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

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      Author
      Erdag, B.
      Koray Balcioglu, B.
      Ozdemir Bahadir, A.
      Serhatli, M.
      Kacar O.
      Bahar, A.
      Seker, U.O.S.
      Akgun, E.
      Ozkan, A.
      Kilic, T.
      Tamerler, C.
      Baysal, K.
      Date
      2011
      Source Title
      Biotechnology and Applied Biochemistry
      Print ISSN
      0885-4513
      Volume
      58
      Issue
      6
      Pages
      412 - 422
      Language
      English
      Type
      Article
      Item Usage Stats
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      134
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      Abstract
      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. © 2011 International Union of Biochemistry and Molecular Biology, Inc.
      Keywords
      antiangiogenic
      corneal angiogenesis assay
      HUVEC
      phage display
      single-chain variable fragment (scFv)
      vascular endothelial growth factor receptor-2 (VEGFR-2)
      Angiogenesis
      Antiangiogenic
      HUVEC
      phage display
      Single chain variable fragments
      Vascular endothelial growth factor
      Animal cell culture
      Assays
      Cell proliferation
      Endothelial cells
      Peptides
      Plants (botany)
      Surface plasmon resonance
      Antibodies
      vasculotropin antibody
      vasculotropin receptor 2
      angiogenesis
      animal cell
      animal experiment
      article
      binding affinity
      biopanning
      cell proliferation
      controlled study
      cornea
      DNA fingerprinting
      DNA sequence
      enzyme linked immunosorbent assay
      human
      human cell
      human tissue
      in vitro study
      in vivo study
      male
      mouse
      nonhuman
      phage display
      protein binding
      protein domain
      rat
      spleen cell
      surface plasmon resonance
      Amino Acid Sequence
      Angiogenesis Inhibitors
      Animals
      Base Sequence
      Cell Proliferation
      Cornea
      Dose-Response Relationship, Immunologic
      Enzyme-Linked Immunosorbent Assay
      Human Umbilical Vein Endothelial Cells
      Humans
      Male
      Mice
      Mice, Inbred BALB C
      Molecular Sequence Data
      Peptide Library
      Rats
      Recombinant Proteins
      Single-Chain Antibodies
      Spleen
      Surface Plasmon Resonance
      Vascular Endothelial Growth Factor A
      Vascular Endothelial Growth Factor Receptor-2
      Permalink
      http://hdl.handle.net/11693/21723
      Published Version (Please cite this version)
      http://dx.doi.org/10.1002/bab.61
      Collections
      • Institute of Materials Science and Nanotechnology (UNAM) 1891
      • Nanotechnology Research Center (NANOTAM) 1049
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