Generation And Characterization Of Cynomolgus Macaque Monoclonal Antibodies Targeting Hepatitis B Surface Antigen By Memory B Cell Culture With Stimulation In Vitro

Hepatitis B virus(HBV)infection remains one of the main global public health issues.At present,there are still about 240 million people with HBV infection globally,being cause of about 600 thousand annual deaths from liver cirrhosis,liver cancer and other HBV-associated diseases.However,current antiviral treatments including interferon therapy and nucleoside analogues hardly eradicate HBV.In 2015,reseachers found a novel monoclonal antibody(mAb)E6F6,recognizing a conserved linear SA epitope(GPCKTCT,aa118-125)on HBsAg,exhibited striking the most therapeutic effects.Futhermore,a novel epitope-based vaccine was deleveloped,designed based on this sequence.In this study,we aimed at investigating the antibody responses and functionality of antibodies following immunization of this vaccine.Due to the relative similarity in immune systems,similar adaptive responses elicited by a vaccine in humans and NHPs.Therefore,NHPs,especially Macaca fascicularis,are widely used for vaccine evaluation studies.Firstly,we aimed to develop a platform to generate cynomolgus macaque monoclonal antibodies using memory B cell culture with stimulation in vitro and generate anti-HBsAg specific antibodies.Memory B cells were enriched by the combination of EasySepTM Human PanB Cell Enrichment Kit and IgM&IgD Negative Selection Cocktail kit.Then,we defined the robust system for memory B cell culture,that is memory B cells together with CD40L feeder cells were seeded in a 96-well cell culture in RPMI-1640 with 10%FBS and hIL21(50ng/mL).The density of memory B cells is 5-10 cells/well and the feeder cells are 10,000 cells/well.The validation of PCR primers for the amplification and cloning of the genes encoding antibody fragments was done.By using these platforms,we successfully generated 10 anti-HBsAg specific antibodies from 28 positive B cell culture wells.Secondly,we aimed to investiga.te and characterize the functionality of these antibodies.We found that antibody 1-12H4,1-23,3-13,3-23H1 and 3-23H4 have excellent HBsAg binding activity and we used antibody E6F6 which recognizes an SA epitope to compete with our monkey antibodies against HBsAg,the result showed that antibody 1-12H4,1-23,3-13,3-23H1 and 3-23H4 recognized the same epitope as E6F6.Furthermore,serum HBsAg and HBV DNA were significantly lowered in antibodies-treated HBV transgenic mice,especially in antibody 1-23 and 3-23H1 treated mice.Thirdly,a further degree of humanization was achieved by matuting the different monkey amino acid residues back into human ones in the franmework regions of these two antibodies.Then,the function of these new humanized antibodies were characterized by comparing with their parent antibodies and hu-E6F6(162).Finally,MID(parental antibody 1-23;the degree of humanness is 98.33%)and M3D(parental antibody 3-23H1;the degree of humanness is 97.78%)were chosen as candidate molecules.MID and M3D also showed a broad-spectrum activity of anti three main genotypes of HBV,including genotype A,genotype B and genotype C.Finally,we aimed to investigate the residues for MID and M3D interacting with HBsAg.We then identified the key CDR region and key amino acids contributing to the interaction in the antibodies,and the core epitopes recongnized by these two antibodies were identified as well.In conclusion,this study preliminarily explored the antibody response induced by the SA linear epitope based therapeutic vaccine in the cynomolgus monkeys.We obtained two humanlized antibodies exhibited striking therapeutic effects in HBV transgenic mice.MID and M3D recognized the same epitope as E6F6 and their core epitopes overlapped with that of E6F6,suggesting that antibodies recognizes the SA linear epitopes could mediate efficient HBV virus clearance in vivo.The results also provided important information for study the efficacy evaluation of this vaccine.It can provide candidate molecules for hepatitis B therapeutic antibody drugs development as well.In addition,the research of NHP antibody provides a method for the development of human-like antibody with excellent performance.