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Andreas Plückthun - One of the best experts on this subject based on the ideXlab platform.
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Abstract A75: Designed Ankyrin Repeat Proteins (DARPins) as recognition motifs in chimeric antigen receptors
New CARs, 2017Co-Authors: Ashwini Balakrishnan, Andreas Plückthun, Alexander I. Salter, Stanley R. RiddellAbstract:Chimeric antigen receptors (CARs) consist primarily of single chain variable fragments (scFv) linked to the T cell activation (CD3ζ) and costimulatory domains. We investigated whether alternative recognition motifs selected for ligand binding from synthetic libraries of Designed Ankyrin Repeat proteins (DARPins) could be incorporated into CARs. As proof of principle, we linked a DARPin (DARPin E01) specific for EGFR to the CAR signaling domain and compared anti-tumor function with a high affinity CAR containing the Cetuximab scfv, which recognizes an overlapping epitope. Our in vitro results show that DARPin CARs designed with flexible linkers and with short and long spacer domains exhibit specific cytotoxicity, cytokine secretion and proliferation against EGFR + targets that is comparable to the Cetuximab CAR. We tested DARPins CARs in an in vivo breast cancer tumor model with EGFR + MDA-MB-231 tumor cells and found enhanced anti-tumor clearance with both E01 DARPin and Cetuximab scFV CARs. We show that an EpCAM-specific DARPin chimeric costimulatatory receptor (CCR) can also be used in conjunction with a scFV CAR targeting ROR1 to provide combinatorial antigen recognition and enhance T cell function against tumor cells expressing both targets. The EpCAM-specific DARPin CCR combined with first or second generation ROR1 scFv CARs enhanced T cell cytokine secretion and proliferation. The compact stable structure of DARPins should facilitate linking of multiple DARPins targeting different antigens reducing the likelihood of antigen loss. In summary, the stable structural scaffold of DARPins and the presence of diverse binder libraries make them attractive recognition motifs to incorporate into CARs. Citation Format: Ashwini Balakrishnan, Alexander I. Salter, Andreas Pluckthun, Stanley R. Riddell. Designed Ankyrin Repeat Proteins (DARPins) as recognition motifs in chimeric antigen receptors. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A75.
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Designed Ankyrin Repeat Proteins (DARPins): Binding Proteins for Research, Diagnostics, and Therapy
Annual review of pharmacology and toxicology, 2015Co-Authors: Andreas PlückthunAbstract:Designed Ankyrin Repeat proteins (DARPins) can recognize targets with specificities and affinities that equal or surpass those of antibodies, but because of their robustness and extreme stability, they allow a multitude of more advanced formats and applications. This review highlights recent advances in DARPin design, illustrates their properties, and gives some examples of their use. In research, they have been established as intracellular, real-time sensors of protein conformations and as crystallization chaperones. For future therapies, DARPins have been developed by advanced, structure-based protein engineering to selectively induce apoptosis in tumors by uncoupling surface receptors from their signaling cascades. They have also been used successfully for retargeting viruses. In ongoing clinical trials, DARPins have shown good safety and efficacy in macular degeneration diseases. These developments all ultimately exploit the high stability, solubility, and aggregation resistance of these molecules, pe...
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Designed Ankyrin Repeat Proteins (DARPins)
Methods in Enzymology, 2012Co-Authors: Rastislav Tamaskovic, Nikolas Stefan, Manuel Simon, Martin Schwill, Andreas PlückthunAbstract:Designed Ankyrin Repeat proteins (DARPins) have been developed into a robust and versatile scaffold for binding proteins. High-affinity binders are routinely selected by ribosome display and phage display. DARPins have entered clinical trials and have found numerous uses in research, due to their high stability and robust folding, allowing many new molecular formats. We summarize the DARPin properties and highlight some biomedical applications. Protocols are given for labeling with dyes and polyethylene glycol, for quantitatively measuring binding to cell surface receptors by kinetics and thermodynamics, and for exploiting new engineering opportunities from using "click chemistry" with nonnatural amino acids.
