Abstract To harness the cytotoxic capacity of T cells for the treatment of EGFRvIII+ cancers, we developed a humanized bispecific tetravalent antibody, with two binding sites for CD3 (or CD16A) and EGFRvIII, the EGFRvIII/CD3 (and EGFRvIII/CD16A) RECRUIT-TandAb. EGFR dysregulation has been linked to numerous cancers, and both small molecules and EGFR targeting antibodies have successfully reached the clinic. The deletion variant III (EGFRvIII), the most common mutant form of EGFR is expressed exclusively in cancer tissues contributing to oncogenic transformation. The restricted EGFRvIII expression on various solid tumor types provides an opportunity to develop cytotoxic antibodies that solely target cancer, sparing normal tissues, and substantially reduce the side effects associated with EGFR therapy. Using phage display libraries, we identified scFvs that selectively bound to the mutated and not the native form of EGFR. T cells are potent tumor-killing effector cells that cannot be recruited by native antibodies; hence we engineered a panel of bispecific EGFRvIII/CD3 TandAbs, capable of T cell-recruitment, with a broad range of binding and cytotoxic properties. The TandAbs' binding properties, T cell-mediated cytotoxic activity, and target-mediated T cell activation were characterized in a panel of in vitro assays. They exhibited exquisite specificity towards EGFRvIII in ELISA and FACS assays, with no wild-type EGFR binding observed, up to the maximally evaluated TandAb concentration of 1 μM. The most potent TandAbs displayed cytotoxicity towards EGFRvIII expressing F98 glioma and CHO cells with EC50 = 25 pM. We also assayed the cytotoxicity of these TandAbs towards EGFR+ cells as a more sensitive probe of residual binding to the native form. No cytotoxicity was observed up to the maximally evaluated TandAb concentration of 0.5 μM. The cytotoxic potency of the EGFRvIII/CD3 TandAb was higher than that of a comparator bivalent bispecific antibody, constructed from different anti-EGFRvIII/CD3 moieties or of EGFRvIII/CD16A TandAbs, which recruit NK cells instead of cytotoxic T cells. EGFRvIII/CD3 TandAbs with high affinity binding to CD3 were most efficacious for T cell recruitment and tumor cell killing and yet, in the absence of EGFRvIII+ target cells in vitro, TandAbs did not elicit T cell activation, as measured by their lack of proliferation, contributing to a good preclinical safety profile. In vivo EGFRvIII/CD3 TandAbs demonstrated a robust dose-dependent growth retardation of EGFRvIII+ subcutaneous Xenograft tumors in NOD/scid mice reconstituted with human PBMC; this tumor growth inhibition was more pronounced than that associated with cetuximab. In summary, our data demonstrate the strict specificity and high potency of the anti-tumor cytotoxicity mediated by the EGFRvIII/CD3 TandAb, a novel highly potent drug candidate for the treatment of EGFRvIII+ malignancies. Citation Format: Eugene A. Zhukovsky, Kristina Ellwanger, Uwe Reusch, Markus Eser, Fionnuala McAleese, Ivica Fucek, Carmen Burkhardt, Stefan Knackmuss. A highly cytotoxic EGFRvIII/CD3 TandAb recruits T cells to specifically and potently kill several types of solid tumor cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2643. doi:10.1158/1538-7445.AM2014-2643