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Review articles
Methods
Antibody engineering
Repeat Proteins (general)
DARPins
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Directed Evolution
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2021

# Reference PDF
  • Mysore, V. P., Zhou, Z. W., Ambrogio, C., Li, L., Kapp, J. N., Lu. C., Wang. Q., Tucker. M. R., Okoro. J. J., Nagy-Davidescu, G., Bai, X., Plückthun, A., Jänne, P. A., Westover, K. D., Shan, Y., and Shaw, D.E. (2021). A structural model of a Ras-Raf signalosome. Nature Struct. Mol. Biol. 28, 847-857.

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    Abstract

    The protein K-Ras functions as a molecular switch in signaling pathways regulating cell growth. In the human mitogen-activated protein kinase (MAPK) pathway, which is implicated in many cancers, multiple K-Ras proteins are thought to assemble at the cell membrane with Ras effector proteins from the Raf family. Here we propose an atomistic structural model for such an assembly. Our starting point was an asymmetric guanosine triphosphate-mediated K-Ras dimer model, which we generated using unbiased molecular dynamics simulations and verified with mutagenesis experiments. Adding further K-Ras monomers in a head-to-tail fashion led to a compact helical assembly, a model we validated using electron microscopy and cell-based experiments. This assembly stabilizes K-Ras in its active state and presents composite interfaces to facilitate Raf binding. Guided by existing experimental data, we then positioned C-Raf, the downstream kinase MEK1 and accessory proteins (Galectin-3 and 14-3-3σ) on and around the helical assembly. The resulting Ras-Raf signalosome model offers an explanation for a large body of data on MAPK signaling.

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  • Cucuzza, S., Güntert, P., Plückthun, A., and Zerbe, O. (2021) An automated iterative approach for protein structure refinement using pseudocontact shifts. J. Biomol. NMR 75, 319–334.

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    Abstract

    NMR structure calculation using NOE-derived distance restraints requires a considerable number of assignments of both backbone and sidechains resonances, often difficult or impossible to get for large or complex proteins. Pseudocontact shifts (PCSs) also play a well-established role in NMR protein structure calculation, usually to augment existing structural, mostly NOE-derived, information. Existing refinement protocols using PCSs usually either require a sizeable number of sidechain assignments or are complemented by other experimental restraints. Here, we present an automated iterative procedure to perform backbone protein structure refinements requiring only a limited amount of backbone amide PCSs. Already known structural features from a starting homology model, in this case modules of repeat proteins, are framed into a scaffold that is subsequently refined by experimental PCSs. The method produces reliable indicators that can be monitored to judge about the performance. We applied it to a system in which sidechain assignments are hardly possible, designed Armadillo repeat proteins (dArmRPs), and we calculated the solution NMR structure of YM4A, a dArmRP containing four sequence-identical internal modules, obtaining high convergence to a single structure. We suggest that this approach is particularly useful when approximate folds are known from other techniques, such as X-ray crystallography, while avoiding inherent artefacts due to, for instance, crystal packing.

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  • Stephens, E. A., Ludwicki, M. B., Meksiriporn, S., Li, M., Ye, T., Monticello, C., Forsythe, K. J. Kummer, L., Zhou, P., Plückthun, A., and DeLisa, M. P. (2021) Engineering single pan-specific Ubiquibodies for targeted degradation of all forms of endogenous ERK protein kinase. ACS Synth. Biol. 10, 2396-2408.

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    Abstract

    Ubiquibodies (uAbs) are a customizable proteome editing technology that utilizes E3 ubiquitin ligases genetically fused to synthetic binding proteins to steer otherwise stable proteins of interest (POIs) to the 26S proteasome for degradation. The ability of engineered uAbs to accelerate the turnover of exogenous or endogenous POIs in a post-translational manner offers a simple yet robust tool for dissecting diverse functional properties of cellular proteins as well as for expanding the druggable proteome to include tumorigenic protein families that have yet-to-be successfully drugged by conventional inhibitors. Here, we describe the engineering of uAbs composed of human carboxyl-terminus of Hsc70-interacting protein (CHIP), a highly modular human E3 ubiquitin ligase, tethered to differently designed ankyrin repeat proteins (DARPins) that bind to nonphosphorylated (inactive) and/or doubly phosphorylated (active) forms of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Two of the resulting uAbs were found to be global ERK degraders, pan-specifically capturing all endogenous ERK1/2 protein forms and redirecting them to the proteasome for degradation in different cell lines, including MCF7 breast cancer cells. Taken together, these results demonstrate how the substrate specificity of an E3 ubiquitin ligase can be reprogrammed to generate designer uAbs against difficult-to-drug targets, enabling a modular platform for remodeling the mammalian proteome.

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  • Becker, L., Singh Badwal, J., Brandl, F., Verdurmen, W. P. R., and Plückthun, A. (2021). Thermodynamic stability is a strong predictor for the delivery of DARPins to the cytosol via anthrax toxin. Pharmaceutics 13, 1285.

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    Abstract

    Anthrax toxin has evolved to translocate its toxic cargo proteins to the cytosol of cells carrying its cognate receptor. Cargo molecules need to unfold to penetrate the narrow pore formed by its membrane-spanning subunit, protective antigen (PA). Various alternative cargo molecules have previously been tested, with some showing only limited translocation efficiency, and it may be assumed that these were too stable to be unfolded before passing through the anthrax pore. In this study, we systematically and quantitatively analyzed the correlation between the translocation of various designed ankyrin repeat proteins (DARPins) and their different sizes and thermodynamic stabilities. To measure cytosolic uptake, we used biotinylation of the cargo by cytosolic BirA, and we measured cargo equilibrium stability via denaturant-induced unfolding, monitored by circular dichroism (CD). Most of the tested DARPin cargoes, including target-binding ones, were translocated to the cytosol. Those DARPins, which remained trapped in the endosome, were confirmed by CD to show a high equilibrium stability. We could pinpoint a stability threshold up to which cargo DARPins still get translocated to the cytosol. These experiments have outlined the requirements for translocatable binding proteins, relevant stability measurements to assess translocatable candidates, and guidelines to further engineer this property if needed.

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  • Kosareva, A., Punjabi, M., Ochoa-Espinosa, A., Xu, L., Schaefer, J. V., Dreier, B., Plückthun, A, and Kaufmann B. A. (2021). Designed ankyrin repeat proteins as novel binders for ultrasound molecular imaging. Ultrasound Med. Biol. 47, 2664-2675.

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    Abstract

    Clinical translation of ultrasound molecular imaging will depend on the development of binders that can easily be generated, manufactured and coupled, and that are compatible with in vivo use. We describe targeted microbubbles (MBs) using designed ankyrin repeat proteins (DARPins) as a novel class of such translatable binders. Candidate DARPin binders for vascular cell adhesion molecule 1, an endothelial cell adhesion molecule involved in inflammatory processes, were selected using ribosome display and coupled to MBs. Flow-chamber assays of five MBs carrying high-affinity binders showed selective retention on endothelial cells activated by tumor necrosis factor-a for two binders compared with a MB carrying a control DARPin. In vivo ultrasound molecular imaging in a murine hind-limb inflammation model demonstrated up to a fourfold signal enhancement for three of the five MBs versus control. However, there was no correlation between results from flow-chamber assays and in vivo imaging. Thus, we conclude that ultrasound molecular imaging of inflammation using DARPin binders is feasible per se, but that screening of candidates cannot be accomplished with flow-chamber assays as used in our study.

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  • Nemergut, M., Škrabana, R., Berta, M., Plückthun, A., Sedlák, E. (2021). Purification of MBP fusion proteins using engineered DARPin affinity matrixInt. J. Biol. Macromol. 187, 105-112

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    Abstract

    Maltose binding protein (MBP) has a long history as an expression tag with the ability to increase the solubility of fused proteins. A critical step for obtaining a sufficient amount of the MBP fusion protein is purification. Commercially available amylose matrix for the affinity purification of MBP fusion proteins has two main issues: (i) low (micromolar) affinity and (ii) the limited number of uses due to the cleavage of polysaccharide matrix by the amylases, present in the crude cell extract. Here, we present a new affinity purification approach based on the protein-protein interaction. We developed the affinity matrix which contains immobilized Designed Ankyrin Repeat Protein off7 (DARPin off7) - previously identified MBP binder with nanomolar affinity. The functionality of the DARPin affinity matrix was tested on the purification of MBP-tagged green fluorescent protein and flavodoxin. The affinity purification of the MBP fusion proteins, based on the MBP-DARPin off7 interaction, enables the purification of the fusion proteins in a simple two-steps procedure. The DARPin affinity matrix - easy to construct, resistant to amylase, insensitive to maltose contamination, and reusable for multiple purification cycles - provides an alternative approach to commercially available affinity matrices for purification of proteins containing the MBP tag.

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  • Merten, M., Brandl, F., Zimmermann, M., Schaefer, J. V., Irpinio, L., Sand, K. M. K., Nilsen, J., Andersen, J.T., Zangemeister-Wittke, U., and Plückthun, A. (2021) Half-life extension of efficiently produced DARPin serum albumin fusionsas a function of FcRn affinity and recycling. Eur. J. Pharm. Biopharm. 167, 104-113.

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    Abstract

    Serum albumin shows slow clearance from circulation due to neonatal Fc receptor (FcRn)-mediated recycling and has been used for half-life extension. We report here fusions to a high-affinity DARPin, binding to Epithelial Cell Adhesion Molecule (EpCAM). We developed a novel, efficient expression system for such fusion proteins in Pichia pastoris with titers above 300 mg/L of lab-scale shake-flask culture. Since human serum albumin (HSA) does not bind to the murine FcRn, half-lives of therapeutic candidates are frequently measured in human FcRn transgenic mice, limiting useable tumor models. Additionally, serum albumins with extended half-life have been designed. We tested HSA7, motivated by its previously claimed extraordinarily long half-life in mice, which we could not confirm. Instead, we determined a half-life of only 29 h for HSA7, comparable to MSA. The fusion of HSA7 to a DARPin showed a similar half-life. To rationalize these findings, we measured binding kinetics and affinities to murine and human FcRn. Briefly, HSA7 showed affinity to murine FcRn only in the micromolar range, comparable to MSA to its cognate murine FcRn, and an affinity in the nanomolar range only to the human FcRn. This explains the comparable half-life of MSA and HSA7 in mice, while wild-type-HSA has a half-life of only 21 h, as it does not bind the murine FcRn and is not recycled. Thus, HSA-fusions with improved FcRnaffinity, such as HSA7, can be used for preclinical experiments in mice when FcRn transgenes cannot be used, as they reflect better the complex FcRn-mediated recycling and distribution mechanisms.

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  • Bradbury, A. R. M., Dübel, S., Knappik, A., and Plückthun, A. (2021). Animal- versus in vitro-derived antibodies: avoiding the extremes. mAbs 13, e1950265.

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    Abstract

    Recent recommendations from the European Union Reference Laboratory regarding the generation of antibodies using animals have stimulated significant debate. Here, four of the scientists who served on the Scientific Advisory Committee provide clarification of their views regarding the use of animals and in vitro platforms in antibody generation.

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  • Radom, F., Vonrhein, C., Mittl, P. R. E., and Plückthun A. (2021). Crystal structures of HER3 extracellular domain 4 in complex with the designed ankyrin-repeat protein D5. Acta Cryst. Section F 77, 192–201.

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    Abstract

    The members of the human epidermal growth factor receptor (HER) family are among the most intensely studied oncological targets. HER3 (ErbB3), which had long been neglected, has emerged as a key oncogene, regulating the activity of other receptors and being involved in progression and tumor escape in multiple types of cancer. Designed ankyrin-repeat proteins (DARPins) serve as antibody mimetics that have proven to be useful in the clinic, in diagnostics and in research. DARPins have previously been selected against EGFR (HER1), HER2 and HER4. In particular, their combination into bivalent binders that separate or lock receptors in their inactive conformation has proved to be a promising strategy for the design of potent anticancer therapeutics. Here, the selection of DARPins targeting extracellular domain 4 of HER3 (HER3d4) is described. One of the selected DARPins, D5, in complex with HER3d4 crystallized in two closely related crystal forms that diffracted to 2.3 and 2.0 Å resolution, respectively. The DARPin D5 epitope comprises HER3d4 residues 568–577. These residues also contribute to interactions within the tethered (inactive) and extended (active) conformations of the extracellular domain of HER3.

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  • Stüber, J. C., Richter, C. P., Sotolongo Bellón, J., Schwill, M., König, I., Schuler, B., Piehler, J., and Plückthun, A. (2021). Apoptosis-inducing anti-HER2 agents operate through oligomerization-induced receptor immobilization. Commun. Biol. 4, 762.

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    Abstract

    Overexpression of the receptor tyrosine kinase HER2 plays a critical role in the development of various tumors. Biparatopic designed ankyrin repeat proteins (bipDARPins) potently induce apoptosis in HER2-addicted breast cancer cell lines. Here, we have investigated how the spatiotemporal receptor organization at the cell surface is modulated by these agents and is distinguished from other molecules, which do not elicit apoptosis. Binding of conventional antibodies is accompanied by moderate reduction of receptor mobility, in agreement with HER2 being dimerized by the bivalent IgG. In contrast, the most potent apoptosis-inducing bipDARPins lead to a dramatic arrest of HER2. Dual-color single-molecule tracking revealed that the HER2 “lockdown” by these bipDARPins is caused by the formation of HER2-DARPin oligomer chains, which are trapped in nanoscopic membrane domains. Our findings establish that efficient neutralization of receptor tyrosine kinase signaling can be achieved through intermolecular bipDARPin crosslinking alone, resulting in inactivated, locked-down bipDARPin-HER2 complexes.

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  • Kast, F., Schwill, M., Stüber, J. C., Pfundstein, S., Nagy-Davidescu, G., Monné Rodriguez, J. M., Seehusen, F., Richter, C. P., Honegger, A., Hartmann, K. P., Weber, T. G., Kroener, F., Ernst, P., Piehler, J., and Plückthun, A. (2021). Engineering an anti-HER2 biparatopic antibody with a multimodal mechanism of action. Nature Comm. 12, 3790

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    Abstract

    The receptor tyrosine kinase HER2 acts as oncogenic driver in numerous cancers. Usually, the gene is amplified, resulting in receptor overexpression, massively increased signaling and unchecked proliferation. However, tumors become frequently addicted to oncogenes and hence are druggable by targeted interventions. Here, we design an anti-HER2 biparatopic and tetravalent IgG fusion with a multimodal mechanism of action. The molecule first induces HER2 clustering into inactive complexes, evidenced by reduced mobility of surface HER2. However, in contrast to our earlier binders based on DARPins, clusters of HER2 are thereafter robustly internalized and quantitatively degraded. This multimodal mechanism of action is found only in few of the tetravalent constructs investigated, which must target specific epitopes on HER2 in a defined geometric arrangement. The inhibitory effect of our antibody as single agent surpasses the combination of trastuzumab and pertuzumab as well as its parental mAbs in vitro and it is effective in a xenograft model.

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  • Vulovic, I., Yao, Q., Park, Y. J., Courbet, A., Norris, A., Busch, F., Sahasrabuddhe, A., Merten, H., Sahtoe, D. D., Ueda, G., Fallas, J. A., Weaver, S. J., Hsia, Y., Langan, R. A., Plückthun, A., Wysocki, V. H., Veesler, D., Jensen, G. J., and Baker, D. (2021). Generation of ordered protein assemblies using rigid three-body fusion. Proc. Natl. Acad. Sci. U. S. A. 118, e2015037118.

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    Abstract

    Protein nanomaterial design is an emerging discipline with applications in medicine and beyond. A long-standing design approach uses genetic fusion to join protein homo-oligomer subunits via α-helical linkers to form more complex symmetric assemblies, but this method is hampered by linker flexibility and a dearth of geometric solutions. Here, we describe a general computational method for rigidly fusing homo-oligomer and spacer building blocks to generate user-defined architectures that generates far more geometric solutions than previous approaches. The fusion junctions are then optimized using Rosetta to minimize flexibility. We apply this method to design and test 92 dihedral symmetric protein assemblies using a set of designed homodimers and repeat protein building blocks. Experimental validation by native mass spectrometry, small-angle X-ray scattering, and negative-stain single-particle electron microscopy confirms the assembly states for 11 designs. Most of these assemblies are constructed from designed ankyrin repeat proteins (DARPins), held in place on one end by α-helical fusion and on the other by a designed homodimer interface, and we explored their use for cryogenic electron microscopy (cryo-EM) structure determination by incorporating DARPin variants selected to bind targets of interest. Although the target resolution was limited by preferred orientation effects and small scaffold size, we found that the dual anchoring strategy reduced the flexibility of the target-DARPIN complex with respect to the overall assembly, suggesting that multipoint anchoring of binding domains could contribute to cryo-EM structure determination of small proteins.

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  • Smith, S. N., Schubert, R., Simic, B.,Brücher, D., Schmid, M., Kirk, N., Freitag, P. C., Gradinaru, V., and Plückthun, A. (2021). The SHREAD gene therapy platform for paracrine delivery improves tumor localization and intratumoral effects of a clinical antibody. Proc. Natl. Acad. Sci. U. S. A. 118, e2017925118.

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    Abstract

    The goal of cancer-drug delivery is to achieve high levels of therapeutics within tumors with minimal systemic exposure that could cause toxicity. Producing biologics directly in situ where they diffuse and act locally is an attractive alternative to direct administration of recombinant therapeutics, as secretion by the tumor itself provides high local concentrations that act in a paracrine fashion continuously over an extended duration (paracrine delivery). We have engineered a SHielded, REtargeted ADenovirus (SHREAD) gene therapy platform that targets specific cells based on chosen surface markers and converts them into biofactories secreting therapeutics. In a proof of concept, a clinically approved antibody is delivered to orthotopic tumors in a model system in which precise biodistribution can be determined using tissue clearing with passive CLARITY technique (PACT) with high-resolution three-dimensional imaging and feature quantification within the tumors made transparent. We demonstrate high levels of tumor cell–specific transduction and significant and durable antibody production. PACT gives a localized quantification of the secreted therapeutic and allows us to directly observe enhanced pore formation in the tumor and destruction of the intact vasculature. In situ production of the antibody led to an 1,800-fold enhanced tumor-to-serum antibody concentration ratio compared to direct administration. Our detailed biochemical and microscopic analyses thus show that paracrine delivery with SHREAD could enable the use of highly potent therapeutic combinations, including those with systemic toxicity, to reach adequate therapeutic windows.

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  • Incaviglia, I., Frutiger, A.,  Blickenstorfer, Y.,  Treindl, F., Ammirati, G., Lüchtefeld, I., Dreier, B., Plückthun, A.,  Vörös, J., and  Reichmuth A. M. (2021). An approach for the real-time quantification of cytosolic protein–protein interactions in living cells. ACS Sens. 6, 1572-1582.

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    Abstract

    In recent years, cell-based assays have been frequently used in molecular interaction analysis. Cell-based assays complement traditional biochemical and biophysical methods, as they allow for molecular interaction analysis, mode of action studies, and even drug screening processes to be performed under physiologically relevant conditions. In most cellular assays, biomolecules are usually labeled to achieve specificity. In order to overcome some of the drawbacks associated with label-based assays, we have recently introduced “cell-based molography” as a biosensor for the analysis of specific molecular interactions involving native membrane receptors in living cells. Here, we expand this assay to cytosolic protein−protein interactions. First, we created a biomimetic membrane receptor by tethering one cytosolic interaction partner to the plasma membrane. The artificial construct is then coherently arranged into a two-dimensional pattern within the cytosol of living cells. Thanks to the molographic sensor, the specific interactions between the coherently arranged protein and its endogenous interaction partners become visible in real time without the use of a fluorescent label. This method turns out to be an important extension of cell-based molography because it expands the range of interactions that can be analyzed by molography to those in the cytosol of living cells.

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  • Waltenspühl, Y., Jeliazkov, J. R., Kummer, L., and Plückthun, A. (2021). Directed evolution for high functional production and stability of a challenging G protein-coupled receptorSci. Rep. 11, 8630.

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    Abstract

    Membrane proteins such as G protein-coupled receptors (GPCRs) carry out many fundamental biological functions, are involved in a large number of physiological responses, and are thus important drug targets. To allow detailed biophysical and structural studies, most of these important receptors have to be engineered to overcome their poor intrinsic stability and low expression levels. However, those GPCRs with especially poor properties cannot be successfully optimised even with the current technologies. Here, we present an engineering strategy, based on the combination of three previously developed directed evolution methods, to improve the properties of particularly challenging GPCRs. Application of this novel combination approach enabled the successful selection for improved and crystallisable variants of the human oxytocin receptor, a GPCR with particularly low intrinsic production levels. To analyse the selection results and, in particular, compare the mutations enriched in different hosts, we developed a Next-Generation Sequencing (NGS) strategy that combines long reads, covering the whole receptor, with exceptionally low error rates. This study thus gave insight into the evolution pressure on the same membrane protein in prokaryotes and eukaryotes. Our long-read NGS strategy provides a general methodology for the highly accurate analysis of libraries of point mutants during directed evolution.

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  • Waltenspühl, Y., Ehrenmann, J., Klenk, C., Plückthun, A. (2021) Engineering of challenging G protein-coupled receptors for structure determination and biophysical studies. Molecules 26, 1465.

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    Abstract

    Membrane proteins such as G protein-coupled receptors (GPCRs) exert fundamental biological functions and are involved in a multitude of physiological responses, making these receptors ideal drug targets. Drug discovery programs targeting GPCRs have been greatly facilitated by the emergence of high-resolution structures and the resulting opportunities to identify new chemical entities through structure-based drug design. To enable the determination of high-resolution structures of GPCRs, most receptors have to be engineered to overcome intrinsic hurdles such as their poor stability and low expression levels. In recent years, multiple engineering approaches have been developed to specifically address the technical difficulties of working with GPCRs, which are now beginning to make more challenging receptors accessible to detailed studies. Importantly, successfully engineered GPCRs are not only valuable in X-ray crystallography, but further enable biophysical studies with nuclear magnetic resonance spectroscopy, surface plasmon resonance, native mass spectrometry, and fluorescence anisotropy measurements, all of which are important for the detailed mechanistic understanding, which is the prerequisite for successful drug design. Here, we summarize engineering strategies based on directed evolution to reduce workload and enable biophysical experiments of particularly challenging GPCRs.

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  • Zhang, M., Gui, M., Wang, Z. F., Gorgulla, C., Yu, J. J., Wu, H., Sun, Z. Y. J., Klenk, C., Merklinger, L., Morstein, L., Hagn, F., Plückthun, A., Brown, A., Nasr, M. L., and Wagner, G. (2021). Cryo-EM structure of an activated GPCR-G protein complex in lipid nanodiscs. Nature Struct. Mol. Biol. 28, 258-267.

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    Abstract

    G-protein-coupled receptors (GPCRs) are the largest superfamily of transmembrane proteins and the targets of over 30% of currently marketed pharmaceuticals. Although several structures have been solved for GPCR-G protein complexes, few are in a lipid membrane environment. Here, we report cryo-EM structures of complexes of neurotensin, neurotensin receptor 1 and Gαi1β1γ1 in two conformational states, resolved to resolutions of 4.1 and 4.2 Å. The structures, determined in a lipid bilayer without any stabilizing antibodies or nanobodies, reveal an extended network of protein-protein interactions at the GPCR-G protein interface as compared to structures obtained in detergent micelles. The findings show that the lipid membrane modulates the structure and dynamics of complex formation and provide a molecular explanation for the stronger interaction between GPCRs and G proteins in lipid bilayers. We propose an allosteric mechanism for GDP release, providing new insights into the activation of G proteins for downstream signaling.

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  • Brücher, D., Kirchhammer, N., Smith, S. N., Schumacher, J., Schumacher, N., Kolibius, J., Freitag, P. C., Schmid, M. Weiss, F., Keller, C., Grove, M., Greber, U. F., Zippelius, A., and Plückthun, A. (2021). iMATCH: an integrated modular assembly system for therapeutic combination high-capacity adenovirus gene therapy. Mol. Therapy Meth. Clin. Dev. 20, 572-586.

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    Abstract

    Adenovirus-mediated combination gene therapies have shown promising results in vaccination or treating malignant and genetic diseases. Nevertheless, an efficient system for the rapid assembly and incorporation of therapeutic genes into high-capacity adenoviral vectors (HCAdVs) is still missing. In this study, we developed the iMATCH (integrated modular assembly for therapeutic combination HCAdVs) platform, which enables the generation and production of HCAdVs encoding therapeutic combinations in high quantity and purity within 3 weeks. Our modular cloning system facilitates the efficient combination of up to four expression cassettes and the rapid integration into HCAdV genomes with defined sizes. Helper viruses (HVs) and purification protocols were optimized to produce HCAdVs with distinct capsid modifications and unprecedented purity (0.1 ppm HVs). The constitution of HCAdVs, with adapters for targeting and a shield of trimerized single-chain variable fragment (scFv) for reduced liver clearance, mediated cell- and organ-specific targeting of HCAdVs. As proof of concept, we show that a single HCAdV encoding an anti PD-1 antibody, interleukin (IL)-12, and IL-2 produced all proteins, and it led to tumor regression and prolonged survival in tumor models, comparable to a mixture of single payload HCAdVs in vitro and in vivo. Therefore, the iMATCH system provides a versatile platform for the generation of high-capacity gene therapy vectors with a high potential for clinical development.

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  • Deluigi, M., Klipp, A., Klenk, C., Merklinger, L., Eberle, S. A., Morstein, L., Heine, P., Mittl, P. R. E., Ernst, P., Kamenecka, T. M., He, Y., Vacca, S., Egloff, P., Honegger, A., and Plückthun, A. (2021). Complexes of the neurotensin receptor 1 with small-molecule ligands reveal structural determinants of full, partial, and inverse agonism. Science Adv. 7eabe5504.

