Abstract 1690: Mechanistic characterization of MM-131, a bispecific antibody that blocks c-Met signaling through concurrent targeting of EpCAM

Abstract

Abstract Purpose: MM-131 is a purely antagonistic, bispecific antibody that potently inhibits HGF/c-Met signaling by co-targeting the widely expressed tumor antigen EpCAM. The purpose of these studies is to uncover the mechanism by which MM-131 exhibits potent inhibition of both HGF-dependent and HGF-independent c-Met signaling in EpCAM positive tumor cells. Methods: To assess the role of EpCAM in mediating avid binding of MM-131 to c-Met, we quantified the cell surface levels of c-Met and EpCAM in a panel of cancer cell lines using flow cytometry and determined how potently MM-131 inhibits HGF-dependent c-Met signaling in each cell line. Using these data, we built a computational model to explain and quantify the effect of EpCAM targeting. We then tested this model by (1) predicting the activity of MM-131 in other cell lines, based on their EpCAM:c-Met ratios; (2) knocking down EpCAM in cell lines by RNA interference; and (3) comparing MM-131 to a variant of MM-131 in which its EpCAM-targeting arm was mutated to impair binding. To uncover the mechanism by which MM-131 inhibits HGF-independent c-Met signaling, we monitored the effect of MM-131 on c-Met levels, using quantitative imaging and immunoblotting. Results: Consistent with its design, we found that MM-131 is more potent at inhibiting HGF-dependent c-Met signaling, cell viability, and cell migration in EpCAM-high cells than in EpCAM-low cells. In addition, MM-131 potency is reduced when EpCAM levels are knocked down by RNA interference. By mutating the EpCAM-targeting arm of MM-131, the observed potency in EpCAM-high cells is noticeably reduced. Further cell biological characterization of MM-131 revealed two distinct mechanisms of action: (1) MM-131 blocks ligand binding to c-Met; and (2) MM-131 induces downregulation of c-Met. In side-by-side comparison studies, MM-131 was found to be more potent at inhibiting HGF-dependent signaling, cell migration, and cell viability than one-armed-5D5 (MetMab), and uniquely effective at inhibiting HGF-independent signaling through downregulation of c-Met. Consistent with the design criteria, MM-131 did not exhibit any discernable agonistic activity characteristic of bivalent c-Met antibodies such as LY2875358. The molecular effects of MM-131 observed in vitro were also observed in vivo: MM-131 inhibited tumor growth in models of ligand-dependent and ligand-independent c-Met signaling and induced downregulation of c-Met. Conclusions: MM-131 is a bispecific antibody designed to overcome HGF-dependent and HGF-independent c-Met pathway activation by concurrently targeting EpCAM. MM-131 shows no agonistic activity in preclinical models and exhibits two principal mechanisms of action: it antagonizes HGF binding and it induces downregulation of c-Met. These findings support the clinical development of MM-131 in c-Met-driven epithelial tumors that also express EpCAM. Citation Format: Adnan O. Abu-Yousif, Jessica B. Casaletto, Kristina Masson, Aaron Fulgham, Melissa Geddie, Birgit Schoeberl, Ulrik Nielsen, Gavin MacBeath. Mechanistic characterization of MM-131, a bispecific antibody that blocks c-Met signaling through concurrent targeting of EpCAM. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1690. doi:10.1158/1538-7445.AM2015-1690