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Xiangdan Wang, Jihong Yang; Analysis of the binding affinity of vascular endothelial growth factor A (VEGF) to ranibizumab, aflibercept and bevacizumab. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1961. doi: https://doi.org/.
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To evaluate affinities and kinetics of recombinant human VEGF (rhVEGF) binding to anti-VEGF therapeutics in a comprehensive Biacore study.
The binding affinities of anti-VEGF therapeutics to rhVEGF were evaluated using Biacore T200 at 37 oC. Three assay formats were used (Figure). To evaluate the 3 anti-VEGF molecules in their native conformation, rhVEGF was directly immobilized onto a sensor chip while the anti-VEGF molecules were injected at different concentrations as analytes in the liquid phase (Format 1). To minimize any potential bias on the binding that may result from rhVEGF immobilization, the anti-VEGF molecules were immobilized as ligands and different concentrations of rhVEGF were injected as analyte (Format 2). Finally the anti-VEGF molecules were indirectly captured onto a sensor chip, and rhVEGF was used as the analyte (Format 3).
In the direct comparison of binding (Format 1) ranibizumab, aflibercept, and bevacizumab showed average dissociation equilibrium constant (KD) values of 67, 9263, and 4456 pM, respectively. A much slower dissociation rate constant was obtained for ranibizumab than for aflibercept or bevacizumab, resulting in a higher affinity for rhVEGF by ranibizumab. This observation was consistent with data from Format 2, although the extremely slow dissociation of ranibizumab from rhVEGF, and challenges in data fitting for aflibercept precluded an accurate determination of kinetics parameters for the binding of these molecules to rhVEGF. Using Format 3, aflibercept had a KD of 1.9 pM for rhVEGF. However this format was found to be unsuitable for evaluating ranibizumab binding to rhVEGF because a significant amount of ranibizumab dissociated from the capture antibody during sample analysis.
In a direct comparison under uniform conditions, ranibizumab had a higher binding affinity for rhVEGF than aflibercept. The Biacore assay format profoundly affected the binding of aflibercept to VEGF and the indirect capture format was not suitable for evaluating ranibizumab binding to rhVEGF. This study highlights the importance of rigorous experimental design and careful execution of in vitro binding studies. In addition, it suggested that biological implications of the binding affinity/kinetics results need to be interpreted within broader context including potency, pharmacokinetics and clinical efficacy data.
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