MHC Class II Antigen Presentation In Melanoma: Impact on Immune Recognition

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Regulation of tumor-specific CD4 T cell responses holds great promise for improving cancer immunotherapy with immune checkpoint inhibition, cancer vaccines, and adoptive T cell therapy. Effective response to immunotherapy requires T cell recognition of mutated peptide epitopes from cancer cells, called neoantigens. Effective response to immunotherapy requires neoantigens capable of stimulating both CD4 and CD8 T cells. Tumor-specific CD4 T cells recognize neoantigens presented by MHC class II. However, experimental or computational methods for identification of MHC class II neoantigens that are immunogenic (elicit a T cell response) have not been well-established. While tumor-specific CD4 T cell epitopes are necessary to generate effective anti-tumor T cell responses to immunotherapy, CD4 regulatory T (Treg) cells that develop within tumors suppress the proliferation and function of other T cells. Destroying CD4 Treg cells is a promising immunotherapeutic approach. However, the lack of a method to specifically target and destroy tumor-specific CD4 Treg cells prevents depletion of Treg cells from advancing further in clinical trials for cancer immunotherapy. Peptide-MHC tetramers can be used to identify tumor-specific T cells. However, peptide-MHC class II tetramers cannot be generated for many peptide antigens that are known to elicit T cell responses due to low affinity or stability of the peptide interaction with MHC class II. This proposal will advance the field of cancer immunology by addressing three gaps in knowledge: 1) inability to use tetramers to identify most tumor- specific CD4 T cells, 2) inability to target tumor-specific Treg cells for destruction, and 3) lack of experimental and computational tools to prioritize immunogenic MHC class II neoantigens. This proposal will utilize a new, patented technology of a biomimetic 5-module chimeric antigen receptor (5MCAR). The 5MCAR consists of the ectodomains of peptide-MHC class II fused to the transmembrane and cytoplasmic domains of the T cell receptor, which facilitates assembly with the CD3 signaling complex, and a surrogate coreceptor composed of CD80 fused to the Src kinase Lck. The 5MCAR is expressed on a cytotoxic T cell. Signaling through the 5MCAR allows identification and killing of CD4 T cells expressing a T cell receptor than recognizes the peptide-MHC class II of the 5MCAR. The central hypothesis is that 5MCAR will facilitate determination of immunogenic MHC class II melanoma neoantigens and effectively treat melanoma tumors by destruction of tumor-specific CD4 Treg cells. The aim of this proposal is to identify immunogenic MHC class II neoantigens and test the efficacy of depleting neoantigen-specific CD4 cells in treating melanoma, using a clinically relevant, immunogenic mouse melanoma model. A cell based assay with 5MCAR will be used in high throughput screening with a peptide library to identify the MHC class II neoantigens which elicit CD4 conventional T cell and regulatory T cell responses in melanoma tumors. The efficacy of tumor-specific 5MCAR T cells in treating established melanoma tumors through depletion of neoantigen-specific Treg cells alone and in combination with immune checkpoint inhibition with anti-PD-1 will be assessed. Using the comprehensive, unbiased dataset generated from testing the immunogenicity of all missense mutations, a computational model to predict immunogenic MHC class II neoantigens will be created and validated. The impact of this proposal is to 1) establish a collaboration with Dr. Kuhns at the University of Arizona, a Hispanic Serving Institution, 2) incorporate a new technological innovation to allow high throughput screening for identification of immunogenic MHC class II neoantigens, targeted destruction of tumor-specific CD4 Treg cells, and improved computational methods for prioritization of immunogenic MHC class II neoantigens, 3) increase the impact of the Merit award by addressing gaps in the current knowledge that prevent advancement of cancer immunotherapy and ultimately improve cancer care for Veterans, and 4) assist underrepresented investigators engage in VA Research.