Anti-HER2/neu TCR-T Cells in Action: linking transcriptional signatures, secretomics, and In Vivo tumor suppression

Introduction T cell receptor-engineered T cell therapy has emerged as a promising approach in cancer immunotherapy, leveraging the ability of T cells to recognize tumor antigens presented on major histocompatibility complex molecules, offering a targeted approach for treating cancers. This study advances previous research conducted at the Laboratory of Molecular Immunology at RIFCI, where the full repertoire of HER2/neu-specific TCRs was identified. Specifically, here we are functionally validating a distinct TCR clonotype targeting the KIFGSLAFL peptide of HER2/neu protein presented by the HLA-A*02. Methods We employed an integrated approach combining in vitro cytotoxicity assays, single-cell RNA sequencing via BD Rhapsody, secretome profiling via LegendPlex, and in vivo HER2/neu-expressing xenograft models in SCID mice. Results Anti-HER2/neu TCR-T cells exhibited robust antigen-specific cytotoxicity in vitro , preferentially targeting tumor cells with high HER2/neu expression. Single-cell RNA sequencing revealed a unique double-positive (CD4+CD8+) T cell population emerging upon antigen engagement, characterized by a cytotoxic transcriptome with elevated granzyme B, granulysin, perforin, and TNF-α gene expression. Secretome profiling confirmed significantly enhanced production of effector molecules, including IL-2, granzyme B, TNF-α, and IFN-γ, supporting potent T cell activation and function. In vivo , anti-HER2/neu TCR-T cells achieved sustained and significant suppression of tumor growth in HER2/neu-expressing xenograft models, underscoring their therapeutic potential. Discussion These findings validate the broader utility of the previously identified HER2/neu-specific TCR repertoire and elucidate the molecular mechanisms driving its therapeutic efficacy, demonstrating the potential of TCR-T cells for treating solid tumors through robust cytotoxic activity and the emergence of a favorable CD4+CD8+ T cell population. This study offers critical mechanistic insights, establishing a foundation for advancing TCR-engineered therapies toward clinical use in HER2/neu-positive cancers.