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Identification of a Neutralizing scFv Binding to Human Vascular Endothelial Growth Factor 165 (VEGF165) Using a Phage Display Antibody Library

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Abstract

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 × 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.

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Acknowledgments

This work was partially supported by grants from the Scientific and Technological Program of Zhejiang Province (2006C33075; to Prof. Zhihua Lin), Jiangsu Province International Cooperation Promotion Program (to Dr. Peng Cao), and Startup Foundation of Jiangsu Province Institute of Traditional Chinese Medicine (KC0602; BZ2007078; to Dr. Peng Cao).

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Authors and Affiliations

  1. Department of Biology, School of Chemistry and Life Sciences, Lishui University, Lishui, 323000, Zhejiang, People’s Republic of China

    Zhihua Lin & Huanzong Lei

  2. Laboratory of Molecular Medicine, Jiangsu Province Institute of Traditional Chinese Medicine, 210028, Nanjing, People’s Republic of China

    Peng Cao

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  1. Zhihua Lin
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  2. Peng Cao
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  3. Huanzong Lei
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Correspondence to Zhihua Lin.

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Lin, Z., Cao, P. & Lei, H. Identification of a Neutralizing scFv Binding to Human Vascular Endothelial Growth Factor 165 (VEGF165) Using a Phage Display Antibody Library. Appl Biochem Biotechnol 144, 15–26 (2008). https://doi.org/10.1007/s12010-007-8011-4

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  • DOI: https://doi.org/10.1007/s12010-007-8011-4

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