Identification of novel neutralizing single-chain antibodies against vascular endothelial growth factor receptor 2
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
2011Source Title
Biotechnology and Applied Biochemistry
Print ISSN
0885-4513
Volume
58
Issue
6
Pages
412 - 422
Language
English
Type
ArticleItem Usage Stats
118
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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
antiangiogeniccorneal 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/11693/21723Published Version (Please cite this version)
http://dx.doi.org/10.1002/bab.61Collections
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