The experiments described herein provide a comprehensive assessment of the ability of VEGF Trap, ranibizumab and bevacizumab to bind and block the activity of VEGF family ligands in vitro, under identical experimental conditions. The data demonstrate that VEGF Trap binds human VEGF-A with higher affinity and a significantly faster association rate, thus neutralizing VEGF-A with greater potency than ranibizumab or bevacizumab. In addition, the studies show that VEGF Trap has the unique ability to bind the additional VEGF family ligands, VEGF-B and PlGF. Moreover, VEGF Trap also bound VEGF-A and PlGF isoforms from all mammalian species tested with similar high affinity, while neither ranibizumab nor bevacizumab efficiently bind and neutralize mouse or rat VEGF-A [46
Several published papers have provided binding affinity data for ranibizumab’s interactions with human VEGF-A [28
]. However, to date, binding affinity and specificity data have been provided only for the monovalent Fab fragment of bevacizumab (Fab-12), and not the full bivalent bevacizumab molecule itself. The equilibrium dissociation constant (KD
) for Fab-12 has been variously reported as 1.8 nM [36
] or 20 nM [28
], indicating an affinity improvement of ranibizumab over Fab-12 of 10–100-fold. Likewise, ranibizumab has been reported to be 30–100-fold more potent than Fab-12 in bioassays measuring VEGF-induced endothelial cell mitogenesis [26
]. However, measuring the kinetic binding parameters or in vitro activity of the Fab-12 fragment does not take into account potential avidity interactions of bivalent antibodies, especially when the binding partner is a dimeric ligand such as VEGF-A. These types of avidity driven interactions can significantly increase binding affinity, and potentially the potency of the bivalent antibody relative to that of the monovalent antigen binding fragment in cell-based assays and in vivo.
In the present study, Biacore and KinExA analyses have demonstrated that the equilibrium dissociation constants for VEGF Trap binding VEGF-A121
, were less than 1 pM, in close agreement with earlier reports [34
]. In contrast, ranibizumab exhibited a KD
of 46 pM for VEGF-A165
. While this represents an approximately 3–4-fold greater affinity for VEGF-A relative to SPR Biacore values previously reported for ranibizumab (KD
≤ 140 pM, [28
]; ≤179 pM, [37
]), it is nevertheless an ~94-fold weaker binding for VEGF-A165
relative to VEGF Trap (0.490 pM) (Table ). Similarly, the KD
of soluble VEGF Trap for VEGF-A165
, as determined by KinExA was 0.66 pM, while that of ranibizumab was 20.6 pM, approximately 30-fold lower than that of VEGF Trap.
Relative VEGF binding affinities and potency of VEGFR signaling blockade
Interestingly, the KD
of bevacizumab for VEGF-A165
as determined by Biacore was 58 pM, markedly lower than that reported previously for Fab-12 [28
] and within twofold of the binding affinity of ranibizumab. This was also the case for soluble equilibrium binding of bevacizumab in the Kinexa assay (KD
of 35.1 pM for bevacizumab and 20.6 pM for ranibizumab), and most likely reflects avidity interactions of the bivalent, full antibody molecule. However, like other conventional antibodies that bind dimeric targets, bevacizumab has the potential to form higher order complexes with VEGF, which under some conditions may act as immune complexes [53
]. In contrast, each molecule of VEGF Trap forms an inert 1 to 1 complex with VEGF, and cannot form higher order complexes [35
for VEGF Trap binding of VEGF-A documented in the SPR Biacore and KinExA assays translated into increased potency relative to ranibizumab and bevacizumab in all of the bioassays employed. Specifically, VEGF Trap was ~33–71-fold more potent than ranibizumab at inhibiting VEGF-A induced receptor activation in cell lines expressing either VEGFR1 or VEGR2 (Table ). Moreover, VEGF Trap was highly effective at reducing VEGF-A-induced calcium signaling in HUVEC, where it was ~130-fold more potent than ranibizumab (Table ). In addition to promoting endothelial cell proliferation and vascular permeability, VEGF-A is powerful mediator of endothelial cell migration [25
]. Consistent with the high potency of VEGF Trap to neutralize VEGF receptor activation, VEGF Trap was highly effective at blocking HUVEC migration induced by VEGF-A165
. In agreement with previous reports [38
], ranibizumab and bevacizumab were also effective at decreasing HUVEC migration, though they were less potent than VEGF Trap, such that a 10- to 100-fold molar excess of ranibizumab or bevacizumab was required to completely block VEGF-induced HUVEC migration, while VEGF Trap was effective at equimolar concentrations.
In the present studies, the ability of ranibizumab to neutralize VEGF-A activity in cell-based assays was only moderately better than that of bevacizumab. For example, the IC50 values for inhibition of activation of VEGFR1 and VEGFR2 by 20 pM VEGF-A were less than twofold lower for ranibizumab than bevacizumab (Table ). This corresponded closely to the observed differences in the binding kinetics of ranibizumab and the full length bivalent bevacizumab antibody, where the KD of bevacizumab for VEGF-A was within twofold of that of ranibizumab, as determined by both Biacore and KinExA assays (Tables , , ). Interestingly, bevacizumab was ~fivefold more potent than ranibizumab at neutralizing VEGF-A induced calcium influx in HUVEC. This finding may reflect the ~threefold faster association rate of bevacizumab (Table ), as ka is a critical determinant of potency in relatively acute cell-based assays.
