ADAM29, ADAM2, ADAM I 2. uPAR, LINE-1, ACTL8. In some embodiments, the cancer expresses somatic mutations such as RAS pathway mutations (KRAS, NRAS), BRAF, JAK, TET2, p53, fusion gene products and other mutations individual to the patient.

[0116]

[0086] In some embodiments, the cancer is small cell lung cancer, colorectal cancer, testicular cancer, ovarian cancer, melanoma, lymphoma, leukemia, multiple myeloma, prostate cancer, breast cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, liver cancer, kidney cancer, head & neck cancer, glioblastoma, neuroblastoma, soft tissue sarcoma, uterine cancer, brain cancer, skin cancer, renal cancer, bladder cancer, pancreatic cancer, thyroid cancer, eye cancer, gastrointestinal cancer, carcinoma, or sarcoma.

[0117]

[0087] In some embodiments, the T cells initially provided for the method of producing the antigen-specific CD4+ cytotoxic T cells are autologous to a subject later administered the antigenspecific CD4+ cytotoxic T cells as a composition or population. In some embodiments, the cells initially provided for the method of producing the antigen-specific CD4+ cytotoxic T cells comprise T cells obtained from a tumor of a subject. In some embodiments, the cells initially provided for the method of producing the antigen-specific CD4+ cytotoxic T cells comprise T cells obtained from blood of a subject. In some embodiments, the cells initially provided for the method of producing the antigen-specific CD4+ cytotoxic T cells comprise T cells obtained from leukapheresis of a subject. In some embodiments, the T cells initially provided for the method of producing the antigen-specific CD4+ cytotoxic T cells are allogeneic (e.g., derived from a healthy donor) to a subject who is later administered the allogeneic antigen-specific CD4+ cytotoxic T cells, e.g., as part of a pharmaceutical composition comprising a population of the cells.

[0118]

[0088] In some embodiments, the antigen-specific CD4+ cytotoxic T cells generated described herein are purified and / or enriched (e.g., based on their binding abilities or other characteristics).

[0119] 15

[0120] 4916-0122-4820v.8 In some embodiments, the antigen-specific CD4+ cytotoxic T cells are a component in a non- naturally occurring composition, for example, a pharmaceutical composition comprising additional components (e.g., carriers, excipients, or solutions). In some embodiments, the composition contains a therapeutic amount of antigen-specific CD4+ cytotoxic T cells. In some embodiments, the composition comprises from 107to 109antigen-specific CD4+ cytotoxic T cells. In some embodiments the composition is a pharmaceutical composition in a dosage form of 5- lOOmL prepared for administration to a subject, e.g. by infusion.

[0121]

[0089] In some embodiments of the methods involving administration to a subject, the administration is via infusion. In some embodiments, administration can be auricular, buccal, conjunctival, cutaneous, subcutaneous, endocervical, endosinusial, endotracheal, enteral, epidural, via hemodialysis, interstitial, intrabdominal, intraamniotic, intra-arterial, intra-articular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavemous, intracavitary, intracerebral, intracisternal, intracorneal, intracoronary, intradermal, intradiscal, intraductal, intraepidermal, intraesophagus, intragastric, intravaginal, intragingival, intraileal, intraluminal, intralesional, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intraepicardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathecal, intrathoracic, intratubular, intratumor, intratympanic, intrauterine, intravascular, intravenous, intraventricular, intravesical, intravitreal, laryngeal, nasal, nasogastric, ophthalmic, oral, oropharyngeal, parenteral, percutaneous, periarticular, peridural, rectal, inhalationally, retrobulbar, subarachnoid, subconjuctival, sublingual, submucosal, topically, transdermal, transmucosal, transplacental, transtracheal, ureteral, uretheral, and vaginal.

[0122] Definitions

[0123]

[0090] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0124] 16

[0125] 4916-0122-4820v.8

[0091] In the discussion unless otherwise stated, adjectives such as “substantially” and “about” modifying a condition or relationship characteristic of a feature or features of an embodiment of the invention, are understood to mean that the condition or characteristic is defined to within tolerances that are acceptable for operation of the embodiment for an application for which it is intended. In embodiments, about means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to + / - 10% of the specified value. In embodiments, about includes the specified value. Unless otherwise indicated, the word “or” in the specification and claims is considered to be the inclusive “or” rather than the exclusive or, and indicates at least one of and any combination of items it conjoins.

[0126]

[0092] It should be understood that the terms “a” and “an” as used above and elsewhere herein refer to “one or more” of the enumerated components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Therefore, the terms “a,” “an” and “at least one” are used interchangeably in this application.

[0127]

[0093] For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0128]

[0094] In the description and claims of the present application, each of the verbs, “comprise,” “include” and “have” and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb. Other terms as used herein are meant to be defined by their well-known meanings in the art.

