A Vascular Endothelial Growth Factor High Affinity Receptor 1-specific Peptide with Antiangiogenic Activity Identified Using a Phage Display Peptide Library

Vascular endothelial growth factor (VEGF) is known to play a predominant role in tumor angiogenesis and metastasis formation that is mediated by its interactions with two tyrosine kinase receptors, VEGFRI (Flt-1) and VEGFRII (KDR). Inhibition of VEGF-dependent events in tumor tissues is known to enhance apoptosis and to suppress tumor growth. A novel peptide, SP5.2, which selectively binds Flt-1 and inhibits a broad range of VEGF-mediated events, was identified using a phage-display library screening. The fluorescein-labeled SP5.2 specifically bound to VEGF-stimulated primary human cerebral endothelial cells (HCECs), whereas non-stimulated HCECs, as well as human neuroblastoma cells (ShyY) did not show any interaction with the peptide. SP5.2 prevented proliferation of cultured primary human umbilical vein endothelial cells induced by recombinant human VEGF165 with an IC50 of 5 μm. SP5.2 was also shown to antagonize VEGF- and PLGF-induced, but not basic fibroblast growth factor-induced proliferation of HCECs. In contrast to “scrambled” peptide, SP5.2 was also found to selectively inhibit VEGF-stimulated migration of HCECs. The in vitro analysis of antiangiogenic activity of SP5.2 using a capillary-like tube formation assay showed that VEGF-induced angiogenesis of HCECs grown on Matrigel™ was completely inhibited in the presence of 10 μm SP5.2. Further studies demonstrated that SP5.2 prevented VEGF-induced permeability increase in HCECs monolayers. To explore whether SP5.2 can be used as a targeting agent, chemical and recombinant conjugates of SP5.2 with reporter proteins (peroxidase and β-galactosidase) were produced. The resulting products showed significant increases (200-fold for SP5.2-β-gal and 400-fold for SP5.2-peroxidase) in binding affinity to recombinant Flt-1 compared with the original synthetic SP5.2, suggesting that conjugate with therapeutic activity in nanomolar range could potentially be developed based on SP5.2 structure. Vascular endothelial growth factor (VEGF) is known to play a predominant role in tumor angiogenesis and metastasis formation that is mediated by its interactions with two tyrosine kinase receptors, VEGFRI (Flt-1) and VEGFRII (KDR). Inhibition of VEGF-dependent events in tumor tissues is known to enhance apoptosis and to suppress tumor growth. A novel peptide, SP5.2, which selectively binds Flt-1 and inhibits a broad range of VEGF-mediated events, was identified using a phage-display library screening. The fluorescein-labeled SP5.2 specifically bound to VEGF-stimulated primary human cerebral endothelial cells (HCECs), whereas non-stimulated HCECs, as well as human neuroblastoma cells (ShyY) did not show any interaction with the peptide. SP5.2 prevented proliferation of cultured primary human umbilical vein endothelial cells induced by recombinant human VEGF165 with an IC50 of 5 μm. SP5.2 was also shown to antagonize VEGF- and PLGF-induced, but not basic fibroblast growth factor-induced proliferation of HCECs. In contrast to “scrambled” peptide, SP5.2 was also found to selectively inhibit VEGF-stimulated migration of HCECs. The in vitro analysis of antiangiogenic activity of SP5.2 using a capillary-like tube formation assay showed that VEGF-induced angiogenesis of HCECs grown on Matrigel™ was completely inhibited in the presence of 10 μm SP5.2. Further studies demonstrated that SP5.2 prevented VEGF-induced permeability increase in HCECs monolayers. To explore whether SP5.2 can be used as a targeting agent, chemical and recombinant conjugates of SP5.2 with reporter proteins (peroxidase and β-galactosidase) were produced. The resulting products showed significant increases (200-fold for SP5.2-β-gal and 400-fold for SP5.2-peroxidase) in binding affinity to recombinant Flt-1 compared with the original synthetic SP5.2, suggesting that conjugate with therapeutic activity in nanomolar range could potentially be developed based on SP5.2 structure. Vascular endothelial growth factor (VEGF) 1The abbreviations used are: VEGFvascular endothelial growth factorFlt-1tyrosine kinase receptor VEGFRIKDRtyrosine kinase receptor VEGFRIINRP-1neuropillin-1HRPhorseradish peroxidaseHCEChuman cerebral endothelial cellHUVEChuman umbilical vein endothelial cellBSAbovine serum albuminPBSphosphate-buffered salineFlt-MPFlt-1-coated magnetic particleELISAenzyme-linked immunosorbent assaycfucolony-forming unit(s)bFGFbasic fibroblast growth factorCMVcytomegalovirusSPDPN-succinimidy 1,3-(2-pyridylthio)propionateECendothelial cellABantibodyABTS2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)PLGFplacenta growth factor. and its receptors are the focus of intense interest because of their role in blood vessel formation (angiogenesis and vasculogenesis) in a variety of physiological and pathophysiological processes, including embryogenesis, development of the fetal cardiovascular system, wound healing, tumor growth, proliferative retinopathies, and chronic inflammatory diseases such as rheumatoid arthritis (1Folkman J. 