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Chapter five – Designed Ankyrin Repeat Proteins (DARPins): From Research to Therapy
Methods in enzymology, 2012Co-Authors: Rastislav Tamaskovic, Nikolas Stefan, Manuel Simon, Martin Schwill, Andreas PlückthunAbstract:Designed Ankyrin Repeat proteins (DARPins) have been developed into a robust and versatile scaffold for binding proteins. High-affinity binders are routinely selected by ribosome display and phage display. DARPins have entered clinical trials and have found numerous uses in research, due to their high stability and robust folding, allowing many new molecular formats. We summarize the DARPin properties and highlight some biomedical applications. Protocols are given for labeling with dyes and polyethylene glycol, for quantitatively measuring binding to cell surface receptors by kinetics and thermodynamics, and for exploiting new engineering opportunities from using “click chemistry” with nonnatural amino acids.
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designed Ankyrin Repeat proteins darpins from research to therapy
Methods in Enzymology, 2012Co-Authors: Rastislav Tamaskovic, Nikolas Stefan, Manuel Simon, Martin Schwill, Andreas PlückthunAbstract:Designed Ankyrin Repeat proteins (DARPins) have been developed into a robust and versatile scaffold for binding proteins. High-affinity binders are routinely selected by ribosome display and phage display. DARPins have entered clinical trials and have found numerous uses in research, due to their high stability and robust folding, allowing many new molecular formats. We summarize the DARPin properties and highlight some biomedical applications. Protocols are given for labeling with dyes and polyethylene glycol, for quantitatively measuring binding to cell surface receptors by kinetics and thermodynamics, and for exploiting new engineering opportunities from using "click chemistry" with nonnatural amino acids.
Rachelle Gaudet - One of the best experts on this subject based on the ideXlab platform.
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differential regulation of trpv1 trpv3 and trpv4 sensitivity through a conserved binding site on the Ankyrin Repeat domain
Journal of Biological Chemistry, 2010Co-Authors: Christopher B Phelps, Ruiqi Rachel Wang, Shelly Seungah Choo, Rachelle GaudetAbstract:Transient receptor potential vanilloid (TRPV) channels, which include the thermosensitive TRPV1–V4, have large cytoplasmic regions flanking the transmembrane domain, including an N-terminal Ankyrin Repeat domain. We show that a multiligand binding site for ATP and calmodulin previously identified in the TRPV1 Ankyrin Repeat domain is conserved in TRPV3 and TRPV4, but not TRPV2. Accordingly, TRPV2 is insensitive to intracellular ATP, while, as previously observed with TRPV1, a sensitizing effect of ATP on TRPV4 required an intact binding site. In contrast, ATP reduced TRPV3 sensitivity and potentiation by Repeated agonist stimulations. Thus, ATP and calmodulin, acting through this conserved binding site, are key players in generating the different sensitivity and adaptation profiles of TRPV1, TRPV3, and TRPV4. Our results suggest that competing interactions of ATP and calmodulin influence channel sensitivity to fluctuations in calcium concentration and perhaps even metabolic state. Different feedback mechanisms likely arose because of the different physiological stimuli or temperature thresholds of these channels.
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a primer on Ankyrin Repeat function in trp channels and beyond
Molecular BioSystems, 2008Co-Authors: Rachelle GaudetAbstract:Transient receptor potential (TRP) channels are rapidly gaining attention as important receptors and transducers of diverse sensory and environmental cues. Recent progress in the field has provided new insights into the structure and function of the Ankyrin Repeat motifs present in the N-terminal cytosolic domain of many TRP channels. The topics addressed in this Highlight include the structural features of canonical Ankyrin Repeats, new clues into the functions these Repeats perform in cells, and how this information can be applied to develop further experiments on TRP channels and other proteins containing Ankyrin Repeats.
