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.

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Designed Ankyrin Repeat Proteins (DARPins): Binding Proteins for Research, Diagnostics, and Therapy

The Annual Review of Pharmacology and Toxicology ◽

10.1146/annurev-pharmtox-010611-134654 ◽

2015 ◽

Vol 55 (1) ◽

pp. 489-511 ◽

Cited By ~ 268

Author(s):

Andreas Plückthun

Keyword(s):

Binding Proteins ◽

Ankyrin Repeat ◽

Repeat Proteins ◽

Ankyrin Repeat Proteins ◽

Diagnostics And Therapy

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Designed Ankyrin Repeat Proteins as Novel Binders for Ultrasound Molecular Imaging

Ultrasound in Medicine & Biology ◽

10.1016/j.ultrasmedbio.2021.04.027 ◽

2021 ◽

Author(s):

Alexandra Kosareva ◽

Mukesh Punjabi ◽

Amanda Ochoa-Espinosa ◽

Lifen Xu ◽

Jonas V. Schaefer ◽

...

Keyword(s):

Molecular Imaging ◽

Ankyrin Repeat ◽

Repeat Proteins ◽

Ultrasound Molecular Imaging ◽

Ankyrin Repeat Proteins

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Thermostable designed ankyrin repeat proteins (DARPins) as building blocks for innovative drugs

10.1101/2021.04.27.441521 ◽

2021 ◽

Author(s):

Johannes Schilling ◽

Christian Jost ◽

Ioana Mariuca Ilie ◽

Joachim Schnabl ◽

Oralea Buechi ◽

...

Keyword(s):

Melting Temperature ◽

Building Blocks ◽

Ankyrin Repeat ◽

Internal Repeat ◽

Repeat Proteins ◽

Repeat Domain ◽

Ankyrin Repeat Proteins ◽

Classical Immunoglobulin ◽

Dynamics Simulations ◽

Innovative Drugs

AbstractDesigned Ankyrin Repeat Proteins (DARPins) are a class of antibody mimetics with a high and mostly unexplored potential in drug development. They are clinically validated and thus represent a true alternative to classical immunoglobulin formats. In contrast to immunoglobulins, they are built from solenoid protein domains comprising an N-terminal capping repeat, one or more internal repeats and a C-terminal capping repeat. By using in silico analysis and a rationally guided Ala-Scan, we identified position 17 of the N-terminal capping repeat to play a key role for the overall protein thermostability. The melting temperature of a DARPin domain with a single full-consensus internal repeat was increased by about 8°C to 10°C when the original Asp17 was replaced by Leu, Val, Ile, Met, Ala or Thr, as shown by high-temperature unfolding experiments at equilibrium. We then transferred the Asp17Leu mutation to various backgrounds, including different N- and C-terminal capping repeats and clinically validated DARPin domains, such as the VEGF-binding ankyrin repeat domain of abicipar pegol. In all cases, the proteins remained monomeric and showed improvements in the thermostability of about 8°C to 16°C. Thus, the replacement of Asp17 seems to be generically applicable to this drug class. Molecular dynamics simulations show that the Asp17Leu mutation reduces electrostatic repulsion and improves van-der-Waals packing, rendering the DARPin domain less flexible and more stable. Interestingly, such a beneficial Asp17Leu mutation is present in the N-terminal caps of three of the five DARPin domains of ensovibep, a SARS-CoV-2 entry inhibitor currently in clinical development. This mutation is likely responsible, at least in part, for the very high melting temperature (>90°C) of this promising anti-Covid-19 drug. Overall, such N-terminal capping repeats with increased thermostability seem to be beneficial for the development of innovative drugs based on DARPins.

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Site-Selective Enzymatic Labeling of Designed Ankyrin Repeat Proteins Using Protein Farnesyltransferase

Methods in Molecular Biology - Bioconjugation ◽

10.1007/978-1-4939-9654-4_14 ◽

2019 ◽

pp. 207-219 ◽

Cited By ~ 1

Author(s):

Yi Zhang ◽

Shelby Auger ◽

Jonas V. Schaefer ◽

Andreas Plückthun ◽

Mark D. Distefano

Keyword(s):

Ankyrin Repeat ◽

Protein Farnesyltransferase ◽

Repeat Proteins ◽

Ankyrin Repeat Proteins ◽

Site Selective

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Rigid fusions of designed helical repeat binding proteins efficiently protect a binding surface from crystal contacts

Scientific Reports ◽

10.1038/s41598-019-52121-9 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Patrick Ernst ◽

Annemarie Honegger ◽

Floor van der Valk ◽

Christina Ewald ◽

Peer R. E. Mittl ◽

...

Keyword(s):

Protein Design ◽

Binding Proteins ◽

Peptide Binding ◽

Binding Mode ◽

Equilibrium Structure ◽

Repeat Proteins ◽

Binding Surface ◽

Crystal Contacts ◽

Armadillo Repeat ◽

Ankyrin Repeat Proteins

Abstract Designed armadillo repeat proteins (dArmRPs) bind extended peptides in a modular way. The consensus version recognises alternating arginines and lysines, with one dipeptide per repeat. For generating new binding specificities, the rapid and robust analysis by crystallography is key. Yet, we have previously found that crystal contacts can strongly influence this analysis, by displacing the peptide and potentially distorting the overall geometry of the scaffold. Therefore, we now used protein design to minimise these effects and expand the previously described concept of shared helices to rigidly connect dArmRPs and designed ankyrin repeat proteins (DARPins), which serve as a crystallisation chaperone. To shield the peptide-binding surface from crystal contacts, we rigidly fused two DARPins to the N- and C-terminal repeat of the dArmRP and linked the two DARPins by a disulfide bond. In this ring-like structure, peptide binding, on the inside of the ring, is very regular and undistorted, highlighting the truly modular binding mode. Thus, protein design was utilised to construct a well crystallising scaffold that prevents interference from crystal contacts with peptide binding and maintains the equilibrium structure of the dArmRP. Rigid DARPin-dArmRPs fusions will also be useful when chimeric binding proteins with predefined geometries are required.

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