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    Abstract

    Neurotensin receptor 1 (NTSR1) and related G protein–coupled receptors of the ghrelin family are clinically unexploited, and several mechanistic aspects of their activation and inactivation have remained unclear. Enabled by a new crystallization design, we present five new structures: apo-state NTSR1 as well as complexes with nonpeptide inverse agonists SR48692 and SR142948A, partial agonist RTI-3a, and the novel full agonist SRI-9829, providing structural rationales on how ligands modulate NTSR1. The inverse agonists favor a large extracellular opening of helices VI and VII, undescribed so far for NTSR1, causing a constriction of the intracellular portion. In contrast, the full and partial agonists induce a binding site contraction, and their efficacy correlates with the ability to mimic the binding mode of the endogenous agonist neurotensin. Providing evidence of helical and side-chain rearrangements modulating receptor activation, our structural and functional data expand the mechanistic understanding of NTSR1 and potentially other peptidergic receptors.

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  • Goba, I., Goricanec, D., Schum, D., Hillenbrand, M., Plückthun, A., and Hagn, F. (2021). Probing the conformation states of neurotensin receptor 1 variants by NMR site-directed methyl labeling. ChemBioChem 22, 139-146.

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    Abstract

    G protein-coupled receptors (GPCRs) are key players in mediating signal transduction across the cell membrane. However, due to their intrinsic instability, many GPCRs are not suitable for structural investigations. Various approaches have been developed in recent years to remedy this situation, ranging from the use of more native membrane mimetics to protein-stabilization methods. The latter approach typically results in GPCRs that contain various numbers of mutations. However, probing the functionality of such variants by in vitro and in vivo assays is often time consuming. In addition, to validate the suitability of such GPCRs for structural investigations, an assessment of their conformation state is required. NMR spectroscopy has been proven to be suitable to probe the conformation state of GPCRs in solution. Here, by using chemical labeling with an isotope-labeled methyl probe, we show that the activity and the conformation state of stabilized neurotensin receptor 1 variants obtained from directed evolution can be efficiently assayed in 2D NMR experiments. This strategy enables the quantification of the active and inactive conformation states and the derivation of an estimation of the basal as well as agonist-induced activity of the receptor. Furthermore, this assay can be used as a readout when re-introducing agonist-dependent signaling into a highly stabilized, and thus rigidified, receptor by mutagenesis. This approach will be useful in cases where low production yields do not permit the addition of labeled compounds to the growth medium and where 1D NMR spectra of selectively 19 F-labeled receptors are not sufficient to resolve signal overlap for a more detailed analysis.

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  • Teale, W. D., Pasternak, T., Dal Bosco, C., Dovzhenko, A., Kratzat, K., Bildl, W., Schwörer, M., Falk, T., Ruperti, B., Schaefer, J. V., Shahriari, M., Pilgermayer, L., Li, X., Lübben, F., Plückthun, A., Schulte, U., and Palme, K. (2021). Flavonol-mediated stabilization of PIN efflux complexes regulates polar auxin transport. EMBO J. 40, e104416

     

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    Abstract

    The transport of auxin controls the rate, direction and localization of plant growth and development. The course of auxin transport is defined by the polar subcellular localization of the PIN proteins, a family of auxin efflux transporters. However, little is known about the composition and regulation of the PIN protein complex. Here, using blue-native PAGE and quantitative mass spectrometry, we identify native PIN core transport units as homo- and heteromers assembled from PIN1, PIN2, PIN3, PIN4 and PIN7 subunits only. Furthermore, we show that endogenous flavonols stabilize PIN dimers to regulate auxin efflux in the same way as does the auxin transport inhibitor 1-naphthylphthalamic acid (NPA). This inhibitory mechanism is counteracted both by the natural auxin indole-3-acetic acid and by phosphomimetic amino acids introduced into the PIN1 cytoplasmic domain. Our results lend mechanistic insights into an endogenous control mechanism which regulates PIN function and opens the way for a deeper understanding of the protein environment and regulation of the polar auxin transport complex.

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2020

# Reference PDF
  • Gray, A., Bradbury, A. R. M., Knappik, A., Plückthun, A., Borrebaeck, C. A. K., and Dübel, S. (2020) Animal-free alternatives and the antibody iceberg. Nature Biotechnol. 38, 1234-1239.

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    Abstract

    No abstract

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  • Nowak, K., Rosenthal, F., Karlberg, T., Bütepage, M., Thorsell, A.-G., Dreier, B., Grossmann, J., Sobek, J., Imhof, R., Lüscher, B., Schüler, H., Plückthun, A., Pedrioli, D. M. L., and Hottiger, M. O. (2020). Engineering Af1521 improves ADP-ribose binding and identification of ADP-ribosylated proteins. Nature Commun. 11, 5199.

     

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    Abstract

    Protein ADP-ribosylation is a reversible post-translational modification that regulates important cellular functions. The identification of modified proteins has proven challenging and has mainly been achieved via enrichment methodologies. Random mutagenesis was used here to develop an engineered Af1521 ADP-ribose binding macro domain protein with 1000-fold increased affinity towards ADP-ribose. The crystal structure reveals that two point mutations K35E and Y145R form a salt bridge within the ADP-ribose binding domain. This forces the proximal ribose to rotate within the binding pocket and, as a consequence, improves engineered Af1521 ADPr-binding affinity. Its use in our proteomic ADP-ribosylome workflow increases the ADP-ribosylated protein identification rates and yields greater ADP-ribosylome coverage. Furthermore, generation of an engineered Af1521 Fc fusion protein confirms the improved detection of cellular ADP-ribosylation by immunoblot and immunofluorescence. Thus, this engineered isoform of Af1521 can also serve as a valuable tool for the analysis of cellular ADP-ribosylation under in vivo conditions.

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  • Becker, L., Verdurmen, W. P. R., and Plückthun, A. (2020). Reengineering anthrax toxin protective antigen for improved receptor-specific protein delivery. BMC Biology 18, 100.

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    Abstract

    Background: To increase the size of the druggable proteome, it would be highly desirable to devise efficient methods to translocate designed binding proteins to the cytosol, as they could specifically target flat and hydrophobic protein-protein interfaces. If this could be done in a manner dependent on a cell surface receptor, two layers of specificity would be obtained: one for the cell type and the other for the cytosolic target. Bacterial protein toxins have naturally evolved such systems. Anthrax toxin consists of a pore-forming translocation unit (protective antigen (PA)) and a separate protein payload. When engineering PA to ablate binding to its own receptor and instead binding to a receptor of choice, by fusing a designed ankyrin repeat protein (DARPin), uptake in new cell types can be achieved. Results: Prepore-to-pore conversion of redirected PA already occurs at the cell surface, limiting the amount of PA that can be administered and thus limiting the amount of delivered payload. We hypothesized that the reason is a lack of a stabilizing interaction with wild-type PA receptor. We have now reengineered PA to incorporate the binding domain of the anthrax receptor CMG2, followed by a DARPin, binding to the receptor of choice. This construct is indeed stabilized, undergoes prepore-to-pore conversion only in late endosomes, can be administered to much higher concentrations without showing toxicity, and consequently delivers much higher amounts of payload to the cytosol. Conclusion: We believe that this reengineered system is an important step forward to addressing efficient cell-specific delivery of proteins to the cytosol.

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  • Brandl, F., Busslinger, S., Zangemeister-Wittke, U., and Plückthun, A. (2020). Optimizing the anti-tumor efficacy of protein-drug conjugates by engineering the molecular size and half-life. J. Control. Release 327, 186-197.

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    Abstract

    Despite some approvals of antibody-drug conjugates for cancer therapy, their clinical success rate is unsatisfactory because of very small therapeutic windows, influenced by on-target and off-target toxicities of conjugate and liberated toxin. Additional formats with systematically investigated molecular parameters must therefore be explored to increase their therapeutic window. Here we focused on the effective molecular weight. To generate conjugates with exactly defined drug loads and tunable pharmacokinetics, we used Designed Ankyrin Repeat Proteins (DARPins), fused to unstructured polypeptides of different lengths, to produce proteins with any desired half-life, to identify those with the best efficacy. We generated an EpCAM-targeting DARPin-MMAF conjugate, fused to PAS or XTEN of different lengths, and a matched series of controls of a non-binding DARPin to account for the enhanced permeability and retention (EPR) effect, covering half-lives of minutes to 20.6 h in mice. All conjugates were produced at high purity, and demonstrated high specificity and cytotoxicity in human tumor cell cultures, with IC50 values in the low nM range, independent of the polypeptide type and length. Due to their more facile purification, the PASylated conjugates were tested in nude mice bearing HT29 tumor xenografts. Independent of their size, all PASylated conjugates were very well tolerated after repeated systemic administration of 300 nmol/kg. We found that the conjugates with intermediate size and half-life showed the strongest anti-tumor effects, and deduced that this effect is a compromise of serum half-life and diffusion within the tumor, as on-rates and affinities are essentially identical, with extravasation playing only a very minor role.

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  • Brücher, D., Vojtech, F., Smith, S. N., Heck, A. J. R., and Plückthun, A. (2020). Malignant tissues produce divergent antibody glycosylation of relevance for cancer gene therapy effectiveness. mAbs 12, e1792084

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    Abstract

    Gene therapy approaches now allow for the production of therapeutic antibodies by healthy or cancerous human tissues directly in vivo, and, with an increasing number of gene delivery methods available, the cell type for expression can be chosen. Yet, little is known about the biophysical changes introduced by expressing antibodies from producer cells or tissues targeted by gene therapy approaches, nor about the consequences for the type of glycosylation. The effects of different glycosylation on therapeutic antibodies have been well studied by controlling their glycan compositions in non-human mammalian production cells, i.e., Chinese hamster ovary cells. Therefore, we investigated the glycosylation state of clinically approved antibodies secreted from cancer tissues frequently targeted by in vivo gene therapy, using native mass spectrometry and glycoproteomics. We found that antibody sialylation and fucosylation depended on the producer tissue and the antibody isotype, allowing us to identify optimal producer cell types according to the desired mode of action of the antibody. Furthermore, we discovered that high amounts (>20%) of non-glycosylated antibodies were produced in cells sensitive to the action of the produced antibodies. Different glycosylation in different producer cells can translate into an altered potency of in-vivo produced antibodies, depending on the desired mode of action, and can affect their serum half-lives. These results increase our knowledge about antibodies produced from cells targeted by gene therapy, enabling development of improved cancer gene therapy vectors that can include in vivo glycoengineering of expressed antibodies to optimize their efficacies, depending on the desired mode of action.

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  • Voskuil, J. L. A., Bandrowski, A., Begley, C. G., Bradbury, A. R. M., Chalmers, A. D., Gomes, A. V., Hardcastle, T., Lund-Johansen, F., Plückthun, A., Roncador, G., Solache, A., Taussig, M. J., Trimmer, J. S., Williams, C., and Goodman, S. L. (2020). The Antibody Society’s antibody validation webinar series. mAbs 12, e1794421

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    Abstract

    In the wake of the reproducibility crisis and numerous discussions on how commercially available antibodies as research tool contribute to it, The Antibody Society developed a series of 10 webinars to address the issues involved. The webinars were delivered by speakers with both academic and commercial backgrounds. This report highlights the problems, and offers solutions to help the scientific community appropriately identify the right antibodies and to validate them for their research and development projects. Despite the various solutions proposed here, they must be applied on a case-by-case basis. Each antibody must be verified based on the content of the product sheet, and subsequently through experimentation to confirm integrity, specificity and selectivity. Verification needs to focus on the precise application and tissue/cell type for which the antibody will be used, and all verification data must be reported openly. The various approaches discussed here all have caveats, so a combination of solutions must be considered.

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  • Gray, A. C., Bradbury, A. R. M., Knappik, A., Plückthun, A., Borrebaeck, C. A. K., and Dübel, S. (2020). Animal-derived-antibody generation faces strict reform in accordance with European Union policy on animal use. Nat. Methods 17, 755-756.

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    Abstract

    No abstract

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  • Waltenspühl, Y., Schöppe, J., Ehrenmann, J., Kummer, L., and Plückthun, A. (2020). Crystal structure of the human oxytocin receptor. Science Advances 6, eabb5419.

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    Abstract

    The peptide hormone oxytocin modulates socioemotional behavior and sexual reproduction via the centrally ex-pressed oxytocin receptor (OTR) across several species. Here, we report the crystal structure of human OTR in complex with retosiban, a nonpeptidic antagonist developed as an oral drug for the prevention of preterm labor. Our structure reveals insights into the detailed interactions between the G protein–coupled receptor (GPCR) and an OTR-selective antagonist. The observation of an extrahelical cholesterol molecule, binding in an unexpected location between helices IV and V, provides a structural rationale for its allosteric effect and critical influence on OTR function. Further-more, our structure in combination with experimental data allows the identification of a conserved neurohypophyseal receptor-specific coordination site for Mg2+ that acts as potent, positive allosteric modulator for agonist binding. Together, these results further our molecular understanding of the oxytocin/vasopressin receptor family and will facilitate structure-guided development of new therapeutics.

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  • Chabloz, A., Schaefer, J. V., Kozieradzki, I., Cronin, S. J. F., Strebinger, D., Macaluso, F., Wald, J., Rabbitts, T. H., Plückthun, A., Marlovits, T. C., and Penninger, J. M. (2020). Salmonella-based platform for efficient delivery of functional binding proteins to the cytosol. Commun. Biol. 3, 342.

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    Abstract

    Protein-based affinity reagents (like antibodies or alternative binding scaffolds) offer wide-ranging applications for basic research and therapeutic approaches. However, whereas small chemical molecules efficiently reach intracellular targets, the delivery of macromolecules into the cytosol of cells remains a major challenge; thus cytosolic applications of protein-based reagents are rather limited. Some pathogenic bacteria have evolved a conserved type III secretion system (T3SS) which allows the delivery of effector proteins into eukaryotic cells. Here, we enhance the T3SS of an avirulent strain of Salmonella typhimurium to reproducibly deliver multiple classes of recombinant proteins into eukaryotic cells. The efficacy of the system is probed with both DARPins and monobodies to functionally inhibit the paradigmatic and largely undruggable RAS signaling pathway. Thus, we develop a bacterial secretion system for potent cytosolic delivery of therapeutic macromolecules.

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  • Persson, B. D., Lenman, A., Frangsmyr, L., Schmid, M., Ahlm, C., Plückthun, A., Jenssen, H., and Arnberg, N. (2020). Lactoferrin-hexon interactions mediate CAR-independent adenovirus infection of human respiratory cells. J. Virol.94, e00542-00520.

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    Abstract

    Virus entry into host cells is a complex process that is largely regulated by access to specific cellular receptors. Human adenoviruses (HAdVs) and many other viruses use cell adhesion molecules such as the coxsackievirus and adenovirus receptor (CAR) for attachment to and entry into target cells. These molecules are rarely expressed on the apical side of polarized epithelial cells, which raises the question of how adenoviruses-and other viruses that engage cell adhesion molecules-enter polarized cells from the apical side to initiate infection. We have previously shown that species C HAdVs utilize lactoferrin-a common innate immune component secreted to respiratory mucosa-for infection via unknown mechanisms. Using a series of biochemical, cellular, and molecular biology approaches, we mapped this effect to the proteolytically cleavable, positively charged, N-terminal 49 residues of human lactoferrin (hLF) known as human lactoferricin (hLfcin). Lactoferricin (Lfcin) binds to the hexon protein on the viral capsid and anchors the virus to an unknown receptor structure of target cells, resulting in infection. These findings suggest that HAdVs use distinct cell entry mechanisms at different stages of infection. To initiate infection, entry is likely to occur at the apical side of polarized epithelial cells, largely by means of hLF and hLfcin bridging HAdV capsids via hexons to as-yet-unknown receptors; when infection is established, progeny virions released from the basolateral side enter neighboring cells by means of hLF/hLfcin and CAR in parallel.IMPORTANCE Many viruses enter target cells using cell adhesion molecules as receptors. Paradoxically, these molecules are abundant on the lateral and basolateral side of intact, polarized, epithelial target cells, but absent on the apical side that must be penetrated by incoming viruses to initiate infection. Our study provides a model whereby viruses use different mechanisms to infect polarized epithelial cells depending on which side of the cell-apical or lateral/basolateral-is attacked. This study may also be useful to understand the biology of other viruses that use cell adhesion molecules as receptors.

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  • Schuster, M., Deluigi, M., Pantic, M., Vacca, S., Baumann, C., Scott, D. J., Plückthun, A., and Zerbe, O. (2020). Optimizing the alpha1B- adrenergic receptor for solution NMR studies. Biochim. Biophys. Acta Biomembr. 1862, 183354.

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    Abstract

    Sample preparation for NMR studies of G protein-coupled receptors faces special requirements: Proteins need to be stable for prolonged measurements at elevated temperatures, they should ideally be uniformly labeled with the stable isotopes (13)C, (15)N, and all carbon-bound protons should be replaced by deuterons. In addition, certain NMR experiments require protonated methyl groups in the presence of a perdeuterated background. All these requirements are most easily satisfied when using Escherichia coli as the expression host. Here we describe a workflow, starting from a temperature-stabilized mutant of the alpha1B-adrenergic receptor, obtained using the CHESS methodology, into an even more stable species, in which flexible parts from termini were removed and the intracellular loop 3 (ICL3) was stabilized against proteolytic cleavage. The yield after purification corresponds to 1-2 mg/L of D2O culture. The final purification step is ligand-affinity chromatography to ensure that only well-folded ligand-binding protein is isolated. Proper selection of detergent has a remarkable influence on the quality of NMR spectra. All optimization steps of sequence and detergent are monitored on a small scale by monitoring the melting temperature and long-term thermal stability to allow for screening of many conditions. The stabilized mutant of the alpha1B-adrenergic receptor was additionally incorporated in nanodiscs, but displayed slightly inferior spectra compared to a sample in detergent micelles. Finally, both [(15)N,(1)H]- as well as [(13)C,(1)H]-HSQC spectra are shown highlighting the high quality of the final NMR sample. Importantly, the quality of [(13)C,(1)H]-HSQC spectra indicates that the so prepared receptor could be used for studying side-chain dynamics.

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  • Gray, A. C., Bradbury, A., Dübel, S., Knappik, A., Plückthun, A. and Borrebaeck, C. A. K. (2020). Reproducibility: bypass animals for antibody production. Nature [Correspondence] 581, 262 .

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    Abstract

    No abstract

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  • Andres, F., Schwill, M., Boersma, Y. L., and Plückthun, A. (2020) High-throughput generation of bispecific binding proteins by sortase A-mediated coupling for direct functional screening in cell culture. Mol. Cancer Ther. 19, 1080-1088.

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    Abstract

    High-throughput construction of multivalent binders and subsequent screening for biological activity represents a fundamental challenge: a linear increase of monovalent components translates to the square of possible bivalent combinations. Even high-efficiency cloning and expression methods become limiting when thousands of bispecific binders need to be screened for activity. In this study, we present an in vitro method for the efficient production of flexibly linked bispecific binding agents from individually expressed and purified monovalent binders. We established a sortase A-mediated coupling reaction to generate bispecific Designed Ankyrin Repeat Proteins (DARPins), with an optimized reaction maximizing the bivalent coupling product with low levels of monovalent side-products. These one-pot reaction mixtures could be used directly, without further purification, in cell-based assays. We generated a matrix of 441 different bispecific DARPins against the extracellular domains of the cancer-associated receptors EGFR, ErbB2, ErbB3, ErbB4, EpCAM and c-MET and screened on two different ErbB2-positive cancer cells lines for growth-inhibitory effects. We identified not only known but also novel biologically active biparatopic DARPins. Furthermore, we found that the cancer cell lines respond in a highly reproducible and defined manner to the treatment with the 441 different bivalent binding agents. The generated response profiles can thus be used for functional characterization of cell lines since they are strongly related to the cell line-specific surface receptor landscape. Thus, our method not only represents a robust tool for screening and lead identification of bispecific binding agents, but additionally offers an orthogonal approach for the functional characterization of cancer cell lines.

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  • Petrencakova, M., Filandr, F., Hovan, A., Yassaghi, G., Man, P., Kozar, T., Schwer, M. S., Jancura, D., Plückthun, A., Novak, P., Miskovsky, P., Bano, G., and Sedlak, E. (2020) Photoinduced damage of AsLOV2 domain is accompanied by increased singlet oxygen production due to flavin dissociation. Sci. Rep. 10, 4119.

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    Abstract

    Flavin mononucleotide (FMN) belongs to the group of very efficient endogenous photosensitizers producing singlet oxygen, (1)O2, but with limited ability to be targeted. On the other hand, in genetically-encoded photosensitizers, which can be targeted by means of various tags, the efficiency of FMN to produce (1)O2 is significantly diminished due to its interactions with surrounding amino acid residues. Recently, an increase of (1)O2 production yield by FMN buried in a protein matrix was achieved by a decrease of quenching of the cofactor excited states by weakening of the protein-FMN interactions while still forming a complex. Here, we suggest an alternative approach which relies on the blue light irradiation-induced dissociation of FMN to solvent. This dissociation unlocks the full capacity of FMN as (1)O2 producer. Our suggestion is based on the study of an irradiation effect on two variants of the LOV2 domain from Avena sativa; wild type, AsLOV2 wt, and the variant with a replaced cysteine residue, AsLOV2 C450A. We detected irradiation-induced conformational changes as well as oxidation of several amino acids in both AsLOV2 variants. Detailed analysis of these observations indicates that irradiation-induced increase in (1)O2 production is caused by a release of FMN from the protein. Moreover, an increased FMN dissociation from AsLOV2 wt in comparison with AsLOV2 C450A points to a role of C450 oxidation in repelling the cofactor from the protein.

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  • Ernst, P., Zosel, F., Reichen, C., Nettels, D., Schuler, B., and Plückthun, A. (2020) Structure-guided design of a peptide lock for modular peptide binders. ACS Chem. Biol. 15, 457-468.

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    Abstract

    Peptides play an important role in intermolecular interactions and are frequent analytes in diagnostic assays, also as unstructured, linear epitopes in whole proteins. Yet, due to the many different sequence possibilities even for short peptides, classical selection of binding proteins from a library, one at a time, is not scalable to proteomes. However, moving away from selection to a rational assembly of preselected modules binding to predefined linear epitopes would split the problem into smaller parts. These modules could then be reassembled in any desired order to bind to, in principle, arbitrary sequences, thereby circumventing any new rounds of selection. Designed Armadillo repeat proteins (dArmRPs) are modular, and they do bind elongated peptides in a modular way. Their consensus sequence carries pockets that prefer arginine and lysine. In our quest to select pockets for all amino acid side chains, we had discovered that repetitive sequences can lead to register shifts and peptide flipping during selections from libraries, hindering the selection of new binding specificities. To solve this problem, we now created an orthogonal binding specificity by a combination of grafting from beta-catenin, computational design and mutual optimization of the pocket and the bound peptide. We have confirmed the design and the desired interactions by X-ray structure determination. Furthermore, we could confirm the absence of sliding in solution by a single-molecule Forster resonance energy transfer. The new pocket could be moved from the N-terminus of the protein to the middle, retaining its properties, further underlining the modularity of the system.

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  • Mittl, P. R., Ernst, P. and Plückthun, A. (2020) Chaperone-assisted structure elucidation with DARPins. Curr. Opin. Struct. Biol. 60, 93-100.

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    Abstract

    Designed ankyrin repeat proteins (DARPins) are artificial binding proteins that have found many uses in therapy, diagnostics and biochemical research. They substantially extend the scope of antibody-derived binders. Their high affinity and specificity, rigidity, extended paratope, and facile bacterial production make them attractive for structural biology. Complexes with simple DARPins have been crystallized for a long time, but particularly the rigid helix fusion strategy has opened new opportunities. Rigid DARPin fusions expand crystallization space, enable recruitment of targets in a host lattice and reduce the size limit for cryo-EM. Besides applications in structural biology, rigid DARPin fusions also serve as molecular probes in cells to investigate spatial restraints in targets.

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2019

# Reference PDF
  • Stüber, J. C. and Plückthun, A. (2019) Labeling surface proteins with high specificity: Intrinsic limitations of phosphopantetheinyl transferase systems. PLoS One 14, e0226579.

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    Abstract

    OBJECTIVE: Fluorescent labeling of specific cell-surface proteins enables a manifold of techniques to study their function in health and disease. A frequently cited family of methods employs phosphopantetheinyl transferases (PPTases) to attach probes, provided as conjugates of Coenzyme A. This method appears attractive, as only short peptide tags genetically fused to the protein of interest are needed as conjugation sites. Here, we describe observations we made when evaluating such protocols for delicate single-molecule applications where we require a particular combination of dyes, low background binding or low labeling of other proteins, and a high degree of labeling. RESULTS: When we tested a PPTase-acceptor peptide couple with several experimental protocols and various CoA conjugates for labeling of a protein on the cell surface, we noticed substantial non-specific labeling. For the first time, we provide here a quantification of the non-specific fraction of the signals obtained using appropriate controls. We further present evidence that this background is due to CoA-dye conjugates entering the cell, where they may be covalently attached to endogenous proteins. However, when studying cell-surface proteins, most fluorescent readouts require that labeling is strictly limited to the protein of interest located at the cell surface. While such data have so far been missing in the literature, they suggest that for applications where labeling of unwanted molecules would affect the conclusions, researchers need to be aware of this potential non-specificity of PPTase methods when selecting a labeling strategy. We show, again by quantitative comparison, that the HaloTag is a viable alternative.

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  • Ehrenmann, J., Schöppe, J., Klenk, C. and Plückthun, A. (2019) New views into class B GPCRs from the crystal structure of PTH1R. FEBS J. 286, 4852-4860.

