Abstract:Triple-negative breast cancer (TNBC) is an aggressive subtype with poor prognosis and limited treatment options. Chimeric antigen receptor (CAR)-T cell therapy holds promise, but its efficacy is hindered by tumor antigen escape and heterogeneity. To address these challenges, we developed a novel bispecific T cell engagers CAR-T (BiTEs CAR-T) targeting Mesothelin (MSLN) and secreting NKG2D-Bispecific T cell Engagers (BiTEs) to engage NKG2D ligands (NKG2DL). Analysis of TNBC tissues using The Cancer Genome Atlas and tumor microarrays revealed high but weakly correlated expression of MSLN and NKG2DL, making them ideal targets for dual engagement. To reduce immunogenicity and enhance stability, we used a nanobody and the natural receptor NKG2D as antigen-binding domains instead of traditional scFvs in the CAR construct. The secreted BiTEs could promote the cytotoxicity of untransduced T cells against NKG2DL + tumor cells. In vitro, BiTEs CAR-T cells exhibited superior cytotoxicity, T cell activation, and cytokines production against heterogeneous target cells compared to MSLN CAR-T. In vivo, BiTEs CAR-T cells demonstrated potent antitumor activity in zebrafish and murine TNBC models, significantly reducing tumor burden and prolonging survival without detectable toxicity. These findings suggest that BiTE CAR-T cells offer a highly promising therapeutic strategy for TNBC by addressing antigen heterogeneity and immune escape mechanisms, with promising translational potential for clinical application.