J Gynecol Oncol.  2015 Oct;26(4):327-335. 10.3802/jgo.2015.26.4.327.

Identification of high-affinity VEGFR3-binding peptides through a phage-displayed random peptide library

• Shi LF
• Wu Y
• Li CY

• KMID: 2345924
• DOI: http://doi.org/10.3802/jgo.2015.26.4.327

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.

Keyword

Bacteriophages; Enzyme-Linked Immunosorbent Assay; Immunohistochemistry; Ovarian Neoplasms; Peptides; Vascular Endothelial Growth Factor

MeSH Terms

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
Peptide Library
Vascular Endothelial Growth Factor A
Vascular Endothelial Growth Factor Receptor-3

Figure

• 

  Fig. 1 Interaction of phages with recombinant human vascular endothelial growth factor receptor-3 (rhVEGFR-3). (A) The interaction of all 10 phage clones with rhVEGFR-3 was determined by enzyme-linked immunosorbent assay. Negative controls consisted of bovine serum albumin (BSA) as well as uncoated wells. Interaction was determined at an optical density (OD) 410 nmol/L. S and T had no specific meanings; they are just different clone names. (B) The binding affinity of all 10 clones for VEGFR-3 was much higher than that of both BSA and blank control (both p<0.001).

• 

  Fig. 2 Agarose gel electrophoresis of single-stranded DNA isolated from the phage clones. Lanes 1 to 10: single-stranded DNA of phage clones; lane M, DNA marker IV.

• 

  Fig. 3 Interaction of (A) peptides I, (B) II, and (C) III with vascular endothelial growth factor receptor-3 (VEGFR-3). Interaction of peptides I, II, and III with VEGFR-3 was analyzed by non-competitive enzyme-linked immunosorbent assay. A, B, C, and D are the various concentrations of VEGFR-3 analyzed (20, 10, 5, and 2.5 µg/mL, respectively). Interaction was determined at an optical density (OD) 410 nmol/L. One representative experimental curve plot from three experiments of the Kaff (affinity constant) of peptides I, II, and III was shown. The Kaff of peptides I to III was 16.4±8.6, 9.2±2.1, and 174.8±31.1 µg/mL, respectively.

• 

  Fig. 4 Interaction of vascular endothelial growth factor (VEGF)-D with vascular endothelial growth factor receptor-3 (VEGFR-3)/Fc. Different concentrations of VEGF-D (1,000, 500, 250, 125, 62.5, and 31.25) were coated on the enzyme-linked immunosorbent assay plates and incubated with VEGFR-3/Fc chimera and immunoglobulin G Fc antibody to generate the binding curves. Interaction was determined at an optical density (OD) 410 nmol/L. One representative experimental curve plot from three experiments of the Kaff (affinity constant) of peptides I, II, and III was shown. The Ka was 264.01±56.7 µg/mL.

• 

  Fig. 5 Immunohistochemical and immunofluorescence staining of ovarian carcinoma tissue sections. Tissue sections were analyzed for vascular endothelial growth factor receptor-3 (A, C, E) and peptide III (B, D) as determined by immunohistochemistry and immunofluorescence staining, respectively. Both were detected on the endothelial cells of lymphatic vessel. (F) The fluorescein isothiocyanate (FITC)-labeled negative peptide [FITC-(aminohexanoic)-WHNSLKANYTWG] was used as negative controls for immunofluorescence (A, B, E, F: ×100; C, D: ×200; A-D: scale bar, 20 µm; E, F: scale bar, 10 µm).

• 

  Fig. 6 Localization of peptide III to ovarian tumors in vivo. (A) Technetium-99 (99Tc)-labeled peptide (phage-WHWLPNLRHYAS) interacted with the ovarian tumor in vivo (arrow). (B) Free probe (99Tc) accumulated in the liver, kidney, and bladder, but not at the tumor (arrow).

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