A Novel Multipurpose Monoclonal Antibody for Evaluating Human c-Met Expression in Preclinical and Clinical Settings

Abstract

The inappropriate expression of the c-MET cell surface receptor in many human solid tumors necessitates the development of companion diagnostics to identify those patients who could benefit from c-MET targeted therapies. Tumor tissues are formalin-fixed and paraffin embedded (FFPE) for histopathological evaluation, making the development of an antibody against c-MET that accurately and reproducibly detects the protein in FFPE samples an urgent need. We have developed a monoclonal antibody, designated MET4, from a panel of MET-avid monoclonal antibodies, based on its specific staining pattern in FFPE preparations of normal human prostate tissues. The accuracy of MET4 immunohistochemistry (MET4-IHC) was assessed by comparing MET4-IHC in FFPE cell pellets with immunoblotting analysis. The technical reproducibility of MET4-IHC possessed a percentage coefficient of variability (%CV) of 6.25% in intra-assay and inter-assay testing.Comparison with other commercial c-MET antibody detection reagents demonstrated equal specificity and increased sensitivity for c-MET detection in prostate tissues. In two cohorts of ovarian cancers and gliomas, MET4 reacted with ovarian cancers of all histological subtypes (strong staining in 25%) and with 63% of gliomas. In addition, MET4 bound c-Met on the surfaces of cultured human cancer cells and tumor xenografts. In summary, the MET4 monoclonal antibody accurately and reproducibly measures c-MET expression by IHC in FFPE tissues and can be used for molecular imaging in-vivo. These properties encourage further development of MET4 as a multipurpose

In vitro indeterminate teleost myogenesis appears to be dependent on Pax3

“…Primary myoblasts adhere to the culture substratum and therefore are not suspended in the culture medium. Previous c-Met localization demonstrates a halo-like or punctuated expression patterns surrounding the nuclei in suspended cells (Knudsen et al 2009), suggesting an important potential feature of relative association of c-Met and the nucleus. The relative lack of cytoplasm in these cells may lead to a potential in vitro artifact in primary myoblasts that appears as nuclear staining, when in fact the staining is more likely membranous with close proximity to the nucleus.…”

Pharmacodynamic Response of the MET/HGF Receptor to Small-Molecule Tyrosine Kinase Inhibitors Examined with Validated, Fit-for-Clinic Immunoassays

“…The pY1235-specific antibody could be a key reagent in a new diagnostic test for selecting the right patients to receive MET-targeted therapies because it substitutes the phosphorylated MET kinase domain (48) in place of elevated MET, mRNA, gene copy number, colocalized proteins, or multiply phosphorylated epitopes (49,50). However, unlike the validated immunoassay, cross-reactivity to MET fragments with undefined biologic significance (27) will confound the reported immunohistochemical tests of MET signaling (26,37,(51)(52)(53).…”

“…Sample load was 25 to 50 mg protein per gel lane. The numbers "2" and "3" indicate the specimen numbers described in Supplementary Table 2. MET expression (36)(37)(38)(39), but these assessments were made without a validated pharmacodynamic biomarker for activated MET or knowledge of the effect of warm ischemia on full-length MET degradation. Therefore, the trials to date unfortunately represent missed opportunities to understand clinical pharmacodynamic responses elicited by current MET inhibitors that could guide their future clinical development (40).…”

Novel Therapeutic Inhibitors of the c-Met Signaling Pathway in Cancer

“…Recently, however, a monoclonal antibody, MET4, that is directed against the extracellular domain of human MET has been described. MET4 has been shown to accurately and reproducibly detect c-MET in FFPE tissues, and the MET4 reagent displays high sensitivity with low background (42).…”