ID #268 Brain-sensing synNotch-CAR T-cells enable precise targeting and clearance of diffuse midline glioma in multiple preclinical models

Abstract Background Diffuse midline glioma (DMG), including diffuse intrinsic pontine glioma (DIPG), is a fatal pediatric brain tumor with limited therapeutic options. Although early-phase clinical trials indicate promise for chimeric antigen receptor (CAR) T-cell therapy, the lack of tumor-specific antigens, poor trafficking, and limited T-cell persistence remain significant barriers to achieving meaningful clinical benefit. Methods We engineered a synthetic Notch (synNotch)–based “prime-and-kill” CAR T-cell circuit in which recognition of the brain-specific proteoglycan, Brevican (BCAN) induces expression of a tandem CAR targeting B7-H3 and IL13Rα2, antigens highly expressed in DMG. This brain-sensing strategy restricts CAR expression to the tumor microenvironment while minimizing peripheral off-tumor activity. Results α-BCAN synNotch→α-B7-H3/IL13Rα2 (B-SYNC-B/I) T-cells demonstrated potent, BCAN-dependent cytotoxicity against DMG/DIPG cell lines in vitro. A single intravenous infusion significantly prolonged survival in immunodeficient mice bearing orthotopic DMG xenografts, with complete tumor eradication observed in approximately 50% of treated animals. In contrast, intracerebroventricular (ICV) delivery of B-SYNC-B/I T-cells caused significant neurotoxicity in both tumor and non-tumor-bearing mice, even at low doses, consistent with on-target, off-tumor recognition of B7-H3 expressed on normal brain tissue. In immunocompetent DMG models, ICV infusion of B-SYNC-B7-H3 T-cells with or without lymphodepletion led to durable tumor eradication without neurotoxicity and conferred protection against tumor rechallenge. Conversely, constitutively active B7-H3/IL13Rα2 CAR T cells failed to persist, traffic to the brainstem, or improve survival in immunodeficient models; however, lymphodepletion allowed effective tumor clearance following ICV delivery of constitutive B7-H3 CAR T-cells in immunocompetent mice. Conclusions Brain-sensing synNotch-CAR T-cells provide spatially restricted activation and robust anti-tumor efficacy in preclinical DMG models, supporting their clinical evaluation as a promising therapeutic strategy for pediatric patients with DMG/DIPG.