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1.
Mol Cancer Ther ; 16(12): 2780-2791, 2017 Dec.
Article in English | MEDLINE | ID: mdl-28802255

ABSTRACT

Increased MET activity is linked with poor prognosis and outcome in several human cancers currently lacking targeted therapies. Here, we report on the characterization of Sym015, an antibody mixture composed of two humanized IgG1 antibodies against nonoverlapping epitopes of MET. Sym015 was selected by high-throughput screening searching for antibody mixtures with superior growth-inhibitory activity against MET-dependent cell lines. Synergistic inhibitory activity of the antibodies comprising Sym015 was observed in several cancer cell lines harboring amplified MET locus and was confirmed in vivo Sym015 was found to exert its activity via multiple mechanisms. It disrupted interaction of MET with the HGF ligand and prompted activity-independent internalization and degradation of the receptor. In addition, Sym015 induced high levels of CDC and ADCC in vitro The importance of these effector functions was confirmed in vivo using an Fc-effector function-attenuated version of Sym015. The enhanced effect of the two antibodies in Sym015 on both MET degradation and CDC and ADCC is predicted to render Sym015 superior to single antibodies targeting MET. Our results demonstrate strong potential for use of Sym015 as a therapeutic antibody mixture for treatment of MET-driven tumors. Sym015 is currently being tested in a phase I dose escalation clinical trial (NCT02648724). Mol Cancer Ther; 16(12); 2780-91. ©2017 AACR.


Subject(s)
Epitopes/genetics , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Animals , Cell Line, Tumor , Cell Proliferation , Disease Models, Animal , Humans , Mice , Mice, Nude , Xenograft Model Antitumor Assays
2.
PLoS One ; 11(6): e0157850, 2016.
Article in English | MEDLINE | ID: mdl-27359329

ABSTRACT

OBJECTIVE: Metabolic dysfunctions, such as fatty liver, obesity and insulin resistance, are among the most common contemporary diseases worldwide, and their prevalence is continuously rising. Mimp/Mtch2 is a mitochondrial carrier protein homologue, which localizes to the mitochondria and induces mitochondrial depolarization. Mimp/Mtch2 single-nucleotide polymorphism is associated with obesity in humans and its loss in mice muscle protects from obesity. Our aim was to study the effects of Mimp/Mtch2 overexpression in vivo. METHODS: Transgenic mice overexpressing Mimp/Mtch2-GFP were characterized and monitored for lipid accumulation, weight and blood glucose levels. Transgenic mice liver and kidneys were used for gene expression analysis. RESULTS: Mimp/Mtch2-GFP transgenic mice express high levels of fatty acid synthase and of ß-oxidation genes and develop fatty livers and kidneys. Moreover, high-fat diet-fed Mimp/Mtch2 mice exhibit high blood glucose levels. Our results also show that Mimp/Mtch2 is involved in lipid accumulation and uptake in cells and perhaps in human obesity. CONCLUSIONS: Mimp/Mtch2 alters lipid metabolism and may play a role in the onset of obesity and development of insulin resistance.


Subject(s)
Blood Glucose/metabolism , Gene Expression Profiling/methods , Kidney/pathology , Liver/pathology , Mitochondrial Membrane Transport Proteins/genetics , Obesity/genetics , Oligonucleotide Array Sequence Analysis/methods , Animals , Diet, High-Fat , Fatty Acid Synthases/genetics , Gene Expression Regulation , Kidney/metabolism , Lipid Metabolism , Liver/metabolism , Membrane Potential, Mitochondrial , Mice , Mice, Transgenic , Mitochondrial Membrane Transport Proteins/metabolism
3.
Proc Natl Acad Sci U S A ; 110(32): E2987-96, 2013 Aug 06.
Article in English | MEDLINE | ID: mdl-23882082

