A phenotypic signature-based strategy that identifies tumor-specific TCR in fresh human colorectal cancers

Despite advances in cancer immunotherapy, solid tumors remain challenging due to antigenic heterogeneity and immunosuppressive microenvironments. T-cell receptor-engineered T cells (TCR-T) hold promises by targeting intracellular antigens, yet identifying tumor-specific TCRs is hindered by the rarity of tumor-reactive T cells. Here, we developed a phenotypic signature-based strategy to efficiently isolate tumor-specific TCRs from colorectal cancer (CRC) tumor-infiltrating lymphocytes (TILs). We leveraged the co-expression of surface markers CD39, CD103, and PD-1 to enrich CXCL13 + CD8 + T cells, a subset linked to tumor reactivity and improved patient survival. Single-cell RNA sequencing and flow cytometry of a small cohort CRC patients revealed that triple-positive (CD39 + CD103 + PD-1 + ) CD8 + TILs exhibited the highest CXCL13 enrichment, providing a basis for subsequent TCR clonotype identification. High-throughput TCR sequencing of the enriched cells identified dominant clonotypes, which were validated using autologous tumor organoids. A top candidate TCR (TCR-1A7) demonstrated robust tumor-specific cytotoxicity, as evidenced by upregulated activation markers (CD69, CD137), cytokine secretion (IFN-γ, granzyme B), and tumor organoid apoptosis, while sparing normal tissues. MHC blockade confirmed antigen-specific recognition. Combining phenotypic profiling, TCR screening, and organoid-based validation, this integrative approach proposes a potential framework for developing precision TCR-T therapies against solid tumors, addressing critical bottlenecks in efficacy and safety.