Ji Min Lee

A new anti-c-met antibody selected by a mechanism-based dual-screening method: Therapeutic potential in cancer

Abstractc-Met, the high affinity receptor for hepatocyte growth factor (HGF), is one of the most frequently activated tyrosine kinases in many human cancers and a target for cancer therapy. However, inhibitory targeting of c-Met with antibodies has proven difficult, because most antibodies have intrinsic agonist activity. Therefore, the strategy for reducing the agonism is critical for successful development of cancer therapies based on anti-c-Met antibodies. Here we developed a mechanism-based assay method for rapid screening of anti-c-Met antibodies, involving the determination of Akt phosphorylation and c-Met degradation for agonism and efficacy, respectively. Using the method, we identified an antibody, F46, that binds to human c-Met with high affinity (Kd = 2.56 nM) and specificity, and induces the degradation of c-Met in multiple cancer cells (including MKN45, a gastric cancer cell line) with minimal activation of c-Met signaling. F46 induced c-Met internalization in both HGF-dependent and HGF-independent cells, suggesting that the degradation of c-Met results from antibody-mediated receptor internalization. Furthermore, F46 competed with HGF for binding to c-Met, resulting in the inhibition of both HGF-mediated invasion and angiogenesis. Consistently, F46 inhibited the proliferation of MKN45 cells, in which c-Met is constitutively activated in an HGF-independent manner. Xenograft analysis revealed that F46 markedly inhibits the growth of subcutaneously implanted gastric 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.

The Dual Inhibition of Met and EGFR by ME22S, a Novel Met/EGFR Bispecific Monoclonal Antibody, Suppresses the Proliferation and Invasion of Laryngeal Cancer

PurposeIt has been reported that the abnormal activation of receptor tyrosine kinases is associated with the development of many human carcinomas and the high activation of EGFR and Met mediates the tumorigenicity of laryngeal carcinoma. In this study, we have done the therapeutic efficacy of ME22S (a novel EGFR/Met bispecific antibody) in laryngeal carcinoma in vitro and in vivo was thoroughly evaluated.MethodsThe effects of ME22S on cell viability was assessed through MTT assays, and then Western blotting and immunocytochemistry were used to determine the expression of EGFR and Met. Also, wound healing and invasion assays were performed to observe the inhibitory effects of ME22S.ResultsWe found the ability of ME22S reducing the expression of both EGFR and Met and significantly inhibiting the cell migration, invasion, and proliferation of SNU899 and HN3 in vitro. Also, the notably reduced levels of p-Met, p-ERK, and p-AKT were found when the cells were treated with only ME22S alone or with HGF together. Meanwhile, ME22S, interestingly enough, caused caspase-3-dependent apoptotic cell death when HN3 cells were treated with ME22S for 72xa0h, decreased the HGF-induced Slug expression, and also inhibited the tumor growth of HN3 cells in a xenograft model in vivo.ConclusionsTaken together, our findings suggest that the dual inhibition of EGFR and Met through ME22S largely suppresses the invasion and growth of laryngeal carcinoma both in vitro and in vivo, hence, can be a practical approach as a novel therapeutic strategy for the treatment of laryngeal carcinoma.

Abstract 662: USP8 modulates ubiquitination of LRIG1 for Met degradation

The Met receptor tyrosine kinase is an attractive target for cancer therapy as it promotes invasive tumor growth. SAIT301 is a novel anti-Met antibody, which induces LRIG1-mediated Met degradation and inhibits tumor growth. However, detailed downstream mechanism by which LRIG1 mediates target protein down-regulation is unknown. In the present study, we discovered that SAIT301 induces ubiquitination of LRIG1, which in turn promotes recruitment of Met and LRIG1 complex to the lysosome through its interaction with Hrs, resulting in concomitant degradation of both LRIG1 and Met. We also identified USP8 as a LRIG1-specific deubiquitinating enzyme, reporting the interaction between USP8 and LRIG1 for the first time. SAIT301 triggers degradation of LRIG1 by inhibiting the interaction of LRIG1 and USP8, which regulates ubiquitin modification and stability of LRIG1. In summary, SAIT301, a Met targeting antibody, employs ubiquitination of LRIG1 for its highly effective Met degradation. This unique feature of SAIT301 enables it to function as a fully antagonistic antibody without Met activation. We found that USP8 is involved in deubiquitination of LRIG1, influencing the efficiency of Met degradation. The relation of Met, LRIG1 and USP8 strongly supports the potential clinical benefit of a combination treatment of a USP8 inhibitor and a Met inhibitor, such as SAIT301. Citation Format: Ji Min Lee, Bogyou Kim, Kyung-Ah Kim. USP8 modulates ubiquitination of LRIG1 for Met degradation. [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 662. doi:10.1158/1538-7445.AM2015-662

Abstract 1036: Cbl-independent degradation of Met: Ways to avoid agonism of bivalent Met targeting antibody.

The Met receptor tyrosine kinase, found to be constitutively activated in many tumors, has become a leading target for cancer therapy. Disruptions in Met down-regulation have been associated with aggressive tumor progression with several therapeutic strategies addressing this aspect of Met biology. Cbl E3 ligase-mediated degradation, which attenuates Met signaling via ligand-dependent Met internalization, is a major negative regulator of Met expression. It is believed that one of the mechanisms by which the therapeutic anti-Met antibodies induce cancer cell death in Met over-expressing tumors is via internalization and subsequent degradation of Met from the cell surface. However, a previously reported Met targeting antibody demonstrated intrinsic agonistic activity while capable of inducing Cbl-mediated degradation of Met, suggesting that Cbl-mediated degradation requires receptor activation and impedes therapeutic application. We have developed a potent and selective bivalent Met targeting antibody (SAIT301) that invokes Met degradation using an alternative regulator LRIG1. In this report, we demonstrate that LRIG1 mediates degradation of Met by SAIT301 and this degradation does not require Met activation. Furthermore, SAIT301 was able to down-regulate Met and dramatically inhibit growth of tumors with low or no Cbl expression, as well as tumors with Met exon 14 deletion that prevents Met binding to Cbl. In summary, we demonstrate the enhanced therapeutic potential of a novel tumor-inhibiting anti-Met antibody, SAIT301, which utilizes a Cbl-independent, LRIG1-mediated Met degradation pathway and thereby avoids the agonism that limits the effectiveness of previously reported anti-Met antibodies. Citation Format: Ji Min Lee, Bogyou Kim, Kyung-Ah Kim. Cbl-independent degradation of Met: Ways to avoid agonism of bivalent Met targeting 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 1036. doi:10.1158/1538-7445.AM2013-1036