Directed Evolution and Characterisaton of an Ang2-Selective Ligand Trap

The angiopoietin (Ang) ligands and their Tie2 receptor play a role in vascular growth, maintenance of adult vasculature and vascular remodelling. Many studies showed that Ang1 ligand has a protective effect for controlling of vascular morphogenesis and homeostasis whereas excess Ang2 has more deleterious effect on the vascular system. Upregulated expression of Ang2 is associated with several pathologies such as inflammation and tumour angiogenesis, and blocking Ang2 with antibodies or using transgenic approaches has been shown to be improve outcomes in preclinical models of these conditions. Ang2 inhibitors therefore have significant potential as therapeutics. Ligand traps are alternatives to antibodies for blocking the action of ligands. This study aims to use directed evolution to modify Tie2 extracellular domain to a form that selectively binds Ang2 binding, and test its ability to supress Ang2-mediated effects. Such a selective ectodomain would be a candidate Ang2 ligand trap. Directed evolution was attempted using a method that combines in-cell mutagenesis, utilizing somatic hypermutation, with cell surface display. Despite several attempts evolution was not successful. However, an evolved ectodomain was produced by others. This evolved ectodomain was analysed for binding specificity, cellular, in vivo effects. The ectodomain was found to be selective for Ang2 binding, and unable to bind Ang1 and Ang4. Furthermore, the evolved ectodomain was found to inhibit the antagonistic and agonistic effects of Ang2 on endothelial cell Akt signalling. Studies were also found that the evolved ectodomain was able to inhibit endothelial cell migration in response to high concentrations of Ang2. Preliminary in vivo work showed that the ectodomain was able to block localized oedema in a mouse model of lipopolysaccharide-induced inflammation. These findings suggest the evolved ectodomain would be a good candidate for development into an Ang2-ligand-trap.