A Rationally Designed Fully Human EGFRvIII:CD3-Targeted Bispecific Antibody Redirects Human T Cells to Treat Patient-derived Intracerebral Malignant Glioma

Purpose: Conventional therapy for malignant glioma (MG) fails to specifically target tumor cells. In contrast, substantial evidence indicates that if appropriately redirected, T cells can precisely eradicate tumors. Here we report the rational development of a fully-human bispecific antibody (hEGFRvIII-CD3 bi-scFv) that redirects human T cells to lyse MG expressing a tumor-specific mutation of the epidermal growth factor receptor (EGFRvIII). Experimental Design: We generated a panel of bispecific single-chain variable-fragments and optimized design through successive rounds of screening and refinement. We tested the ability of our lead construct to re-direct naive T cells and induce target-cell specific lysis. To test for efficacy, we evaluated tumor growth and survival in xenogeneic and syngeneic models of glioma. Results: A highly-expressed bispecific antibody with specificity to CD3 and EGFRvIII was generated (hEGFRvIII-CD3 bi-scFv). Antibody-induced T cell activation, secretion of pro-inflammatory cytokines, and proliferation was robust and occurred exclusively in the presence of target antigen. hEGFRvIII-CD3 bi-scFv was potent and target-specific, mediating significant lysis of multiple MG cell lines and patient-derived MG samples. In both subcutaneous and orthotopic models, well-engrafted, patient-derived MG was effectively treated despite heterogeneity of EGFRvIII expression; intravenous hEGFRvIII-CD3 bi-scFv administration caused significant regression of tumor burden (p<0.0001) and significantly extended survival (p<0.0001). Similar efficacy was obtained in highly-infiltrative, syngeneic glioma models. Conclusions: We have developed a clinically-translatable bispecific antibody that redirects human T cells to safely and effectively treat MG. On the basis of these results, we have developed a clinical study of hEGFRvIII-CD3 bi-scFv for patients with EGFRvIII-positive MG.