An antibody targeted to VEGFR-2 Ig domains 4-7 inhibits VEGFR-2 activation and VEGFR-2-dependent angiogenesis without affecting ligand binding

Mol Cancer Ther. 2011 May;10(5):770-83. doi: 10.1158/1535-7163.MCT-10-0876. Epub 2011 Mar 9.


Inhibition of VEGFR-2 signaling reduces angiogenesis and retards tumor growth. Current biotherapeutics that inhibit VEGFR-2 signaling by either sequestering VEGF ligand or inhibiting VEGF binding to VEGFR-2 may be compromised by high VEGF concentrations. Here we describe a biotherapeutic that targets VEGFR-2 signaling by binding to Ig domains 4-7 of VEGFR-2 and therefore has the potential to work independently of ligand concentration. 33C3, a fully human VEGFR-2 antibody, was generated using XenoMouse technology. To elucidate the mechanism of action of 33C3, we have used a number of competition and binding assays. We show that 33C3 binds VEGFR-2 Ig domains 4-7, has no impact on VEGF-A binding to VEGFR-2, and does not compete with an antibody that interacts at the ligand binding site. 33C3 has a high affinity for VEGFR-2 (K(D) < 1 nmol/L) and inhibits VEGF-A induced phosphorylation of VEGFR-2 with an IC(50) of 99 ± 3 ng/mL. In vitro, in a 2D angiogenesis assay, 33C3 potently inhibits both tube length and number of branch points, and endothelial tubule formation in a 3D assay. In vivo, 33C3 is a very effective inhibitor of angiogenesis in both a human endothelial angiogenesis assay and in a human skin chimera model. These data show targeting VEGFR-2 outside of the ligand binding domain results in potent inhibition of VEGFR-2 signaling and inhibition of angiogenesis in vitro and in vivo.

MeSH terms

  • Angiogenesis Inhibitors / metabolism*
  • Angiogenesis Inhibitors / pharmacology
  • Animals
  • Antibodies / metabolism*
  • Antibodies / pharmacology
  • Antibody Specificity / immunology
  • Cell Line
  • Endothelial Cells / drug effects
  • Endothelial Cells / metabolism
  • Humans
  • Ligands*
  • Mice
  • Mice, SCID
  • Neovascularization, Physiologic / drug effects*
  • Protein Binding / drug effects
  • Protein Structure, Tertiary
  • Skin / blood supply*
  • Skin / drug effects
  • Swine
  • Vascular Endothelial Growth Factor A / metabolism
  • Vascular Endothelial Growth Factor Receptor-2 / antagonists & inhibitors*
  • Vascular Endothelial Growth Factor Receptor-2 / chemistry
  • Vascular Endothelial Growth Factor Receptor-2 / metabolism*


  • Angiogenesis Inhibitors
  • Antibodies
  • Ligands
  • Vascular Endothelial Growth Factor A
  • Vascular Endothelial Growth Factor Receptor-2