[0129] General

[0130]

[0095] For the foregoing embodiments, each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments.

[0131] 17

[0132] 4916-0122-4820v.8

[0096] As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections. All combinations of the various elements disclosed herein are within the scope of the invention.

[0133]

[0097] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0134]

[0098] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0135]

[0099] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only.

[0136] EXAMPLES

[0137] Example 1: Enhancing Adoptive Immunotherapy: Targeting Epstein-Barr Virus with Highly Active, Durable and Immunosuppression-resistant Cytotoxic CD4+T Cells.

[0138]

[0100] Background: Prolonged immunosuppression in solid organ transplant (SOT) recipients often triggers reactivation of latent viruses like Epstein-Barr Virus (EBV). This reactivation initiates EBV's growth-transforming program, driving uncontrolled B-cell proliferation manifesting as post-transplant lymphoproliferative disease (PTLD). Rapid restoration of immune competency through adoptive transfer of EBV-specific cytotoxic T cells (EBV-CTLs) is a common treatment strategy. Although EBV-CTLs have shown promise in hematopoietic stem

[0139] 18

[0140] 4916-0122-4820v.8 cell transplant (HSCT) patients, their efficacy in SOT recipients remains limited due to T-cell exhaustion driven by ongoing immunosuppression. This study explores whether the adoptive transfer of EBV-specific Thl / 17 polarized CD4+-CTLs (Thl.l7-CTLs), characterized by their sternness, enhanced self-renewal capacity, and resistance to immunosuppression, could provide a more effective therapeutic approach.

[0141]

[0101] Methods: In this study, we developed a method for ex-vivo generation of novel, highly potent immunosuppression resistant EBV specific Thl .17-CTLs by simultaneously targeting EBV antigens covering all EBV latency origins (Latency EO-III). CD4+T cells from healthy donors were stimulated with PBMCs loaded with overlapping peptide libraries (15-mers) spanning LMP1, LMP2A, and EBNA1 antigens and expanded ex vivo in the presence of pro-inflammatory (Thl / 17- CTLs) cytokines for 12-14 days. Standard Thl-CTLs cells, or EBV-CTLs produced from expansion of Total T cells in neutral conditions for the same period for comparison. Fig. 1 shows schematic of the method in comparison to standard Thl expansion. Following the expansion for 12-14 days, the reactivity against EBV antigens and MDR1 expression, efflux and ability to resist immunosuppression were assessed by flow cytometry. The Cytotoxic ability of Thl.17-CTLs against autologous EBV-Lymphoblastoid cell lines (LCLs) were assessed in vitro and in vivo upon adoptive transfer into tumor-bearing NSG mice. To understand the bioenergetics profde for the T cell persistence assay was performed in Seahorse machine following 14-days of Thl. l7-CTL expansion.

[0142]

[0102] Results: In comparison to EBV-CTLs generated from unselected PBMCs (standard conditions), purified CD4+ expanded in the presence of pro-inflammatory cytokines (Thl.17- CTLs) exhibited significantly higher frequency of antigen specific T cells against all EBV antigens (Fig 2). Additionally, Thl.17 exhibit a robust polyfunctional response concurrently secreting Tumor Necrosis Factor alpha (TNF-a), Interferon Gamma (ZFN-y), Interleukin 2 (IL-2), and inducible Granzyme B (GZMB). Unlike the Thl-CTLs, this polyfunctional response was further amplified following a second stimulation, indicating resistance to functional exhaustion (Data not shown). The Thl.17-CTLs are highly proliferative and functionally, Thl / 17-CTLs demonstrated enhanced recognition and cytotoxicity against autologous EBV lymphoblastoid cell lines (EBV- LCLs) in vitro. Adoptive transfer of Thl.17-CTLs in NSG mice systemically inoculated with EBV-LCLs resulted in significantly reduced tumor burden, correlating with their superior persistence and self-renewal in vivo (Fig. 3). Analysis of the phenotypic markers revealed that

[0143] 19

[0144] 4916-0122-4820v.8 Thl / 17-CTLs contained subsets of cells with less differentiated central memory (TCM) (CCR7hl, CD27hl) and tissue-resident memory (TRM) (CD103hl, CD69hl) phenotype, in contrast to predominantly effector memory (TEM) phenotype of the Thl-CTLs (CCR7lowCD103low) (Data not shown). More importantly, Th.l7-CTLs display elevated expression of Multi-Drug Resistance Protein 1 (MDR1), resulting in elevated drug-efflux which conferred resistance to immunosuppression by tacrolimus at therapeutic levels making them clinically relevant in transplantation setting (Fig 4). The less exhausted Thl.l7-CTL phenotype is further elucidated in their superior mitochondrial health and robust bioenergetic profile. As elucidated by the T cell persistence assay, Thl. l7-CTLs have robust respiratory profile including higher ATP production rate, basal, maximal and spare respiratory capacity (Fig 11).

