Highly potent VEGF-A-antagonistic DARPins as anti-angiogenic agents for topical and intravitreal applications

Abstract

The next-generation ophthalmic anti-VEGF therapeutics must aim at being superior to the currently available agents with regard to potency and improved drug delivery, while still being stable and safe to use at elevated concentrations. We show here the generation of a set of highly potent VEGF-A antagonistic DARPins (designed ankyrin repeat proteins) delivering these properties. DARPins with single-digit picomolar affinity to human VEGF-A were generated using ribosome display selections. Specific and potent human VEGF-A binding was confirmed by ELISA and endothelial cell sprouting assays. Cross-reactivity with VEGF-A of several species was confirmed by ELISA. Intravitreally injected DARPin penetrated into the retina and reduced fluorescein extravasation in a rabbit model of vascular leakage. In addition, topical DARPin application was found to diminish corneal neovascularization in a rabbit suture model, and to suppress laser-induced neovascularization in a rat model. Even at elevated doses, DARPins were safe to use. The fact that several DARPins are highly active in various assays illustrates the favorable class behavior of the selected binders. Anti-VEGF-A DARPins thus represent a novel class of highly potent and specific drug candidates for the treatment of neovascular eye diseases in both the posterior and the anterior eye chamber.

Fig. 1

Screening for potent VEGF-A-binding DARPins and characterization of their affinities by sandwich inhibition ELISA (Quantikine; see “Materials and methods” section). a Screening for quantitative VEGF-A-inhibiting DARPins. Numbers 1–10 represent 10 different VEGF-A binding DARPins used in this screening assay. L represents Lucentis (ranibizumab), A represents Avastin (bevacizumab), I1 and I2 represent two non-binding DARPin isotype controls. V1, V2, V3, and V0 represent the signals obtained for 25, 12.5, 6.25, and 0 pM VEGF-A, respectively, applied without any inhibitor. The dashed line represents the signal obtained with free 12.5 pM VEGF-A, corresponding approximately to the equivalent of 50 % inhibition. b IC50 analysis of DARPin #6 in the identical assay as a. DARPin #6 is displayed as one VEGF-binding DARPin representative; similar curves were obtained for other anti-VEGF DARPins identified in a

Fig. 2

Cellular screening for DARPins inhibiting VEGF-induced phosphorylation of VEGFR-2. VEGF-A (1.4 nM) was pre-incubated with 1.4 nM of different purified DARPins (#1–#10), isotype (negative) control DARPins (I1 and I2), as well as the controls ranibizumab (Lucentis, L), bevacizumab (Avastin, A). The signal of PBS (NI non-inhibited) and the signal obtained without addition of VEGF-A (NV) were used as controls

Fig. 3

HUVEC spheroid sprouting inhibition with DARPins. HUVEC spheroids were incubated with VEGF-A, mixed with either PBS, or test substances. Test substances were DARPins #2 and #4, and ranibizumab (2, 4, and L (Lucentis), respectively). a Average accumulated total sprout length per spheroid. All groups (L, 2 and 4) are statistically different from PBS controls using ANOVA analysis and Tukey Multiple Comparison post hoc test (p < 0.001). b Representative pictures of sprouting spheroids. The isotype control DARPin I2 showed equivalent results as the PBS control

Fig. 4

Inhibition of VEGF-A induced vascular leakage in rabbit eyes by intravitreal DARPin injection. The ratios of vascular leakage (fluorescein leakage in the eye) of the right (study) eyes in comparison to the left (untreated) eyes are shown. 50 μg DARPin #4 or PBS (in 100 μl volume each) were injected. The dotted line represents a ratio of ‘treated versus untreated eye’ of 1, which would be expected at complete inhibition of VEGF-A induced vascular leakage. Representative fluorescence scans are shown in Supplementary Figure 4

Fig. 5

Prevention of blood vessel growth using DARPins in a rabbit corneal suture model. a Two eyes treated with DARPin #4 eight times daily in 50 μl drops at 20 mg/ml for 20 days. b Corresponding PBS-treated eyes

Fig. 6

The effect of DARPin eye-drops on rat choroidal neovascularization (CNV) in comparison to Triamcinolon (positive control, ivt) [45] or PBS (buffer control, ivt). a Comparison of mean CNV lesion size of the different study groups. Neovascularization in eyes treated with Triamcinolon and topical DARPins was significantly reduced compared to PBS 24 days after Laser injury (p = 0.0065 for Triamcinolon and p = 0.025 for DARPin #6). b Representative pictures of CNV formation at day 15 taken with a Heidelberg retinal angiograph