Targeting Precision in Cancer Immunotherapy: Naturally-Occurring Antigen-Specific TCR Discovery with Single-Cell Sequencing.

: Adoptive cell therapy is the most promising approach for battling cancer, with T cell receptor-engineered T (TCR-T) cell therapy emerging as the most viable option for treating solid tumors. Current techniques for preparing TCR-T cell therapy provide a limited number of candidates TCRs, missing the comprehensive view of the repertoire, which may hinder the identification of the most effective TCRs. : Dendritic cells were primed with immunogenic peptides of the antigen of interest to expand antigen-specific CD8 T lymphocytes from peripheral blood. Following that, the entire repertoire of naturally occurring antigen-specific TCRs was analyzed using single-cell RNA sequencing, alongside the assessment of the dominancy, transcriptome, and binding specificity of the obtained clonotypes, utilizing the TCRscape tool and ERGO-II neural network to identify the most effective candidate for TCR-T cell therapy development. Finally, TCR-T cells with the candidate TCR were obtained, followed by assessing their functionality and selectivity. : The developed protocol achieved a remarkable increase in the percentage of antigen-specific T cells by more than 200-fold, with more than 100 antigen-specific TCR clonotypes identified. The resulting TCR-T cells demonstrated high cytotoxicity and selectivity for the targeted antigen, indicating their potential to preferentially target tumor cells. : This study offers a comprehensive approach for the discovery and analysis of not only few, but the entire repertoire of naturally occurring antigen-specific TCRs for TCR-T cell therapy development. Additionally, the proposed approach can be tailored to accommodate different types of antigens and MHC variants, making it a highly versatile tool for both research and clinical applications.