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WO2025072717A1 - Traitement du cancer - Google Patents

Traitement du cancer Download PDF

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Publication number
WO2025072717A1
WO2025072717A1 PCT/US2024/048918 US2024048918W WO2025072717A1 WO 2025072717 A1 WO2025072717 A1 WO 2025072717A1 US 2024048918 W US2024048918 W US 2024048918W WO 2025072717 A1 WO2025072717 A1 WO 2025072717A1
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aire
polypeptide
cancer
mammal
cells
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Richard G. Vile
Jose S. Pulido
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Mayo Foundation for Medical Education and Research
Mayo Clinic in Florida
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Mayo Foundation for Medical Education and Research
Mayo Clinic in Florida
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/19—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/24—Antigen-presenting cells [APC]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/32—T-cell receptors [TCR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • a level of an autoimmune regulator (AIRE) polypeptide within a mammal can be altered (e.g., increased or decreased) to expose one or more epitopes of a tumor-specific antigen within a cancer cell present in the mammal (e g., such that the mammal produces an immune response against the cancer cell).
  • AIRE autoimmune regulator
  • one or more modulators of an AIRE polypeptide level can be administered to a mammal having cancer (e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors) to treat the mammal.
  • TSA tissue-specific self-antigens
  • mTECs medullary thymic epithelial cells
  • a cancer within in a patient that presents to a treatment clinic is, by definition, heavily selected for immune evasion. This is, in part at least, due to the profile of polypeptide epitopes displayed by the major histocompatibility complex (MHC) Class I molecules of the cancer cells.
  • MHC major histocompatibility complex
  • some cancers respond to immune checkpoint inhibitors while other cancers do not (Jhunjhunwala et al., Nat. Rev. Cancer, 21(5):298-312 (2021)).
  • AIRE plays a fundamental role in the immuno-tolerance process by inducing expression of TSA in mTECs.
  • the relationship between the level of an AIRE polypeptide and T cell targeting can depend on the nature of the antigen be targeted.
  • a level of an AIRE polypeptide within a mammal can be altered (e.g., increased or decreased) to expose one or more epitopes of a tumor-specific antigen (e.g., an antigenic substance produced by a cancer cell) within a cancer cell (e.g., a cancer cell that exhibits little or no response to treatment with immune checkpoint inhibitors) present in the mammal (e.g., such that the mammal produces an immune response against the cancer cell).
  • a tumor-specific antigen e.g., an antigenic substance produced by a cancer cell
  • a cancer cell e.g., a cancer cell that exhibits little or no response to treatment with immune checkpoint inhibitors
  • one or more modulators of an AIRE polypeptide level can have the ability to induce immune responses against cancer cells (e.g., cancer cells that exhibit little or no response to treatment with immune checkpoint inhibitors) within a mammal (e.g., a human).
  • cancer cells e.g., cancer cells that exhibit little or no response to treatment with immune checkpoint inhibitors
  • a mammal e.g., a human
  • one or more modulators of an AIRE polypeptide level can be administered to a mammal having cancer (e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors) to treat the mammal.
  • cancer e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors
  • an AIRE polypeptide can alter the profde of epitopes displayed on MHC molecules of the cancer cell and presented to T cells. Also as demonstrated herein, altering (e.g., increasing or decreasing) a level of an AIRE polypeptide can alter the immunogenicity of the cancer cell.
  • one or more AIRE polypeptides can be administered to a mammal (e.g., a human) having cancer (e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors) to treat the mammal.
  • a mammal e.g., a human
  • cancer e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors
  • one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) can be administered to a mammal (e.g., a human) having cancer including one or more cancer cells that express one or more AIRE- regulated antigens to increase the presentation of epitopes of one or more AIRE-regulated antigens, thereby treating the mammal.
  • one or more inhibitors of an AIRE polypeptide can be administered to a mammal (e.g., a human) having cancer (e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors) to treat the mammal.
  • a mammal e.g., a human
  • cancer e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors
  • one or more inhibitors of an AIRE polypeptide can be administered to a mammal (e.g., a human) having cancer including one or more cancer cells that do not express AIRE-regulated antigens to increase the presentation of epitopes of one or more AIRE-independent antigens, thereby treating the mammal.
  • the ability to expose one or more epitopes of a tumor antigen in a cancer cell of a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors as described herein e.g., by administering one or more modulators of an AIRE polypeptide level (e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide) to the mammal) provides a unique opportunity to induce an immune response against cancer cells that are typically undetectable by the immune system as well as an opportunity to use immunotherapy to target such cancer cells.
  • an AIRE polypeptide level e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • one aspect of this document features methods for treating a mammal having cancer
  • the methods can include, or consist essentially of, administering an AIRE polypeptide or nucleic acid designed to express said AIRE polypeptide to said mammal, wherein said cancer comprises cancer cells expressing an AIRE-regulated antigen.
  • the mammal can be a human.
  • the cancer can be a diffuse midline glioma (DMG), a melanoma, a hepatobiliary cancer, a breast cancer, a lung cancer, or a prostate cancer.
  • the method can include administering said AIRE polypeptide to said mammal.
  • the method can include administering said nucleic acid designed to express said AIRE polypeptide to said mammal.
  • the AIRE-regulated antigen can be a TYRP2 polypeptide, a CSDE1 polypeptide, or a gplOO polypeptide.
  • the method can include administering an immune checkpoint inhibitor to said mammal.
  • the immune checkpoint inhibitor can be an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-CTL4A antibody, an anti -LAG-3 antibody, an anti-TIM3 antibody, or an anti-TIGIT antibody.
  • the immune checkpoint inhibitor can be pembrolizumab, nivolumab, cemiplimab, ipilimumab, tremelimumab, durvalumab, dostarlimab, avelumab, atezolizumab, or relatlimab.
  • the immune checkpoint inhibitor can be BMS-8, BMS-37, BMS-202, BMS-230, BMS-242, BMS-1001, BMS-1166, SB415286, vorinostat, decitabine, entitostat, JQ1, BET151, GSK503, panobinostat, ACY-241, azacytidine, DB36, DB71, DB15, CVN, MGCD0103, SNDX-275, IMP32, BMS986016, TSR-022, Sym023, ATIK2a, or DZNep.
  • this document features methods for converting a mammal having cancer resistant to treatment with an immune checkpoint inhibitor into a mammal having cancer susceptible to treatment with said immune checkpoint inhibitor
  • the methods can include, or consist essentially of, administering an AIRE polypeptide or nucleic acid designed to express said AIRE polypeptide to said mammal, wherein said cancer comprises cancer cells expressing an AIRE-regulated antigen.
  • the mammal can be a human.
  • the cancer can be a DMG, a melanoma, a hepatobiliary cancer, a breast cancer, a lung cancer, or a prostate cancer.
  • the method can include administering said AIRE polypeptide to said mammal.
  • the method can include administering said nucleic acid designed to express said AIRE polypeptide to said mammal.
  • the AIRE-regulated antigen can be a TYRP2 polypeptide, a CSDE1 polypeptide, or a gplOO polypeptide.
  • the immune checkpoint inhibitor can be an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-CTL4A antibody, an anti-LAG-3 antibody, an anti-TIM3 antibody, or an anti-TIGIT antibody.
  • the immune checkpoint inhibitor can be pembrolizumab, nivolumab, cemiplimab, ipilimumab, tremelimumab, durvalumab, dostarlimab, avelumab, atezolizumab, or relatlimab.
  • the immune checkpoint inhibitor can be BMS-8, BMS-37, BMS-202, BMS-230, BMS-242, BMS-1001, BMS-1166, SB415286, vorinostat, decitabine, entitostat, JQ1, BET151, GSK5O3, panobinostat, ACY-241, azacytidine, DB36, DB71, DB15, CVN, MGCD0103, SNDX-275, IMP32, BMS986016, TSR-022, Sym023, ATIK2a, or DZNep.
  • this document features methods for treating a mammal having cancer
  • the methods can include, or consist essentially of, (a) administering an AIRE polypeptide or nucleic acid designed to express said AIRE polypeptide to said mammal, and (b) administering an immune checkpoint inhibitor to said mammal; wherein said cancer comprises cancer cells expressing an AIRE-regulated antigen.
  • the mammal can be a human.
  • the cancer can be a DMG, a melanoma, a hepatobiliary cancer, a breast cancer, a lung cancer, or a prostate cancer.
  • the method can include administering said AIRE polypeptide to said mammal.
  • the method can include administering said nucleic acid designed to express said AIRE polypeptide to said mammal.
  • the AIRE-regulated antigen can be a TYRP2 polypeptide, a CSDE1 polypeptide, or a gplOO polypeptide.
  • the immune checkpoint inhibitor can be an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-CTL4A antibody, an anti-LAG-3 antibody, an anti-TIM3 antibody, or an anti-TIGIT antibody.
  • the immune checkpoint inhibitor can be pembrolizumab, nivolumab, cemiplimab, ipilimumab, tremelimumab, durvalumab, dostarlimab, avelumab, atezolizumab, or relatlimab.
  • the immune checkpoint inhibitor can be BMS-8, BMS-37, BMS-202, BMS-230, BMS-242, BMS-1001, BMS-1166, SB415286, vorinostat, decitabine, entitostat, JQ1, BET151, GSK5O3, panobinostat, ACY-241, azacytidine, DB36, DB71, DB15, CVN, MGCD0103, SNDX-275, IMP32, BMS986016, TSR-022, Sym023, ATIK2a, or DZNep.
  • this document features methods for treating a mammal having cancer
  • the methods can include, or consist essentially of, administering an inhibitor of an AIRE polypeptide to said mammal, wherein said cancer comprises cancer cells that do not express an AIRE-regulated antigen selected from the group consisting of a TYRP2 polypeptide, a CSDE1 polypeptide, and a gplOO polypeptide.
  • the mammal can be a human.
  • the cancer can be a DMG, a melanoma, a hepatobiliary cancer, a breast cancer, a lung cancer, or a prostate cancer.
  • the inhibitor of an AIRE polypeptide can include an anti-AIRE antibody.
  • the inhibitor of an AIRE polypeptide can include a short hairpin RNA (shRNA) targeting nucleic acid encoding said AIRE polypeptide.
  • the method can include administering an immune checkpoint inhibitor to said mammal.
  • the immune checkpoint inhibitor can be an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-CTL4A antibody, an anti-LAG-3 antibody, an anti-TIM3 antibody, or an anti-TIGIT antibody.
  • the immune checkpoint inhibitor can be pembrolizumab, nivolumab, cemiplimab, ipilimumab, tremelimumab, durvalumab, dostarlimab, avelumab, atezolizumab, or relatlimab.
  • the immune checkpoint inhibitor can be BMS-8, BMS-37, BMS-202, BMS-230, BMS-242, BMS-1001, BMS-1166, SB415286, vorinostat, decitabine, entitostat, JQ1, BET151, GSK5O3, panobinostat, ACY-241, azacytidine, DB36, DB71, DB15, CVN, MGCD0103, SNDX-275, IMP32, BMS986016, TSR-022, Sym023, ATIK2a, or DZNep.
  • this document features methods for converting a mammal having cancer resistant to treatment with an immune checkpoint inhibitor into a mammal having cancer susceptible to treatment with said immune checkpoint inhibitor
  • the methods can include, or consist essentially of, administering an inhibitor of an AIRE polypeptide to said mammal, wherein said cancer comprises cancer cells that do not express an AIRE- regulated antigen selected from the group consisting of a TYRP2 polypeptide, a CSDE1 polypeptide, and a gplOO polypeptide.
  • the mammal can be a human.
  • the cancer can be a DMG, a melanoma, a hepatobiliary cancer, a breast cancer, a lung cancer, or a prostate cancer.
  • the inhibitor of an AIRE polypeptide can include an anti-AIRE antibody.
  • the inhibitor of an AIRE polypeptide can include a shRNA targeting nucleic acid encoding said AIRE polypeptide.
  • the immune checkpoint inhibitor can be an anti-PD-1 antibody, an anti- PD-L1 antibody, an anti-CTL4A antibody, an anti-LAG-3 antibody, an anti-TIM3 antibody, or an anti-TIGIT antibody.
  • the immune checkpoint inhibitor can be pembrolizumab, nivolumab, cemiplimab, ipilimumab, tremelimumab, durvalumab, dostarlimab, avelumab, atezolizumab, or relatlimab.