The above findings stand in contrast to those recently described by Yu et al. [40
]. Specifically, ranibizumab and VEGF Trap were reported to be equally effective in blocking endothelial cell proliferation and migration in HUVEC, while bevacizumab was approximately tenfold less potent. Evaluation of MAPK phosphorylation, which reflects activation of intracellular signaling pathways downstream of the VEGF receptors, showed that all three agents completely blocked MAPK phosphorylation when the VEGF inhibitors were pre-incubated with VEGF-A overnight, before addition to the cells, while VEGF Trap was more potent than either ranibizumab or bevacizumab when preincubated with VEGF-A for shorter time periods (5 and 30 min). The apparent discrepancies with findings of the present study are likely attributable to the fact that Yu et al. [40
] utilized higher concentrations of exogenous VEGF-A in all of their cell-based assays, in the range of 0.15–1.25 nM. In other words, the concentration of ligand was above the KD
values for ranibizumab and bevacizumab, as well as VEGF Trap (Table ); under these assay conditions the IC50
is determined primarily by the concentration of ligand relative to that of the blocker, rather than by the binding affinity. Therefore, precise evaluation of the relative activity of different inhibitors in bioassays requires utilization of the lowest amount of VEGF-A practicable, so that the IC50
can reflect differences in binding affinity and not simply inhibition of activity at stoichiometric concentrations of inhibitor, which predominates under conditions where both antibody and ligand concentrations are well above the KD
For example, several studies published to date have reported that ranibizumab and bevacizumab are equally effective in neutralizing VEGF-induced endothelial cell proliferation at ‘clinically relevant’ concentrations, i.e., those that obtain in the eye shortly following intravitreal injection [38
], which are well above the equilibrium dissociation constants for both antibodies. Differences in activity emerge only when lower concentrations of drug are evaluated, or where acute bioassay readouts reflect differences in association rate constants. For example, Klettner et al. [39
], reported that at lower concentrations ranibizumab more efficiently neutralized VEGF secreted from retinal-choroidal cultures than did bevacizumab. Costa et al. [54
] also reported that ranibizumab was moderately more effective at inhibiting endothelial cell proliferation than bevacizumab, while in an acute assay bevacizumab more effectively inhibited VEGF-stimulated VEGFR2 and MAPK phosphorylation in human microvascular endothelial cells.
Binding kinetics and affinity are key determinants of the biological activity of antibody-like drugs. In addition to binding affinity, the activity of a drug is also influenced by the concentration present at the site of target activity, which is in turn dependent on tissue distribution and clearance, with larger molecules typically having longer half-lives. With respect to ocular delivery, it was estimated that biologically active concentrations of ranibizumab would be maintained in the vitreous for approximately 4 weeks following intravitreal injections of 0.5 mg [26
]. Indeed, monthly injection of 0.5 mg ranibizumab has proven to be the most effective regimen for the treatment of neovascular AMD, based on the outcomes of several phase III clinical trials [29
], and is the currently approved regimen for treating this disease. Using mathematical modeling, and the then available information on intravitreal clearance and binding affinities, Stewart [61
] predicted that the anti-VEGF bioactivity present in the vitreous 30 days following intravitreal (IVT) injection of 0.5 mg ranibizumab would be equivalent to that present at 27–38 days following an injection of 1.25 mg bevacizumab. More recently, using the same modeling approach, Stewart and Rosenfeld [62
] predicted the intraocular biological activity comparable to that of 0.5 mg ranibizumab at 30 days post-injection would be maintained for approximately twice that time following injection of 0.5 mg VEGF Trap, and potentially as long as 12 weeks following IVT injection of 2 mg VEGF Trap. This substantial theoretical increase in the relative duration of VEGF neutralizing activity was driven primarily by the higher binding affinity of VEGF Trap for VEGF-A compared to ranibizumab, with a lesser contribution of the predicted longer intravitreal half-life of VEGF Trap (e.g. 4.7 days in rabbits, compared to ~2.9 days for ranibizumab, [63
]. Thus, modeling studies suggested that intravitreal administration of the current clinical doses of ranibizumab and bevacizumab would result in effective VEGF-A inhibition of relatively similar duration, while VEGF Trap might be as efficacious as ranibizumab, but with less frequent dosing.
