Objectives The increasing success of Chimeric Antigen Receptor (CAR) T cell therapy in haematological malignancies is reinvigorating its application in many other cancer types and with renewed focus on its application to solid tumors. We present a novel CAR against glioblastoma, an aggressive, malignant glioma, with a dismal survival rate for which treatment options have remained unchanged for over a decade. Methods We use the human Retained Display (ReD) antibody platform (Myrio Therapeutics) to identify a novel single‐chain variable fragment (scFv) that recognises epidermal growth factor receptor mutant variant III (EGFRvIII), a common and tumor‐specific mutation found in glioblastoma. We use both in vitro functional assays and an in vivo orthotopic xenograft model of glioblastoma to examine the function of our novel CAR, called GCT02, targeted using murine CAR T cells. Results Our EGFRvIII‐specific scFv was found to be of much higher affinity than reported comparators reverse‐engineered from monoclonal antibodies. Despite the higher affinity, GCT02 CAR T cells kill equivalently but secrete lower amounts of cytokine. In addition, GCT02‐CAR T cells also mediate rapid and complete tumor elimination in vivo . Conclusion We present a novel EGFRvIII‐specific CAR, with effective antitumor functions both in in vitro and in a xenograft model of human glioblastoma.

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Background Diffuse intrinsic pontine glioma (DIPG) and other diffuse midline gliomas (DMG) of the thalamus and spinal cord are rare but devastating high-grade glial tumors of childhood with no curative treatment. Despite aggressive treatment attempts the prognosis has remained poor. Chimeric antigen receptor (CAR) T cell therapy has been identified as a promising new approach in the treatment of DMG tumors; however, additional targets are urgently required given known tumor heterogeneity and the prospect of antigen escape of this cancer. Methods Using cell surface mass spectrometry, we detected high HER2 cell surface protein across a panel of patient-derived DIPG cells, thereby identifying an existing CAR T cell therapy for use in DIPG. Primary human T cells were transduced to express a second-generation HER2 CAR and interrogated for efficacy against patient-derived DIPG cells. Results HER2 CAR T cells demonstrated potent and antigen-specific cytotoxicity and cytokine secretion when co-cultured with patient-derived DIPG cells. Furthermore, HER2 CAR T cells provided a significant regression in intracranial DIPG xenograft tumors. Conclusions HER2 CAR T cells are already in clinic development and are well tolerated in pediatric patients. Here we provide strong preclinical evidence for the inclusion of DIPG patients in future pediatric CNS tumor HER2 CAR T cell clinical trials.

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.