EXTH-39 - D2C7-CAR: A novel, dual-specific chimeric antigen receptor T cell with high specificity for EGFR and EGFRvIII improves survival in EGFR/EGFRvIII glioblastoma and EGFR over-expressing medulloblastoma

Background: GBM is the most common primary malignant brain tumor found in adults. Despite strenuous efforts at improving outcomes, overall survival remains dismal (15-20 months). D2C7-CAR is a dual specific CAR T cell that targets EGFR and EGFRvIII. We performed pre-clinical analysis of D2C7-CAR glioblastoma and other EGFR aberrant tumor models to assessed efficacy and safety.

Methods: U87 and U87vIII glioblastoma and DAOY medulloblastoma cell lines were characterized by flow cytometry to evaluate for EGFR and EGFRvIII expression. Cytotoxicity assays were performed utilizing flow cytometry on serially diluted effector: target ratios of U87, U87vIII, and DAOY mixed with D2C7 CAR. U87 and U87vIII was orthotopically implanted into the frontal lobe and DAOY was orthotopically implanted into the frontal lobe and posterior fossa of NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice. After tumor implantation, D2C7 CAR, vIII CAR, or Mock CAR was introduced within the tumor bed. Survival was measured and analyzed using the Log-rank test. Bioluminescence with IVIS imaging of CT2AVIII with luciferase was utilized to perform time point analysis of IC tumor response to treatment vs control. Toxicity against somatic tissue EGFR expression was assessed by in vitro cytotoxicity assay as described above.

Results: EGFR was detected in U87, U87vIII, and DAOY on flow analysis. EGFRvIII was expressed only on U87vIII. D2C7 CAR demonstrated effective in vitro cytotoxicity against all three cell lines with >90% cytotoxicity achieved at an effector: target ratio of 2.5:1. In vivo, D2C7 CAR significantly prolonged survival compared to treatment with mock and vIII CAR when tumor was implanted intracranially. Bioluminescence imaging demonstrated robust tumor response. D2C7 was not cytotoxic when co-cultured with EGFR expressing human keratinocytes.

Conclusion: D2C7-CAR demonstrates effective preclinical results both in vitro and in vivo. D2C7-CAR is not cytotoxic against human keratinocytes, suggesting it will be well tolerated in clinical trials.