Humanization Of Therapeutic Antibody Against Hepatitis B Virus

6D11 mAb,which is a mouse monoclonal antibody binding to a special epitope on the hepatitis B surface(HBsAg),was discovered in our lab by using the high-replicative HBV transgenic mice.It has been demonstrated that in vivo administration of 6D11 profoundly and persistently suppressed the levels of HBsAg and HBV DNA for several weeks.In previous work,we have constructed a chimeric antibody of 6D11(6D11 cAb).However,murine variable region of chimeric antibody is still possiblely to induce HAMA response.For further therapeutic antibody development based on 6D11,it is necessary for us to humanize its variable region.In this study,we accomplished the humanization of 6D11 by CDR-grafting and framework region optimization to keep the original biological function using phage display technology.First,human germline genes 4-28-02 for VH and 2D-28-01 for VL were selected as the humanized templates.By analyzing the characteristic and spatial location of the different amino acids between 6D11 and the humanized templates,we retained the key residues of murine while the unimportant residues were humanized.The other different residues including 20 amino acids in VH and 6 ones in VL were designed as human and murine alternatives in the combinatorial library.The full length humanized 6D11 scFv genes were synthesized using splicing overlap extension polymerase chain reaction(SOE-PCR).A humanized combinatorial library was constructed successfully by phage display technique,and the real content of the library was 5×107.After three rounds of panning with antigen-coated enzyme-linked immunosorbent assay and antibody-coated assay,we achieved 20 unique sequences of positive ScFvs.The humanized genes of 10 positive clones were selected to be constructed into PTT5 vector,then the recombinant antibodies were transiently expressed in CHO-S cells.Their biological activities were detected by antigen specific reactivity,neutralizing activity and virus clearing capability.B-S3-45 which was identified to be the best one maintained the biological activities of 6D11 cAb,and its humanized degree was 89.94%.Its half maximal effective concentration(EC50)was about 3 ng/mL,and its efficacy period to inhibit HBV DNA amplification and HBsAg infection was up to five days in HBV transgenic mice.In HepaRG cells,B-S3-45 inhibited 50%viral infection at concentration of 290 ng/mL,and 90%inhibition required 2500 ng/mL of antibody.Furthermore,compared to original mouse antibody,6D11 cAb was found to have weaker neutralizing activity.Constant region back replacement was implemented by construction of B45 humanized variable region and mouse constant region(B45-mC)to explore which influenced 6D11 cAb bio-reactivity reduction.The result showed us that B45-mC indeed behaved better than B-S3-45 humanized antibody either in binding or neutralizing activities.It indicated that the constant region is major factor affecting the biological activity of 6D11 and the humanized antibodies,therefore the costant region is important to be taken into consideration to improve the affinity of humanized antibodies.