  • the immune checkpoint inhibitor can be BMS-8, BMS-37, BMS-202, BMS-230, BMS-242, BMS-1001, BMS-1166, SB415286, vorinostat, decitabine, entitostat, JQ1, BET151, GSK5O3, panobinostat, ACY-241, azacytidine, DB36, DB71, DB15, CVN, MGCD0103, SNDX-275, IMP32, BMS986016, TSR-022, Sym023, ATIK2a, or DZNep.
  • this document features methods for treating a mammal having cancer
  • the methods can include, or consist essentially of, (a) administering an inhibitor of an AIRE polypeptide to said mammal, and (b) administering an immune checkpoint inhibitor to said mammal; wherein said cancer comprises cancer cells that do not express an AIRE-regulated antigen selected from the group consisting of a TYRP2 polypeptide, a CSDE1 polypeptide, and a gplOO polypeptide.
  • the mammal can be a human.
  • the cancer can be a DMG, a melanoma, a hepatobiliary cancer, a breast cancer, a lung cancer, or a prostate cancer.
  • the inhibitor of an AIRE polypeptide can include an anti-AIRE antibody.
  • the inhibitor of an AIRE polypeptide can include a shRNA targeting nucleic acid encoding said AIRE polypeptide.
  • the immune checkpoint inhibitor can be an anti-PD-1 antibody, an anti- PD-L1 antibody, an anti-CTL4A antibody, an anti -LAG-3 antibody, an anti-TIM3 antibody, or an anti-TIGIT antibody.
  • the immune checkpoint inhibitor can be pembrolizumab, nivolumab, cemiplimab, ipilimumab, tremelimumab, durvalumab, dostarlimab, avelumab, atezolizumab, or relatlimab.
  • the immune checkpoint inhibitor can be BMS-8, BMS-37, BMS-202, BMS-230, BMS-242, BMS-1001, BMS-1166, SB415286, vorinostat, decitabine, entitostat, JQ1, BET151, GSK5O3, panobinostat, ACY-241, azacytidine, DB36, DB71, DB15, CVN, MGCD0103, SNDX-275, IMP32, BMS986016, TSR-022, Sym023, ATIK2a, or DZNep.
  • FIG. 1 AIRE expression inhibited immunogenicity/presentation of a non-self neoantigen.
  • Figure 2. AIRE expression enhanced immunogenicity/presentation of self tumor associated antigen.
  • Figure 3. AIRE expression enhanced immunogenicity/presentation of neoantigens derived from VSV-escape - CSDE1* derived from a self peptide mutated to reduce viral replication.
  • Figure 4 AIRE expression inhibited immunogenicity/presentation of a self derived neoantigen when expressed from a non-self promoter.
  • FIG. 5 AIRE expression inhibited immunogenicity/presentation of non-AIRE regulated neoantigens induced by APOBEC3B mutagenesis.
  • AIRE expression enhanced immunogenicity/presentation of AIRE -regulated neoantigens induced by APOBEC3B mutagenesis.
  • FIG. 6 AIRE expression inhibited immunogenicity/presentation of a non self neoantigen.
  • APOBEC mutation of the OVA gene inhibited SIINFEKL (SEQ ID NO:3) presentation.
  • FIG. 1 AIRE-mediated MHC I occupancy controlled presentation of foreign epitopes and selfness.
  • FIG. 1 AIRE expression in tumor cell lines.
  • FIG. 1 Experimental protocol to determine immunogenicity of AIRE-modified cells.
  • FIGS 16A-16D Vaccination with parental PKC cells was not-immunogenic against parental PKC DMG cells.
  • FIGs 17A-17D Vaccination with PKC (shRNA-AIRE) cells revealed immunogenic neoantigens against parental PKC DMG cells, as well as neoantigens exposed by closing down chromatin by restoration of K27.
  • Figures 18A-18D Closing down chromatin by restoration of K27 in DMG cells revealed immunogenic neoantigens against parental PKC DMG cells, an effect which was mimicked by inhibiting AIRE expression.
  • FIG. 20 Overexpression of AIRE enhanced the immunogenicity of self antigen expressing tumor cells. gpl OO was more visible with overexpression of AIRE and less visible with less AIRE.
  • FIG. 21 Overexpression of AIRE in an AIRE-ve tumor vaccine exposed self antigens to confer sensitivity to immune checkpoint blockade (ICB; administration of one or more immune checkpoint inhibitors) to an otherwise ICB-insensitive tumor.
  • ICB immune checkpoint blockade
  • Figure 22 Western blot analysis of 10 7 murine melanoma B l 6-F 10 cells, murine melanoma Bl 6-F1 -OVA cells, human DIPG-XIII cells, or human DIPG-SOH DMG cells for expression of AIRE using a rabbit polyclonal 22517-1AP antibody.
  • FIG. 23 Levels of AIRE expression in B16-OVA, B16-OVA-(shRNA-AIRE) (B16-OVA stably transfected with shRNA against murine AIRE), or B16-OVA-(AIRE) (B16-OVA stably transfected with CMV-murine AIRE expression plasmid) cells were measured by quantitative reverse transcriptase polymerase chain reaction (qrtPCR). Expression of AIRE in two independent mouse thymi was used as a control.
  • qrtPCR quantitative reverse transcriptase polymerase chain reaction
  • Figures 24A-24B Mean CT levels, measured by qrtPCR, of GAPDH (Figure 24A) and OVA ( Figure 24B) are shown for Bl 6-0 VA, B16-OVA-(shRNA-AIRE), and B16-0VA- (AIRE) cells. Mean values represent three biological replicates. CT values of two independent mouse thymi were used as a control.
  • FIGS 25A-25D Levels of TYRP2 ( Figures 25A and 25B) and CSDE1 ( Figures 25C and 25D) expression relative to OVA in B16-0VA, B16-OVA-(shRNA-AIRE), and B 16-0 VA-( AIRE) cells were measured by qrtPCR. Levels of TYRP2 and CSDE1 expression in B16-0VA vs. B 16-0VA-(shRNA-AIRE) were not statistically significant using ANOVA using the 2' AACt method for comparative analysis ( Figures 25A and 25C); however, levels of TYRP2 and CSDE1 expression in B16-0VA vs. B16-0VA-(shRNA-AIRE) were both highly significant using an unpaired t-test ( Figures 25B and 25D).
  • FIGS 26A-26C Flow cytometry was performed on B16-0VA, B16-0VA-(shRNA- AIRE), or B16-0VA-(AIRE) tumor cells labelled with the 25-D1.16 antibody which recognizes the H-2Kb MHC Class I molecule bound to the OVA-derived peptide SIINFEKL (Kb-SIINFEKL; SEQ ID NO:3).
  • Tumor cells were untreated (Figure 26A) or pretreated for 24 hours with 50 units (U) of rlFN-y (to increase surface expression of H-2Kb MHC class I molecules) (Figure 26B) or 50 U of rlFN-y and 1 pg/mL of SIINFEKL (SEQ ID NO:3) peptide ( Figure 26C) prior to quantification of surface-expressed Kb-SIINFEKL (SEQ ID NO:3). Histograms show mean and standard deviation of three separate biological triplicates.
  • FIGS 27A-27C OVA-specific IFN-y positive spots in IxlO 5 B16-OVA B16-OVA- (scrambled shRNA), B16-OVA-(shRNA-AIRE), or B16-OVA-(AIRE) cells, pre-treated for 24 hours with 50 U of rIFN-y and co-cultured with OT-I CD8 + T cells that were activated for 4 - 5 days in vitro (Figure 27A), PMEL CD8 + T cells that were activated for 4 - 5 days in vitro (Figure 27B), or tumor experienced CD8 + T cells (T cells recovered from mice which had rejected B16-TK tumors in a CD8 + T cell dependent mechanism) (Figure 27C). All groups were cultured at an Effector: Target ratio of 1 :1 in 96-well ELISPOT plates (3 biological triplicates shown) for 48 hours at 37°C and were quantified by computer assisted image analyzer.
  • FIG. 28 10 6 B16-OVA cells, pre-treated for 24 hours with 50 U of rlFN-y, were cocultured with OT-I or PMEL CD8 + T cells that were activated for 4 - 5 days in vitro at an Effector: Target ratio of 10: 1 (3 biological triplicates shown) for 21 days at 37°C.
  • Surviving B16-OVA cells were pooled and expanded for 2 weeks in vitro to give B16-OVA-OT-I-ESC or B16-OVA-PMEL-ESC populations.
  • AIRE expression, as measured by qrtPCR, in Bl 6- OVA, B16-OVA-OT-I-ESC, or B16-OVA-PMEL-ESC cells is shown.
  • FIG. 29 C57B1/6 mice with 10 days established subcutaneous B16-OVA, B16- OVA-(shRNA-AIRE), or B16-OVA-(AIRE) tumors were treated intravenously with 10 7 naive OT-I CD8 + T cells. Two 2 days later (day 12) mice were treated with VSV-OVA, VSV-hgplOO, or VSV-GFP as shown (5xl0 6 pfu virus/inj ection, i.v.). This cycle of adoptive T cell transfer and virus boost was repeated on days 14 and 17 and on days 19 and 21. Tumor diameters were measured thrice weekly in two dimensions using calipers, and mice were sacrificed when tumor size was — 1.0 cm. 2 . Survival with time is shown.
  • FIG. 30 C57B1/6 mice with 10 days established subcutaneous B16-OVA, B16- OVA-(shRNA-AIRE), or B16-OVA-(AIRE) tumors were treated intravenously with 10 7 PMEL CD8 + T cells. Two days later (day 12) mice were treated with VSV-OVA, VSV- hgplOO, or VSV-GFP as shown (5xl0 6 pfu virus/inj ection, i.v.). This cycle of adoptive T cell transfer and virus boost was repeated on days 14 and 17 and on days 19 and 21. Tumor diameters were measured thrice weekly in two dimensions using calipers, and mice were sacrificed when tumor size was —1.0 cm. 2 . Survival with time is shown.
  • FIG. 31 C57B1/6 mice with 10 day s-establi shed B16-F10, B16-F10-(shRNA- AIRE), or B16-F10-(AIRE) tumors were treated with dendritic cells (DCs) loaded in vitro with lysates of PBS (no lysate), Bl 6-F10-(shRNA-AIRE), or B 16-F 10-(AIRE) cells (10 6 DCs loaded with lysate equivalent of 10 7 tumor cells per injection) on days 10, 12, and 14. Mice were then treated with anti-PD-1 antibody or isotype IgG control as shown on days 17, 19, 21 and on days 24, 26, and 28. Survival (tumor size) with time is shown.
  • DCs dendritic cells
  • FIGS 32A-32C CD8 T cells (10 6 /well) were isolated at the endpoint of the experiment described in Figure 31 from mice that had been treated with DC vaccines loaded with lysates of Bl 6-F 10, B16-F10-(shRNA-AIRE), or B16-F10-(AIRE) and co-treated with anti-PD-1 or control IgG.
  • CD8 + T cells were re-stimulated in 96-well ELISPOT plates (4 biological triplicates shown) with 5xl0 5 live target cells (B16-F10 ( Figure 32A), B16-F10- (AIRE) ( Figure 32B), or CT2A (pre-treated with IFN-y for 24 hours to increase MHC class 1 expression) ( Figure 32C)) for 48 hours at 37°C. Tumor-specific IFN-y positive spots were quantified by computer assisted image analyzer.
  • FIG. 33 Splenocytes from naive C57B1/6 mice were co-cultured with live PKC, PKC-(shRNA-AIRE), or PKC-(AIRE) cells pre-treated for 24 hours with IFN-y to enhance MHC Class I presentation at a ratio of 10: 1 for three days with IL-2. On days 6/7 and days 9/10, co-cultures were replenished with live, IFN-y-pre-treated PKC variant tumor cells. After 2 weeks of in vitro priming with PKC, PKC-(shRNA-AIRE), or PKC-(AIRE) cells, purified CD8 1 T cells were co-cultured with 10’ parental PKC tumor cells at a ratio of -10: 1. Cell-free supernatants were collected after 48 hours and tested by ELISA for IFN-y. Mean of three biological triplicates is shown.
  • Figure 34 Exemplery in vitro protocol for priming/educating human CD8 + T cells with human DMG cell variants.
