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BackgroundT cell receptor (TCR)-based therapies offer a powerful approach to target intracellular tumor antigens that are inaccessible to modalities such as antibody-drug conjugates (ADCs) and conventional antibodies. Central to these therapies are TCRs that can specifically recognize tumor antigen (Ag)-derived peptides presented on MHC molecules. However, identifying TCRs with both high affinity and high specificity remains a significant bottleneck in therapeutic development. Tumor-infiltrating lymphocytes (TILs), which often contain clonally expanded, antigen-experienced T cells, may offer an enriched pool of candidates with ideal characteristics to develop TCR- based T cell engagers.MethodsWe utilized Repertoire’s proprietary DECODE™ Platform to identify antigen-specific TCRs from TILs and peripheral blood mononuclear cells (PBMCs) derived from melanoma, head and neck, and lung cancer patients. Multiplexed, barcoded peptide-MHC class I tetramer libraries were used to stain TILs and PBMCs, which were then subjected to single-cell sequencing to identify and characterize T cells reactive to tumor associated antigens. The functional avidity of each TCR identified was assessed via cloning and transduction into Jurkat reporter cells with subsequent co-culture with T2 cells across a range of peptide concentrations. TCR specificity was characterized via an alanine scan. Furthermore, soluble TCR-bispecifics were expressed in mammalian cells and purified, and affinity was assessed using biolayer interferometry. Finally, we employed yeast surface display to assess the ability of the TCRs to be used in yeast systems for affinity optimization, characterizing surface expression, and pMHC binding characteristics.ResultsOur study identified antigen-specific TCRs targeting peptides derived from tumor-associated antigens including gp100, MAGE family members, and other non-canonical, ‘dark’ antigens. Notably, we identified several TCRs specific for the gp100 YLEP peptide presented in HLA-A*02:01. These TCRs, derived from a variety of anatomical locations, donors, and disease contexts, exhibited high similarity in their CDR3 sequences and shared VJ gene usage in both α and β chains, suggesting convergent selection. Tumor-infiltrating T cells exhibited an exhausted phenotype, whereas peripheral T cells displayed an effector memory phenotype. Functionally, TCRs from tumors or inflamed tissues demonstrated over 10,000-fold significantly avidity compared to TCRs from healthy donors. These TCRs also exhibited higher specificity, recognizing only the cognate peptide. Finally, tumor-derived TCRs demonstrated improved yeast display and pMHC binding.ConclusionsTogether, these findings highlight tumor-derived TCRs as optimal candidates for the development of TCR-based T cell engagers, combining high affinity, specificity, and favorable biophysical properties.

BackgroundIdentification 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.MethodsTo 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.ResultsBy 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.ConclusionsThese 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.