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structure of the n terminal Ankyrin Repeat domain of the trpv2 ion channel
Journal of Biological Chemistry, 2006Co-Authors: Xiangshu Jin, Jason Touhey, Rachelle GaudetAbstract:The TRPV ion channels mediate responses to many sensory stimuli including heat, low pH, neuropeptides, and chemical ligands. All TRPV subfamily members contain an intracellular N-terminal Ankyrin Repeat domain (ARD), a prevalent protein interaction motif. The 1.6-A crystal structure of the TRPV2-ARD, with six Ankyrin Repeats, reveals several atypical structural features. Repeats one through three display unusually long and flexible fingers with a large number of exposed aromatic residues, whereas Repeats five and six have unusually long outer helices. Furthermore, a large counterclockwise twist observed in the stacking of Repeats four and five breaks the regularity of the domain, altering the shape of surfaces available for interactions with proteins or other cellular ligands. Both solution studies and crystal packing interactions indicate that the TRPV2-ARD does not form homo-oligomers, suggesting that the ARD of TRPV ion channels may be used for interactions with regulatory factors rather than in promoting tetrameric assembly of the ion channels.
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Structure of the N-terminal Ankyrin Repeat Domain of the TRPV2 Ion Channel *
The Journal of biological chemistry, 2006Co-Authors: Xiangshu Jin, Jason Touhey, Rachelle GaudetAbstract:The TRPV ion channels mediate responses to many sensory stimuli including heat, low pH, neuropeptides, and chemical ligands. All TRPV subfamily members contain an intracellular N-terminal Ankyrin Repeat domain (ARD), a prevalent protein interaction motif. The 1.6-A crystal structure of the TRPV2-ARD, with six Ankyrin Repeats, reveals several atypical structural features. Repeats one through three display unusually long and flexible fingers with a large number of exposed aromatic residues, whereas Repeats five and six have unusually long outer helices. Furthermore, a large counterclockwise twist observed in the stacking of Repeats four and five breaks the regularity of the domain, altering the shape of surfaces available for interactions with proteins or other cellular ligands. Both solution studies and crystal packing interactions indicate that the TRPV2-ARD does not form homo-oligomers, suggesting that the ARD of TRPV ion channels may be used for interactions with regulatory factors rather than in promoting tetrameric assembly of the ion channels.
Jenny Yeung - One of the best experts on this subject based on the ideXlab platform.
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development of the designed Ankyrin Repeat protein darpin g3 for her2 molecular imaging
European Journal of Nuclear Medicine and Molecular Imaging, 2015Co-Authors: Robert Goldstein, Jane K Sosabowski, Maria Livanos, Julius Leyton, K Vigor, Gaurav Bhavsar, Gabriela Nagydavidescu, Mohammed Rashid, Enrique Miranda, Jenny YeungAbstract:Purpose Human epidermal growth factor receptor-2 (HER2) overexpression is a predictor of response to anti-HER2 therapy in breast and gastric cancer. Currently, HER2 status is assessed by tumour biopsy, but this may not be representative of the larger tumour mass or other metastatic sites, risking misclassification and selection of suboptimal therapy. The designed Ankyrin Repeat protein (DARPin) G3 binds HER2 with high affinity at an epitope that does not overlap with trastuzumab and is biologically inert. We hypothesized that radiolabelled DARPin G3 would be capable of selectively imaging HER2-positive tumours, and aimed to identify a suitable format for clinical application.
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Development of the designed Ankyrin Repeat protein (DARPin) G3 for HER2 molecular imaging
European journal of nuclear medicine and molecular imaging, 2014Co-Authors: Robert Goldstein, Gabriela Nagy-davidescu, Jane K Sosabowski, Maria Livanos, Julius Leyton, K Vigor, Gaurav Bhavsar, Mohammed Rashid, Enrique Miranda, Jenny YeungAbstract:Purpose Human epidermal growth factor receptor-2 (HER2) overexpression is a predictor of response to anti-HER2 therapy in breast and gastric cancer. Currently, HER2 status is assessed by tumour biopsy, but this may not be representative of the larger tumour mass or other metastatic sites, risking misclassification and selection of suboptimal therapy. The designed Ankyrin Repeat protein (DARPin) G3 binds HER2 with high affinity at an epitope that does not overlap with trastuzumab and is biologically inert. We hypothesized that radiolabelled DARPin G3 would be capable of selectively imaging HER2-positive tumours, and aimed to identify a suitable format for clinical application.
