Binding and neutralization of vascular endothelial growth factor (VEGF) and related ligands by VEGF Trap, ranibizumab and bevacizumab

Abstract

Pharmacological inhibition of VEGF-A has proven to be effective in inhibiting angiogenesis and vascular leak associated with cancers and various eye diseases. However, little information is currently available on the binding kinetics and relative biological activity of various VEGF inhibitors. Therefore, we have evaluated the binding kinetics of two anti-VEGF antibodies, ranibizumab and bevacizumab, and VEGF Trap (also known as aflibercept), a novel type of soluble decoy receptor, with substantially higher affinity than conventional soluble VEGF receptors. VEGF Trap bound to all isoforms of human VEGF-A tested with subpicomolar affinity. Ranibizumab and bevacizumab also bound human VEGF-A, but with markedly lower affinity. The association rate for VEGF Trap binding to VEGF-A was orders of magnitude faster than that measured for bevacizumab and ranibizumab. Similarly, in cell-based bioassays, VEGF Trap inhibited the activation of VEGFR1 and VEGFR2, as well as VEGF-A induced calcium mobilization and migration in human endothelial cells more potently than ranibizumab or bevacizumab. Only VEGF Trap bound human PlGF and VEGF-B, and inhibited VEGFR1 activation and HUVEC migration induced by PlGF. These data differentiate VEGF Trap from ranibizumab and bevacizumab in terms of its markedly higher affinity for VEGF-A, as well as its ability to bind VEGF-B and PlGF.

Electronic supplementary material: The online version of this article (doi:10.1007/s10456-011-9249-6) contains supplementary material, which is available to authorized users.

Fig. 1

The effects of VEGF Trap, ranibizumab and bevacizumab on luciferase activation induced by VEGF-A₁₂₁, VEGF-A₁₆₅, human PlGF-2 (hPlGF-2) or mouse PlGF-2 (mPLGF-2) in HEK293/VEGFR1 cells. a Dose response curves for VEGF-A₁₂₁, VEGF-A₁₆₅ and hPlGF-2 yielded EC₅₀ values of 13, 17, and 29 pM, respectively. b Serial dilutions of VEGF Trap (open box), ranibizumab (triangle), or bevacizumab (closed circle) were added to HEK293/VEGFR1 cells along with 20 pM of VEGF-A₁₂₁. c Serial dilutions of VEGF Trap (open box), ranibizumab (triangle), or bevacizumab (closed circle) were added to HEK293/VEGFR1 cells along with 20 pM of VEGF-A₁₆₅. d Serial dilutions of VEGF Trap (open box), ranibizumab (triangle), or bevacizumab (closed circle) were added to HEK293/VEGFR1 cells along with 40 pM of human PlGF-2. e Dose response curve for mPlGF-2 yielded an EC₅₀ value of 10 pM (f). Serial dilutions of VEGF Trap were added to HEK293/VEGFR1 cells along with 20 pM of mPlGF-2. The cells were incubated for 6 h and OneGlo luciferase substrate was then added to each well. The plates were read on a luminometer and the data were plotted using a four parameter curve fit with GraphPad Prism. Each point represents a replica of 3 wells at each concentration

Fig. 2

The effects of VEGF Trap, ranibizumab and bevacizumab on luciferase activation induced by VEGF-A₁₂₁ and VEGF-A₁₆₅ in HEK293/VEGFR2 cells. a Dose response curves for VEGF-A₁₂₁ and VEGF-A₁₆₅ with EC₅₀ values of 70 and 30 pM, respectively. PlGF-2 was not active in this assay. b Serial dilutions of VEGF Trap (open box), ranibizumab (triangle) or bevacizumab (closed circle) were added to HEK293/VEGFR2 cells along with 20 pM of VEGF-A₁₂₁. c Serial dilutions of VEGF Trap (open box), ranibizumab (triangle) or bevacizumab (closed circle) were added to HEK293/VEGFR2 cells along with 20 pM of VEGF-A₁₆₅. The cells were incubated for 6 h and OneGlo luciferase substrate was then added to each well. The plates were read on a luminometer and the data were plotted using a four parameter curve fit with GraphPad Prism. Each point represents a replica of 3 wells at each concentration

Fig. 3

The effects of VEGF Trap, ranibizumab and bevacizumab on calcium mobilization induced byVEGF-A₁₆₅ in HUVEC. a A dose–response curve generated using serial dilutions of VEGF-A₁₆₅ (4.0 nM–0.023 pM) resulted in an EC₅₀ value of 5 pM. b Serial dilutions of VEGF Trap (open box), ranibizumab (triangle) or bevacizumab (closed circle) were added to HUVEC along with 20 pM of VEGF-A₁₆₅. The VEGF-A₁₆₅ was preincubated with the inhibitors for 10 min at 25°C. The solution was added to HUVEC preloaded with fluo-4 and the fluorescence of the well was determined on a FLIPR instrument. The data were plotted using a four parameter curve fit with GraphPad Prism. Each point represents duplicate wells at each concentration

Fig. 4

The effects of VEGF Trap, ranibizumab and bevacizumab on HUVEC migration. a HUVEC were placed in the upper compartment of the Boyden chamber and allowed to migrate towards basal media containing 0.1% fetal bovine serum with or without VEGF-A₁₆₅ or VEGF-A₁₆₅ mixed with four concentrations each of VEGF Trap (circles, solid line), ranibizumab (triangles, dotted line) or bevacizumab (squares, dashed line) ranging from 0.013 to 13 nM. The percentage of total migration (y-axis) was calculated as (F _Drug − F _Basal)/(F _Total− F _Basal) × 100; where F _Total is fluorescence in the presence of VEGF-A₁₆₅, F _Basal is fluorescence in the absence of VEGF-A₁₆₅, and F _Drug is fluorescence in the presence of VEGF-A₁₆₅ mixed with drug at a specific molar ratio (x-axis). b HUVEC migration was assessed in the absence and presence of human PLGF-2 (hPLGF-2) or mouse PLGF-2 (mPLGF-2) with and without a 100-fold molar excess of VEGF Trap (VGT), ranibizumab (RAN) or bevacizumab (BEV). Fold migration (y-axis) was calculated as the ratio F/F _Basal; where F is the total fluorescence measured for the indicated condition (x-axis) and F _Basal is the fluorescence in the absence of either hPLGF-2 or mPLGF-2. Statistical significance: *P < 0.05; **P < 0.01; ns, no significance. Values and error bars represent the average value and standard error of the mean from at least three independent experiments with each experiment containing four biological replicates per condition (total n = 12–16 per condition) for all conditions tested. AU arbitrary units