Abstract 9773: Phage Display of Designed Ankyrin Repeat Proteins (DARPins) against the I Domain Of Mac-1 Results in Novel Diagnostic and Therapeutic Agents Specifically Acting on Activated Monocytes
Introduction: Designed Ankyrin Repeat Proteins (DARPins) are a novel class of highly stable binding proteins (≈10-20kDa) and represent a valuable source for specific binders with picomolar affinities. The I domain is the activation-dependent binding site of the leukocyte integrin Mac-1 that is a central mediator of inflammation. Hypothesis: We assessed whether phage selection against the I domain results in DARPins that specifically bind to activated leukocytes without affecting non-activated ones.
Methods: Selection was performed on purified mouse I domain using a DARPin phage library with a high diversity (1010).
Results: After 3 panning rounds, specific phages were selected and DARPins expressed with a high yield (≈ 200 mg/L). PMA activation revealed that DARPins bind stronger to activated than to non-activated mouse monocytes (Fig.1). Static and dynamic adhesion assays proved that selected DARPins inhibit binding of Mac-1 to its ligands (Fig.2). Using a peritonitis model, DARPins inhibited leukocyte migration into inflammatory areas, thereby indicating strong therapeutic, anti-inflammatory effects (control vs DARPin: 940 vs. 230 cells, p<0.02). DARPins showed strong crossreactivity to activated human Mac-1.
Conclusion: Selected DARPins specifically bind to activated Mac-1 and can be used for direct monitoring of monocyte activation both in mice and humans. Thereby, based on the novel DARPin format we established a potential anti-monocyte drug that exerts strong anti-inflammatory effects in in vivo. Cross-reactivity with human Mac-1 promises translation into a broad spectrum of inflammation driven diseases.
- © 2012 by American Heart Association, Inc.