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

ABSTRACT 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.