DARPins: A true alternative to antibodies
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.
Available from: Maksym Tsytlonok
- "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). "
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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.
Available from: Agata Zieba
- "Designed ankyrin repeat proteins (DARPins) are potent alternatives to conventional antibodies. They detect antigens with high specificity and picomolar affinity, are independent of target immunogenicity and possess attractive molecular properties such as small size and high stability (111). They are synthetic, non-immunoglobulin binding proteins that form scaffolds containing tandem repeats of an elementary, structural motif, typically composed of 33 amino acid residues folded into a β-turn followed by two antiparallel α-helices. "
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ABSTRACT: The emergence of targeted therapies for cancer has created a need for the development of companion diagnostic tests. Assays developed in recent years are aimed at determining both the effectiveness and safety of specific drugs for a defined group of patients, thus, enabling the more efficient design of clinical trials and also supporting physicians when making treatment-related decisions. Immunohistochemistry (IHC) is a widely accepted method for protein expression analyses in human tissues. Immunohistochemical assays, used to localize and quantitate relative protein expression levels within a morphological context, are frequently used as companion diagnostics during clinical trials and also following drug approval. Herein, we describe established immunochemistry-based methods and their application in routine diagnostics. We also explore the possibility of using IHC to detect specific protein mutations in addition to DNA-based tests. Finally, we review alternative protein binders and proximity ligation assays and discuss their potential to facilitate the development of novel, targeted therapies against cancer.
Available from: Thomas Egli
- "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. "
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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.
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