title: PEGylation potentiates the effectiveness of an antagonistic peptide that targets the EphB4 receptor with nanomolar affinity.
abstract: The EphB4 receptor tyrosine kinase together with its preferred ligand, ephrin-B2, regulates a variety of physiological and pathological processes, including tumor progression, pathological forms of angiogenesis, cardiomyocyte differentiation and bone remodeling. We previously reported the identification of TNYL-RAW, a 15 amino acid-long peptide that binds to the ephrin-binding pocked of EphB4 with low nanomolar affinity and inhibits ephrin-B2 binding. Although ephrin-B2 interacts promiscuously with all the EphB receptors, the TNYL-RAW peptide is remarkably selective and only binds to EphB4. Therefore, this peptide is a useful tool for studying the biological functions of EphB4 and for imaging EphB4-expressing tumors. Furthermore, TNYL-RAW could be useful for treating pathologies involving EphB4-ephrin-B2 interaction. However, the peptide has a very short half-life in cell culture and in the mouse blood circulation due to proteolytic degradation and clearance by the kidneys and reticuloendothelial system. To overcome these limitations, we have modified TNYL-RAW by fusion with the Fc portion of human IgG1, complexation with streptavidin or covalent coupling to a 40 KDa branched polyethylene glycol (PEG) polymer. These modified forms of TNYL-RAW all have greatly increased stability in cell culture, while retaining high binding affinity for EphB4. Furthermore, PEGylation most effectively increases peptide half-life in vivo. Consistent with increased stability, submicromolar concentrations of PEGylated TNYL-RAW effectively impair EphB4 activation by ephrin-B2 in cultured B16 melanoma cells as well as capillary-like tube formation and capillary sprouting in co-cultures of endothelial and epicardial mesothelial cells. Therefore, PEGylated TNYL-RAW may be useful for inhibiting pathological forms of angiogenesis through a novel mechanism involving disruption of EphB4-ephrin-B2 interactions between endothelial cells and supporting perivascular mesenchymal cells. Furthermore, the PEGylated peptide is suitable for other cell culture and in vivo applications requiring prolonged EphB4 receptor targeting.
authors.affiliation: Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America.
authors.forename: Roberta
authors.initials: R
authors.lastname: Noberini
authors.forename: Sayantan
authors.initials: S
authors.lastname: Mitra
authors.forename: Ombretta
authors.initials: O
authors.lastname: Salvucci
authors.forename: Fatima
authors.initials: F
authors.lastname: Valencia
authors.forename: Srinivas
authors.initials: S
authors.lastname: Duggineni
authors.forename: Natalie
authors.initials: N
authors.lastname: Prigozhina
authors.forename: Ke
authors.initials: K
authors.lastname: Wei
authors.forename: Giovanna
authors.initials: G
authors.lastname: Tosato
authors.forename: Ziwei
authors.initials: Z
authors.lastname: Huang
authors.forename: Elena B
authors.initials: EB
authors.lastname: Pasquale
mesh_terms: D000818:Animals; D002460:Cell Line; D018920:Coculture Techniques; D003470:Culture Media / Q000494:pharmacology; D036388:Ephrin-B2 / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006207:Half-Life; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D051379:Mice; D008832:Microchemistry; D018919:Neovascularization, Physiologic / Q000187:drug effects; D010455:Peptides / Q000097:blood / Q000494:pharmacology*; D010496:Pericardium / Q000166:cytology; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D011092:Polyethylene Glycols / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D036224:Receptor, EphB4 / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D003470:Culture Media; D036388:Ephrin-B2; D010455:Peptides; C509441:TNYL-RAW peptide; D019000:Phosphotyrosine; D011092:Polyethylene Glycols; D036224:Receptor, EphB4