Abstract C163: A highly cytotoxic EGFRvIII/CD3 TandAb recruits T cells to specifically and potently kill several types of solid tumor cancers.
Authors: Zhukovsky Eugene A. , Ellwanger Kristina , Reusch Uwe , Eser Markus , Fucek Ivica , Burkhardt Carmen 
Published: August 2014

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 and EGFRvIII, the EGFRvIII/CD3 RECRUIT-TandAb. EGFR dysregulation has been linked to numerous cancers, and both small molecules and EGFR targeting antibodies have successfully reached the clinic. However, the antibodies approved for clinical use, and those in development, all share a severe side effect profile due to the broad normal tissue-expression of EGFR. The deletion variant III of EGFR (EGFRvIII), with truncated extracellular domain and ensuing ligand-independent constitutive activity, is the most common mutant form associated with oncogenic transformation. EGFRvIII is expressed exclusively in cancer tissues and is associated with various solid tumor types. The restricted EGFRvIII expression on cancer cells 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 anti-EGFRvIII antibodies 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; moreover, the comparator was not strictly specific for EGFRvIII and had residual cytotoxicity, EC50 = 1 nM, towards EGFR+ cell lines. High affinity binding to CD3 was essential for efficacious T cell recruitment 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 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 Information: Mol Cancer Ther 2013;12(11 Suppl):C163.
Citation Format: Eugene A. Zhukovsky, Kristina Ellwanger, Uwe Reusch, Markus Eser, Ivica Fucek, Carmen Burkhardt. 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 AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C163.