Expression of a bispecific dsFv–dsFv′ antibody fragment in Escherichia coli

A bispecific disulfide-stabilized Fv antibody fragment (dsFv-dsFv') consisting of two different disulfide-stabilized Fv antibody fragments connected by flexible linker peptides was produced by secretion of three polypeptide chains into the periplasm of Escherichia coli. The dsFv-dsFv' molecules were enriched by immobilized metal affinity chromatography and further purified by anion-exchange chromatography. The recombinant antibody constructs retained the two parental antigen binding specificities and were able to cross-link the two different antigens. The described dsFv-dsFv' design might be of particular value for therapeutic in vivo applications since improved stability is expected to be combined with minimal immunogenicity.