Construction and expression of anti-HBsAg and anti-RBC bispecific minibody / 中国免疫学杂志

ABSTRACT

Objective:To construct bispecific minibody by using anti HBsAg and anti RBC single chain Fv(ScFv).Methods:A “knob” variant T366W was first obtained by replacement of a small amino acid with a larger one in the human IgG1 CH 3 domain.The knob was designed to insert into a “hole” in another CH 3 domain which was created by replacement of three large residues with three smaller residues:T366S:L368A:Y407V.The “knob into hole” mutation:S354C:T366W/Y349C:T366S:L368A:Y407V.Then a disulfide bond was engineered in combination with previously designed “knob” or “hole” CH 3 was connected to anti HBsAg or anti RBC ScFv genes respectively.Then the two genes were combined together to form a bispecific minibody expression vector.The bispecific minibodies were expressed in E.coli.Results:Three different form of anti HBsAg and anti RBC minibody expression vectors were constructed.They contained wild type CH 3,“knob into hole”CH 3 or “knob into hole” plus disulfide bond CH 3 respectively.The results indicated that these three different types of bacterially expressed minibodies had similar HBsAg and RBC binding activities.The second and third type of minibody could cause agglutination of human RBC when HBsAg was present,which demonstrated bispecific function.Conclusion:Engineered interface of CH 3 can promote formation of heterodimers of different antibodies and faciliates the formation of bispecific antibodies(bispecific minibody) in E.coli expression system.