Abstract 2724: Development of a sensitive assay for measuring pharmacodynamic modulation of c-Met in biopsies
Authors: Bladt Friedhelm , Jaehrling Frank , Friese-Hamim Manja , Falchook Gerald S. , Amin Hesham M. , Klevesath Manfred B. , Blaukat Andree 
Published: May 2015

Abstract
Introduction: MSC2156119J, a highly selective, potent, reversible, ATP-competitive c-Met inhibitor currently under clinical testing, efficiently inhibits c-Met phosphorylation and downstream signaling in vivo and induces regression of established tumors in xenograft models in preclinical studies (Bladt et al. Clin Cancer Res. 2013;19:2941-51). One initial event following c-Met activation is Y1234/35 phosphorylation in the activation loop of the kinase domain. This results in kinase activation and triggers phosphorylation of tyrosine residues in the c-Met C-terminal tail (eg, Y1349), creating multifunctional docking sites for intracellular adapters. The development of an assay allowing the measurement of these phospho-c-Met epitopes in tumor biopsies will be crucial for establishing an optimal biologic dose for MSC2156119J. Methods: We describe here the development of a highly sensitive Luminex assay that can reliably and reproducibly measure the phosphorylation state of Y1234/35 and Y1349 residues of the c-Met receptor. The high sensitivity of the assay was confirmed by measuring the phospho-c-Met levels in biopsies from patients before and during MSC2156119J treatment. Results: We first assessed the stability of phosphorylated Y1234/35 and Y1349 epitopes in preclinical tumor samples. Analyses revealed that the total c-Met protein is rather stable over time, while the phospho-c-Met epitopes Y1234/35 and Y1349 are relatively unstable; their detection requires rapid processing of tumor samples derived from preclinical tumors or from patients. In tumor samples derived from human xenografts treated with MSC2156119J, Y1349 phosphorylation inhibition varied in different tumor models and did not show strong dose dependence. In contrast, phosphorylation of Y1234/35 residues was effectively inhibited by MSC2156119J in all tumor models tested. Based on these findings, phosphorylation of c-Met Y1234/35 was also assessed in tumor biopsies from patients treated with MSC2156119J in the first-in-man trial (NCT01014936; Falchook et al. J Clin Oncol. 2013;31(suppl):2506). When compared to pretreatment tumor biopsies, on-treatment samples showed effective, dose-dependent inhibition of &gt;90% of c-Met Y1234/35 phosphorylation. Conclusions: We successfully developed an assay capable of detecting phosphorylation of the c-Met Y1234/35 epitope, which is crucial for c-Met activation. Using this assay on tumor samples from xenograft models and on patient-derived tumor biopsies, we demonstrated that MSC2156119J inhibits phosphorylation of c-Met Y1234/35 in a dose-dependent fashion. Therefore, phosphorylation of c-Met Y1234/35 can be used as a pharmacodynamic biomarker of c-Met inhibition and will be an important and valuable element for the selection of the optimal biologic dose of MSC2156119J.
Citation Format: Friedhelm Bladt, Frank Jaehrling, Manja Friese-Hamim, Gerald S. Falchook, Hesham M. Amin, Manfred B. Klevesath, Andree Blaukat. Development of a sensitive assay for measuring pharmacodynamic modulation of c-Met in biopsies. [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 2724. doi:10.1158/1538-7445.AM2014-2724