Efficacy of allogeneic B7-H3 CAR γδ T cells with comparative target assessment across preclinical models in glioblastoma

Glioblastoma, the most common and aggressive primary brain malignancy, remains a lethal disease with limited therapeutic progress despite multimodal treatment. Although chimeric antigen receptor (CAR) T-cell therapy has shown promise in several solid tumors, its broader application in glioblastoma is limited by manufacturing hurdles of autologous T cells and the lack of validated tumor-specific antigens. As an alternative, gamma-delta (γδ) T cells have emerged as an attractive cellular platform, offering MHC-independent tumor recognition, robust ex vivo expansion, and suitability for allogeneic use. However, studies applying CAR engineering to γδ T cells in glioblastoma are extremely limited, and an optimal target antigen has not been clearly defined. In this context, our study identified B7-H3 as a clinically relevant target and demonstrated that B7-H3 CAR γδ T cells exhibited superior antitumor efficacy compared to unmodified γδ T cells across patient-derived and orthotopic glioblastoma models. To identify an optimal antigen for γδ CAR-T therapy in glioblastoma, we compared the expression and clinical relevance of HER2, GD2, and B7-H3 through analyses of transcriptomic datasets and patient tumor samples. Based on this, human γδ T cells from healthy donors were expanded ex vivo, engineered with a B7-H3–specific CAR, and evaluated for antitumor activity across patient-derived glioblastoma cell lines, organoids, and orthotopic xenograft models. B7-H3 was identified as the most clinically relevant antigen, showing high expression in glioblastoma and correlated with poor prognosis and immunosuppressive gene signatures. B7-H3 CAR γδ T cells demonstrated potent cytotoxicity and enhanced cytokine production in B7-H3–high patient-derived glioblastoma models. In patient-derived organoids, CAR γδ T cells significantly reduced tumor viability and invasion. In orthotopic xenografts, a single intracranial administration induced tumor control and prolonged survival, with long-term survivors observed only in the CAR γδ T cell treated group. Across all preclinical models, B7-H3 CAR γδ T cells exerted superior antitumor efficacy compared with parental γδ T cells. These findings suggest B7-H3 as a clinically meaningful and therapeutically targetable antigen in glioblastoma and demonstrate the potent preclinical efficacy of allogeneic B7-H3 CAR γδ T cells, supporting their development as a novel immunotherapeutic strategy for glioblastoma.