Kyung-ah Kim

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.

Microfabricated neural thermocouple arrays probe for brain research

A microfabricated neural thermocouple arrays probe (NTAP) is proposed for measuring and comparing the temperature of local brain area in real time sensing. Four junctions of T type thin film thermocouple arrays were fabricated on a sharp silicon probe tip. Each junction size was 20μm by 20 μm. The average seebeck coefficient was 15.12μV/ºC and the dynamic response time was 0.78sec. The temperature of both inside and outside the mouses thalamus was measured. The result showed that the temperature inside the mouses thalamus was about 4°C higher than that of the outside.

Abstract 661: Increasing sensitivity of SAIT301, a specific monoclonal antibody of c-Met, with paclitaxel combination in c-Met positive gastric cancer

The c-Met is frequently overexpressed and related to a worse prognosis in gastric carcinoma (GC). This study aimed to determine a c-Met targeting monoclonal antibody (SAIT301) with effects on the proliferation and to increase efficacy of SAIT301 administered in combination with paclitaxel in gastric cancer cell lines. Anticancer effect of SAIT301 was assessed using CCK-8 assay in 52 GC cell lines. c-Met status was identified using RTK array and qPCR. Cell cycle distribution was determined with flow cytometry. Expression levels of c-Met and cleaved PARP, apoptosis induction marker, were determined by western blot analysis. Combination index (CI) evaluated by CalcuSyn software. We screened 52 cell lines for growth inhibition by SAIT301.Among 11 c-Met positive GC cells, 5 cell lines were sensitive to SAIT301 (5/11, 45%) with 40∼60% inhibition rate at 5 μg/ml.SAIT301 sensitivity of 5 cell lines has a tendency of correlation between MET amplification, c-Met/p-Met co-expression, low Cbl level or exon14 deletion of MET. Next, we performed combination treatment with SAIT301 and paclitaxel in 5 cell lines. The results exhibited increasing anti-proliferative effects with synergism. In cell cycle analysis, SAIT301 increased the percentage of cells in the G1 phase, while paclitaxel increased the G2/M phase(3-19%). Combination of SAIT301 and paclitaxel significantly increased in sub G1(2-17%) and G2/M arrest(4-19%). Western blot analysis showed paclitaxel induced c-Met expression. In addition, SAIT301 and paclitaxel combination down-regulated c-Met expression and enhanced PARP cleavage more than monotherapy. Our results indicate that the SAIT301 combined with paclitaxel therapy had synergistic effects in the treatment of c-Met positive gastric cancer. Citation Format: Sun Kyoung Kang, Jeong Min Kim, Won Suk Lee, Woo Sun Kwon, Tae Soo Kim, Seon-hui Shim, Kyung-Ah Kim, Ho-Yeong Lim, Hyun Cheol Chung, Sun Young Rha. Increasing sensitivity of SAIT301, a specific monoclonal antibody of c-Met, with paclitaxel combination in c-Met positive gastric cancer. [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 661. doi:10.1158/1538-7445.AM2015-661

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 2483: Met degradation by SAIT301, a Met monoclonal antibody, reduces the invasion and migration of nasopharyngeal cancer cells via inhibition of EGR-1 expression

Nasopharyngeal carcinoma (NPC) is a common malignant tumor with high invasive and metastatic potential. The hepatocyte growth factor (HGF)-Met signaling pathway has a critical role in mediating the invasive growth of many different types of cancer, including head and neck squamous cell carcinoma. HGF also stimulates NPC cell growth and invasion in the cell line model. In this study, we determined the inhibitory effect of Met, using a Met-targeting monoclonal antibody (SAIT301), on the invasive and growth potential of NPC cell lines. Met inhibition by SAIT301 resulted in highly significant inhibition of cell migration and invasion in both the HONE1 and HNE1 cell lines. In addition, we also found that co-treatment of SAIT301 and HGF decreased the anchorage-independent growth induced by HGF in HNE1 cell lines. After SAIT301 treatment, Met, together with its downstream signaling proteins, showed downregulation of p-Met and p-ERK, but not p-AKT, in both HONE1 and HNE1 cell lines. Interestingly, we found that HGF treatment of NPC cell lines induced early growth response protein (EGR-1) expression, which is involved in cell migration and invasion. In addition, co-treatment with SAIT301 and HGF inhibited the HGF-induced expression of EGR-1. Next, knockdown of EGR-1 using small-interfering RNA inhibited HGF-induced cell invasion in NPC cell lines, suggesting that the expression level of EGR-1 is important in HGF-induced cell invasion of NPC cells. Therefore, the results support that SAIT301 inhibited Met activation as well as the downstream EGR-1 expression and could have therapeutic potential in NPC. Taken together, we suggest that Met is an anticancer therapeutic target for NPC that warrants further investigation and clinical trials and SAIT301 may be a promising tool for NPC therapy. Citation Format: Bok-Soon Lee, Sam Kang, Kyung-Ah Kim, Yun-Jeong Song, Kwang Ho Cheong, Hyun-Young Cha, Haeng-Jun Kim, Hye Sook Hwang, Yeon Soo Kim, Chul-Ho Kim. Met degradation by SAIT301, a Met monoclonal antibody, reduces the invasion and migration of nasopharyngeal cancer cells via inhibition of EGR-1 expression. [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 2483. doi:10.1158/1538-7445.AM2015-2483

