Identification and validation of tumor-specific T cell receptors from tumor infiltrating lymphocytes using tumor organoid co-cultures

Background：T cell receptor-engineered T cells (TCR-Ts) therapy is promising for cancer immunotherapy. Most studies have focused on identifying tumor-specific T cell receptors (TCRs) through predicted tumor neoantigens. However, current prediction algorithms for tumor neoantigens are not reliable and many tumor neoantigens are derive from non-coding regions. Thus, the technological platform for identifying tumor-specific TCRs using natural antigens expressed on tumor cells is urgently need.Methods：In this study, tumor organoids-enriched tumor infiltrating lymphocytes (oeT) were obtained by repeatedly stimulating of autologous patient-derived organoids (PDO) in vitro. The oeT cells specifically responded to autologous tumor PDO by detecting CD137 expression and the secretion of IFN-γ using enzyme-linked immunospot (ELISPOT) assay. The measurement of oeT cell-mediated killing of three-dimensional organoids was conducted using a caspase3/7 flow cytometry assay kit. Subsequently, tumor-specific T cells were isolated based on CD137 expression and their TCRs were identified through single-cell RT-PCR analysis. The specificity cytotoxic of TCRs were confirmed by transferring to primary peripheral blood T cells.Results: The co-culture system proved highly effective in generating CD8+ tumor-specific oeT cells. These oeT cells effectively induced IFN-γ secretion and exhibited specificity in killing autologous tumors, while not eliciting a cytotoxic response against normal organoids. The analysis conducted by TCRs revealed a significant expansion of T cells within a specific subset of TCRs. Subsequently, the TCRs were cloned and transferred to peripheral blood T cells generation engineered TCR-Ts, which adequately recognized and killed tumor cell in a patient-specific manner.Conclusions: The co-culture system provided an approach to generate tumor-specific TCRs from tumor-infiltrating lymphocytes (TILs) of patients with colorectal cancer (CRC), and tumor-specific TCRs can potentially be used for personalized TCR-Ts therapy.