bioRxiv2025
BackgroundDiffuse intrinsic pontine glioma (DIPG) is a fatal pediatric brain tumor affecting over 300 children annually in the United States. Chimeric antigen receptor (CAR) T cells are a targeted immune effector cell therapy with substantial clinical benefit against hematologic cancers. Against CNS tumors, CAR T cells targeting B7-H3, a protein highly expressed on DIPG, have rapidly advanced from preclinical studies to clinical trials. BrainChild-03 (NCT04185038), a phase 1 trial of repeatedly delivered intracerebroventricular (ICV) B7-H3-targeting CAR T cells (B7-H3 CAR T cells), demonstrated tolerability and potential efficacy for children and young adults with DIPG. However, clinical benefits were not uniformly seen, and multi-agent treatment strategies may be required against such an aggressive disease. Here, we combined B7-H3 CAR T cells with ONC206, an imipridone molecule also under clinical investigation. MethodsWe tested B7-H3 CAR T cells combined with ONC206 across multiple DIPG cell cultures and orthotopic xenograft mouse models. ResultsB7-H3 CAR T cell monotherapy induced robust cytotoxicity while ONC206 treatment resulted in significant mitochondrial dysfunction against DMG/DIPG cells. The combination of low effector-to-target ratios of B7-H3 CAR T cells and IC50 concentrations of ONC206 led to significantly enhanced cytotoxicity in vitro (p<0.003) and increased IL-2, IL-29, VEGF-A, and Granzyme B levels. In vivo combinatorial studies of ONC206 and a single ICV dose of B7-H3 CAR T cells significantly extended survival in multiple DIPG xenograft mouse models (p<0.05). ConclusionsB7-H3 CAR T cells combined with ONC206 is a feasible and efficacious multi-agent approach against multiple DIPG models. Importance of the studyDiffuse intrinsic pontine glioma (DIPG) is a fatal pediatric brain tumor. While B7-H3 CAR T cells have shown tolerability and potential benefit in early trials, combinatorial regimens may be required for consistent cures against this aggressive disease. This study demonstrates that a preclinical therapeutic regimen of B7-H3 CAR T cells with ONC206, a second-generation imipridone, increases anti-tumor efficacy in vitro and in orthotopic DIPG mouse models. To our knowledge, this is the first study to evaluate ONC206 in combination with CAR T cells. Our findings provide a preclinical roadmap for evaluating small molecules with CAR T cells to interrogate both their combined benefit and the effect of small molecules on T cells themselves. This work offers a biologically-informed, clinically translatable strategy integrating small molecule therapeutics with CAR T cell therapy and support the development of multi-agent immunotherapy trials for children with DIPG and other high-grade brain and spinal cord tumors. Key PointsO_LIB7-H3 CAR T cells are cytotoxic against preclinical DMG models. C_LIO_LIONC206 causes metabolic apoptosis in preclinical DMG models. C_LIO_LIB7-H3 CAR T cells and ONC206 have combinatorial efficacy against DMG. C_LI