| Infectious disease refer to that invasion of human body by pathogenic microorganisms,such as bacteria,viruse,etc.causing the organism to take on a series of diseases with infectious clinical symptom,which seriously threaten the life and health of mankind.In recent years,the research on anti-infective antibody drugs has been developing rapidly.Currently,25 anti-infective antibody drugs are under clinical study.Among them,the humanized monoclonal antibody Palivizumab against respiratory syncytial virus(RSV)was approved by FDA for the prevention of respiratory syncytial virus infection,anti-human immunodeficiency virus(HIV)antibodies in clinical research,anti-hepatitis C virus(HCV),anti-human macrophage virus(CMV)antibodies in research,and so on.HBV infection can lead to chronic diseases such as hepatitis B,liver cirrhosis and hepatocellular carcinoma(HCC),the world each year about 600000 people died of acute or chronic hepatitis B infection.Although the successful development of a preventive hepatitis B vaccines have effectively reduced new cases of HBV infection globally,there are still 250 million people who are persistently infected with HBV,some of them need effective treatment to prevent the complications of the disease.Approved anti-HBV drugs,whether interferon or nucleoside analogues,can only alleviate the disease but do not effectively clear the virus.The ideal end point of anti-hbv therapy is the loss of HBsAg.However,treatment regimens based on existing drugs rarely result in clear HBsAg.Therefore,more effective new drugs need to be developed to improve clinical management of HBV-related diseases.Although the development of full-human antibody technology greatly increased the number of full-human antibodies as clinical candidate strains,most of the therapeutic antibodies approved by FDA are still mainly humanized antibodies.Humanization of antibody mainly includes the CDR grafting,surface reshaping,phage display and other antibody library technologies,as well as computer-aided design.However,existing technologies have the problems of reducing affinity of derived antibodies,having great immunogenicity,heavy workload,unvisibility of screening,and inconsistency in the activity of conversion into complete antibodies,which have prolonged the time of humanization of antibodies,so it is imperative to develop new and rapid humanization methods.The purpose of this study was to establish a new method of rapid point mutation and humanization,and to achieve the rapid acquisition of humanized antibodies with high degree of humanization and the maintenance of parental antibody activity.Using this method,HBV mouse antibody E6F6 was modified to obtain humanized antibody with clinical therapeutic potential.This research was divided into two parts,the first part was the establishment of rapid point mutation humanized platform.Through the comparison and analysis of the CDR partition method and the selection method of humanized template,the CDR region was defined by combining Kabat and Contact,and the germLine genes with the highest homology was selected as the template.By analyzing a large number of available or preclinical antibody sequences and structures and summarizing some previous humanization work experience,the position and function of each amino acid in antibody sequences can be found with certain regularity.Summary rules include:(1)some amino acids in FR exist on the CDR contact surface,directly or indirectly participate in the binding of antibodies to antigens or play an important role in maintaining the conformation of CDR of antibodies.(2)some FR residues exist at the interface of VH-VK,and they are usually retained during mutation.(3)some of the FR residues belong to the embedded amino acids in the antibody sequences,which are selectively retained when mutated.(4)the last remaining FR residues are amino acids that have little effect on antibody activity and can be mutated directly.Whether belong to which kind of function of FR residues,its position is relatively fixed.We start with a Kabat number for its light and heavy chain variable area,and then classify the CDR area according to the CDR region defined by Kabat and Contact.12G6 was a broadly murine monoclonal antibody that can efficiently neutralize influenza B virus(Flu-B).Then,the IMGT website was used to search and compare,and 2-3 human germLine genes with the highest homology were found,and the sequence was compared.Finally,according to the amino acid rule summarized above,the differential amino acids between in FR genes and human germLine genes were mutated selectively.Four heavy chains and four light chains were designed,and any two of them were paired with each other for eukaryotic expression.The expression,binding activity and neutralization activity of sixteen humanized antibodies were detected with parental mouse antibodies as control.Finally,for 12G6,we selected six superior humanized antibodies.No matter in the binding,neutralization and HI experiments of HA proteins of influenza B virus in 2 different subspecies,six human antibodies retained the biological activity of 12G6.5B11 and 7G5 are mouse-derived monoclonal antibodies that can efficiently neutralize A/B respiratory syncytial