IMMU-14. Secretion of GD2-CD3 BiTEs from B7H3 CAR T-Cells Enhances their Anti-Tumor Activity in Pediatric High-Grade Gliomas

Abstract Pediatric high-grade gliomas (pHGG) have seen limited therapeutic progress over decades. While CAR T-cell therapies have shown promise in hematologic malignancies, CNS tumors pose additional challenges. Major barriers to the lasting benefit of CAR T-cell therapy in pHGG are their immunosuppressive tumor microenvironment (TME) dominated by tumor-associated macrophages (TAMs) and poor T cell infiltration. Macrophages should facilitate anti-tumor immunity, but pHGG tumors also resist macrophage phagocytosis through overexpression of ‘Do Not Eat Me’ cell surface signals like CD47 and GD2. We have developed B7H3-directed CAR T-cells capable of secreting GD2-CD3 bispecific T-cell engagers (BiTEs) to recruit nearby T-cells and enhance tumor killing. A mutated GD2 scFv with high binding affinity was fused with CD3 scFv to generate GD2-CD3 BiTEs. B7H3 CAR T-cells were engineered to secrete GD2-CD3 BiTEs, enabling dual-targeting of tumor cells and increased engagement of T-cells. Anti-tumor activity was assessed in vitro using GD2+ pHGG cell lines (KNS42, CHLA200) via co-culture and transwell assays. In vivo efficacy was evaluated in an orthotopic NSG murine model using MRI and bioluminescence imaging. Anti-CD47 was added to CAR T-cell constructs to assess its impact on macrophage phagocytosis and survival outcomes. GD2-CD3 BiTEs showed significant in vitro cytotoxicity against pHGG cells with untransduced T-cells. GD2-CD3.B7H3 BiTE-secreting CAR T-cells recruited bystander T-cells, enhancing tumor cell killing. Although the combination of GD2-CD3 BiTEs and anti-CD47 antibody increased phagocytosis in macrophage assays, its in vivo combination with CAR T-cells led to reduced survival, likely due to unintended T-cell phagocytosis. In contrast, GD2-CD3.B7H3 BiTE-secreting CAR T-cells demonstrated superior survival in the pHGG murine model compared to B7H3 CAR T-cells. This dual-targeting approach shows promise for pHGG treatment by enhancing T-cell recruitment and tumor cytotoxicity. While anti-CD47 therapy improved phagocytosis in vitro, its in vivo application requires caution due to potential adverse effects.