Research In Novel Antibody For Efficient And Sustained Clearance Of Hepatitis B Virus

Monoclonal antibodies have become the main type of antibody drug because of their high specificity and strong affinity to antigen.However,with the intensive study of the natural monoclonal antibody,many defects have faced,such as the limit times of binding to antigen,the unanticipated antibody clearance and antigen accumulation.Therefore,studies are no longer limited to the natural antibody screening,but rather to improve the efficiency of antibody drugs by engineering.In recent years,it is hot to study in maxim the binding times of antibody and antigen,prolong the half-life of antibody.In this study,we try to gain a sweeping antibody that can prolong the half-life in vivo on two anti-HBV antibodies by variable region(pH-dependent antigen binding)engineering and Fc region engineering.The main research includes the following four parts:Firstly,the construction,selection and screening of pH dependent antigen binding antibody library.24 or 26 histidine mutation sites were considered by the comprehensive analysis results of structure simulation and reasonable phage storage in this rearch.After four or three rounds of amplification and screening of the amino acid combinatorial mutagenesis phage library that constructed based on phage display,5 variants were obtained.All of them maintain the high antigen binding activity comparable to their parents in pH 7.4,while show weak binding affinity to antigen in pH 6.0.Secondly,expression,purification and function evaluation of variant.After the construction of eukaryotic express vector,5 variants were purificated,property identificated and function evaluated by FreeStyleTM 293F cell.The binding affinity to antigen of these antibody were similar to parent antibody in neutral pH,but significantly weaker than that of parent antibody in acid pH.HBV transgenic mice were used to evaluate the effect of variant in vivo,results show that the pH-dependent binding antibody has 50%longer antigen restrain time while maintain the antigen eliminate ability similar to that of the parent.Thirdly,the construction of sweeping antibody and function identification based on cell.1)Induce M252Y?N286E and N434Y mutation to Fc region of two pH-dependent antigen binding variant,who perform excellent on HBV clearance,for enhance the affinity with hFcRn in neutral pH.The MDCK cell lines that stable express hFcRn fused with EGFP were used for the evaluation of cell phagocytosis in vitro,the results indicated the sweeping antibody with V4 mutation can enhance the phagocytosis of HBsAg by MDCK cell notably.2)Induce K326D and L328Y mutation to Fc region of one pH-dependent antigen binding variant,for enhance the affinity with mF cyR II in neutral pH.The THP-1 cell and mouse macrophage were used for the evaluation of cell phagocytosis in vitro,the results indicated the sweeping antibody with DY mutation can enhance the phagocytosis of HBsAg by the used cell notably compare to parent.Fourthly,the effect evaluation of sweeping antibody in vivo.The hFcRn transgenic mice infected with HBV adeno-associated virus and HBV transgenic mice were used.The results shown the sweeping antibody with V4 mutation maintained the antigen eliminate equivalent to that of parents,shown 100%extension of antigen restrain time.The sweeping antibody with DY mutation shown greater antigen eliminate than parent over an order of magnitude and has the 12 d prolonged antigen restrain time.In conclusion,the sweeping antibody obtained in this study has a significantly prolonged antigen restrain time and greater antigen clearance efficiency than that of parent,have potentiality to develop into the second-generation therapeutic antibody of HBV,it has a far-reaching influence on the therapeutic of hepatitis B virus.