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

Cancer Res. 2001 Oct 1;61(19):7002-8.

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