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WO-2026096415-A1 - EX VIVO GENERATED ANTIGEN-SPECIFIC CD4+ T CELLS WITH ENHANCED SELF-RENEWAL AND CYTOTOXIC ACTIVITY FOR IMMUNOTHERAPY OF CANCER

WO2026096415A1WO 2026096415 A1WO2026096415 A1WO 2026096415A1WO-2026096415-A1

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.

Inventors

  • MURANSKI, Pawel
  • SONI, Mithil
  • KHATIWADA, Prabesh

Assignees

  • THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF NEW YORK

Dates

Publication Date
20260507
Application Date
20251028
Priority Date
20241028

Claims (20)

  1. 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. 2. The method of claim 1, wherein the stimulating comprises exposing the CD4 + T cells to an overlapping peptide library.
  3. 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, 28 4916-0122-4820v.8 0TP5, 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. 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. 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), 29 4916-0122-4820v.8 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.
  6. 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. 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. 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. 9. The method of any one of claims 1-8, wherein the provided plurality of CD4 + T cells are naive.
  10. 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. 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. 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. 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. 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. 30 4916-0122-4820v.8
  15. 15. 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. 16. A pharmaceutical composition comprising an antigen-specific CD4 + cytotoxic T cell generated by the method of any one of claims 1-15.
  17. 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. 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. 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. 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. 31 4916-0122-4820v.8

Description

Docket: 93597/7387 (92420-A-PCT) EX VIVO GENERATED ANTIGEN-SPECIFIC CD4+ T CELLS WITH ENHANCED SELF-RENEWAL AND CYTOTOXIC ACTIVITY FOR IMMUNOTHERAPY OF CANCER AND VIRAL DISEASES [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. CROSS-REFERENCE TO RELATED APPLICATIONS [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. BACKGROUND OF THE INVENTION [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. BRIEF SUMMARY OF THE INVENTION [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. 1 4916-0122-4820v.8 [5] A pharmaceutical composition comprising an antigen-specific CD4+ cytotoxic T cell generated by a method described herein. [6] A population of cells comprising the antigen-specific CD4+ cytotoxic T cells generated by a method described herein. [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. [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. [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. [10] 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. BRIEF DESCRIPTION OF THE DRAWINGS [11] 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. [12] 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 2 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). [13] 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. [14] 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. [15] 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