title: Comparison of binding characteristics and in vitro activities of three inhibitors of vascular endothelial growth factor A.
abstract: The objectives of this study were to evaluate the relative binding and potencies of three inhibitors of vascular endothelial growth factor A (VEGF), used to treat neovascular age-related macular degeneration, and assess their relevance in the context of clinical outcome. Ranibizumab is a 48 kDa antigen binding fragment, which lacks a fragment crystallizable (Fc) region and is rapidly cleared from systemic circulation. Aflibercept, a 110 kDa fusion protein, and bevacizumab, a 150 kDa monoclonal antibody, each contain an Fc region. Binding affinities were determined using Biacore analysis. Competitive binding by sedimentation velocity analytical ultracentrifugation (SV-AUC) was used to support the binding affinities determined by Biacore of ranibizumab and aflibercept to VEGF. A bovine retinal microvascular endothelial cell (BREC) proliferation assay was used to measure potency. Biacore measurements were format dependent, especially for aflibercept, suggesting that biologically relevant, true affinities of recombinant VEGF (rhVEGF) and its inhibitors are yet to be determined. Despite this assay format dependency, ranibizumab appeared to be a very tight VEGF binder in all three formats. The results are also very comparable to those reported previously.1-3 At equivalent molar ratios, ranibizumab was able to displace aflibercept from preformed aflibercept/VEGF complexes in solution as assessed by SV-AUC, whereas aflibercept was not able to significantly displace ranibizumab from preformed ranibizumab/VEGF complexes. Ranibizumab, aflibercept, and bevacizumab showed dose-dependent inhibition of BREC proliferation induced by 6 ng/mL VEGF, with average IC50 values of 0.088 ± 0.032, 0.090 ± 0.009, and 0.500 ± 0.091 nM, respectively. Similar results were obtained with 3 ng/mL VEGF. In summary Biacore studies and SV-AUC solution studies show that aflibercept does not bind with higher affinity than ranibizumab to VEGF as recently reported,4 and both inhibitors appeared to be equipotent with respect to their ability to inhibit VEGF function.
authors.affiliation: Genentech, Inc. , 1 DNA Way, MS 56-2A, South San Francisco, California 94080, United States.
authors.forename: Jihong
authors.initials: J
authors.lastname: Yang
authors.forename: Xiangdan
authors.initials: X
authors.lastname: Wang
authors.forename: Germaine
authors.initials: G
authors.lastname: Fuh
authors.forename: Lanlan
authors.initials: L
authors.lastname: Yu
authors.forename: Eric
authors.initials: E
authors.lastname: Wakshull
authors.forename: Mehraban
authors.initials: M
authors.lastname: Khosraviani
authors.forename: Eric S
authors.initials: ES
authors.lastname: Day
authors.forename: Barthélemy
authors.initials: B
authors.lastname: Demeule
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Shire
authors.forename: Napoleone
authors.initials: N
authors.lastname: Ferrara
authors.forename: Sandeep
authors.initials: S
authors.lastname: Yadav
mesh_terms: D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000378:metabolism*; D000068258:Bevacizumab; D002417:Cattle; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects; D011485:Protein Binding; D000069579:Ranibizumab; D040262:Receptors, Vascular Endothelial Growth Factor / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D014461:Ultracentrifugation; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors* / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D011993:Recombinant Fusion Proteins; D042461:Vascular Endothelial Growth Factor A; C533178:aflibercept; D000068258:Bevacizumab; D040262:Receptors, Vascular Endothelial Growth Factor; D000069579:Ranibizumab
keywords: VEGF; affinity; aflibercept; analytical ultracentrifugation; bevacizumab; ranibizumab