ABSTRACT: Diffuse midline glioma (DMG) is a lethal pediatric brain tumor with limited therapeutic options and a poor prognosis. The ultimate success of chimeric antigen receptor (CAR) T-cell therapy requires improving T-cell tumor infiltration, and persistence, and reducing the risk of on-target/off-tumor toxicity. Here, we adapt and advance a brain-restricted CAR T-cell platform, previously validated in glioblastoma, to address the distinct anatomical and biological challenges of DMG. We engineer we test whether a strategy developed for glioblastoma can also be effective against DMG. We evaluated a T- cell circuit in which a brain-specific sensor -- the synthetic Notch (synNotch) receptor recognizingagainst Brevican (BCAN) -- induces CAR the expression against of a CAR targeting either EphA2 or IL-13Rα2 (“B-SYNC”), enabling multi-antigen tumor recognition tumor recognition through multiple antigens while restricting CAR expression to the CNS. In immunodeficient DMG models, a single infusion of B-SYNC T- cells, either by intravenousintravenously (IV) or intracerebroventricularly (ICV) infusion, led to tumor infiltration, sustained persistence, robust anti-tumor activity, and significantly extended survival. An alternative BCAN-primed circuit CAR inducing a CAR targeting eithertargeting B7-H3 or IL-13Rα2 (“B-SYNC-B/I”) similarly improved therapeutic efficacy when delivered IV. However, ICV administration triggered fatal neurotoxicity due to low-level B7-H3 expression in non-tumor 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. , supporting superior functional durability in the DMG microenvironment. These findings demonstrate that brain-sensing synNotch CAR T circuits represent a generalizable and translatable strategy for targeting multiple CNS malignancies, including DMG.
