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Background Identification and characterization of the antigen specificity of tumor-infiltrating lymphocytes (TILs) are key to understand anti-tumor immunity and can guide the development of novel and effective immunotherapies. Our DECODE® platform enables the identification of TCR and peptide-MHC (pMHC) across the immune synapse of TILs.Methods To investigate the T cell response directed against the melanoma antigen landscape, we performed a comprehensive analysis of tumor-infiltrating lymphocytes (TILs) derived from 21 melanoma patients. We employed two complementary approaches that support our DECODE ® platform: (1) an engineered reporter cell system expressing chimeric pMHC-TCR (MCR) hybrid molecules, loaded with tens of thousands of peptides derived from antigens highly expressed in melanoma; and (2) a high-throughput TIL screening platform using barcoded peptide-HLA tetramers. These approaches incorporated diverse libraries that allowed us to broadly screen hundreds of antigens across 68 HLA alleles, enabling the in-depth discovery, validation, and application of TCR-epitope pairs. Immunogenicity of the identified epitopes was tested by assessing their ability to activate T cells in vitro.Results By using our TCR-pMHC DECODE® technologies, we identified a broad repertoire of pMHC-TCR pairs from TILs derived from melanoma patients. The epitopes originated from tumor-associated antigens (TAAs), tumor-specific antigens (TSAs), personalized neoantigens, and Dark Antigens, a group of cancer targets derived from otherwise non-coding genetic regions. In-depth analysis of one patient revealed tumor-specific dark antigens recognized by TIL-derived TCRs, which were also detectable in peripheral blood, suggesting systemic immunologic prevalence.Epitopes from these antigens were tested for immunogenicity using in vitro expansion approaches; we observed Dark Antigen epitope-specific T cell expansion, supporting a role for integration of these epitopes into cancer vaccine approaches. Likewise, decoded TCRs were expressed in primary T cells, and the recombinant TCR-T cells were activated by cell lines derived from multiple solid tumor types, highlighting the therapeutic potential of these TCRs for both cell therapy and bispecific engager formats.Conclusions These findings showcase the power of our DECODE® technology to uncover clinically relevant, TIL-derived epitopes and TCRs from TAAs, TSAs, personalized neoantigens and, dark antigens for next-generation cancer immunotherapies that include TCR bispecifics and cancer vaccines. Application of this approach will provide more effective immunotherapies for many solid tumor indications.