  • FIG. 35 Human CD8 + T cells from three different donors were primed with human DMG DIPG-XIII cell lines. CD8 + T cells were re-stimulated in 96-well ELISPOT plates (3 biological triplicates shown) with live, parental DIPG-XIII target cells (pre-treated with IFN- y for 24 hours to increase MHC class I expression) for 72 hours. Tumor-specific IFN-y positive spots were quantified.
  • FIG. 36 Human CD8 + T cells from three different donors were primed with human DMG DIPG-SOH cell lines. CD8 + T cells were re-stimulated in 96-well ELISPOT plates (3 biological triplicates shown) with live, DIPG-SOH target cells (pre-treated with IFN-y for 24 hours to increase MHC class I expression) for 72 hours. Tumor-specific IFN-y positive spots were quantified.
  • FIG. 37 Human CD8 + T cells from three different donors were primed by DIPG- XIII, DIPG-XIII-(AIRE), DIPG-XIII-(shRNA-AIRE), DIPG-XIII-(AP0BEC3B), DIPG- XIII-(AP0BEC3B)-(AIRE), or DIPG-XIII-(AP0BEC3B)-(shRNA-AIRE) cells and restimulated in IFN-y ELISPOT plates for 3 days with parental DIPG-XIII cells.
  • FIG 38 C57B1/6 mice with 5 day s-establi shed PKC tumors were treated with DCs loaded in vitro with lysates of PKC cells alone, PKC-(shRNA AIRE), or PKC-(AIRE) cells (10 6 DCs loaded with lysate equivalent of 10 7 tumor cells per injection) on days 5, 7, and 9. Mice were then treated with anti-PD-1 antibody or isotype IgG control as shown on days 12, 14, and 16 and on days 19, 21, and 23. Survival (tumor size) with time is shown.
  • FIGs 39A-39C CD8 T cells (10 6 /well) were isolated at the endpoint of the experiment described in Figure 38 from mice (3 per group) that had been treated with DC vaccines loaded with lysates of parental PKC cells or with DC vaccines loaded with lysates of PKC-(shRNA-AIRE) cells and were re-stimulated with 5x10’ live target PKC ( Figure 39 A), PKC-(shRNA-AIRE) ( Figure 39B), or PKC-(AIRE) (pre-treated with IFN-y for 24 hours to increase MHC class I expression) (Figure 39C) cells for 48 hours at 37°C (3 biological triplicates per group). IFN-y in the supernatants was measured.
  • Figure 40 Schematic of exemplary AAV-8-AIRE and AAV-8-GFP vectors.
  • FIG 41 Tumor cells were transfected with CMV-AIRE plasmid (lane 1), left untransfected (lane 2), or infected with AAV-8-AIRE (lane 3) or AAV-8-Control (lane 4) (IxlO 9 genome copies). 48 hours later, cells were harvested and assayed by western blot for AIRE and B-actin.
  • Figure 42 Schematic showing 5 days established B16-F10 subcutaneous tumors were injected with either virus (AAV-8-AIRE or AAV-8-GFP) or with anti-PD-1 ICB or control IgG antibody (200 pg/inj ection) in three cycles: EARLY (days 5, 7, and 9), MIDDLE (days 12, 14, and 16), and LATE (days 19, 21, and 23).
  • virus AAV-8-AIRE or AAV-8-GFP
  • anti-PD-1 ICB or control IgG antibody 200 pg/inj ection
  • FIGS 44A-44B Spleens were recovered from the first three mice euthanized in each treatment group.
  • CD8 + T cells were isolated by magnetic bead sorting and co-cultured at an EffectonTarget ratio of 5:1 in 96-well ELISPOT plates with live target B16-F10 cells pre-treated for 24 hours with IFN-y for 48 hours at 37° C to increase MHC Class I expression.
  • Levels of IFN-y were measured from the supernatants by ELISA ( Figure 44 A) and B16-F10- specific IFN-y ELISPOT positive spots ( Figure 44B). Three biological replicates per group were quantified by computer assisted image analyzer.
  • Figure 45 A survival curve showing that AIRE-mediated tumor therapy was dependent upon CD4 + and CD8 + T cells.
  • Figure 46 Schematic showing MHC Class I immunoprecipitation from B16-F10 cells using an anti-H-2K b antibody (Clone Y-3).
  • FIG 47 C57B1/6 mice with 5 day s-establi shed B16-F10 tumors were treated with DCs loaded in vitro with SIINFEKL (SEQ ID NO:3) peptide (1 pg per 10 6 DCs/inj ection) with PBS or with peptide sets of the Unique, Shared, and Longer peptides (Table 13) (at 0.25 pg per peptide, total 1 pg per 10 6 DCs/inj ection) on days 5, 7, and 9. Mice were then treated with anti-PD-1 antibody or isotype IgG control as shown on days 12, 14, and 16 and on days 19, 21, and 23. Survival (tumor size) with time is shown for groups treated with control IgG (no ICB).
  • SIINFEKL SEQ ID NO:3 peptide (1 pg per 10 6 DCs/inj ection) with PBS or with peptide sets of the Unique, Shared, and Longer peptides (Table 13) (at 0.25 p
  • FIG. 48 C57B1/6 mice with 5 day s-establi shed B16-F10 tumors were treated with DCs loaded in vitro with SIINFEKL (SEQ ID NO:3) peptide (1 pg per 10 6 DCs/inj ection) with PBS or with peptide sets of the Unique, Shared, and Longer peptides (Table 13) (at 0.25 pg per peptide, total 1 pg per 10 6 DCs/inj ection) on days 5, 7, and 9. Mice were then treated with anti-PD-1 antibody or isotype IgG control as shown on days 12, 14, 16 and 19, 21, 23. Survival (tumor size) with time is shown for groups treated with ICB.
  • SIINFEKL SEQ ID NO:3 peptide (1 pg per 10 6 DCs/inj ection) with PBS or with peptide sets of the Unique, Shared, and Longer peptides (Table 13) (at 0.25 pg per peptide, total 1 p
  • FIGS 49A-49F CD8 + T cells (10 6 per well) were isolated at endpoint in Figures 47 and 48 from mice (4 per group) that had been treated with DC vaccines loaded with different peptides and co-treated with anti-PD-1. 10 6 CD8 + T cells were re-stimulated in 96-well ELISPOT plates (4 biological triplicates shown) with either 5xl0 5 live target cells (B16-F10 ( Figure 49A) or B 16-F 10-(AIRE) (pre-treated with fFN-y for 24 hours to increase MHC Class I expression) (Figure 49B)) or with either 5xl0 5 DCs loaded with the Unique (Figure 49C), Shared (Figure 49D), or Longer (Figure 49E) peptide sets or SIINFEKL (SEQ ID NO:3) peptide ( Figure 49F) (total of 1 pg peptide perlO 6 DCs) for 48 hours at 37°C. IFN-y positive spots were quantified by computer assisted image analyzer.
  • FIGS 50A-50C CD4 T cells were isolated by magnetic bead sorting at the endpoint of the experiment described in Figures 47 and 48 from mice (3 per group) that had been treated with DC vaccines loaded with different peptides and co-treated with anti-PD-1. 5x10 5 CD4 + T cells were re-stimulated in 96-well plates (3 biological triplicates shown) with 5xl0 5 DCs loaded with the Longer (Figure 50C), Shared ( Figure 50B), or Unique (Figure 50A) peptide sets (Table 13) (total of 1 pg peptide per 10 6 DCs) for 48 hours at 37°C. IL-15 in the supernatant 48 hours later, measured by ELISA, is shown.
  • a level of an AIRE polypeptide within a mammal can be altered (e.g., increased or decreased) to expose one or more epitopes of a tumor antigen within a cancer cell (e.g., a cancer cell that exhibits little or no response to treatment with immune checkpoint inhibitors) present in the mammal (e.g., such that the mammal produces an immune response against the cancer cell).
  • a cancer cell e.g., a cancer cell that exhibits little or no response to treatment with immune checkpoint inhibitors
  • one or more modulators of an AIRE polypeptide level can have the ability to induce immune responses against cancer cells (e.g., cancer cells that exhibit little or no response to treatment with immune checkpoint inhibitors) within a mammal (e.g., a human).
  • cancer cells e.g., cancer cells that exhibit little or no response to treatment with immune checkpoint inhibitors
  • a mammal e.g., a human
  • one or more modulators of an AIRE polypeptide level can be administered to a mammal having cancer (e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors) to treat the mammal.
  • cancer e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors
  • administering one or more modulators of an AIRE polypeptide level e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • a mammal e.g., a human
  • administering one or more modulators of an AIRE polypeptide level e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • a mammal e.g., a human
  • one or more modulators of an AIRE polypeptide level can be administered to a mammal (e.g., a human) to expose one or more epitopes of a tumor antigen present on a cancer cell within the mammal.
  • a mammal e.g., a human
  • T cells present within the mammal can mediate an immune response against cancer cells containing the newly exposed epitope(s) such that those cancer cells are targeted (e.g., targeted and destroyed).
  • an AIRE polypeptide level e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • T cells present within the mammal can mediate an immune response against cancer cells containing the newly exposed epitope(s) such that those cancer cells are targeted (e.g., targeted and destroyed).
  • T cells present within a mammal that can target (e.g., target and destroy) cancer cells containing the newly exposed epitope(s) can be cross-reactive against parent cancer cells.
  • T cells present within a mammal e.g., a human
  • that can target (e.g., target and destroy) cancer cells that expresses a tumor antigen but did not have one or more epitopes of the tumor antigen exposed as described herein (e.g., by administering one or more modulators of an AIRE polypeptide level (e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide).
  • an AIRE polypeptide level e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide.
  • administering one or more modulators of an AIRE polypeptide level e g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • a mammal e.g., a human
  • administering one or more modulators of an AIRE polypeptide level can be effective to sensitize cancer cells (e.g., cancer cells that exhibit little or no response to treatment with immune checkpoint inhibitors) present within the mammal to one or more immune checkpoint inhibitors.
  • one or more modulators of an AIRE polypeptide level can be administered to a mammal (e.g., a human) to sensitize cancer cells (e.g., cancer cells that exhibit little or no response to treatment with immune checkpoint inhibitors) present within the mammal to one or more immune checkpoint inhibitors, and one or more immune checkpoint inhibitors can be administered to the mammal to treat the mammal.
  • a mammal e.g., a human
  • cancer cells e.g., cancer cells that exhibit little or no response to treatment with immune checkpoint inhibitors
  • immune checkpoint inhibitors can be administered to the mammal to treat the mammal.
  • Any appropriate mammal having cancer can be treated as described herein (e.g., by administering one or more modulators of an AIRE polypeptide level (e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide) and, optionally, administering one or more immune checkpoint inhibitors).
  • modulators of an AIRE polypeptide level e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • immune checkpoint inhibitors e.g., anti-associated antigene, antigen, antigen, antigen, antigen, antigen, antigen, antigen, antigen, antigen, antigen, antigen, antigen, antigen, antigen, antigen, antigen, antigen, antigen, antigen, antigen, antigen, antigen, antigen, antigen, antigen, antigen
  • a human having cancer e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors
  • a human having cancer can be treated by administering (a) one or more modulators of an AIRE polypeptide level (e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide) and (b) one or more immune checkpoint inhibitors to the human.
  • one or more modulators of an AIRE polypeptide level e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • a mammal having any type of cancer can be treated as described herein (e.g., by administering one or more modulators of an AIRE polypeptide level (e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide) and, optionally, administering one or more immune checkpoint inhibitors).
  • a cancer that can be treated as described herein can include one or more solid tumors.
  • a cancer that can be treated as described herein can be a blood cancer.
  • a cancer treated as described herein can be resistant to one or more immune checkpoint inhibitors.
  • a cancer treated as described herein can be a primary cancer.
  • a cancer treated as described herein can be a metastatic cancer.
  • a cancer treated as described herein can be a refractory cancer.
  • Examples of cancers that can be treated as described herein include, without limitation, brain cancers (e.g., diffuse midline gliomas, skin cancers (e.g., melanomas), hepatobiliary cancers, breast cancers, lung cancers, and prostate cancers.
  • the methods described herein also can include identifying a mammal as having cancer.
  • methods for identifying a mammal as having cancer include, without limitation, physical examination, laboratory tests (e.g., blood and/or urine), biopsy, imaging tests (e.g., X-ray, PET/CT, MRI, and/or ultrasound), nuclear medicine scans (e.g., bone scans), endoscopy, genetic tests, and/or histopathology.