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    Abstract

    The parathyroid hormone 1 receptor (PTH1R) is a major regulator of mineral ion homeostasis and bone metabolism and is thus considered an attractive drug target for the treatment of disorders in calcium metabolism and bone-related diseases such as osteoporosis. PTH1R is a member of the class B of GPCRs, which all share a dynamic multidomain binding mechanism to the peptide hormone. For a long time, these complexes have been recalcitrant to structural studies despite their great therapeutic relevance. Through extensive engineering of both the receptor and the peptide agonist ligand, we were able to determine the first high-resolution structure of a PTH1R-agonist complex. Comparisons of the PTH1R crystal structure with subsequently reported cryo-electron microscopy structures of the same receptor in complex with a G protein, as well as with other class B GPCR structures bound to antagonists, reveal new insights into the two-step activation mechanism of class B GPCRs and extend our understanding of the precise molecular rearrangements during receptor activation.

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  • Balakrishnan, A., Rajan, A., Salter, A. I., Kosasih, P. L., Wu, Q., Voutsinas, J., Jensen, M. C., Plückthun, A. and Riddell, S. R. (2019) Multispecific targeting with synthetic ankyrin repeat motif chimeric antigen receptors. Clin. Cancer Res. 25, 7506-7516.

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    Abstract

    PURPOSE: The outgrowth of antigen-negative variants is a significant challenge for adoptive therapy with T cells that target a single specificity. Chimeric antigen receptors (CAR) are typically designed with one or two scFvs that impart antigen specificity fused to activation and costimulation domains of T-cell signaling molecules. We designed and evaluated the function of CARs with up to three specificities for overcoming tumor escape using Designed Ankyrin Repeat Proteins (DARPins) rather than scFvs for tumor recognition. EXPERIMENTAL DESIGN: A monospecific CAR was designed with a DARPin binder (E01) specific for EGFR and compared with a CAR designed using an anti-EGFR scFv. CAR constructs in which DARPins specific for EGFR, EpCAM, and HER2 were linked together in a single CAR were then designed and optimized to achieve multispecific tumor recognition. The efficacy of CAR-T cells bearing a multispecific DARPin CAR for treating tumors with heterogeneous antigen expression was evaluated in vivo. RESULTS: The monospecific anti-EGFR E01 DARPin conferred potent tumor regression against EGFR(+) targets that was comparable with an anti-EGFR scFv CAR. Linking three separate DARPins in tandem was feasible and in an optimized format generated a single tumor recognition domain that targeted a mixture of heterogeneous tumor cells, each expressing a single antigen, and displayed synergistic activity when tumor cells expressed more than one target antigen. CONCLUSIONS: DARPins can serve as high-affinity recognition motifs for CAR design, and their robust architecture enables linking of multiple binders against different antigens to achieve functional synergy and reduce antigen escape.

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  • Chernyavska, M., Schmid, M., Freitag, P. C., Palacio-Castaneda, V., Piruska, A., Huck, W. T. S., Plückthun, A. and Verdurmen, W. P. R. (2019) Unravelling receptor and RGD motif dependence of retargeted adenoviral vectors using advanced tumor model systems. Sci. Rep. 9, 18568.

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    Abstract

    Recent advances in engineering adenoviruses are paving the way for new therapeutic gene delivery approaches in cancer. However, there is limited knowledge regarding the impact of adenoviral retargeting on transduction efficiency in more complex tumor architectures, and the role of the RGD loop at the penton base in retargeting is unclear. To address this gap, we used tumor models of increasing complexity to study the role of the receptor and the RGD motif. Employing tumor-fibroblast co-culture models, we demonstrate the importance of the RGD motif for efficient transduction in 2D through the epithelial cell adhesion molecule (EpCAM), but not the epidermal growth factor receptor (EGFR). Via optical clearing of co-culture spheroids, we show that the RGD motif is required for transduction via both receptors in 3D tumor architectures. We subsequently employed a custom-designed microfluidic model containing collagen-embedded tumor spheroids, mimicking the interplay between interstitial flow, extracellular matrix and adenoviral transduction. Image analysis of on-chip cleared spheroids indicated the importance of the RGD motif for on-chip adenoviral transduction. Together, our results show the interrelationship between receptor characteristics, the RGD motif, the 3D tumor architecture and retargeted adenoviral transduction efficiency. The findings are important for the rational design of next-generation therapeutic adenoviruses.

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  • Ernst, P., Honegger, A., van der Valk, F., Ewald, C., Mittl, P. R. E. and Plückthun, A. (2019) Rigid fusions of designed helical repeat binding proteins efficiently protect a binding surface from crystal contacts. Sci. Rep. 9, 16162.

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    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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  • Dunne, M., Rupf, B., Tala, M., Qabrati, X., Ernst, P., Shen, Y., Sumrall, E., Heeb, L., Plückthun, A., Loessner, M. J. and Kilcher, S. (2019) Reprogramming bacteriophage host range through structure-guided design of chimeric receptor binding proteins. Cell Rep. 29, 1336-1350.

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    Abstract

    Bacteriophages provide excellent tools for diagnostics, remediation, and targeted microbiome manipulation, yet isolating viruses with suitable host specificity remains challenging. Using Listeria phage PSA, we present a synthetic biology blueprint for host-range engineering through targeted modification of serovar-specific receptor binding proteins (RBPs). We identify Gp15 as the PSA RBP and construct a synthetic phage library featuring sequence-randomized RBPs, from which host range mutants are isolated and subsequently integrated into a synthetic, polyvalent phage with extended host range. To enable rational design of chimeric RBPs, we determine the crystal structure of the Gp15 receptor-binding carboxyl terminus at 1.7-A resolution and employ bioinformatics to identify compatible, prophage-encoded RBPs targeting different Listeria serovars. Structure-guided design enables exchange of heterologous RBP head, neck, or shoulder domains to generate chimeric phages with predictable and extended host ranges. These strategies will facilitate the development of phage biologics based on standardized virus scaffolds with tunable host specificities.

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  • Ernst, P., Plückthun, A. and Mittl, P. R. E. (2019) Structural analysis of biological targets by host:guest crystal lattice engineering. Sci. Rep. 9, 15199.

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    Abstract

    To overcome the laborious identification of crystallisation conditions for protein X-ray crystallography, we developed a method where the examined protein is immobilised as a guest molecule in a universal host lattice. We applied crystal engineering to create a generic crystalline host lattice under reproducible, predefined conditions and analysed the structures of target guest molecules of different size, namely two 15-mer peptides and green fluorescent protein (sfGFP). A fusion protein with an N-terminal endo-alpha-N-acetylgalactosaminidase (EngBF) domain and a C-terminal designed ankyrin repeat protein (DARPin) domain establishes the crystal lattice. The target is recruited into the host lattice, always in the same crystal form, through binding to the DARPin. The target structures can be determined rapidly from difference Fourier maps, whose quality depends on the size of the target and the orientation of the DARPin.

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  • Renko, M., Fiedler, M., Rutherford, T. J., Schaefer, J. V., Pluckthun, A. and Bienz, M. (2019) Rotational symmetry of the structured Chip/LDB-SSDP core module of the Wnt enhanceosome. Proc. Natl. Acad. Sci. U. S. A. 116, 20977-20983.

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    Abstract

    The Chip/LIM-domain binding protein (LDB)-single-stranded DNA-binding protein (SSDP) (ChiLS) complex controls numerous cell-fate decisions in animal cells, by mediating transcription of developmental control genes via remote enhancers. ChiLS is recruited to these enhancers by lineage-specific LIM-domain proteins that bind to its Chip/LDB subunit. ChiLS recently emerged as the core module of the Wnt enhanceosome, a multiprotein complex that primes developmental control genes for timely Wnt responses. ChiLS binds to NPFxD motifs within Pygopus (Pygo) and the Osa/ARID1A subunit of the BAF chromatin remodeling complex, which could synergize with LIM proteins in tethering ChiLS to enhancers. Chip/LDB and SSDP both contain N-terminal dimerization domains that constitute the bulk of their structured cores. Here, we report the crystal structures of these dimerization domains, in part aided by DARPin chaperones. We conducted systematic surface scanning by structure-designed mutations, followed by in vitro and in vivo binding assays, to determine conserved surface residues required for binding between Chip/LDB, SSDP, and Pygo-NPFxD. Based on this, and on the 4:2 (SSDP-Chip/LDB) stoichiometry of ChiLS, we derive a highly constrained structural model for this complex, which adopts a rotationally symmetrical SSDP2-LDB2-SSDP2 architecture. Integrity of ChiLS is essential for Pygo binding, and our mutational analysis places the NPFxD pockets on either side of the Chip/LDB dimer, each flanked by an SSDP dimer. The symmetry and multivalency of ChiLS underpin its function as an enhancer module integrating Wnt signals with lineage-specific factors to operate context-dependent transcriptional switches that are pivotal for normal development and cancer.

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  • Heine, P., Witt, G., Gilardi, A., Gribbon, P., Kummer, L. and Plückthun, A. (2019) High-throughput fluorescence polarization assay to identify ligands using purified G-protein-coupled receptor. SLAS Discov. 24, 915-927.

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    Abstract

    The development of cell-free high-throughput (HT) methods to screen and select novel lead compounds remains one of the key challenges in G protein-coupled receptor (GPCR) drug discovery. Mutational approaches have allowed the stabilization of GPCRs in a purified and ligand-free state. The increased intramolecular stability overcomes two major drawbacks for usage in in vitro screening, the low receptor density on cells and the low stability in micelles. Here, an HT fluorescence polarization (FP) assay for the neurotensin receptor type 1 (NTS1) was developed. The assay operates in a 384-well format and is tolerant to DMSO. From a library screen of 1272 compounds, 12 (~1%) were identified as primary hits. These compounds were validated in orthogonal assay formats using surface plasmon resonance (SPR), which confirmed binding of seven compounds (0.6%). One of these compounds showed a clear preference for the orthosteric binding pocket with submicromolar affinity. A second compound revealed binding at a nonorthosteric binding region and showed specific biological activity on NTS1-expressing cells. A search of analogs led to further enhancement of affinity, but at the expense of activity. The identification of GPCR ligands in a cell-free assay should allow the expansion of GPCR pharmaceuticals with antagonistic or agonistic activity.

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  • Brandl, F., Merten, H., Zimmermann, M., Behe, M., Zangemeister-Wittke, U. and Plückthun, A. (2019) Influence of size and charge of unstructured polypeptides on pharmacokinetics and biodistribution of targeted fusion proteins. J. Control. Release 307, 379-392.

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    Abstract

    Alternative non-IgG binding proteins developed for therapy are small in size and, thus, are rapidly cleared from the circulation by renal filtration. To avoid repeated injection or continuous infusion for the maintenance of therapeutic serum concentrations, extensions of unfolded polypeptides have been developed to prolong serum half-life, but systematic, comparative studies investigating the influence of their size and charge on serum half-life, extravasation, tumor localization and excretion mechanisms have so far been lacking. Here we used a high-affinity Designed Ankyrin Repeat Protein (DARPin) targeting the tumor marker epithelial cell adhesion molecule (EpCAM) in a preclinical tumor xenograft model in mice, and fused it with a series of defined unstructured polypeptides. We used three different sizes of two previously described polypeptides, an uncharged one consisting of only Pro, Ala and Ser (termed PAS) and a charged one consisting of Pro, Ala, Ser, Thr, Gly, Glu (termed XTEN) and performed for the first time a precise comparative localization, distribution and extravasation study. Pharmacokinetic analysis showed a clear linear relationship between hydrodynamic radius and serum half-life across both polypeptides, reaching a half-life of up to 21h in mice. Tumor uptake was EpCAM-dependent and directly proportional to half-life and size, showing an even tumor penetration for all fusion proteins without unspecific accumulation in non-target tissue. Unexpectedly, charge had no influence on any parameter, neither tumor nor tissue accumulation nor kidney elimination kinetics. Thus, both polypeptide types have a very similar potential for precise half-life modification and tumor targeting.

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  • Merten, H., Schaefer, J. V., Brandl, F., Zangemeister-Wittke, U. and Plückthun, A. (2019) Facile site-specific multiconjugation strategies in recombinant proteins produced in bacteria. Methods Mol. Biol. 2033, 253-273.

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    Abstract

    For biomedical applications, proteins may require conjugation to small and large molecules. Typical examples are dyes for imaging, cytotoxic effector molecules for cell killing, or half-life extension modules for optimized pharmacokinetics. Although many conjugation strategies are straightforward to apply, most of them do not enable site-specific and orthogonal conjugation, and do not yield a defined stoichiometry. Moreover, techniques offering these desirable features often rely on complex expression procedures and suffer from low production yields. A more promising manufacturing strategy for flexible, site-specific and stoichiometrically defined payloading of proteins is the combination of click chemistry and thiol-maleimide conjugation, which even enables dual labeling when used consecutively. Here, we describe as an example the production of Designed Ankyrin Repeat Proteins (DARPins), a non-IgG binding scaffold, in a specific E. coli strain to obtain high yields of protein carrying both a thiol and an azide group. We provide straightforward protocols for strain-promoted azide-alkyne cycloaddition (SPAAC) and thiol-maleimide conjugation, and furthermore compare these conjugation chemistries with existing alternatives like copper-catalyzed azide-alkyne cycloaddition (CuAAC). Finally, detailed instructions for reactivity analysis and yield estimations of the reactions are provided.

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  • Zhang, Y., Auger, S., Schaefer, J. V., Plückthun, A. and Distefano, M. D. (2019) Site-selective enzymatic labeling of designed ankyrin repeat proteins using protein farnesyltransferase. Methods Mol. Biol. 2033, 207-219.

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    Abstract

    Affinity agents coupled to a functional moiety play an ever-increasing role in modern medicine, ranging from radiolabeled selective binders in diagnosis to antibody-drug conjugates in targeted therapies. In biomedical research, protein coupling to fluorophores, surfaces and nanoparticles has become an integral part of many procedures. In addition to antibodies, small scaffold proteins with similar target binding properties are being widely explored as alternative targeting moieties. To label these binders of interest with different functional moieties, conventional chemical coupling methods can be employed, but often result in heterogeneously modified protein products. In contrast, enzymatic labeling methods are highly site-specific and efficient. Protein farnesyltransferase (PFTase) catalyzes the transfer of an isoprenoid moiety from farnesyl diphosphate (FPP) to a cysteine residue in a C-terminal CaaX motif at the C-terminus of a protein substrate. The addition of only four amino acid residues minimizes the influence on the native protein structure. In addition, a variety of isoprenoid analogs containing different bioorthogonal functional groups, including azides, alkynes, and aldehydes, have been developed to enable conjugation to various cargos after being incorporated onto the target protein by PFTase. In this protocol, we present a detailed procedure for labeling Designed Ankyrin Repeat Proteins (DARPins) engineered with a C-terminal CVIA sequence using an azide-containing FPP analog by yeast PFTase (yPFTase). In addition, procedures to subsequently conjugate the labeled DARPins to a TAMRA fluorophore using strained-promoted alkyne-azide cycloaddition (SPAAC) reactions as well as the sample preparation to evaluate the target binding ability of the conjugates by flow cytometry are described.

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  • Stüber, J. C., Kast, F. and Plückthun, A. (2019) High-throughput quantification of surface protein internalization and degradation. ACS Chem. Biol. 14, 1154-1163.

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    Abstract

    Cell surface proteins are key regulators of fundamental cellular processes and, therefore, often at the root of human diseases. Thus, a large number of targeted drugs which are approved or under development act upon cell surface proteins. Although down-regulation of surface proteins by many natural ligands is well-established, the ability of drug candidates to cause internalization or degradation of the target is only recently moving into focus. This property is important both for the pharmacokinetics and pharmacodynamics of the drug but may also constitute a potential resistance mechanism. The enormous numbers of drug candidates targeting cell surface molecules, comprising small molecules, antibodies, or alternative protein scaffolds, necessitate methods for the investigation of internalization and degradation in high throughput. Here, we present a generic high-throughput assay protocol, which allows the simultaneous and independent quantification of internalization and degradation of surface proteins on a single-cell level. Because we fuse a HaloTag to the cell surface protein of interest and exploit the differential cell permeability of two fluorescent HaloTag ligands, no labeling of the molecules to be screened is required. In contrast to previously described approaches, our homogeneous assay is performed with adherent live cells in a 96-well format. Through channel rescaling, we are furthermore able to obtain true relative abundances of surface and internal protein. We demonstrate the applicability of our procedure to three major drug targets, EGFR, HER2, and EpCAM, examining a selection of well-investigated but also novel small molecule ligands and protein affinity reagents.

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  • Radom, F., Paci, E. and Plückthun, A. (2019) Computational modeling of designed ankyrin repeat protein complexes with their targets. J. Mol. Biol. 431, 2852-2868.

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    Abstract

    Recombinant therapeutic proteins are playing an ever-increasing role in the clinic. High-affinity binding candidates can be produced in a high-throughput manner through the process of selection and evolution from large libraries, but the structures of the complexes with target protein can only be determined for a small number of them in a costly, low-throughput manner, typically by x-ray crystallography. Reliable modeling of complexes would greatly help to understand their mode of action and improve them by further engineering, for example, by designing bi-paratopic binders. Designed ankyrin repeat proteins (DARPins) are one such class of antibody mimetics that have proven useful in the clinic, in diagnostics and research. Here we have developed a standardized procedure to model DARPin-target complexes that can be used to predict the structures of unknown complexes. It requires only the sequence of a DARPin and a structure of the unbound target. The procedure includes homology modeling of the DARPin, modeling of the flexible parts of a target, rigid body docking to ensembles of the target and docking with a partially flexible backbone. For a set of diverse DARPin-target complexes tested it generated a single model of the complex that well approximates the native state of the complex. We provide a protocol that can be used in a semi-automated way and with tools that are freely available. The presented concepts should help to accelerate the development of novel bio-therapeutics for scaffolds with similar properties.

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  • Lorenzer, C., Streussnig, S., Tot, E., Winkler, A. M., Merten, H., Brandl, F., Sayers, E. J., Watson, P., Jones, A. T., Zangemeister-Wittke, U., Plückthun, A. and Winkler, J. (2019) Targeted delivery and endosomal cellular uptake of DARPin-siRNA bioconjugates: Influence of linker stability on gene silencing. Eur. J. Pharm. Biopharm. 141, 37-50.

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    Abstract

    Specific cell targeting and efficient intracellular delivery are major hurdles for the widespread therapeutic use of nucleic acid technologies, particularly siRNA mediated gene silencing. To enable receptor-mediated cell-specific targeting, we designed a synthesis scheme that can be generically used to engineer Designed Ankyrin Repeat Protein (DARPin)-siRNA bioconjugates. Different linkers, including labile disulfide-, and more stable thiol-maleimide- and triazole- (click chemistry) tethers were employed. Crosslinkers were first attached to a 3'-terminal aminohexyl chain on the siRNA sense strands. On the protein side thiols of a C-terminal cysteine were used as anchoring sites for disulfide- and thiol-maleimide conjugate formations, while strain-promoted azido-alkyne cycloadditions were carried out at a metabolically introduced N-terminal azidohomoalanine. After establishing efficient purification methods, highly pure products were obtained. Bioconjugates of EpCAM-targeted DARPins with siRNA directed at the luciferase gene were evaluated for cell-specific binding, uptake and gene silencing. As shown by flow cytometry and fluorescence microscopy, all constructs retained the highly specific and high-affinity antigen recognition properties of the native DARPin. As expected, internalization was observed only in EpCAM-positive cell lines, and predominantly endolysosomal localization was detected. Disulfide linked conjugates showed lower serum stability against cleavage at the linker and thus lower internalization into endosomes compared to thiol-maleimide- and triazole-linked conjugates, yet induced more pronounced gene silencing. This indicates that the siRNA payload needs to be liberated from the protein in the endosome. Our data confirm the promise of DARPin-siRNA bioconjugates for tumor targeting, but also identified endosomal retention and limited cytosolic escape of the siRNA as the rate-limiting step for more efficient gene silencing.

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  • Campanacci, V., Urvoas, A., Cantos-Fernandes, S., Aumont-Nicaise, M., Arteni, A. A., Velours, C., Valerio-Lepiniec, M., Dreier, B., Plückthun, A., Pilon, A., Pous, C., Minard, P. and Gigant, B. (2019) Insight into microtubule nucleation from tubulin-capping proteins. Proc. Natl. Acad. Sci. U. S. A. 116, 9859-9864.

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    Abstract

    Nucleation is one of the least understood steps of microtubule dynamics. It is a kinetically unfavorable process that is templated in the cell by the gamma-tubulin ring complex or by preexisting microtubules; it also occurs in vitro from pure tubulin. Here we study the nucleation inhibition potency of natural or artificial proteins in connection with their binding mode to the longitudinal surface of alpha- or beta-tubulin. The structure of tubulin-bound CopN, a Chlamydia protein that delays nucleation, suggests that this protein may interfere with two protofilaments at the (+) end of a nucleus. Designed ankyrin repeat proteins that share a binding mode similar to that of CopN also impede nucleation, whereas those that target only one protofilament do not. In addition, an alphaRep protein predicted to target two protofilaments at the (-) end does not delay nucleation, pointing to different behaviors at both ends of the nucleus. Our results link the interference with protofilaments at the (+) end and the inhibition of nucleation.

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  • Meksiriporn, B., Ludwicki, M. B., Stephens, E. A., Jiang, A., Lee, H. C., Waraho-Zhmayev, D., Kummer, L., Brandl, F., Plückthun, A. and DeLisa, M. P. (2019) A survival selection strategy for engineering synthetic binding proteins that specifically recognize post-translationally phosphorylated proteins. Nature Comm. 10, 1830.

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    Abstract

    There is an urgent need for affinity reagents that target phospho-modified sites on individual proteins; however, generating such reagents remains a significant challenge. Here, we describe a genetic selection strategy for routine laboratory isolation of phospho-specific designed ankyrin repeat proteins (DARPins) by linking in vivo affinity capture of a phosphorylated target protein with antibiotic resistance of Escherichia coli cells. The assay is validated using an existing panel of DARPins that selectively bind the nonphosphorylated (inactive) form of extracellular signal-regulated kinase 2 (ERK2) or its doubly phosphorylated (active) form (pERK2). We then use the selection to affinity-mature a phospho-specific DARPin without compromising its selectivity for pERK2 over ERK2 and to reprogram the substrate specificity of the same DARPin towards non-cognate ERK2. Collectively, these results establish our genetic selection as a useful and potentially generalizable protein engineering tool for studying phospho-specific binding proteins and customizing their affinity and selectivity.

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  • Andres, F., Iamele, L., Meyer, T., Stüber, J. C., Kast, F., Gherardi, E., Niemann, H. H. and Plückthun, A. (2019) Inhibition of the MET kinase activity and cell growth in MET-addicted cancer cells by bi-paratopic linking. J. Mol. Biol. 431, 2020-2039.

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    Abstract

    MET, the product of the c-MET proto-oncogene, and its ligand hepatocyte growth factor/scatter factor (HGF/SF) control survival, proliferation and migration during development and tissue regeneration. HGF/SF-MET signaling is equally crucial for growth and metastasis of a variety of human tumors, but resistance to small-molecule inhibitors of MET kinase develops rapidly and therapeutic antibody targeting remains challenging. We made use of the designed ankyrin repeat protein (DARPin) technology to develop an alternative approach for inhibiting MET. We generated a collection of MET-binding DARPins covering epitopes in the extracellular MET domains and created comprehensive sets of bi-paratopic fusion proteins. This new class of molecules efficiently inhibited MET kinase activity and downstream signaling, caused receptor downregulation and strongly inhibited the proliferation of MET-dependent gastric carcinoma cells carrying MET locus amplifications. MET-specific bi-paratopic DARPins may represent a novel and potent strategy for therapeutic targeting of MET and other receptors, and this study has elucidated their mode of action.

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  • Deprey, K., Becker, L., Kritzer, J. and Plückthun, A. (2019) Trapped! A critical evaluation of methods for measuring total cellular uptake versus cytosolic localization. Bioconjug. Chem. 30, 1006-1027.

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    Abstract

    Biomolecules have many properties that make them promising for intracellular therapeutic applications, but delivery remains a key challenge because large biomolecules cannot easily enter the cytosol. Furthermore, quantification of total intracellular versus cytosolic concentrations remains demanding, and the determination of delivery efficiency is thus not straightforward. In this review, we discuss strategies for delivering biomolecules into the cytosol and briefly summarize the mechanisms of uptake for these systems. We then describe commonly used methods to measure total cellular uptake and, more selectively, cytosolic localization, and discuss the major advantages and drawbacks of each method. We critically evaluate methods of measuring ""cell penetration"" that do not adequately distinguish total cellular uptake and cytosolic localization, which often lead to inaccurate interpretations of a molecule's cytosolic localization. Finally, we summarize the properties and components of each method, including the main caveats of each, to allow for informed decisions about method selection for specific applications. When applied correctly and interpreted carefully, methods for quantifying cytosolic localization offer valuable insight into the bioactivity of biomolecules and potentially the prospects for their eventual development into therapeutics.

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  • Tomatis, P. E., Schütz, M., Umudumov, E. and Plückthun, A. (2019) Mutations in sigma 70 transcription factor improves expression of functional eukaryotic membrane proteins in Escherichia coli. Sci. Rep. 9, 2483.

