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Background Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide, with oncogenic KRAS mutations playing a critical role in tumor initiation and progression. In recent years, T cell-based therapies have emerged as one of the most promising approaches of cancer treatment. To expand the therapeutic potential of TCR-T cell therapy for solid tumors, we aim to directly identify and characterize the phenotypic profiles of CD8 + T cells that express endogenous tumor antigen-specific T cell receptors (TCRs) from patient bloods. In the future, we also aim to validate TCR sequences from these CD8+ T cells in their ability of tumor-killing.Methods To overcome limitations of conventional approaches, we utilized a real-time, stimulation-free method to identify and characterize TCR clonotypes in the peripheral blood of patients. CD8 + T cells were isolated from six patients with microsatellite-stable colorectal adenocarcinoma (CRA) harboring KRAS G12V or G12D mutations, collected at multiple time points during chemotherapy. Patients were pre-screened for HLA-A02:01 or HLA-A11:01 expression. Neoantigen-specific CD8+ T cells were profiled using BEAM (Barcoded-pMHC Multimer) single-cell RNA and TCR sequencing to simultaneously capture gene expression and paired TCR sequences.Results We analyzed 119,915 cells from six patients treated with fluorouracil-based chemotherapy. Three patients had blood collected at two time points, and three at a single time point, resulting in 12 unique specimens. Neoantigen-specific TCR clonotypes were detected in all 12 specimens and exhibited variable functional states—some with high cytotoxic potential (GZMB, NKG7 expression) and others showing naïve-like markers (LEF1, TCF7, SELL, CCR7 expression). In each sample, 1–9 tumor antigen-specific CD8 + T cells were identified per 10,000 CD8+ T cells using BEAM-T combined with single-cell RNA sequencing and TCR sequencing. Most of these tumor antigen-specific cells fell into the naïve-like cluster, with a naïve-like to effector-like ratio of approximately 5.2—substantially higher than that observed in dominant viral CD8+ T cell clones.Conclusions This study demonstrates that antigen-mapping single-cell RNA/TCR sequencing is an effective approach for identifying endogenous neoantigen-specific TCRs. Our findings suggest that TCR structure, particularly in the CDR3 region, may influence T cell functional state. Ongoing work in our lab focuses on evaluating the cytotoxic function of KRAS-specific TCRs and investigating TCR-pMHC complex structures to better understand TCR affinity and cross-reactivity.Consent Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.