  • a mammal can be treated as described herein (e.g., by administering one or more modulators of an AIRE polypeptide level (e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide) and, optionally, administering one or more immune checkpoint inhibitors).
  • a mammal can be administered or instructed to selfadminister one or more modulators of an AIRE polypeptide level (e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide).
  • a mammal can be administered or instructed to self-administer one or more modulators of an AIRE polypeptide level (e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide) and one or more immune checkpoint inhibitors.
  • one or more modulators of an AIRE polypeptide level e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • immune checkpoint inhibitors e.g., one or more modulators of an AIRE polypeptide level
  • An immune checkpoint inhibitor can be any appropriate immune checkpoint inhibitor.
  • An immune checkpoint inhibitor can inhibit one or more polypeptides involved in an immune checkpoint pathway. Examples of immune checkpoint pathways include, without limitation, PD-1/PD-L1 pathways, PD-1/PD-L2 pathways, CTLA-4 pathways, TRAIL pathways, TIM3 pathways, and TIGIT pathways.
  • An immune checkpoint inhibitor can inhibit any polypeptide involved in an immune checkpoint pathway. Examples of polypeptides involved in an immune checkpoint pathway that can be inhibited by an immune checkpoint inhibitor as described herein include, without limitation, PD-1 polypeptides, PD-L1 polypeptides, CTLA4 polypeptides, LAG-3 polypeptides, TIM3 polypeptides, and TIGIT polypeptides.
  • An immune checkpoint inhibitor can inhibit polypeptide activity of a polypeptide involved in an immune checkpoint pathway or can inhibit polypeptide expression of a polypeptide involved in an immune checkpoint pathway.
  • Examples of compounds that can inhibit polypeptide activity of a polypeptide involved in an immune checkpoint pathway include, without limitation, antibodies (e.g., neutralizing antibodies) that target (e.g., target and bind) to a polypeptide involved in an immune checkpoint pathway and small molecules that target (e.g., target and bind) to a polypeptide involved in an immune checkpoint pathway.
  • Examples of compounds that can inhibit polypeptide expression of a polypeptide involved in an immune checkpoint pathway include, without limitation, nucleic acid molecules designed to induce RNA interference of polypeptide expression of a polypeptide involved in an immune checkpoint pathway (e g., a siRNA molecule or a shRNA molecule), antisense molecules that can target (e.g., are complementary to) nucleic acid encoding a polypeptide involved in an immune checkpoint pathway, and miRNAs that can target (e.g., are complementary to) nucleic acid encoding a polypeptide involved in an immune checkpoint pathway.
  • nucleic acid molecules designed to induce RNA interference of polypeptide expression of a polypeptide involved in an immune checkpoint pathway e g., a siRNA molecule or a shRNA molecule
  • antisense molecules that can target (e.g., are complementary to) nucleic acid encoding a polypeptide involved in an immune checkpoint pathway
  • miRNAs that can target (e.g., are
  • immune checkpoint inhibitors that can be administered to mammal (e.g., a human) having cancer (e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors) include, without limitation, anti -PD-1 antibodies, anti-PD-Ll antibodies, anti-CTL4A antibodies, anti -LAG-3 antibodies, anti- TIM3 antibodies, and anti-TIGIT antibodies.
  • an immune checkpoint inhibitor that can be administered to mammal (e.g., a human) having cancer e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors) as described herein can be as shown in Table 1. Table 1. Exemplary immune checkpoint inhibitors.
  • an immune checkpoint inhibitor can be as described elsewhere (see, e.g., Smith et al., Am. J. Transl. Res., 11(2):529-541 (2019) at, for example, Table 1; and Terranova-Barberio et al., Immunotherapy, 8(6):705-719 (2016) at, for example, Table 1).
  • one or more AIRE polypeptides can be administered to a mammal (e.g., a human) having cancer (e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors) to treat the mammal.
  • a mammal e.g., a human
  • cancer e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors
  • one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) can be administered to a mammal (e.g., a human) having cancer including one or more cancer cells that express one or more AIRE- regulated antigens to treat the mammal.
  • AIRE polypeptides and/or nucleic acids designed to express an AIRE polypeptide
  • a mammal e.g., a human
  • cancer e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors
  • the cancer cells can express any appropriate AIRE-regulated antigen(s).
  • AIRE- regulated antigens include, without limitation, TYRP2 polypeptides, CSDE1 polypeptides, and gplOO polypeptides.
  • an AIRE-regulated antigen can be as described elsewhere (see, e.g., Perniola, Front. Immunol, 9:98 (2016)).
  • AIRE polypeptide or fragment thereof can be administered to a mammal (e.g., a human) having cancer including one or more cancer cells that express one or more AIRE-regulated antigens as described herein.
  • a mammal e.g., a human
  • AIRE polypeptides and nucleic acids encoding AIRE polypeptides include, without limitation, those set forth in the National Center for Biotechnology Information (NCBI) databases at, for example, accession no. NM_000383, NM_000658, and NM_000659.
  • an AIRE polypeptide can have an amino acid sequence as set forth in SEQ ID NO: 1 (see, e.g., Example 3).
  • a nucleic acid encoding an AIRE polypeptide can have a nucleotide sequence as set forth in SEQ ID NO:2 (see, e.g., Example 2).
  • an AIRE polypeptide provided herein can include the amino acid sequence set forth in SEQ ID NO: 1 with zero, one, or two amino acid substitutions within the articulated sequence, with zero, one, two, three, four, or five amino acid residues preceding the articulated sequence of the sequence identifier, and/or with zero, one, two, three, four, or five amino acid residues following the articulated sequence of the sequence identifier, provided that the AIRE polypeptide retains at least some activity exhibited by the an AIRE polypeptide set forth in SEQ ID NO: 1 (e.g., the ability to bind to chromatin and drive expression of one or more AIRE-regulated antigens).
  • any appropriate AIRE polypeptide fragment (and/or nucleic acid designed to express an AIRE polypeptide fragment) can be administered to a mammal (e.g., a human) having cancer including one or more cancer cells that express one or more AIRE-regulated antigens as described herein.
  • a mammal e.g., a human
  • an AIRE polypeptide fragment can be derived from the amino acid sequence set forth in SEQ ID NO:1.
  • an AIRE polypeptide fragment can be derived from the amino acid sequence of SEQ ID NO: 1, provided that it maintains at least some function of a full-length AIRE polypeptide (e.g., the ability to bind to chromatin and drive expression of one or more AIRE-regulated antigens).
  • An AIRE polypeptide fragment can be any appropriate length (e.g., can include any number of amino acids) provided that it maintains at least some function of a naturally- occurring AIRE polypeptide (e.g., the ability to bind to chromatin and drive expression of one or more AIRE-regulated antigens).
  • an AIRE polypeptide or fragment thereof provided herein e.g., a polypeptide that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 1.
  • an AIRE polypeptide or fragment thereof provided herein e.g., a polypeptide that comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 1 can be obtained by synthesizing the polypeptide of interest using appropriate polypeptide synthesizing techniques such as solid phase peptide synthesis (SPPS).
  • SPPS solid phase peptide synthesis
  • nucleic acid when one or more nucleic acids designed to express an AIRE polypeptide and/or a fragment thereof are administered to a mammal (e.g., a human), the nucleic acid can be in the form of a vector (e.g., a viral vector or a non-viral vector).
  • a vector e.g., a viral vector or a non-viral vector
  • nucleic acid encoding an AIRE polypeptide and/or a fragment thereof When nucleic acid encoding an AIRE polypeptide and/or a fragment thereof is administered to a mammal, the nucleic acid can be used for transient expression of an AIRE polypeptide and/or a fragment thereof or for stable expression of an AIRE polypeptide and/or a fragment thereof.
  • the nucleic acid encoding an AIRE polypeptide and/or a fragment thereof can be engineered to integrate into the genome of a cell.
  • Nucleic acid can be engineered to integrate into the genome of a cell using any appropriate method. For example, gene editing techniques (e.g., CRISPR or TALEN gene editing) can be used to integrate nucleic acid designed to express an AIRE polypeptide and/or a fragment thereof into the genome of a cell.
  • a vector used to deliver nucleic acid encoding an AIRE polypeptide and/or a fragment thereof to a mammal is a viral vector
  • any appropriate viral vector can be used.
  • a viral vector can be derived from a positive-strand virus or a negative-strand virus.
  • a viral vector can be derived from a virus with a DNA genome or a RNA genome.
  • a viral vector can be a chimeric viral vector.
  • a viral vector can infect dividing cells. In some cases, a viral vector can infect non-dividing cells.
  • virus-based vectors that can be used to deliver nucleic acid encoding an AIRE polypeptide and/or a fragment thereof to a mammal (e.g., a human) include, without limitation, virusbased vectors based on adenoviruses, adeno-associated viruses (AAVs), Sendai viruses, retroviruses, lentiviruses, vesicular stomatitis viruses (VSVs), measles viruses, vaccinia viruses, or herpes viruses.
  • AAVs adeno-associated viruses
  • Sendai viruses Sendai viruses
  • retroviruses retroviruses
  • lentiviruses lentiviruses
  • VSVs vesicular stomatitis viruses
  • measles viruses vaccinia viruses
  • herpes viruses herpes viruses.
  • a vector used to deliver nucleic acid encoding an AIRE polypeptide and/or a fragment thereof to a mammal is a non-viral vector
  • any appropriate non-viral vector can be used.
  • a non-viral vector can be an expression plasmid (e.g., a cDNA expression vector).
  • a vector in addition to nucleic acid encoding an AIRE polypeptide and/or a fragment thereof, can contain one or more regulatory elements operably linked to the nucleic acid encoding an AIRE polypeptide and/or a fragment thereof.
  • regulatory elements can include promoter sequences, enhancer sequences, response elements, signal peptides, internal ribosome entry sequences, polyadenylation signals, terminators, and inducible elements that modulate expression (e.g., transcription or translation) of a nucleic acid.
  • the choice of regulatory element(s) that can be included in a vector depends on several factors, including, without limitation, inducibility, targeting, and the level of expression desired.
  • a promoter can be included in a vector to facilitate transcription of a nucleic acid encoding an AIRE polypeptide.
  • a promoter can be a naturally occurring promoter or a recombinant promoter.
  • a promoter can be ubiquitous or inducible (e.g., in the presence of tetracycline), and can affect the expression of a nucleic acid encoding a polypeptide in a general or tissue-specific manner.
  • promoters that can be used to drive expression of an AIRE polypeptide and/or a fragment thereof in cells include, without limitation, CMV promoters, Rous Sarcoma virus promoters; murine leukemia virus LTR promoters, and adenoviral E1A promoters.
  • operably linked refers to positioning of a regulatory element in a vector relative to a nucleic acid encoding a polypeptide in such a way as to permit or facilitate expression of the encoded polypeptide.
  • a vector can contain a promoter and nucleic acid encoding an AIRE polypeptide and/or a fragment thereof.
  • the promoter is operably linked to a nucleic acid encoding an AIRE polypeptide and/or a fragment thereof such that it drives expression of the AIRE polypeptide and/or a fragment thereof in cells.
  • nucleic acid encoding an AIRE polypeptide and/or a fragment thereof can contain nucleic acid encoding a detectable label.
  • a vector can include nucleic acid encoding an AIRE polypeptide and/or a fragment thereof and nucleic acid encoding a detectable label positioned such that the encoded polypeptide is a fusion polypeptide that includes an AIRE polypeptide and/or a fragment thereof fused to a detectable polypeptide.
  • a detectable label can be a peptide tag.
  • a detectable label can be a fluorescent molecule (e.g., a fluorescent polypeptide).
  • detectable labels examples include, without limitation, an HA tag, a Myc-tag, a FLAG-tag, green fluorescent polypeptides (GFPs; e.g., enhanced GFPs), and mCherry polypeptides.
  • GFPs green fluorescent polypeptides
  • mCherry polypeptides examples include, without limitation, an HA tag, a Myc-tag, a FLAG-tag, green fluorescent polypeptides (GFPs; e.g., enhanced GFPs), and mCherry polypeptides.
  • Nucleic acid encoding an AIRE polypeptide and/or a fragment thereof can be produced by techniques including, without limitation, common molecular cloning, polymerase chain reaction (PCR), chemical nucleic acid synthesis techniques, and combinations of such techniques.
