Treating cancer

Abstract

This document provides methods and materials involved in treating cancer. For example, 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). In some cases, 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.

Description

[0001] TREATING CANCER

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Patent Application Serial No. 63 / 540,750, filed on September 27, 2023. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.

[0004] SEQUENCE LISTING

[0005] This application contains a Sequence Listing that has been submitted electronically as an XML file named “07039-2254W01.xml.” The XML file, created on September 20, 2024, is 152,023 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0006] TECHNICAL FIELD

[0007] This document relates to methods and materials involved in treating cancer. For example, 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). In some cases, 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.

[0008] BACKGROUND

[0009] Central tolerance is achieved by purging self-reactive lymphocytes through promiscuous expression of genes encoding tissue-specific self-antigens (TSA) by medullary thymic epithelial cells (mTECs).

[0010] A cancer (e.g., a cancer including one or more solid tumors) 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. However, some cancers respond to immune checkpoint inhibitors while other cancers do not (Jhunjhunwala et al., Nat. Rev. Cancer, 21(5):298-312 (2021)).

[0011] SUMMARY

[0012] AIRE plays a fundamental role in the immuno-tolerance process by inducing expression of TSA in mTECs. However, the relationship between the level of an AIRE polypeptide and T cell targeting can depend on the nature of the antigen be targeted.

[0013] This document relates to methods and materials involved in treating cancer (e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors). For example, 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). In some cases, 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 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). In some cases, 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 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.

[0014] As demonstrated herein, 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.

[0015] In some cases, 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 (e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors) to treat the mammal. For example, 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.

[0016] In some cases, 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. For example, 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.

[0017] 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.

[0018] In general, one aspect of this document features methods for treating a mammal having cancer where 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.

[0019] In another aspect, 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 where 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.

[0020] In another aspect, this document features methods for treating a mammal having cancer where 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.

[0021] In another aspect, this document features methods for treating a mammal having cancer where 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.

[0022] In another aspect, 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 where 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.

[0023] In another aspect, this document features methods for treating a mammal having cancer where 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.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0025] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0026] BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1. AIRE expression inhibited immunogenicity / presentation of a non-self neoantigen.

[0028] 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.

[0029] Figure 4. AIRE expression inhibited immunogenicity / presentation of a self derived neoantigen when expressed from a non-self promoter.

[0030] Figure 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.

[0031] Figure 6. AIRE expression inhibited immunogenicity / presentation of a non self neoantigen. APOBEC mutation of the OVA gene inhibited SIINFEKL (SEQ ID NO:3) presentation.

[0032] Figure 7. AIRE-mediated MHC I occupancy controlled presentation of foreign epitopes and selfness.

[0033] Figure 8. AIRE expression in tumor cell lines.

[0034] Figure 9. Cytotoxic T lymphocyte (CTL) assays against parental XIII DIPG cells.

[0035] Figure 10. CTL assays against parental SF7761 DIPG cells.

[0036] Figure 11. CTL assays against parental SOH DIPG cells.

[0037] Figure 12. CTL assays against parental SF7761 DIPG cells.

[0038] Figure 13. Inhibition of AIRE expression in DIPG / DMG tumor cells enhanced the immunogenicity of dendritic cell loaded tumor lysates.

[0039] Figure 14. The K27M mutation of DIPG / DMG enhanced selfness in part through AIRE expression which was reversed by reversion to K27 status.

[0040] Figure 15. Experimental protocol to determine immunogenicity of AIRE-modified cells.

[0041] Figures 16A-16D. Vaccination with parental PKC cells was not-immunogenic against parental PKC DMG cells.

[0042] Figures 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.

[0043] Figure 19. Overexpression of AIRE decreased the immunogenicity of non-self antigen expressing tumor cells. OVA(SIINFEKL; SEQ ID NO:3) was less visible with AIRE overexpression and more visible with loss of AIRE.

[0044] Figure 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.

[0045] Figure 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.

[0046] Figure 22. Western blot analysis of 107murine 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.

[0047] Figures 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.

[0048] 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.

[0049] Figures 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'AACtmethod 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).

[0050] Figures 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.

[0051] Figures 27A-27C. OVA-specific IFN-y positive spots in IxlO5B16-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.

[0052] Figure 28. 106B16-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.

[0053] Figure 29. C57B1 / 6 mice with 10 days established subcutaneous B16-OVA, B16- OVA-(shRNA-AIRE), or B16-OVA-(AIRE) tumors were treated intravenously with 107naive OT-I CD8+T cells. Two 2 days later (day 12) mice were treated with VSV-OVA, VSV-hgplOO, or VSV-GFP as shown (5xl06pfu 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.

[0054] Figure 30. C57B1 / 6 mice with 10 days established subcutaneous B16-OVA, B16- OVA-(shRNA-AIRE), or B16-OVA-(AIRE) tumors were treated intravenously with 107PMEL CD8+T cells. Two days later (day 12) mice were treated with VSV-OVA, VSV- hgplOO, or VSV-GFP as shown (5xl06pfu 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.

[0055] Figure 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 (106DCs loaded with lysate equivalent of 107tumor 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.

[0056] Figures 32A-32C. CD8 T cells (106 / 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 5xl05live 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.

[0057] Figure 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 CD81T 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.

[0058] Figure 34. Exemplery in vitro protocol for priming / educating human CD8+T cells with human DMG cell variants.

[0059] Figure 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.

[0060] Figure 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.

[0061] Figure 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.

[0062] Figure 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 (106DCs loaded with lysate equivalent of 107tumor 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.

[0063] Figures 39A-39C. CD8 T cells (106 / 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.

[0064] Figure 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) (IxlO9genome copies). 48 hours later, cells were harvested and assayed by western blot for AIRE and B-actin.

[0065] 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).

[0066] Figure 43. When tumors in mice reached an endpoint of 1.0 cm tumor diameter in any direction, mice were euthanized. Survival by tumor size endpoint is shown.

[0067] Figures 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.

[0068] Figure 45. A survival curve showing that AIRE-mediated tumor therapy was dependent upon CD4+and CD8+T cells.

[0069] Figure 46. Schematic showing MHC Class I immunoprecipitation from B16-F10 cells using an anti-H-2Kbantibody (Clone Y-3).

[0070] Figure 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 106DCs / 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 106DCs / 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). Figure 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 106DCs / 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 106DCs / 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.

[0071] Figures 49A-49F. CD8+T cells (106per 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. 106CD8+T cells were re-stimulated in 96-well ELISPOT plates (4 biological triplicates shown) with either 5xl05live 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 5xl05DCs 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 perlO6DCs) for 48 hours at 37°C. IFN-y positive spots were quantified by computer assisted image analyzer.

[0072] Figures 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. 5x105CD4+T cells were re-stimulated in 96-well plates (3 biological triplicates shown) with 5xl05DCs loaded with the Longer (Figure 50C), Shared (Figure 50B), or Unique (Figure 50A) peptide sets (Table 13) (total of 1 pg peptide per 106DCs) for 48 hours at 37°C. IL-15 in the supernatant 48 hours later, measured by ELISA, is shown.

[0073] DETAILED DESCRIPTION

[0074] This document relates to methods and materials involved in treating cancer (e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors). For example, 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). In some cases, 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 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). In some cases, 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 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.

[0075] In some cases, 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 a mammal (e.g., a human) can be effective to stimulate a cell-mediated immune response within the mammal. For example, 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 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. Once one or more epitopes of a tumor antigen present on a cancer cell within a mammal have been 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) to the mammal), 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). In some cases, T cells present within a mammal (e.g., a human) that can target (e.g., target and destroy) cancer cells containing the newly exposed epitope(s) can be cross-reactive against parent cancer cells. For example, 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).

[0076] In some cases, 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 a mammal (e.g., a human) 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. For example, 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 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.

