Immu-13. preclinical efficacy of multi-antigen (her2/egfr806/b7-h3/il13ra2) targeting quad-car t cells against diffuse midline glioma

Abstract BACKGROUND Diffuse midline glioma (DMG), including diffuse intrinsic pontine glioma (DIPG), remains a fatal disease. We have published the preclinical efficacy of HER2, EGFR806, and B7-H3 CAR-T cells and opened 3 trials (BrainChild-01, -02, and -03, respectively) that have delivered >350 intracranial CAR T doses to >70 enrolled children with recurrent/refractory CNS tumors and DIPG/DMG. While these trials have demonstrated the feasibility and tolerability of repeated, locoregional CAR-T cell dosing, responses have not been uniform in part due to tumor heterogeneity. METHODS Seattle Children’s Therapeutics generated multi-antigen (HER2, EGFR806, B7-H3, IL13ra2) quad-CAR T cells and we performed IND-enabling preclinical studies using CNS tumor models including DMG models (PBT-22, PBT-27, and PBT-29). RESULTS Quad-CAR T cells were generated by transducing T cells with a mixture of four vectors. The specificity of these CARs is conferred by scFv binding domains derived from the monoclonal antibodies Herceptin, mAb806, and MGA271 for HER2, EGFR806, and B7-H3, respectively. The IL13-zetakine, aligned with NCT00730613, carries an E13Y mutation in place of an scFv for specificity. In vitro, the quad-CAR T cells demonstrated significant in vitro cytolysis and cytokine (IL-2, IFNy, TNFa) production compared to mock (untransduced) CAR-Ts. In vivo, pilot studies in a GFP:ffLuc+Her2 U87 orthoptic xenograft mouse model showed significant survival (p<0.001). To validate against DMG, we are conducting a larger study of single dose intracranial quad-CAR T cells in an orthotopic xenograft PBT-29 DMG mouse model. While ongoing, at day 19 post-treatment there is already a significant bioluminescence decrease (p<0.001). In vivo studies will be completed and complimented by tumor spatial transcriptomics. CONCLUSION Quad-CAR T cell targeting of DMG is highly effective preclinically. A phase I trial of repeated, locoregional quad-CAR T cells for children with recurrent/refractory CNS tumors and DIPG/DMG is FDA-approved and will open in 2023.