  • PCR polymerase chain reaction
  • RT-PCR can be used with oligonucleotide primers designed to amplify nucleic acid (e.g., genomic DNA or RNA) encoding an AIRE polypeptide and/or a fragment thereof.
  • one or more inhibitors of an AIRE polypeptide can be administered to a mammal (e.g., a human) having cancer (e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors) to treat the mammal.
  • a mammal e.g., a human
  • cancer e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors
  • one or more inhibitors of an AIRE polypeptide can be administered to a mammal (e.g., a human) having cancer including one or more cancer cells that do not express AIRE-regulated antigens to treat the mammal.
  • an AIRE polypeptide When one or more inhibitors of an AIRE polypeptide are administered to a mammal (e.g., a human) having cancer (e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors) including one or more cancer cells that do not express AIRE-regulated antigens, the cancer cells can express one or more antigens that are not regulated by an AIRE polypeptide.
  • antigens that are not regulated by an AIRE polypeptide include, without limitation, Epstein-Barr virus (EBV) cancer associated antigens and human papillomavirus (HPV) cancer associated antigens.
  • EBV Epstein-Barr virus
  • HPV human papillomavirus
  • An inhibitor of an AIRE polypeptide can be an inhibitor of AIRE polypeptide activity (e.g., anti-AIRE antibodies such as neutralizing anti-AIRE antibodies and small molecules that target an AIRE polypeptide) or an inhibitor of AIRE polypeptide expression (e.g., nucleic acid molecules designed to induce RNA interference (RNAi) of AIRE polypeptide expression such as antisense oligonucleotides (ASOs), siRNA molecules, and shRNA molecules).
  • RNAi RNA interference
  • Additional inhibitors of an AIRE polypeptide can be designed based on any appropriate nucleic acid (e.g., a mRNA) encoding an AIRE polypeptide sequence.
  • nucleic acids encoding an AIRE polypeptide sequence include, without limitation, those set forth in NCBI accession nos. NM_000383, NM_000658, and NM_000659.
  • one or more modulators of an AIRE polypeptide level can be formulated into a composition (e.g., a pharmaceutical composition) for administration to a mammal (e.g., a human).
  • a composition e.g., a pharmaceutical composition
  • a mammal e.g., a human
  • one or more modulators of an AIRE polypeptide level can be formulated into a pharmaceutically acceptable composition for administration to a mammal (e.g., a human) having cancer (e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors).
  • a mammal e.g., a human
  • cancer e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors.
  • one or more modulators of an AIRE polypeptide level can be formulated together with one or more pharmaceutically acceptable carriers (additives), excipients, and/or diluents.
  • Examples of pharmaceutically acceptable carriers, excipients, and diluents that can be used in a composition described herein include, without limitation, sucrose, lactose, starch (e.g., starch glycolate), cellulose, cellulose derivatives (e.g., modified celluloses such as microcrystalline cellulose and cellulose ethers like hydroxypropyl cellulose (HPC) and cellulose ether hydroxypropyl methylcellulose (HPMC)), xylitol, sorbitol, mannitol, gelatin, polymers (e.g., polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), crosslinked polyvinylpyrrolidone (crospovidone), carboxymethyl cellulose, polyethylene-polyoxypropylene-block polymers, and crosslinked sodium carboxymethyl cellulose (croscarmellose sodium)), titanium oxide, azo dyes, silica gel, fumed silica, talc, magnesium carbonate, vegetable stearin,
  • a composition containing one or more modulators of an AIRE polypeptide level (e g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide) can be formulated into any appropriate dosage form.
  • dosage forms include solid or liquid forms including, without limitation, gels, liquids, suspensions, solutions (e.g., sterile solutions), sustained-release formulations, and delayed-release formulations.
  • a composition containing one or more modulators of an AIRE polypeptide level (e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide) can be designed for parenteral (e.g., intratumoral, intracerebral, intraperitoneal, and subcutaneous) administration.
  • modulators of an AIRE polypeptide level e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • parenteral e.g., intratumoral, intracerebral, intraperitoneal, and subcutaneous
  • compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations can be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.
  • a composition e.g., a pharmaceutical composition
  • one or more modulators of an AIRE polypeptide level e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • a composition can be administered locally or systemically.
  • composition containing one or more modulators of an AIRE polypeptide level can be administered locally by direct injection (e.g., an intratumoral injection) to one or more solid tumors present within a mammal (e.g., a human).
  • direct injection e.g., an intratumoral injection
  • solid tumors present within a mammal e.g., a human.
  • An effective amount of a composition containing one or more modulators of an AIRE polypeptide level (e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide) can be any amount that can treat the mammal without producing significant toxicity to the mammal.
  • the effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal’s response to treatment.
  • Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition may require an increase or decrease in the actual effective amount administered.
  • the frequency of administration of a composition containing one or more modulators of an AIRE polypeptide level (e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide) can be any frequency that can treat a mammal (e.g., a human) having cancer (e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors) without producing significant toxicity to the mammal.
  • the frequency of administration can be from about once a day to about once a month, from about twice a week to about twice a month, or from about once a month to about twice a year.
  • the frequency of administration can remain constant or can be variable during the duration of treatment.
  • a course of treatment with a composition containing one or more modulators of an AIRE polypeptide level e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • the effective amount various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition may require an increase or decrease in administration frequency.
  • An effective duration for administering a composition containing one or more modulators of an AIRE polypeptide level (e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide) can be any duration that treat a mammal (e.g., a human) having cancer (e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors) without producing significant toxicity to the mammal.
  • the effective duration can vary from several days to several weeks, months, or years. Multiple factors can influence the actual effective duration used for a particular treatment.
  • an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, route of administration, and severity of the condition being treated.
  • both one or more modulators of an AIRE polypeptide level e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • the one or more modulators of an AIRE polypeptide level e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • the one or more immune checkpoint inhibitors can be administered to a mammal at the same time (e.g., in a single composition).
  • both one or more modulators of an AIRE polypeptide level e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • the one or more modulators of an AIRE polypeptide level e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • the one or more immune checkpoint inhibitors can be administered to a mammal separately.
  • one or more modulators of an AIRE polypeptide level e g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • one or more immune checkpoint inhibitors can be administered to a mammal at the same time (e.g., concurrently) as independent compositions.
  • the one or more modulators of an AIRE polypeptide level e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • the one or more modulators of an AIRE polypeptide level e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • the one or more immune checkpoint inhibitors can be administered to a mammal within from about 1 second to about 5 minutes (e.g., about 2 seconds to about 5 minutes, about 5 seconds to about 5 minutes, about 10 seconds to about 5 minutes, about 15 seconds to about 5 minutes, about 1 second to about 2 minutes, about 1 second to about 3 minutes, or about 5 seconds to about 4 minutes) of each other.
  • both one or more modulators of an AIRE polypeptide level e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • the one or more modulators of an AIRE polypeptide level e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • the one or more immune checkpoint inhibitors can be administered to a mammal at different times.
  • the one or more modulators of an AIRE polypeptide level e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • the one or more modulators of an AIRE polypeptide level e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • the one or more immune checkpoint inhibitors can be administered to a mammal with from about 5 minutes to about 7 days (e.g., from about 5 minutes to about 6 days, from about 5 minutes to about 5 days, from about 5 minutes to about 4 days, from about 5 minutes to about 3 days, from about 5 minutes to about 2 days, from about 5 minutes to about 1 day, from about 30 minutes to about 7 days, from about 60 minutes to about 7 days, from about 1 day to about 7 days, from about 3
  • each composition can be administered to a mammal by any appropriate route.
  • one or more modulators of an AIRE polypeptide level e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • one or more modulators of an AIRE polypeptide level e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • one or more immune checkpoint inhibitors can be administered by the same route.
  • one or more modulators of an AIRE polypeptide level e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • one or more immune checkpoint inhibitors can be administered by different routes.
  • One or more immune checkpoint inhibitors described herein can be administered to a mammal by any appropriate route.
  • one or more immune checkpoint inhibitors described herein can be administered locally or systemically.
  • one or more immune checkpoint inhibitors described herein can be designed for oral or parenteral (e.g., subcutaneous, intramuscular, intravenous, intraperitoneal, and intradermal) administration.
  • one or more immune checkpoint inhibitors described herein can be administered via an intra-tumoral administration.
  • compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostats, and solutes which render the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the composition can be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.
  • the population of T cells can be administered first, and the one or more immune checkpoint inhibitors administered second, or vice versa.
  • one or more modulators of an AIRE polypeptide level e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • one or more immune checkpoint inhibitors can be administered to a mammal (e.g., a human) having cancer (e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors) as the sole active agent(s) to induce immune responses against cancer cells (e.g., cancer cells that exhibit little or no response to treatment with immune checkpoint inhibitors) within the mammal (e.g., to treat the mammal).
  • a combination therapy used to treat a mammal e.g., a human having cancer (e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors) can include administering to the mammal one or more modulators of an AIRE polypeptide level (e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide) and, optionally, one or more immune checkpoint inhibitors, and can include performing one or more (e.g., one, two, three, or more) therapies used to treat cancer.
  • an AIRE polypeptide level e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • immune checkpoint inhibitors e.g., one, two, three, or more
  • additional therapies that can be used to treat a mammal (e.g., a human) having cancer (e g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors) include, without limitation, administering one or more anti-angiogenesis agents, administering one or more targeted therapies (e.g., one or more antibody therapies), administering one or more cancer vaccines, radiation therapies, and/or surgeries.
  • a mammal e.g., a human
  • cancer e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors
  • additional therapies include, without limitation, administering one or more anti-angiogenesis agents, administering one or more targeted therapies (e.g., one or more antibody therapies), administering one or more cancer vaccines, radiation therapies, and/or surgeries.
  • one or more modulators of an AIRE polypeptide level e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • one or more immune checkpoint inhibitors are used in combination with one or more therapies used to treat a mammal (e.g., a human) having cancer (e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors)
  • the one or more additional therapies can be performed at the same time or independently of the administration of the one or more modulators of an AIRE polypeptide level (e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide) and, optionally, the one or more immune checkpoint inhibitors.
  • one or more modulators of an AIRE polypeptide level e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • one or more immune checkpoint inhibitors can be administered before, during, or after the one or more additional therapies are performed.
  • the materials and methods provided herein can be used to improve survival of a mammal (e.g., a human) having cancer.
  • a mammal in need thereof e.g., a mammal having cancer such as a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors
  • a mammal in need thereof e.g., a mammal having cancer such as a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors
  • can be administered one or more modulators of an AIRE polypeptide level e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • an AIRE polypeptide level e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • immune checkpoint inhibitors e.g., one or more immune
  • the materials and methods described herein can be used to improve the survival of a mammal having cancer by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
  • the materials and methods described herein can be used to improve the survival of a mammal having cancer by, for example, at least 6 months (e.g., about 6 months, about 8 months, about 10 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 4 years, about 5 years, or more).
  • the materials and methods provided herein can be used to reduce the size of the cancer in the mammal.
  • a mammal in need thereof e.g., a mammal having cancer such as a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors
  • a mammal in need thereof can be administered one or more modulators of an AIRE polypeptide level (e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide) and, optionally, one or more immune checkpoint inhibitors to reduce the size of the cancer in the mammal.
  • modulators of an AIRE polypeptide level e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide
  • immune checkpoint inhibitors e.g., one or more immune checkpoint inhibitors to reduce the size of the cancer in the ma
  • the methods and materials provided herein can be used as described herein to reduce the number of cancer cells in the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some cases, the methods and materials provided herein can be used as described herein to reduce the volume of one or more tumors in the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
  • the materials and methods provided herein can include monitoring the mammal (e.g., the human) being treated as described herein (e.g., by administering one or more modulators of an AIRE polypeptide level (e.g., one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) or one or more inhibitors of an AIRE polypeptide) and, optionally, one or more immune checkpoint inhibitors).
  • the size of the cancer e.g., the number of cancer cells and/or the volume of one or more tumors
  • Any appropriate method can be used to determine whether or not the size of the cancer present within a mammal is reduced.
  • imaging techniques can be used to assess the size of the cancer present within a mammal (e.g., a human).
