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.Electronic supplementary materialThe online version of this article (doi:10.1007/s10456-012-9302-0) contains supplementary material, which is available to authorized users.

Antagonism of PDGF-BB suppresses subretinal neovascularization and enhances the effects of blocking VEGF-A

“…A VEGF-A antagonistic DARPin that selectively binds VEGF-A with an apparent IC 50 of 10 pM has been shown to suppress suture-induced corneal NV in rabbits and laser-induced choroidal NV in rats after topical administration [14]. In the current study, we have extended those findings to show that systemic administration of an anti-VEGF-A DARPin reduces laser-induced choroidal NV in mice and reduces the incidence and severity of exudative retinal detachments in a particularly severe model, Tet/opsin/VEGF double transgenic mice.…”

“…Libraries have been constructed to produce DARPins with random surface that can be screened for binding to target proteins [12, 13]. A DARPin that selectively binds VEGF-A with a Kd of 2 pM has been characterized and when injected into the vitreous cavity of rabbits, it suppresses VEGF-induced leakage [14]. It also suppresses laser-induced choroidal NV in rats after topical administration to the cornea.…”

Recent developments in the treatment of age-related macular degeneration

“…Animal studies indicate that dosing frequency in patients may be reduced compared with current therapy (67). A phase I dose escalation study demonstrated the overall safety and efficacy of MP0112 (NCT01086761) (68).…”

Biobetters

“…From such combinatorial libraries DARPins with high affi nities have been selected, normally via ribosome display, against a variety of proteins, including several medically relevant targets: for example, HIV gp120 (Mann et al 2013 ;Schweizer et al 2008 ), EpCAM (Stefan et al 2011 ), Alzheimer amyloid-β peptide (Aβ) (Hanenberg et al 2014 ) and, as will be described in greater detail below, VEGF-A (Stahl et al 2013 ). Since the advantageous biophysical properties of the parental ankyrin scaffold such as high-level expression, solubility, and stability are often retained in these DARPins, they are also well suited for the generation of multispecifi c constructs (Molecular Partners, http://www.molecularpartners.com ).…”