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    Abstract

    Eukaryotic integral membrane proteins (IMPs) are difficult to study due to low functional expression levels. To investigate factors for efficient biogenesis of eukaryotic IMPs in the prokaryotic model organism Escherichia coli, important, e.g., for isotope-labeling for NMR, we selected for E. coli cells expressing high levels of functional G protein-coupled receptors (GPCRs) by FACS. Utilizing an E. coli strain library with all non-essential genes systematically deleted, we unexpectedly discovered upon whole-genome sequencing that the improved phenotype was not conferred by the deleted genes but by various subtle alterations in the ""housekeeping"" sigma 70 factor (RpoD). When analyzing effects of the rpoD mutations at the transcriptome level we found that toxic effects incurred on wild-type E. coli during receptor expression were diminished by two independent and synergistic effects: a slower but longer-lasting GPCR biosynthesis and an optimized transcriptional pattern, augmenting growth and expression at low temperature, setting the basis for further bacterial strain engineering.

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  • Michel, E., Plückthun, A. and Zerbe, O. (2019) Peptide binding affinity redistributes preassembled repeat protein fragments. Biol. Chem. 400, 395-404.

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    Abstract

    Designed armadillo repeat proteins (dArmRPs) are modular peptide binders composed of N- and C-terminal capping repeats Y and A and a variable number of internal modules M that each specifically recognize two amino acids of the target peptide. Complementary fragments of dArmRPs obtained by splitting the protein between helices H1 and H2 of an internal module show conditional and specific assembly only in the presence of a target peptide (Michel, E., Pluckthun, A., and Zerbe, O. (2018). Peptide-guided assembly of repeat protein fragments. Angew. Chem. Int. Ed. 57, 4576-4579). Here, we investigate dArmRP fragments that already spontaneously assemble with high affinity, e.g. those obtained from splits between entire modules or between helices H2 and H3. We find that the interaction of the peptide with the assembled fragments induces distal conformational rearrangements that suggest an induced fit on a global protein level. A population analysis of an equimolar mixture of an N-terminal and three C-terminal fragments with various affinities for the target peptide revealed predominant assembly of the weakest peptide binder. However, adding a target peptide to this mixture altered the population of the protein complexes such that the combination with the highest affinity for the peptide increased and becomes predominant when adding excess of peptide, highlighting the feasibility of peptide-induced enrichment of best binders from inter-modular fragment mixtures.

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  • Schwill, M., Tamaskovic, R., Gajadhar, A. S., Kast, F., White, F. M. and Plückthun, A. (2019) Systemic analysis of tyrosine kinase signaling reveals a common adaptive response program in a HER2-positive breast cancer. Science Signal. 12, eaau2875.

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    Abstract

    Drug-induced compensatory signaling and subsequent rewiring of the signaling pathways that support cell proliferation and survival promote the development of acquired drug resistance in tumors. Here, we sought to analyze the adaptive kinase response in cancer cells after distinct treatment with agents targeting human epidermal growth factor receptor 2 (HER2), specifically those that induce either only temporary cell cycle arrest or, alternatively, apoptosis in HER2-overexpressing cancers. We compared trastuzumab, ARRY380, the combination thereof, and a biparatopic, HER2-targeted designed ankyrin repeat protein (DARPin; specifically, 6L1G) and quantified the phosphoproteome by isobaric tagging using tandem mass tag liquid chromatography/tandem mass spectrometry (TMT LC-MS/MS). We found a specific signature of persistently phosphorylated tyrosine peptides after the nonapoptotic treatments, which we used to distinguish between different treatment-induced cancer cell fates. Next, we analyzed the activation of serine/threonine and tyrosine kinases after treatment using a bait peptide chip array and predicted the corresponding active kinases. Through a combined system-wide analysis, we identified a common adaptive kinase response program that involved the activation of focal adhesion kinase 1 (FAK1), protein kinase C-delta (PRKCD), and Ephrin (EPH) family receptors. These findings reveal potential targets to prevent adaptive resistance to HER2-targeted therapies.

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  • Kumar, A. and Plückthun, A. (2019) In vivo assembly and large-scale purification of a GPCR – G-alpha fusion with G-beta-gamma, and characterization of the active complex. PLoS One 14, e0210131.

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    Abstract

    G protein coupled receptors (GPCRs) are central players in recognizing a variety of stimuli to mediate diverse cellular responses. This myriad of functions is accomplished by their modular interactions with downstream intracellular transducers, such as heterotrimeric G proteins and arrestins. Assembling a specific GPCR-G protein pair as a purified complex for their structural and functional investigations remains a challenging task, however, because of the low affinity of the interaction. Here, we optimized fusion constructs of the Galpha subunit of the heterotrimeric G protein and engineered versions of rat Neurotensin receptor 1 (NTR1), coexpressed and assembled in vivo with Gbeta and Ggamma. This was achieved by using the baculovirus-based MultiBac system. We thus generated a functional receptor-G protein fusion complex, which can be efficiently purified using ligand-based affinity chromatography on large scales. Additionally, we utilized a purification method based on a designed ankyrin repeat protein tightly binding to Green Fluorescent Protein (GFP-DARPin) that may be used as a generic approach for a large-scale purification of GPCR-G protein fusion complexes for which no ligands column can be generated. The purification methods described herein will support future studies that aim to understand the structural and functional framework of GPCR activation and signaling.

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  • Schöppe, J., Ehrenmann, J., Klenk, C., Rucktooa, P., Schütz, M., Dore, A. S. and Plückthun, A. (2019) Crystal structures of the human neurokinin 1 receptor in complex with clinically used antagonists. Nature Comm. 10, 17.

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    Abstract

    Neurokinins (or tachykinins) are peptides that modulate a wide variety of human physiology through the neurokinin G protein-coupled receptor family, implicated in a diverse array of pathological processes. Here we report high-resolution crystal structures of the human NK1 receptor (NK1R) bound to two small-molecule antagonist therapeutics - aprepitant and netupitant and the progenitor antagonist CP-99,994. The structures reveal the detailed interactions between clinically approved antagonists and NK1R, which induce a distinct receptor conformation resulting in an interhelical hydrogen-bond network that cross-links the extracellular ends of helices V and VI. Furthermore, the high-resolution details of NK1R bound to netupitant establish a structural rationale for the lack of basal activity in NK1R. Taken together, these co-structures provide a comprehensive structural basis of NK1R antagonism and will facilitate the design of new therapeutics targeting the neurokinin receptor family.

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2018

# Reference PDF
  • Ehrenmann, J., Schöppe, J., Klenk, C., Rappas, M., Kummer, L., Dore, A. S. and Plückthun, A. (2018) High-resolution crystal structure of parathyroid hormone 1 receptor in complex with a peptide agonist. Nature Struct. Mol. Biol. 25, 1086-1092.

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    Abstract

    Parathyroid hormone 1 receptor (PTH1R) is a class B multidomain G-protein-coupled receptor (GPCR) that controls calcium homeostasis. Two endogenous peptide ligands, parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP), activate the receptor, and their analogs teriparatide and abaloparatide are used in the clinic to increase bone formation as an effective yet costly treatment for osteoporosis. Activation of PTH1R involves binding of the peptide ligand to the receptor extracellular domain (ECD) and transmembrane domain (TMD), a hallmark of class B GPCRs. Here, we present the crystal structure of human PTH1R in complex with a peptide agonist at 2.5-A resolution, allowing us to delineate the agonist binding mode for this receptor and revealing molecular details within conserved structural motifs that are critical for class B receptor function. Thus, this study provides structural insight into the function of PTH1R and extends our understanding of this therapeutically important class of GPCRs.

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  • Vigano, M. A., Bieli, D., Schaefer, J. V., Jakob, R. P., Matsuda, S., Maier, T., Plückthun, A. and Affolter, M. (2018) DARPins recognizing mTFP1 as novel reagents for in vitro and in vivo protein manipulations. Biology Open 7, bio036749.

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    Abstract

    Over the last few years, protein-based affinity reagents have proven very helpful in cell and developmental biology. While many of these versatile small proteins can be expressed both in the intracellular and extracellular milieu in cultured cells and in living organisms, they can also be functionalized by fusing them to different protein domains in order to regulate or modulate their target proteins in diverse manners. For example, protein binders have been employed to degrade, trap, localize or enzymatically modify specific target proteins. Whereas binders to many endogenous proteins or small protein tags have been generated, several affinity reagents against fluorescent proteins have also been created and used to manipulate target proteins tagged with the corresponding fluorescent protein. Both of these approaches have resulted in improved methods for cell biological and developmental studies. While binders against GFP and mCherry have been previously isolated and validated, we now report the generation and utilization of designed ankyrin repeat proteins (DARPins) against the monomeric teal fluorescent protein 1 (mTFP1). Here we use the generated DARPins to delocalize Rab proteins to the nuclear compartment, in which they cannot fulfil their regular functions anymore. In the future, such manipulations might enable the production of acute loss-of-function phenotypes in different cell types or in living organisms based on direct protein manipulation rather than on genetic loss-of-function analyses.

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  • ElGamacy, M., Coles, M., Ernst, P., Zhu, H., Hartmann, M. D., Plückthun, A. and Lupas, A. N. (2018) An interface-driven design strategy yields a novel, corrugated protein architecture. ACS Synth. Biol. 7, 2226-2235.

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    Abstract

    Designing proteins with novel folds remains a major challenge, as the biophysical properties of the target fold are not known a priori and no sequence profile exists to describe its features. Therefore, most computational design efforts so far have been directed toward creating proteins that recapitulate existing folds. Here we present a strategy centered upon the design of novel intramolecular interfaces that enables the construction of a target fold from a set of starting fragments. This strategy effectively reduces the amount of computational sampling necessary to achieve an optimal sequence, without compromising the level of topological control. The solenoid architecture has been a target of extensive protein design efforts, as it provides a highly modular platform of low topological complexity. However, none of the previous efforts have attempted to depart from the natural form, which is characterized by a uniformly handed superhelical architecture. Here we aimed to design a more complex platform, abolishing the superhelicity by introducing internally alternating handedness, resulting in a novel, corrugated architecture. We employed our interface-driven strategy, designing three proteins and confirming the design by solving the structure of two examples.

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  • Schaefer, J. V., Sedlak, E., Kast, F., Nemergut, M. and Plückthun, A. (2018) Modification of the kinetic stability of immunoglobulin G by solvent additives. MAbs 10, 607-623.

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    Abstract

    Biophysical properties of antibody-based biopharmaceuticals are a critical part of their release criteria. In this context, finding the appropriate formulation is equally important as optimizing their intrinsic biophysical properties through protein engineering, and both are mutually dependent. Most previous studies have empirically tested the impact of additives on measures of colloidal stability, while mechanistic aspects have usually been limited to only the thermodynamic stability of the protein. Here we emphasize the kinetic impact of additives on the irreversible denaturation steps of immunoglobulins G (IgG) and their antigen-binding fragments (Fabs), as these are the key committed steps preceding aggregation, and thus especially informative in elucidating the molecular parameters of activity loss. We examined the effects of ten additives on the conformational kinetic stability by differential scanning calorimetry (DSC), using a recently developed three-step model containing both reversible and irreversible steps. The data highlight and help to rationalize different effects of the additives on the properties of full-length IgG, analyzed by onset and aggregation temperatures as well as by kinetic parameters derived from our model. Our results further help to explain the observation that stabilizing mutations in the antigen-binding fragment (Fab) significantly affect the kinetic parameters of its thermal denaturation, but not the aggregation properties of the full-length IgGs. We show that the proper analysis of DSC scans for full-length IgGs and their corresponding Fabs not only helps in ranking their stability in different formats and formulations, but provides important mechanistic insights for improving the conformational kinetic stability of IgGs.

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  • Dreier, B. and Plückthun, A. (2018) Rapid selection of high-affinity antibody scFv fragments using ribosome display. Methods Mol. Biol. 1827, 235-268.

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    Abstract

    Ribosome display has proven to be a powerful in vitro selection and evolution method for generating high-affinity binders from libraries of folded proteins. It works entirely in vitro, and this has two important consequences. First, since no transformation of any cells is required, libraries with much greater diversity can be handled than with most other techniques. Second, since a library does not have to be cloned and transformed, it is very convenient to introduce random errors in the library by PCR-based methods and select improved binders. Thus, a true directed evolution, an iteration between randomization and selection over several generations, can be conveniently carried out, e.g., for affinity maturation, either on a given clone or on the whole library. Ribosome display has been successfully applied to antibody single-chain Fv fragments (scFv), which can be selected not only for specificity but also for stability and catalytic activity. High-affinity binders with new target specificity can be obtained from highly diverse libraries in only a few selection rounds. In this protocol, the selection from the library and the process of affinity maturation and off-rate selection are explained in detail.

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  • Mikula, K. M., Krumwiede, L., Plückthun, A. and Iwai, H. (2018) Segmental isotopic labeling by asparaginyl endopeptidase-mediated protein ligation. J. Biomol. NMR 71, 225-235.

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    Abstract

    Segmental isotopic labeling can facilitate NMR studies of large proteins, multi-domain proteins, and proteins with repetitive sequences by alleviating NMR signal overlaps. Segmental isotopic labeling also allows us to investigate an individual domain in the context of a full-length protein by NMR. Several established methods are available for segmental isotopic labeling such as intein-mediated ligation, but each has specific requirements and limitations. Here, we report an enzymatic approach using bacterially produced asparagine endopeptidase from Oldenlandia affinis for segmental isotopic labeling of a protein with repetitive sequences, a designed armadillo repeat protein, by overcoming some of the shortcomings of enzymatic ligation for segmental isotopic labeling.

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  • Hartmann, J., Münch, R. C., Freiling, R. T., Schneider, I. C., Dreier, B., Samukange, W., Koch, J., Seeger, M. A., Plückthun, A. and Buchholz, C. J. (2018) A library-based screening strategy for the identification of DARPins as ligands for receptor-targeted AAV and lentiviral vectors. Mol. Ther. Methods Clin. Dev. 10, 128-143.

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    Abstract

    Delivering genes selectively to the therapeutically relevant cell type is among the prime goals of vector development. Here, we present a high-throughput selection and screening process that identifies designed ankyrin repeat proteins (DARPins) optimally suited for receptor-targeted gene delivery using adeno-associated viral (AAV) and lentiviral (LV) vectors. In particular, the process includes expression, purification, and in situ biotinylation of the extracellular domains of target receptors as Fc fusion proteins in mammalian cells and the selection of high-affinity binders by ribosome display from DARPin libraries each covering more than 10(12) variants. This way, DARPins specific for the glutamate receptor subunit GluA4, the endothelial surface marker CD105, and the natural killer cell marker NKp46 were generated. The identification of DARPins best suited for gene delivery was achieved by screening small-scale vector productions. Both LV and AAV particles displaying the selected DARPins transduced only cells expressing the corresponding target receptor. The data confirm that a straightforward process for the generation of receptor-targeted viral vectors has been established. Moreover, biochemical analysis of a panel of DARPins revealed that their functional cell-surface expression as fusion proteins is more relevant for efficient gene delivery by LV particles than functional binding affinity.

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  • Yen, H. Y., Hoi, K. K., Liko, I., Hedger, G., Horrell, M. R., Song, W., Wu, D., Heine, P., Warne, T., Lee, Y., Carpenter, B., Plückthun, A., Tate, C. G., Sansom, M. S. P. and Robinson, C. V. (2018) PtdIns(4,5)P2 stabilizes active states of GPCRs and enhances selectivity of G-protein coupling. Nature 559, 423-427.

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    Abstract

    G-protein-coupled receptors (GPCRs) are involved in many physiological processes and are therefore key drug targets(1). Although detailed structural information is available for GPCRs, the effects of lipids on the receptors, and on downstream coupling of GPCRs to G proteins are largely unknown. Here we use native mass spectrometry to identify endogenous lipids bound to three class A GPCRs. We observed preferential binding of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) over related lipids and confirm that the intracellular surface of the receptors contain hotspots for PtdIns(4,5)P2 binding. Endogenous lipids were also observed bound directly to the trimeric Galphasbetagamma protein complex of the adenosine A2A receptor (A2AR) in the gas phase. Using engineered Galpha subunits (mini-Galphas, mini-Galphai and mini-Galpha12)(2), we demonstrate that the complex of mini-Galphas with the beta1 adrenergic receptor (beta1AR) is stabilized by the binding of two PtdIns(4,5)P2 molecules. By contrast, PtdIns(4,5)P2 does not stabilize coupling between beta1AR and other Galpha subunits (mini-Galphai or mini-Galpha12) or a high-affinity nanobody. Other endogenous lipids that bind to these receptors have no effect on coupling, highlighting the specificity of PtdIns(4,5)P2. Calculations of potential of mean force and increased GTP turnover by the activated neurotensin receptor when coupled to trimeric Galphaibetagamma complex in the presence of PtdIns(4,5)P2 provide further evidence for a specific effect of PtdIns(4,5)P2 on coupling. We identify key residues on cognate Galpha subunits through which PtdIns(4,5)P2 forms bridging interactions with basic residues on class A GPCRs. These modulating effects of lipids on receptors suggest consequences for understanding function, G-protein selectivity and drug targeting of class A GPCRs.

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  • Radom, F., Plückthun, A. and Paci, E. (2018) Assessment of ab initio models of protein complexes by molecular dynamics. PLoS Comput. Biol. 14, e1006182.

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    Abstract

    Determining how proteins interact to form stable complexes is of crucial importance, for example in the development of novel therapeutics. Computational methods to determine the thermodynamically stable conformation of complexes from the structure of the binding partners, such as RosettaDock, might potentially emerge to become a promising alternative to traditional structure determination methods. However, while models virtually identical to the correct experimental structure can in some cases be generated, the main difficulty remains to discriminate correct or approximately correct models from decoys. This is due to the ruggedness of the free-energy landscape, the approximations intrinsic in the scoring functions, and the intrinsic flexibility of proteins. Here we show that molecular dynamics simulations performed starting from a number top-scoring models can not only discriminate decoys and identify the correct structure, but may also provide information on an initial map of the free energy landscape that elucidates the binding mechanism.

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  • Wu, Y., Honegger, A., Batyuk, A., Mittl, P. R. E. and Plückthun, A. (2018) Structural basis for the selective inhibition of c-Jun N-terminal kinase 1 determined by rigid DARPin-DARPin fusions. J. Mol. Biol. 430, 2128-2138.

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    Abstract

    To untangle the complex signaling of the c-Jun N-terminal kinase (JNK) isoforms, we need tools that can selectively detect and inhibit individual isoforms. Because of the high similarity between JNK1, JNK2 and JNK3, it is very difficult to generate small-molecule inhibitors with this discriminatory power. Thus, we have recently selected protein binders from the designed ankyrin repeat protein (DARPin) library which were indeed isoform-specific inhibitors of JNK1 with low nanomolar potency. Here we provide the structural basis for their isotype discrimination and their inhibitory action. All our previous attempts to generate crystal structures of complexes had failed. We have now made use of a technology we recently developed which consists of rigid fusion of an additional special DARPin, which acts as a crystallization enhancer. This can be rigidly fused with different geometries, thereby generating a range of alternative crystal packings. The structures reveal the molecular basis for isoform specificity of the DARPins and their ability to prevent JNK activation and may thus form the basis of further investigation of the JNK family as well as novel approaches to drug design.

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  • Yong, K. J., Vaid, T. M., Shilling, P. J., Wu, F. J., Williams, L. M., Deluigi, M., Plückthun, A., Bathgate, R. A. D., Gooley, P. R. and Scott, D. J. (2018) Determinants of ligand subtype-selectivity at alpha1A-adrenoceptor revealed using saturation transfer difference (STD) NMR. ACS Chem. Biol. 13, 1090-1102.

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    Abstract

    alpha1A- and alpha1B-adrenoceptors (alpha1A-AR and alpha1B-AR) are closely related G protein-coupled receptors (GPCRs) that modulate the cardiovascular and nervous systems in response to binding epinephrine and norepinephrine. The GPCR gene superfamily is made up of numerous subfamilies that, like alpha1A-AR and alpha1B-AR, are activated by the same endogenous agonists but may modulate different physiological processes. A major challenge in GPCR research and drug discovery is determining how compounds interact with receptors at the molecular level, especially to assist in the optimization of drug leads. Nuclear magnetic resonance spectroscopy (NMR) can provide great insight into ligand-binding epitopes, modes, and kinetics. Ideally, ligand-based NMR methods require purified, well-behaved protein samples. The instability of GPCRs upon purification in detergents, however, makes the application of NMR to study ligand binding challenging. Here, stabilized alpha1A-AR and alpha1B-AR variants were engineered using Cellular High-throughput Encapsulation, Solubilization, and Screening (CHESS), allowing the analysis of ligand binding with Saturation Transfer Difference NMR (STD NMR). STD NMR was used to map the binding epitopes of epinephrine and A-61603 to both receptors, revealing the molecular determinants for the selectivity of A-61603 for alpha1A-AR over alpha1B-AR. The use of stabilized GPCRs for ligand-observed NMR experiments will lead to a deeper understanding of binding processes and assist structure-based drug design.

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  • Michel, E., Plückthun, A. and Zerbe, O. (2018) Peptide-guided assembly of repeat protein fragments. Angew. Chem. Int. Ed. Engl. 57, 4576-4579.

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    Abstract

    Herein, we present the peptide-guided assembly of complementary fragments of designed armadillo repeat proteins (dArmRPs) to create proteins that bind peptides not only with high affinity but also with good selectivity. We recently demonstrated that complementary N- and C-terminal fragments of dArmRPs form high-affinity complexes that resemble the structure of the full-length protein, and that these complexes bind their target peptides. We now demonstrate that dArmRPs can be split such that the fragments assemble only in the presence of a templating peptide, and that fragment mixtures enrich the combination with the highest affinity for this peptide. The enriched fragment combination discriminates single amino acid variations in the target peptide with high specificity. Our results suggest novel opportunities for the generation of new peptide binders by selection from dArmRP fragment mixtures.

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  • Stichling, N., Suomalainen, M., Flatt, J. W., Schmid, M., Pacesa, M., Hemmi, S., Jungraithmayr, W., Maler, M. D., Freudenberg, M. A., Plückthun, A., May, T., Köster, M., Fejer, G. and Greber, U. F. (2018) Lung macrophage scavenger receptor SR-A6 (MARCO) is an adenovirus type-specific virus entry receptor. PLoS Pathog. 14, e1006914.

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    Abstract

    Macrophages are a diverse group of phagocytic cells acting in host protection against stress, injury, and pathogens. Here, we show that the scavenger receptor SR-A6 is an entry receptor for human adenoviruses in murine alveolar macrophage-like MPI cells, and important for production of type I interferon. Scavenger receptors contribute to the clearance of endogenous proteins, lipoproteins and pathogens. Knockout of SR-A6 in MPI cells, anti-SR-A6 antibody or the soluble extracellular SR-A6 domain reduced adenovirus type-C5 (HAdV-C5) binding and transduction. Expression of murine SR-A6, and to a lower extent human SR-A6 boosted virion binding to human cells and transduction. Virion clustering by soluble SR-A6 and proximity localization with SR-A6 on MPI cells suggested direct adenovirus interaction with SR-A6. Deletion of the negatively charged hypervariable region 1 (HVR1) of hexon reduced HAdV-C5 binding and transduction, implying that the viral ligand for SR-A6 is hexon. SR-A6 facilitated macrophage entry of HAdV-B35 and HAdV-D26, two important vectors for transduction of hematopoietic cells and human vaccination. The study highlights the importance of scavenger receptors in innate immunity against human viruses.

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  • Hansen, S., Ernst, P., König, S. L. B., Reichen, C., Ewald, C., Nettels, D., Mittl, P., Schuler, B. and Plückthun, A. (2018) Curvature of designed armadillo repeat proteins allows modular peptide binding. J. Struct. Biol. 201, 108-117.

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    Abstract

    Designed armadillo repeat proteins (dArmRPs) were developed to create a modular peptide binding technology where each of the structural repeats binds two residues of the target peptide. An essential prerequisite for such a technology is a dArmRP geometry that matches the peptide bond length. To this end, we determined a large set (n =27) of dArmRP X-ray structures, of which 12 were previously unpublished, to calculate curvature parameters that define their geo-metry. Our analysis shows that consensus dArmRPs exhibit curvatures close to the optimal range for modular peptide recognition. Binding of peptide ligands can induce a curvature within the desired range, as confirmed by single-molecule FRET experiments in solution. On the other hand, computationally designed ArmRPs, where side chains have been chosen with the intention to optimally fit into a geometrically optimized backbone, turned out to be more divergent in reality, and thus not suitable for continuous peptide binding. Furthermore, we show that the formation of a crystal lattice can induce small but significant deviations from the curvature adopted in solution, which can interfere with the evaluation of repeat protein sca_olds when high accuracy is required. This study corroborates the suitability of consensus dArmRPs as a sca_old for the development of modular peptide binders.

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  • Schmid, M., Ernst, P., Honegger, A., Suomalainen, M., Zimmermann, M., Braun, L., Stauffer, S., Thom, C., Dreier, B., Eibauer, M., Kipar, A., Vogel, V., Greber, U. F., Medalia, O. and Plückthun, A. (2018) Adenoviral vector with shield and adapter increases tumor specificity and escapes liver and immune control. Nature Comm. 9, 450.

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    Abstract

    Most systemic viral gene therapies have been limited by sequestration and degradation of virions, innate and adaptive immunity, and silencing of therapeutic genes within the target cells. Here we engineer a high-affinity protein coat, shielding the most commonly used vector in clinical gene therapy, human adenovirus type 5. Using electron microscopy and crystallography we demonstrate a massive coverage of the virion surface through the hexon-shielding scFv fragment, trimerized to exploit the hexon symmetry and gain avidity. The shield reduces virion clearance in the liver. When the shielded particles are equipped with adaptor proteins, the virions deliver their payload genes into human cancer cells expressing HER2 or EGFR. The combination of shield and adapter also increases viral gene delivery to xenografted tumors in vivo, reduces liver off-targeting and immune neutralization. Our study highlights the power of protein engineering for viral vectors overcoming the challenges of local and systemic viral gene therapies.

