Abstract
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.
MeSH terms
-
3T3 Cells
-
Animals
-
Antibodies, Bispecific / genetics
-
Antibodies, Bispecific / immunology*
-
Antibodies, Bispecific / metabolism
-
Cell Division / drug effects
-
Cell Movement / drug effects
-
Cloning, Molecular
-
Endothelial Growth Factors / antagonists & inhibitors*
-
Endothelial Growth Factors / metabolism
-
Endothelial Growth Factors / pharmacology
-
Endothelium, Vascular / cytology
-
HL-60 Cells / cytology
-
Humans
-
Immunoglobulin Heavy Chains / genetics
-
Immunoglobulin Heavy Chains / immunology
-
Immunoglobulin Light Chains / genetics
-
Immunoglobulin Light Chains / immunology
-
Lymphokines / antagonists & inhibitors*
-
Lymphokines / metabolism
-
Lymphokines / pharmacology
-
Mice
-
Proto-Oncogene Proteins / antagonists & inhibitors
-
Proto-Oncogene Proteins / immunology*
-
Proto-Oncogene Proteins / metabolism
-
RNA, Messenger / genetics
-
Receptor Protein-Tyrosine Kinases / antagonists & inhibitors
-
Receptor Protein-Tyrosine Kinases / immunology*
-
Receptor Protein-Tyrosine Kinases / metabolism
-
Receptors, Growth Factor / antagonists & inhibitors
-
Receptors, Growth Factor / immunology*
-
Receptors, Growth Factor / metabolism
-
Receptors, Vascular Endothelial Growth Factor
-
Recombinant Proteins / genetics
-
Recombinant Proteins / immunology
-
Recombinant Proteins / metabolism
-
Vascular Endothelial Growth Factor A
-
Vascular Endothelial Growth Factor Receptor-1
-
Vascular Endothelial Growth Factors
Substances
-
Antibodies, Bispecific
-
Endothelial Growth Factors
-
Immunoglobulin Heavy Chains
-
Immunoglobulin Light Chains
-
Lymphokines
-
Proto-Oncogene Proteins
-
RNA, Messenger
-
Receptors, Growth Factor
-
Recombinant Proteins
-
Vascular Endothelial Growth Factor A
-
Vascular Endothelial Growth Factors
-
Receptor Protein-Tyrosine Kinases
-
Receptors, Vascular Endothelial Growth Factor
-
Vascular Endothelial Growth Factor Receptor-1