Identification of novel neutralizing single-chain antibodies against vascular endothelial growth factor receptor 2

Biotechnol Appl Biochem. 2011 Nov-Dec;58(6):412-22. doi: 10.1002/bab.61. Epub 2011 Nov 15.


Human vascular endothelial growth factor (VEGF) and its receptor (VEGFR-2/kinase domain receptor [KDR]) play a crucial role in angiogenesis, which makes the VEGFR-2 signaling pathway a major target for therapeutic applications. In this study, a single-chain antibody phage display library was constructed from spleen cells of mice immunized with recombinant human soluble extracellular VEGFR-2/KDR consisting of all seven extracellular domains (sKDR D1-7) to obtain antibodies that block VEGF binding to VEGFR-2. Two specific single-chain antibodies (KDR1.3 and KDR2.6) that recognized human VEGFR-2 were selected; diversity analysis of the clones was performed by BstNI fingerprinting and nucleotide sequencing. The single-chain variable fragments (scFvs) were expressed in soluble form and specificity of interactions between affinity purified scFvs and VEGFR-2 was confirmed by ELISA. Binding of the recombinant antibodies for VEGFR-2 receptors was investigated by surface plasmon resonance spectroscopy. In vitro cell culture assays showed that KDR1.3 and KDR2.6 scFvs significantly suppressed the mitogenic response of human umbilical vein endothelial cells to recombinant human VEGF(165) in a dose-dependent manner, and reduced VEGF-dependent cell proliferation by 60% and 40%, respectively. In vivo analysis of these recombinant antibodies in a rat cornea angiogenesis model revealed that both antibodies suppressed the development of new corneal vessels (p < 0.05). Overall, in vitro and in vivo results disclose strong interactions of KDR1.3 and KDR2.6 scFvs with VEGFR-2. These findings indicate that KDR1.3 and KDR2.6 scFvs are promising antiangiogenic therapeutic agents.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Angiogenesis Inhibitors / immunology
  • Angiogenesis Inhibitors / pharmacology
  • Animals
  • Base Sequence
  • Cell Proliferation / drug effects
  • Cornea / blood supply
  • Cornea / drug effects
  • Dose-Response Relationship, Immunologic
  • Enzyme-Linked Immunosorbent Assay
  • Human Umbilical Vein Endothelial Cells / drug effects
  • Human Umbilical Vein Endothelial Cells / immunology
  • Humans
  • Male
  • Mice
  • Mice, Inbred BALB C
  • Molecular Sequence Data
  • Peptide Library
  • Rats
  • Recombinant Proteins / immunology
  • Recombinant Proteins / metabolism
  • Recombinant Proteins / pharmacology*
  • Single-Chain Antibodies / genetics
  • Single-Chain Antibodies / immunology*
  • Single-Chain Antibodies / metabolism
  • Spleen / immunology
  • Surface Plasmon Resonance
  • Vascular Endothelial Growth Factor A / metabolism
  • Vascular Endothelial Growth Factor Receptor-2 / genetics*
  • Vascular Endothelial Growth Factor Receptor-2 / immunology*


  • Angiogenesis Inhibitors
  • Peptide Library
  • Recombinant Proteins
  • Single-Chain Antibodies
  • Vascular Endothelial Growth Factor A
  • Vascular Endothelial Growth Factor Receptor-2