DMG-27. Brain-sensing synNotch-CAR T cells demonstrate robust anti-tumor activity against diffuse midline glioma

Abstract Diffuse midline glioma (DMG) is an aggressive brain tumor in children, with limited treatment options. Recent phase 1 clinical trials have shown promising results for chimeric antigen receptor (CAR) T cell therapy in DMG patients. However, several challenges such as the absence of tumor-specific antigens, tumor-antigen heterogeneity, limited trafficking to the tumor sites, and poor persistence impede the full therapeutic potential of CAR T cells. To overcome these obstacles, we adopted a novel synthetic Notch (synNotch) receptor system and engineered T cell circuits employing a “prime-and-kill” strategy. In this system, a synNotch receptor recognizing the priming antigen, Brevican (BCAN), exclusively expressed on cells in the central nervous system (CNS) but not on non-CNS tissues, locally induces the expression of a tandem CAR against glioma-associated antigens, ephrin type A receptor (EphA2) and interleukin-13 receptor a2 (IL13Ra2), resulting in the effective elimination of DMG cells. In preclinical DMG models, a single intravenous (IV) infusion of 6 × 106 a-BCAN synNotch-a-EphA2/IL13Ra2 CAR (B-SYNC) T cells significantly (P 0.001) prolonged the survival of immunodeficient mice-bearing aggressive, orthotopic SF8628 (n = 8) and BT245 (n = 8) xenografts, and completely eradicated the tumor in 50% of mice. Furthermore, a single intracerebroventricular (ICV) administration of 2 × 106 B-SYNC T cells mediated complete tumor regression in all the mice-bearing SF8628 (n = 7) and BT245 xenografts (n = 7), and provided protection against tumor rechallenge. Notably, B-SYNC T cells administered either IV or ICV, displayed exceptional homing, priming, activation, killing, and persistence in the brain stem of mice bearing DMG xenografts. In contrast, constitutively active α-EphA2/IL-13Rα2 CAR T cells or untransduced T cells delivered either IV or ICV failed to persist, inhibit tumor growth or improve survival. Taken together, these compelling findings strongly support the development of clinical trials to evaluate the efficacy of B-SYNC T cells in treating DMG patients.