Complete inhibition of vascular endothelial growth factor (VEGF) activities with a bifunctional diabody directed against both VEGF kinase receptors, fms-like tyrosine kinase receptor and kinase insert domain-containing receptor.
Vascular endothelial growth factor (VEGF) binds to and mediates its activity mainly through two tyrosine kinase receptors, VEGF receptor 1 [or fms-like tyrosine kinase receptor (Flt-1)] and VEGF receptor 2 [or kinase insert domain-containing receptor (KDR)]. Numerous studies have shown that overexpression of VEGF and its receptor plays an important role in tumor-associated angiogenesis and hence in both tumor growth and metastasis. We demonstrated previously that antagonistic antibodies to KDR specifically inhibited VEGF-stimulated receptor activation, cell migration, and endothelial cell mitogenesis. Here we constructed a recombinant bifunctional diabody that is capable of blocking both Flt-1 and KDR from binding to their ligands, including VEGF and placenta growth factor (PlGF). The diabody was expressed in Escherichia coli and purified by single-step affinity chromatography. The diabody retained the capacity to bind both KDR and Flt-1 and effectively blocked interaction between KDR and VEGF, Flt-1 and VEGF, and Flt-1 and PlGF. Furthermore, the diabody is a stronger inhibitor than its parent antibodies to VEGF-stimulated mitogenesis of human endothelial cells, as well as both VEGF- and PlGF-induced migration of human leukemia cells. Taken together, our results suggest that dual receptor blockade with the bifunctional diabody may prove to be a more efficient approach in inhibiting VEGF-stimulated angiogenesis.
Lu
D
D
Department of Molecular and Cell Biology, ImClone Systems Inc., New York, New York 10014, USA.
Jimenez
X
X
Zhang
H
H
Wu
Y
Y
Bohlen
P
P
Witte
L
L
Zhu
Z
Z
D016475:3T3 Cells; D000818:Animals; D018033:Antibodies, Bispecific / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D003001:Cloning, Molecular; D016228:Endothelial Growth Factors / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology; D018922:HL-60 Cells / Q000166:cytology; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology; D008222:Lymphokines / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000378:metabolism; D017978:Receptors, Growth Factor / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000378:metabolism; D040262:Receptors, Vascular Endothelial Growth Factor; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A; D040281:Vascular Endothelial Growth Factor Receptor-1; D042442:Vascular Endothelial Growth Factors
D018033:Antibodies, Bispecific; D016228:Endothelial Growth Factors; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D008222:Lymphokines; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D017978:Receptors, Growth Factor; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor; D040281:Vascular Endothelial Growth Factor Receptor-1