[0077] Any appropriate mammal having cancer (e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors) 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). Examples of mammals that can have cancer and can be treated as described herein include, without limitation, humans, non-human primates (e.g., monkeys), horses, bovine species, porcine species, dogs, cats, mice, and rats. In some cases, a human having cancer (e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors) 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.

[0078] A mammal (e.g., a human) 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). In some cases, a cancer that can be treated as described herein can include one or more solid tumors. In some cases, a cancer that can be treated as described herein can be a blood cancer. In some cases, a cancer treated as described herein can be resistant to one or more immune checkpoint inhibitors. In some cases, a cancer treated as described herein can be a primary cancer. In some cases, a cancer treated as described herein can be a metastatic cancer. In some cases, 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.

[0079] In some cases, the methods described herein also can include identifying a mammal as having cancer. Examples of 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.

[0080] Once identified as having cancer, 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). For example, 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). For example, 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.

[0081] 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.

[0082] 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. Examples of 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. In some cases, 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.

[0083] In some cases, 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).

[0084] In some cases, 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 (e.g., a cancer that exhibits little or no response to treatment with immune checkpoint inhibitors) to treat the mammal. For example, 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.

[0085] When one or more AIRE polypeptides (and / or nucleic acids designed to express 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 express one or more AIRE-regulated antigens, the cancer cells can express any appropriate AIRE-regulated antigen(s). Examples of AIRE- regulated antigens include, without limitation, TYRP2 polypeptides, CSDE1 polypeptides, and gplOO polypeptides. In some cases, an AIRE-regulated antigen can be as described elsewhere (see, e.g., Perniola, Front. Immunol, 9:98 (2018)).

[0086] Any appropriate AIRE polypeptide or fragment thereof (and / or nucleic acid designed to express an AIRE polypeptide or a 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. Examples of 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.

[0087] In some cases, an AIRE polypeptide can have an amino acid sequence as set forth in SEQ ID NO: 1 (see, e.g., Example 3). In some cases, 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).

[0088] In some cases, 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).

[0089] When the materials and methods provided herein use a fragment of an AIRE polypeptide, 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. In some cases, an AIRE polypeptide fragment can be derived from the amino acid sequence set forth in SEQ ID NO:1. For example, 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).

[0090] 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).

[0091] Any appropriate method can be used to obtain 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). For example, 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).

[0092] 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).

[0093] 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. In cases where a nucleic acid encoding an AIRE polypeptide and / or a fragment thereof is used 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.

[0094] When a vector used to deliver nucleic acid encoding an AIRE polypeptide and / or a fragment thereof to a mammal (e.g., a human) 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. In some cases, a viral vector can be a chimeric viral vector. In some cases, a viral vector can infect dividing cells. In some cases, a viral vector can infect non-dividing cells. Examples 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.

[0095] When a vector used to deliver nucleic acid encoding an AIRE polypeptide and / or a fragment thereof to a mammal (e.g., a human) is a non-viral vector, any appropriate non-viral vector can be used. In some cases, a non-viral vector can be an expression plasmid (e.g., a cDNA expression vector).

[0096] In addition to nucleic acid encoding an AIRE polypeptide and / or a fragment thereof, a vector (e.g., a viral vector or a non-viral vector) can contain one or more regulatory elements operably linked to the nucleic acid encoding an AIRE polypeptide and / or a fragment thereof. Such 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. For example, 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. Examples of 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. As used herein, “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. For example, a vector can contain a promoter and nucleic acid encoding an AIRE polypeptide and / or a fragment thereof. In this case, 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.

[0097] In some cases, nucleic acid encoding an AIRE polypeptide and / or a fragment thereof can contain nucleic acid encoding a detectable label. For example, 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. In some cases, a detectable label can be a peptide tag. In some cases, a detectable label can be a fluorescent molecule (e.g., a fluorescent polypeptide). Examples of detectable labels that can be used as described herein include, without limitation, an HA tag, a Myc-tag, a FLAG-tag, green fluorescent polypeptides (GFPs; e.g., enhanced GFPs), and mCherry polypeptides.

[0098] 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. For example, PCR or 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.

[0099] In some cases, 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. For example, 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.

[0100] 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. Examples of 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.

[0101] Any appropriate inhibitor of an AIRE polypeptide can be used as described herein. 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). Additional inhibitors of an AIRE polypeptide (e.g., nucleic acid molecules designed to induce RNAi against AIRE polypeptide expression) can be designed based on any appropriate nucleic acid (e.g., a mRNA) encoding an AIRE polypeptide sequence. Examples of nucleic acids encoding an AIRE polypeptide sequence include, without limitation, those set forth in NCBI accession nos. NM_000383, NM_000658, and NM_000659.

[0102] In some cases, 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, can be formulated into a composition (e.g., a pharmaceutical composition) for administration to a mammal (e.g., a human). For example, 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 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). In some cases, 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 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, magnesium stearate, aluminum stearate, stearic acid, antioxidants (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, and selenium), citric acid, sodium citrate, parabens (e.g., methyl paraben and propyl paraben), petrolatum, dimethyl sulfoxide, mineral oil, serum proteins (e.g., human serum albumin), glycine, sorbic acid, potassium sorbate, water, salts or electrolytes (e.g., saline, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts), colloidal silica, magnesium trisilicate, polyacrylates, waxes, wool fat, and lecithin.

[0103] A composition (e.g., a pharmaceutical 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. Examples of 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.

[0104] A composition (e.g., a pharmaceutical 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. 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.

[0105] A composition (e.g., a pharmaceutical 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 administered locally or systemically. For example, 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 administered locally by direct injection (e.g., an intratumoral injection) to one or more solid tumors present within a mammal (e.g., a human).

[0106] An effective amount of a composition (e.g., a pharmaceutical 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.

[0107] The frequency of administration of a composition (e.g., a pharmaceutical 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. For example, 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) provided herein can include rest periods. As with 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 (e.g., a pharmaceutical 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. For example, 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. For example, 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.

[0108] In some cases when a mammal is administered 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) and one or more immune checkpoint inhibitors, 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 the one or more immune checkpoint inhibitors can be administered to a mammal at the same time (e.g., in a single composition).

[0109] In some cases when a mammal is administered 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) and one or more immune checkpoint inhibitors, 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 the one or more immune checkpoint inhibitors can be administered to a mammal separately. For example, 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 can be administered to a mammal at the same time (e.g., concurrently) as independent compositions. When 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 are administered concurrently, 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 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.

[0110] In some cases when a mammal is administered 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) and one or more immune checkpoint inhibitors, 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 the one or more immune checkpoint inhibitors can be administered to a mammal at different times. When 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 are administered 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) and 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 days to about 7 days, or from about 5 days to about 7 days) between each administration. When 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 are administered as separate compositions, each composition can be administered to a mammal by any appropriate route. In some cases, 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 can be administered by the same route. In some cases, 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 can be administered by different routes.

[0111] One or more immune checkpoint inhibitors described herein (e.g., a composition including one or more immune checkpoint inhibitors) can be administered to a mammal by any appropriate route. For example, one or more immune checkpoint inhibitors described herein can be administered locally or systemically. In some cases, one or more immune checkpoint inhibitors described herein can be designed for oral or parenteral (e.g., subcutaneous, intramuscular, intravenous, intraperitoneal, and intradermal) administration. In some cases, 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.

[0112] When 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 are administered as separate compositions, the population of T cells can be administered first, and the one or more immune checkpoint inhibitors administered second, or vice versa.

[0113] In some cases, 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 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).

[0114] In some cases, 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. Examples of 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. In cases where 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 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. For example, 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 can be administered before, during, or after the one or more additional therapies are performed.

[0115] In some cases, the materials and methods provided herein can be used to improve survival of a mammal (e.g., a human) having cancer. For example, 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) and, optionally, one or more immune checkpoint inhibitors to improve survival of the mammal. For example, 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. For example, 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).

