Real-time discovery of neoantigen-specific T cell receptor clonotypes reveals variable CD8+ T cell functional states in patients with KRAS-mutant colorectal cancer 4704

Abstract Description Traditional methods for identifying neoantigen-specific TCRs rely on labor-intensive in vitro co-cultures, which can alter T cell characteristics. We aim to identify endogenous neoantigen-specific TCRs in their natural state using antigen-mapping single-cell RNA and TCR sequencing. We profiled circulating CD8+ T cells from patients with KRAS-mutant metastatic colorectal adenocarcinoma (CRA) to identify TCRs that target HLA-A restricted (*02:01 or *11:01) mutant KRAS epitopes (G12D or G12V) using Barcode Enabled Antigen Mapping (BEAM; 10x Genomics) and performed cluster analysis to decipher T cell function. CD8+ T cells were isolated from peripheral blood and stained with peptide-MHC complexes for KRAS G12D/V epitopes (HLA-A02:01 and HLA-A11:01). Sequencing data were processed with Cell Ranger and Seurat. We analyzed 12 specimens from 6 patients who were receiving fluorouracil-based chemotherapy for metastatic CRA and detected neoantigen-specific TCR clonotypes in 11 specimens, suggesting their persistent presence in peripheral blood. TCR clonotypes displayed varying functional states, with some showing cytotoxic effector markers (GZMB, NKG7) and others showing markers of Naïve T cells (LEF1, TCF7, SELL, CCR7). These findings show that neoantigen-specific TCRs have diverse functional states and evolve over time. This approach is a promising tool for identifying endogenous neoantigen-specific TCRs and provides insights into TCR structure and function for adoptive therapy. Funding Sources This project is supported by the Eric & Wendy Schmidt Fund for AI Research & Innovation at Mayo Clinic (MZ and HD), and Mayo Clinic Comprehensive Cancer Center Distinguished Senior Scientist Award (HD). Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)