title: Identification of high-affinity VEGFR3-binding peptides through a phage-displayed random peptide library.
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 K(aff) (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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Obstetrics and Gynecology, No.117 Center Military Hospital, Hangzhou, China. lifeng_shi@163.com.
authors.forename: Li Feng
authors.initials: LF
authors.lastname: Shi
authors.affiliation: Department of Obstetrics and Gynecology, No.117 Center Military Hospital, Hangzhou, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Department of Obstetrics and Gynecology, No.117 Center Military Hospital, Hangzhou, China.
authors.forename: Cai Yun
authors.initials: CY
authors.lastname: Li
mesh_terms: D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D010051:Ovarian Neoplasms / Q000628:therapy*; D019151:Peptide Library; D017422:Sequence Analysis, DNA; D015398:Signal Transduction / Q000502:physiology; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D040321:Vascular Endothelial Growth Factor Receptor-3 / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D019151:Peptide Library; D042461:Vascular Endothelial Growth Factor A; C581309:FLT4 protein, human; D040321:Vascular Endothelial Growth Factor Receptor-3
keywords: Bacteriophages; Enzyme-Linked Immunosorbent Assay; Immunohistochemistry; Ovarian Neoplasms; Peptides; Vascular Endothelial Growth Factor