[0116] In some cases, the materials and methods provided herein can be used to reduce the size of the cancer in the mammal. For example, 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) and, optionally, one or more immune checkpoint inhibitors to reduce the size of the cancer in the mammal. In some cases, 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.

[0117] In some cases, 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). For example, the size of the cancer (e.g., the number of cancer cells and / or the volume of one or more tumors) present within a mammal can be monitored. Any appropriate method can be used to determine whether or not the size of the cancer present within a mammal is reduced. For example, imaging techniques can be used to assess the size of the cancer present within a mammal (e.g., a human).

[0118] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0119] EXAMPLES

[0120] Example 1: AIRE and Tumor Immunogenicity

[0121] 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.

[0122] The term “self’, as used in this Example with respect to antigens refers to antigens that present as self-antigens, such that the immune system tolerates cells expressing these antigens and does not attack them.

[0123] The term “non-self ’, as used in this Example with respect to antigens refers to antigens that present as foreign antigens such that the immune system attacks cells expressing these antigens.

[0124] AIRE regulates MHC display of Tissue Specific Antigen (TSA)-derived epitopes

[0125] Expression of GAPDH, OVA (a foreign, non- AIRE regulated antigen), TYRP2 (self), and CSDE1 (self) was examined in B16ova cells with or without modulation of AIRE expression (Table 2). 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.

[0126] 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.

[0127] Table 2. qrtPCR for levels of OVA and TYP2

[0128] AIRE Inhibits Immunogenicity / Presentation of Non-Self Neoantigens

[0129] Splenocytes from either OT-1 or cancer experienced T cells (splenocytes from mice vaccinated with B16 tumor cells (NO OVA)) were co-cultured with:

[0130] 1). B16ova targets;

[0131] 2). 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);

[0132] 3). 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).

[0133] An effector cell to target cell (E:T) 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.

[0134] When 5-day activated OT-1 effector cells and 24-hour pre-treated B16ova target cells were co-cultured, AIRE expression inhibited immunogenicity / presentation of non-self neoantigens (Figure 1).

[0135] When cancer experienced effector T cells and 24-hour pre-treated B16ova target cells were co-cultured, AIRE expression enhanced immunogenicity / presentation of cancer associated self antigens (Figure 2).

[0136] When cancer experienced effector T cells and 24-hour pre-treated B16-VSV-ESC target cells were co-cultured, AIRE expression enhanced immunogenicity / presentation of neoantigens derived from VSV-escape (Figure 3).

[0137] When cancer experienced effector T cells and 24-hour pre-treated Bl 6-CSDE1* transfected target cells were co-cultured, AIRE expression inhibited immunogenicity / presentation of a self-derived neoantigen when expressed from a non-self promoter (Figure 4).

[0138] When cancer experienced effector T cells and 24-hour pre-treated B 16ova- AP0BEC3B target cells were co-cultured, AIRE expression inhibited immunogenicity / presentation of non-AIRE regulated neoantigens induced by AP0BEC3B mutagenesis and enhanced immunogenicity / presentation of AIRE-regulated neoantigens induced by APOBEC3B mutagenesis (Figure 5).

[0139] When 5-day activated OT-1 effector cells and 24-hour pre-treated B16ova- AP0BEC3B target cells were co-cultured, AIRE expression inhibited immunogenicity / presentation of a non-self neoantigens (Figure 6). APOBEC mutation of the OVA gene inhibits SIINFEKL (SEQ ID NO:3) presentation (Evgin et al., Cancer Immunol. Res., 7(5): 828-840 (2019)). AIRE-mediated MHC I Occupancy Controls Presentation of Foreign Epitopes and SELFness

[0140] AIRE expression makes cancer cells look immunologically self through increasing the occupancy of MHC molecules with self TSA.

[0141] Together, these results suggest that inhibiting AIRE in tumor cells can reduce their self-ness (e.g., can make the tumor cells recognizable as foreign by the immune system).

[0142] Immunogenic Identity of Gliomas Is Determined by Cancer Cell Expression of AIRE

[0143] Human Donor PBMC: Autologous DC and autologous CD8

[0144] Day 1: CD8 Education

[0145] CD8+DC+Target cells co-cultured at CD8:DC;T ratio of 10: 10: l(10A5 cells) for 10 day;

[0146] Target cells:

[0147] DIPG XIII; XIII+ CMV-AIRE or XIII-shRNA AIRE

[0148] SF7761; SF7761+CMV-AIRE; SF7761+shRNA AIRE

[0149] SOH; SOH+CMV-AIRE; SOH-shRNA AIRE

[0150] CD8 and DC re-added at days 4 and 8;

[0151] Day 10: CD8 Effector:

[0152] CD8+ T cells isolated by Magnetic bead and incubated with PARENTAL cells E:T 10: 1 (10A6: 10A5)

[0153] Day 12: IFNy ELISPOT developed

[0154] Donor #1: M24

[0155] Donor #2: M31 Donor #3: M32

[0156] Human Donor PBMC: Autologous DC and autologous CD8

[0157] Day 1: CD8 Education

[0158] CD8+DC+Target cells co-cultured at CD8:DC;T ratio of 10: 10: l(10A5 cells) for 10 day;

[0159] Target cells:

[0160] SF7761; SF 7761+CMV- AIRE; SF7761+shRNA AIRE

[0161] SF7761 / APOBEC3B; SF7761 / APOBEC3B+CMV-AIRE; SF7761 / APOBEC3B+shRNA AIRE

[0162] CD8 and DC re-added at days 4 and 8;

[0163] Day 10: CD8 Effector:

[0164] CD8+ T cells isolated by Magnetic bead and incubated with PARENTAL cells E:T 10:1 (10A6: 10A5)

[0165] Day 12: fFNy ELISPOT developed

[0166] Donor #1 : M24

[0167] Diffuse midline gliomas (DMG) tumor cells with reduced expression of AIRE were more immunogenic for human CD8+ T cells than parental DMG.

[0168] 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

[0169] 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.

[0170] To examine whether reduced AIRE expression in DMG cells increased the appearance of non self, PKC DMG cells were loaded with lysates of 107cell equivalents of PKC, with or without anti-PDl ICB, were administered to mice. The treatment regimen was as follows:

[0171] Day 0- Implant PKC cells, intracranially in 2 pL PBS; 2xl04cells per mouse

[0172] Day 7- Inject vaccine intravenously in 100 pL PBS

[0173] Day 10- Inject vaccine intravenously in 100 pL PBS

[0174] Day 12- Inject vaccine intravenously in 100 pL PBS

[0175] Day 15- Inject vaccine intravenously in 100 pL PBS

[0176] Day 17- Inject vaccine intravenously in 100 pL PBS

[0177] Day 19- Inject vaccine intravenously in 100 pL PBS

[0178] Day 21 -Inject 100 pg anti-PDl in 100 pL PBS, intraperitoneally

[0179] Day 23- Inject 100 pg anti-PDl in 100 pL PBS, intraperitoneally

[0180] Day 25- Inject 100 pg anti-PDl in 100 pL PBS, intraperitoneally

[0181] In a first set of experiments, PKC DMG cells were injected intracranially (ic) with 106DCs loaded with lysates of 107cell equivalents of PKC (murine DMG cells), with or without anti-PDl ICB as shown below.

[0182] Table 3. Treatment conditions.

[0183] Inhibition of AIRE expression in DIPG / DMG cancer cells significantly enhanced the immunogenicity of DC loaded cancer lysates (Figure 13).

[0184] In another set of experiments, PKC DMG cells were injected ic with 106DCs loaded with lysates of 107cell 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.

[0185] Table 4. Treatment conditions.

[0186] 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).

[0187] Survival of mice having DMG cancers was also evaluated. A schematic of an exemplary experimental protocol is shown in Figure 15. Compositions containing 107splenocytes and 106target 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.

