AN ENGINEERED BIVALENT SINGLE-CHAIN ANTIBODY FRAGMENT THAT INCREASES ANTIGEN-BINDING ACTIVITY

Bivalent single chain Fv (scFv) was constructed by fusing a polypeptid e extension containing one or two cysteines to the COOH-terminus of an scFv antibody fragment, The scFv protein was expressed and secreted i n a recombinant Pichia pastoris system as a dimer with a C-terminal di sulfide bridge, as determined by Western blot analysis under non-reduc ing conditions, We found that the scFv construct with one cysteine in the C-extension (scFv-1Cys) exhibited a much higher dimer/monomer rati o than the two cysteine counterpart (scFv-2Cys). Binding activity meas urements performed by means of a competitive radioimmunoassay showed t hat scFv-1Cys exhibited specific antigen binding activity, which was a lmost the same as that of the parental MAb, and approximately four- an d fortyfold higher than those of the control scFv monomer and scFv-2Cy s.