Combined targeting of mesothelin and tenascin-C enhances CAR-T cell function and tumor microenvironment modulation in ovarian cancer

BACKGROUND: The efficacy of chimeric antigen receptor T (CAR-T) cell therapy in solid tumors is limited by dense extracellular matrix deposition and an immunosuppressive tumor microenvironment. Tenascin-C (TNC), an extracellular matrix protein enriched in ovarian cancer stroma, may provide a stromal cue that restricts T-cell engagement while offering a complementary target to mesothelin (MSLN). This study evaluated whether combined targeting of MSLN and TNC could enhance CAR-T-cell function and remodel the ovarian cancer microenvironment. METHODS: CAR-T cells targeting MSLN and/or TNC were generated using single lentiviral vectors encoding second-generation CAR constructs, including tandem dual-target CARs and MSLN-directed CAR-T cells secreting an anti-TNC single-chain variable fragment with or without a membrane-tethered PD-L1-binding module. CAR expression and function were evaluated across three healthy-donor batches. Target expression on SKOV3 cells and ovarian cancer-associated fibroblasts (CAFs) was validated by flow cytometry and qPCR. Functional studies included NFAT reporter assays, cytokine release, cytotoxicity, immune synapse quantification, adhesion assays, CAF co-culture, and xenograft studies with randomized treatment allocation and blinded tumor measurements. RESULTS: Dual-target TNC+MSLN CAR-T cells demonstrated reproducible CAR expression across donors, enhanced NFAT activation in TNC-rich conditions, increased CD69/CD25 upregulation, and greater IFN-γ, TNF-α, and IL-2 secretion than single-target controls. Compared with MSLN-CAR-T cells, dual-target CAR-T cells showed improved tumor-cell killing, larger and more polarized immune synapses, and stronger tumor-cell adhesion. In CAF co-culture, anti-TNC-secreting CAR-T cells partially reversed suppression, reduced PD-1 and Tim-3 expression, and showed concordant reductions in LAG-3 and TIGIT in exploratory analyses. In vivo, combination stromal targeting delayed tumor growth, increased intratumoral CD4 + and CD8 + infiltration, improved CAR-T persistence, and reduced extracellular matrix deposition and CAF-associated markers. CONCLUSIONS: Combined targeting of MSLN and TNC was associated with improved CAR-T-cell activation and broader microenvironmental remodeling in ovarian cancer models. These data support further evaluation of stromal co-targeting as an adjunct strategy for CAR-T therapy in ovarian cancer, while additional validation of target dependence, safety, and long-term persistence remains necessary.