Antagonists to Human and Mouse Vascular Endothelial Growth Factor Receptor 2 Generated by Directed Protein Evolution In Vitro Original Research Article

Using directed in vitro protein evolution, we generated proteins that bound and antagonized the function of vascular endothelial growth factor receptor 2 (VEGFR2). Binders to human VEGFR2 (KDR) with 10–200 nM affinities were selected by using mRNA display from a library (1013 variants) based on the tenth human fibronectin type III domain (10Fn3) scaffold. Subsequently, a single KDR binding clone (Kd = 11 nM) was subjected to affinity maturation. This yielded improved KDR binding molecules with affinities ranging from 0.06 to 2 nM. Molecules with dual binding specificities (human/mouse) were also isolated by using both KDR and Flk-1 (mouse VEGFR2) as targets in selection. Proteins encoded by the selected clones bound VEGFR2-expressing cells and inhibited their VEGF-dependent proliferation. Our results demonstrate the potential of these inhibitors in the development of anti-angiogenesis therapeutics.