Consistent with differences in binding affinity, abicipar and aflibercept were significantly more potent in the HUVEC calcium mobilization assay than ranibizumab and bevacizumab (
Fig. 1B). Similarly, Papadopoulos et al.
56 demonstrated that aflibercept was more potent than ranibizumab and bevacizumab in a HUVEC calcium mobilization assay. However, in that study, the anti-VEGF compounds were preincubated with VEGF-A
165 for 1 hour. Previous results with ranibizumab and bevacizumab, molecules with slower VEGF-A
165-binding on-rates, demonstrated that potency measurements can be modulated by preincubating these inhibitors with VEGF-A
165 for different lengths of time prior to assessing potency.
50 In the studies presented here, we used a 4-hour preincubation to minimize the effect of differences in the association constants of various inhibitors when comparing their VEGF-A
165 neutralization potencies. Under these conditions, the potencies of abicipar and aflibercept were still significantly higher than those of ranibizumab and bevacizumab. These results differ from those reported by Yu et al.
50,57 In their experiments measuring effects on MAP kinase phosphorylation, HUVEC migration, and bovine retinal endothelial cell (BREC) proliferation, no differences were observed between ranibizumab and aflibercept. However, in contrast to the experiments presented here as well as those of Papadopoulos et al.,
56 the concentrations of VEGF used to drive cellular responses in those experiments (1.25 nM for MAP kinase phosphorylation, 500 pM for HUVEC migration, and 150 pM for BREC proliferation) far exceeded the K
d values for the compounds. Thus, the efficacy observed may largely reflect stoichiometric effects rather than differences in potency. More recently, Yang et al.
57 compared the potencies of ranibizumab, bevacizumab, and aflibercept in a BREC proliferation assay using 75 pM VEGF-A
165 and similarly did not detect differences between ranibizumab and aflibercept. The discrepancy between these results and ours, as well as those of Papadopoulos et al., could again reflect the concentrations of VEGF-A
165 used (75 pM versus 26 pM in the results presented here and 20 pM in the study by Papdopoulos et al.) and/or the extended assay times required to measure cellular proliferation (6 days).