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Purpose: MET, the receptor for hepatocyte growth factor (HGF), has been implicated in driving tumor proliferation and metastasis. High MET expression is correlated with poor prognosis in multiple cancers. Activation of MET can be induced either by HGF-independent mechanisms such as gene amplification, specific genetic mutations, and transcriptional upregulation or by HGF-dependent autocrine or paracrine mechanisms. Experimental Design/Results: Here, we report on LY2875358, a novel humanized bivalent anti-MET antibody that has high neutralization and internalization activities, resulting in inhibition of both HGF-dependent and HGF-independent MET pathway activation and tumor growth. In contrast to other bivalent MET antibodies, LY2875358 exhibits no functional agonist activity and does not stimulate biologic activities such as cell proliferation, scattering, invasion, tubulogenesis, or apoptosis protection in various HGF-responsive cells and no evidence of inducing proliferation in vivo in a monkey toxicity study. LY2875358 blocks HGF binding to MET and HGF-induced MET phosphorylation and cell proliferation. In contrast to the humanized one-armed 5D5 anti-MET antibody, LY2875358 induces internalization and degradation of MET that inhibits cell proliferation and tumor growth in models where MET is constitutively activated. Moreover, LY2875358 has potent antitumor activity in both HGF-dependent and HGF-independent (MET-amplified) xenograft tumor models. Together, these findings indicate that the mechanism of action of LY2875358 is different from that of the one-armed MET antibody. Conclusions: LY2875358 may provide a promising therapeutic strategy for patients whose tumors are driven by both HGF-dependent and HGF-independent MET activation. LY2875358 is currently being investigated in multiple clinical studies. Clin Cancer Res; 20(23); 6059–70. ©2014 AACR.

Development of novel targeted therapies directed against hepatocyte growth factor (HGF) or its receptor (MET) necessitates the availability of quality diagnostics to facilitate their safe and effective use. Limitations of some commercially available anti-MET antibodies have prompted development of the highly sensitive and specific clone A2H2-3. Here we report its analytical properties when applied by an automated immunohistochemistry method.Excellent antibody specificity was demonstrated by immunoblot, ELISA, and IHC evaluation of characterised cell lines including NIH3T3 overexpressing the related kinase MST1R (RON). Sensitivity was confirmed by measurements of MET in cell lines or characterised tissues. IHC correlated well with FISH and quantitative RT-PCR assessments of MET (P 0.001). Good total agreement (89%) was observed with the anti-MET antibody clone SP44 using whole-tissue sections, but poor positive agreement (21-47%) was seen in tissue microarray cores. Multiple lots displayed appropriate reproducibility (R(2) 0.9). Prevalence of MET positivity by IHC was higher in non-squamous cell NSCLC, MET or EGFR amplified cases, and in tumours harbouring abnormalities in EGFR exon 19 or 21.The anti-MET antibody clone A2H2-3 displays excellent specificity and sensitivity. These properties make it suitable for clinical trial investigations and development as a potential companion diagnostic.

MET is involved in many mechanisms of cancer proliferation and metastasis. Inappropriate activation of MET can be induced by HGF-independent mechanisms such as gene amplification, specific genetic mutations, and transcriptional up-regulation, or by HGF-dependent autocrine or paracrine mechanisms. LY2875358 is a novel humanized bivalent MET antibody currently in phase I clinical testing (trial NCT01287546). LY2875358 has high neutralization and internalization activities against MET for inhibiting HGF-dependent and HGF-independent MET pathway activation and tumor growth. In HGF-dependent MET activation, LY2875358 blocks HGF binding to MET, HGF-induced MET phosphorylation and tumor growth both in cell culture and in mouse xenograft models, resembling activities of a humanized one-armed 5D5 MET antibody (monovalent antibody similar to Onartuzumab). In tumors with HGF-independent MET activation through MET gene amplification, LY2875358 induces internalization and degradation of MET, which results in decreased pMET and total MET, inhibition of cell proliferation and tumor growth in MKN45 and SNU5 gastric tumor lines and EBC-1 and H1993 NSCLC tumor lines. Moreover, LY2875358 enhances antitumor activity in combination with cisplatin or 5-FU in vitro and in vivo in MET amplified tumor cells. However, under the same ligand-independent conditions, the one-armed 5D5 antibody did not have anti-tumor activities. When HGF is added to tumor cells with high MET gene amplification, LY2875358 decreases cell proliferation, while the one-armed 5D5 antibody does not. In contrast to other bivalent MET antibodies, LY2875358 has no or otherwise negligible agonist activity and does not stimulate biological activities such as cell proliferation, scattering, invasion, tubulogenesis, apoptosis protection or angiogenesis in various HGF responsive cells. These findings indicate that LY2875358 has a different mechanism of action from the humanized one-armed 5D5 MET antibody. LY2875358 may be a promising therapy for treatment of patients whose tumors are driven by HGF-dependent and HGF-independent MET activation. Citation Format: Wei Zeng, Victoria Peek, Mark Wortinger, Jonathan Tetreault, Jinqi Xia, Jennifer Stephens, Kelly Credille, Darryl Ballard, Trish Brown-Augsburger, Jirong Lu, Chi-Kin Chow, Peter Vaillancourt, Ying Tang, Sau-Chi B. Yan, Ling Liu. LY2875358, a bivalent MET antibody with anti-tumor activity through blocking HGF as well as inducing degradation of MET, differentiates from a one-armed 5D5 MET antibody. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5465. doi:10.1158/1538-7445.AM2013-5465