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2017

# Reference PDF
  • Hansen, S., Stüber, J. C., Ernst, P., Koch, A., Bojar, D., Batyuk, A. and Plückthun, A. (2017) Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity. Sci. Rep. 7, 16292.

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    Abstract

    Green fluorescent protein (GFP) fusions are pervasively used to study structures and processes. Specific GFP-binders are thus of great utility for detection, immobilization or manipulation of GFP-fused molecules. We determined structures of two designed ankyrin repeat proteins (DARPins), complexed with GFP, which revealed different but overlapping epitopes. Here we show a structure-guided design strategy that, by truncation and computational reengineering, led to a stable construct where both can bind simultaneously: by linkage of the two binders, fusion constructs were obtained that ""wrap around"" GFP, have very high affinities of about 10-30 pM, and extremely slow off-rates. They can be natively produced in E. coli in very large amounts, and show excellent biophysical properties. Their very high stability and affinity, facile site-directed functionalization at introduced unique lysines or cysteines facilitate many applications. As examples, we present them as tight yet reversible immobilization reagents for surface plasmon resonance, as fluorescently labelled monomeric detection reagents in flow cytometry, as pull-down ligands to selectively enrich GFP fusion proteins from cell extracts, and as affinity column ligands for inexpensive large-scale protein purification. We have thus described a general design strategy to create a ""clamp"" from two different high-affinity repeat proteins, even if their epitopes overlap.

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  • Nemergut, M., Zoldak, G., Schaefer, J. V., Kast, F., Miskovsky, P., Plückthun, A. and Sedlak, E. (2017) Analysis of IgG kinetic stability by differential scanning calorimetry, probe fluorescence and light scattering. Protein Sci. 26, 2229-2239.

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    Abstract

    Monoclonal antibodies of the immunoglobulin G (IgG) type have become mainstream therapeutics for the treatment of many life-threatening diseases. For their successful application in the clinic and a favorable cost-benefit ratio, the design and formulation of these therapeutic molecules must guarantee long-term stability for an extended period of time. Accelerated stability studies, e.g., by employing thermal denaturation, have the great potential for enabling high-throughput screening campaigns to find optimal molecular variants and formulations in a short time. Surprisingly, no validated quantitative analysis of these accelerated studies has been performed yet, which clearly limits their application for predicting IgG stability. Therefore, we have established a quantitative approach for the assessment of the kinetic stability over a broad range of temperatures. To this end, differential scanning calorimetry (DSC) experiments were performed with a model IgG, testing chaotropic formulations and an extended temperature range, and they were subsequently analyzed by our recently developed three-step sequential model of IgG denaturation, consisting of one reversible and two irreversible steps. A critical comparison of the predictions from this model with data obtained by an orthogonal fluorescence probe method, based on 8-anilinonaphthalene-1-sulfonate binding to partially unfolded states, resulted in very good agreement. In summary, our study highlights the validity of this easy-to-perform analysis for reliably assessing the kinetic stability of IgGs, which can support accelerated formulation development of monoclonal antibodies by ranking different formulations as well as by improving colloidal stability models.

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  • Verdurmen, W. P. R., Mazlami, M. and Plückthun, A. (2017) A quantitative comparison of cytosolic delivery via different protein uptake systems. Sci. Rep. 7, 13194.

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    Abstract

    Over many years, a variety of delivery systems have been investigated that have the capacity to shuttle macromolecular cargoes, especially proteins, into the cytosol. Due to the lack of an objective way to quantify cytosolic delivery, relative delivery efficiencies of the various transport systems have remained unclear. Here, we demonstrate the use of the biotin ligase assay for a quantitative comparison of protein transport to the cytosol via cell-penetrating peptides, supercharged proteins and bacterial toxins in four different cell lines. The data illustrate large differences in both the total cellular internalization, which denotes any intracellular location including endosomes, and in the cytosolic uptake of the transport systems, with little correlation between the two. Also, we found significant differences between the cell lines. In general, protein transport systems based on cell-penetrating peptides show a modest total uptake, and mostly do not deliver cargo to the cytosol. Systems based on bacterial toxins show a modest receptor-mediated internalization but an efficient delivery to the cytosol. Supercharged proteins, on the contrary, are not receptor-specific and lead to massive total internalization into endosomes, but only low amounts end up in the cytosol.

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  • Hansen, S., Kiefer, J. D., Madhurantakam, C., Mittl, P. R. E. and Plückthun, A. (2017) Structures of designed armadillo repeat proteins binding to peptides fused to globular domains. Protein Sci. 26, 1942-1952.

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    Abstract

    Designed armadillo repeat proteins (dArmRP) are alpha-helical solenoid repeat proteins with an extended peptide binding groove that were engineered to develop a generic modular technology for peptide recognition. In this context, the term ""peptide"" not only denotes a short unstructured chain of amino acids, but also an unstructured region of a protein, as they occur in termini, loops, or linkers between folded domains. Here we report two crystal structures of dArmRPs, in complex with peptides fused either to the N-terminus of Green Fluorescent Protein or to the C-terminus of a phage lambda protein D. These structures demonstrate that dArmRPs bind unfolded peptides in the intended conformation also when they constitute unstructured parts of folded proteins, which greatly expands possible applications of the dArmRP technology. Nonetheless, the structures do not fully reflect the binding behavior in solution, that is, some binding sites remain unoccupied in the crystal and even unexpected peptide residues appear to be bound. We show how these differences can be explained by restrictions of the crystal lattice or the composition of the crystallization solution. This illustrates that crystal structures have to be interpreted with caution when protein-peptide interactions are characterized, and should always be correlated with measurements in solution.

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  • Wu, Y., Batyuk, A., Honegger, A., Brandl, F., Mittl, P. and Plückthun, A. (2017) Rigidly connected multispecific artificial binders with adjustable geometries. Sci. Rep. 7, 1 – 11.

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    Abstract

    Multivalent binding proteins can gain biological activities beyond what is inherent in the individual binders, by bringing together different target molecules, restricting their conformational flexibility or changing their subcellular localization. In this study, we demonstrate a method to build up rigid multivalent and multispecific scaffolds by exploiting the modular nature of a repeat protein scaffold and avoiding flexible linkers. We use DARPins (Designed Ankyrin Repeat Proteins), synthetic binding proteins based on the Ankyrin-repeat protein scaffold, as binding units. Their ease of in vitro selection, high production yield and stability make them ideal specificity-conferring building blocks for the design of more complex constructs. C- and N-terminal DARPin capping repeats were re-designed to be joined by a shared helix in such a way that rigid connector modules are formed. This allows us to join two or more DARPins in predefined geometries without compromising their binding affinities and specificities. Nine connector modules with distinct geometries were designed; for eight of these we were able to confirm the structure by X-ray crystallography, while only one did not crystallize. The bispecific constructs were all able to bind both target proteins simultaneously.

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  • Jost, C., Stüber, J. C., Honegger, A., Wu, Y., Batyuk, A. and Plückthun, A. (2017) Rigidity of the extracellular part of HER2: Evidence from engineering subdomain interfaces and shared-helix DARPin-DARPin fusions. Protein Sci. 26, 1796-1806.

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    Abstract

    The second member of the human ErbB family of receptor tyrosine kinases, HER2/hErbB2, is regarded as an exceptional case: The four extracellular subdomains could so far only be found in one fixed overall conformation, designated ""open"" and resembling the ligand-bound form of the other ErbB receptors. It thus appears to be different from the extracellular domains of the other family members that show inter-subdomain flexibility and exist in a ""tethered"" form in the absence of ligand. For HER2, there was so far no direct evidence for such a tethered conformation on the cell surface. Nonetheless, alternative conformations of HER2 in vivo could so far not be excluded. We now demonstrate the rigidity of HER2 on the surface of tumor cells by employing two orthogonal approaches of protein engineering: To directly test the potential of the extracellular domain of HER2 to adopt a pseudo-tethered conformation on the cell surface, we first designed HER2 variants with a destabilized interface between extracellular subdomains I and III that would favor deviation from the ""open"" conformation. Secondly, we used differently shaped versions of a Designed Ankyrin Repeat Protein (DARPin) fusion, recognizing subdomain I of HER2, devised to work as probes for a putative pseudo-tethered extracellular domain of HER2. Combining our approaches, we exclude, on live cells and in vitro, that significant proportions of HER2 deviate from the ""open"" conformation.

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  • Parren, Pwhi, Carter, P. J. and Plückthun, A. (2017) Changes to International Nonproprietary Names for antibody therapeutics 2017 and beyond: of mice, men and more. MAbs 9, 898-906.

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    Abstract

    Active pharmaceutical substances require an International Nonproprietary Name (INN) assigned by the World Health Organization (WHO) to obtain market authorization as a medicinal product. INNs are selected to represent a unique, generic name for a drug enabling unambiguous identification by stakeholders worldwide. INNs may be requested after initiating clinical development of an investigational drug. Pharmaceutical classes are indicated by a common stem or suffix. Currently, INNs for monoclonal antibody-based drugs are recognized by the suffix, -mab, preceded by a source infix such as -xi- (chimeric), -zu- (humanized) or -u- (human) designating the species from which the antibody was derived. However, many technological advances have made it increasingly difficult to accurately capture an antibody's source in its name. In 2014, the WHO and the United States Adopted Names (USAN) Council approached this challenge by implementing changes to antibody source infix definitions. Unfortunately, gaps and ambiguities in the definitions and procedures resulted in inconsistent source category assignments and widespread confusion. The Antibody Society, extensively supported by academic and industry scientists, voiced concerns leading to constructive dialog during scheduled consultations with WHO and USAN Council representatives. In June 2017, the WHO announced that use of the source infix will be discontinued for new antibody INNs effective immediately. We fully support this change as it better aligns antibody INNs with current and foreseeable future innovations in antibody therapeutics. Here we review the changes implemented. Additionally, we analyzed antibody INNs recently assigned under the previous 2014 definitions and provide recommendations for further alignment.

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  • Afroz, T., Hock, E. M., Ernst, P., Foglieni, C., Jambeau, M., Gilhespy, L. A. B., Laferriere, F., Maniecka, Z., Plückthun, A., Mittl, P., Paganetti, P., Allain, F. H. T. and Polymenidou, M. (2017) Functional and dynamic polymerization of the ALS-linked protein TDP-43 antagonizes its pathologic aggregation. Nature Comm. 8, 45.

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    Abstract

    TDP-43 is a primarily nuclear RNA-binding protein, whose abnormal phosphorylation and cytoplasmic aggregation characterizes affected neurons in patients with amyotrophic lateral sclerosis and frontotemporal dementia. Here, we report that physiological nuclear TDP-43 in mouse and human brain forms homo-oligomers that are resistant to cellular stress. Physiological TDP-43 oligomerization is mediated by its N-terminal domain, which can adopt dynamic, solenoid-like structures, as revealed by a 2.1 A crystal structure in combination with nuclear magnetic resonance spectroscopy and electron microscopy. These head-to-tail TDP-43 oligomers are unique among known RNA-binding proteins and represent the functional form of the protein in vivo, since their destabilization results in loss of alternative splicing regulation of known neuronal RNA targets. Our findings indicate that N-terminal domain-driven oligomerization spatially separates the adjoining highly aggregation-prone, C-terminal low-complexity domains of consecutive TDP-43 monomers, thereby preventing low-complexity domain inter-molecular interactions and antagonizing the formation of pathologic aggregates.TDP-43 aggregation is observed in amyotrophic lateral sclerosis. Here the authors combine X-ray crystallography, nuclear magnetic resonance and electron microscopy studies and show that physiological oligomerization of TDP-43 is mediated through its N-terminal domain, which forms functional and dynamic oligomers antagonizing pathologic aggregation.

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  • Huber, S., Casagrande, F., Hug, M. N., Wang, L., Heine, P., Kummer, L., Plückthun, A. and Hennig, M. (2017) SPR-based fragment screening with neurotensin receptor 1 generates novel small molecule ligands. PLoS One 12, e0175842.

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    Abstract

    The neurotensin receptor 1 represents an important drug target involved in various diseases of the central nervous system. So far, the full exploitation of potential therapeutic activities has been compromised by the lack of compounds with favorable physicochemical and pharmacokinetic properties which efficiently penetrate the blood-brain barrier. Recent progress in the generation of stabilized variants of solubilized neurotensin receptor 1 and its subsequent purification and successful structure determination presents a solid starting point to apply the approach of fragment-based screening to extend the chemical space of known neurotensin receptor 1 ligands. In this report, surface plasmon resonance was used as primary method to screen 6369 compounds. Thereby 44 hits were identified and confirmed in competition as well as dose-response experiments. Furthermore, 4 out of 8 selected hits were validated using nuclear magnetic resonance spectroscopy as orthogonal biophysical method. Computational analysis of the compound structures, taking the known crystal structure of the endogenous peptide agonist into consideration, gave insight into the potential fragment-binding location and interactions and inspires chemistry efforts for further exploration of the fragments.

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  • Ranganathan, A., Heine, P., Rudling, A., Plückthun, A., Kummer, L. and Carlsson, J. (2017) Ligand discovery for a peptide-binding GPCR by structure-based screening of fragment- and lead-like chemical libraries. ACS Chem. Biol. 12, 735-745.

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    Abstract

    Peptide-recognizing G protein-coupled receptors (GPCRs) are promising therapeutic targets but often resist drug discovery efforts. Determination of crystal structures for peptide-binding GPCRs has provided opportunities to explore structure-based methods in lead development. Molecular docking screens of two chemical libraries, containing either fragment- or lead-like compounds, against a neurotensin receptor 1 crystal structure allowed for a comparison between different drug development strategies for peptide-binding GPCRs. A total of 2.3 million molecules were screened computationally, and 25 fragments and 27 leads that were top-ranked in each library were selected for experimental evaluation. Of these, eight fragments and five leads were confirmed as ligands by surface plasmon resonance. The hit rate for the fragment screen (32%) was thus higher than for the lead-like library (19%), but the affinities of the fragments were approximately 100-fold lower. Both screens returned unique scaffolds and demonstrated that a crystal structure of a stabilized peptide-binding GPCR can guide the discovery of small-molecule agonists. The complementary advantages of exploring fragment- and lead-like chemical space suggest that these strategies should be applied synergistically in structure-based screens against challenging GPCR targets.

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  • Verdurmen, W. P., Mazlami, M. and Plückthun, A. (2017) A biotin ligase-based assay for the quantification of the cytosolic delivery of therapeutic proteins. Methods Mol. Biol. 1575, 223-236.

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    Abstract

    The efficient delivery of external proteins from the external milieu to the cytosol of mammalian cells has great potential for both scientific investigations and future therapies. However, when assessing the cellular uptake of proteins, it is often difficult to distinguish between proteins that are stuck in the endosomes and those that have escaped into the cytosol. Here, we describe a method employing the prokaryotic enzyme biotin ligase that overcomes this problem and which can be employed for a highly sensitive quantification of cytosolic protein delivery.

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  • Anders, U., Schaefer, J. V., Hibti, F. E., Frydman, C., Suckau, D., Plückthun, A. and Zenobi, R. (2017) SPRi-MALDI MS: characterization and identification of a kinase from cell lysate by specific interaction with different designed ankyrin repeat proteins. Anal. Bioanal. Chem. 409, 1827-1836.

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    Abstract

    We report on the direct coupling of surface plasmon resonance imaging (SPRi) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) for the investigation of specific, non-covalent interactions, using the example of designed ankyrin repeat proteins (DARPins) and ribosomal protein S6 kinase 2 (RPS6KA2) directly from lysate of SH-SY5Y cells, derived from human bone marrow. Due to an array format, tracing of binding kinetics of numerous DARPins simultaneously and in real time becomes possible. By optimizing both the proteolytic digest directly on the SPRi chip (amount of trypsin, incubation time, and temperature) as well as the MALDI matrix application (concentration of matrix and number of spray cycles), we are able to identify the specific interaction with RPS6KA2 directly from the cell lysate at a surface coverage of only 0.8 fmol/mm2. Graphical Abstract Workflow of the direct coupling of SPRi with MALDI mass spectrometry.

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  • Syafrizayanti, Lueong, S. S., Di, C., Schaefer, J. V., Plückthun, A. and Hoheisel, J. D. (2017) Personalised proteome analysis by means of protein microarrays made from individual patient samples. Sci. Rep. 7, 39756.

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    Abstract

    DNA sequencing has advanced to a state that permits studying the genomes of individual patients as nearly a matter of routine. Towards analysing a tissue's protein content in a similar manner, we established a method for the production of microarrays that represent full-length proteins as they are encoded in individual specimens, exhibiting the particular variations, such as mutations or splice variations, present in these samples. From total RNA isolates, each transcript is copied to a specific location on the array by an on-chip polymerase elongation reaction, followed by in situ cell-free transcription and translation. These microarrays permit parallel analyses of variations in protein structure and interaction that are specific to particular samples.

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  • Nasr, M. L., Baptista, D., Strauss, M., Sun, Z. J., Grigoriu, S., Huser, S., Plückthun, A., Hagn, F., Walz, T., Hogle, J. M. and Wagner, G. (2017) Covalently circularized nanodiscs for studying membrane proteins and viral entry. Nature Methods 14, 49-52.

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    Abstract

    We engineered covalently circularized nanodiscs (cNDs) which, compared with standard nanodiscs, exhibit enhanced stability, defined diameter sizes and tunable shapes. Reconstitution into cNDs enhanced the quality of nuclear magnetic resonance spectra for both VDAC-1, a beta-barrel membrane protein, and the G-protein-coupled receptor NTR1, an alpha-helical membrane protein. In addition, we used cNDs to visualize how simple, nonenveloped viruses translocate their genomes across membranes to initiate infection.

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  • Ernst, P. and Plückthun, A. (2017) Advances in the design and engineering of peptide-binding repeat proteins. Biol. Chem. 398, 23-29.

      |  

    Abstract

    The specific recognition of peptides, which we define to include unstructured regions or denatured forms of proteins, is an intrinsic part of a multitude of biochemical assays and procedures. Many cellular interactions are also based on this principle as well. While it would be highly desirable to have a stockpile of sequence-specific binders for essentially any sequence, a de novo selection of individual binders against every possible target peptide sequence would be rather difficult to reduce to practice. Modular peptide binders could overcome this problem, as preselected and/or predesigned modules could be reused for the generation of new binders and thereby revolutionize the generation of binding proteins. This minireview summarizes advances in the development of peptide binders and possible scaffolds for their design.

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2016

# Reference PDF
  • Reichen, C., Hansen, S., Forzani, C., Honegger, A., Fleishman, S. J., Zhou, T., Parmeggiani, F., Ernst, P., Madhurantakam, C., Ewald, C., Mittl, P. R., Zerbe, O., Baker, D., Caflisch, A. and Plückthun, A. (2016) Computationally designed armadillo repeat proteins for modular peptide recognition. J. Mol. Biol. 428, 4467-4489.

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    Abstract

    Armadillo repeat proteins (ArmRPs) recognize their target peptide in extended conformation and bind, in a first approximation, two residues per repeat. Thus, they may form the basis for building a modular system, in which each repeat is complementary to a piece of the target peptide. Accordingly, preselected repeats could be assembled into specific binding proteins on demand and thereby avoid the traditional generation of every new binding molecule by an independent selection from a library. Stacked armadillo repeats, each consisting of 42 aa arranged in three alpha-helices, build an elongated superhelical structure. Here, we analyzed the curvature variations in natural ArmRPs and identified a repeat pair from yeast importin-alpha as having the optimal curvature geometry that is complementary to a peptide over its whole length. We employed a symmetric in silico design to obtain a uniform sequence for a stackable repeat while maintaining the desired curvature geometry. Computationally designed ArmRPs (dArmRPs) had to be stabilized by mutations to remove regions of higher flexibility, which were identified by molecular dynamics simulations in explicit solvent. Using an N-capping repeat from the consensus-design approach, two different crystal structures of dArmRP were determined. Although the experimental structures of dArmRP deviated from the designed curvature, the insertion of the most conserved binding pockets of natural ArmRPs onto the surface of dArmRPs resulted in binders against the expected peptide with low nanomolar affinities, similar to the binders from the consensus-design series.

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  • Ahmad, S., Pecqueur, L., Dreier, B., Hamdane, D., Aumont-Nicaise, M., Plückthun, A., Knossow, M. and Gigant, B. (2016) Destabilizing an interacting motif strengthens the association of a designed ankyrin repeat protein with tubulin. Sci. Rep. 6, 28922.

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    Abstract

    Affinity maturation by random mutagenesis and selection is an established technique to make binding molecules more suitable for applications in biomedical research, diagnostics and therapy. Here we identified an unexpected novel mechanism of affinity increase upon in vitro evolution of a tubulin-specific designed ankyrin repeat protein (DARPin). Structural analysis indicated that in the progenitor DARPin the C-terminal capping repeat (C-cap) undergoes a 25 degrees rotation to avoid a clash with tubulin upon binding. Additionally, the C-cap appears to be involved in electrostatic repulsion with tubulin. Biochemical and structural characterizations demonstrated that the evolved mutants achieved a gain in affinity through destabilization of the C-cap, which relieves the need of a DARPin conformational change upon tubulin binding and removes unfavorable interactions in the complex. Therefore, this specific case of an order-to-disorder transition led to a 100-fold tighter complex with a subnanomolar equilibrium dissociation constant, remarkably associated with a 30% decrease of the binding surface.

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  • Goricanec, D., Stehle, R., Egloff, P., Grigoriu, S., Plückthun, A., Wagner, G. and Hagn, F. (2016) Conformational dynamics of a G-protein alpha subunit is tightly regulated by nucleotide binding. Proc. Natl. Acad. Sci. U. S. A. 113, E3629-3638.

      |  

    Abstract

    Heterotrimeric G proteins play a pivotal role in the signal-transduction pathways initiated by G-protein-coupled receptor (GPCR) activation. Agonist-receptor binding causes GDP-to-GTP exchange and dissociation of the Galpha subunit from the heterotrimeric G protein, leading to downstream signaling. Here, we studied the internal mobility of a G-protein alpha subunit in its apo and nucleotide-bound forms and characterized their dynamical features at multiple time scales using solution NMR, small-angle X-ray scattering, and molecular dynamics simulations. We find that binding of GTP analogs leads to a rigid and closed arrangement of the Galpha subdomain, whereas the apo and GDP-bound forms are considerably more open and dynamic. Furthermore, we were able to detect two conformational states of the Galpha Ras domain in slow exchange whose populations are regulated by binding to nucleotides and a GPCR. One of these conformational states, the open state, binds to the GPCR; the second conformation, the closed state, shows no interaction with the receptor. Binding to the GPCR stabilizes the open state. This study provides an in-depth analysis of the conformational landscape and the switching function of a G-protein alpha subunit and the influence of a GPCR in that landscape.

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  • Bender, R. R., Muth, A., Schneider, I. C., Friedel, T., Hartmann, J., Plückthun, A., Maisner, A. and Buchholz, C. J. (2016) Receptor-targeted Nipah virus glycoproteins improve cell-type selective gene delivery and reveal a preference for membrane-proximal cell attachment. PLoS Pathog. 12, e1005641.

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    Abstract

    Receptor-targeted lentiviral vectors (LVs) can be an effective tool for selective transfer of genes into distinct cell types of choice. Moreover, they can be used to determine the molecular properties that cell surface proteins must fulfill to act as receptors for viral glycoproteins. Here we show that LVs pseudotyped with receptor-targeted Nipah virus (NiV) glycoproteins effectively enter into cells when they use cell surface proteins as receptors that bring them closely enough to the cell membrane (less than 100 A distance). Then, they were flexible in receptor usage as demonstrated by successful targeting of EpCAM, CD20, and CD8, and as selective as LVs pseudotyped with receptor-targeted measles virus (MV) glycoproteins, the current standard for cell-type specific gene delivery. Remarkably, NiV-LVs could be produced at up to two orders of magnitude higher titers compared to their MV-based counterparts and were at least 10,000-fold less effectively neutralized than MV glycoprotein pseudotyped LVs by pooled human intravenous immunoglobulin. An important finding for NiV-LVs targeted to Her2/neu was an about 100-fold higher gene transfer activity when particles were targeted to membrane-proximal regions as compared to particles binding to a more membrane-distal epitope. Likewise, the low gene transfer activity mediated by NiV-LV particles bound to the membrane distal domains of CD117 or the glutamate receptor subunit 4 (GluA4) was substantially enhanced by reducing receptor size to below 100 A. Overall, the data suggest that the NiV glycoproteins are optimally suited for cell-type specific gene delivery with LVs and, in addition, for the first time define which parts of a cell surface protein should be targeted to achieve optimal gene transfer rates with receptor-targeted LVs.

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  • Tamaskovic, R., Schwill, M., Nagy-Davidescu, G., Jost, C., Schaefer, D. C., Verdurmen, W. P. R., Schaefer, J. V., Honegger, A. and Plückthun, A. (2016) Intermolecular biparatopic trapping of ErbB2 prevents compensatory activation of PI3K/AKT via RAS–p110 crosstalk. Nature Comm. 7, 11672.

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    Abstract

    Compensatory mechanisms, such as relief of AKT-ErbB3-negative feedback, are known to desensitize ErbB2-dependent tumours to targeted therapy. Here we describe an adaptation mechanism leading to reactivation of the PI3K/AKT pathway during trastuzumab treatment, which occurs independently of ErbB3 re-phosphorylation. This signalling bypass of phospho-ErbB3 operates in ErbB2-overexpressing cells via RAS-PI3K crosstalk and is attributable to active ErbB2 homodimers. As demonstrated by dual blockade of ErbB2/RAS and ErbB3 by means of pharmacological inhibition, RNA interference or by specific protein binders obstructing the RAS–p110a interaction, both routes must be blocked to prevent reactivation of the PI3K/AKT pathway. Applying these general principles, we developed biparatopic designed ankyrin repeat proteins (DARPins) trapping ErbB2 in a dimerizationincompetent

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  • Batyuk, A., Wu, Y., Honegger, A., Heberling, M. M. and Plückthun, A. (2016) DARPin-based crystallization chaperones exploit molecular geometry as a screening dimension in protein crystallography. J. Mol. Biol. 428, 1574-1588.

