Expression of a bispecific dsFv-dsFv ' antibody fragment in Escherichia coli

A bispecific disulfide-stabilized Fv antibody fragment (dsFv-dsFv') consist ing of two different disulfide-stabilized Fv antibody fragments connected b y flexible linker peptides was produced by secretion of three polypeptide c hains 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 reta ined the two parental antigen binding specificities and were able to cross- link the two different antigens. The described dsFv-dsFv' design might be o f particular value for therapeutic in vivo applications since improved stab ility is expected to be combined with minimal immunogenicity.