IMMU-45. Allogeneic B7-H3 γδ CAR-T Cells Effectively Eradicate Glioblastoma in Preclinical Models

Abstract BACKGROUND γδ T cells (GDTs) represent a promising allogeneic immune cell platform due to their MHC-unrestricted cytotoxicity. In this study, we developed a novel B7-H3-specific CAR vector (CD3ζ-CD28-based) and evaluated the therapeutic efficacy of B7-H3-directed γδ CAR-T (B7H3-GD CAR-T) cells in preclinical glioblastoma models. METHODS We constructed a lentiviral vector encoding a B7-H3-specific CAR and transduced it into human allogeneic GDT cells. B7-H3 expression was assessed in glioblastoma cell lines (U87, U373, U138) and 10 patient-derived primary cell lines. Formalin-fixed paraffin-embedded (FFPE) tumor specimens were analyzed using immunohistochemistry. Additionally, TCGA data were analyzed for B7-H3 expression and survival correlation. In vitro cytotoxicity and cytokine secretion were evaluated. In vivo efficacy was tested in an orthotopic U87 glioblastoma model in NOG mice. RESULTS Bioinformatic analysis of the TCGA database confirmed that higher B7-H3 expression correlates with poorer overall survival in glioma patients. IHC of FFPE specimens demonstrated increased B7-H3 expression in high-grade gliomas, surpassing HER2 and GD2 expression. We validate that B7-H3 is overexpressed in high-grade gliomas and associated with poor prognosis, suggesting its value as a therapeutic target. In vitro, B7H3-GD CAR-T cells exhibited markedly enhanced cytotoxicity and IFN-γ release compared to unmodified GDTs, with killing strongly correlating with B7-H3 expression and abrogated by B7-H3 antibody blockade. In vivo, a single intratumoral injection of 1×10⁶ B7H3-GD CAR-T cells resulted in complete tumor regression in >80% of mice. Furthermore, intraventricular injection of B7H3-GD CAR-T cells also demonstrated comparable efficacy, indicating the feasibility of clinically applicable delivery routes. CONCLUSION This is the first study to demonstrate potent, target-dependent antitumor activity of B7H3-targeted γδ CAR-T cells in glioblastoma. Our results validate B7-H3 as a clinically relevant target and support the use of allogeneic GDTs as a next-generation platform for glioblastoma immunotherapy.