Fc Engineering Of An Anti-HBV Therapeutic Antibody For Longer Half-life

In recent years,the extension of the therapeutic antibody half-life has become the focus of antibody engineering.It can not only improve the efficacy of antibody,but also reduce delivery frequency and the cost of treatment.Genetical modification of Fc to improve the affinity for human neonatal Fc receptor(hFcRn)has been widely used in extending antibody half-life.However,for the same Fc mutation,the effect of half-life improvement varies from different antibodies.In this study,we try to obtain the Fc modified variant which can prolong the half-life of an anti-HBV therapeutic humanized antibody,huE6F6,by screening different Fc mutations which have been reported to be effective.The main contents include the following three parts:Firstly,the expression and characterization of Fc engineered antibodies.In this study,three Fc mutants known to improve half-life of antibody were selected,and the variants including huE6F6-YTE(M252Y/S254T/T256E),huE6F6-QL(T250Q/M428L)and huE6F6-LS(M428L/M434S)were constructed and expressed.From the results of he thermal stability decteted by DSC,we found that the Tml value of all antibodies was higher than 60?,and the Tm2 value was maintained at the same level in the range of 81 ??-82?,which indicated that the conformation of the engineered antibodies were stable.The HBsAg binding activity of the engineeried antibodies showed that the point mutations in the Fc region did not affect the antigen specificity.Among the hFcRn binding activity of these three engineered antibodies,huE6F6-YTE showed about 60 times increase compared with huE6F6-WT at pH 6.0,and maintained the pH dependency.While the binding activity of the other two antibodies increased in less than 10 times,and the pH dependency is not obvious.Secondly,cell-based hFcRn binding activity of engineered antibodies.Through the construction of MDCK cell line stably expressing EGFP-hFcRn,competitive binding of engineered antibodies and human IgG to hFcRn at pH 6.0 were tested,we found that the binding ability of huE6F6-YTE was the strongest.Thus,huE6F6-YTE was selected as the candidate molecule for the evaluation of in vivo half-life.Thirdly,the in vivo half-life and antigen clearance of huE6F6-YTE were detected.The results showed that the serum half-life of huE6F6-YTE in FcRn transgenic mice and cynomolgus monkeys was 1.5 times and 2.5 times of the wild type,respectively.In the HBV transgenic mice,the maximum clearance of huE6F6-YTE injection group was less than that of huE6F6-WT,but the duration of huE6F6-YTE was longer and lasted about 4 days.In the HBsAg clearance test of cynomolgus monkeys,the half-life of antigen in the huE6F6-YTE injection group was 4 times higher than that of the huE6F6-WT injection group,and the antibody half-life of huE6F6-YTE was about 1.5 times of huE6F6-WT.In conclusion,we successfully improved the half-life of HBV therapeutic antibody by Fc engineering,which is of great significance for the preclinical study of chronic hepatitis B.