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

Angiogenesis. 2013 Jan;16(1):101-11. doi: 10.1007/s10456-012-9302-0. Epub 2012 Sep 15.


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.

MeSH terms

  • Administration, Topical
  • Angiogenesis Inhibitors / administration & dosage
  • Angiogenesis Inhibitors / pharmacology*
  • Angiogenesis Inhibitors / therapeutic use
  • Animals
  • Ankyrin Repeat*
  • Blood Vessels / drug effects
  • Blood Vessels / growth & development
  • Choroidal Neovascularization / drug therapy
  • Choroidal Neovascularization / pathology
  • Disease Models, Animal
  • Drug Evaluation, Preclinical
  • Eye / blood supply
  • Eye / drug effects
  • Eye / pathology
  • Human Umbilical Vein Endothelial Cells / drug effects
  • Human Umbilical Vein Endothelial Cells / metabolism
  • Humans
  • Intravitreal Injections
  • Mice
  • Ophthalmic Solutions / pharmacology
  • Ophthalmic Solutions / therapeutic use
  • Protein Binding / drug effects
  • Rabbits
  • Rats
  • Rats, Inbred BN
  • Recombinant Proteins / administration & dosage
  • Recombinant Proteins / pharmacology*
  • Recombinant Proteins / therapeutic use
  • Vascular Endothelial Growth Factor A / antagonists & inhibitors*
  • Vascular Endothelial Growth Factor A / metabolism


  • Angiogenesis Inhibitors
  • Ophthalmic Solutions
  • Recombinant Proteins
  • Vascular Endothelial Growth Factor A