ABSTRACT

Binding of hepatocyte growth factor (HGF) to the receptor tyrosine kinase MET is implicated in the malignant process of multiple cancers, making disruption of this interaction a promising therapeutic strategy. However, targeting MET with bivalent antibodies can mimic HGF agonism via receptor dimerization. To address this limitation, we have developed onartuzumab, an Escherichia coli-derived, humanized, and affinity-matured monovalent monoclonal antibody against MET, generated using the knob-into-hole technology that enables the antibody to engage the receptor in a one-to-one fashion. Onartuzumab potently inhibits HGF binding and receptor phosphorylation and signaling and has antibody-like pharmacokinetics and antitumor activity. Biochemical data and a crystal structure of a ternary complex of onartuzumab antigen-binding fragment bound to a MET extracellular domain fragment, consisting of the MET Sema domain fused to the adjacent Plexins, Semaphorins, Integrins domain (MET Sema-PSI), and the HGF ß-chain demonstrate that onartuzumab acts specifically by blocking HGF α-chain (but not ß-chain) binding to MET. These data suggest a likely binding site of the HGF α-chain on MET, which when dimerized leads to MET signaling. Onartuzumab, therefore, represents the founding member of a class of therapeutic monovalent antibodies that overcomes limitations of antibody bivalency for targets impacted by antibody crosslinking.


Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Antibodies, Monoclonal/pharmacology , Immunoglobulin Fab Fragments/pharmacology , Neoplasms/drug therapy , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Xenograft Model Antitumor Assays , Amino Acid Sequence , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/metabolism , Antibodies, Monoclonal, Humanized/chemistry , Antibodies, Monoclonal, Humanized/genetics , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Crystallography, X-Ray , Drug Design , Hepatocyte Growth Factor/chemistry , Hepatocyte Growth Factor/metabolism , Hepatocyte Growth Factor/pharmacology , Humans , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Nude , Mice, SCID , Mice, Transgenic , Models, Molecular , Molecular Sequence Data , Neoplasms/pathology , Protein Binding/drug effects , Protein Structure, Tertiary , Proto-Oncogene Proteins c-met/chemistry , Proto-Oncogene Proteins c-met/metabolism , Sequence Homology, Amino Acid
4.
Mol Cancer Ther ; 12(8): 1429-41, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23720767

ABSTRACT

The MET and EGFR receptor tyrosine kinases (RTK) are often coexpressed and may cross-talk in driving the development and progression of non-small cell lung carcinoma (NSCLC). In addition, MET amplification is an alternative resistance mechanism for escaping EGFR-targeted therapy. To assess the benefits of combined targeting of MET and EGFR for treating NSCLCs, we investigated the activities of these two RTK pathways in NSCLC cell lines and evaluated their responses to SGX523 and erlotinib, the small-molecule kinase inhibitors of MET and EGFR, respectively. We showed that MET interacts with and cross-activates EGFR in MET-amplified or -overexpressed cells. The inhibition of both MET and EGFR results in maximal suppression of downstream signaling and of cell proliferation when their ligands are present. Furthermore, we showed that SGX523 plus erlotinib strengthens anticancer activity in vivo in a cellular context-dependent manner. The combination led to the regression of H1993 tumors by enhancing the suppression of proliferation and inducing apoptosis, whereas H1373 tumor growth was significantly reduced by the combination via suppression of proliferation without inducing apoptosis. SGX523 alone was sufficient to achieve near-complete regression of EBC-1 tumors; its combination with erlotinib strongly inhibited the viability of a population of insensitive cells emerging from an SGX523-treated EBC-1 tumor recurrence. Our data suggest that inhibition of both MET and EGFR can enhance anticancer effects against NSCLCs in a context-dependent manner and thus provide a strong rationale for combining MET and EGFR inhibitors in treating NSCLCs.


