104P Leveraging endogenous TIL responses to develop noncanonical peptide-directed TCR therapies

Background: Noncanonical peptides (NCPs) are germline-encoded and tumorrestricted antigens, offering mutation-independent targets with potential cross-patient applicability.Recent studies provide detailed pipelines for discovery, but therapeutic translation remains limited.Here, we identify candidate NCPs in melanoma, demonstrate endogenous TIL responses, and provide a translational foundation.Methods: NCP identification in commercial and primary cell lines involved bulk RNA sequencing, RNAscope, and mass spectrometry (MS)-based immunopeptidomics.TIL responses were evaluated using ELISpot.10X Genomics single cell RNA sequencing was used for discovery of antigen-specific TCRs from melanoma derived TILs.Healthy donor T cells were engineered with an NCP-targeting TCR by lentiviral transduction and functionally assessed using intracellular cytokine stain, chromium release, and impedance-based cytotoxicity assays.Results: Among 23 tumor cell lines, 69.5% expressed at least one of three candidate NCP transcripts.The most abundant transcript, EVA003, was highly prevalent in HLA-A*03:01-positive tumor cell lines expressed 87% compared to HLA-A*03:01-negative, 60%.Histology of malignant and healthy tissue confirmed tumor restriction of EVA003.Two EVA003-derived peptides LLMRPLRIK (LLM) and VLLMRPLRIK (VLL) demonstrated HLA-A*03:01-restriction. TILs from two patients were activated in response to stimulation with both peptides with preference towards LLM.Single cell sequencing of LLM-stimulated TILs revealed the TCR clone, NCP-TCR-1.Reactivity of this TCR with LLM and VLL peptides was confirmed by its transduction into healthy donor T cells (TCR-Ts), demonstrating significant activation and cytotoxicity compared to non-transduced (NTD) cells.A melanoma cell line with MS-confirmed endogenous LLM expression also elicited activation in TCR-Ts compared to NTD cells and were susceptible to specific lysis.Conclusions: Here, we provide direct evidence of naturally primed TIL immunity to NCPs and establish a foundation for therapeutic application.This study highlights NCPs as promising therapeutic targets, as well as emphasizes the need to further define their contribution in TIL-mediated immunity.