Study On Identification And Anti-tumor Activities Of Recombinant Humanized Anti-HGF Monoclonal Antibody

Hepatocyte growth factor(HGF)is a glycoprotein secreted by interstitial cells that elicits a broad spectrum of biological responses.It can specifically bind to the C-Met receptor on the cell surface,induce the C-Met conformation to change,activate its tyrosine kinase(PTK)activity,and undergo a series of signal amplification to activate a variety of downstream signal pathway.This signal pathway not only participates in regulating the cell proliferation and survival,but also correlates with the migration,invasion and other behavior.Though meanwhile the signal pathway is also subject to C-Met endocytosis,degradation,secretion of paracrine and other mechanisms of strict regulation.Despite the existence of multiple regulatory mechanisms,a variety of tumor cells occure imbalance phenomenon about this signal pathway.The continued activation of this signaling pathway leads to rapid proliferation of tumor cells and avoidment of apoptosis.The continuous activation of the signaling pathway leads to rapid proliferation of tumor cells and avoid apoptosis,promote tumor cell invasion and migration,and can promote tumor angiogenesis.So the role of drug blocking the signal pathway will become one of the effective means of anti-tumor therapy.At present,there are no monoclonal antibody drugs listed at home and abroad.In order to develop therapeutic drugs with potential for clinical application,the related research work has been carried out,including the following three parts.For the first part,we identified the properties of two monoclonal antibodies.The concentration of HGF was detected by UV-vis,and the molecular weight and purity of the antibody were identified by SDS-PAGE and SEC-HPLC.The binding abilities of antibodies with HGF were measured by indirect Elisa,which was equally used to evaluate the inhibition of binding of HGF with its receptor by developed antibodies.The results showed that the two antibodies had a high concentration,the molecular weight of the antibody was consistent with the theoretical value,and had high purity,almost no impurities and multimers.Two antibodies have a strong ability to bind to HGF,and can inhibit the binding of C-Met receptor to HGF,suggesting that it has a strong neutralizing activity.For the second part,the anti-tumor activity of two monoclonal antibodies was studied in vitro and in vivo.The binding abilities of antibodies with HGF were measured by indirect Elisa,which was equally used to evaluate the inhibition of binding of HGF with its receptor by developed antibodies.The regulatory effect of antibodies on U87 MG cells proliferation was further tested by MTT.The regulatory effects of antibodies on PC-3 cell migration were tested by transwell assay.The in vivo antitumor effects of antibodies were evaluated by using established xenograft animal model of U87 MG.In vitro experiments showed that both of them had strong ability to inhibit cell proliferation.No significant difference in anti-tumor activity was observed for humanized and non-humanized antibodies.They had the ability to inhibit the promoting effect on PC-3 cell migration by HGF.In vivo pharmacological results showed that the two antibodies can significantly inhibit the growth of tumor tissue,and HE19-12 antibody showed a stronger anti-tumor activity,tumor growth inhibition rate of 80%.For the third one,we first established the method of Elisa to detect the concentration of HGF antibody in mouse serum,and the method was validated by the method.The pharmacokinetic parameters of the two antibodies were detected by using the established method to detect the antibody concentration of serum samples at different time points.The pharmacokinetic properties of the two antibodies were evaluated.The results show that we have successfully established the Elisa method to detect the concentration of HGF antibody,which has good sensitivity,stability and anti-interference ability.The results showed that HE19-12 antibody had longer in vivo half-life and higher drug exposure compared with HD19-27 antibody.This result is consistent with the experimental results in mice.Two humanized antibodies were identified to investigate their anti-tumor activities.Our studies suggest that antibodies targeting HGF/C-Met pathway demonstrate good therapeutic potential against glioma with optimal pharmacokinetics characteristics.