Construction and expression of an anti-EGFR/anti-KDR bispecific single-chain diabody

Bispecific antibodies have been exploited as both cancer immunodiagnostics and cancer therapeutics, which have shown promises in clinical trials in cancer imaging and therapy.  To improve the anti-tumor effect, an scDb (bispecific single-chain diabody) was constructed from the variable domain genes of two scFvs (single-chain variable fragment antibodies) directed against human EGFR (epidermal growth factor receptor) and VEGFR2 (vascular endothelial growth factor receptor 2) extracellular domains.  The anti-EGFR/ anti-KDR scDb was constructed into pHEN2 plasmid and expressed in Escherichia coli HB2151 host.  After purification by one-step affinity chromatography of IMAC, scDb protein was characterized by Western blotting.  The yield of scDb protein was 570 μg per liter medium. scDb bound to EGFR as efficiently as the parental antibody scFv-E10, while a little bit weaker than the parental antibody scFv-AK404R when bound to KDR.  In conclusion, the scDb protein could bind both EGFR and KDR specifically and could be applied for further anti-tumor research.