Subject(s)
Antineoplastic Agents/pharmacology , ErbB Receptors/antagonists & inhibitors , Lung Neoplasms/metabolism , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Small Cell Lung Carcinoma/metabolism , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Models, Animal , Drug Synergism , ErbB Receptors/metabolism , Erlotinib Hydrochloride , Humans , Lung Neoplasms/drug therapy , Mice , Molecular Targeted Therapy , Proto-Oncogene Proteins c-met/metabolism , Pyridazines/pharmacology , Quinazolines/pharmacology , Signal Transduction/drug effects , Small Cell Lung Carcinoma/drug therapy , Triazoles/pharmacology , Xenograft Model Antitumor Assays
5.
Proc Natl Acad Sci U S A ; 109(2): 570-5, 2012 Jan 10.
Article in English | MEDLINE | ID: mdl-22203985

ABSTRACT

Because oncogene MET and EGF receptor (EGFR) inhibitors are in clinical development against several types of cancer, including glioblastoma, it is important to identify predictive markers that indicate patient subgroups suitable for such therapies. We investigated in vivo glioblastoma models characterized by hepatocyte growth factor (HGF) autocrine or paracrine activation, or by MET or EGFR amplification, for their susceptibility to MET inhibitors. HGF autocrine expression correlated with high phospho-MET levels in HGF autocrine cell lines, and these lines showed high sensitivity to MET inhibition in vivo. An HGF paracrine environment may enhance glioblastoma growth in vivo but did not indicate sensitivity to MET inhibition. EGFRvIII amplification predicted sensitivity to EGFR inhibition, but in the same tumor, increased copies of MET from gains of chromosome 7 did not result in increased MET activity and did not predict sensitivity to MET inhibitors. Thus, HGF autocrine glioblastoma bears an activated MET signaling pathway that may predict sensitivity to MET inhibitors. Moreover, serum HGF levels may serve as a biomarker for the presence of autocrine tumors and their responsiveness to MET therapeutics.


Subject(s)
Autocrine Communication/physiology , Biomarkers/metabolism , ErbB Receptors/metabolism , Glioblastoma/metabolism , Hepatocyte Growth Factor/metabolism , Proto-Oncogene Proteins c-met/metabolism , Biomarkers/blood , Blotting, Western , Cell Line, Tumor , Cluster Analysis , Comparative Genomic Hybridization , DNA Primers/genetics , Enzyme-Linked Immunosorbent Assay , Gene Expression Regulation, Neoplastic/drug effects , Glioblastoma/blood , Hepatocyte Growth Factor/blood , Humans , In Situ Hybridization, Fluorescence , Microarray Analysis , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Pyridazines/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Triazoles/pharmacology
6.
Cancer Res ; 70(17): 6880-90, 2010 Sep 01.
Article in English | MEDLINE | ID: mdl-20643778

ABSTRACT

The hepatocyte growth factor (HGF)-MET pathway supports several hallmark cancer traits, and it is frequently activated in a broad spectrum of human cancers (http://www.vai.org/met/). With the development of many cancer drugs targeting this pathway, there is a need for relevant in vivo model systems for preclinical evaluation of drug efficacy. Here, we show that production of the human HGF ligand in transgenic severe combined immunodeficient mice (hHGF(tg)-SCID mice) enhances the growth of many MET-expressing human carcinoma xenografts, including those derived from lung, breast, kidney, colon, stomach, and pancreas. In this model, the MET-specific small-molecule kinase inhibitor SGX523 partially inhibits the HGF-dependent growth of lung, breast, and pancreatic tumors. However, much greater growth suppression is achieved by combinatorial inhibition with the epidermal growth factor receptor (EGFR) kinase inhibitor erlotinib. Together, these results validate the hHGF(tg)-SCID mouse model for in vivo determination of MET sensitivity to drug inhibition. Our findings also indicate that simultaneously targeting the MET and EGFR pathways can provide synergistic inhibitory effects for the treatment of cancers in which both pathways are activated.