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    Abstract

    DARPin libraries, based on a Designed Ankyrin Repeat Protein consensus framework, are a rich source of binding partners for a wide variety of proteins. Their modular structure, stability, ease of in vitro selection and high production yields make DARPins an ideal starting point for further engineering. The X-ray structures of around 30 different DARPin complexes demonstrate their ability to facilitate crystallization of their target proteins by restricting flexibility and preventing undesired interactions of the target molecule. However, their small size (18kDa), very hydrophilic surface and repetitive structure can limit the DARPins' ability to provide essential crystal contacts and their usefulness as a search model for addressing the crystallographic phase problem in molecular replacement. To optimize DARPins for their application as crystallization chaperones, rigid domain-domain fusions of the DARPins to larger proteins, proven to yield high-resolution crystal structures, were generated. These fusions were designed in such a way that they affect only one of the terminal capping repeats of the DARPin and do not interfere with residues involved in target binding, allowing to exchange at will the binding specificities of the DARPin in the fusion construct. As a proof of principle, we designed rigid fusions of a stabilized version of Escherichia coli TEM-1 beta-lactamase to the C-terminal capping repeat of various DARPins in six different relative domain orientations. Five crystal structures representing four different fusion constructs, alone or in complex with the cognate target, show the predicted relative domain orientations and prove the validity of the concept.

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  • Schütz, M., Batyuk, A., Klenk, C., Kummer, L., de Picciotto, S., Gülbakan, B., Wu, Y., Newby, G. A., Zosel, F., Schöppe, J., Sedlàk, E., Mittl, P. R., Zenobi, R., Wittrup, K. D. and Plückthun, A. (2016) Generation of fluorogen-activating Designed Ankyrin Repeat Proteins (FADAs) as versatile sensor tools. J. Mol. Biol. 428, 1272-1289.

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    Abstract

    Fluorescent probes constitute a valuable toolbox to address a variety of biological questions and they have become irreplaceable for imaging methods. Commonly, such probes consist of fluorescent proteins or small organic fluorophores coupled to biological molecules of interest. Recently, a novel class of fluorescence-based probes, fluorogen-activating proteins (FAPs), has been reported. These binding proteins are based on antibody single-chain variable fragments and activate fluorogenic dyes, which only become fluorescent upon activation and do not fluoresce when free in solution. Here we present a novel class of fluorogen activators, termed FADAs, based on the very robust designed ankyrin repeat protein scaffold, which also readily folds in the reducing environment of the cytoplasm. The FADA generated in this study was obtained by combined selections with ribosome display and yeast surface display. It enhances the fluorescence of malachite green (MG) dyes by a factor of more than 11,000 and thus activates MG to a similar extent as FAPs based on single-chain variable fragments. As shown by structure determination and in vitro measurements, this FADA was evolved to form a homodimer for the activation of MG dyes. Exploiting the favorable properties of the designed ankyrin repeat protein scaffold, we created a FADA biosensor suitable for imaging of proteins on the cell surface, as well as in the cytosol. Moreover, based on the requirement of dimerization for strong fluorogen activation, a prototype FADA biosensor for in situ detection of a target protein and protein-protein interactions was developed. Therefore, FADAs are versatile fluorescent probes that are easily produced and suitable for diverse applications and thus extend the FAP technology.

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  • Hansen, S., Tremmel, D., Madhurantakam, C., Reichen, C., Mittl, P. R. and Plückthun, A. (2016) Structure and energetic contributions of a designed modular peptide-binding protein with picomolar affinity. J. Am. Chem. Soc. 138, 3526-3532.

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    Abstract

    Natural armadillo repeat proteins (nArmRP) like importin-alpha or beta-catenin bind their target peptides such that each repeat interacts with a dipeptide unit within the stretched target peptide. However, this modularity is imperfect and also restricted to short peptide stretches of usually four to six consecutive amino acids. Here we report the development and characterization of a regularized and truly modular peptide-specific binding protein, based on designed armadillo repeat proteins (dArmRP), binding to peptides of alternating lysine and arginine residues (KR)n. dArmRP were obtained from nArmRP through cycles of extensive protein engineering, which rendered them more uniform. This regularity is reflected in the consistent binding of dArmRP to (KR)-peptides, where affinities depend on the lengths of target peptides and the number of internal repeats in a very systematic manner, thus confirming the modularity of the interaction. This exponential dependency between affinity and recognition length suggests that each module adds a constant increment of binding energy to sequence-specific recognition. This relationship was confirmed by comprehensive mutagenesis studies that also reveal the importance of individual peptide side chains. The 1.83 A resolution crystal structure of a dArmRP with five identical internal repeats in complex with the cognate (KR)5 peptide proves a modular binding mode, where each dipeptide is recognized by one internal repeat. The confirmation of this true modularity over longer peptide stretches lays the ground for the design of binders with different specificities and tailored affinities by the assembly of dipeptide-specific modules based on armadillo repeats.

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  • Schütz, M., Schöppe, J., Sedlak, E., Hillenbrand, M., Nagy-Davidescu, G., Ehrenmann, J., Klenk, C., Egloff, P., Kummer, L. and Plückthun, A. (2016) Directed evolution of G protein-coupled receptors in yeast for higher functional production in eukaryotic expression hosts. Sci. Rep. 6, 21508.

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    Abstract

    Despite recent successes, many G protein-coupled receptors (GPCRs) remained refractory to detailed molecular studies due to insufficient production yields, even in the most sophisticated eukaryotic expression systems. Here we introduce a robust method employing directed evolution of GPCRs in yeast that allows fast and efficient generation of receptor variants which show strongly increased functional production levels in eukaryotic expression hosts. Shown by evolving three different receptors in this study, the method is widely applicable, even for GPCRs which are very difficult to express. The evolved variants showed up to a 26-fold increase of functional production in insect cells compared to the wild-type receptors. Next to the increased production, the obtained variants exhibited improved biophysical properties, while functional properties remained largely unaffected. Thus, the presented method broadens the portfolio of GPCRs accessible for detailed investigations. Interestingly, the functional production of GPCRs in yeast can be further increased by induced host adaptation.

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  • Hanauer, J. R., Gottschlich, L., Riehl, D., Rusch, T., Koch, V., Friedrich, K., Hutzler, S., Prüfer, S., Friedel, T., Hanschmann, K. M., Münch, R. C., Jost, C., Plückthun, A., Cichutek, K., Buchholz, C. J. and Mühlebach, M. D. (2016) Enhanced lysis by bispecific oncolytic measles viruses simultaneously using HER2/neu or EpCAM as target receptors. Mol. Ther. 3, 16003.

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    Abstract

    To target oncolytic measles viruses (MV) to tumors, we exploit the binding specificity of designed ankyrin repeat proteins (DARPins). These DARPin-MVs have high tumor selectivity while maintaining excellent oncolytic potency. Stability, small size, and efficacy of DARPins allowed the generation of MVs simultaneously targeted to tumor marker HER2/neu and cancer stem cell (CSC) marker EpCAM. For optimization, the linker connecting both DARPins was varied in flexibility and length. Flexibility had no impact on fusion helper activity whereas length had. MVs with bispecific MV-H are genetically stable and revealed the desired doubletarget specificity. In vitro, the cytolytic activity of bispecific MVs was superior or comparable to mono-targeted viruses depending on the target cells. In vivo, therapeutic efficacy of the bispecific viruses was validated in an orthotopic ovarian carcinoma model revealing an effective reduction of tumor mass. Finally, the power of bispecific targeting was demonstrated on cocultures of different tumor cells thereby mimicking tumor heterogeneity in vitro, more closely reflecting real tumors. Here, bispecific excelled monospecific viruses in efficacy. DARPin-based targeting domains thus allow the generation of efficacious oncolytic viruses with double specificity, with the potential to handle intratumoral variation of antigen expression and to simultaneously target CSCs and the bulk tumor mass.

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  • Klenk, C., Ehrenmann, J., Schütz, M. and Plückthun, A. (2016) A generic selection system for improved expression and thermostability of G protein-coupled receptors by directed evolution. Sci. Rep. 6, 21294.

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    Abstract

    Structural and biophysical studies as well as drug screening approaches on G protein-coupled receptors (GPCRs) have been largely hampered by the poor biophysical properties and low expression yields of this largest class of integral membrane proteins. Thermostabilisation of GPCRs by introduction of stabilising mutations has been a key factor to overcome these limitations. However, labelled ligands with sufficient affinity, which are required for selective binding to the correctly folded receptor, are often not available. Here we describe a novel procedure to improve receptor expression and stability in a generic way, independent of specific ligands, by means of directed evolution in E. coli. We have engineered a homogenous fluorescent reporter assay that only detects receptors which are correctly integrated into the inner cell membrane and, thus, discriminates functional from non-functional receptor species. When we combined this method with a directed evolution procedure we obtained highly expressing mutants of the neurotensin receptor 1 with greatly improved thermostability. By this procedure receptors with poor expression and/or low stability, for which no ligands or only ones with poor binding properties are available, can now be generated in quantities allowing detailed structural and biophysical analysis.

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  • Jones, T. D., Carter, P. J., Plückthun, A., Vasquez, M., Holgate, R. G., Hotzel, I., Popplewell, A. G., Parren, P. W., Enzelberger, M., Rademaker, H. J., Clark, M. R., Lowe, D. C., Dahiyat, B. I., Smith, V., Lambert, J. M., Wu, H., Reilly, M., Haurum, J. S., Dübel, S., Huston, J. S., Schirrmann, T., Janssen, R. A., Steegmaier, M., Gross, J. A., Bradbury, A. R., Burton, D. R., Dimitrov, D. S., Chester, K. A., Glennie, M. J., Davies, J., Walker, A., Martin, S., McCafferty, J. and Baker, M. P. (2016) The INNs and outs of antibody nonproprietary names. MAbs 8, 1-9.

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    Abstract

    An important step in drug development is the assignment of an International Nonproprietary Name (INN) by the World Health Organization (WHO) that provides healthcare professionals with a unique and universally available designated name to identify each pharmaceutical substance. Monoclonal antibody INNs comprise a -mab suffix preceded by a substem indicating the antibody type, e.g., chimeric (-xi-), humanized (-zu-), or human (-u-). The WHO publishes INN definitions that specify how new monoclonal antibody therapeutics are categorized and adapts the definitions to new technologies. However, rapid progress in antibody technologies has blurred the boundaries between existing antibody categories and created a burgeoning array of new antibody formats. Thus, revising the INN system for antibodies is akin to aiming for a rapidly moving target. The WHO recently revised INN definitions for antibodies now to be based on amino acid sequence identity. These new definitions, however, are critically flawed as they are ambiguous and go against decades of scientific literature. A key concern is the imposition of an arbitrary threshold for identity against human germline antibody variable region sequences. This leads to inconsistent classification of somatically mutated human antibodies, humanized antibodies as well as antibodies derived from semi-synthetic/synthetic libraries and transgenic animals. Such sequence-based classification implies clear functional distinction between categories (e.g., immunogenicity). However, there is no scientific evidence to support this. Dialog between the WHO INN Expert Group and key stakeholders is needed to develop a new INN system for antibodies and to avoid confusion and miscommunication between researchers and clinicians prescribing antibodies.

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  • Reichen, C., Madhurantakam, C., Hansen, S., Grütter, M. G., Plückthun, A. and Mittl, P. R. (2016) Structures of designed armadillo-repeat proteins show propagation of inter-repeat interface effects. Acta Crystallogr. D Biol. Crystallogr. 72, 168-175.

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    Abstract

    The armadillo repeat serves as a scaffold for the development of modular peptide-recognition modules. In order to develop such a system, three crystal structures of designed armadillo-repeat proteins with third-generation N-caps (YIII-type), four or five internal repeats (M-type) and second-generation C-caps (AII-type) were determined at 1.8 A ˚ (His-YIIIM4AII), 2.0 A ˚ (His-YIIIM5AII) and 1.95 A ˚ (YIIIM5AII) resolution and compared with those of variants with thirdgeneration C-caps. All constructs are full consensus designs in which the internal repeats have exactly the same sequence, and hence identical conformations of the internal repeats are expected. The N-cap and internal repeats M1 to M3 are indeed extremely similar, but the comparison reveals structural differences in internal repeats M4 and M5 and the C-cap. These differences are caused by longrange effects of the C-cap, contacting molecules in the crystal, and the intrinsic design of the repeat. Unfortunately, the rigid-body movement of the C-terminal part impairs the regular arrangement of internal repeats that forms the putative peptide-binding site. The second-generation C-cap improves the packing of buried residues and thereby the stability of the protein. These considerations are useful for future improvements of an armadillo-repeat-based peptiderecognition system.Department of Biochemistry, University of Zu¨ rich, Winterthurerstrasse 190, 8057 Zu¨ rich,

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2015

# Reference PDF
  • König, I., Zarrine-Afsar, A., Aznauryan, M., Soranno, A., Wunderlich, B., Dingfelder, F., Stüber, J. C., Plückthun, A., Nettels, D. and Schuler, B. (2015) Single-molecule spectroscopy of protein conformational dynamics in live eukaryotic cells. Nature Methods 12, 773-779.

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    Abstract

    Single-molecule methods have become widely used for quantifying the conformational heterogeneity and structural dynamics of biomolecules in vitro. Their application in vivo, however, has remained challenging owing to shortcomings in the design and reproducible delivery of labeled molecules, the range of applicable analysis methods, and suboptimal cell culture conditions. By addressing these limitations in an integrated approach, we demonstrate the feasibility of probing protein dynamics from milliseconds down to the nanosecond regime in live eukaryotic cells with confocal single-molecule Forster resonance energy transfer (FRET) spectroscopy. We illustrate the versatility of the approach by determining the dimensions and submicrosecond chain dynamics of an intrinsically disordered protein; by detecting even subtle changes in the temperature dependence of protein stability, including in-cell cold denaturation; and by quantifying the folding dynamics of a small protein. The methodology opens possibilities for assessing the effect of the cellular environment on biomolecular conformation, dynamics and function.

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  • Merten, H., Brandl, F., Plückthun, A. and Zangemeister-Wittke, U. (2015) Antibody-drug conjugates for tumor targeting-novel conjugation chemistries and the promise of non-IgG binding proteins. Bioconjug. Chem. 26, 2176-2185.

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    Abstract

    Antibody-drug conjugates (ADCs) have emerged as a promising class of anticancer agents, combining the specificity of antibodies for tumor targeting and the destructive potential of highly potent drugs as payload. An essential component of these immunoconjugates is a bifunctional linker capable of reacting with the antibody and the payload to assemble a functional entity. Linker design is fundamental, as it must provide high stability in the circulation to prevent premature drug release, but be capable of releasing the active drug inside the target cell upon receptor-mediated endocytosis. Although ADCs have demonstrated an increased therapeutic window, compared to conventional chemotherapy in recent clinical trials, therapeutic success rates are still far from optimal. To explore other regimes of half-life variation and drug conjugation stoichiometries, it is necessary to investigate additional binding proteins which offer access to a wide range of formats, all with molecularly defined drug conjugation. Here, we delineate recent progress with site-specific and biorthogonal conjugation chemistries, and discuss alternative, biophysically more stable protein scaffolds like Designed Ankyrin Repeat Proteins (DARPins), which may provide such additional engineering opportunities for drug conjugates with improved pharmacological performance.

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  • Bradbury, A. R. and Plückthun, A. (2015) Getting to reproducible antibodies: the rationale for sequenced recombinant characterized reagents. Protein Eng. Des. Sel. 28, 303-305.

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    Abstract

    No abstract

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  • Sedlak, E., Schaefer, J. V., Marek, J., Gimeson, P. and Plückthun, A. (2015) Advanced analyses of kinetic stabilities of IgGs modified by mutations and glycosylation. Protein Sci. 24, 1100-1113.

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    Abstract

    The stability of Immunoglobulin G (IgG) affects production, storage and usability, especially in the clinic. The complex thermal and isothermal transitions of IgGs, especially their irreversibilities, pose a challenge to the proper determination of parameters describing their thermodynamic and kinetic stability. Here, we present a reliable mathematical model to study the irreversible thermal denaturations of antibody variants. The model was applied to two unrelated IgGs and their variants with stabilizing mutations as well as corresponding non-glycosylated forms of IgGs and Fab fragments. Thermal denaturations of IgGs were analyzed with three transitions, one reversible transition corresponding to CH 2 domain unfolding followed by two consecutive irreversible transitions corresponding to Fab and CH 3 domains, respectively. The parameters obtained allowed us to examine the effects of these mutations on the stabilities of individual domains within the full-length IgG. We found that the kinetic stability of the individual Fab fragment is significantly lowered within the IgG context, possibly because of intramolecular aggregation upon heating, while the stabilizing mutations have an especially beneficial effect. Thermal denaturations of non-glycosylated variants of IgG consist of more than three transitions and could not be analyzed by our model. However, isothermal denaturations demonstrated that the lack of glycosylation affects the stability of all and not just of the CH 2 domain, suggesting that the partially unfolded domains may interact with each other during unfolding. Investigating thermal denaturation of IgGs according to our model provides a valuable tool for detecting subtle changes in thermodynamic and/or kinetic stabilities of individual domains.

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  • Ewald, C., Christen, M. T., Watson, R. P., Mihajlovic, M., Zhou, T., Honegger, A., Plückthun, A., Caflisch, A. and Zerbe, O. (2015) A combined NMR and computational approach to investigate peptide binding to a designed armadillo repeat protein. J. Mol. Biol. 427, 1919-1933.

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    Abstract

    The specific recognition of peptide sequences by proteins plays an important role both in biology and in diagnostic applications. Here we characterize the relatively weak binding of the peptide neurotensin (NT) to the previously developed Armadillo repeat protein VG_328 by a multidisciplinary approach based on solution NMR spectroscopy, mutational studies, and molecular dynamics (MD) simulations, totaling 20mus for all MD runs. We describe assignment challenges arising from the repetitive nature of the protein sequence, and we present novel approaches to address them. Partial assignments obtained for VG_328 in combination with chemical shift perturbations allowed us to identify the repeats not involved in binding. Their subsequent elimination resulted in a reduced-size binder with very similar affinity for NT, for which near-complete backbone assignments were achieved. A binding mode suggested by automatic docking and further validated by explicit solvent MD simulations is consistent with paramagnetic relaxation enhancement data collected using spin-labeled NT. Favorable intermolecular interactions are observed in the MD simulations for the residues that were previously shown to contribute to binding in an Ala scan of NT. We further characterized the role of residues within the N-cap for protein stability and peptide binding. Our multidisciplinary approach demonstrates that an initial low-resolution picture for a low-micromolar-peptide binder can be refined through the combination of NMR, protein design, docking, and MD simulations to establish its binding mode, even in the absence of crystallographic data, thereby providing valuable information for further design.

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  • Friedel, T., Hanisch, L. J., Muth, A., Honegger, A., Abken, H., Plückthun, A., Buchholz, C. J. and Schneider, I. C. (2015) Receptor-targeted lentiviral vectors are exceptionally sensitive toward the biophysical properties of the displayed single-chain Fv. Protein Eng. Des. Sel. 28, 93-106.

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    Abstract

    An increasing number of applications require the expression of single-chain variable fragments (scFv) fusion proteins in mammalian cells at the cell surface membrane. Here we assessed the CD30-specific scFv HRS3, which is used in immunotherapy, for its ability to retarget lentiviral vectors (LVs) to CD30 and to mediate selective gene transfer into CD30-positive cells. Fused to the C-terminus of the type-II transmembrane protein hemagglutinin (H) of measles virus and expressed in LV packaging cells, gene transfer mediated by the released LV particles was inefficient. A series of point mutations in the scFv framework regions addressing its biophysical properties, which substantially improved production and increased the melting temperature without impairing its kinetic binding behavior to CD30, also improved the performance of LV particles. Gene transfer into CD30-positive cells increased approximately 100-fold due to improved transport of the H-scFv protein to the plasma membrane. Concomitantly, LV particle aggregation and syncytia formation in packaging cells were substantially reduced. The data suggest that syncytia formation can be triggered by trans-cellular dimerization of H-scFv proteins displayed on adjacent cells. Taken together, we show that the biophysical properties of the targeting ligand have a decisive role for the gene transfer efficiency of receptor-targeted LVs.

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  • Hillenbrand, M., Schori, C., Schöppe, J. and Plückthun, A. (2015) Comprehensive analysis of heterotrimeric G-protein complex diversity and their interactions with GPCRs in solution. Proc. Natl. Acad. Sci. U. S. A. 112, E1181-90.

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    Abstract

    Agonist binding to G-protein-coupled receptors (GPCRs) triggers signal transduction cascades involving heterotrimeric G proteins as key players. A major obstacle for drug design is the limited knowledge of conformational changes upon agonist binding, the details of interaction with the different G proteins, and the transmission to movements within the G protein. Although a variety of different GPCR/G protein complex structures would be needed, the transient nature of this complex and the intrinsic instability against dissociation make this endeavor very challenging. We have previously evolved GPCR mutants that display higher stability and retain their interaction with G proteins. We aimed at finding all G-protein combinations that preferentially interact with neurotensin receptor 1 (NTR1) and our stabilized mutants. We first systematically analyzed by coimmunoprecipitation the capability of 120 different G-protein combinations consisting of alphai1 or alphasL and all possible betagamma-dimers to form a heterotrimeric complex. This analysis revealed a surprisingly unrestricted ability of the G-protein subunits to form heterotrimeric complexes, including betagamma-dimers previously thought to be nonexistent, except for combinations containing beta5. A second screen on coupling preference of all G-protein heterotrimers to NTR1 wild type and a stabilized mutant indicated a preference for those Galphai1betagamma combinations containing gamma1 and gamma11. Heterotrimeric G proteins, including combinations believed to be nonexistent, were purified, and complexes with the GPCR were prepared. Our results shed new light on the combinatorial diversity of G proteins and their coupling to GPCRs and open new approaches to improve the stability of GPCR/G-protein complexes.

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  • Egloff, P., De Luigi, M., Heine, P., Balada, S. and Plückthun, A. (2015) A cleavable ligand column for the rapid isolation of large quantities of homogeneous and functional neurotensin receptor 1 variants from E. coli. Protein Expr. Purif. 108, 106-114.

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    Abstract

    G protein-coupled receptors (GPCRs) are key players of cell signaling, thus representing important drug targets for the treatment of human diseases. Since inherent difficulties in receptor production and handling have precluded the application of many in vitro experiments, major questions about GPCR mechanisms and dynamics remain elusive to date. We recently used directed evolution in Escherichia coli on neurotensin receptor 1 (NTR1) for the generation of GPCR variants with greatly elevated functional expression levels and with excellent stability in detergent micelles. In this work we outline a highly efficient purification method for our evolved receptor variants, which is based on the application of an inexpensive, disposable high-affinity ligand column as the initial purification step. The ligand resin allows isolation of correctly folded GPCR variants directly from whole E. coli cell lysates at the scale of 10mg and it permits preparations of agonist- and antagonist-bound receptor samples. The purification principle presented here was key to the first structures of signaling-active NTR1 variants (Egloff et al., 2014). Since E. coli is uniquely suitable for the production of fully deuterated proteins, our method provides the basis for an array of NMR experiments that were not feasible for GPCRs to date, but which will shed light on novel aspects of receptor function and dynamics.

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  • Oude Blenke, E., Klaasse, G., Merten, H., Plückthun, A., Mastrobattista, E. and Martin, N. I. (2015) Liposome functionalization with copper-free «»click chemistry»». J. Control. Release 202, 14-20.

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    Abstract

    The modification of liposomal surfaces is of interest for many different applications and a variety of chemistries are available that makes this possible. A major disadvantage of commonly used coupling chemistries (e.g. maleimide-thiol coupling) is the limited control over the site of conjugation in cases where multiple reactive functionalities are present, leading to heterogeneous products and in some cases dysfunctional conjugates. Bioorthogonal coupling approaches such as the well-established copper-catalyzed azide-alkyne cycloaddition (CuAAC) ""click"" reaction are attractive alternatives as the reaction kinetics are favorable and azide-containing reagents are widely available. In the work described here, we prepared lipids containing a reactive cyclooctyne group and, after incorporation into liposomes, demonstrated successful conjugation of both a small molecule dye (5'-TAMRA-azide) as well as a larger azide-containing model protein based upon a designed ankyrin repeat protein (azido-DARPin). By applying the strain-promoted azido-alkyne cycloaddition (SPAAC) the use of Cu(I) as a catalyst is avoided, an important advantage considering the known deleterious effects associated with copper in cell and protein studies. We demonstrate complete control over the number of ligands coupled per liposome when using a small molecule azide with conjugation occurring at a reasonable reaction rate. By comparison, the conjugation of a larger azide-modified protein occurs more slowly, however the number of protein ligands coupled was found to be sufficient for liposome targeting to cells. Importantly, these results provide a strong proof of concept for the site-specific conjugation of protein ligands to liposomal surfaces via SPAAC. Unlike conventional approaches, this strategy provides for the homogeneous coupling of proteins bearing a single site-specific azide modification and eliminates the chance of forming dysfunctional ligands on the liposome. Furthermore, the absence of copper in the reaction process should also make this approach much more compatible with cell-based and in vivo applications.