[0145]

[0103] Conclusion: Ex vivo priming with EBV antigens in the presence of pro-inflammatory cytokines reliably induces EBV-specific Thl. l7-CTLs with markedly enhanced antigenspecificity, polyfunctionality, cytotoxic potential, preserved sternness, proliferative capacity, and superior in vivo persistence and anti-tumor activity. These findings show how improved adoptive immunotherapies can target EBV-associated diseases, especially of Latency-I origin, and can be generalized as a strategy for targeting other viral oncoproteins expressed by human cancers. More importantly, this novel approach of making highly persistent and active immune effectors with enhanced resistance to immunosuppression offers considerable promise to combat key limitations of existing T-cell therapies, paving the way for durable, effective immunotherapies in immunocompromised patients and suggesting broader applications for targeting other viral oncoproteins.

[0146] Example 2: Multi-epitope specific cytotoxic CD4+T cells for adoptive immunotherapy of myelodysplastic syndrome.

[0147]

[0104] Background: Myelodysplastic Syndromes (MDS) are heterogeneous clonal hematological disorders marked by ineffective multi-lineage hematopoiesis and frequent transformation into acute myeloid leukemia (AML).

[0148]

[0105] Patients with MDS have limited therapeutic options, as curative hematopoietic stem cell transplant (HSCT) is reserved only for younger patients and hypomethylating agents offer modest benefit to HSCT-ineligible patients. Thus, more effective therapies are needed.

[0149] 20

[0150] 4916-0122-4820v.8

[0106] CD4+ T cells are often present in tumor infiltrating lymphocytes (TILs) and TIL-like products specific for tumor associated antigens (TAAs), displaying antigen-specific activity but have not been formally studied despite their central role in orchestrating the immune response.

[0151]

[0107] Here, the feasibility of generating autologous TIL-like multi-TAA-specific CD4+ cells (Thl / 17 CTLs) with enhanced potency for high-risk MDS and AML Patients was studied.

[0152]

[0108] Aim: Generation of multi-TAA specific polyfunctional, cytotoxic autologous CD4+ T cells from MDS / AML patients using novel pro-inflammatory priming strategy that generates cell product with highly desirable stem-cell like properties and robust proliferative capacity, necessary for immunotherapy.

[0153]

[0109] Methods: Purified Naive or Total CD4 + T cells were stimulated using monocyte- derived dendritic cells (MoDCs) pulsed with overlapping peptide libraries spanning PRAME, WT- 1, NYESO-1, MAGE-A3, MAGE A4 and CT45-A1.

[0154]

[0110] Cultured under standard conditions or proinfl ammatory cytokines for 24-28 days with two rounds of stimulation.

[0155]

[0111] Phenotypes, reactivity, and antigen recognition capability of final CD4+CTLs were assessed.

[0156]

[0112] Ability to reduce leukemic tumor burden was tested using fully HL A matched patient and donor via adoptive transfer in NSG mice.

[0157]

[0113] A schematic of the methods is shown in Fig. 1.

[0158]

[0114] Results:

[0159]

[0115] 1) Expansion of CD4+T cells in presence of pro-inflammatory cytokines produces TAA specific Thl / 17-CTLs with superior reactivity, as shown in Fig. 5.

[0160]

[0116] 2) Thl / 17-CTLs maintain polyfunctionality despite repetitive stimulations, as shown in Fig. 6.

[0161]

[0117] 3) Thl / 17-CTLs have superior proliferative capacity, as shown in Fig. 7

[0162]

[0118] 4) Thl / 17-CTLs have less differentiated Central (TCM) and Resident (TRM) memory phenotype, as shown in Fig. 8.

[0163] 21

[0164] 4916-0122-4820v.8

[0119] 5) Thl / 17-CTLs recognize naturally processed in vitro and in vivo, as shown in Fig. 9.

[0165]

[0120] 6) Successful generation of autologous Multi-TAA specific Thl / 17-CTLs from MDS patients, as shown in Fig. 10.

[0166]

[0121] Conclusions: Our priming strategy reliably induces TAA-reactive Thl / 17-CTLs with markedly enhanced polyfunctionality and cytotoxicity.

[0167]

[0122] The strategy generates a cell product that differs from natural examples, or those produced under neutral cytokine conditions, by exhibiting less differentiated phenotype and robust proliferative capacity despite repetitive stimulation.

[0168]

[0123] These cells are clinically relevant and can recognize their target antigens in vitro and in vivo.

[0169]

[0124] Since the strategy is epitope- and MHC-agnostic, it is applicable to diverse group of patients, and easily tailored based on patient’s mutational landscape.

