Comparison of Binding Characteristics and In Vitro Activities of Three Inhibitors of Vascular Endothelial Growth Factor A

We report the results of the determination of affinity constants for binding of vascular endothelial growth factor A to ranibizumab, aflibercept, and bevacizumab by surface plasmon resonance using different assay formats as well as the potency of inhibition of VEGF

Jihong Yang; Xiangdan Wang; Germaine Fuh; Lanlan Yu; Eric Wakshull; Mehraban Khosraviani; Eric S. Day; Barthélemy Demeule; Jun Liu; Steven J. Shire; Napoleone Ferrara; Sandeep Yadav

2014

Scholarcy highlights

  • The determination of binding affinity of a therapeutic protein to a target is an integral part of pharmaceutical development
  • The results are very comparable to those reported previously.1−3 At equivalent molar ratios, ranibizumab was able to displace aflibercept from preformed aflibercept/vascular endothelial growth factor A complexes in solution as assessed by sedimentation velocity analytical ultracentrifugation), whereas aflibercept was not able to significantly displace ranibizumab from preformed ranibizumab/VEGF complexes
  • Article competition experiments were conducted using a preformed inhibitor/VEGF complex challenged with a different VEGF inhibitor to assess whether the previously reported ∼100-fold higher affinity of aflibercept to Recombinant human VEGF165 compared with ranibizumab4 is valid in free solution, i.e., no binding to a surface as in surface plasmon resonance measurements
  • In the present study, when we consider all Biacore data, it is evident that binding affinities and kinetics of all three inhibitors of homodimeric rhVEGF are highly assay format dependent
  • One hypothesis is that the molecular structure of aflibercept might have a different flexibility than a conventional antibody or antibody fragment such as bevacizumab or ranibizumab
  • As a result the overall molecular conformation/orientation of aflibercept on a sensor chip might be quite dependent on how the molecule is immobilized and result in very different VEGF binding kinetics and affinities using various assay formats
  • While optimizing therapeutic candidates to enhance their binding affinities is often useful in developing efficacious drugs, the contribution of affinity alone should always be interpreted in the context of mechanism of action of the drug and disease biology.32 it has been reported that a lower affinity anti-TfR variant showed improved brain uptake of the molecule compared with a high affinity variant, resulting in successful development of a therapeutic candidate for treating Alzheimer’s disease.33 Taking all into consideration, including results of the bovine retinal microvascular endothelial cell assay, as well as recent clinical trial results in AMD,24,34 it appears that the affinities of ranibizumab and aflibercept binding to their target, vascular endothelial growth factor A, are

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