cMet is a member of the receptor tyrosine kinase family and is the receptor for hepatocyte growth factor (HGF). cMet has been implicated in the initiation and progression of cancer due to the range of activities that cMet stimulates including proliferation, migration, morphogenesis, and survival. Inappropriate activation of c-Met can be induced by ligand-independent mechanisms such as gene amplification, specific genetic mutations, and transcriptional upregulation, or by ligand-dependent autocrine or paracrine mechanisms. Indeed, amplification of the c-Met gene, with consequent protein overexpression and constitutive kinase activation, has been reported in a number of human cancers, including gastric, esophageal and non-small-cell lung carcinomas. It has been reported that ∼10-20% of gastric tumors have increased copy numbers of the MET gene and overexpression of c-Met significantly correlates with poor prognosis in gastric cancer. c-Met antibody LY2875358 treatment reduces proliferation of gastric cancer cell lines with ligand-independent activation of c-Met resulting from gene amplification. The ability of LY2875358 to internalize and deplete cell surface c-Met is implicated in its activity against ligand-independent driven gastric cell lines. Here, we demonstrate that the pre-clinical combination of c-Met antibody LY2875358 with gastric cancer standard-of-care treatment has better efficacy than either treatment alone, both in vitro and in vivo. These data suggest that LY2875358 in combination with standard-of-care may be a promising treatment for gastric cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2738. doi:1538-7445.AM2012-2738

c-Met is a member of the receptor tyrosine kinase family and is the receptor for hepatocyte growth factor (HGF). c-Met is involved in many mechanisms of cancer proliferation and metastasis. Inappropriate activation of c-Met can be induced by ligand-independent mechanisms such as gene amplification, specific genetic mutations, and transcriptional up-regulation, or by ligand-dependent autocrine or paracrine mechanisms. Lung cancer is the leading cause of cancer death worldwide. Despite the successful development of EGFR- or EML4-ALK-targetd therapies, treatment options remain limited for patients with advanced lung cancer, making the identification of new therapeutic targets essential. c-Met expression was reported in 41-72% non-small cell lung cancer (NSCLC), amplification of c-Met occurs in 5-10 % of patients, and c-Met mutations have been detected in 8-13% of patients. We have developed a bi-valent c-Met antibody LY2875358 (LA480), which blocks ligand-dependent and ligand-independent c-Met activations. It is currently in clinical development. Here, we have demonstrated that LY2875358 alone or in combination with standard-of-care (SOC) affected cell proliferation, migration and signal transduction in NSCLC cells with c-Met gene amplification, mutations and overexpression. In vitro, LY2875358 induces wild type and mutant c-Met internalization and degradation. In vivo, LY2875358 alone shows a marked antitumor activity in Met amplification NSCLC xenograft models. The combination of LY2875358 with SOC chemotherapeutics treatments has better efficacy than either treatment alone, both in vitro and in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2734. doi:1538-7445.AM2012-2734