LS Itzhaki - One of the best experts on this subject based on the ideXlab platform.
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Effects of ligand binding on the mechanical properties of Ankyrin Repeat protein gAnkyrin.
PLoS computational biology, 2013Co-Authors: Giovanni Settanni, David Serquera, Piotr E. Marszalek, Emanuele Paci, LS ItzhakiAbstract:Ankyrin Repeat proteins are elastic materials that unfold and refold sequentially, Repeat by Repeat, under force. Herein we use atomistic molecular dynamics to compare the mechanical properties of the 7-Ankyrin-Repeat oncoprotein GAnkyrin in isolation and in complex with its binding partner S6-C. We show that the bound S6-C greatly increases the resistance of GAnkyrin to mechanical stress. The effect is specific to those Repeats of GAnkyrin directly in contact with S6-C, and the mechanical ‘hot spots’ of the interaction map to the same Repeats as the thermodynamic hot spots. A consequence of stepwise nature of unfolding and the localized nature of ligand binding is that it impacts on all aspects of the protein's mechanical behavior, including the order of Repeat unfolding, the diversity of unfolding pathways accessed, the nature of partially unfolded intermediates, the forces required and the work transferred to the system to unfold the whole protein and its parts. Stepwise unfolding thus provides the means to buffer Repeat proteins and their binding partners from mechanical stress in the cell. Our results illustrate how ligand binding can control the mechanical response of proteins. The data also point to a cellular mechano-switching mechanism whereby binding between two partner macromolecules is regulated by mechanical stress.
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Mechanical Unfolding of an Ankyrin Repeat Protein
Biophysical journal, 2010Co-Authors: David Serquera, Whasil Lee, Giovanni Settanni, Piotr E. Marszalek, Emanuele Paci, LS ItzhakiAbstract:Ankryin Repeat proteins comprise tandem arrays of a 33-residue, predominantly α-helical motif that stacks roughly linearly to produce elongated and superhelical structures. They function as scaffolds mediating a diverse range of protein-protein interactions, and some have been proposed to play a role in mechanical signal transduction processes in the cell. Here we use atomic force microscopy and molecular-dynamics simulations to investigate the natural 7-Ankyrin Repeat protein gAnkyrin. We find that gAnkyrin unfolds under force via multiple distinct pathways. The reactions do not proceed in a cooperative manner, nor do they always involve fully stepwise unfolding of one Repeat at a time. The peeling away of half an Ankyrin Repeat, or one or more Ankyrin Repeats, occurs at low forces; however, intermediate species are formed that are resistant to high forces, and the simulations indicate that in some instances they are stabilized by nonnative interactions. The unfolding of individual Ankyrin Repeats generates a refolding force, a feature that may be more easily detected in these proteins than in globular proteins because the refolding of a Repeat involves a short contraction distance and incurs a low entropic cost. We discuss the origins of the differences between the force- and chemical-induced unfolding pathways of Ankyrin Repeat proteins, as well as the differences between the mechanics of natural occurring Ankyrin Repeat proteins and those of designed consensus ankyin Repeat and globular proteins.
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Shifting transition states in the unfolding of a large Ankyrin Repeat protein
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Nicolas D. Werbeck, Pamela J. E. Rowling, Vasuki Ranjani Chellamuthu, LS ItzhakiAbstract:The 33-amino-acid Ankyrin motif comprises a β-turn followed by two anti-parallel α-helices and a loop and tandem arrays of the motif pack in a linear fashion to produce elongated structures characterized by short-range interactions. In this article we use site-directed mutagenesis to investigate the kinetic unfolding mechanism of D34, a 426-residue, 12-Ankyrin Repeat fragment of the protein AnkyrinR. The data are consistent with a model in which the N-terminal half of the protein unfolds first by unraveling progressively from the start of the polypeptide chain to form an intermediate; in the next step, the C-terminal half of the protein unfolds via two pathways whose transition states have either the early or the late C-terminal Ankyrin Repeats folded. We conclude that the two halves of the protein unfold by different mechanisms because the N-terminal moiety folds and unfolds in the context of a folded C-terminal moiety, which therefore acts as a “seed” and confers a unique directionality on the process, whereas the C-terminal moiety folds and unfolds in the context of an unfolded N-terminal moiety and therefore behaves like a single-domain Ankyrin Repeat protein, having a high degree of symmetry and consequently more than one unfolding pathway accessible to it.
