No 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

Purpose: Aberrant c-Met expression has been implicated in most types of human cancer.We are developing Met-directed imaging and therapeutic agents. Experimental Design: To seek peptides that bind specifically to receptor Met, the Metexpressing cell lines S114 and SK-LMS-1were used for biopanning with a random peptide phage display library. Competition ELISA, fluorescence-activated cell sorting analysis, an internalization assay, and a cell proliferation assay were used to characterize a Met-binding peptide in vitro. To evaluate the utility of the peptide as a diagnostic agent in vivo, 125

Inappropriate expression of the receptor tyrosine kinase Met and its ligand hepatocyte growth factor (HGF)/scatter factor (SF) is usually associated with an aggressive solid tumor phenotype (angiogenesis, invasiveness, and metastasis) and poor clinical prognosis. We report here the design and construction of a large, human naïve antigen-binding fragment (Fab) phage-display library with a diversity of 2.0 x 109, which allows rapid isolation of antigen-specific human antibody fragments. A Fab fragment specifically against Met (designated hFab-Met-1) was successively selected from this library by using biopanning on Met-transfected cell line S114. The specificity of hFab-Met-1 was characterized by immunoprecipitation, Western blotting, and flow cytometry. The results demonstrate that hFab-Met-1 reacts with the extracellular domain of Met in its native conformation. Moreover, functional analysis by Madine-Darby canine kidney cell scattering and urokinase-type plasminogen activator assays demonstrated that hFab-Met-1 is not an agonist to HGF/Met signaling compared with a murine intact monoclonal antibody (MAb) Met5. To confirm that hFab-Met-1 interacts with Met-expressing tumors in vivo, I-125-labeled hFab-Met-1 was nuclear-imaged in a mouse xenograft of Met- and HGF/SF-expressing human leiomyosarcoma. Total body scintigrams were obtained between 1 and 48 h postinjection (PI). Tumor-associated activity was imaged as early as 1 h PI, and remained visible in some animals as late as 24 h PI. As expected, activity was highest in the kidneys in early images, whereas thyroid activity became predominant in later images. In conclusion, hFab-Met-1 interacts with Met both in vitro and in vivo, and is a promising candidate for clinical diagnosis and therapeutics.

Purpose: Met, an oncogene product and receptor tyrosine kinase, is a keystone molecule for malignant progression in solid human tumors. We are developing Met-directed imaging and therapeutic agents, including anti-Met monoclonal antibodies (MetSeek TM ). In this study, we compared two antibodies, Met5 and Met3, for nuclear imaging of human and canine Metexpressing tumor xenografts in nude mice. Experimental Design: Xenografts representing cancers of three different human tissue origins and metastatic canine prostate cancer were raised s.c. in host athymic nude mice. Animals were injected i.v. with I-125-Met5 or I-125-Met3, posterior total body gamma camera images were acquired for several days postinjection, and quantitative region-of-interest activity analysis was done. Results: PC-3, SK-LMS-1/HGF, and CNE-2 xenografts imaged with I-125-Met5 were compared with PC-3, SK-LMS-1/HGF, and DU145 xenografts imaged with I-125-Met3. Nuclear imaging contrast was qualitatively similar for I-125-Met5 and I-125-Met3 in PC-3 and SK-LMS-1/HGF host mice. However, by region-of-interest analysis, the set of human tumors imaged with I-125-Met3 exhibited a pattern of rapid initial tumor uptake followed by a continuous decline in activity, whereas the set of human tumors imaged with I-125-Met5 showed slow initial uptake, peak tumorassociated activity at 1 day postinjection, and persistence of activity in xenografts for at least 5 days. GN4 canine prostate cancer xenografts were readily imaged with I-125-Met5. Conclusions: We conclude that radioiodinated Met3 and Met5 offer qualitatively similar nuclear images in xenograft-bearing mice, but quantitative considerations indicate that Met5 might be more useful for radioimmunotherapy. Moreover, canine prostate cancer seems to be a suitable model for second-stage preclinical evaluation of Met5.Met, a receptor tyrosine kinase and oncogene product, is a keystone molecule for the initiation and malignant progression of solid neoplasms in humans and in experimental animal models of cancer (1). The myriad sequelae of Met activation, either following interaction with its native ligand hepatocyte growth factor/scatter factor (HGF/SF), with surrogate ligands (2, 3), or through ligand-independent pathways, include cell proliferation, migration, differentiation, and survival. In normal cells, these events are responsible for such phenomena as the epithelial-to-mesenchymal transformation responsible for limb muscle and diaphragm formation, and the formation of ductular glands. In neoplastic cells, these events are recapitulated as tumor formation, invasion, metastasis, and the prevention of apoptosis. Indeed, the plasticity involved in the initial seeding and subsequent growth of tumor metastases-which requires switching cells from an invasive to a proliferative state-can be accounted for in some experimental systems by ligand-dependent activation of Met (4).Virtually any type of solid tumor can express Met and/or its ligand abnormally (by overabundance, mutation, or dysregulation; see...