Identification of high-affinity VEGFR3-binding peptides through a phage-displayed random peptide library.
10.3802/jgo.2015.26.4.327
- Author:
Li Feng SHI
1
,
2
;
Yan WU
;
Cai Yun LI
Author Information
1. Department of Obstetrics and Gynecology, No.117 Center Military Hospital, Hangzhou, China. lifeng_shi@
2. com
- Publication Type:Original Article ; Research Support, Non-U.S. Gov't
- Keywords:
Bacteriophages;
Enzyme-Linked Immunosorbent Assay;
Immunohistochemistry;
Ovarian Neoplasms;
Peptides;
Vascular Endothelial Growth Factor
- MeSH:
Enzyme-Linked Immunosorbent Assay;
Female;
Humans;
Ovarian Neoplasms/*therapy;
*Peptide Library;
Sequence Analysis, DNA;
Signal Transduction/physiology;
Vascular Endothelial Growth Factor A/metabolism;
Vascular Endothelial Growth Factor Receptor-3/antagonists & inhibitors/*metabolism
- From:Journal of Gynecologic Oncology
2015;26(4):327-335
- CountryRepublic of Korea
- Language:English
-
Abstract:
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 microg/mL (n=3), 9.2+/-2.1 microg/mL (n=3), and 174.8+/-31.1 microg/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.