While it remains to be unequivocally determined whether the durations of bioactivity of these VEGF blockers predicted by the above modeling studies will be confirmed by clinical experience, data available to date suggest that the results of these modeling studies may prove reasonably accurate. For example, several clinical studies have investigated alternative strategies to monthly ranibizumab injection, including quarterly (every 3 months) or pro renata (PRN) injections following a treatment initiation phase comprising 3 monthly loading doses. Most large, well-controlled studies conducted to date have found that improvements in visual acuity attained during the initiation phase are lost during the quarterly or PRN maintenance phases [58
]. The recent CATT Trial produced the best results obtained to date using PRN dosing of ranibizumab, which was statistically non-inferior to that of monthly ranibizumab. This may reflect the fact that in the CATT study patients were followed monthly and rigorous criteria were established for retreatment [32
]. Nevertheless, the mean improvement in visual acuity attained in CATT using PRN ranibizumab was 1.6 letters below that of monthly ranibizumab, at the end of 1 year. Importantly, the effect of bevacizumab given monthly on visual outcomes was within 0.4 letters of that obtained with ranibizumab given monthly. However, bevacizumab administered PRN failed non-inferiority comparisons to monthly regimens for both antibodies, despite the fact that it was administered more frequently than ranibizumab PRN. These findings are in line with the predictions of modeling studies, as well as the results of the present report, which indicate that the binding affinity and in vitro activity of bevacizumab are moderately less than those of ranibizumab. Several additional large scale controlled trials are currently in progress to evaluate the effects of these two antibodies in patients with neovascular AMD, using both fixed and PRN dosing schedules [31
]. These studies, together with outcomes from the CATT trial following longer-term treatment, should provide a clearer picture of the relative clinical activity, and safety, of ranibizumab and bevacizumab.
Although fewer clinical trials have been conducted to date with VEGF Trap-Eye, the available data suggest that, as predicted in modeling studies, the increased affinity of VEGF Trap for VEGF-A may be reflected in clinical activity. For example, in a recent double masked phase 2 trial (CLEAR-IT 2) patients with exudative AMD were randomized to an initiation phase of either a single, or monthly IVT injections of VEGF Trap for 12 weeks at doses of either 0.5 or 2 mg. Patients were then switched to a PRN regimen at their originally assigned doses. Reports of the 1 year results described maintenance of statistically significant improvements in vision, retinal thickness and size of the CNV lesions [66
]. Here, patients initially dosed on a 2.0 mg monthly schedule received, on average, only 1.6 additional injections during the 40 week PRN period, and those initially dosed on a 0.5 mg monthly schedule received, on average, 2.5 injections. More recently, 1 year results have been reported from two phase 3 clinical trials (VIEW 1 and VIEW 2) in which VEGF Trap-Eye was dosed monthly at 0.5 or 2.0 mg in patients with wet AMD, or at 2.0 mg every other month following an initiation phase of 3 monthly doses. All VEGF Trap-Eye treatment arms, including the 2.0 mg every other month treatment regimen, produced improvements in visual acuity that were equivalent to that obtained in patients dosed with 0.5 mg ranibizumab monthly [68
The development of ranibizumab has demonstrated that binding multiple VEGF-A isoforms is of substantial benefit in the treatment of neovascular AMD, compared to treatment with pegaptanib, which binds only the 165 isoform of VEGF-A [23
]. Recent studies have implicated additional VEGF family members, notably PlGF and VEGF-B, in the pathology of ocular vascular diseases as well as some cancers [8
]. Therefore, a unique potential advantage of VEGF Trap relative to ranibizumab and bevacizumab is that it also binds VEGF-B and PlGF with high affinity. PlGF in particular has been shown to act in concert with VEGF-A to promote pathological angiogenesis, vascular leak and inflammation [8
], and like VEGF-A, levels of PlGF are elevated in the eyes of patients with diverse ocular vascular diseases, including wet AMD [15
]. Furthermore, genetic deletion or pharmacological inhibition of PlGF has been shown to inhibit choroidal neovascularization and inflammation, and to enhance the activity of VEGF-A targeted molecules in animal models of choroidal neovascularization [13
]. More recently, it has been reported that overexpression of VEGF-B in the murine retina, via adeno-associated virus gene transfer, also promotes retinal and choroidal neovascularization and blood-retinal barrier breakdown [76
]. These studies suggest that targeting PlGF and VEGF-B, in addition to VEGF-A, could be of added benefit in treating angiogenic ocular disorders.
Similarly, targeting these additional factors may be important in the oncology setting. First, these VEGF family ligands, most notably PlGF, have been implicated in promoting tumor growth [8
], therefore inhibiting these factors, in addition to VEGF-A, may prove therapeutically beneficial in treating cancer. Bevacizumab, which inhibits only VEGF-A, is approved for use in various cancer treatment settings. VEGF Trap, while not currently approved for use, has also exhibited efficacy in the oncology setting. Most recently it was reported to have an overall survival benefit in metastatic colorectal cancer [77
]. Changes in the levels of PlGF and other factors have been observed in patients with metastatic colorectal cancer treated with bevacizumab, during and following cessation of treatment [78
], and the authors of both studies suggested that increases in other pro-angiogenic factors may be one mechanism underlying the development of resistance to anti-VEGF therapy. However, further prospective evaluations are needed to confirm these hypotheses.
In summary, VEGF Trap demonstrated higher binding affinity for VEGF-A isoforms and greater potency in vitro than ranibizumab or bevacizumab. These attributes, in addition to its ability to bind VEGF-B and PlGF, could be of added benefit in treating various ocular disorders and cancers.