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Biophysical characterisation of the small Ankyrin Repeat protein myotrophin
J Mol Biol, 2007Co-Authors: LS ItzhakiAbstract:The 118 residue protein myotrophin is composed of four Ankyrin Repeats that stack linearly to form an elongated, predominantly alpha-helical structure. The protein folds via a two-state mechanism at equilibrium. The free energy change of unfolding in water (DeltaG(U-N)(H(2)O)) is 5.8 kcal.mol(-1). The chevron plot reveals that the folding reaction has a broad energy barrier and that it conforms to a two-state mechanism. The rate of folding in water (k(f)(H(2)O)) of 95 s(-1) is several orders of magnitude slower than the value predicted by topological calculations. Proline mutants were used to show that the minor kinetic phases observed for myotrophin arise from heterogeneity of the ground states due to cis-trans isomerisation of prolyl as well as non-prolyl peptide bonds. Myotrophin is the first example of a naturally occurring Ankyrin Repeat protein that conforms to an apparent two-state mechanism at equilibrium and under kinetic conditions, making it highly suitable for high resolution protein folding studies.
Rastislav Tamaskovic - One of the best experts on this subject based on the ideXlab platform.
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Designed Ankyrin Repeat Proteins (DARPins)
Methods in Enzymology, 2012Co-Authors: Rastislav Tamaskovic, Nikolas Stefan, Manuel Simon, Martin Schwill, Andreas PlückthunAbstract:Designed Ankyrin Repeat proteins (DARPins) have been developed into a robust and versatile scaffold for binding proteins. High-affinity binders are routinely selected by ribosome display and phage display. DARPins have entered clinical trials and have found numerous uses in research, due to their high stability and robust folding, allowing many new molecular formats. We summarize the DARPin properties and highlight some biomedical applications. Protocols are given for labeling with dyes and polyethylene glycol, for quantitatively measuring binding to cell surface receptors by kinetics and thermodynamics, and for exploiting new engineering opportunities from using "click chemistry" with nonnatural amino acids.
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Chapter five – Designed Ankyrin Repeat Proteins (DARPins): From Research to Therapy
Methods in enzymology, 2012Co-Authors: Rastislav Tamaskovic, Nikolas Stefan, Manuel Simon, Martin Schwill, Andreas PlückthunAbstract:Designed Ankyrin Repeat proteins (DARPins) have been developed into a robust and versatile scaffold for binding proteins. High-affinity binders are routinely selected by ribosome display and phage display. DARPins have entered clinical trials and have found numerous uses in research, due to their high stability and robust folding, allowing many new molecular formats. We summarize the DARPin properties and highlight some biomedical applications. Protocols are given for labeling with dyes and polyethylene glycol, for quantitatively measuring binding to cell surface receptors by kinetics and thermodynamics, and for exploiting new engineering opportunities from using “click chemistry” with nonnatural amino acids.
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designed Ankyrin Repeat proteins darpins from research to therapy
Methods in Enzymology, 2012Co-Authors: Rastislav Tamaskovic, Nikolas Stefan, Manuel Simon, Martin Schwill, Andreas PlückthunAbstract:Designed Ankyrin Repeat proteins (DARPins) have been developed into a robust and versatile scaffold for binding proteins. High-affinity binders are routinely selected by ribosome display and phage display. DARPins have entered clinical trials and have found numerous uses in research, due to their high stability and robust folding, allowing many new molecular formats. We summarize the DARPin properties and highlight some biomedical applications. Protocols are given for labeling with dyes and polyethylene glycol, for quantitatively measuring binding to cell surface receptors by kinetics and thermodynamics, and for exploiting new engineering opportunities from using "click chemistry" with nonnatural amino acids.