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  • Münch, R. C., Muth, A., Muik, A., Friedel, T., Schmatz, J., Dreier, B., Trkola, A., Plückthun, A., Büning, H. and Buchholz, C. J. (2015) Off-target-free gene delivery by affinity-purified receptor-targeted viral vectors. Nature Comm. 6, 6246.

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    Abstract

    We describe receptor-targeted adeno-associated viral (AAV) vectors that allow genetic modification of rare cell types ex vivo and in vivo while showing no detectable off-targeting. Displaying designed ankyrin repeat proteins (DARPins) on the viral capsid and carefully depleting DARPin-deficient particles, AAV vectors were made specific for Her2/neu, EpCAM or CD4. A single intravenous administration of vector targeted to the tumour antigen Her2/neu was sufficient to track 75% of all tumour sites and to extend survival longer than the cytostatic antibody Herceptin. CD4-targeted AAVs hit human CD4-positive cells present in spleen of a humanized mouse model, while CD8-positive cells as well as liver or other off-target organs remained unmodified. Mimicking conditions of circulating tumour cells, EpCAM-AAV detected single tumour cells in human blood opening the avenue for tumour stem cell tracking. Thus, the approach developed here delivers genes to target cell types of choice with antibody-like specificity.

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  • Goldstein, R., Sosabowski, J., Livanos, M., Leyton, J., Vigor, K., Bhavsar, G., Nagy-Davidescu, G., Rashid, M., Miranda, E., Yeung, J., Tolner, B., Plückthun, A., Mather, S., Meyer, T. and Chester, K. (2015) Development of the designed ankyrin repeat protein (DARPin) G3 for HER2 molecular imaging. Eur. J. of Nucl. Med. Mol. Imaging 42, 288-301.

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    Abstract

    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. METHODS: G3 DARPins tagged with hexahistidine (His6) or with histidine glutamate (HE)3 and untagged G3 DARPins were manufactured using a GMP-compatible Pichia pastoris protocol and radiolabelled with (125)I, or with (111)In via DOTA linked to a C-terminal cysteine. BALB/c mice were injected with radiolabelled G3 and tissue biodistribution was evaluated by gamma counting. The lead construct ((HE)3-G3) was assessed in mice bearing HER2-positive human breast tumour (BT474) xenografts. RESULTS: For both isotopes, (HE)3-G3 had significantly lower liver uptake than His6-G3 and untagged G3 counterparts in non-tumour-bearing mice, and there was no significantly different liver uptake between His6-G3 and untagged G3. (HE)3-G3 was taken forward for evaluation in mice bearing HER2-positive tumour xenografts. The results demonstrated that radioactivity from (111)In-(HE)3-G3 was better maintained in tumours and cleared faster from serum than radioactivity from (125)I-(HE)3-G3, achieving superior tumour-to-blood ratios (343.7 +/- 161.3 vs. 22.0 +/- 11.3 at 24 h, respectively). On microSPECT/CT, (111)In-labelled and (125)I-labelled (HE)3-G3 could image HER2-positive tumours at 4 h after administration, but there was less normal tissue uptake of radioactivity with (111)In-(HE)3-G3. Preadministration of trastuzumab did not affect the uptake of (HE)3-G3 by HER2-positive tumours. CONCLUSION: Radiolabelled DARPin (HE)3-G3 is a versatile radioligand with potential to allow the acquisition of whole-body HER2 scans on the day of administration.

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  • Bradbury, A. and Plückthun, A. (2015) Standardize antibodies used in research. Nature 518, 27-29.

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    Abstract

    No abstract

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  • Salvati Manni, L., Zabara, A., Osornio, Y. M., Schöppe, J., Batyuk, A., Plückthun, A., Siegel, J. S., Mezzenga, R. and Landau, E. M. (2015) Phase behavior of a designed cyclopropyl analogue of monoolein: implications for low-temperature membrane protein crystallization. Angew. Chem. 54, 1027-1031.

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    Abstract

    Lipidic cubic phases (LCPs) are used in areas ranging from membrane biology to biodevices. Because some membrane proteins are notoriously unstable at room temperature, and available LCPs undergo transformation to lamellar phases at low temperatures, development of stable low-temperature LCPs for biophysical studies of membrane proteins is called for. Monodihydrosterculin (MDS) is a designer lipid based on monoolein (MO) with a configurationally restricted cyclopropyl ring replacing the olefin. Small-angle X-ray scattering (SAXS) analyses revealed a phase diagram for MDS lacking the high-temperature, highly curved reverse hexagonal phase typical for MO, and extending the cubic phase boundary to lower temperature, thereby establishing the relationship between lipid molecular structure and mesophase behavior. The use of MDS as a new material for LCP-based membrane protein crystallization at low temperature was demonstrated by crystallizing bacteriorhodopsin at 20 degrees C as well as 4 degrees C.

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  • Plückthun, A. (2015) Designed ankyrin repeat proteins (DARPins): binding proteins for research, diagnostics, and therapy. Annu. Rev. Pharmacol. Toxicol. 55, 489-511.

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    Abstract

    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, permitting a wide range of conjugates and fusions to be produced and purified.

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  • Verdurmen, W. P., Luginbühl, M., Honegger, A. and Plückthun, A. (2015) Efficient cell-specific uptake of binding proteins into the cytoplasm through engineered modular transport systems. J. Control. Release 200, 13-22.

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    Abstract

    Through advances in protein scaffold engineering and selection technologies, highly specific binding proteins, which fold under reducing conditions, can be generated against virtually all targets. Despite tremendous therapeutic opportunities, intracellular applications are hindered by difficulties associated with achieving cytosolic delivery, compounded by even correctly measuring it. Here, we addressed cytosolic delivery systematically through the development of a biotin ligase-based assay that objectively quantifies cytosolic delivery in a generic fashion. We developed modular transport systems that consist of a designed ankyrin repeat protein (DARPin) for receptor targeting and a different DARPin for intracellular recognition and a bacterial toxin-derived component for cytosolic translocation. We show that both anthrax pores and the translocation domain of Pseudomonas exotoxin A (ETA) efficiently deliver DARPins into the cytosol. We found that the cargo must not exceed a threshold thermodynamic stability for anthrax pores, which can be addressed by engineering, while the ETA pathway does not appear to have this restriction.

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2014

# Reference PDF
  • Stefan, N., Zimmermann, M., Simon, M., Zangemeister-Wittke, U. and Plückthun, A. (2014) Novel prodrug-like fusion toxin with protease-sensitive bioorthogonal PEGylation for tumor targeting. Bioconjug. Chem. 25, 2144-2156.

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    Abstract

    Highly potent biotoxins like Pseudomonas exotoxin A (ETA) are attractive payloads for tumor targeting. However, despite replacement of the natural cell-binding domain of ETA by tumor-selective antibodies or alternative binding proteins like designed ankyrin repeat proteins (DARPins) the therapeutic window of such fusion toxins is still limited by target-independent cellular uptake, resulting in toxicity in normal tissues. Furthermore, the strong immunogenicity of the bacterial toxin precludes repeated administration in most patients. Site-specific modification to convert ETA into a prodrug-like toxin which is reactivated specifically in the tumor, and at the same time has a longer circulation half-life and is less immunogenic, is therefore appealing. To engineer a prodrug-like fusion toxin consisting of the anti-EpCAM DARPin Ec1 and a domain I-deleted variant of ETA (ETA''), we used strain-promoted azide alkyne cycloaddition for bioorthogonal conjugation of linear or branched polyethylene glycol (PEG) polymers at defined positions within the toxin moiety. Reversibility of the shielding was provided by a designed peptide linker containing the cleavage site for the rhinovirus 3C model protease. We identified two distinct sites, one within the catalytic domain and one close to the C-terminal KDEL sequence of Ec1-ETA'', simultaneous PEGylation of which resulted in up to 1000-fold lower cytotoxicity in EpCAM-positive tumor cells. Importantly, the potency of the fusion toxin was fully restored by proteolytic unveiling. Upon systemic administration in mice, PEGylated Ec1-ETA'' was much better tolerated than Ec1-ETA''; it showed a longer circulation half-life and an almost 10-fold increased area under the curve (AUC). Our strategy of engineering prodrug-like fusion toxins by bioorthogonal veiling opens new possibilities for targeting tumors with more specificity and efficacy.

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  • Brauchle, M., Hansen, S., Caussinus, E., Lenard, A., Ochoa-Espinosa, A., Scholz, O., Sprecher, S. G., Plückthun, A. and Affolter, M. (2014) Protein interference applications in cellular and developmental biology using DARPins that recognize GFP and mCherry. Biology Open 3, 1252-1261.

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    Abstract

    Protein-protein interactions are crucial for cellular homeostasis and play important roles in the dynamic execution of biological processes. While antibodies represent a well-established tool to study protein interactions of extracellular domains and secreted proteins, as well as in fixed and permeabilized cells, they usually cannot be functionally expressed in the cytoplasm of living cells. Non-immunoglobulin protein-binding scaffolds have been identified that also function intracellularly and are now being engineered for synthetic biology applications. Here we used the Designed Ankyrin Repeat Protein (DARPin) scaffold to generate binders to fluorescent proteins and used them to modify biological systems directly at the protein level. DARPins binding to GFP or mCherry were selected by ribosome display. For GFP, binders with KD as low as 160 pM were obtained, while for mCherry the best affinity was 6 nM. We then verified in cell culture their specific binding in a complex cellular environment and found an affinity cut-off in the mid-nanomolar region, above which binding is no longer detectable in the cell. Next, their binding properties were employed to change the localization of the respective fluorescent proteins within cells. Finally, we performed experiments in Drosophila melanogaster and Danio rerio and utilized these DARPins to either degrade or delocalize fluorescently tagged fusion proteins in developing organisms, and to phenocopy loss-of-function mutations. Specific protein binders can thus be selected in vitro and used to reprogram developmental systems in vivo directly at the protein level, thereby bypassing some limitations of approaches that function at the DNA or the RNA level.

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  • Reichen, C., Madhurantakam, C., Plückthun, A. and Mittl, P. R. (2014) Crystal structures of designed armadillo repeat proteins: Implications of construct design and crystallization conditions on overall structure. Protein Sci. 23, 1572-1583.

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    Abstract

    Designed armadillo repeat proteins (dArmRP) are promising modular proteins for the engineering of binding molecules that recognize extended polypeptide chains. We determined the structure of a dArmRP containing five internal repeats and 3rd generation capping repeats in three different states by X-ray crystallography: without N-terminal His6 -tag and in the presence of calcium (YM5 A/Ca(2+) ), without N-terminal His6 -tag and in the absence of calcium (YM5 A), and with N-terminal His6 -tag and in the presence of calcium (His-YM5 A/Ca(2+) ). All structures show different quaternary structures and superhelical parameters. His-YM5 A/Ca(2+) forms a crystallographic dimer, which is bridged by the His6 -tag, YM5 A/Ca(2+) forms a domain-swapped tetramer, and only in the absence of calcium and the His6 -tag, YM5 A forms a monomer. The changes of superhelical parameters are a consequence of calcium binding, because calcium ions interact with negatively charged residues, which can also participate in the modulation of helix dipole moments between adjacent repeats. These observations are important for further optimizations of dArmRPs and provide a general illustration of how construct design and crystallization conditions can influence the exact structure of the investigated protein.

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  • Hanenberg, M., McAfoose, J., Kulic, L., Welt, T., Wirth, F., Parizek, P., Strobel, L., Cattepoel, S., Spani, C., Derungs, R., Maier, M., Plückthun, A. and Nitsch, R. M. (2014) Amyloid-beta peptide-specific DARPins as a novel class of potential therapeutics for Alzheimer disease. J. Biol. Chem. 289, 27080-27089.

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    Abstract

    Passive immunization with anti-amyloid-beta peptide (Abeta) antibodies is effective in animal models of Alzheimer disease. With the advent of efficient in vitro selection technologies, the novel class of designed ankyrin repeat proteins (DARPins) presents an attractive alternative to the immunoglobulin scaffold. DARPins are small and highly stable proteins with a compact modular architecture ideal for high affinity protein-protein interactions. In this report, we describe the selection, binding profile, and epitope analysis of Abeta-specific DARPins. We further showed their ability to delay Abeta aggregation and prevent Abeta-mediated neurotoxicity in vitro. To demonstrate their therapeutic potential in vivo, mono- and trivalent Abeta-specific DARPins (D23 and 3xD23) were infused intracerebroventricularly into the brains of 11-month-old Tg2576 mice over 4 weeks. Both D23 and 3xD23 treatments were shown to result in improved cognitive performance and reduced soluble Abeta levels. These findings demonstrate the therapeutic potential of Abeta-specific DARPins for the treatment of Alzheimer disease.

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  • Yan, J., Gu, G. J., Jost, C., Hammond, M., Plückthun, A., Landegren, U. and Kamali-Moghaddam, M. (2014) A universal approach to prepare reagents for DNA-assisted protein analysis. PLoS One 9, e108061.

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    Abstract

    The quality of DNA-labeled affinity probes is critical in DNA-assisted protein analyses, such as proximity ligation and extension assays, immuno-PCR, and immuno-rolling circle amplification reactions. Efficient, high-performance methods are therefore required for isolation of pure conjugates from reactions where DNA strands have been coupled to antibodies or recombinant affinity reagents. Here we describe a universal, scalable approach for preparing high-quality oligonucleotide-protein conjugates by sequentially removing any unconjugated affinity reagents and remaining free oligonucleotides from conjugation reactions. We applied the approach to generate high-quality probes using either antibodies or recombinant affinity reagents. The purified high-grade probes were used in proximity ligation assays in solution and in situ, demonstrating both augmented assay sensitivity and improved signal-to-noise ratios.

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  • Kiuchi, T., Ortiz-Zapater, E., Monypenny, J., Matthews, D. R., Nguyen, L. K., Barbeau, J., Coban, O., Lawler, K., Burford, B., Rolfe, D. J., de Rinaldis, E., Dafou, D., Simpson, M. A., Woodman, N., Pinder, S., Gillett, C. E., Devauges, V., Poland, S. P., Fruhwirth, G., Marra, P., Boersma, Y. L., Plückthun, A., Gullick, W. J., Yarden, Y., Santis, G., Winn, M., Kholodenko, B. N., Martin-Fernandez, M. L., Parker, P., Tutt, A., Ameer-Beg, S. M. and Ng, T. (2014) The ErbB4 CYT2 variant protects EGFR from ligand-induced degradation to enhance cancer cell motility. Science Signal. 7, ra78.

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    Abstract

    The epidermal growth factor receptor (EGFR) is a member of the ErbB family that can promote the migration and proliferation of breast cancer cells. Therapies that target EGFR can promote the dimerization of EGFR with other ErbB receptors, which is associated with the development of drug resistance. Understanding how interactions among ErbB receptors alter EGFR biology could provide avenues for improving cancer therapy. We found that EGFR interacted directly with the CYT1 and CYT2 variants of ErbB4 and the membrane-anchored intracellular domain (mICD). The CYT2 variant, but not the CYT1 variant, protected EGFR from ligand-induced degradation by competing with EGFR for binding to a complex containing the E3 ubiquitin ligase c-Cbl and the adaptor Grb2. Cultured breast cancer cells overexpressing both EGFR and ErbB4 CYT2 mICD exhibited increased migration. With molecular modeling, we identified residues involved in stabilizing the EGFR dimer. Mutation of these residues in the dimer interface destabilized the complex in cells and abrogated growth factor-stimulated cell migration. An exon array analysis of 155 breast tumors revealed that the relative mRNA abundance of the ErbB4 CYT2 variant was increased in ER(+) HER2(-) breast cancer patients, suggesting that our findings could be clinically relevant. We propose a mechanism whereby competition for binding to c-Cbl in an ErbB signaling heterodimer promotes migration in response to a growth factor gradient.

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  • Scholz, O., Hansen, S. and Plückthun, A. (2014) G-quadruplexes are specifically recognized and distinguished by selected designed ankyrin repeat proteins. Nucleic Acids Res. 42, 9182-94.

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    Abstract

    We introduce designed ankyrin repeat binding proteins (DARPins) as a novel class of highly specific and structure-selective DNA-binding proteins, which can be functionally expressed within all cells. Human telomere quadruplex was used as target to select specific binders with ribosome display. The selected DARPins discriminate the human telomere quadruplex against the telomeric duplex and other quadruplexes. Affinities of the selected binders range from 3 to 100 nM. CD studies confirm that the quadruplex fold is maintained upon binding. The DARPins show different specificity profiles: some discriminate human telomere quadruplexes from other quadruplex-forming sequences like ILPR, c-MYC and c-KIT, while others recognize two of the sequences tested or even all quadruplexes. None of them recognizes dsDNA. Quadruplex-binding DARPins constitute valuable tools for specific detection at very small scales and for the in vivo investigation of quadruplex DNA.

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  • Scott, D. J., Kummer, L., Egloff, P., Bathgate, R. A. and Plückthun, A. (2014) Improving the apo-state detergent stability of NTS with CHESS for pharmacological and structural studies. Biochim. Biophys. Acta 1838, 2817-2824.

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    Abstract

    The largest single class of drug targets is the G protein-coupled receptor (GPCR) family. Modern high-throughput methods for drug discovery require working with pure protein, but this has been a challenge for GPCRs, and thus the success of screening campaigns targeting soluble, catalytic protein domains has not yet been realized for GPCRs. Therefore, most GPCR drug screening has been cell-based, whereas the strategy of choice for drug discovery against soluble proteins is HTS using purified proteins coupled to structure-based drug design. While recent developments are increasing the chances of obtaining GPCR crystal structures, the feasibility of screening directly against purified GPCRs in the unbound state (apo-state) remains low. GPCRs exhibit low stability in detergent micelles, especially in the apo-state, over the time periods required for performing large screens. Recent methods for generating detergent-stable GPCRs, however, offer the potential for researchers to manipulate GPCRs almost like soluble enzymes, opening up new avenues for drug discovery. Here we apply cellular high-throughput encapsulation, solubilization and screening (CHESS) to the neurotensin receptor 1 (NTS1) to generate a variant that is stable in the apo-state when solubilized in detergents. This high stability facilitated the crystal structure determination of this receptor and also allowed us to probe the pharmacology of detergent-solubilized, apo-state NTS1 using robotic ligand binding assays. NTS1 is a target for the development of novel antipsychotics, and thus CHESS-stabilized receptors represent exciting tools for drug discovery.

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  • Jost, C. and Plückthun, A. (2014) Engineered proteins with desired specificity: DARPins, other alternative scaffolds and bispecific IgGs. Curr. Opin. Struct. Biol. 27, 102-112.

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    Abstract

    Specific binding proteins have become essential for diagnostic and therapeutic applications, and traditionally these have been antibodies. Nowadays an increasing number of alternative scaffolds have joined these ranks. These additional folds have raised a lot of interest and expectations within the last decade. It appears that they have come of age and caught up with antibodies in many fields of applications. The last years have seen an exploration of possibilities in research, diagnostics and therapy. Some scaffolds have received further improvements broadening their fields of application, while others have started to occupy their respective niche. Protein engineering, the prerequisite for the advent of all alternative scaffolds, remains the driving force in this process, for both non-immunoglobulins and immunoglobulins alike.

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  • Watson, R. P., Christen, M. T., Ewald, C., Bumbak, F., Reichen, C., Mihajlovic, M., Schmidt, E., Guntert, P., Caflisch, A., Plückthun, A. and Zerbe, O. (2014) Spontaneous self-assembly of engineered armadillo repeat protein fragments into a folded structure. Structure 22, 985-995.

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    Abstract

    Repeat proteins are built of modules, each of which constitutes a structural motif. We have investigated whether fragments of a designed consensus armadillo repeat protein (ArmRP) recognize each other. We examined a split ArmRP consisting of an N-capping repeat (denoted Y), three internal repeats (M), and a C-capping repeat (A). We demonstrate that the C-terminal MA fragment adopts a fold similar to the corresponding part of the entire protein. In contrast, the N-terminal YM2 fragment constitutes a molten globule. The two fragments form a 1:1 YM2:MA complex with a nanomolar dissociation constant essentially identical to the crystal structure of the continuous YM3A protein. Molecular dynamics simulations show that the complex is structurally stable over a 1 mus timescale and reveal the importance of hydrophobic contacts across the interface. We propose that the existence of a stable complex recapitulates possible intermediates in the early evolution of these repeat proteins.

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  • Schilling, J., Schöppe, J., Sauer, E. and Plückthun, A. (2014) Co-crystallization with conformation-specific designed ankyrin repeat proteins explains the conformational flexibility of BCL-W. J. Mol. Biol. 426, 2346-2361.

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    Abstract

    BCL-W is a member of the BCL-2 family of anti-apoptotic proteins. A key event in the regulation of apoptosis is the heterodimerization between anti-apoptotic and pro-apoptotic family members, which involves a conserved surface-exposed groove on the anti-apoptotic proteins. Crystal structures of the ligand binding-competent conformation exist for all anti-apoptotic family members, with the exception of BCL-W, due to the flexibility of the BCL-W groove region. Existing structures had suggested major deviations of the BCL-W groove region from the otherwise structurally highly related remaining anti-apoptotic family members. To capture its ligand binding-competent conformation by counteracting the conformational flexibility of the BCL-W groove, we had selected high-affinity groove-binding designed ankyrin repeat proteins (DARPins) using ribosome display. We now determined two high-resolution crystal structures of human BCL-W in complex with different DARPins at resolutions 1.5 and 1.85A, in which the structure of BCL-W is virtually identical, and BCL-W adopts a conformation extremely similar to the ligand-free conformation of its closest relative BCL-XL in both structures. However, distinct differences to all previous BCL-W structures are evident, notably in the ligand-binding region. We provide the first structural explanation for the conformational flexibility of the BCL-W groove region in comparison to other BCL-2 family members. Due to the importance of the anti-apoptotic BCL-2 family as drug targets, the presented crystal structure of ligand binding-competent BCL-W may serve as a valuable basis for structure-based drug design in the future and provides a missing piece for the structural characterization of this protein family.

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  • Egloff, P., Hillenbrand, M., Klenk, C., Batyuk, A., Heine, P., Balada, S., Schlinkmann, K. M., Scott, D. J., Schütz, M. and Plückthun, A. (2014) Structure of signaling-competent neurotensin receptor 1 obtained by directed evolution in Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 111, E655-662.

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    Abstract

    Crystallography has advanced our understanding of G protein-coupled receptors, but low expression levels and instability in solution have limited structural insights to very few selected members of this large protein family. Using neurotensin receptor 1 (NTR1) as a proof of principle, we show that two directed evolution technologies that we recently developed have the potential to overcome these problems. We purified three neurotensin-bound NTR1 variants from Escherichia coli and determined their X-ray structures at up to 2.75 A resolution using vapor diffusion crystallization experiments. A crystallized construct was pharmacologically characterized and exhibited ligand-dependent signaling, internalization, and wild-type-like agonist and antagonist affinities. Our structures are fully consistent with all biochemically defined ligand-contacting residues, and they represent an inactive NTR1 state at the cytosolic side. They exhibit significant differences to a previously determined NTR1 structure (Protein Data Bank ID code 4GRV) in the ligand-binding pocket and by the presence of the amphipathic helix 8. A comparison of helix 8 stability determinants between NTR1 and other crystallized G protein-coupled receptors suggests that the occupancy of the canonical position of the amphipathic helix is reduced to various extents in many receptors, and we have elucidated the sequence determinants for a stable helix 8. Our analysis also provides a structural rationale for the long-known effects of C-terminal palmitoylation reactions on G protein-coupled receptor signaling, receptor maturation, and desensitization.

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  • Reichen, C., Hansen, S. and Plückthun, A. (2014) Modular peptide binding: From a comparison of natural binders to designed armadillo repeat proteins. J. Struct. Biol. 185, 147-162.

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    Abstract

    Several binding scaffolds that are not based on immunoglobulins have been designed as alternatives to traditional monoclonal antibodies. Many of them have been developed to bind to folded proteins, yet cellular networks for signaling and protein trafficking often depend on binding to unfolded regions of proteins. This type of binding can thus be well described as a peptide–protein interaction. In this review, we compare different peptide-binding scaffolds, highlighting that armadillo repeat proteins (ArmRP) offer an attractive modular system, as they bind a stretch of extended peptide in a repeat-wise manner. Instead of generating each new binding molecule by an independent selection, preselected repeats – each complementary to a piece of the target peptide – could be designed and assembled on demand into a new protein, which then binds the prescribed complete peptide. Stacked armadillo repeats (ArmR), each typically consisting of 42 amino acids arranged in three a-helices, build an elongated superhelical structure which enables binding of peptides in extended conformation. A consensus-based design approach, complemented with molecular dynamics simulations and rational engineering, resulted in well-expressed monomeric proteins with high stability. Peptide binders were selected and several structures were determined, forming the basis for the future development of modular peptide-binding scaffolds.

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  • Simon, M., Stefan, N., Borsig, L., Plückthun, A. and Zangemeister-Wittke, U. (2014) Increasing the antitumor effect of an EpCAM-targeting fusion toxin by facile click PEGylation. Mol. Cancer Ther. 13, 375-385.

