Humanized and Charge-Optimized CSPG4-Specific CAR-T Cells show Enhanced Efficacy against Head and Neck Squamous Cell...

Major challenges of developing CAR‑T cell therapy for head and neck squamous cell carcinoma (HNSCC) include identifying a robust tumor antigen and a suitable CAR design. Here, we validate chondroitin sulfate proteoglycan 4 (CSPG4) as a highly expressed, prognostic antigen in HPV‑negative HNSCC that drives tumor proliferation. By grafting the murine single-chain variable fragment (scFv) 763.74 complementarity determining regions (CDRs) onto a human antibody framework engineered to minimize surface positive‑charge patches and immunogenic epitopes, we generated humanized CSPG4 (CSPG4^Hu) CAR‑T cells with reduced tonic signaling and alleviated exhaustion. Transcriptomic and metabolic profiling reveal that this biophysical refinement reprograms CAR-T cells away from a glycolytic, terminal exhaustion state toward a PI3K/Akt-driven stem-like state. Consequently, CSPG4^Hu CAR-T cells demonstrate superior persistence and potent antitumor efficacy across systemic xenograft and patient-derived xenograft models. Our study establishes a rational engineering framework that links biophysical CAR design to transcriptomic and metabolic rejuvenation, offering a promising therapeutic candidate for advanced HNSCC.