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.

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.

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

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 K_aff (affinity constant) of peptides I, II, and III was shown. The K_aff 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 K_aff (affinity constant) of peptides I, II, and III was shown. The K_a 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).