2013steelekhphd.pdf (20.42 MB)
Directed Evolution of Angiopoietin-Binding Proteins by Somatic Hypermutation and Cell Surface Display
thesisposted on 2013-06-27, 15:50 authored by Kathryn Helen Steele
Angiopoietin-2 (Ang2) is a secreted ligand that promotes blood vessel destabilisation, remodelling, leakage and inflammation in response to pro-inflammatory activators. In binding to its primary receptor, Tie2, it functions as a competitive antagonist of the anti-inflammatory and pro-quiescent agonist angiopoietin-1 (Ang1). Ang2 is markedly elevated in conditions associated with vascular dysfunction and therefore development of an Ang2 inhibitor has multiple potential clinical applications. Ligand traps are developed from native receptor ectodomain fragments and offer improved pharmacokinetics over monoclonal antibodies. However production of an Ang2-trap requires manipulation of the endogenous Tie2 receptor to permit Ang2 but not Ang1 binding, and this objective formed the inspiration for this study. The aim was to test whether the somatic hypermutation (SHM) gene diversification activity of B cells could be combined with cell surface display to create a streamlined system for evolving binding proteins. The data presented demonstrates that a cell surface-expressed Tie2 mutant library can be generated via single transfection of a chimeric Tie2 ectodomain-cell surface display construct into the hypermutating DT40 cell line. Subsequently selection of Ang2-binding phenotypes via fluorescently labelled-angiopoietin binding assay and Fluorescence Activated Cell Sorting (FACS) was repeated iteratively prior to sequencing ofTie2 from recovered cells. This approach was successful in isolating mutant forms of Tie2 with altered binding characteristics. Directed evolution is one of the most powerful approaches for manipulating binding properties of proteins but traditionally has involved laborious techniques including iterative cycles of mutagenesis, expression and selection with inter-species translation issues. This combination of vertebrate 'in-cell' diversification and 'on-cell' binding and selection demonstrates a sustainable approach for evolution of any protein.
Supervisor(s)Brindle, Nicholas; London, Nicholas
Date of award2013-06-01
Awarding institutionUniversity of Leicester