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Chem. 2000; 275: 15905-15911Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). vascular endothelial growth factor tyrosine kinase receptor VEGFRI tyrosine kinase receptor VEGFRII neuropillin-1 horseradish peroxidase human cerebral endothelial cell human umbilical vein endothelial cell bovine serum albumin phosphate-buffered saline Flt-1-coated magnetic particle enzyme-linked immunosorbent assay colony-forming unit(s) basic fibroblast growth factor cytomegalovirus N-succinimidy 1,3-(2-pyridylthio)propionate endothelial cell antibody 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) placenta growth factor. VEGF activities are mediated through binding to two high affinity receptors, human kinase domain receptor (KDR) and Fms-like tyrosine kinase receptor (Flt-1) (5Shibuya M. Yamaguchi S. Yamane A. Ikeda T. Tojo A. Matsushime H. Sato M. 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Biochem. 1998; PubMed Scopus Google Scholar). were and by were on using and The was the by of with were from the with and were by with products were by high on a using a of to for a The of was by III for Flt-1 in was and of an with Flt-1 In the of or VEGF165 were and were 10 with and the bound was using a Flt-1 were grown on in a for was cells were two in and with for 10 were in in serum in for and to the primary antibody Flt-1 in for in cells were to the antibody of in and for were with and with and in for of A and with for 5 were a SP5.2 to SP5.2 was to HCECs cells and and for The neuroblastoma cell was used as a were and cells were in and a Cell were in cells well in of with fetal bovine were for in HCECs were in and to for were by and in a and for to suppress cell growth. Both HCECs and were to fetal bovine VEGF165 in the or presence of SP5.2 the was used as of of was in and were for an The cells were on with fetal bovine and for 10 in with cells were and was in of was by Cell of 10 μm for HCECs by VEGF165 were in the or presence of SP5.2 or using a of a and a as in a T.A. D.B. R. J. 1993; PubMed Scopus Google Scholar). the of the were with or and by The was on and HCECs were in of or was on the of The was for and the of endothelial cells the was by the of in a in were on a matrix, Matrigel™ and to were with 10 VEGF165 or cell for in the or presence of SP5.2 or for HCECs were with a for were in and capillary-like tube a of in vitro angiogenesis, was using a with an and a for were to a of μm. The was through a to using an of were and the was was to enhance and contrast using SP5.2-β-gal expression from was used to the SP5.2 on the and on the of the from was with the resulting was from and by as to the for of which has by of with and The SP5.2 the the and the the was by the two by and the was used to The resulting were to the and of a was grown in of and to an of The was induced with for and the resulting was The expression of and the presence of were by using and antibody The was and in 10 and was to a of for of was to the and The was a with 10 and the SP5.2-β-gal was with the The was by to and by to a of SP5.2-β-gal binding for were used to the receptor antibody 10 in were well of The were for in a and for with human in was were in to receptor to receptor was with of well was with of in and for a with antibody were with in SP5.2-β-gal and recombinant from used as were to well and for were with and the of bound SP5.2-β-gal was with and conjugate were in of of peroxidase was in of 1,3-(2-pyridylthio)propionate was in in a of of of The of was to the of peroxidase and The of peroxidase was to and with of of were and a of and 5 The peroxidase were and of SP5.2 was in to and for The was by The conjugate was on for and the conjugate were and of conjugate was by peroxidase activity using to peroxidase was to be used as a binding for were used to the receptor antibody 10 in was well of The were for in a and for with human in was were in a to receptor to receptor was with of well was with of in and for a with antibody were with in conjugate