Identification of a neutralizing scFv binding to human vascular endothelial growth factor 165 (VEGF165) using a phage display antibody library.
Vascular endothelial growth factor (VEGF) is a multifunctional cytokine that plays a major role in angiogenesis. Alternative splicing causes the production of several different isoforms (VEGF121, 145, 165, 183, 189, 206). VEGF is essential for tumor angiogenesis, and several studies have correlated elevated VEGF levels with tumor stage, metastases, and progression. We now report the isolation by phage display of human single-chain antibody fragment (scFv) anti-VEGF165. After four rounds of panning against VEGF165, 40 out of 90 phage clones displayed VEGF165-binding activity. One of the positive clones, designated B8, bound to VEGF165 with relatively high affinity and neutralized VEGF165 bioactivity in vitro. The B8 clone was expressed in the soluble form in Escherichia coli HB2151 and purified by immobilized metal affinity chromatography. The purified scFv recognized VEGF165 with the K(D) of 1.80 x 10(-8) M without cross-reaction to VEGF121. In addition to binding, the purified scFv could does-dependently inhibit VEGF165-induced human umbilical vein-derived endothelial cells proliferation. Together with its fully human mature, B8 scFv may have therapeutic implications in therapy of angiogenesis-dependent diseases.
Lin
Zhihua
Z
Department of Biology, School of Chemistry and Life Sciences, Lishui University, Lishui, 323000 Zhejiang, People's Republic of China. zhihualin1015@126.com
Cao
Peng
P
Lei
Huanzong
H
D000595:Amino Acid Sequence; D000937:Antigen-Antibody Reactions; D001483:Base Sequence; D001709:Biotechnology; D049109:Cell Proliferation; D002478:Cells, Cultured; D017931:DNA Primers / Q000235:genetics; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D019151:Peptide Library; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology; D020349:Surface Plasmon Resonance; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000276:immunology*
D017931:DNA Primers; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A