Subject(s)
Carcinoma/drug therapy , ErbB Receptors/antagonists & inhibitors , Hepatocyte Growth Factor/metabolism , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Pyridazines/pharmacology , Quinazolines/pharmacology , Receptors, Growth Factor/antagonists & inhibitors , Triazoles/pharmacology , Animals , Carcinoma/metabolism , Carcinoma/pathology , Cell Cycle/drug effects , Cell Growth Processes/drug effects , Drug Synergism , ErbB Receptors/metabolism , Erlotinib Hydrochloride , Female , Humans , Mice , Mice, Inbred C3H , Mice, SCID , Protein Kinase Inhibitors/pharmacology , Xenograft Model Antitumor Assays
7.
Neoplasia ; 8(5): 344-52, 2006 May.
Article in English | MEDLINE | ID: mdl-16790083

ABSTRACT

Molecular functional and metabolic imaging allows visualization of disease-causing processes in living organisms. Here we present a new approach for the functional molecular imaging (FMI) of endogenous tyrosine kinase receptor activity using Met and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), as a model. HGF/SF and Met play significant roles in the biology and pathogenesis of a wide variety of cancers and, therefore, may serve as potential targets for cancer prognosis and therapy. We have previously shown that Met activation by HGF/SF increases oxygen consumption in vitro and results in substantial alteration of blood oxygenation levels in vivo, as measured by blood oxygenation level-dependent magnetic resonance imaging. Using contrast medium (CM) ultrasound imaging, we demonstrate here that HGF/SF induces an increase in tumor blood volume. This increase is evident in small vessels, including vessels that were not detected before HGF/SF treatment. The specificity of the effect was validated by its inhibition using anti-HGF/SF antibodies. This change in tumor hemodynamics, induced by HGF/SF and measured by CM ultrasound, is further used as a tool for Met FMI in tumors. This novel noninvasive molecular imaging technique may be applied for the in vivo diagnosis, prognosis, and therapy of Met-expressing tumors.


Subject(s)
Diagnostic Imaging/methods , Hepatocyte Growth Factor/metabolism , Neoplasms/pathology , Neovascularization, Pathologic , Proto-Oncogene Proteins c-met/metabolism , Animals , Cell Line, Tumor , Contrast Media/pharmacology , Female , Humans , Mice , Mice, Nude , Neoplasm Transplantation , Neoplasms/blood supply , Oxygen/metabolism , Oxygen Consumption , Prognosis
8.
Neoplasia ; 8(5): 353-63, 2006 May.
Article in English | MEDLINE | ID: mdl-16790084

ABSTRACT

The tyrosine kinase receptor Met and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), play an important role in normal developmental processes, as well as in tumorigenicity and metastasis. We constructed a green fluorescent protein (GFP) Met chimeric molecule that functions similarly to the wild-type Met receptor and generated GFP-Met transgenic mice. These mice ubiquitously expressed GFP-Met in specific epithelial and endothelial cells and displayed enhanced GFP-Met fluorescence in sebaceous glands. Thirty-two percent of males spontaneously developed adenomas, adenocarcinomas, and angiosarcomas in their lower abdominal sebaceous glands. Approximately 70% of adenocarcinoma tumors metastasized to the kidneys, lungs, or liver. Quantitative subcellular-resolution intravital imaging revealed very high levels of GFP-Met in tumor lesions and in single isolated cells surrounding them, relative to normal sebaceous glands. These single cells preceded the formation of local and distal metastases. Higher GFP-Met levels correlated with earlier tumor onset and aggressiveness, further demonstrating the role of Met-HGF/SF signaling in cellular transformation and acquisition of invasive and metastatic phenotypes. Our novel mouse model and high-resolution intravital molecular imaging create a powerful tool that enables direct real-time molecular imaging of receptor expression and localization during primary events of tumorigenicity and metastasis at single-cell resolution.


Subject(s)
Diagnostic Imaging/methods , Gene Transfer Techniques , Green Fluorescent Proteins/metabolism , Microscopy, Fluorescence/methods , Proto-Oncogene Proteins c-met/genetics , Transgenes , Animals , Diagnostic Imaging/instrumentation , Disease Progression , Female , Green Fluorescent Proteins/genetics , Male , Mice , Mice, Transgenic , Microscopy, Confocal , Neoplasm Metastasis , Phenotype
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