  • This Example describes the discovery that modulation of AIRE expression in cancer cells can alter (e.g., increase or decrease) the immunogenicity of the cancer.
  • self refers to antigens that present as self-antigens, such that the immune system tolerates cells expressing these antigens and does not attack them.
  • non-self refers to antigens that present as foreign antigens such that the immune system attacks cells expressing these antigens.
  • TSA Tissue Specific Antigen
  • AIRE expression was decreased by short-hairpin RNA (shRNA) targeting nucleic acid encoding an AIRE polypeptide. Knocking down AIRE (shRNA) decreased expression of GAPDH, TYRP2, and CSDE1. The level of OVA was not changed.
  • AIRE expression was increased by expressing a nucleic acid encoding an AIRE polypeptide under the control of a CMV promoter (CMV-AIRE). Overexpressing AIRE (CMV-AIRE) increased expression of GAPDH, TYRP2, and CSDE1. The level of OVA was not changed.
  • Splenocytes from either OT-1 or cancer experienced T cells (splenocytes from mice vaccinated with B16 tumor cells (NO OVA)) were co-cultured with:
  • B16-VSV-ESC cells B16 cells selected in vitro for escape from VSV in which the self CSDE1 protein is -90% mutated to CSDE1* and expressed from the endogenous CSDE1 promoter such that the CSDE1* protein acts as a neoantigen in the C57B1/6 model;
  • B16-CSDE1* transfected cells cells in which the CSDE1* neoantigen is overexpressed in B16 cells from the CMV promoter; or 4).
  • B16ova-APOBEC3B cells B16ova cells infected with a viral vector expressing a AP0BEC3B polypeptide.
  • E:T effector cell to target cell ratio of 1 :1 was used for all experiments unless otherwise noted.
  • Target cells were treated with 1) mock shRNA; 2) anti-AIRE shRNA lentiviral particles; or 3) plasmid containing CMV-AIRE. JFN-y expression was assessed by ELISA.
  • AIRE expression makes cancer cells look immunologically self through increasing the occupancy of MHC molecules with self TSA.
  • CD8+ T cells isolated by Magnetic bead and incubated with PARENTAL cells E:T 10: 1 (10 A 6: 10 A 5)
  • CD8+ T cells isolated by Magnetic bead and incubated with PARENTAL cells E:T 10:1 (10 A 6: 10 A 5)
  • Diffuse midline gliomas (DMG) tumor cells with reduced expression of AIRE were more immunogenic for human CD8+ T cells than parental DMG.
  • APOBE3B mutation co-operated with both enhanced AIRE expression (e.g., for neoepitopes in self antigens) and with decreased AIRE expression (e.g., for neo-epitopes in nonself antigens).
  • Example 2 Reduction of AIRE In Vivo
  • This Example examined whether a vaccine from AIRE down-regulated DMG was more immunogenic than a vaccine from self DMG cells having normal AIRE expression.
  • PKC DMG cells were loaded with lysates of 10 7 cell equivalents of PKC, with or without anti-PDl ICB, were administered to mice.
  • the treatment regimen was as follows:
  • PKC DMG cells were injected intracranially (ic) with 10 6 DCs loaded with lysates of 10 7 cell equivalents of PKC (murine DMG cells), with or without anti-PDl ICB as shown below.
  • PKC DMG cells were injected ic with 10 6 DCs loaded with lysates of 10 7 cell equivalents of PKC (murine DMG cells) that were transfected with nucleic acid encoding a histone H3 polypeptide having a lysine to methionine substitution of residue 27 on histone H3 (K27M), with or without anti-PDl ICB as shown below.
  • the K27M mutation of DIPG/DMG enhanced self-ness, at least in part through AIRE expression, which could be reversed by reversion to K27 status ( Figure 14).
  • FIG. 15 A schematic of an exemplary experimental protocol is shown in Figure 15. Compositions containing 10 7 splenocytes and 10 6 target cancer cells were pre-treated with IFNy for 24 hours and administered to mice. After 48 hours, ELISA was used to examine IFNy levels. For each experiment, three technical triplicates and three biological triplicates were performed.
  • Vaccination with PKC(shRNA-AIRE) cells revealed immunogenic neoantigens against parental PKC DMG cells ( Figure 17A). There was a further subset of neoantigens generated by vaccination with PKC(shRNA-AIRE) cells which were not recognized on PKC parental cells ( Figure 17B). Reducing AIRE expression in DMG cells exposed neoantigens which were also exposed by closing the chromatin by restoring K27 expression ( Figure 17D).
  • cancer cells expressing self antigens as the immunogens or cancer cells expressing non-AIRE regulated antigens were seeded in mice, and then the mice were treated with naive OT-I cells (murine CD8+ T cells with a transgenic T cell receptor that recognizes an immunodominant epitope of an OVA polypeptide) or PMEL cells (murine CD8+ T cells with a transgenic T cell receptor that recognizes an immunodominant epitope of a human gplOO polypeptide), with ICB.
  • Treatment groups were as shown below.
  • VSV Boost VSV-ova, VSV-hgplOO or VSV-GFP 5xl0 6 pfu/injection
  • AIRE-ve cancer vaccine e.g., a cell line that does not express AIRE as analyzed by western blot
  • AIRE-ve cancer vaccine exposes self antigens to confer sensitivity to immune checkpoint blockade to an otherwise ICB-insensitive tumor ( Figure 21).
  • Example 3 Cancel Immunotherapy Using AIRE Conditioning of the Tumor Epitopeome
  • AIRE steady state levels reset the selfness of cancer cells and convert a highly tolerized T cell compartment into a heteroclitic tumor-reactive T cell population, thus eliminating the need to identify specific TAAs as targets for generation of de novo CD8 + and helper CD4 + T cell responses that can clear tumors.
  • one or more AIRE polypeptides or nucleic acid designed to express an AIRE polypeptide can be used to treat a cancer having cancer cells that express an AIRE-regulated antigen and such treatment can be supported by additional immunotherapy interventions.
  • B16 murine melanoma cells were obtained from ATCC prior to being modified with the relevant transgenes.
  • Cell lines were authenticated by morphology, growth characteristics, PCR for melanoma specific gene expression (gplOO, TYRP- and TYRP-T), and biologic behavior. The cells tested mycoplasma-free and were frozen. Cells were cultured for less than 3 months after thawing.
  • the B16-OVA cell line was derived from a B16-F10 clone transfected with a pcDNA3.1ova plasmid. B16-0VA cells were grown in DMEM
  • B16-OVA-(AIRE), B16-F10-(AIRE), PKC-(AIRE), DIPG-XIII-(AIRE), and SOH-(AIRE) cells were generated by stable transduction of B16-OVA or B16-F10 cells with pCMV-Entry AIRE (Accession Number NM009646, Origene, Rockville, USA CAT#: MC218789).
  • B16-OVA- (shRNA-AIRE), B16-F10-(shRNA-AIRE), PKC-(shRNA-AIRE), DIPG-XIII-(shRNA- AIRE), and SOH-(shRNA-AIRE) cells were generated by transduction with shRNA lentiviral particles (4 unique 29mer target-specific shRNA) or with a scramble control (Origene, Rockville, USA Catalogue #TL510188V) followed by selection in puromycin (1.25 pg/mL).
  • B16-TK cells were derived from a B16-Fl clone transfected with a plasmid expressing the HSV-1 TK gene in 1997/1998 (Sanchez-Perez et al., Gene Ther, 14:998-1009 (2007); Vile et al., Int J Cancer, 71 :267-274 (1997); Vile et al., Cancer Res, 54:6228-6234 (1994); Evgin et al., Cancer Immunol Res, 7:828-840 (2019)). Following stable selection in 1.25 pg/mL puromycin, these cells were shown to be sensitive to ganciclovir (CYMEVENE®) at 5 pg/mL.
  • CYMEVENE® ganciclovir
  • the PKC cell line was derived from a genetically engineered mouse model that closely mirrors human DMG. This model makes use of an RCAS tumor virus system to induce PDGFp and H3.3K27M overexpression in the context of p53 loss and is targeted to neonatal neural progenitor cells by the expression of the virus receptor under the control of the Nestin promoter (Becher, et al., Cancer Res, 50:2548-2557 (2010); Misuraca et al., Front Oncol, 5: 172 (2015)).
  • brainstem tumors were established by implanting DF-1 producer cells transfected with the RCAS plasmids (Becher, et al., Cancer Res, 50:2548- 2557 (2010); Misuraca et al., Front Oncol, 5: 172 (2015)) into Nestin tv-a/p53 floxed mice.
  • the PKC cell line was established by explanting an established tumor from this model. K27M status of PKC was confirmed by sequence analysis.
  • DIPG-XIII and SOH are pediatric DIPG/DMG cell lines that were cultured in TSM media, which consists of 50% NeurobasalTM-A Medium, 50% DMEM/F-12, 10 mM HEPES solution, 1 mM MEM sodium pyruvate solution, lx GlutaMAXTM Supplement, l x antibiotic/antimycotic solution, lx B-27TM Supplement minus Vitamin A, 20 ng/mL human epidermal growth factor (Shenandoah Biotech), 20 ng/mL human fibroblast growth factor basic-154 (Shenandoah Biotech), 10 ng/mL human PDGF-AA (Shenandoah Biotech), 10 ng/mL human PDGF-BB (Shenandoah Biotech), and 2 pg/mL heparin solution (StemCell Technologies). Cells were tested for mycoplasma using the MycoAlert® Mycoplasma Detection Kit (Lonza Rockland, Inc., ME
  • the OT-I mouse strain is on a C57B1/6 background and expresses a transgenic T-cell receptor Va2/V05 specific for the SIINFEKL (SEQ ID NO:3) peptide of ovalbumin in the context of MHC class I, H-2Kb as described.
  • the PMEL mouse strain is on a C57B1/6 background and express a transgenic T-cell receptor Val/Vpi3 that recognizes amino acids 25-33 of gplOO presented by H2-D b .
  • spleen and lymph nodes from OT-I or PMEL transgenic mice were combined and crushed through a 100-pm filter to prepare a single cell suspension.
  • RBC were removed by a 2-minute incubation in ACK buffer (sterile distilled H2O containing 0.15 mol/L NH4CI, 1.0 mmol/L KHCO3, and 0.1 mmol/L EDTA adjusted to pH 7.2-7.4).
  • ACK buffer sterile distilled H2O containing 0.15 mol/L NH4CI, 1.0 mmol/L KHCO3, and 0.1 mmol/L EDTA adjusted to pH 7.2-7.4
  • CD8 1 T cells were isolated using the MACS CD8a(Ly-2) Microbead magnetic cell sorting system (Miltenyi Biotec, Auburn, CA) and stained with CFSE dye (Molecular Probes, Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.
  • nonadherent and loosely adherent cells were harvested following one activation cycle of 3 to 5 days and viable cells were purified by density gradient centrifugation using Lympholyte®- M (Cedarlane Laboratories) according to the manufacturer's instructions. More than 90% of the cells expressed the V012 chain of the transgenic OT-I T cell receptor.
  • CD8 + T cells were co-cultured with target tumor cells at various effector to target ratios.
  • Supernatants were assayed for IFN-y by ELISA as directed in the manufacturer’s instructions (Mouse IFN-y ELISA Kit, OptEIA, BD Biosciences, San Diego, CA).
  • VSV-GFP, VSV-hgplOO, and VSV-ova were generated by cloning the appropriate cDNAs into the plasmid pVSV-XN2, as described elsewhere (Fernandez et al., J Virol, 76:895-904 (2002)).
  • Monoclonal VSVs were obtained by plaque purification on BHK-21 cells. Concentration and purification were done by sucrose gradient centrifugation.
  • Virus stock titers were measured by standard plaque assays of serially diluted samples on BHK-21 cells (Diaz et al., Cancer Res, 67:2840-2848 (2007); Rommelfanger et al., Cancer Res, 72:4753-4764 (2012); Fernandez et al., J Virol, 76:895-904 (2002)).
  • CD8 + T cells were prepared from C57BL/6 mice that had been cured of subcutaneous B16-TK tumors following three weekly courses of GCV (50 mg/kg on days 5- 9, 12-16, and 19- 23). Cells were harvested between 60- and 80-days post tumor implantation.
