Abstract 583: Evaluation of EphA2 as a therapeutic target for redirected T-cell killing by DART® bispecific molecules

Abstract

Abstract Introduction: EphA2 is a receptor tyrosine kinase that plays a critical role in cancer progression through both ligand-dependent and independent mechanisms. The broad overexpression in tumors but limited normal tissue expression of EphA2 makes it an attractive therapeutic target amenable for redirected T-cell killing via an EphA2 x CD3 Dual-Affinity Re-Targeting (DART®) molecules designed to co-engage cytotoxic T cells with EphA2 expressing tumor cells. Methods: Anti-EphA2 monoclonal antibodies (mAbs) were identified by target-specific screening of a library generated by whole-cell immunization with proprietary cancer cell lines, including models of cancer stem cells. The binding and signaling properties of the antibodies were characterized by ELISA, SPR, flow cytometry and phosphorylation assays. Receptor binding regions were determined by ELISA based competition assays and by utilizing human-mouse chimeric EphA2 molecules. Immunohistochemistry (IHC) was performed on frozen normal and tumor tissues. In vitro functional studies were performed with various cancer as well as transfected cell lines, and primary human T cells or PBMCs. In vivo activity was evaluated in xenograft models in immune deficient mice. Results: EphA2 mAbs encompassing diversity in binding kinetics and effects on receptor phosphorylation were classified in 5 discrete binding groups. The majority interacted with the N-terminal ligand-binding domain of EphA2 and most mAbs within that group interfered with ligand binding. The majority of mAbs displayed little IHC reactivity with normal tissue, while strong staining of cancer tissues was observed, including colon, lung, pancreas, ovary, bladder and breast cancers. Seven mAbs recognizing independent epitopes were selected for conversion into EphA2 x CD3 DART molecules that showed a range of potency in redirecting T cells to kill EphA2-expressing target cells. A lead EphA2 x CD3 DART molecule was selected based on potency and cross-reactivity with the cynomolgus monkey ortholog; this lead was engineered with a human Fc domain to confer an extended circulating half-life. The resulting Fc-bearing EphA2 x CD3 DART molecule demonstrated in vivo anti-tumor activity at doses as low as 20 mcg/kg in NOD/SCID/IL-2 gamma chain null mice co-implanted with activated human T cells and MDA-MB231 breast cancer cells. Conclusion: EphA2 is a potential cancer target for redirected T-cell killing applications independent of ligand mediated mechanisms. Further preclinical assessment of EphA2 x CD3 DART molecules as a strategy for targeting EphA2-expressing malignancies is warranted. Citation Format: Claudia B. Fieger, Kalpana Shah, Gurunadh Chichili, Jonathan Li, Thomas Son, Jeffrey Hooley, Francine Chen, Sergey Gorlatov, Steve Burke, Valentina Ciccarone, Ralph Alderson, Deryk Loo, Syd Johnson, Ezio Bonvini, Paul Moore. Evaluation of EphA2 as a therapeutic target for redirected T-cell killing by DART® bispecific molecules. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 583.