title: Novel affinity binders for neutralization of vascular endothelial growth factor (VEGF) signaling.
abstract: Angiogenesis denotes the formation of new blood vessels from pre-existing vasculature. Progression of diseases such as cancer and several ophthalmological disorders may be promoted by excess angiogenesis. Novel therapeutics to inhibit angiogenesis and diagnostic tools for monitoring angiogenesis during therapy, hold great potential for improving treatment of such diseases. We have previously generated so-called biparatopic Affibody constructs with high affinity for the vascular endothelial growth factor receptor-2 (VEGFR2), which recognize two non-overlapping epitopes in the ligand-binding site on the receptor. Affibody molecules have previously been demonstrated suitable for imaging purposes. Their small size also makes them attractive for applications where an alternative route of administration is beneficial, such as topical delivery using eye drops. In this study, we show that decreasing linker length between the two Affibody domains resulted in even slower dissociation from the receptor. The new variants of the biparatopic Affibody bound to VEGFR2-expressing cells, blocked VEGFA binding, and inhibited VEGFA-induced signaling of VEGFR2 over expressing cells. Moreover, the biparatopic Affibody inhibited sprout formation of endothelial cells in an in vitro angiogenesis assay with similar potency as the bivalent monoclonal antibody ramucirumab. This study demonstrates that the biparatopic Affibody constructs show promise for future therapeutic as well as in vivo imaging applications.
authors.affiliation: Division of Protein Technology, School of Biotechnology, KTH, Royal Institute of Technology, AlbaNova University Center, 106 91, Stockholm, Sweden.
authors.forename: Filippa
authors.initials: F
authors.lastname: Fleetwood
authors.affiliation: Division of Protein Technology, School of Biotechnology, KTH, Royal Institute of Technology, AlbaNova University Center, 106 91, Stockholm, Sweden.
authors.forename: Rezan
authors.initials: R
authors.lastname: Güler
authors.affiliation: Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Dag Hammarskjöldsv. 20, Uppsala, Sweden.
authors.forename: Emma
authors.initials: E
authors.lastname: Gordon
authors.affiliation: Division of Protein Technology, School of Biotechnology, KTH, Royal Institute of Technology, AlbaNova University Center, 106 91, Stockholm, Sweden.
authors.forename: Stefan
authors.initials: S
authors.lastname: Ståhl
authors.affiliation: Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Dag Hammarskjöldsv. 20, Uppsala, Sweden.
authors.forename: Lena
authors.initials: L
authors.lastname: Claesson-Welsh
authors.affiliation: Division of Protein Technology, School of Biotechnology, KTH, Royal Institute of Technology, AlbaNova University Center, 106 91, Stockholm, Sweden. lofblom@kth.se.
authors.forename: John
authors.initials: J
authors.lastname: Löfblom
mesh_terms: D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D061067:Antibodies, Monoclonal, Humanized; D000915:Antibody Affinity / Q000276:immunology; D001665:Binding Sites; D002460:Cell Line; D049109:Cell Proliferation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005434:Flow Cytometry; D057809:HEK293 Cells; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D009369:Neoplasms / Q000188:drug therapy / Q000473:pathology; D009389:Neovascularization, Pathologic / Q000473:pathology*; D010766:Phosphorylation; D011485:Protein Binding / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000276:immunology / Q000378:metabolism*; D000096662:Ramucirumab
chemical_list: D020533:Angiogenesis Inhibitors; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D048049:Extracellular Signal-Regulated MAP Kinases; D011993:Recombinant Fusion Proteins; D042461:Vascular Endothelial Growth Factor A; D040301:Vascular Endothelial Growth Factor Receptor-2; D000096662:Ramucirumab; C467484:VEGFA protein, human; C000588866:KDR protein, human
keywords: Affibody molecules; Angiogenesis; Biparatopic affinity protein; Bispecific; VEGF; VEGFR2