Feng Ji

Dual-target CAR-T therapy for ovarian cancer: synergistic targeting of MSLN and B7H3 enhances anti-tumor efficacy and overcomes antigen heterogeneity

Ovarian cancer (OC) remains a lethal malignancy with limited treatment options owing to antigen heterogeneity and an immunosuppressive tumor microenvironment (TME). Here, we designed a unique chimeric Antigen Receptor T-Cell (CAR-T) construct (B4M3) that integrates an anti-MSLN scFv linked to the CD3ζ activation domain and an anti-B7H3 scFv linked to the 4-1BB co-stimulatory domain. In vitro, B4M3 CAR-T cells exhibited robust cytotoxicity against OC cell lines with enhanced degranulation (CD107a) and efficient tumor cell killing, even at low effector-to-target ratios. In vivo, B4M3 CAR-T cells significantly inhibited tumor growth and prolonged survival and demonstrated superior tumor infiltration and persistence in OC xenograft models. Imaging mass cytometry (IMC) revealed that B4M3 treatment reshaped the TME, increased cytotoxic T lymphocyte (CTL) infiltration, and reduced regulatory T cells (Tregs). Mechanistically, B4M3 therapy upregulated TGF-β, promoting Th17 differentiation and CTL recruitment, thereby enhancing anti-tumor immunity. Our findings demonstrate that B4M3 CAR-T cells effectively address antigen heterogeneity and enhance therapeutic efficacy in OC, thereby offering a promising strategy for solid tumor immunotherapy.