[0188] Vaccination with parental PKC cells was not-immunogenic against parental PKC DMG cells (Figures 16A - 16D).

[0189] 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).

[0190] Closing down chromatin expression in DMG cells (vaccination with PKC(K27) cells) revealed immunogenic neoantigens against parental PKC DMG Cells (Figure 18A). There was a further subset of neoantigens generated by closing down chromatin byK27 which were not recognized on PKC parental cells (Figure 18D). Reducing AIRE expression in DMG cells exposed a subset of neoantigens which were also recognized as a result of closing the chromatin down by restoring expression of K27 (Figure 18B).

[0191] To examine whether modulation of AIRE expression affected the display of epitopes presented by cancer cells, 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.

[0192] Table 5. Treatment conditions.

[0193] And the treatment regimen was as follows:

[0194] Day 0: subcutaneous B16-ova challenge

[0195] Day +10: IV OT-I or PMEL (naive) 107cells per mouse Day +12: IV VSV Boost (VSV-ova, VSV-hgplOO or VSV-GFP) 5xl06pfu / injection Day + 14: IV OT-I or PMEL (naive) 107cells per mouse

[0196] Day +17: IV VSV Boost (VSV-ova, VSV-hgplOO or VSV-GFP) 5xl06pfu / injection

[0197] Day +19: anti-PDl

[0198] Day +21 : anti-PDl

[0199] Day +24: anti-PDl

[0200] Day +26: anti-PDl

[0201] Day +28: anti-PDl

[0202] Overexpression of AIRE decreased the immunogenicity of non-self antigen expressing cancer cells, and OVA (SIINFEKL; SEQ ID NO:3) was less visible with AIRE overexpression and more visible with loss of AIRE (Figure 19).

[0203] Overexpression of AIRE enhanced the immunogenicity of self antigen expressing cancer cells, and mgplOO was more visible with overexpression of AIRE and less visible with less AIRE (Figure 20).

[0204] To examine whether enhancement of AIRE expression in B16.F10 (AIRE negative) cells increased the appearance of AIRE controlled epitopes, a vaccine from AIRE up regulated B16 F10 cells was used. Treatment included administering 106DCs loaded with lysates of 107cell equivalents of the treatment groups were as shown below.

[0205] Table 6. Treatment conditions. The treatment regimen was as follows:

[0206] Day 0: subcutaneous B16-EGFRvIII-OVA challenge 2xl05cells per mouse

[0207] Day +7: Injection of DC

[0208] Day +10: Injection of DC anti-PD-1 or isotype IgG control

[0209] Day + 12: Injection of DC anti-PD-1 or isotype IgG control

[0210] Day +14: Injection of DC anti-PD-1 or isotype IgG control

[0211] Day +17: anti-PD-1 or isotype IgG control

[0212] Day +19: anti-PD-1 or isotype IgG control

[0213] Day +21 : anti-PD-1 or isotype IgG control

[0214] Overexpression of AIRE in an AIRE-ve cancer vaccine (e.g., a cell line that does not express AIRE as analyzed by western blot) exposes self antigens to confer sensitivity to immune checkpoint blockade to an otherwise ICB-insensitive tumor (Figure 21).

[0215] Example 3: Cancel Immunotherapy Using AIRE Conditioning of the Tumor Epitopeome

[0216] This example describes the discovery that changes in 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. Accordingly, 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.

[0217] Materials and Methods

[0218] Cell lines

[0219] 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

[0220] (Hy Clone™, Cytivia, Logan, UT, USA) + 10% FBS (Life Technologies) + 5 mg / mL G418 (Mediatech, Manassas, VA, USA) until challenge. 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). Cells were co-transfected with pBabePuro at a 10: 1 ratio followed by selection in puromycin (1.25 pg / mL). 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.

[0221] 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)). Briefly, 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.

[0222] DIPG-XIII and SOH are pediatric DIPG / DMG cell lines that were cultured in TSM media, which consists of 50% Neurobasal™-A Medium, 50% DMEM / F-12, 10 mM HEPES solution, 1 mM MEM sodium pyruvate solution, lx GlutaMAX™ Supplement, l x antibiotic / antimycotic solution, lx B-27™ 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, USA).

[0223] Mice

[0224] Six to eight-week-old female C57BL / 6 mice were purchased from Jackson Laboratories (Bar Harbor, Maine). 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-Db.

[0225] CD8+T-cell preparation

[0226] For the preparation of naive OT-I or PMEL T cells, 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). When indicated, CD81T 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.

[0227] For preparation of activated OT-I T cells, single cell suspensions from spleen and lymph nodes were adjusted to 1.0 x 106cells / mL in Iscove's modified Dulbecco's medium plus 5% FCS, 10-5 mol / L of 2-ME, 100 units / mL of penicillin, and 100 pg / mL of streptomycin and stimulated with 1 pg / mL of SIINFEKL (SEQ ID NO:3) peptide and 50 lU / mL of human interleukin 2. Every 2 to 3 days, one-half of the medium was removed and replaced with fresh medium containing 50 lU / mL of interleukin 2. For use in vivo, 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).

[0228] Viruses

[0229] 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)).

[0230] Generation of tumor experienced B16-TK (T.E.) CD8+T cells

[0231] 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.

[0232] Quantitative RT-PCR and Sequencing

[0233] 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 SYBR™ 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.

[0234] In Vivo Studies

[0235] C57B1 / 6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME) at 6 to 8 weeks of age. To establish subcutaneous tumors, 5* 1O5B16-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 (ChromPure™ 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.

[0236] For immune checkpoint blockade, 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. For adoptive transfer experiments, mice were given naive OT-I or PMEL T cells intravenously (107cells in 100 pL per injection) as described after tumor injection. For survival studies, tumor diameters were measured thrice weekly in two dimensions using calipers, and mice were sacrificed when tumor size was —1.0 cm2.

[0237] ELI SPOT and ELISA analysis for IFN-y secretion

[0238] Spleens or tumor draining lymph nodes were removed from mice at the indicated times. For ELISA, IxlO6cells 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-2i8o-i88SVYDFFVWL (SEQ ID NO: 14), ova SIINFEKL (SEQ ID NO:3)) or cells as indicated. 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 OptEIA™ Mouse IFN-y ELISA Set; BD Biosciences Pharmingen, San Diego, CA, USA). For ELISPOT assays (Mouse IFN-y ELISpotPlus, MABTECH AB, Nacka Strand, Sweden), IxlO5cells 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.

[0239] Generation of murine bone marrow dendritic cells and vaccine preparation

[0240] Murine bone marrow DCs (BMDC) 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 106cells 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. 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 107cells 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 106mature DCs loaded with the equivalent of 107tumor cells in 100 pL administered intravenously to mice.

[0241] Murine T cell in vitro education and restimulation

[0242] 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 OptEIA™ Mouse IFN-y ELISA Set; BD Biosciences Pharmingen).

[0243] APOBEC3 Overexpression

[0244] Human DMG DIPG-XIII or SOH cell lines, or their AIRE over-expressing or knocked down variants, were infected with a retroviral vector encoding either full length functional APOBEC3B (APOBEC3BACTIVE) or a mutated, catalytically inactive form of APOBEC3B (APOBEC3BINACTIX E) as a negative control. Forty-eight hours post transduction with either pBABE-Hygro APOBEC3BACTIVEor pBABE-Hygro APOBEC3BINACTIVEviruses, bulk populations of cells were selected in hygromycin for no more than 2 weeks and used for experiments. 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)).

[0245] Human T cell in vitro education and restimulation

[0246] Fresh PBMCs from a healthy donor were acquired. 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.

[0247] 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). On day 7, 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). On each of the first three days of co-culture, 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. Two days later, DCs were harvested for co-incubation with activated T cells at a ratio of 1: 10.

[0248] Ten days after initial DC (Lysate) / CD8+T cell co-culture, 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).

