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Selection of designed ankyrin repeat proteins (DARPins) from a phage library against activated αᴍβ₂ integrin
thesisposted on 09.02.2017, 03:40 by Diehl, Philipp
Introduction: Designed Ankyrin Repeat Proteins (DARPins) are a novel class of highly stable binding proteins and represent a valuable source for specific binders with picomolar affinities. The αᴍ I domain is the activation-dependent binding site of the leukocyte integrin Mac-1 (αᴍβ₂) that is a central mediator of inflammation. A DARPin that binds with a high affinity and specificity to Mac-1 would be of major benefit both as a diagnostic tool for the detection of monocyte activation as well as a therapeutic agent with a potential selectivity for activated monocytes. Aims: Phage display with selection against the αᴍ I domain was employed to generate DARPins that specifically bind to activated monocytes without affecting non-activated monocytes. Methods: Selection was performed on purified mouse αᴍ I domain using a DARPin phage library with a high diversity (10₁₀). Results: After three panning rounds, specific phages were selected and DARPins produced in E.co/i bacteria with a high yield (=100 mg/L). PMA activation revealed that DARPins bind stronger to activated than to non-activated mouse monocytes. Static and dynamic adhesion assays proved that selected DARPins inhibit binding of Mac-1 to its ligands. In addition, some DARPins showed cross-reactivity to activated human Mac-1. Conclusion: Selected DARPins specifically bind to activated monocytes and can be used for direct monitoring of monocyte activation both in mice and humans. In addition, using the novel DARPin format we established a potential anti-monocyte drug that presumably exerts strong anti-inflammatory effects. Cross-reactivity with human Mac-1 suggests successful translation towards generation of a new pharmaceutical reagent for the treatment of a broad spectrum of inflammatory diseases.