[0170]

[0125] Summary: Here, we studied the feasibility of generating autologous TIL-like multi- TAA-specific CD4 T cells with enhanced potency for high-risk patients with MDS and AML. We evaluated a novel ex vivo priming strategy to generate highly active CD4 cytotoxic T cells (CD4 CTLs) simultaneously targeting multiple TAAs. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors or MDS patients collected under the IRB-approved protocol (AAAF2693). Total CD4 T cells or naive CD4 T cells were purified and cocultured with autologous monocyte derived dendritic cells (MoDCs) pulsed with overlapping peptide libraries spanning PRAME, WT1, NYESO-1, and MAGE- A3 and expanded with conventional cytokines or in the presence of pro-inflammatory cytokines for 10-14 days. Two rounds of stimulation were performed. The reactivity, expansion and proliferative capacity, phenotyping upon repetitive stimulation were assessed. The ability of the final CD4 CTLs to recognize naturally processed targets was tested by loading autologous antigen presenting cells with full length recombinant proteins. T cells cultured under Type 1 / 17 conditions displayed significantly increased reactivity against TAAs compared to T cells cultured under conventional conditions (defined here as Thl). Furthermore, Thl / 17 antigen-specific reactivity was markedly enhanced by repetitive stimulation while Thl cells showed gradual reduction in the secretion of effector cytokines. Thl / 17 cells also expanded significantly better than Thl cells (displayed enhanced polyfunctionality evidenced by

[0171] 22

[0172] 4916-0122-4820v.8 increased IL-2 and Granzyme B secretion indicative of self-renewal and cytotoxicity, respectively). In CFSE assay Thl / 17 cells showed marked proliferative capacity despite long term culture and repetitive stimulation. These cells also recognized naturally processed antigen indicating these cells are clinically relevant. Next, we successfully generated multi- TAA CTLs cells from the PBMCs of multiple MDS patients where Thl / 17 cells displayed superior reactivity, polyfunctionality and proliferative capacity, as seen in healthy subjects. In conclusion, ex vivo priming with TAAs in the presence of pro-inflammatory cytokines reliably induces multi-TAA CD4 CTLs.

[0173] Example 3: Additional data showing the generation of TILs from resected tumor fragments, and the differentiation status of the manufactured T cells.

[0174]

[0126] Tumor infiltrating lymphocytes (TILs) made using the strategy described herein is shown in Fig. 13. Specifically, generation of TILs from head and neck cancer are shown. Thl.17 TILs were generated from viably frozen tumor fragments in presence of IL-la, IL-7, IL-6, IL-21, TGFpi. Media was also supplemented with low concentration of IL-2 (100-200IU / ml). Standard (control Thl) TILs were generated in presence of media supplemented with IL-2 at 3000IU / ml.

[0175]

[0127] Memory phenotype and exhaustion marker expression status of the T cells manufactured using the method described herein is shown.

[0176]

[0128] Example 4: Targeting EBV viral oncoproteins with novel ex vivo generated highly active CD4+T cells displaying enhanced cytotoxicity and self-renewal properties.

[0177]

[0129] Background: Epstein-Barr virus (EBV), infects a majority of humans, establishing a lifelong latency controlled by immune surveillance. Impaired cellular immunity can lead to viral reactivation and B cell lymphoproliferation manifesting as post-transplant lymphoproliferative disease (PTLD) and malignant transformation into B-cell lymphomas and some epithelial tumors. Adoptive transfer of virus-specific cytotoxic T cells targeting viral oncoproteins of EBV (EBV- CTLs) may lead to rapid reconstitution of T cell immune surveillance and may efficiently control PTLD and some EBV-driven lymphomas. However, success of this approach is not universal and might be hampered by suboptimal / variable potency of donor-derived EBV-CTLs and possibly their terminally differentiated / exhausted phenotype associated with limited in vivo persistence and activity. We hypothesized that that EBV-specific CD4+T cells differentiated using novel pro-

[0178] 23

[0179] 4916-0122-4820v.8 inflammatory conditions might efficiently target viral oncoproteins concurrently retaining highly desirable stem-cell like properties required for successful eradication of tumor cells in vivo.

[0180]

[0130] Methods: We developed a method for generating novel and highly potent CD4+cytotoxic T cells (CD4+-CTLs) simultaneously targeting EBNA1, LMP1, and LMP2A. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors, and either total T cells or CD4+T cells were purified. These cells were then stimulated with PBMCs loaded with overlapping peptide libraries spanning LMP1, LMP2A, and EBNA1 antigens, either under neutral conditions (IL2 and IL-7) or in the presence of pro-inflammatory cytokines ex vivo for 12-14 days. The phenotypes, reactivity, and cytotoxic capability of these CD4+-CTLs against autologous EBV- Lymphoblastoid cell lines (LCLs) were assessed in vitro and in vivo upon adoptive transfer into tumor-bearing NSG mice.

