The New Antibody Met4 Used For Immunohistochemical Evaluation Of Breast Cancer, The Met Expression Level Of The Study Of Human Anti Met Genetically Engineered Antibody Scfv Transformation And Features

The Met RTK family is the only known high-affinity receptor for hepatocyte growth factor (HGF), also known as scatter factor (SF). Met is widely expressed in a variety of tissues。It is one of the most frequently genetically altered or otherwise deregulated receptor tyrosine kinases (RTK) in advanced human cancers.More and more researches have proved that Met is overexpressed in breast cancer cells. Clinical studies have provided the most direct evidence that Met is a powerful factor associated with the progression and prognosis of patients with breast cancer.Given the clinical importance of Met, evaluating the expression of Met will have strong clinical significance.Polyclonal antisera are commonly affected by lot-to-lot variability ,which is the limitation of available antibodies to measure Met expression in immunohistochemical.Therefore we developed a mouse monoclonal antibody, named Met4, for measurement of Met protein expression in formalin-fixed tissues.Purpose:Inappropriate expression of the receptor tyrosine kinase Met and its ligand is associated with an aggressive phenotype and the poor clinical prognosis in breast cancer, and it is important to develop the companion diagnostics for identifying those patients who could benefit from Met-targeted therapies. We have developed a diagnostic agent named Met4 that targets Met for the immunohistochemical assay in formalin-fixed and paraffin-embedded (FFPE) tissues.In this study, we want to evaluate the sensitivity and specificity of this anti-Met monoclonal antibody in human breast cancer tissue by immunohistochemistry.Methods:We recruited a homogeneous cohort of 70 patients from 2001 to 2006. The expressions of Met in these patients were analyzed using immunohistochemistry by Met4, compared with polyclonal antibody C28. The correlation of Met overexpression in invasive breast cancer with clinicopathological characteristics was evaluated.And Met expression in corresponding lymph node metastasis and adjacent normal cells were also detected in 17 cases.Results:Met4 gave strongly specific cytoplasmic and cytomembrane staining in breast cancer, and the results is consistent . Immunohistochemistry ,using C28, showed Met in the cytoplasm and nucleus rather than the previously reported predominantly membrane staining. Eleven cases (16%) tumors had high cytoplasmic Met expression, and19 cases (27%) had high membrane Met expression using Met4. No significant difference was found between C28 group and Met4 group in cytoplasm staining. Met overexpression was found in primary tumor tissue and corresponding lymph node metastasis. Furthermore, Met overexpression in the both sites was stronger than that in adjacent normal tissue.Conclusion:1)Met is overexpressed in breast cancer2)It is feasible for Met4 as an immunohistochemistry diagnostic agent in human breast cancer. Objective To generate an anti-Met single-chain antibody fragment(scFv) and human IgG Fc fusion protein to increase the solubility of scFv.Methods The human IgG Fc gene was amplified by RT-PCR and cloned into the expression vector pBAD-scFv which had been constructed previously. The anti-Met scFv-Fc expressing vector was transferred into E.coli.Top10 and fusion protein expression was induced by L-arabinose.The soluble protein was purified by His affinity chromatography and characterized by SDS-PAGE, Western blot. The scFv-Fc fusion protein specificity was confirmed by ELISA.Result DNA sequence result showed that the cloned scFv-Fc gene sequence was corrected which corresponded to the data of GenBank. SDS-PAGE and Western blot showed that the molecular weight of fusion protein was about 60 ku.The ELISA results confirmed the scFv-Fc fusion protein could bind to Met protein specifically.Conclusion The solubilty of scFv-Fc fusion protein was increased and stability which compared to the scFv fragment.