virus(RSV)simultaneously.According to the experimental results of 12G6,a humanized scheme was designed for 5B11 and 7G5 respectively to obtain the humanized antibody of N-5B11 and N-7G5.The two humanified antibodies both showed good reactive activity and neutralization activity,and the activity was similar to parental.So far,the humanization of mouse antibodies of 12G6,5B11 and 7G5 have been preliminarily completed,the feasibility of the design scheme has been confirmed,and the construction of the humanization platform of rapid point mutation has been completed.The second part of this study was to use the new humanized platform established in the first part to humanize HBV mouse antibody E6F6,and to verify and evaluate the feasibility as a drug of its humanized antibody.E6F6 is a mouse monoclonal antibody against a specific epitope of HBV outer membrane protein(HBsAg).It was iscovered in our lab.It can effectively and persistently clean HBV and HBsAg in transgenic mice and has that potential to develop into the therapeutic antibody of HBV.In the previous study,we used phage display technology to humanize and mature E6F6.The humanized degree of E6F6 was 89%and the activity of humanized antibody 162 in vitro and in vivo was comparable to that of E6F6.This research use BIAcore T200 affinity measurement for 162,and its affinity was 4.18 x10-10 m.Through the crystal culture and structure analysis of 162 Fab,the three-dimensional structure of 162 crystals was obtained.162 was docked with HBsAg short epitope to obtain important amino acids of antigen-antibody interaction.In order to verify the feasibility of the first part of the established humanization platform and improve the humanized degree of 162,in the second part of this research the humanization platform of rapid point mutation established in the first part,162 amino acid sequence,162 docking data with HBsAg short peptide and Discovery Studio simulation platform was used to humanize E6F6,and three humanized antibodies,huAb1,huAb2 and huAb3,were obtained.Transient expression and purification in CHO-S cells and were analyzed comprehensively from the aspects of homogeneity of antibodies,reaction with antigens,neutralization activity,virus clearance effect in vivo and drug formation evaluation.The humanized degree of huAbl,huAb2 and huAb3 were up to about 97%,in vitro binding and neutralization activity were comparable to 162,but the ability of persistently clear HBsAg was reduced in HBV transgenic mice.In C56b1/6 mice,huAb1 with 97%humanized degree was found to have the fastest clearance rate.However,the in vitro binding,,neutralization and in vivo activity of reverse chimeric antibodies of huAb 1,huAb 2 and huAb 3 were similar to that of E6F6.The solubility of 162,huAb1,huAb2 and huAb3 were above 140 mg/mL,the viscosity were less than 14,and the isoelectric point were between 8 and 9,which meet the standard of antibody drugs.The TM value were above 80 ?,which proved that they have good thermal stability.They also had good stability under simulated conditions of human body environment and drug storage environment.Pharmacokinetic experiments in cynomolgus monkeys were conducted to evaluate the half-life of 162,huAb1,huAb2 and huAb3,all of which were consistent and effective in clearing HBsAg in cynomolgus monkeys.It was found unexpectedly that the half-life of huAb3 was longer,which was related to the surface charge distribution.Pharmacokinetic parameters and blood routine and biochemical results of cynomolgus monkey suggest that 162,huAb1,huAb2 and huAb3 have good efficacy and safety.Further research has revealed that 162,huAb1,huAb2 and huAb3 and huAb3 and huAb3 have formed smaller immune complexes with HBsAg,which was conducive to efficient phagocytosis.By mutating in the Fc region of human IgGl,a 162-IgGl-KD mutant was obtained with the same in vitro activity as 162 but significantly enhanced phagocytic activity.The effect of the distance between the arms of the antibody Fab on phagocytosis was first discovered.The introduction of flexible linke(Gly4Ser)3 at the end of the variable region of 162 heavy chain could increase the distance between the arms of Fab and reduce the phagocytic function of the antibody.In conclusion,the rapid point mutation humanization method was established,and the three mouse antibodies against different targets were used to complete the comprehensive verification,which greatly shortened the time of transformation,and laid a solid foundation for the humanization of mouse antibodies,which is expected to develop into a new method of humanization of antibodies.It has enriched the humanification techniques,and has provided a new option for antibody humanization.The human antibody molecules 162,huAb1,huAb2 and huAb3,which have patent protection for the treatment of HBV infection-related diseases,have been obtained,and a relatively comprehensive evaluation of in vivo and in vitro activity and drug analysis have been completed,to improve the humanization platform.It lays a firm foundation for the research and development of humanized antibody for the treatment of corresponding diseases and is expected to become a new drug for the treatment of chronic hepatitis B. |
PDF Full Text Request