Abstract 413: Designed Ankyrin Repeat Proteins Against Activation Specific Binding Sites of The Leukocyte Integrin αmβ2: Novel Strategies in Vascular Inflammation
Introduction: Vascular inflammation is the underlying condition of several cardiovascular diseases and is mainly mediated by activated leukocytes. The leukocyte integrin αMβ2 with its activation specific epitope (I domain) is strongly involved in leukocyte adhesion to endothelial cells and thus represents an interesting therapeutic target. Designed Ankyrin Repeat Proteins (DARPins) are a novel class of linear, thermostable, highly specific recombinant binding proteins that overcome several limitations of immunoglobulins.
Hypothesis: DARPins selected against the mouse I domain (mId) of αMβ2 bind specifically to activated leukocytes and can be used as a novel diagnostic tool as well as a therapeutic, anti-inflammatory agent.
Methods: Using phage display, binding proteins were selected against recombinant I domain. Specific binding behavior to only activated leukocytes was assessed in FACS. Docking studies were used to define specific interaction sites of selected DARPins with the I domain. Therapeutic, anti-inflammatory effects of anti-mId DARPins was assessed in a sepsis mouse model.
Results: DARPins selected against the I domain bind in FACS specifically to activated monocytes (activated vs. non-activated 61±4 % vs. 19±6 %, p<0.05). Docking studies revealed amino acid positions responsible for the specific binding behavior. Mutagenesis of these residues showed significantly reduced binding of the mutated DARPin using FACS analysis (anti-mId DARPin vs. mutated anti-mId DARPin 61±4 vs. 29±7, p<0.05) proving that binding of the wild type DARPin to its target is specific. Furthermore, anti- I domain DARPins showed anti-inflammatory effects in a mouse sepsis model (peritoneal cells: anti-mId DARPin vs control: 2,049±189 103/ml vs. 3,382±213 103/ml, p<0.01).
Conclusions: DARPins selected against the I domain of αMβ2 bind specifically to activated leukocytes and inhibit leukocyte function as a new class of anti-inflammatory agents under in vivo conditions.
Author Disclosures: P. Diehl: None. P. Siegel: None. J. Holien: None. N. Bassler: Employment; Modest; employee of Molecular Partners. U. Flierl: None. X. Tonnar: None. C. Olivier: None. N. Anto Michel: None. D. Wolf: None. A. Zirlik: None. D. Steiner: Employment; Modest; employee of Molecular Partners. C. Bode: None. M. Moser: None. M. Parker: None. K. Peter: None.
- © 2015 by American Heart Association, Inc.