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KMID : 1137020150260040327
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Journal of Gynecologic Oncology
2015 Volume.26 No. 4 p.327 ~ p.335
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Identification of high-affinity VEGFR3-binding peptides through a phage-displayed random peptide library
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Shi Li-Feng
Wu Yan
Li Cai-Yun
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Abstract
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Objective: Vascular endothelial growth factor (VEGF) interaction with its receptor, VEGFR-3/Flt-4, regulates lymphangiogenesis. VEGFR-3/Flt-4 expression in cancer cells has been correlated with clinical stage, lymph node metastasis, and lymphatic invasion. The objective of this study is to identify a VEGFR-3/Flt-4-interacting peptide that could be used to inhibit VEGFR-3 for ovarian cancer therapy.
Methods: The extracellular fragment of recombinant human VEGFR-3/Flt-4 (rhVEGFR-3/Flt-4) fused with coat protein pIII was screened against a phage-displayed random peptide library. Using affinity enrichment and enzyme-linked immunosorbent assay (ELISA) screening, positive clones of phages were amplified. Three phage clones were selected after four rounds of biopanning, and the specific binding of the peptides to rhVEGFR-3 was detected by ELISA and compared with that of VEGF-D. Immunohistochemistry and immunofluorescence analyses of ovarian cancer tissue sections was undertaken to demonstrate the specificity of the peptides.
Results: After four rounds of biopanning, ELISA confirmed the specificity of the enriched bound phage clones for rhVEGFR-3. Sequencing and translation identified three different peptides. Non-competitive ELISA revealed that peptides I, II, and III had binding affinities for VEGFR-3 with Kaff (affinity constant) of 16.4±8.6 μg/mL (n=3), 9.2±2.1 μg/mL (n=3), and 174.8±31.1 μg/mL (n=3), respectively. In ovarian carcinoma tissue sections, peptide III (WHWLPNLRHYAS), which had the greatest binding affinity, also co-localized with VEGFR-3 in endothelial cells lining lymphatic vessels; its labeling of ovarian tumors in vivo was also confirmed.
Conclusion: These finding showed that peptide III has high specificity and activity and, therefore, may represent a potential therapeutic approach to target VEGF-VEGFR-3 signaling for the treatment or diagnosis of ovarian cancer.
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KEYWORD
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Bacteriophages, Enzyme-Linked Immunosorbent Assay, Immunohistochemistry, Ovarian Neoplasms, Peptides, Vascular Endothelial Growth Factor
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