The Study Of A Broad-spectrum Nanobody For Chronic Hepatitis B Infection Therapy

Hepatitis B virus(HBV)is a highly infectious liver-specific virus,with more than 250 million chronic hepatitis B(CHB)carriers and more than 0.9 million deaths worldwide.However,the functional cure of CHB patients using approved anti-HBV drugs remains elusive,which poses great pressure on both patients and public health systems.The successful clinical application of monoclonal antibodys(mAbs)has aroused extensive research on mAbs to prevent and treat CHB.However,the reported HBV therapeutic antibodies often have some problems,such as high dosage or frequency of treatment,poor clearance of HBV surface antigen(HBsAg)with high initial titer,quick rebound of HBsAg and ineffective response to HBV mutations.The purpose of this study is to obtain HBV therapeutic nanobodies with broad-spectrum and long-lasting viral suppression activity,which can help CHB infected organisms overcome immune tolerance,and provide novel candidates for clinical treatment of CHB.In the first part of this study,39 anti-HBsAg nanobodies with abundant amino acid sequence diversity were obtained through the nanobody immunolibrary.8 nanobodies of them were selected and fused with human Fc(hFc),then 8 heavy chain antibodies(HcAbs)were obtained through eukaryotic expression system.According to the competitive binding relationship,these 8 HcAbs identified 6 different HBsAg conformation epitopes and 6 of them was selected as representative HcAbs.These 6 HcAbs showed good thermal stability,5 of them could form small and dispersed immune complexes with HBsAg,and 2 HcAbs(containing 125s-hFc)showed neutralizing activity.These 6 HcAbs showed broad-spectrum binding activity to 9 genotypes(A-H,J)of HBsAg particles and broad-spectrum therapeutic activity in four HBV genotypes(A-D)carrying mouse models.125S-hFc stood out from these HcAbs by its efficient HBsAg clearance and persistent HBsAg inhibition.In the single injection regimen of the mouse model,125s-mFc showed better therapeutic effect than 125s-hFc due to the better match between mouse Fc(mFc)and mouse Fc receptor.In chronic HBV/AAV carrier mouse model,125s-mFc-based long-term passive immunotherapy could continuously control of viremia and induce cellular immune responses.In the second part of this study,in order to further optimize the therapeutic activity of 125s and improve its transformation value for clinical application,we carried out antibody engineering modification in two aspects based on the parent HcAb(125s-hFc).The first aspect is humanization of 125 s to reduce immunogenicity and improve safety.One humanized HcAb hu125s-1-S40G with 94.56%humanization degree and unchanged HBsAg binding activity was obtained by CDR transplantation and restoration mutation technique.The second aspect is to carry out site-directed mutation modification on the Fc part of 125s-hFc to improve the Fc effector function.We obtained 125s-YTE and 125s-DY variants respectively by construction,expression and purification.125s-YTE and 125s-DY maintained comparable HBsAg binding activity to that of parent 125s-hFc in vitro.Next,we evaluated the cytophagocytosis activity,antibody half-life and therapeutic activity of hul25s-1-S40G,125s-yte and 125s-DY,respectively.The results showed that the therapeutic activity of hu125s-1-S40G was inferior to that of its parent 125s-hFc.The metabolic kinetics of 125s-YTE were consistent with that of parent 125s-hFc not achieving prolonging HcAb half-life,and the therapeutic effect of 125s-YTE was significantly inferior to that of parent 125s—hFc.We were excited to find that 125s-DY showed strong HBsAg clearance(Serum HBsAg was lower than the detection limit)and remarkable long-lasting HBsAg suppression effects(Within 15 days after treatment,serum HBsAg remained below the detection limit)in FcRn/HBV double transgenic mouse model that received a single administration.Subsequently,we further explored the epitope of 125s on HBsAg.We characterized a highly conserved epitope(named Cter)at the C-terminus of the HBsAg surface motif from amino acids 157 to 174.Cter differs from the PreS or Major hydrophilic region(MHR)epitopes that have been recognized by reported therapeutic antibodies for HB V.In summary,we found a HBV therapeutic nanobody 125 s with great potential for clinical application.125s based HcAb has neutralizing activity and show broadspectrum therapeutic activity in vivo(HBV genotype A-D).Even at high initial HBsAg titers(>1E4 IU/mL),125s-mFc showed significant long-term HBsAg inhibition in mouse models.In addition,our study also revealed that 125s-based long-term passive immunotherapy could induce cellular immune responses.These results suggested that 125s-based antibody has potential to achieve seroconversion and clearance of HBsAg in CHB infection.We successfully obtained 125 s-DY through antibody engineering which showed strong therapeutic activity in FcRn/HBV mice.In addition,we identified a novel and conserved epitope of 125s on HBsAg.This study advances our understanding of antibody-based immunotherapy strategies,provide a nanobody candidate with clinical transformation value for the immunotherapy of CHB,and provide a new strategy for the treatment of CHB infection.