[0249] Flow Cytometry

[0250] For analysis of phenotype, 1 x 106cells 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 CellQuest™ software (BD Biosciences, San Jose, CA, USA) or FlowJo (Tree Star, Inc., Ashland, OR, USA).

[0251] MHC immunoprecipitation and mass spectrometry

[0252] MHC Class I immunoprecipitation from B16-F10 cells was performed as described elsewhere using the anti-H-2Kb antibody (Clone Y-3) (Purcell et al., Nat Protoc, 14:1687- 1707 (2019)). Briefly, B16-F10 cells were propagated to IxlO9total 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 (BioRad) 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. 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.

[0253] Statistics

[0254] Survival curves were analyzed by the Log-Rank test. Student’s T tests, one-way ANOVA, and two-way ANOVA were applied for in vitro assays as appropriate. Statistical significance was set at p<0.05 for all experiments.

[0255] Results

[0256] AIRE expression in tumor cells

[0257] 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). Because of the availability of reagents to monitor T cell responses to the immunodominant 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). In contrast, levels of OVA RNA were very similar between all three cell lines (Figure 24B) thus suggesting that OVA is a more suitable reference gene for comparison of expression levels of AIRE than GAPDH. It was also shown that the levels of two well characterized SELF proteins, TYRP2 and CSDE1, were significantly decreased in B16- OVA-(shRNA-AIRE) cells and increased in B 16-0 VA-( AIRE) cells relative to levels of OVA expression (Figures 25A-25D). Consistent with the reported dependency of SELF melanoma associated antigen expression upon AIRE ((Trager et al., PloS one, 7:e35005 (2012); Zhu et al., Cancer Res, 73:2104-2116 (2013)), three well-characterized SELF proteins, GAPDH, TYRP2 and CSDE1, were regulated by AIRE in Bl 6-0 VA cells. Expression of stably transfected, CMV-driven OVA protein was independent of AIRE.

[0258] AIRE-mediated MHC I Occupancy Controls Presentation of Foreign Epitopes and SELFNESS

[0259] Knockdown of AIRE significantly increased levels of SIINFEKL (SEQ ID NO:3) presented by MHC Class I in B16-OVA-(shRNA-AIRE) cells compared to parental Bl 6- OVA cells (Figure 26A). In contrast, over-expression of AIRE reduced the levels of H2Kb / SIINFEKL (SEQ ID NO:3) occupancy compared to parental B16-0VA cells (Figure 26A). These effects were amplified when the B16-0VA cells were pre-treated with IFN-y to increase levels of MHC Class I expression (Figure 26B) and increased even further when additional SIINFEKL (SEQ ID NO:3) peptide was supplied along with IFN-y pre-treatment (Figure 26C). As such, by changing levels of AIRE, the relative balance of SELF, AIRE- regulated compared to non-AIRE-regulated, proteins available for MHC Class I occupancy can be reset.

[0260] 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)). While 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-Db-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). Unlike with OT-I T cells, B 16-0 VA-( AIRE) cells were significantly more immunogenic to PMEL T cells than the parental B16-OVA cells (Figure 27B). Thus, MHC occupancy by SELF, AIRE-regulated proteins, such as mgplOO, is enhanced by over-expression of AIRE and reduced by AIRE knockdown.

[0261] It has been shown that clearance of B16-TK tumors (B l 6 tumor cells engineered to express the Herpes Simplex Virus thymidine kinase gene) by ganciclovir is dependent upon CD8+T cells and that tumor-cured mice have CD8+T cell responses (Tumor Experienced (T.E.) CD8+T cells) against multiple B16 tumor associated antigens (TAA) (Figure 27C) (Sanchez-Perez et al., Gene Ther, 14:998-1009 (2007); Vile et a\., 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)). Similar to PMEL recognition of B16-OVA cell variants (Figure 27B), 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. It was also observed that 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). In contrast, 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

[0262] 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)). When the naive transgenic T cells were not supported by the VSV- TAA adjuvant (replaced by VSV-GFP), therapy of established B16-OVA tumors was significantly reduced (Figures 29 and30) (Diaz et al., Cancer Res, 67:2840-2848 (2007); Rommelfanger et al., Cancer Res, 72:4753-4764 (2012)). Tumors formed by 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. Consistent with the in vitro data of Figures 26-28, the converse was true for treatment with PMEL T cells in which tumors derived from B16-OVA-(AIRE) cells were recognized and cleared as effectively by naive PMEL T cells as if they had been activated in vivo by VSV-hgplOO (Figure 30). Taken together, Figures 29 and 30 suggest that in vivo modulation of AIRE expression in tumors can be used to enhance adoptive T cell therapies with a close dependency upon whether or not the T cell target antigen is AIRE regulated.

[0263] AIRE mediated regulation of TAA can be exploited for vaccine-based immunotherapy

[0264] Whether changes in AIRE expression in tumor cells, either through local delivery or in the vaccine setting, would generate T cell responses that can target parental tumors not modified for AIRE -over- or -under-expression was investigated. First, whether changes in the constellation of epitopes presented by AIRE-modified tumor cells can be faithfully transferred through a DC vaccine to the activation of de novo T cell responses against the modified epitope profile which would also include heteroclitic T cell specificities against a subset of TAA expressed by the parental, non-AIRE-modified tumors was tested. B16-F10 tumors were almost completely unresponsive to ICB with anti-PD-1, even with DC -Bl 6- F10- or B16-F10-(shRNA-AIRE)-loaded vaccines (Figure 31). In contrast, DCs loaded with lysates of B16-F10-(AIRE) cells were extremely effective at vaccinating against established B16-F10 tumors either alone or in combination with anti-PD-1 ICB (Figure 31). Very low levels of B16-F10-reactive CD8+T cells were detected from spleens of mice treated with anti-PD-1 ICB and a DC vaccine loaded with lysates of B16-F10 cells when they were restimulated with Bl 6-F10-( AIRE) target cells (Figures 32A) thus suggesting that overexpression of AIRE exposes higher levels of epitopes which can be targeted by low levels of CD8+ T cells stimulated in vivo by vaccination with B16-F10 + ICB. In contrast, significantly higher levels of B 16-F 10-reactive CD8+T cells were present in spleens of mice treated with DCs loaded with lysates of B16-F10-(AIRE) cells both with and without combination ICB therapy (Figure 32B). Higher numbers of reactive CD8+T cells were observed in spleens from those groups when re-stimulated with B 16-F 10-(AIRE) target cells (Figure 32B). There was no significant cross reactivity of these Bl 6-F 10-reactive T cells induced by over-expression of AIRE in B l 6-F 10 cells with an unrelated CT2A glioma cell line of the same MHC background (Figure 32C). Therefore, 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.

[0265] AIRE in DMG

[0266] 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). In contrast, 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). Similarly, 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). Of 5 donors tested, two generated very potent allogeneic T cell responses (IFN-y >500 IFN-y spots per well) against the DIPG-XIII cell line upon co-culture of DIPG-Xin cells with donor CD8+T cells (no priming / education phase (Figure 34)). Of three different donors where minimal allogeneic reactivity was observed, over-expression of AIRE could not enhance immunogenicity (Figure 35). However, when AIRE expression was knocked down, priming / education of CD8+T cells against the unmodified parental DIPG- XIII cell line was possible (Figure 35). Priming / education with parental SOH DMG cells generated significant CD8+T cell reactivity against the parental SOH cells (Figure 36). Overexpression of AIRE significantly inhibited the ability of the SOH tumor cells to prime CD8+T cells against themselves (Figure 36). Conversely, priming with SOH cells in which AIRE was knocked down uncovered a more potent T cell response against the parental tumor cells (Figure 36).

