Modification Of HBV Specific Engineering Antibody And Evaluation Of Its Therapeutic Effect In Animals

HBV infection is still a serious public health problem for the whole world.In 2015,there were about 257 million CHB infected people in the world,accounting for 3.6%of the total population.Every year,887,000 people died of liver cirrhosis and liver cancer caused by HBV infection,the most important of which was the death caused by CHB-related liver diseases.The ideal therapeutic endpoint for CHB patients is to realize HBsAg seroconversion(HBsAg loss).However,due to the high titer of HBsAg,chronic HBV infected patients are often in immune tolerance state,and treatment based on existing drugs(Interferon or Nucleoside/Nucleotide analogues)is difficult to achieve the ideal therapeutic endpoint.Therefore,it is urgent and necessary to develop innovative therapeutic drugs and methods for chronic HBV infected patients.As an extremely effective means,antibodies have been widely used in the treatment of tumors,autoimmune diseases and inflammation.Therefore,more and more scientists are turning their attention to the research of anti-infection antibodies,hoping to effectively prevent and treat infectious diseases.Antibodies for Chronic Virus Infection,such as HIV,suggest that antibody-mediated immunotherapy is expected to become a new strategy for CHB patients to resist HBV continuous infection.However,with the deepening of research,many defects of natural antibodies,such as limited binding times with antigen and antigen accumulation effect,have surfaced.Therefore,the research focus is no longer limited to the screening of natural antibodies,but to improve the efficacy of antibody drugs through modification.The purpose of this study is to systematically evaluate the therapeutic potential of the existing sweeping antibody,complete the conceptual verification of the therapeutic sweeping antibody,and reform the therapeutic humanized antibody of HBV in order to obtain the sweeping antibody with clinical therapeutic potential.This study is divided into two parts.In the first part,a modified antibody 73-DY obtained in the early stage of the laboratory is taken as a model molecule,and its function is systematically evaluated through a series of experiments such as affinity determination,ADCP phagocytosis,transgenic animal treatment,AAV/HBV mouse long-term treatment,etc.The experimental results show that the modification of sweeping antibody does not affect the size of antigen-antibody complex.The affinity between sweeping antibody and FcyRIIb receptor was greatly increased,and the phagocytosis efficiency mediated by antibody was significantly improved.The sweeping antibody has outstanding therapeutic effect in HBV-Tg mice and AAV/HBV mouse models,which not only effectively reduces the expression of HBsAg in mouse serum and liver,breaks through the limitation of traditional antibodies,and realizes low-dose and low-frequency treatment,but also,more importantly,after long-term treatment,it can activate the humoral immune response in mice to generate Anti-HBs.So far,we have completed the systematic evaluation of 73-DY and the conceptual verification of HBV therapeutic sweeping antibody.The second part of this study is to modify HBV humanized antibody 162 with broad-spectrum binding activity,and to verify the properties of the obtained sweeping antibody and evaluate the therapeutic effect.The histidine mutation site of 162 was optimized through new three-dimensional structure simulation and comprehensive analysis of mutation law of 162 CDR region,and a pH-dependent antigen binding phage antibody library was constructed.After multiple rounds of screening,detection and analysis,6 candidate molecules were obtained at the phage level.They were expressed as IgG and evaluated in vitro activities such as pH-dependent effect detection,electron microscopy analysis of antigen-antibody complexes,and broad-spectrum binding activity detection.Finally,B179 candidate molecules were determined for subsequent sweeping antibody construction.On the basis of B179,the degree of humanization was improved,and B179A and B179C were obtained.Four antibodies including the parent antibody 162 were modified in Fc region,and the obtained four molecules were preliminarily identified in nature,among which 162-DY and B179-DY molecules were finally selected for subsequent therapeutic effect evaluation.The therapeutic effect of HBV-Tg mice showed that DY modification enhanced antigen clearance ability,and pH-dependent antigen binding prolonged antigen clearance time.To sum up,through systematic evaluation of the modified antibody 73-DY,this study has completed the conceptual verification of HBV therapeutic sweeping antibody and modified HBV humanized antibody with broad-spectrum binding activity.The finally obtained sweeping antibody B179-DY can effectively remove high titer HBsAg in the circulatory system,inhibit the HBsAg level from rising for a long time,reduce the dosage of antibody drugs,and is expected to reduce the administration frequency and improve the drug effect..