[0181]

[0131] Results: In comparison to EBV-CTLs generated from otherwise unselected PBMCs (standard conditions) and Th, purified CD41expanded in the presence of pro-inflammatory cytokines exhibited significantly higher frequency of antigen specific T cells with a marked skewing towards EBNA1 antigen and displaying polyfunctionality, concurrently secreting tumor necrosis factor-a (TNF-a), interferon-v (IFN-y), and inducible granzyme B (GzmB), while maintaining interleukin-2 (IL -2) secretion capacity. Antigen-reactive CD4+-CTLs induced in pro- inflammatory conditions retained a significant population with central memory (TCM) phenotype (CCR7hl, CD27hl), while those expanded in neutral conditions predominantly displayed an effector memory (TEM) phenotype. These CD4 -CTLs demonstrated enhanced cytotoxicity against autologous EBV-LCLs and superior proliferative capacity in vitro upon secondary stimulation, whereas standard EBV-CTLs proliferated poorly. In the LCL-injected NSG mice, CD4+-CTLs expanded under pro-inflammatory conditions exhibited superior self-renewal and long-term persistence, resulting in a significant reduction / eradi cation of LCL burden when compared to standard EBV-CTLs.

[0182]

[0132] Conclusion: This is a reliable strategy for ex vivo expansion of highly active EBV- specific CD4+-CTLs. The approach induces EBV-reactive CD4+-CTLs with enhanced antigenspecificity, polyfunctionality, cytotoxic potential, preserved sternness, proliferative capacity, and superior in vivo persistence and anti-tumor activity.

[0183] 24

[0184] 4916-0122-4820v.8 Example 5: Thl / 17-like CD4+Cytotoxic T lymphocytes: A Novel Approach of Producing Highly Functional, Durable, and Immunosuppression Resistant Immune Effectors Targeting Epstein-Bar Virus.

[0185]

[0133] Solid organ transplantation (SOT) requires prolonged / life-long immunosuppression, often causing Epstein-Barr Virus (EBV) reactivation and post-transplant lymphoproliferative disease (EBV-PTLD). Adoptive transfer of EBV-specific cytotoxic T cells (EBV-CTLs) may rapidly restore immune competency and control EBV-PTLD. However, clinical efficacy is often limited by exhaustion of in vitro expanded CTLs (mostly CD8+) and / or by ongoing immunosuppression. Here, we hypothesized that EBV-specific Thl / 17-like CD4+-CTLs programmed to maintain high potency, self-renewal, and resistance to immunosuppression could be a superior therapeutic strategy.

[0186]

[0134] Ex vivo generated EBV-specific CD4+-CTLs (Thl / 17-CTLs) demonstrated superior recognition and polyfunctional responses, concurrently secreting TNF-a, IFN-y, IL-2, and inducible Granzyme B against EBV-antigens compared to the standard Thl-CTLs. Thl / 17-CTLs displayed less differentiated central memory (TCM) (CCR7hl, CD27hl) and resident memory (TRM) (CD103111, CD69hl) phenotype, unlike the predominantly effector memory (TEM) (CCR71OW, CD103low) Thl-CTLs. Paradoxically, a second stimulation further enhanced Thl / 17-CTLs’ polyfunctionality, robust proliferative capacity, superior bioenergetic / mitochondrial stability, and spare respiratory capacity, unlike the Thl-CTLs.

[0187]

[0135] Importantly, Thl / 17-CTLs overexpressed Multi -Drug Resistance Protein 1 (MDR1) and displayed resistance to tacrolimus-mediated immunosuppression. Functionally, Thl / 17-CTLs exhibited superior recognition and cytotoxicity against autologous EBV lymphoblastoid cell lines (EBV-LCLs). Adoptive transfer of Thl / 17-CTLs led to the eradication of systemic PTLD-like disease in humanized mice, demonstrating in vivo persistence and superior self-renewal ability without cytokine support. This study introduces a novel, human-made, highly functional, nonexhausted oncoprotein-specific T cell population with superior cytotoxic capability and resistance to immunosuppression, addressing key prior art limitations of existing T-cell therapies in immunocompromised patients.

[0188] 25

[0189] 4916-0122-4820v.8 Example 6: Decoding and Targeting the CMML Antigenic Landscape with novel, highly effective multi-epitope specific cytotoxic CD4+ T cells.

