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.

Association between ultrasound findings and serum levels of vascular endothelial growth factor in ampullary pregnancy

“…Inhibitors of VEGF, such as ranibizumab, aflibercept, and bevacizumab, are already used in patients with certain diseases, such as wet, age-related macular degeneration, retinal vein occlusion, and diabetic macular edema, among others (32). This treatment could be an alternative to surgical intervention in ectopic cases with higher serum VEGF concentrations.…”

Solubility Challenges in High Concentration Monoclonal Antibody Formulations: Relationship with Amino Acid Sequence and Intermolecular Interactions

“…SEDFIT has been previously used for antibody reversible interaction systems as recently documented. 22 All s values obtained with the c(s) distribution were converted to S 20,w with SEDNTERP (http://sednterp.unh.edu) using the measured density (ρ) and viscosity (η) of buffer and protein partial specific volume (ν ̅ = 0.73 mL/g) estimated from amino acid composition as follows: 23,24 where T in the subscript stands for experimental temperature, B for buffer, w for water, and 20,w for standard conditions of water at 20°C. The extrapolation to zero concentration of the S 20,w versus concentration plot yields a value for S°2 0,w , which is the sedimentation coefficient in the limit of infinite dilution, thus representative of size of a single molecule, unaffected by intermolecular interactions.…”

Impact of SPR biosensor assay configuration on antibody: Neonatal Fc receptor binding data

“…When different assay formats yield consistent results, this suggests that the biological activity of the assay reagents has not been compromised by immobilization. Although discrepant results from biosensor assays using different immobilization formats have been reported, 51,52,53 in their meta-analysis benchmarking study of optical biosensor data from over 1200 publications, Rich and Myszka 54 state that only a few investigators have directly compared multiple immobilization and capture methods to address the potential caveats associated with protein immobilization methods.…”