[0267] Transduction of tumor cells with the cytidine deaminase APOBEC3B induces genome mutations which generate immunogenic neo-epitopes in both human and murine tumor model systems (Driscoll et al., Nat Comm, 11 :790 (2020)). To test the effects of AIRE expression on priming against DIPG cells expressing a higher mutational load than in the parental cells, DIPG-XIII cells transduced with an APOBEC3B-expressing vector (Driscoll et al., Nat Comm, 11 :790 (2020)) were tested in the assay of Figures 35 and 36. As before, only 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)). Increased AIRE expression in APOBEC-modified / mutated cells was significantly more effective in priming T cell responses against parental DIPG-XIII cells than the DIPG-XIII-(AIRE) cells (Figure 37) thus suggesting that an increased mutational load can generate potentially heteroclitic neo-epitopes in AIRE-regulated proteins. Moreover, knockdown of AIRE in DIPG-XIII-(APOBEC3B) cells generated the most potent CD8+T cell responses against parental DIPG-XIII cells of all the DIPG-XIII cell variants (Figure 37), thus suggesting that (APOBEC3B-induced) neo-epitopes in non- AIRE-regulated genes are produced and are potentially highly heteroclitic and immunogenic. Taken together, these data suggest that AIRE routinely maintains the high levels of SELFNESS of DMG tumors and that by inhibiting AIRE and / or introducing a higher mutational load, these tumors may become more amenable to T cell mediated immunotherapy. Inhibition of AIRE expression enables immunotherapy of DMG

[0268] Typical of DMG, PKC tumors were both non immunogenic themselves in the context of DC vaccination and were almost completely unresponsive to ICB with anti-PD-1 (Figure 38). Dendritic cells loaded with lysates from PKC-(AIRE) cells were also unable to immunize against PKC tumors (Figure 38) thus confirming that AIRE over-expression was not revealing new SELF epitopes for de novo T cell recognition even with ICB (Figure 38). However, DCs loaded with lysates of PKC-(shRNA-AIRE) cells significantly improved survival times of PKC tumor-bearing mice, an effect which was significantly further improved in combination with anti-PD-1 ICB (Figure 38). 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). However, consistent with Figure 38, 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). Although vaccination with PKC- (shRNA-AIRE) cells raised de novo T cell responses against parental PKC cells (Figure 39A), those T cells were unable to be re-stimulated in vitro with PKC-(AIRE) cells (Figure 39C). These results imply that levels (as well as identity) of SELF proteins / epitopes expressed by DMG may be important in determining SELFNESS (lack of T cell immunogenicity). Overall, Figures 33-39 suggest that expression of AIRE plays a key role in the inhibition of T cell recognition of DMG cells, and by inhibiting AIRE, novel T cell targets for immune attack on these tumors may be revealed.

[0269] In vivo delivery of AIRE stimulates potent T cell-mediated tumor rejection

[0270] An AAV-8-AIRE vector led to high levels of AIRE transduction of cells in vitro (Figures 40 and 41). This vector was used to test whether direct in vivo delivery of AIRE in combination with anti-PD-1 ICB could generate a therapeutic effect by inducing de novo T cell responses that either early or late ICB could augment (Figure 42). Anti-PD-1 ICB prior to AIRE delivery was completely ineffective (Figure 43). In vivo delivery of early AAV-8- AIRE was significantly more therapeutic against established B16-F10 tumors than the PBS control, AAV-GFP, or anti-PD-1 ICB treatment (Figure 43). Addition of anti-PD-1 ICB subsequent to early AAV-8-AIRE gave a trend toward improved survival which was not significant (Figure 43). However, multiple injections of AAV-8-AIRE alone led to significant numbers of tumor cures (Figure 43). Addition of subsequent anti-PD-1 ICB significantly improved time of survival but did not reach significance in overall cure rate (Figure 43). All 9 mice that were tumor free at day 96 following treatment with AAV-8- AIRE in Figure43, with or without anti-PD-1 ICB, rejected a subsequent challenge with 2xl 05parental B16-F10 cells. In contrast, 5 control mice succumbed to tumor by Day 25 thus indicating generation of immunological memory by AAV-8-AIRE therapy. Consistent with this, ELISPOT analysis showed that very similar numbers of anti-B16-F10 CD8+T cells were generated by AAV-8-AIRE therapy alone as by AAV-8-AIRE + anti-PD-1 ICB (Figures 44A and 44B). However, the activity of those CD8+T cells (amount of IFN-y / CD8+T cell) was significantly greater in mice that had received anti-PD-1 ICB in addition to the AAV-8-AIRE treatment (Figures 44A-44B). The relevance of these anti-B16-F10 CD8+T cell responses to the overall therapy in Figure 43 was confirmed by antibody depletion studies. These studies showed that AAV-8-AIRE-mediated therapy of B16-F10 tumors was dependent upon both CD4+and CD8+T cells but not NK cells (Figure 45).

[0271] AIRE over-expression in melanoma cells induces a novel set of epitope expression

[0272] Mass spectrometry of the peptides eluted from the MHC Class I molecules of Bl 6- F10, B 16-F 10-(shRNA-AIRE), and B 16-F 10-( AIRE) cells (Figure 46) identified a total of 8858, 4497, and 10217 peptides, respectively (Table 8). These results suggest that AIRE is a major controller of the absolute number of MHC-Class I-presented epitopes. Changes in the total numbers of epitopes from the well characterized melanoma TAA TYRP1 between Bl 6- F10 cell lines expressing different levels of AIRE (Table 9) reflected the overall data set (Table 8) in that over-expression of AIRE in Bl 6-F 10 cells increased the total number of TYRP1 epitopes while, in contrast, knock down of AIRE dramatically decreased the number of TYRP1 epitopes (Table 9). Within this data set, three classes of TYRP1 epitopes that varied between the Bl 6-F 10, B 16-F 10-(shRNA- AIRE), and B 16-F 10-( AIRE) cell lines were observed. In the first, some 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. In this respect, 35 peptides were unique to B 16-F 10-(AIRE) cells and not expressed by either parental B16-F10 or B16-F10-(shRNA-AIRE) cells (Table 11), whereas 97 peptides were eluted from both B16-F10-(AIRE) and Bl 6-F 10 parental cells (Table 12). Only 6 peptides were exclusively present in B16-F10 parental cells. 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).

[0273] Table 8. Total Number of Different Peptides Eluted from B16-F10 Parental, B16-F10- (AIRE), or B16-F10-(shRNA-AIRE) Cells.

[0274] 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.

[0275]

[0276] Table 11. TYRP1 Epitopes Uniquely Eluted from Bl 6-F10-( AIRE) Cells.

[0277] Table 12. TYRP1 Epitopes Shared Between Bl 6-F 10 and B16-F10-(AIRE) Cells .

[0278] Table 13. Peptides Prepared for Loading onto DC for Experiments.

[0279] To investigate the biological relevance of these three different classes of TYRP1 peptides, 4 of each of the Shared, B16-F10-(AIRE)-Unique, and B16-F10-(AIRE)-Longer class (Table 13) were tested as vaccines against parental B16-F10 tumors. Dendritic cell vaccines loaded with either the Shared or Unique peptides were not significantly more effective at slowing the growth of B16-F10 tumors than controls (DCs loaded with PBS or SIINFEKL (SEQ ID NO:3) peptide) (Figure 48). However, treatment with DCs loaded with the Longer peptide set significantly increased median survival times of B16-F10 tumor bearing mice compared to controls (Figure 48). In combination with anti-PD-1 ICB, 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). Only treatment with either the Unique or the Longer peptide sets primed recall responses against Bl 6-F 10 parental cells that were significantly greater than the control treatments. 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). The same pattern was observed with the recall response of CD8+T cells against B16-(AIRE) targets in vitro but at higher magnitudes of IFN-y secretion (Figures 50A-50F) thus confirming that the Longer and Unique peptide sets contain AIRE-induced T cell targets that generate T cell responses which can cross react back against parental B16-F10 cells.