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

Abstract 4895: A cancer stem cell instigator pathway revealed by transcriptomics, stem cell mutagenesis, and in-vivo tumor initiation.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC We have developed a multi-stage Cancer Stem Cell (CSC) model that recapitulates the natural process of carcinogenesis. Isogenic human embryonic stem cells (ESCs) exposed to non-biased mutagens were found to undergo rare spontaneous CSC transformation. Progenitor induced CSC (iCSC) clones exhibited classical CSC/SC marker expression, tumorsphere formation, resistance to common chemotherapeutic agents, and a capacity to form serially transplantable tumors in immunocompromised animals. A subset of progenitor clones formed tumors that took on a glioma-like morphology and produced more highly drug resistant mature CSCs that exhibited phenotypic characteristics of the primary patient glioma CSCs. Computational comparison to expression profiles of 313 human tumor lines confirmed that the mature CSCs had adopted a glioma-like expression profile. An integrated molecular model of the instigator pathway was assessed by comprehensive RNASeq transcriptomics, tandem MS proteomics, microRNA profiling, and mutagen-induced molecular polymorphisms. Progenitor iCSCs were found to have an ESC expression phenotype, significantly differing in the expression of only 32 genes, with some bearing direct evidence of mutagenesis in the expressed transcript. The instigator genes formed a proto-oncology seed pathway that was expanded by progressive expression changes to a more classical oncology profile in the glioma-like tumors and mature iCSCs derived in vivo from the progenitor iCSC. Classical oncology targets evident in the late-stage tumor model were absent in the progenitor iCSCs. However, computational repositioning analysis identified a limited set of existing molecules that could act directly to mitigate the progenitor pathway in the iCSCs. Citation Format: Ana Krtolica, Jacob Glanville, Gnanaratnam Giritharan, Eduardo Caceres, Erica Canino, Kyung-Ah Kim. A cancer stem cell instigator pathway revealed by transcriptomics, stem cell mutagenesis, and in-vivo tumor initiation. [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 4895. doi:10.1158/1538-7445.AM2013-4895

Abstract LB-386: Targeting of induced cancer stem cells with iniparib in combination with carboplatin and gemcitabine

BACKGROUND: Mounting evidence exists that human breast cancers include a stem-like cell population with self-renewing, tumorigenic properties. This population of cells, known as cancer stem cells (CSCs) or tumor-initiating cells, shares phenotypic characteristics with embryonic stem cells (ESCs), including activation of certain developmental pathways, and is resistant to many anticancer therapies. Distinguishing features of stem cells include active DNA repair and resistance to apoptosis, which enable their survival in the presence of conventional cytotoxic and cytostatic therapies that target actively proliferating cells. Identification of pharmaceuticals that preferentially target CSCs represents a novel strategy in modern drug discovery and development. We investigated whether treatment with iniparib (BSI-201) can potentiate the anti-proliferative effects of gemcitabine and carboplatin in CSCs. METHODS: A multi-stage stem cell carcinogenesis model that mirrors the natural progression of carcinogenesis was previously developed using in vitro and in vivo selection of mutated ESCs, which exhibit specific tumor-initiating and cancer stem cell-associated behaviors (SLL Sciences). These induced CSCs (iCSCs) were treated with iniparib (3.7, 11, 33, or 100 μM), gemcitabine (1.5 nM), carboplatin (10 μM), or gemcitabine + carboplatin for 72 hours, and effects on iCSC proliferation were evaluated using an ELISA-based bromodeoxyuridine (BrdU) assay and an ATP-based detection assay for measuring cellular metabolism. Effects of iniparib and/or gemcitabine on DNA damage were evaluated by immunofluorescence staining of iCSC using antibodies against γH2AX (a marker of double strand DNA breaks) at 24 hours after treatment. RESULTS: Treatment with iniparib alone resulted in a dose-dependent decrease in cellular proliferation and metabolism, with the IC50 established at 60 μM. Iniparib (60 μM), gemcitabine (1.5 nM), and carboplatin (10 μM) each induced >50% reduction in iCSC proliferation; addition of iniparib to gemcitabine resulted in an 80% reduction in BrdU incorporation compared with gemcitabine alone (P = 0.027). To assess iniparib-associated DNA damage, γH2AX foci were quantified in > 300 cells for each treatment combination. Cells treated with iniparib, gemcitabine, or iniparib + gemcitabine demonstrated 3.7-, 6.8-, and 8.8-fold increases in γH2AX foci, respectively, compared with untreated control (P CONCLUSIONS: These findings demonstrate that iniparib increases the effects of gemcitabine in terms of antiproliferative activity and DNA damage in this CSC model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-386. doi:10.1158/1538-7445.AM2011-LB-386