Novel Short Peptides Isolated From Phage Display Library Inhibit Vascular Endothelial Growth Factor Activity

Abstract

Signal transduction through the vascular endothelial growth factor (VEGF)-VEGF receptor (VEGFR) pathway has a pivotal importance in angiogenesis, and has therefore become a prime target in antitumor therapy. In search for peptides antagonizing VEGF binding to its receptors, we screened a random heptamer library displayed on phage for peptides that bind the whole VEGF165 molecule and inhibit VEGF dependent human umbilical vein endothelial cell (HUVEC) proliferation. Two selected peptides with sequences WHLPFKC and WHKPFRF were synthesized. Biacore and matrix-assisted laser desorption/ionization timeof- flight mass spectrometry analysis indicated that these peptides bind the VEGF homodimer in a concentration- dependent manner, with micromolar affinity, and with a 2:1 peptide:VEGF stoichiometry. They inhibited HUVEC proliferation in vitro by 77 and 55%, respectively. Taken together, our results indicate that these peptides could be potent inhibitors of angiogenesis. Furthermore, we show that the peptide- VEGF binding properties can be quantified, a prerequisite for the further optimization of binders.

Synthesis, Characterisation and in vitro Anti‐Angiogenic Potential of Dendron VEGF Blockers

“…[4] Most of the inhibitors investigated to date, target endothelial cell migration and proliferation either directly through the interaction with VEGF cell receptors (VEGFR1, -R2, -R3) [5] or indirectly through the blockage of growth factors. [6] Drugs and anti-VEGF antibodies have been used to neutralise the proliferative activity of the cells. The inhibition of VEGF activity has also been pursued through the therapeutic administration of soluble VEGF receptors (sFlk-1) that in physiological conditions are secreted by endothelial cells to ensure control of angiogenesis.…”

“…[6] A series of studies has led to the reduction of the peptide length to a minimum of seven amino acids. The reduction of the number of amino acid residues necessary to form complex with the VEGF molecule is considered important in the view of scaling-up the synthesis of these peptides at industrial level as well as for improving the binding affinity of these molecules to VEGF.…”

“…Previously published papers have reported molecular studies by surface plasmon resonance proving the ability of these peptides to form macromolecular complexes with VEGF at different molecular ratios. [6] However, these peptides are likely to have a relatively short half-life in vivo and a limited retention in the target tissues. The present paper shows the synthesis and in vitro anti-angiogenic potential of WHLPFKC when presented at the uppermost branching generation of hyperbranched generation 3 (G3) poly(e-lysine) dendrons.…”

Cost effective filamentous phage based immunization nanoparticles displaying a full-length hepatitis B virus surface antigen

“…This technology has been widely used for the selection of recombinant antibodies, such as Fab, single chain variable fragments or peptides that are able to bind various target molecules such as growth factor receptors, growth factors, viral antigens and even inorganic materials for diagnosis, tumor targeting and drug discovery [10][11][12][13][14]. Phage display has recently been used in new applications for vaccine development by displaying peptides derived from virus antigens on the phage surface [15][16][17].…”

Phage Displayed HBV Core Antigen with Immunogenic Activity

“…DNA sequences of interest are inserted into a location in the genome of a filamentous bacteriophage such that the encoded proteins are expressed or "displayed" on the surface of the filamentous phage as fusion products with pIII or pVIII phage coat protein [11,12]. Phage display technology is widely used for the production of peptides, including recombinant antibodies such as Fabs or single-chain variable fragments, for diagnostic or therapeutic purposes [13,14]. Because of their immunogenic activity, phages are also used as potent carriers for immunization [15].…”