DARPins: A true alternative to antibodies
ABSTRACT Designed ankyrin repeat proteins (DARPins) are a promising class of non-immunoglobulin proteins that can offer advantages over antibodies for target binding in drug discovery and drug development. DARPins have been successfully used, for example, for the inhibition of kinases, proteases and drug-exporting membrane proteins. DARPins specifically targeting the cancer marker HER2 have also been generated and were shown to function in both in vitro diagnostics and in vivo tumor targeting. DARPins are ideally suited for in vivo imaging or delivery of toxins or other therapeutic payloads because of their favorable molecular properties, including small size and high stability. The low-cost production in bacteria and the rapid generation of many target-specific DARPins make the DARPin approach useful for drug discovery. Additionally, DARPins can be easily generated in multispecific formats, offering the potential to target an effector DARPin to a specific organ or to target multiple receptors with one molecule composed of several DARPins.
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- "that make up the energy landscape of D34 and pointing to an interesting mechanism by which the folding and, thereby, the function of tandem-repeat proteins can be regulated. This mechanism could be also be exploited to regulate the binding properties of designed ankyrin-repeat proteins, which are currently in development as alternatives to therapeutic antibodies (Stumpp and Amstutz, 2007). "
ABSTRACT: Here, using single-molecule FRET, we reveal previously hidden conformations of the ankyrin-repeat domain of AnkyrinR, a giant adaptor molecule that anchors integral membrane proteins to the spectrin-actin cytoskeleton through simultaneous binding of multiple partner proteins. We show that the ankyrin repeats switch between high-FRET and low-FRET states, controlled by an unstructured "safety pin" or "staple" from the adjacent domain of AnkyrinR. Opening of the safety pin leads to unravelling of the ankyrin repeat stack, a process that will dramatically affect the relative orientations of AnkyrinR binding partners and, hence, the anchoring of the spectrin-actin cytoskeleton to the membrane. Ankyrin repeats are one of the most ubiquitous molecular recognition platforms in nature, and it is therefore important to understand how their structures are adapted for function. Our results point to a striking mechanism by which the order-disorder transition and, thereby, the activity of repeat proteins can be regulated. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.Structure 01/2015; 23(1):190-8. DOI:10.1016/j.str.2014.10.023 · 6.79 Impact Factor
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- "The limitations of the antibodies caused by the limited range of serogroups may be overcome by using a pool of antibodies, e.g., either covering all Lp serogroups, or even all Ls. In the future antibody alternatives may provide improved binding properties and cost reduction (Stumpp and Amstutz, 2007; Chung et al., 2011). The method developed and tested in the present study might not yet be fit to completely displace the plating methods used in laboratories for routine water analysis. "
ABSTRACT: We developed a rapid detection method for Legionella pneumophila (Lp) by filtration, immunomagnetic separation, double fluorescent staining, and flow cytometry (IMS-FCM method). The method requires 120 min and can discriminate 'viable' and 'membrane-damaged' cells. The recovery is over 85% of spiked Lp SG 1 cells in 1 l of tap water and detection limits are around 50 and 15 cells per litre for total and viable Lp, respectively. The method was compared using water samples from house installations in a blind study with three environmental laboratories performing the ISO 11731 plating method. In 53% of the water samples from different taps and showers significantly higher concentrations of Lp were detected by flow cytometry. No correlation to the plate culture method was found. Since also 'viable but not culturable' (VNBC) cells are detected by our method, this result was expected. The IMS-FCM method is limited by the specificity of the used antibodies; in the presented case they target Lp serogroups 1-12. This and the fact that no Lp-containing amoebae are detected may explain why in 21% of all samples higher counts were observed using the plate culture method. Though the IMS-FCM method is not yet fit to completely displace the established plating method (ISO 11731) for routine Lp monitoring, it has major advantages to plating and can quickly provide important insights into the ecology of this pathogen in water distribution systems.Microbial Biotechnology 11/2012; 5(6):753-63. DOI:10.1111/j.1751-7915.2012.00366.x · 3.21 Impact Factor
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- "The assembled protein consists of two capping repeats intervened by two or more binding modules that are randomly modified on their surface. Combinatorial libraries with DARPins of varying repeat numbers have been constructed and have been used for selections against a wide range of targets       , reviewed recently  . In the field of allergy, we have recently characterized bispecific DARPins that 0165-2478/$ – see front matter © 2010 Elsevier B.V. All rights reserved. "
ABSTRACT: The monoclonal anti-IgE antibody omalizumab (Xolair is mostly used for the treatment of severe allergic asthma. However, the requirement of high doses and suboptimal cost-effectiveness limits the use of the treatment. Here we propose to use a new drug format based on non-immunoglobulin structures, potentially offering increased clinical efficacy while being more cost-effective. For this purpose, DARPins™ (designed ankyrin repeat proteins) against the constant heavy chain region of IgE have been isolated. DARPins were binding to IgE with high specificity and affinities in the low nanomolar range. Selected DARPins antagonized the interaction between IgE and its high-affinity receptor in inhibition assays. Furthermore, anti-IgE DARPins were shown to inhibit proinflammatory mediator release from rat basophilic leukemia cells expressing human high-affinity IgE receptors with higher efficacy than the monoclonal anti-IgE antibody omalizumab. DARPins may thus represent promising future drug candidates for the treatment of allergy.Immunology letters 10/2010; 133(2):78-84. DOI:10.1016/j.imlet.2010.07.005 · 2.37 Impact Factor