Receptor-defined targeting of a genomically unique melanoma-enriched noncanonical antigen.
Effective T cell-based immunotherapies require functional receptors that can be engineered and redeployed to recognize tumor-restricted antigens. Noncanonical peptides arising from transcription outside annotated protein-coding regions expand the antigenic landscape of cancer; however, systematic strategies to biologically prioritize and functionally validate such targets remain underdeveloped. Here, we integrated de novo transcript analysis, exon-resolved quantification, RNA in situ hybridization, and immunopeptidomics to identify melanoma-associated noncanonical transcripts and advance candidates through receptor-level validation. Among three recurrent melanoma-associated transcripts, EVA003 emerged as a lead target based on its distinct repeat-enriched genomic architecture, consistent tumor-enriched exon-level expression across independent datasets, and a genomically unique immunogenic core sequence. We demonstrate endogenous presentation of EVA003-derived peptides on HLA-A*03:01 and detect specific reactivity in patient-derived tumor-infiltrating lymphocytes. Single-cell transcriptomic profiling identified a dominant peptide-reactive clonotype, enabling isolation of a naturally occurring T cell receptor. Transfer of this receptor into healthy donor T cells conferred antigen-dependent activation and cytotoxicity against both peptide-pulsed targets and melanoma cells expressing EVA003 endogenously. Together, these findings establish a biologically informed strategy for prioritizing noncanonical tumor antigens and demonstrate that genomically unique, tumor-enriched noncanonical peptides can be presented to molecularly defined receptors capable of mediating cancer cell killing. These findings support the integration of prioritized noncanonical antigens into engineered T cell therapeutic strategies.