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

Diffuse midline glioma (DMG) is a lethal pediatric brain tumor with limited therapeutic options and a poor prognosis. The success of chimeric antigen receptor (CAR) T cell therapy requires both improving T cell tumor infiltration and persistence and reducing the risk of on-target/off-tumor toxicity. Here, we adapted and advanced a brain-restricted CAR T cell platform previously validated in glioblastoma to address the anatomical and biological challenges of DMG. We engineered a T cell circuit in which a brain-specific synthetic Notch (synNotch) receptor recognizing brevican (BCAN) induced CAR expression against EphA2 or IL-13Rα2 ("B-SYNC"), thereby restricting CAR expression to the central nervous system (CNS). In immunodeficient DMG models, a single infusion of intravenous (iv) or intracerebroventricular (icv) B-SYNC circuit T cells led to tumor infiltration, sustained persistence, antitumor activity, and extended survival. An alternative BCAN-primed CAR targeting B7-H3 or IL-13Rα2 ("B-SYNC-B/I") similarly improved therapeutic efficacy when delivered intravenously. However, icv administration triggered fatal neurotoxicity because of low-level B7-H3 expression in nontumor CNS regions, indicating that rapid CNS exposure via icv delivery can amplify off-tumor toxicity. In contrast, in an immunocompetent syngeneic DMG model, both iv and icv delivery of a murinized BCAN-gated B7-H3 CAR (mB-SYNC-B7-H3) achieved durable tumor clearance and resistance to rechallenge without any detectable neurotoxicity. Mechanistically, synNotch-primed CAR T cells exhibited reduced exhaustion, enhanced activation, and enrichment of naïve and stem cell memory-like subsets. These findings demonstrate that brain-sensing synNotch CAR T circuits represent a generalizable and translatable strategy for targeting multiple CNS malignancies, including DMG.