Immun. c-Met is constitutively activated in an HGF-independent manner. Xenograft analysis revealed that F46 markedly inhibits the growth of subcutaneously implanted gastric Cobimetinib (racemate) and lung tumors. These results indicate that F46, identified by a novel mechanism-based assay, induces c-Met degradation with minimal agonism, implicating a potential role of F46 in therapy of human cancers. Keywords: Akt, anti-c-Met antibody, cancer therapy, c-Met, HGF INTRODUCTION Receptor tyrosine kinases (RTKs) are often dysregulated in human cancer cells associated with genetic alternations, such as mutations, translocations, and amplifications (Cragg et al., 1999; Ferrara et al., 2004; Gschwind et al., 2004; Li et al., 2005). The oncogene and its transforming activity were first identified in a human osteosarcoma cell line. The c-protooncogene product, c-Met tyrosine kinase, is the high affinity receptor for scatter factor/hepatocyte growth factor (SF/HGF), which regulates a wide array of biological activities, including cell proliferation, survival, motility, and differentiation. c-Met is mainly expressed in cells of epithelial origin, while the c-Met ligand (HGF) is secreted by mesenchymal cells. Activation of c-Met coordinates cell proliferation, survival, motility, and differentiation processes, resulting in a complex biological process known as invasive growth (Trusolino and Comoglio, 2002). c-Met activation is essential for the survival of hepatocytes and placental trophoblast cells (Birchmeier and Gherardi, 1998) during normal embryonic development. In adults, the HGF/c-Met pathway is latent, being reactivated under numerous physiological and pathological conditions, such as wound healing (Chmielowiec et al., 2007) and tissue regeneration (Borowiak et al., 2004; Huh et al., 2004). In the case of cancer, however, the pathway is Cobimetinib (racemate) abnormally activated (Birchmeier et al., 2003; Boccaccio and Comoglio 2006; Bussolino et al., 1992; Comoglio and Trusolino 2002). In fact, c-Met is one of the most frequently activated tyrosine kinases in human cancers. Moreover, it is over-expressed in a variety of epithelial RH-II/GuB tumors, and generates signals that trigger several steps critical to oncogenesis, including the epithelial-to-mesenchymal transition, extracellular matrix degradation, tumor invasion, and metastasis (Birchmeier et al., 2003; Comoglio and Trusolino, 2002; Trusolino and Comoglio, 2002). In addition, c-Met has been shown to be tightly Cobimetinib (racemate) associated with the resistance to cancer therapies by targeting the EGFR and VEGF pathways (Bussolino et al., 1992). Reflecting the critical roles Cobimetinib (racemate) in cancer, c-Met and its HGF ligand have become leading candidates for targeted cancer therapies (Burgess et al., 2006; Cao et al., 2001; Jo et al., 2011; Kim et al., 2006). However, generation and development of inhibitory antibodies targeting c-Met has been difficult because the divalent structure of antibodies often activates c-Met signaling receptor dimerization and cross-activation (Ohashi et al., 2000; Prat et al., 1998). Therefore, it is necessary to develop a strategy for generation of therapeutic anti-c-Met antibodies that can efficiently block c-Met action (e.g., by preventing HGF binding to c-Met) without activation of its downstream signals. Many human cancers are featured by gene amplifications or activating mutations where c-Met becomes aberrantly activated and over-expressed. Therefore, therapeutic antibodies should preferentially induce c-Met degradation for its removal from the tumor cell surface and consequent down-regulation. To this end, we devised a novel mechanism-based screening method for selecting anti-c-Met antibodies that can down-regulate c-Met without inducing Akt.