The receptor tyrosine kinase c-Met and its ligand HGF elicit multiple cellular activities including cell proliferation, motility, invasion, tubulogenesis, angiogenesis and anti-apoptosis. Dysregulation of this pathway has emerged as a crucial feature for many human tumors. However, past efforts in developing c-Met therapeutic antibodies that inhibit both ligand-dependent and ligand-independent c-Met activation have been largely unsuccessful due to agonist properties of the antibodies. In fact, c-Met antibodies exhibiting medium to strong agonist activity stimulate proliferation of both normal and tumor cells. We report here that LA480, a bivalent humanized monoclonal c-Met antibody, blocks HGF binding to c-Met and also induces c-Met internalization. Moreover, LA480 inhibits growth of xenograft tumors mediated by HGF-dependent and HGF-independent c-Met pathway activation. In order to evaluate whether LA480 has agonist activity, we tested LA480 in the following HGF-responsive biological assays: proliferation of primary human hepatocytes and tumor cell lines, scattering of DU145 cells, stimulation of HepG2 invasion and tubulogenesis, tube formation in endothelial and stromal cell co-cultures, and protection from apoptosis induced by staurospor in Caki-1 cells. A bivalent c-Met agonist antibody and HGF were included in the assays as positive controls, in addition to antibody isotype negative controls. Our results demonstrate that LA480 does not significantly stimulate cell proliferation, scattering, invasion, tubulogenesis, angiogenesis or apoptosis protection in the above assay systems. Under the same conditions, the agonist positive control c-Met antibody and HGF both significantly induced these biological effects. These unique properties of LA480 suggest that it may be a promising therapeutic reagent for treatment of cancers driven by ligand-dependent and ligand-independent c-Met activation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2583.

Gastric cancer is common in Asia, and advanced disease is typically incurable. Development of effective treatments for gastric cancer is thus a high priority to address this highly unmet clinical need. c-Met gene amplification resulting in c-Met overexpression and pathway activation has been highly linked to tumor progression and metastasis. It has been reported that approximately 10-20% of gastric tumors have increased copy numbers of the MET gene. c-Met- amplified gastric cancer cell lines that are dependent on an activated c-Met pathway for cell growth have been reported to be highly sensitive to growth inhibition by small molecule inhibitors of c-Met. We report that LA480, a humanized monoclonal antibody specific for c-Met, can directly inhibit HGF-independent in vitro cell proliferation of three c-Met- amplified Asian gastric cell lines (MKN45, SNU-5, and NUGC-4), in a dose-dependent manner. After 48 hours of treatment with LA480, 60-70% percent inhibition of 3H-thymidine incorporation was observed. For one of these gastric lines (NUGC-4), sensitivity to LA480 increases with continuous passage of the culture. This increased sensitivity to LA480 over time appears to be associated with increased expression of cell surface c-Met and increased constitutive phospho-Met expression. In addition to inhibiting proliferation of c-Met- amplified Asian gastric lines, LA480 also directly inhibited proliferation of a c-Met- amplified gastric cell line (Hs 746T) from a Caucasian patient. These data suggest that targeting c-Met amplification with LA480 may be a promising treatment for gastric cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2430.

c-Met is a receptor tyrosine kinase that binds hepatocyte growth factor (HGF) and has been implicated in human cancer. Inappropriate activation of c-Met can be induced by ligand-independent mechanisms such as gene amplification, specific genetic mutations, and transcriptional upregulation, or by ligand-dependent autocrine or paracrine mechanisms. Indeed, amplification of the c-Met gene, with consequent protein overexpression and constitutive kinase activation, has been reported in a number of human cancers, including gastric, esophageal and non-small-cell lung carcinomas. Activating mutations of the c-Met gene have been found in a subset of patients with hereditary and sporadic papillary renal cancer, lung cancer, childhood hepatocellular carcinoma and gastric carcinoma. Another common constitutive c-Met activation in human tumors is increased protein expression as a consequence of transcriptional upregulation, in the absence of gene amplification. c-Met expression level has been correlated with poor prognosis in multiple tumor types. In addition, cMet activation by an elevated level of HGF has also been described in gliobastoma, breast carcinomas, rhabdomyosarcoma and osteosarcoma. We report here that LA480, a humanized monoclonal c-Met antibody, inhibits HGF-dependent and HGF-independent c-Met pathway activation and tumor growth. Our data demonstrate that LA480 blocks binding of HGF to c-Met as shown by ELISA, blocks HGF stimulation of phospho-Met in HCT116 cells, and blocks HGF stimulation of primary human hepatocyte proliferation. Moreover, LA480 internalizes and depletes cell surface c-Met, significantly reduces phospho-c-Met and total c-Met in multiple tumor cell lines, and inhibits proliferation of tumor cell lines that have c-Met gene amplification. In addition, LA480 does not stimulate biological activities that are elicited by HGF. Furthermore, LA480 reduces total c-Met and phospho-Met in xenograft tumors and significantly inhibits tumor growth in both ligand-independent and ligand-dependent mouse xenograft models. These findings suggest that LA480 may be a promising therapy for treatment of cancers driven by ligand-dependent and ligand-independent c-Met activation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 695.