Human <scp>EGFRvIII</scp> chimeric antigen receptor T cells demonstrate favorable safety profile and curative responses in orthotopic glioblastoma

Abstract Objectives Glioblastoma is a highly aggressive and fatal brain malignancy, and effective targeted therapies are required. The combination of standard treatments including surgery, chemotherapy and radiotherapy is not curative. Chimeric antigen receptor (CAR) T cells are known to cross the blood–brain barrier, mediating antitumor responses. A tumor‐expressed deletion mutant of the epidermal growth factor receptor (EGFRvIII) is a robust CAR T cell target in glioblastoma. Here, we show our de novo generated, high‐affinity EGFRvIII‐specific CAR; GCT02, demonstrating curative efficacy in human orthotopic glioblastoma models. Methods The GCT02 binding epitope was predicted using Deep Mutational Scanning (DMS). GCT02 CAR T cell cytotoxicity was investigated in three glioblastoma models in vitro using the IncuCyte platform, and cytokine secretion with a cytometric bead array. GCT02 in vivo functionality was demonstrated in two NSG orthotopic glioblastoma models. The specificity profile was generated by measuring T cell degranulation in response to coculture with primary human healthy cells. Results The GCT02 binding location was predicted to be located at a shared region of EGFR and EGFRvIII; however, the in vitro functionality remained exquisitely EGFRvIII specific. A single CAR T cell infusion generated curative responses in two orthotopic models of human glioblastoma in NSG mice. The safety analysis further validated the specificity of GCT02 for mutant‐expressing cells. Conclusion This study demonstrates the preclinical functionality of a highly specific CAR targeting EGFRvIII on human cells. This CAR could be an effective treatment for glioblastoma and warrants future clinical investigation.