  • RNA was prepared with the QIAGEN-RNeasy®-MiniKit (Qiagen, Valencia, CA). One pg total RNA was reverse-transcribed in a 20 pL volume using oligo-(dT) primers using the First Strand cDNA Synthesis Kit (Roche). A cDNA equivalent of 1 ng RNA was amplified by PCR with gene-specific primers using GAPDH as loading control. qRTPCR was carried out using a LightCycler480 SYBRTM Green I Master kit and a LightCycler® 480 instrument (Roche) according to the manufacturer’s instructions. The AACT method was used to calculate the fold change in expression levels of target genes and GAPDH as an endogenous control for all treated samples relative to an untreated calibrator sample. Table 7. Exemplary Primer Sequences.
  • mice C57B1/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME) at 6 to 8 weeks of age.
  • 5* 1O 5 B16-OVA or B16-F10 cells in 100 pL of PBS were injected into the flank of mice.
  • VSV-OVA, VSV-PMEL, and VSV-GFP viral injections (100 pL) were done intravenously at time points as described).
  • AAV-8 injections were administered intratum orally.
  • Immune cell depletions were done by intraperitoneal injections (0.1 mg/mouse) of anti-CD8 (Lyt 2.43) and anti-CD4 (GK1.5), and IgG control (ChromPureTM Rat IgG; Jackson ImmunoResearch, West Grove, PA) at day 4 after tumor implantation and then weekly thereafter. Fluorescence-activated cell sorting analysis of spleens and lymph nodes confirmed subset-specific depletions.
  • mice were treated intravenously or intraperitoneally with anti-PDl (0.25 mg; catalog no. BE0146; Bio X Cell) or isotype control rat IgG antibody (catalog no. 012-000-003; Jackson ImmunoResearch) at times described.
  • anti-PDl 0.25 mg
  • isotype control rat IgG antibody catalog no. 012-000-003; Jackson ImmunoResearch
  • mice were given naive OT-I or PMEL T cells intravenously (10 7 cells in 100 pL per injection) as described after tumor injection.
  • tumor diameters were measured thrice weekly in two dimensions using calipers, and mice were sacrificed when tumor size was —1.0 cm 2 .
  • mice Spleens or tumor draining lymph nodes were removed from mice at the indicated times.
  • IxlO 6 cells were plated (unless otherwise indicated) in 24 well plates and incubated at 37°C with the indicated targets (peptides at 5 pg/mL i.e., H-2Kb-restricted peptides TRP-2i 8 o-i88SVYDFFVWL (SEQ ID NO: 14), ova SIINFEKL (SEQ ID NO:3)) or cells as indicated.
  • targets peptides at 5 pg/mL i.e., H-2Kb-restricted peptides TRP-2i 8 o-i88SVYDFFVWL (SEQ ID NO: 14), ova SIINFEKL (SEQ ID NO:3)
  • B16-OVA cells and variants were treated with 50 U of rIFN-y for 24 hours prior to quantification of surface-expressed K2b/SIINFEKL (SEQ ID NO:3) or co-culture with T cells to increase the frequency of cells expressing MHC Class I Murine rIFN-y (eBioscience, SD, USA, Cat# 14-8311-63). Cell-free supernatants were collected after 48 hours and tested by ELISA for IFN-y (BD OptEIATM Mouse IFN-y ELISA Set; BD Biosciences Pharmingen, San Diego, CA, USA).
  • IxlO 5 cells were plated into each well of a 96-well ELISPOT plate in triplicate and were re-stimulated for 48 hours at 37°C with the relevant targets (peptides or cells). Peptide-specific IFN-y positive spots were detected according to the manufacturer's protocol and were quantified by computer assisted image analyzer.
  • Murine bone marrow DCs were prepared from C57B1/6 mice as described elsewhere (Melcher et al., Cancer Res, 59:2802-2805 (1999); Merrick et al., Cancer Immuol Immunother, 57:897-906 (2008); Ilett et al., Gene Ther, 16:689-699 (2009); Evgin et al., Sci TranslMed, 14:eabn2231 (2022); Gallucci et al., Nat Med, 5: 1249-1255 (1999)). Femurs were collected from C57/B16 mice, and bone marrow was flushed into RPMI media using a 25-gauge needle.
  • Bone marrow was treated with ammonium-chloride-potassium (ACK) lysis buffer, washed with serum-free RPMI, and then resuspended in RPMI supplemented with 10% FBS + l x penicillin/streptomycin + 50 pM 2-mercaptoethanol supplemented with murine granulocyte-macrophage colony-stimulating factor (GM-CSF (20 ng/mL; Peprotech). Cells were seeded at 10 6 cells per well in 2 mL of a 24-well plate. Media were replaced with fresh murine GM-CSF -containing media on day 3. Bone marrow derived dendritic cells (BMDCs) were collected on day 5.
  • BMDCs bone marrow derived dendritic cells
  • B16-F10, B16-F10-(shRNA-AIRE), or B16-F10-(AIRE) tumor cells were expanded in T175 flasks. At 80-90% confluency, cells were trypsinized and washed three times in phosphate-buffered saline (PBS) (HyClone). Aliquots of 5x 10 7 cells were resuspended in a volume of 1 mL PBS and then freeze-thawed for three cycles in liquid nitrogen. Mature DCs were then incubated with the tumor lysates at a ratio of 1 DC to 10 tumor cell equivalents at 37°C for 12 hours. Each vaccine comprised of 10 6 mature DCs loaded with the equivalent of 10 7 tumor cells in 100 pL administered intravenously to mice.
  • PBS phosphate-buffered saline
  • Splenocytes from naive C57B1/6 mice were co-cultured with live PKC, PKC(shRNA- AIRE), or PKC-(CMV-AIRE) cells pre-treated for 24 hours with IFN-y to enhance MHC Class I presentation at a ratio of 10:1 for three days with IL-2. On days 6/7 and 9/10, cocultures were re-plenished with live, ZFN-y -pre-treated PKC variant tumor cells. After 2 weeks of culture, CD8 + T cells were recovered by magnetic bead isolation and co-cultured with 10 ? parental PKC tumor cells at a ratio of between 5: 1 and 10: 1. Cell-free supernatants were collected after 48 hours and tested by ELISA for IFN-y (BD OptEIATM Mouse IFN-y ELISA Set; BD Biosciences Pharmingen).
  • APOBEC3B ACTIVE full length functional APOBEC3B
  • APOBEC3B INACTIX E a mutated, catalytically inactive form of APOBEC3B
  • APOBEC3B Overexpression of APOBEC3B was confirmed by both western blot (using a rabbit monoclonal anti-human APOBEC3B (184990, Abeam, San Francisco, CA)) and qrtPCR as described elsewhere (Driscoll et al., Nat Comm, 11 :790 (2020)). Overexpression of APOBEC3B is toxic. Because mutagenesis by APOBEC3B is tolerable to the cell up to a certain threshold, APOBEC3B cells were used within 14 days to prevent the accumulation of toxic mutations killing the cells (Driscoll et al., Nat Comm, 11 :790 (2020)).
  • CD3 + T cells were isolated using a magnetic sorting kit (Miltenyi Biotech) and activated using CD3/CD28 beads (ThermoFisher). T cells were immediately co-cultured at a ratio of 10: 1 with autologous DCs loaded with DMG cell line lysates.
  • Autologous monocyte-derived DCs were matured by isolating CD14 + cells by magnetic sorting (Miltenyi Biotech), followed by incubation with human GM-CSF (800 U/mL) and IL-4 (1000 U/mL). On days 3 and 5, media was replaced with human GM-CSF (1600 U/mL) and IL-4 (1000 U/mL).
  • non-adherent cells were collected, washed with PBS, and resuspended in medium containing GM-CSF (800 U/mL), IL-4 (1000 U/mL), TNF-alpha (1100 U/mL), IL-lbeta (1870 U/mL), IL-6 (1000 U/mL), and PGE2 (1 pg/mL).
  • GM-CSF 800 U/mL
  • IL-4 1000 U/mL
  • TNF-alpha 1100 U/mL
  • IL-lbeta 1870 U/mL
  • IL-6 1000 U/mL
  • PGE2 (1 pg/mL
  • cell lysates of DMG, DMG-(shRNA-AIRE), or DMG-(CMV-AIRE) were added to the culture at an approximate ratio of DMG cell (lysate):DC of 10: 1.
  • DCs were harvested for co-incubation with activated T cells at a ratio of 1: 10.
  • CD8 + T cells were reisolated using magnetic bead sorting (purchased form Miltenyi Biotech), co-cultured with IFN-y pre-treated (200 U/mL for 12 hours) parental DMG cells for 72 hours, and followed by IFN-y ELISPOT (R&D Systems).
  • phenotype 1 x 10 6 cells were washed in IX PBS containing 0.1 % BSA and 0.01% sodium azide (FACS buffer), re-suspended in 50 pL of FACS buffer, and exposed to fluorochrome-conjugated primary antibodies for 30 minute at 4°C.
  • the mouse IgG125-Dl .16 antibody is specific for the MHC class I molecule Kb bound to the peptide SIINFEKL (SEQ ID NOB) (Kb-SIINFEKL) (Biolegend San Diego, USA).
  • Cells were then washed and resuspended in 500 pL of PBS containing 4% formaldehyde (Shirafkan et al., Front Immunol, 15: 1339714 (2024)). Cells were subjected to flow cytometry and data were analyzed using CellQuestTM software (BD Biosciences, San Jose, CA, USA) or FlowJo (Tree Star, Inc., Ashland, OR, USA).
  • B16-F10 cells were propagated to IxlO 9 total cells in 50, 150 cm dishes. Cells were trypsinized and collected to obtain a cell suspension. Cells were washed twice in PBS, pelleted, flash frozen in liquid nitrogen, and stored at -80°C until prepared for immunoprecipitation.
  • Immunoprecipitation columns were prepared with 4 mL Protein A Sepharose resin (CaptivA® PriMAB) crosslinked with 6 mg anti-H-2KB (Clone Y- 3, BioXCell). Cell pellets were lysed with 20 mL 0.5% IPEGAL® lysis buffer with 2X protease inhibitors (Roche, EDTA free). Lysates were centrifuged at 2,000g for 10 minutes. Supernatant was collected and ultracentrifuged at 100,000g for 75 minutes. Supernatant was collected and filtered through a 0.45 pm filter. Lysates were precleared on columns with 2 mL Sepharose A resin and then loaded onto antibody bound columns.
  • Protein A Sepharose resin CaptivA® PriMAB
  • Lysates were allowed to flow through by gravity and then washed with 100 mL of wash buffer 1 (0.005% IPEGAL®, 50mM Tris, pH 8, 150 mM NaCl, 5 mM EDTA, 100 pM PMSF, and 1 pg/mL pepstatin A), wash buffer 2 (50 mM Tris, pH 8, and 150 mM NaCl), 3 (50 mM Tris, pH 8, and 450 mM NaCl), and wash buffer 4 (50 mM Tris, pH 8). Bound MHC complexes were eluted in 10% v/v acetic acid and LC-MS/MS was performed.
  • wash buffer 1 0.005% IPEGAL®, 50mM Tris, pH 8, 150 mM NaCl, 5 mM EDTA, 100 pM PMSF, and 1 pg/mL pepstatin A
  • wash buffer 2 50 mM Tris, pH 8, and 150 mM NaCl
  • 3 50 mM Tri
  • B16-F10 and B16-F1-OVA murine melanoma cell lines had different patterns of AIRE expression.
  • Two human pediatric DMG cell lines (DIPG-XIII and DIPG-SOH) had similar AIRE profiles ( Figure 22).
  • SIINFEKL SEQ ID NO:3
  • CD8 + T cell epitope of the OVA protein B16-OVA cells lines in which AIRE was either knocked down or overexpressed were generated using stable transfection with a plasmid constitutively expressing murine AIRE by the CMV promoter (Figure 23). Knockdown of AIRE significantly reduced levels of GAPDH while over-expression of AIRE also increased GAPDH levels (Figure 24A).
  • OT-I T cells are transgenic T cells with T Cell Receptor (TCR) specificity for the SIINFEKL (SEQ ID NO:3) epitope of OVA presented in the context of H-2Kb Class I MHC by B16-0VA tumor cells ( Figure 27A) (Hogquist et al., Cell, 76: 17-27 (1994); Clarke et al., Immunol Cell Biol, 78: 110-117 (2000)).
