Developing Somatic Hypermutation as a Protein Engineering Tool to Study Angiopoietin Binding

The angiopoietin (Ang) and Tie families play an important role in the latter stages of vascular development and in adult vasculature. A variety of studies on Ang1 ligand have provided compelling evidence of its therapeutic potential and along with Ang2 plays a major role in various protective and pathological conditions. Understanding the Ang-Tie interaction by manipulation of the angiopoietins is a very desirable prospect for developing it as a protein therapeutic drug. The aim of this project was to examine the molecular basis of Ang binding using a combination of rational mutagenesis and directed evolution. Directed evolution is a powerful strategy for protein engineering. A new method of directed evolution using mammalian surface display combined with mutagenesis driven by somatic hypermutation (SHM) was investigated for engineering Ang proteins with altered binding characteristics (to Tie1 and Tie2). The Ang receptor binding domains (RBD) were cloned linked to the asialoglycoprotein receptor (ASGPR) transmembrane domain. The fusion protein produced was capable of expression on the extracellular cell surface. Activationinduced (cytidine) deaminase (AID) expressing B cells were used for expression of this fusion protein. SHM driven by AID is capable of mutating highly expressed transgenes; this was used to generate the mutant library. The library was screened for enhanced Tie2 binding affinity and acquired Tie1 binding function by Fluorescence Activated Cell Sorting (FACS). Unstable expression of AID in the cell line and non-specific binding of Tie1 to the cell surface proteins in the binding screen prevented efficient selection of desired mutants. However, Ang2 FReD (fibrinogen-related domain) mutants were generated by SHM and rational mutagenesis. These were analysed and compared to the wild-type Ang2 FReD protein for expression by Western blotting and binding affinity determined by flow cytometry.