Humanized and Charge-Optimized CSPG4-Specific CAR-T Cells Show Enhanced Efficacy Against Head and Neck Squamous Cell Carcinoma
Chimeric antigen receptor (CAR) T cell therapy for head and neck squamous cell carcinoma (HNSCC) is hampered by the lack of a robust tumor antigen and a suitable CAR design.CSPG4 is highly expressed in HNSCC clinical samples and cell lines, correlating with poor prognosis. Its knockout markedly reduced tumor cell proliferation in vitro and in vivo, validating CSPG4 as an attractive CAR target. First-generation CSPG4.CAR-T cells bearing the murine scFv 763.74 showed potent in vitro cytotoxicity but failed to control tumor growth in vivo due to excessive tonic signaling and early exhaustion. By humanizing the scFv framework to reduce positive_x001E_charge residues and immunogenicity, we generated CAR_x001E_T cells with low tonic signaling, diminished exhaustion and differentiation, enhanced tumor_x001E_specific killing, and prolonged in vivo persistence, resulting in superior antitumor activity in both xenograft and PDX models. Overall design: We confirmed CSPG4's expression and oncogenic role via qPCR and immunohistochemistry on primary HNSCC specimens, and by CRISPR-mediated knockout in cell lines and xenografts. To optimize CAR design, we grafted the murine scFv 763.74 CDRs onto a human antibody framework engineered to minimize surface positive-charge patches and immunogenic epitopes. Humanized CSPG4.CAR-T cells were then evaluated for tonic signaling, exhaustion marker expression, proliferation, and cytotoxicity in vitro, as well as for antitumor efficacy and persistence in cell-line xenograft and patient-derived xenograft (PDX) models.