Abstract
IntroductionAngiopoietin-2 (Ang2) is a secreted ligand whose concentration is increased in several cardiovascular diseases, and which causes vascular inflammation, microvessel disintegration, cardiac fibrosis and myocardial damage. Ang2 binds the endothelial receptor Tie2, where it competes for binding with the protective ligand Ang1. In this study we aim to develop a ligand-trap to block Ang2 action. To do this we used directed protein evolution to change the binding specificity of the Tie2 ectodomain so that it specifically binds Ang2. The evolved ectodomain will then be used as a soluble trap for introduction into the circulation to bind and sequester Ang2 preventing it from exerting its effects on the endothelium.Methods and resultsTie2 ectodomain was evolved using a novel DT40 cell surface display and evolution system. Evolutions were performed for selective binding to Ang2 by iterative cycles of mutation and selection. Variants evolved for Ang2 binding were then sequenced revealing key residues in the Ang1/2 binding domain determining ligand specificity. Fusion proteins containing the evolved variants were expressed, purified and tested for their ability to selectively bind Ang2, rather than Ang1 and Ang4. These Ang2-selective ligand-traps were found to inhibit Ang2 action on endothelial cells, inhibit adhesion of platelet/leucocyte aggregates to endothelial monolayers and suppress LPS-induced oedema.Conclusions and implicationsUsing a novel method of cell surface display and directed evolution we have evolved Tie2 ectodomain to selectively bind Ang2. This has revealed the key amino acid residues determining ligand-binding specificity of Tie2. In addition we created a series of selective Ang2 ligand-traps. These traps are almost identical to the endogenous receptor ectodomain, differing in four or fewer amino acid residues from endogenous ectodomain, and are able to inhibit the actions of Ang2. These ligand-traps have the potential for development as therapeutics to block the pathogenic and inflammatory actions of Ang2 on the cardiovascular system.