and used as were to well and for were with and the of bound conjugate was with peroxidase were in a of to Flt-1 the was used to that to Flt-1 and inhibit VEGF binding to the A library of was for binding to human Flt-1 receptor with of human antibody In the were by with In used to that with the VEGF binding of 10 was used to the bound A in the to bovine serum albumin used as a target was with the in the of and 400-fold in the of with the the of the a of were and of these showed that two and were with and and were with VEGF and in both were bound to with with in a new The binding of of the to Flt-1 receptor was using enzyme-linked immunosorbent assay on the demonstrated highest affinity to Flt-1 receptor the of and was for the studies To the of for Flt-1 the binding of the of was by to two human endothelial receptors, human low affinity VEGF receptor (KDR) and both with human as well as with the human significant interaction of the with KDR or proteins was In was also found to of Flt-1 to and not with receptor of of the SP5.2 to Flt-1 with the to that of was The SP5.2 was with the The SP5.2 binding to was using assay to that used for the that the was The SP5.2 binding to Flt-1 was with a μm The did not show significant binding The binding of the SP5.2 to Flt-1 was by the analysis of the bound SP5.2 with the A library used the as did not SP5.2 binding The showed that the inhibited SP5.2 binding to Flt-1 by the used in of SP5.2 in the were as a of SP5.2 with a to were were by The binding of these to Flt-1 receptor was by assay as assay demonstrated that the of or with binding of the SP5.2 to Flt-1 to and or on the binding on these the SP5.2 not in the Flt-1 has SP5.2 and VEGF for Flt-1 The of SP5.2 and to inhibit VEGF binding to Flt-1 was using a The for binding to was using 5 or μm fluorescein-labeled SP5.2 and to of human recombinant shown in both SP5.2 and with VEGF for the receptor The of SP5.2 binding was with 5 whereas of VEGF was to inhibit the interaction with Flt-1 receptor by the of to of the are on the that the synthetic SP5.2 has a binding affinity to that of the peptide. of SP5.2 to binding of the SP5.2 to and cells was The Flt-1 expression in the primary HCECs grown in the presence of 10 was by using Flt-1 antibody A and The neuroblastoma cell in which was not by was used as a and SP5.2 bound to VEGF-stimulated HCECs in a as by of the was also in the bound was in cells SP5.2 of μm of SP5.2 on VEGF-mediated of to inhibit induced in endothelial cells by VEGF was using in vitro in vitro were in and formation of capillary-like in the Inhibition of VEGF-mediated of of SP5.2 on endothelial proliferation were in and HCECs cells with VEGF using the as a of cell were with 10 and with of SP5.2 for The inhibited the proliferation of in a with an IC50 of 5 μm In HCECs the of SP5.2 and to the induced by growth and was the of 10 SP5.2 inhibited VEGF- and HCECs proliferation by and and demonstrated on proliferation suggesting that the SP5.2 is the of its interaction with Flt-1 the the did not proliferation Inhibition of VEGF-mediated of of HCECs induced by including VEGF165 and was in the or presence of SP5.2 or as T.A. D.B. R. J. 1993; PubMed Scopus Google Scholar). VEGF migration of HCECs the by and was by SP5.2 but not by the SP5.2 also inhibited migration of HCECs induced by known to high of Inhibition of VEGF-induced Both VEGF and induced capillary tube formation by HCECs grown in Matrigel™ A and SP5.2 was found to completely inhibit both VEGF- and formation of capillary-like whereas was Inhibition of VEGF-induced of VEGF on endothelial permeability was in a in vitro of the VEGF has shown to by endothelial cells in vitro and in W. J. PubMed Google Scholar, S. M. W. PubMed Scopus Google Scholar). A of the to VEGF in permeability of HCECs for the of VEGF was by SP5.2 to the cells the of 5 μm SP5.2-β-gal explore the that the SP5.2 can be used as a targeting for of such as and to a the the and the the of was and by affinity The SP5.2-β-gal affinity and for the Flt-1 receptor was by assay using a recombinant as that SP5.2-β-gal binds to Flt-1 receptor with an of suggesting that the has binding affinity compared with the synthetic SP5.2 peptide. the SP5.2-β-gal showed significant interaction to KDR receptor that SP5.2 can potentially be used as a targeting for therapeutic proteins to be such as tumor or a that the therapeutic can be to SP5.2 was also SP5.2 was with the peroxidase using as a conjugate and 5 of SP5.2 peroxidase The Flt-1 binding of the conjugate was by the peroxidase with was used as a SP5.2 peroxidase conjugate