  • TCR T Cell Receptor
  • B16-OVA-(shRNA-AIRE) cells stimulated OT-I T cells significantly more than the parental B16-0VA cells
  • B16-0VA-(AIRE) cells were significantly less immunogenic to OT-I T cells (Figure 27A) thus confirming that MHC Class I occupancy by epitopes of the non- AIRE-mediated protein OVA is enhanced by lower levels of SELF epitopes (knockdown of AIRE) and inhibited by higher levels of SELF epitopes (AIRE over-expression).
  • PMEL CD8 + T cells are transgenic T cells with TCR specificity for the H2-D b -restricted human pl0025-33 (hgplOO, KVPRNQDWL) epitope but can also be activated through their TCR by the murine homologue of the melanoma- associated antigen mgpl0025-33 (mgplOO, EGSRNQDWL) ( Figure 27B) (Overwijk & Restifo, Crit Rev Immunol, 20:433-450 (2000); Overwijk et al., J Exp Med, 188:277-286 (1998)).
  • GplOO is regulated by AIRE in the thymus (Trager et al., PloS one, 7:e35005 (2012)).
  • B16- OVA-(shRNA-AIRE) cells were almost completely unable to stimulate PMEL T cells to secrete IFN-y (Figure 27B).
  • B 16-0 VA-( AIRE) cells were significantly more immunogenic to PMEL T cells than the parental B16-OVA cells ( Figure 27B).
  • MHC occupancy by SELF, AIRE-regulated proteins, such as mgplOO, is enhanced by over-expression of AIRE and reduced by AIRE knockdown.
  • B16-OVA-(shRNA-AIRE) cells were significantly less able to re-stimulate these T.E. CD8 + T cells to secrete IFN-y (Figure 27C) while Bl 6-OVA-( AIRE) cells were significantly more immunogenic to T.E. CD8 + T cells than were the parental B16-OVA cells ( Figure 27C) thus indicating that T.E. anti-B16-TAA T cells had TCR specificities for SELF, AIRE-regulated proteins, such as mgplOO, TYRP1, TYRP2.
  • B16-OVA cells that escaped OT-I T cells in vitro expressed significantly higher levels of AIRE than the parental B16-OVA prior to T cell pressure ( Figure 28).
  • B16-OVA cells that escaped from PMEL T cell pressure expressed significantly decreased levels of AIRE compared to parental B16-OVA ( Figure 28) thus indicating that changes in AIRE expression can be used to escape from T cell pressure in a manner heavily dependent upon the nature of the antigen targeted by T cells (AIRE-regulated or not).
  • AIRE mediated regulation of TAA can be exploited for adoptive T cell therapies
  • B16-0VA tumors are highly susceptible to adoptive transfer of naive OT-I or PMEL T cells when the OT-I/PMEL T cells are activated in vivo by co-infection with the immunogenic Vesicular Stomatitis Virus (VSV) expressing either OVA or hgplOO ( Figures 29 and 30) (Diaz et al., Cancer Res, 67:2840-2848 (2007); Rommelfanger et al., Cancer Res, 72:4753-4764 (2012)).
  • VSV Vesicular Stomatitis Virus
  • B16-OVA- (shRNA-AIRE) cells were treated very effectively by transfer of naive OT-I T cells in the absence of in vivo activation by VSV-OVA ( Figure 29), however, suggesting that reduced levels of AIRE are associated with both increased levels of (non- AIRE regulated) OVA/SIINFEKL (SEQ ID NO:3) target antigen and the ability of these cells to activate naive T cells in vivo.
  • the constellation of epitopes presented by DCs loaded with B16-F10-(AIRE) lysates raised heteroclitic T cell responses that could cross-react against parental Bl 6-F 10 tumors which were present amongst a larger population of B16-F10-(AIRE)-specific CD8 + T cells.
  • Murine PKC cells derived from a K27M mutant genetically engineered model of DMG, are very poorly immunogenic in C57B1/6 mice and could not induce IFN-y secretion from CD8 + T cells even after extensive in vitro priming/education (Figure 4A).
  • PKC-(CMV- AIRE) cells were equally non-immunogenic ( Figure 33).
  • PKC-(shRNA-AIRE) cells stimulated low but significant levels of IFN-y from CD8 + T cells against parental PKC cells following in vitro priming ( Figure 33).
  • knockdown of AIRE in two different human DMG cell lines significantly enhanced priming of human CD8 + T cells against the parental cells ( Figures 34-36).
  • DIPG-XIII-(shRNA-AIRE) cells effectively educated CD8+ T cells to recognize the parental DIPG-XIII tumor cells ( Figure 37).
  • APOBEC-modified/mutated DIPG-XIII- (APOBEC3B) cells were very slightly but significantly more immunogenic than the unmutated DIPG-XIII cells ( Figure 37), presumably due to an increased mutational load generating immunogenic epitopes (Driscoll et al., Nat Comm, 11 :790 (2020)).
  • CD8 + T cells from spleens of mice treated with DC vaccines loaded with lysates of PKC parental cells did not recognize parental PKC, PKC-(shRNA-AIRE), or PKC-(AIRE) cells as targets upon re-stimulation in vitro ( Figures 39A-C).
  • CD8 + T cells from spleens of mice treated with DC vaccines loaded with lysates of PKC-(shRNA-AIRE) cells could be restimulated with both PKC -(shRNA- AIRE) cells themselves and by parental PKC cells (although to a lesser degree) ( Figures 39A and 39B).
  • AIRE over-expression in melanoma cells induces a novel set of epitope expression
  • TYRP1 epitopes were shared between all three lines, but were more abundantly present in the B16-(AIRE) cells (Table 10). All 13 TYRP1 peptides expressed in the B 16-F 10-(shRNA- AIRE) line were shared between all three cell lines (Table 10) but were present at the lowest abundances. All these peptides were also eluted from both B16-F10 parental and B16-F10-(AIRE) cells but in higher abundances as shown in Table 10 for two representative peptides. The second class of TYRP1 epitopes were those that were unique to one cell line.
  • the third class of TYRP1 peptides comprised those peptides that shared a core sequence between B 16- F10 parental and Bl 6-F 10-( AIRE) cells but were 1-3 amino acids longer in the peptides eluted from the B 16-F 10-( AIRE) line, usually at the carboxy terminal ends of the respective peptides (examples in Table 13).
  • Table 9 Total Number of Different TYRP1 Peptides Eluted from B16-F10 Parental, Bib- Fl O-(AIRE), or B16-F10-(shRNA-AIRE) Cells. Table 10. TYRP1 Peptides Eluted from B16-F10-(shRNA-AIRE) Cells.
  • both the Longer and the Unique peptide set, but not the Shared peptide set significantly enhanced survival times of B16-F10 tumor bearing mice compared to control treatments (Figure 49).
  • Vaccination with the Longer peptides primed recall responses against Bl 6-F 10 parental cells that were significantly greater than any other treatment ( Figures 50A-50F).
  • AIRE-mediated tumor therapy was dependent upon CD4 + and CD8 + T cells (Figure 45). Consistent with a critical role for IL-15-mediated DC activation by CD4 + T cell help in this therapy (Oh et al., Proc Natl Acad Sci USA, 105:5201-5206 (2008); Kutzler et al., J.
  • CD4 + T cells following vaccination with DC/Shared+aPD-1 which was therapeutically ineffective (Figure 49), could not activate DCs to produce IL- 15 even when the DCs presented the Shared peptides as targets, except from a single mouse ( Figure 5 IB).
  • CD4 + T cells from mice vaccinated with DC/Unique+aPD-1 were able to activate weak IL- 15 responses from DCs loaded with the Unique peptides( Figure 51 A). This suggests that these B16-(AIRE) expressed Unique epitopes generated in AIRE-over-expressing cells may contain T helper functions as well ( Figure 51C).
  • AIRE-mediated changes in epitope display by B16-F10 tumor cells can lead to the provision of CD4 + T cell helper epitopes capable of activating DCs against both AIRE- specific and parental expressed epitopes of TAA, such as TYRP1.
  • a human having a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors and contains cancer cells expressing one or more AIRE-regulated antigens is administered one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) to expose one or more epitopes of a tumor antigen within a cancer cell present in the human and induce immune responses against the cancer cells.
  • the administered one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) are effective to reduce the number of cancer cells present within the mammal.
  • a human having a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors and contains cancer cells expressing one or more AIRE-regulated antigens is administered one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) and is administered one or more immune checkpoint inhibitors.
  • the administered one or more AIRE polypeptides (and/or nucleic acids designed to express an AIRE polypeptide) sensitize the cancer cells to the immune checkpoint inhibitors and the immune checkpoint inhibitors are effective to reduce the number of cancer cells present within the mammal.
  • a human having a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors and contains cancer cells that do not express AIRE-regulated antigens is administered one or more inhibitors of an AIRE polypeptide to expose one or more epitopes of a tumor antigen within a cancer cell present in the human and induce immune responses against the cancer cells.
  • the administered one or more inhibitors of an AIRE polypeptide are effective to reduce the number of cancer cells present within the mammal.
  • a human having a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors and contains cancer cells that do not express AIRE-regulated antigens is administered one or more inhibitors of an AIRE polypeptide and is administered one or more immune checkpoint inhibitors.
  • the administered one or more inhibitors of an AIRE polypeptide sensitize the cancer cells to the immune checkpoint inhibitors and the immune checkpoint inhibitors are effective to reduce the number of cancer cells present within the mammal.

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Abstract

Ce document concerne des méthodes et des matériaux impliqués dans le traitement du cancer. Par exemple, un niveau d'un polypeptide régulateur auto-immun (AIRE) à l'intérieur d'un mammifère peut être modifié (par exemple, augmenté ou diminué) pour exposer un ou plusieurs épitopes d'un antigène spécifique d'une tumeur dans une cellule cancéreuse présente chez le mammifère (par exemple, de telle sorte que le mammifère produit une réponse immunitaire contre la cellule cancéreuse). Dans certains cas, un ou plusieurs modulateurs d'un niveau de polypeptide AIRE peuvent être administrés à un mammifère atteint d'un cancer (par exemple, un cancer qui présente peu ou pas de réponse à un traitement avec des inhibiteurs de point de contrôle immunitaire) pour traiter le mammifère.
PCT/US2024/048918 2023-09-27 2024-09-27 Traitement du cancer Pending WO2025072717A1 (fr)

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US20030129169A1 (en) * 2001-05-03 2003-07-10 Kai Krohn Novel expression vectors and uses thereof

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Publication number Priority date Publication date Assignee Title
US20030129169A1 (en) * 2001-05-03 2003-07-10 Kai Krohn Novel expression vectors and uses thereof

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Title
BAKHRU PEARL, ZHU MENG-LEI, WANG HSING-HUI, HONG LEE K., KHAN IMRAN, MOUCHESS MARIA, GULATI AJAY S., STARMER JOSHUA, HOU YAFEI, SA: "Combination central tolerance and peripheral checkpoint blockade unleashes antimelanoma immunity", JCI INSIGHT, AMERICAN SOCIETY FOR CLINICAL INVESTIGATION, vol. 2, no. 18, 21 September 2017 (2017-09-21), XP093300735, ISSN: 2379-3708, DOI: 10.1172/jci.insight.93265 *
TRÄGER ULRIKE, SIERRO SOPHIE, DJORDJEVIC GORDANA, BOUZO BASMA, KHANDWALA SHIVANI, MELONI ANTONELLA, MORTENSEN MONIKA, SIMON ANNA K: "The Immune Response to Melanoma Is Limited by Thymic Selection of Self-Antigens", PLOS ONE, PUBLIC LIBRARY OF SCIENCE, US, vol. 7, no. 4, US , pages e35005, XP093300736, ISSN: 1932-6203, DOI: 10.1371/journal.pone.0035005 *
VILE RICHARD, PULIDO JOSE, CHEN ALEX, KENDALL BENJAMIN, TONNE JASON, METKO MURIEL, THOMPSON JILL, SANGSUWANNUKUL THANICH, YEROVI M: "Cancer Immunotherapy Using AIRE Conditioning of the Tumor Epitopeome", RESEARCH SQUARE, 7 November 2024 (2024-11-07), XP093300737, Retrieved from the Internet <URL:https://pmc.ncbi.nlm.nih.gov/articles/PMC11601838/pdf/nihpp-rs5411393v1.pdf> DOI: 10.21203/rs.3.rs-5411393/v1 *

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