[0190]

[0136] Background: With a median survival of 15-30 months and a lack of effective treatment, novel therapies for chronic myelomonocytic leukemia (CMML) are urgently needed. Current options are rarely curative, as hypomethylating agents offer low response rates and allogeneic stem cell transplantation is not available to most. Adoptive T-cell therapy (ACT) is a potent strategy against other refractory cancers. However, it has not been investigated for CMML. Therefore, for the first time, we investigated the landscape of common TAAs and personalized neoantigens and subsequently established the feasibility of generating novel multi-epitope specific cytotoxic CD4+ T cells directed against identified CMML targets.

[0191]

[0137] Results: Gene expression profiling identified 14 Cancer-Testis Antigens (CTAs) overexpressed in at least 40% of patients, with PRAME, SPACA3, and MAGEA4 being most frequent. Importantly, the top 7 antigens were co-expressed in 50% of subjects, and common immunogenic mutations (e.g., KRAS, NRAS, ASXL1, TET2) were also present, suggesting the possibility of generating multi-TAA products concurrently targeting multiple antigens.

[0192]

[0138] Robust and highly polyfunctional T cell responses against CTAs (including PRAME, MAGE-A1, A3, A4, CT45A1 & WT1) and selected neoantigens were induced under Thl.17 conditions demonstrating superior reactivity, while retaining bioenergetic stability and marked proliferative capacity despite repetitive stimulation vs. Thl counterparts. Moreover, novel Thl.17 T cells also recognized naturally processed tumor targets, and were reliably generated even from pancytopenic MDS patient samples.

[0193]

[0139] Conclusion: This study provides a critical proof-of-concept for T-cell therapy in CMML. We not only identified the landscape of targetable tumor antigens but also developed a superior method for generating potent, multi-specific cytotoxic CD4+ T-cells. The enhanced functionality and durability of our Thl.17 cells, along with the demonstration that they can be generated from even the most vulnerable patients, shows a multi-epitope specific T-cell strategy for therapy.

[0194]

[0140] Example 7: Generation of highly-active neoantigen-specific Thl.17 CD4+ CTLs targeting hotspot mutations in KRAS. Fig. 12 shows superior ability to induce neoantigen CD4+ CTLs using the novel approach.

[0195] 26

[0196] 4916-0122-4820v.8 References:

[0197]

[0141] 1. Solary, E. & Itzykson, R. How I treat chronic myelomonocytic leukemia. Blood, The Journal of the American Society of Hematology 130, 126-136 (2017).

[0198]

[0142] 2. Renneville, A., Patnaik, M. M., Chan, O., Padron, E. & Solary, E. Increasing recognition and emerging therapies argue for dedicated clinical trials in chronic myelomonocytic leukemia. Leukemia 35, 2739-2751 (2021).

[0199]

[0143] 3. Rezazadeh, A., Deininger, M. & Atallah, E. Proposals for Clinical Trials in Chronic Myelomonocytic Leukemia. Curr Treat Options Oncol, doi: 10.1007 / sl 1864-023-01105-z (2023).

[0200]

[0144] 4. Lopez-Bujanda ZA, Obradovic A, Nirschl TR, Crowley L, Macedo R, Papachristodoulou A, O'Donnell T, Laserson U, Zarif JC, Reshef R, Yuan T, Soni MK, Antonarakis ES, Haffner MC, Larman HB, Shen MM, Muranski P, Drake CG. TGM4: an immunogenic prostate-restricted antigen. J Immunother Cancer. 2021 Jun;9(6):e001649. doi: 10.1136 / jitc-2020-001649. PMID: 34193566; PMCID: PMC8246381.

[0201]

[0145] 5. Prabesh Khatiwada, Mithil Soni, Pawel Muranski, Highly Active Cytotoxic CD4 + T Cells Targeting EBV Oncoproteins with Enhanced In Vivo Anti-Tumor and Self-Renewal Properties, Blood, Volume 142, Supplement 1, 2023, Page 3464, ISSN 0006-4971, https: / / doi.org / 10.1182 / blood-2023-190690.

[0202]

[0146] 6. Mithil Soni, Prabesh Khatiwada, Pawel Muranski. Multi-epitope specific cytotoxic CD4+ T cells for adoptive immunotherapy of myelodysplastic syndrome. 27th American Society of Gene & Cell therapy. May 7-11, 2024, Baltimore, MD, USA.

[0203]

[0147] 7. Soni M, Migliori E, Fu j, Assal A, Chan H, Ciubotariu R, Pan J, Mapara M, Sykes M, Muranski P. Reciprocal and non-reciprocal T cell responses against SARS-CoV2 and common human coronaviruses promoting the development of multi-coronavirus specific-T cells for adoptive immunotherapy for immunocompromised patients with COVID-19 and beyond. Front. Immunol, Volume 14, 2023. doi: 10.3389 / fimmu.2023.1212203.