demonstrated binding to whereas binding to Flt-1 of the conjugate was The binding of the SP5.2 peroxidase conjugate was 400-fold that of the The of that SP5.2 can potentially be to therapeutic such as and with a therapeutic studies have shown that antiangiogenic is a for the of D. R. C. N. F. A. Full Text Full Text PDF PubMed Scopus Google Scholar, K. A. T.P. J. Med. 30: PubMed Scopus Google Scholar, M. Google Scholar, S. M. G. G. Curr. Med. Chem. PubMed Scopus Google Scholar). Vascular endothelial growth factor (VEGF) and its receptors are the focus of intense interest because of their role in that VEGF has also shown to play a role in development of vascular in R. J. 2000; PubMed Scopus Google Scholar) and cerebral S. PubMed Scopus Google Scholar). VEGF activities are mediated through its binding to two high affinity receptors Flt-1 and The VEGF-Flt-1 receptor system plays an important role in the of tumor angiogenesis, which makes Flt-1 an important target for antiangiogenic has shown that the interaction VEGF and Flt-1 can in the of and human M. G. M. J. R. M. PubMed Scopus Google Scholar, S. K. S. M. T. H. M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). the of a new VEGF by of a library In of of a library Flt-1 on magnetic in of of binding to The of a the SP5.2 was for SP5.2 was for its to endothelial receptors such as and showed any significant binding compared with was a significant and interaction with the of also to was used to important in the SP5.2 and the binding of the were by on these a of the SP5.2 important for the binding to Flt-1 receptor was identified were not in the Flt-1 have demonstrated that SP5.2 of VEGF-mediated events in cells such as and HCECs. SP5.2 was found to inhibit of in these cells induced by including angiogenesis, and vascular these were and with to VEGF compared with growth SP5.2 was also shown to antagonize of known to with VEGF and to through Flt-1 and receptors J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. D. A. L. D. J. S. De M. F. S. G. K. D. U. M. S. D. C. D.J. Persico G. D. M. D. Nat. Med. PubMed Scopus Google Scholar). to of Flt-1 and KDR receptors, have a that events mediated through Flt-1 are for of VEGF-induced angiogenesis, such as of cell and of endothelial cells, whereas KDR is important in endothelial proliferative K. G. M. S. T. J. PubMed Scopus Google Scholar, U. J. H. S. C. W. 30: PubMed Scopus Google Scholar, W. M. Google Scholar). has recently demonstrated that Flt-1 and KDR can in which Flt-1 in the of proliferative to VEGF S. M. G. G. Curr. Med. Chem. PubMed Scopus Google Scholar). in cells with KDR VEGF165 to the KDR receptor in a that the of signal N. Curr. Opin. 2000; PubMed Scopus Google Scholar). SP5.2, selectively binding to Flt-1 receptor in in vitro was of both events such as capillary-like tube vascular endothelial and endothelial is in with studies suggesting that Flt-1 is in the proliferative of through its with KDR S. M. G. G. Curr. Med. Chem. PubMed Scopus Google Scholar). signal and its activity for endothelial cells can also be inhibited SP5.2 binding to the receptor Further studies are for of the of SP5.2. It is well known that identified during library show as G. S. M. I. F. D. B. C. J. Biochem. 2000; PubMed Scopus Google Scholar, M. S. D. S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: PubMed Scopus Google Scholar). The two for are: synthetic not have in the of is by in which the is and of to in the of and in compared with the synthetic peptide. The synthetic of SP5.2 as a demonstrated a affinity in a variety of binding A to increase binding of the synthetic is to its by to a such as a J. Chem. PubMed Scopus Google Scholar, S. Chem. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, H. J. Sci. Full Text Full Text PDF PubMed Scopus Google Scholar). To explore a of of SP5.2, two of were produced. The was a recombinant of SP5.2 with in which SP5.2 was as a the of the that was used as a The was a chemical conjugate of SP5.2 and peroxidase in which to of the were to the The resulting products showed binding to Flt-1 compared with the original synthetic peptide. The in the Flt-1 binding were (200-fold compared with for the with a of the peptide, and compared with in the of the that SP5.2 can potentially be used as an targeting as an antiangiogenic therapeutic of SP5.2 are being in SP5.2 with therapeutic proteins such as in as well as with with In of the new peptide, SP5.2, which is an and Flt-1 and its have in for and or proliferative with Flt-1 in endothelial and for their

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