[0280] 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. Immunol., 175: 112-123 (2005); Brightman et al., Nat Immunol, 24: 345-1357 (2023); Ahrends et al., Nat Commun, 10:5531 (2019)), CD4+T cells from spleens of mice treated with DC / Longer+aPD- 1 in Figure 49 not only induced strong IL-15 responses in vitro from DCs loaded with the Longer peptides (Figure 51C) but also from DCs loaded with the shorter epitopes which were shared between B16-F10-(AIRE) and Bl 6-F 10 parental cells (Figure 5 IB). 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). CD4+T cells recovered from mice treated with DC / SIINFEKL (SEQ ID NO:3) +aPD-l in Figures 48 and 49 did not induce IL-15 secretion when co-cultured with DCs loaded with any of the peptide sets. These data suggest that 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.

[0281] Example 4: Exemplary Sequences

[0282] Exemplary amino acid sequence of an AIRE polypeptide

[0283] MATDAALRRLLRLHRTEIAVAVDSAFPLLHALADHDWPEDKFQETLHLKEKEGCPQAFHALLSW LLTQDSTAILDFWRVLFKDYNLERYGRLQPILDSFPKDVDLSQPRKGRKPPAVPKALVPPPRLPT KRKASEEARAAAPAALTPRGTASPGSQLKAKPPKKPESSAEQQRLPLGNGIQTMSASVQRAVAMS SGDVPGARGAVEGILIQQVFESGGSKKCIQVGGEFYTPSKFEDSGSGKNKARSSSGPKPLVRAKG AQGAAPGGGEARLGQQGSVPAPLALPSDPQLHQKNEDECAVCRDGGELICCDGCPRAFHLACLSP PLREI PSGTWRCSSCLQATVQEVQPRAEEPRPQEPPVETPLPPGLRSAGEEVRGPPGEPLAGMDT TLVYKHLPAPPSAAPLPGLDSSALHPLLCVGPEGQQNLAPGARCGVCGDGTDVLRCTHCAAAFHW RCHFPAGTSRPGTGLRCRSCSGDVTPAPVEGVLAPSPARLAPGPAKDDTASHEPALHRDDLESLL SEHTFDGILQWAIQSMARPAAPFPS

[0284] (SEQ ID NO: 1)

[0285] Exemplary nucleic acid sequence encoding an AIRE polypeptide

[0286] CGCGGGGGTATAACAGCGGCGCGCGTGGCTCGCAGACCGGGGAGACGGGCGGGCGCACAGCCGGC GCGGAGGCCCCACAGCCCCGCCGGGACCCGAGGCCAAGCGAGGGGCTGCCAGTGTCCCGGGACCC ACCGCGTCCGCCCCAGCCCCGGGTCCCCGCGCCCACCCCATGGCGACGGACGCGGCGCTACGCCG GCTTCTGAGGCTGCACCGCACGGAGATCGCGGTGGCCGTGGACAGCGCCTTCCCACTGCTGCACG CGCTGGCTGACCACGACGTGGTCCCCGAGGACAAGTTTCAGGAGACGCTTCATCTGAAGGAAAAG GAGGGCTGCCCCCAGGCCTTCCACGCCCTCCTGTCCTGGCTGCTGACCCAGGACTCCACAGCCAT CCTGGACTTCTGGAGGGTGCTGTTCAAGGACTACAACCTGGAGCGCTATGGCCGGCTGCAGCCCA TCCTGGACAGCTTCCCCAAAGATGTGGACCTCAGCCAGCCCCGGAAGGGGAGGAAGCCCCCGGCC GTCCCCAAGGCTTTGGTACCGCCACCCAGACTCCCCACCAAGAGGAAGGCCTCAGAAGAGGCTCG AGCTGCCGCGCCAGCAGCCCTGACTCCAAGGGGCACCGCCAGCCCAGGCTCTCAACTGAAGGCCA AGCCCCCCAAGAAGCCGGAGAGCAGCGCAGAGCAGCAGCGCCTTCCACTCGGGAACGGGATTCAG ACCATGTCAGCTTCAGTCCAGAGAGCTGTGGCCATGTCCTCCGGGGACGTCCCGGGAGCCCGAGG GGCCGTGGAGGGGATCCTCATCCAGCAGGTGTTTGAGTCAGGCGGCTCCAAGAAGTGCATCCAGG TTGGCGGGGAGTTCTACACTCCCAGCAAGTTCGAAGACTCCGGCAGTGGGAAGAACAAGGCCCGC

[0287] AGCAGCAGTGGCCCGAAGCCTCTGGTTCGAGCCAAGGGAGCCCAGGGCGCTGCCCCCGGTGGAGG TGAGGCTAGGCTGGGCCAGCAGGGCAGCGTTCCCGCCCCTCTGGCCCTCCCCAGTGACCCCCAGC TCCACCAGAAGAATGAGGACGAGTGTGCCGTGTGTCGGGACGGCGGGGAGCTCATCTGCTGTGAC GGCTGCCCTCGGGCCTTCCACCTGGCCTGCCTGTCCCCTCCGCTCCGGGAGATCCCCAGTGGGAC CTGGAGGTGCTCCAGCTGCCTGCAGGCAACAGTCCAGGAGGTGCAGCCCCGGGCAGAGGAGCCCC GGCCCCAGGAGCCACCCGTGGAGACCCCGCTCCCCCCGGGGCTTAGGTCGGCGGGAGAGGAGGTA

[0288] AGAGGTCCACCTGGGGAACCCCTAGCCGGCATGGACACGACTCTTGTCTACAAGCACCTGCCGGC TCCGCCTTCTGCAGCCCCGCTGCCAGGGCTGGACTCCTCGGCCCTGCACCCCCTACTGTGTGTGG GTCCTGAGGGTCAGCAGAACCTGGCTCCTGGTGCGCGTTGCGGGGTGTGCGGAGATGGTACGGAC GTGCTGCGGTGTACTCACTGCGCCGCTGCCTTCCACTGGCGCTGCCACTTCCCAGCCGGCACCTC CCGGCCCGGGACGGGCCTGCGCTGCAGATCCTGCTCAGGAGACGTGACCCCAGCCCCTGTGGAGG GGGTGCTGGCCCCCAGCCCCGCCCGCCTGGCCCCTGGGCCTGCCAAGGATGACACTGCCAGTCAC GAGCCCGCTCTGCACAGGGATGACCTGGAGTCCCTTCTGAGCGAGCACACCTTCGATGGCATCCT GCAGTGGGCCATCCAGAGCATGGCCCGTCCGGCGGCCCCCTTCCCCTCCTGACCCCAGATGGCCG GGACATGCAGCTCTGATGAGAGAGTGCTGAGAAGGACACCTCCTTCCTCAGTCCTGGAAGCCGGC CGGCTGGGATCAAGAAGGGGACAGCGCCACCTCTTGTCAGTGCTCGGCTGTAAACAGCTCTGTGT TTCTGGGGACACCAGCCATCATGTGCCTGGAAATTAAACCCTGCCCCACTTCTCTACTCTGGAAG TCCCCGGGAGCCTCTCCTTGCCTGGTGACCTACTAAAAATATAAAAATTAGCTGGGTGTGGTGGT GGGTGCCTGTAATCCCAGCTACATGGGAGCCTGAGGCATGAGAATCACTTGAACTCGGGAGGTGG

[0289] AGGTTGCAGTGAGCTGAGATTGCGCCACTGCACTCCAGTCTGGTCGGCAAGAGTGAGACTCCGTC T C AAAAAC AAAACAAAACAAAAAAAC C AC AT AACAT AAAT T T AT CAT C T C GAC C AC T T T T CAG T T CAGTGGCATTCACATCTCATGTAA

[0290] (SEQ ID N0:2)

[0291] Example 5: Treating Cancer

[0292] 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.

[0293] Example 6: Treating Cancer

[0294] 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.