[0204] 27

[0205] 4916-0122-4820v.8

Claims

CLAIMSWhat is claimed is:

1. A method for generating a population of antigen-specific CD4+cytotoxic T cells comprising: a) providing a plurality of CD4+T cells; b) stimulating the CD4+T cells with at least one tumor associated-antigen (TAA) peptide, and / or at least one viral peptide, preferably at least one peptide derived from a viral oncoprotein; and c) expanding the CD4+T cells in the presence of at least one pro-inflammatory cytokine, so as to produce a population of antigen-specific CD4+cytotoxic T cells.

2. The method of claim 1, wherein the stimulating comprises exposing the CD4+T cells to an overlapping peptide library.

3. The method of claim 1 or 2, wherein the at least one tumor associated-antigen (TAA) peptide comprises at least one of a Cancer-Testis Antigen (CTA) peptide, Preferentially Expressed Antigen in Melanoma (PRAME) peptide, New York esophageal squamous cell carcinoma (NYES01) peptide, Cancer / Testis Antigen Family 45 Member Al (CT45A1) peptide, Melanoma-associated antigen 3 (MAGE-A3) peptide, MAGE-A1, MAGE-A4, and Wilms’ tumor 1 (WT-1) peptide, and / or a ACRBP, ZNF165, ZNF645, XAGE5, XAGE3, XAGE2, XAGE1B, WT1, VENTXP1, TULP2, TTK, TSSK6, TSPY3, TSPY2, TSGA10, TPTE, TPPP2, TMPRSS12, TMEM108, TMEFF2, TMEFF1, THEG, TFDP3, TEX15, TEX14, TEX101, TEKT5, TDRD6, TDRD1, TAF7L, SYCP1, SYCE1, SSX9, SSX7, SSX6, SSX5, SSX4B, SSX4, SSX3, SSX2, SSX1, SPO11, SPEF2, SPATA19, SPANXN5, SPANXN4, SPANXN3, SPANXN2, SPANXN1, SPANXD, SPANXC, SPANXB1, SPANXA2, SPANXA1, SPAG9, SPAG8, SPAG6, SPAG4, SPAG17, SPAG1, SPACA3, SPA17, SLCO6A1, SEMG1, SAGE1, R0PN1, RGS22, RBM46, PRSS55, PRSS54, PRM2, PRM1, PRAME, POTEH, POTEG, POTEE, POTED, POTEC, POTEB, POTEA, PLAC1, PIWIL2, PBK, PASD1, PAGE5, PAGE4, PAGE3, PAGE2B, PAGE2, PAGE1, OTO A,284916-0122-4820v.80TP5, 0DF4, 0DF3, 0DF2, 0DF1, NXF2B, NXF2, NR6A1, NOL4, NLRP4, M0RC1 , MAGEC3, MAGEC2, MAGECI, MAGEB6, MAGEB5, MAGEB4, MAGEB3, MAGEB2, MAGEB1, MAGEA9B, MAGEA9, MAGEA8, MAGEA6, MAGEA5, MAGEA4, MAGEA3, MAGEA2B, MAGEA2, MAGEA12, MAGEA11, MAGEA10, MAGEA1, MAEL, LY6K, LUZP4, LIPI, LEMD1, LDHC, KIAA0100, IL13RA2, IGSF11, HSPB9, HORMAD2, HORMAD1, GPATCH2, GPAT2, GAGE2A, GAGE13, GAGE12J, GAGE12H, GAGE12G, GAGE12F, GAGE12E, GAGE12D, GAGE12C, GAGE12B, GAGE1, FTHL17, FMR1NB, FBXO39, FATE1, FAM46D, FAM133A, ELOVL4, DSCR8, DPPA2, DNAJB8, DMRT1, DKKL1, DDX53, DDX43, DCAF12, CTNNA2, CTCFL, CTAGE5, CTAGE1, CTAG2, CTAG1B, CTAG1A, CT83, CT47B1, CT47A9, CT47A8, CT47A7, CT47A6, CT47A5, CT47A4, CT47A3, CT47A2, CT47A11, CT47A10, CT47A1, CT45A6, CT45A5, CT45A3, CT45A2, CT45A1, CSAG2, CSAG1, CRISP2, CPXCR1, COX6B2, CEP55, CEP290, CCDC83, CCDC62, CCDC36, CCDC33, CCDC110, CALR3, CAGE1, CAB YR, BRDT, BIRC5, BAGE2, ATAD2, ARX, ARMC3, ANKRD45, AKAP4, AKAP3, ADAM29, ADAM2, ADAM I 2. uPAR, ACTL8, or Long interspersed nuclear element- 1 (LINE-1) peptide.

