Abstract
We have used protein engineering to generate a stable bivalent Fv molecule of the anti-erbB2 monoclonal antibody e23. The V H and V L domains of the Fv are linked to each other by a disulfide bond and the two Fvs are connected by a flexible 15 amino acid residue (Gly 4-Ser) 3 linker. The e23 (dsFv) 2 molecule is fused to a truncated form of Pseudomonas exotoxin to generate a bivalent disulfide-stabilized, (dsFv) 2, immunotoxin. The immunotoxin was expressed in Escherichia coli, refolded in vitro and purified to about 95% purity. Binding studies demonstrated that the (dsFv) 2 molecule has a much higher affinity for erbB2 than a monovalent dsFv molecule and a similar binding affinity as the parental antibody e23. The (dsFv) 2 immunotoxin was 5 to 20-fold more cytotoxic to two e23 antigen-positive cell lines than the monovalent dsFv immunotoxin. The bivalent dsFv molecule is very stable, retaining 94% of its activity after a 24 hours incubation in human serum at 37°C. Two other molecules with shorter linkers five and ten amino acid residues in length were produced and showed similar activities as the molecule containing a 15 amino acid residue linker. The bivalency, stability and the relative ease of purification makes these e23 (dsFv) 2 molecules valuable reagents for cancer immunotherapy and diagnosis.
Published Version
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