Directed Evolution of an Angiopoietin-2 Ligand Trap by Somatic Hypermutation and Cell Surface Display

Background: The ligand angiopoietin2 contributes to vascular diseases. Results: A new directed evolution method was used to create a specific angiopoietin2-binding protein from a nonspecific angiopoietin receptor. Conclusion: The receptor binding specificity can be switched with just three residue changes. Significance: The new protein has therapeutic potential, and the directed evolution method has advantages for evolving mammalian proteins. Tie2 is a receptor tyrosine kinase that is essential for the development and maintenance of blood vessels through binding the soluble ligands angiopoietin 1 (Ang1) and 2 (Ang2). Ang1 is constitutively produced by perivascular cells and is protective of the adult vasculature. Ang2 plays an important role in blood vessel formation and is normally expressed during development. However, its re-expression in disease states, including cancer and sepsis, results in destabilization of blood vessels contributing to the pathology of these conditions. Ang2 is thus an attractive therapeutic target. Here we report the directed evolution of a ligand trap for Ang2 by harnessing the B cell somatic hypermutation machinery and coupling this to selectable cell surface display of a Tie2 ectodomain. Directed evolution produced an unexpected combination of mutations resulting in loss of Ang1 binding but maintenance of Ang2 binding. A soluble form of the evolved ectodomain binds Ang2 but not Ang1. Furthermore, the soluble evolved ectodomain blocks Ang2 effects on endothelial cells without interfering with Ang1 activity. Our study has created a novel Ang2 ligand trap and provided proof of concept for combining surface display and exogenous gene diversification in B cells for evolution of a non-immunoglobulin target.