Background
Glioblastoma is the most common and aggressive primary brain malignancy, with a dismal prognosis despite multimodal treatment. CAR-T cell therapy holds promise, but its application in glioblastoma is constrained by autologous manufacturing challenges and the lack of validated tumor-specific antigens. Gamma-delta (γδ) T cells offer a compelling platform, combining MHC-independent tumor recognition with robust ex vivo expandability and suitability for allogeneic, off-the-shelf use. However, CAR-engineered γδ T cell strategies for glioblastoma have been minimally explored, and no optimal target antigen has been clearly established. Here, we identify B7-H3 as a therapeutically relevant antigen and evaluate the efficacy of B7-H3 CAR γδ T cells across clinically relevant glioblastoma models.

Methods
Antigen selection was guided by comparative expression and prognostic analyses of HER2, GD2, and B7-H3 across transcriptomic datasets and patient tumor samples. Human γδ T cells from healthy donors were expanded ex vivo and engineered with a B7-H3-specific CAR. Antitumor activity was assessed in patient-derived glioblastoma cell lines, B7-H3 knockdown glioblastoma cell lines, three-dimensional organoids, and orthotopic xenograft models.

Results
Among candidate antigens, B7-H3 showed the highest and most consistent expression in glioblastoma. Elevated B7-H3 expression was associated with poor prognosis and immunosuppressive tumor microenvironment signatures. B7-H3 CAR γδ T cells exhibited superior cytotoxicity compared with BFP-transduced γδ T cells across most patient-derived glioblastoma cell lines, with B7-H3-high tumors showing the greatest susceptibility. B7-H3 knockdown significantly attenuated CAR γδ T cell-mediated killing, confirming that antitumor activity is predominantly antigen-dependent. Residual cytotoxicity and cytokine production against some B7-H3-low lines suggest additional contributions from intrinsic γδ T cell receptor–ligand interactions. In patient-derived organoids, CAR γδ T cells significantly reduced tumor viability and suppressed invasive growth. In orthotopic xenografts, a single intracranial injection induced durable tumor control and prolonged survival. Superior antitumor efficacy of B7-H3 CAR γδ T cells over parental γδ T cells was consistently demonstrated across all preclinical platforms tested.

Conclusions
These results establish B7-H3 as a suitable target in glioblastoma and provide compelling preclinical evidence for allogeneic B7-H3 CAR γδ T cells as a novel, off-the-shelf immunotherapeutic strategy for Glioblastoma.