[0295] 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.

[0296] Example 7: Treating Cancer

[0297] 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.

[0298] The administered one or more inhibitors of an AIRE polypeptide are effective to reduce the number of cancer cells present within the mammal.

[0299] Example 8: Treating Cancer

[0300] 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.

[0301] OTHER EMBODIMENTS It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT Is CLAIMED IS:

1. A method for treating a mammal having cancer, wherein said method comprises 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.

2. The method of claim 1, wherein said mammal is a human.

3. The method of any one of claims 1-2, wherein said cancer is selected from the group consisting of a diffuse midline glioma (DMG), a melanoma, a hepatobiliary cancer, a breast cancer, a lung cancer, and a prostate cancer.

4. The method of any one of claims 1-3, wherein said method comprises administering said AIRE polypeptide to said mammal.

5. The method of any one of claims 1-3, wherein said method comprises administering said nucleic acid designed to express said AIRE polypeptide to said mammal.

6. The method of any one of claims 1-5, wherein said AIRE-regulated antigen is selected from the group consisting of a TYRP2 polypeptide, a CSDE1 polypeptide, and a gplOO polypeptide.

7. The method of any one of claims 1-6, said method further comprises administering an immune checkpoint inhibitor to said mammal.

8. The method of claim 7, wherein said immune checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-CTL4A antibody, an anti-LAG-3 antibody, an anti-TIM3 antibody, and an anti-TIGIT antibody.

9. The method of claim 7, wherein said immune checkpoint inhibitor is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, ipilimumab, tremelimumab, durvalumab, dostarlimab, avelumab, atezolizumab, and relatlimab.

10. The method of claim 7, wherein said immune checkpoint inhibitor is selected from the group consisting of 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, and DZNep.

11. A method 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, wherein said method comprises 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.

12. The method of claim 11, wherein said mammal is a human.

13. The method of any one of claims 11-12, wherein said cancer is selected from the group consisting of a DMG, a melanoma, a hepatobiliary cancer, a breast cancer, a lung cancer, and a prostate cancer.

14. The method of any one of claims 11-13, wherein said method comprises administering said AIRE polypeptide to said mammal.

15. The method of any one of claims 11 -13, wherein said method comprises administering said nucleic acid designed to express said AIRE polypeptide to said mammal.

16. The method of any one of claims 11-15, wherein said AIRE-regulated antigen is selected from the group consisting of a TYRP2 polypeptide, a CSDE1 polypeptide, and a gplOO polypeptide.

17. The method of any one of claims 11-16, wherein said immune checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-CTL4A antibody, an anti-LAG-3 antibody, an anti-TIM3 antibody, and an anti-TIGIT antibody.

18. The method of any one of claims 11-16, wherein said immune checkpoint inhibitor is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, ipilimumab, tremelimumab, durvalumab, dostarlimab, avelumab, atezolizumab, and relatlimab.

19. The method of any one of claims 11-16, wherein said immune checkpoint inhibitor is selected from the group consisting of 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, and DZNep.

20. A method for treating a mammal having cancer, wherein said method comprises(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.

21. The method of claim 20, wherein said mammal is a human.

22. The method of any one of claims 20-21, wherein said cancer is selected from the group consisting of a DMG, a melanoma, a hepatobiliary cancer, a breast cancer, a lung cancer, and a prostate cancer.

23. The method of any one of claims 20-22, wherein said method comprises administering said AIRE polypeptide to said mammal.

24. The method of any one of claims 20-22, wherein said method comprises administering said nucleic acid designed to express said AIRE polypeptide to said mammal.

25. The method of any one of claims 20-24, wherein said AIRE-regulated antigen is selected from the group consisting of a TYRP2 polypeptide, a CSDE1 polypeptide, and a gplOO polypeptide.

26. The method of any one of claims 20-25, wherein said immune checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L l antibody, an anti-CTL4A antibody, an anti-LAG-3 antibody, an anti-TIM3 antibody, and an anti-TIGIT antibody.

27. The method of any one of claims 20-25, wherein said immune checkpoint inhibitor is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, ipilimumab, tremelimumab, durvalumab, dostarlimab, avelumab, atezolizumab, and relatlimab.

28. The method of any one of claims 20-25, wherein said immune checkpoint inhibitor is selected from the group consisting of 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, and DZNep.

29. A method for treating a mammal having cancer, wherein said method comprises 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.

30. The method of claim 29, wherein said mammal is a human.

31. The method of any one of claims 29-30, wherein said cancer is selected from the group consisting of a DMG, a melanoma, a hepatobiliary cancer, a breast cancer, a lung cancer, and a prostate cancer.

32. The method of any one of claims 29-31 , wherein said inhibitor of an AIRE polypeptide comprises an anti-AIRE antibody.

33. The method of any one of claims 29-32, wherein said inhibitor of an AIRE polypeptide comprises a short hairpin RNA (shRNA) targeting nucleic acid encoding said AIRE polypeptide.

34. The method of any one of claims 29-33, wherein said method further comprises administering an immune checkpoint inhibitor to said mammal.

35. The method of claim 34, wherein said immune checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-CTL4A antibody, an anti-LAG-3 antibody, an anti-TIM3 antibody, and an anti-TIGIT antibody.

36. The method of claim 34, wherein said immune checkpoint inhibitor is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, ipilimumab, tremelimumab, durvalumab, dostarlimab, avelumab, atezolizumab, and relatlimab.

37. The method of claim 34, wherein said immune checkpoint inhibitor is selected from the group consisting of 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, MGCD0I03, SNDX-275, IMP32, BMS986016, TSR-022, Sym023, ATIK2a, and DZNep.

38. A method 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, wherein said method comprises 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.

39. The method of claim 38, wherein said mammal is a human.

40. The method of any one of claims 38-39, wherein said cancer is selected from the group consisting of a DMG, a melanoma, a hepatobiliary cancer, a breast cancer, a lung cancer, and a prostate cancer.

41. The method of any one of claims 38-40, wherein said inhibitor of an AIRE polypeptide comprises an anti-AIRE antibody.

42. The method of any one of claims 38-40, wherein said inhibitor of an AIRE polypeptide comprises a shRNA targeting nucleic acid encoding said AIRE polypeptide.

43. The method of any one of claims 38-42, wherein said immune checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-CTL4A antibody, an anti-LAG-3 antibody, an anti-TIM3 antibody, and an anti-TIGIT antibody.

44. The method of any one of claims 38-42, wherein said immune checkpoint inhibitor is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, ipilimumab, tremelimumab, durvalumab, dostarlimab, avelumab, atezolizumab, and relatlimab.

45. The method of any one of claims 38-42, wherein said immune checkpoint inhibitor is selected from the group consisting of 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, and DZNep.

46. A method for treating a mammal having cancer, wherein said method comprises(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.

47. The method of claims 46, wherein said mammal is a human.

48. The method of any one of claims 46-47, wherein said cancer is selected from the group consisting of a DMG, a melanoma, a hepatobiliary cancer, a breast cancer, a lung cancer, and a prostate cancer.

49. The method of any one of claims 46-48, wherein said inhibitor of an AIRE polypeptide comprises an anti-AIRE antibody.

50. The method of any one of claims 46-48, wherein said inhibitor of an AIRE polypeptide comprises a shRNA targeting nucleic acid encoding said AIRE polypeptide.

51. The method of any one of claims 46-50, wherein said immune checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-CTL4A antibody, an anti-LAG-3 antibody, an anti-TIM3 antibody, and an anti-TIGIT antibody.

52. The method of any one of claims 46-50, wherein said immune checkpoint inhibitor is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, ipilimumab, tremelimumab, durvalumab, dostarlimab, avelumab, atezolizumab, and relatlimab.

53. The method of any one of claims 46-50, wherein said immune checkpoint inhibitor is selected from the group consisting of 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, and DZNep.

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