4. The method of any one of claims 1-3, wherein the viral oncoprotein is an Ebstein-Barr Virus oncoprotein, preferably at least one of Epstein-Barr nuclear antigen 1 (EBNA1), Latent Membrane Protein 1 (LMP1), and Latent Membrane Protein 2 A (LMP2A), a papilloma virus (HPV) E6 antigen, an HPV E7 antigens, hepatitis B virus (HBV) Hepatitis B X protein (HBx), a PreS / S protein, a hepatitis C virus (HCV) core protein, NS3 / NS4A, NS5A, Merkel Cell Polyomavirus (MCpyV) Large T (LT) antigen, MCpyV small T (ST) antigen, Human Herpes Virus 8 (HHV8) Latency-Associated Nuclear Antigen (LANA), a vCyclin antigen, a vFLIP antigen, vGPCR antigen, Human T-lymphotropic virus 1 (HTLV-1) Tax antigen, a Human T-lymphotropic virus 2 (HTLV-2) Tax antigen, Simian Virus 40 (SV40) Large T (T-ag) antigen, and SV40 Small T antigen (t-ag).

5. The method of any one of claims 1-4, wherein stimulating the CD4+T cells further comprises co-culturing the CD4+T cells with antigen presenting cells (APCs), peripheral blood mononuclear cells (PBMCs), and / or monocyte-derived dendritic cells (MoDCs),294916-0122-4820v.8wherein said APCs, PBMCs, and / or MoDCs have been exposed to the at least one tumor associated-antigen (TAA) peptide, and / or at least one viral peptide.

6. The method of any one of claims 1-5, wherein the provided CD4+T cells and / or APCs, PBMCs, and / or MoDCs are autologous cells isolated from a single subject.

7. The method of any one of claims 1-6, wherein the at least one pro-inflammatory cytokine is at least one of IL-10, IL-6, IL-7, IL-15, IL-21, IL-23, and / or TGF-0.

8. The method of any one of claims 1-7, wherein the CD4+T cells are exposed to the at least one proinflammatory cytokine about 72 hours after the stimulating.

9. The method of any one of claims 1-8, wherein the provided plurality of CD4+T cells are naive.

10. The method of any one of claims 1-9, wherein the expanding comprises culturing the stimulated CD4+T cells with the at least one proinflammatory cytokine for about 7-35 days, about 10-31 days, about 10-14 days, about 10-12 days, or about 24-28 days.

11. The method of any one of claims 1-10, further comprising repeating the stimulating step at least once, preferably within 7-14 days of first performing the stimulating step, more preferably at about 10 days after first performing the stimulating step.

12. The method of claim 11, wherein the CD4+T cells are exposed to the at least one pro- inflammatory cytokine within about 72 hours of each stimulation.

13. The method of any one of claims 1-12, wherein the population of antigen-specific CD4+cytotoxic T cells produced by the method are tumor infiltrating leukocytes, tumor infiltrating leukocyte (TIL)-like cells, and / or multi-tumor associated-antigen (TAA)- specific CD4+cytotoxic T cells; or wherein the population of antigen-specific CD4+cytotoxic T cells produced by the method are EBV-specific cytotoxic T cells (EBV-CTLs).

14. The method of any one of claims 1-13, further comprising purifying the population of antigen-specific CD4+cytotoxic T cells so as to increase the percentage of cells that are antigen-specific CD4+cytotoxic T cells.304916-0122-4820v.

815. The method of any one of claims 1 -14, wherein the provided CD4+T cells are isolated from a subject suffering from, or at risk of suffering from, a myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML), post-transplant lymphoproliferative disease (PTLD), a B-cell or T cell lymphoma, a Latency 1 associated disease, a Latency 2 associated disease, a Latency 3 associated disease, EBV reactivation or infection, a virus-associated cancer or disease, a urothelial carcinoma, an epithelial cancer, a sarcoma, a glioma, and / or a solid tumor; or or wherein the provided CD4+T cells are isolated from a subject who has undergone, or is expected to undergo, a solid organ transplantation and / or immunosuppression therapy or a subject who is immunodeficient.

16. A pharmaceutical composition comprising an antigen-specific CD4+cytotoxic T cell generated by the method of any one of claims 1-15.

17. A population of cells comprising the antigen-specific CD4+cytotoxic T cells generated by the method of any one of claims 1-15.

18. A method of treating, ameliorating, or reducing development of a cancer or disease in a subject comprising administering the pharmaceutical composition of claim 16, or the population of claim 17, to the subject.

19. A method of treating, ameliorating, or reducing development a disease in an immunosuppressed subject or a subject on active chemotherapy comprising administering the pharmaceutical composition of claim 16, or the population of claim 17, to the subject.

20. An adoptive T-cell therapy for treating a cancer comprising administering the pharmaceutical composition of claim 16, or the population of claim 17, to a subject having the cancer, so as to treat the cancer.314916-0122-4820v.8

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