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    Abstract

    Fusion toxins used for cancer-related therapy have demonstrated short circulation half-lives, which impairs tumor localization and, hence, efficacy. Here, we demonstrate that the pharmacokinetics of a fusion toxin composed of a designed ankyrin repeat protein (DARPin) and domain I-truncated Pseudomonas Exotoxin A (PE40/ETA'') can be significantly improved by facile bioorthogonal conjugation with a polyethylene glycol (PEG) polymer at a unique position. Fusion of the anti-EpCAM DARPin Ec1 to ETA'' and expression in methionine-auxotrophic E. coli enabled introduction of the nonnatural amino acid azidohomoalanine (Aha) at position 1 for strain-promoted click PEGylation. PEGylated Ec1-ETA'' was characterized by detailed biochemical analysis, and its potential for tumor targeting was assessed using carcinoma cell lines of various histotypes in vitro, and subcutaneous and orthotopic tumor xenografts in vivo. The mild click reaction resulted in a well-defined mono-PEGylated product, which could be readily purified to homogeneity. Despite an increased hydrodynamic radius resulting from the polymer, the fusion toxin demonstrated high EpCAM-binding activity and retained cytotoxicity in the femtomolar range. Pharmacologic analysis in mice unveiled an almost 6-fold increase in the elimination half-life (14 vs. 82 minutes) and a more than 7-fold increase in the area under the curve (AUC) compared with non-PEGylated Ec1-ETA'', which directly translated in increased and longer-lasting effects on established tumor xenografts. Our data underline the great potential of combining the inherent advantages of the DARPin format with bioorthogonal click chemistry to overcome the limitations of engineering fusion toxins with enhanced efficacy for cancer-related therapy. Mol Cancer Ther; 13(2); 1-11. (c)2013 AACR.

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  • Schilling, J., Schoeppe, J. and Plückthun, A. (2014) From DARPins to loopDARPins: novel loopDARPin design allows the selection of low picomolar binders in a single round of ribosome display. J. Mol. Biol. 426, 691-721.

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    Abstract

    Antibodies are the most versatile binding proteins in nature with six loops creating a flexible continuous interaction surface. However, in some molecular formats, antibodies are aggregation prone. Designed ankyrin repeat proteins (DARPins) were successfully created as alternative design solutions. Nevertheless, their concave shape, rigidity and incompletely randomized binding surface may limit the epitopes that can be targeted by this extremely stable scaffold. Combining conformational diversity and a continuous convex paratope found in many antibodies with the beneficial biophysical properties of DARPins, we created LoopDARPins, a next generation of DARPins with extended epitope binding properties. We employed X-ray structure determination of a LoopDARPin for design validation. Biophysical characterizations show that the introduction of an elongated loop through consensus design does not decrease the stability of the scaffold, consistent with molecular dynamics simulations. Ribosome-display selections against extracellular signal-regulated kinase 2 (ERK2) and four members of the BCL-2 family (BCL-2, BCL-XL, BCL-W and MCL-1) of anti-apoptotic regulators yielded LoopDARPins with affinities in the mid-picomolar to low nanomolar range against all targets. The BCL-2 family binders block the interaction with their natural interaction partner and will be valuable reagents to test the apoptotic response in functional assays. With the LoopDARPin scaffold, binders for BCL-2 with an affinity of 30 pM were isolated with only a single round of ribosome display, an enrichment that has not been described for any scaffold. Identical stringent one-round selections with conventional DARPins without loop yielded no binders. The LoopDARPin scaffold may become a highly valuable tool for biotechnological high-throughput applications.

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2013

# Reference PDF
  • Simon, M., Frey, R., Zangemeister-Wittke, U. and Plückthun, A. (2013) Orthogonal assembly of a designed ankyrin repeat protein-cytotoxin conjugate with a clickable serum albumin module for half-life extension. Bioconjug. Chem. 24, 1955-1966.

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    Abstract

    The generation of drug conjugates for safe and effective tumor targeting requires binding proteins tolerant to functionalization by rational engineering. Here, we show that Designed Ankyrin Repeat Proteins (DARPins), a novel class of binding proteins not derived from antibodies, can be used as building blocks for facile orthogonal assembly of bioconjugates for tumor targeting with tailored properties. DARPin Ec1, which targets the Epithelial Cell Adhesion Molecule (EpCAM), was genetically modified with a C-terminal cysteine for conjugation of the small molecule cytotoxin monomethylauristatin F (MMAF). In addition, it was N-terminally functionalized by metabolic introduction of the non-natural amino acid azidohomoalanine to enable linkage of site-specifically dibenzocyclooctyne-modified mouse serum albumin (MSA) for half-life extension using Cu(I)-free click chemistry. The conjugate MSA-Ec1-MMAF was assembled to obtain high yields of a pure and stable drug conjugate as confirmed by various analytical methods and in functional assays. The orthogonality of the assembly led to a defined reaction product and preserved the functional properties of all modules, including EpCAM-specific binding and internalization, FcRn binding mediated by MSA, and cytotoxic potency. Linkage of MMAF to the DARPin increased receptor-specific uptake of the drug while decreasing nonspecific uptake, and further coupling of the conjugate to MSA enhanced this effect. In mice, albumin conjugation increased the serum half-life from 11 min to 17.4 h, resulting in a more than 22-fold increase in the area-under-the-curve (AUC). Our data demonstrate the promise of the DARPin format for facile modular assembly of drug conjugates with improved pharmacokinetic performance for tumor targeting.

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  • Jost, C., Schilling, J., Tamaskovic, R., Schwill, M., Honegger, A. and Plückthun, A. (2013) Structural basis for eliciting a cytotoxic effect in HER2-overexpressing cancer cells via binding to the extracellular domain of HER2. Structure 21, 1979-1991.

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    Abstract

    Human epidermal growth factor receptor-2 (HER2) is a receptor tyrosine kinase directly linked to the growth of malignancies from various origins and a validated target for monoclonal antibodies and kinase inhibitors. Utilizing a new approach with designed ankyrin repeat proteins (DARPins) as alternative binders, we show that binding of two DARPins connected by a short linker, one targeting extracellular subdomain I and the other subdomain IV, causes much stronger cytotoxic effects on the HER2-addicted breast cancer cell line BT474, surpassing the therapeutic antibody trastuzumab. We determined crystal structures of these DARPins in complex with the respective subdomains. Detailed models of the full-length receptor, constrained by its rigid domain structures and its membrane anchoring, explain how the bispecific DARPins connect two membrane-bound HER2 molecules, distorting them such that they cannot form signaling-competent dimers with any EGFR family member, preventing any kinase dimerization, and thus leading to a complete loss of signaling.

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  • Gigant, B., Wang, W., Dreier, B., Jiang, Q., Pecqueur, L., Plückthun, A., Wang, C. and Knossow, M. (2013) Structure of a kinesin-tubulin complex and implications for kinesin motility. Nature Struct. Mol. Biol. 20, 1001-1007.

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    Abstract

    The typical function of kinesins is to transport cargo along microtubules. Binding of ATP to microtubule-attached motile kinesins leads to cargo displacement. To better understand the nature of the conformational changes that lead to the power stroke that moves a kinesin's load along a microtubule, we determined the X-ray structure of human kinesin-1 bound to alphabeta-tubulin. The structure defines the mechanism of microtubule-stimulated ATP hydrolysis, which releases the kinesin motor domain from microtubules. It also reveals the structural linkages that connect the ATP nucleotide to the kinesin neck linker, a 15-amino acid segment C terminal to the catalytic core of the motor domain, to result in the power stroke. ATP binding to the microtubule-bound kinesin favors neck-linker docking. This biases the attachment of kinesin's second head in the direction of the movement, thus initiating each of the steps taken.

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  • Kummer, L., Hsu, C. W., Dagliyan, O., Macnevin, C., Kaufholz, M., Zimmermann, B., Dokholyan, N. V., Hahn, K. M. and Plückthun, A. (2013) Knowledge-based besign of a biosensor to quantify localized ERK activation in living cells. Chemistry and Biology 20, 847-856.

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    Abstract

    Investigation of protein activation in living cells is fundamental to understanding how proteins are influenced by the full complement of upstream regulators they experience. Here, we describe the generation of a biosensor based on the DARPin binding scaffold suited for intracellular applications. Combining library selection and knowledge-based design, we created an ERK activity biosensor by derivatizing a DARPin specific for phosphorylated ERK with a solvatochromatic merocyanine dye, whose fluorescence increases upon pERK binding. The biosensor specifically responded to pERK2, recognized by its conformation, but not to ERK2 or other closely related mitogen-activated kinases tested. Activated endogenous ERK was visualized in mouse embryo fibroblasts, revealing greater activation in the nucleus, perinuclear regions, and especially the nucleoli. The DARPin-based biosensor will serve as a useful tool for studying biological functions of ERK in vitro and in vivo.

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  • Scott, D. J., Kummer, L., Tremmel, D. and Plückthun, A. (2013) Stabilizing membrane proteins through protein engineering. Curr. Opin. in Chem. Biol. 17, 427-435.

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    Abstract

    Integral membrane proteins (IMPs) are crucial components of all cells but are difficult to study in vitro because they are generally unstable when removed from their native membranes using detergents. Despite the major biomedical relevance of IMPs, less than 1% of Protein Data Bank (PDB) entries are IMP structures, reflecting the technical gap between studies of soluble proteins compared to IMPs. Stability can be engineered into IMPs by inserting stabilizing mutations, thereby generating proteins that can be successfully applied to biochemical and structural studies when solubilized in detergent micelles. The identification of stabilizing mutations is not trivial, and this review will focus on the methods that have been used to identify stabilized membrane proteins, including alanine scanning and screening, directed evolution and computational design.

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  • Mann, A., Friedrich, N., Krarup, A., Weber, J., Stiegeler, E., Dreier, B., Pugach, P., Robbiani, M., Riedel, T., Moehle, K., Robinson, J. A., Rusert, P., Plückthun, A. and Trkola, A. (2013) Conformation-dependent recognition of HIV gp120 by designed ankyrin repeat proteins provides access to novel HIV entry inhibitors. J. Virol. 87, 5868-5881.

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    Abstract

    Here, we applied the designed ankyrin repeat protein (DARPin) technology to develop novel gp120-directed binding molecules with HIV entry-inhibiting capacity. DARPins are interesting molecules for HIV envelope inhibitor design, as their high-affinity binding differs from that of antibodies. DARPins in general prefer epitopes with a defined folded structure. We probed whether this capacity favors the selection of novel gp120-reactive molecules with specificities in epitope recognition and inhibitory activity that differ from those found among neutralizing antibodies. The preference of DARPins for defined structures was notable in our selections, since of the four gp120 modifications probed as selection targets, gp120 arrested by CD4 ligation proved the most successful. Of note, all the gp120-specific DARPin clones with HIV-neutralizing activity isolated recognized their target domains in a conformation-dependent manner. This was particularly pronounced for the V3 loop-specific DARPin 5m3_D12. In stark contrast to V3-specific antibodies, 5m3_D12 preferentially recognized the V3 loop in a specific conformation, as probed by structurally arrested V3 mimetic peptides, but bound linear V3 peptides only very weakly. Most notably, this conformation-dependent V3 recognition allowed 5m3_D12 to bypass the V1V2 shielding of several tier 2 HIV isolates and to neutralize these viruses. These data provide a proof of concept that the DARPin technology holds promise for the development of HIV entry inhibitors with a unique mechanism of action.

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  • Epa, V. C., Dolezal, O., Doughty, L., Xiao, X., Jost, C., Plückthun, A. and Adams, T. E. (2013) Structural model for the interaction of a designed ankyrin repeat protein with the human epidermal growth factor receptor 2. PloS One 8, e59163.

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    Abstract

    Designed Ankyrin Repeat Proteins are a class of novel binding proteins that can be selected and evolved to bind to targets with high affinity and specificity. We are interested in the DARPin H10-2-G3, which has been evolved to bind with very high affinity to the human epidermal growth factor receptor 2 (HER2). HER2 is found to be over-expressed in 30% of breast cancers, and is the target for the FDA-approved therapeutic monoclonal antibodies trastuzumab and pertuzumab and small molecule tyrosine kinase inhibitors. Here, we use computational macromolecular docking, coupled with several interface metrics such as shape complementarity, interaction energy, and electrostatic complementarity, to model the structure of the complex between the DARPin H10-2-G3 and HER2. We analyzed the interface between the two proteins and then validated the structural model by showing that selected HER2 point mutations at the putative interface with H10-2-G3 reduce the affinity of binding up to 100-fold without affecting the binding of trastuzumab. Comparisons made with a subsequently solved X-ray crystal structure of the complex yielded a backbone atom root mean square deviation of 0.84-1.14 Angstroms. The study presented here demonstrates the capability of the computational techniques of structural bioinformatics in generating useful structural models of protein-protein interactions.

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  • Friedrich, K., Hanauer, J. R., Prüfer, S., Münch, R. C., Völker, I., Filippis, C., Jost, C., Hanschmann, K. M., Cattaneo, R., Peng, K. W., Plückthun, A., Buchholz, C. J., Cichutek, K. and Mühlebach, M. D. (2013) DARPin-targeting of measles virus: unique bispecificity, effective oncolysis, and enhanced safety. Mol. Ther. 21, 849-59.

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    Abstract

    Oncolytic virotherapy is an emerging treatment modality that uses replication-competent viruses to destroy cancers. Many naturally occurring viruses have a preferential, although nonexclusive, tropism for tumors and tumor cells. In addition, specific targeting of cancer cells can be achieved at the virus entry level. We optimized retargeting of cell entry by elongating the measles virus attachment protein with designed ankyrin repeat proteins (DARPins), while simultaneously ablating entry through the natural receptors. DARPin-targeted viruses were strongly attenuated in off-target tissue, thereby enhancing safety, but completely eliminated tumor xenografts. Taking advantage of the unique properties of DARPins of being fused without generating folding problems, we generated a virus simultaneous targeting two different tumor markers. The bispecific virus retained the original oncolytic efficacy, while providing proof of concept for a strategy to counteract issues of resistance development. Thus, DARPin-targeting opens new prospects for the development of personalized, targeted therapeutics.

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  • Simon, M., Stefan, N., Plückthun, A. and Zangemeister-Wittke, U. (2013) Epithelial cell adhesion molecule-targeted drug delivery for cancer therapy. Expert Opinion Drug Delivery 10, 451-468.

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    Abstract

    Introduction: The epithelial cell adhesion molecule (EpCAM) is abundantly expressed in epithelial tumors, on cancer stem cells and circulating tumor cells. Together with its role in oncogenic signaling, this has sparked interest in its potential for tumor targeting with antibodies and drug conjugates for safe and effective cancer therapy. Recent advances in protein engineering, linker design and drug formulations have provided a multitude of EpCAM-targeting anticancer agents, several of them with good perspectives for clinical development. Areas covered: This article reviews the biological, therapeutic and technical aspects of EpCAM-targeted drug delivery for cancer therapy. The authors discuss seminal findings, which distinguish EpCAM as a target with oncogenic function and abundant expression in epithelial tumors. Moreover, recent trends in engineering improved anti-EpCAM antibodies, binding proteins that are not derived from immunoglobulins and drug conjugates derived from them are highlighted and their therapeutic potential based on reported preclinical and clinical data, originality of design and perspectives are critically assessed. Expert opinion: EpCAM has shown promise for safe and efficient targeting of solid tumors using antibodies, alternative binding molecules and novel drug conjugates. Among the myriad of EpCAM-targeting drug delivery systems investigated so far, several could demonstrate therapeutic benefit, other formulations engineered to become tailor-made missiles are on the brink.

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  • Dreier, B., Honegger, A., Hess, C., Nagy-Davidescu, G., Mittl, P. R., Grütter, M. G., Belousova, N., Mikheeva, G., Krasnykh, V. and Plückthun, A. (2013) Development of a generic adenovirus delivery system based on structure-guided design of bispecific trimeric DARPin adapters. Proc. Natl. Acad. Sci. U. S. A. 110, E869-877.

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    Abstract

    Adenoviruses (Ads) have shown promise as vectors for gene delivery in clinical trials. Efficient viral targeting to a tissue of choice requires both ablation of the virus' original tropism and engineering of an efficient receptor-mediated uptake by a specific cell population. We have developed a series of adapters binding to the virus with such high affinity that they remain fully bound for >10 d, block its natural receptor binding site and mediate interaction with a surface receptor of choice. The adapter contains two fused modules, both consisting of designed ankyrin repeat proteins (DARPins), one binding to the fiber knob of adenovirus serotype 5 and the other binding to various tumor markers. By solving the crystal structure of the complex of the trimeric knob with three bound DARPins at 1.95-A resolution, we could use computer modeling to design a link to a trimeric protein of extraordinary kinetic stability, the capsid protein SHP from the lambdoid phage 21. We arrived at a module which binds the knob like a trimeric clamp. When this clamp was fused with DARPins of varying specificities, it enabled adenovirus serotype 5-mediated delivery of a transgene in a human epidermal growth factor receptor 2-, epidermal growth factor receptor-, or epithelial cell adhesion molecule-dependent manner with transduction efficiencies comparable to or even exceeding those of Ad itself. With these adapters, efficiently produced in Escherichia coli, Ad can be converted rapidly to new receptor specificities using any ligand as the receptor-binding moiety. Prefabricated Ads with different payloads thus can be retargeted readily to many cell types of choice.

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  • Scott, D. J. and Plückthun, A. (2013) Direct molecular evolution of detergent-stable G protein-coupled receptors using polymer encapsulated cells. J. Mol. Biol. 425, 662-677.

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    Abstract

    G protein-coupled receptors (GPCRs) are the largest class of pharmaceutical protein targets, yet drug development is encumbered by a lack of information about their molecular structure and conformational dynamics. Most mechanistic and structural studies as well as in vitro drug screening with purified receptors require detergent solubilization of the GPCR, but typically, these proteins exhibit only low stability in detergent micelles. We have developed the first directed evolution method that allows the direct selection of GPCRs stable in a chosen detergent from libraries containing over 100 million individual variants. The crucial concept was to encapsulate single Escherichia coli cells of a library, each expressing a different GPCR variant, to form detergent-resistant, semipermeable nano-containers. Unlike naked cells, these containers are not dissolved by detergents, allowing us to solubilize the GPCR proteins in situ while maintaining an association with the protein's genetic information, a prerequisite for directed evolution. The pore size was controlled to permit GPCR ligands to permeate but the solubilized receptor to remain within the nanocapsules. Fluorescently labeled ligands were used to bind to those GPCR variants inside the nano-containers that remained active in the detergent tested. With the use of fluorescence-activated cell sorting, detergent-stable mutants derived from two different family A GPCRs could be identified, some with the highest stability reported in short-chain detergents. In principle, this method (named cellular high-throughput encapsulation, solubilization and screening) is not limited to engineering stabilized GPCRs but could be used to stabilize other proteins for biochemical and structural studies.

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  • Schlinkmann, K. M. and Plückthun, A. (2013) Directed evolution of G-protein-coupled receptors for high functional expression and detergent stability. Methods Enzymol. 520, 67-97.

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    Abstract

    G-protein-coupled receptors (GPCRs) are cell-surface receptors exhibiting a key role in cellular signal transduction processes, thus making them pharmacologically highly relevant target proteins. However, the molecular mechanisms driving receptor activation by ligand binding and signal transduction are poorly understood, since as integral membrane proteins, most GPCRs are very challenging for functional and structural studies. The biophysical properties of natural GPCRs, usually required by the cell in only low amounts, support their functionality in the lipid bilayer but are insufficient for high-level

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2012

# Reference PDF
  • Gu, G. J., Friedman, M., Jost, C., Johnsson, K., Kamali-Moghaddam, M., Plückthun, A., Landegren, U. and Söderberg, O. (2012) Protein tag-mediated conjugation of oligonucleotides to recombinant affinity binders for proximity ligation. N. Biotechnol. 30, 144-152.

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    Abstract

    While antibodies currently play a dominant role as affinity reagents in biological research and for diagnostics, a broad range of recombinant proteins are emerging as promising alternative affinity reagents in detection assays and quantification. DNA-mediated affinity-based assays, such as immuno-PCR and proximity ligation assays (PLA), use oligonucleotides attached to affinity reagents as reporter molecules. Conjugation of oligonucleotides to affinity reagents generally employs chemistries that target primary amines or cysteines. Because of the random nature of these processes neither the number of oligonucleotides conjugated per molecule nor their sites of attachment can be accurately controlled for affinity reagents with several available amines and cysteines. Here, we present a straightforward and convenient approach to functionalize recombinant affinity reagents for PLA by expressing the reagents as fusion partners with SNAP protein tags. This allowed us to conjugate oligonucleotides in a site-specific fashion, yielding precisely one oligonucleotide per affinity reagent. We demonstrate this method using designed ankyrin repeat proteins (DARPins) recognizing the tumor antigen HER2 and we apply the conjugates in different assay formats. We also show that SNAP or CLIP tags, expressed as fusion partners of transfected genes, allow oligonucleotide conjugations to be performed in fixed cells, with no need for specific affinity reagents. The approach is used to demonstrate induced interactions between the fusion proteins FKBP and FRB by allowing the in situ conjugated oligonucleotides to direct the production of templates for localized rolling circle amplification reactions.

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  • Varadamsetty, G., Tremmel, D., Hansen, S., Parmeggiani, F. and Plückthun, A. (2012) Designed armadillo repeat proteins: library generation, characterization and selection of peptide binders with high specificity. J. Mol. Biol. 424, 68-87.

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    Abstract

    Designed Armadillo repeat proteins (ArmRPs) are a novel class of binding proteins intended for general modular peptide binding and have very favorable expression and stability properties. Using a combination of sequence and structural consensus analyses, we generated a 42-amino-acid designed Armadillo repeat module with six randomized positions, having a theoretical diversity of 9.9x10(6) per repeat. Structural considerations were used to replace cysteine residues, to define less conserved positions and to decide where to introduce randomized amino acid residues for potential interactions with the target peptide. Based on these concepts, combinatorial libraries of designed ArmRPs were assembled. The most stable version of designed ArmRP in library format was the N5C format, with three randomized library repeat modules flanked by full consensus repeat modules on either side and, in turn, flanked by N- and C-terminal capping repeats. Unselected members of this library were well expressed in the Escherichia coli cytoplasm, monomeric and showed the expected CD spectra and cooperative unfolding. N5C libraries were used in ribosome display selections against the peptide neurotensin. Highly specific peptide binders were enriched after four rounds of selections using ribosome display. Four peptide side chains were shown to contribute most of the interaction energy, and single alanine mutants could be discriminated. Thus, designed ArmRP libraries can become valuable sources for peptide binding molecules because of their favorable biophysical properties and with a potential for application in general modular peptide recognition.

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  • Frei, A. P., Jeon, O. Y., Kilcher, S., Moest, H., Henning, L. M., Jost, C., Plückthun, A., Mercer, J., Aebersold, R., Carreira, E. M. and Wollscheid, B. (2012) Direct identification of ligand-receptor interactions on living cells and tissues. Nature Biotechnol. 30, 997-1001.

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    Abstract

    Many cellular responses are triggered by proteins, drugs or pathogens binding to cell-surface receptors, but it can be challenging to identify which receptors are bound by a given ligand. Here we describe TRICEPS, a chemoproteomic reagent with three moieties-one that binds ligands containing an amino group, a second that binds glycosylated receptors on living cells and a biotin tag for purifying the receptor peptides for identification by quantitative mass spectrometry. We validated this ligand-based, receptor-capture (LRC) technology using insulin, transferrin, apelin, epidermal growth factor, the therapeutic antibody trastuzumab and two DARPins targeting ErbB2. In some cases, we could also determine the approximate ligand-binding sites on the receptors. Using TRICEPS to label intact mature vaccinia viruses, we identified the cell surface proteins AXL, M6PR, DAG1, CSPG4 and CDH13 as binding factors on human cells. This technology enables the identification of receptors for many types of ligands under near-physiological conditions and without the need for genetic manipulations.

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  • Schaefer, J. V. and Plückthun, A. (2012) Transfer of engineered biophysical properties between different antibody formats and expression systems. Protein Eng. Des. Sel. 25, 485-506.

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    Abstract

    Recombinant antibodies and their derivatives are receiving ever increasing attention for many applications. Nevertheless, they differ widely in biophysical properties, from stable monomers to metastable aggregation-prone mixtures of oligomers. Previous work from our laboratory presented the combination of structure-based analysis with family consensus alignments as being able to improve the properties of immunoglobulin variable domains. We had identified a series of mutations in the variable domains that greatly influenced both the stability and the expression level of single-chain Fv (scFv) fragments produced in the periplasm of Escherichia coli. We now investigated whether these effects are transferable to Fab fragments and immunoglobulin G (IgG) produced in bacteria, Pichia pastoris, and mammalian cells. Taken together, our data indicate that engineered mutations can increase functional expression levels only for periplasmic expression in prokaryotes. In contrast, stability against thermal and denaturant-induced unfolding is improved by the same mutations in all formats tested, including scFv, Fab and IgG, independent of the expression system. The mutations in V(H) also influenced the structural homogeneity of full-length IgG, and the reducibility of the distant C(H)1-C(L) inter-chain disulfide bond. These results confirm the potential of structure-based protein engineering in the context of full-length IgGs and the transferability of stability improvements discovered with smaller antibody fragments.

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  • Mignot, I., Pecqueur, L., Dorléans, A., Karuppasamy, M., Ravelli, R. B., Dreier, B., Plückthun, A., Knossow, M. and Gigant, B. (2012) Design and characterization of modular scaffolds for tubulin assembly. J. Biol. Chem. 287, 31085-31094.

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    Abstract

    In cells, microtubule dynamics is regulated by stabilizing and destabilizing factors. Whereas proteins in both categories have been identified, their mechanism of action is rarely understood at the molecular level. This is due in part to the difficulties faced in structural approaches to obtain atomic models when tubulin is involved. Here, we design and characterize new stathmin-like domain (SLD) proteins that sequester tubulins in numbers different from two, the number of tubulins bound by stathmin or by the SLD of RB3, two stathmin family members that have been extensively studied. We established rules for the design of tight tubulin-SLD assemblies and applied them to complexes containing one to four tubulin heterodimers. Biochemical and structural experiments showed that the engineered SLDs behaved as expected. The new SLDs will be tools for structural studies of microtubule regulation. The larger complexes will be useful for cryo-electron microscopy, whereas crystallography or nuclear magnetic resonance will benefit from the 1:1 tubulin-SLD assembly. Finally, our results provide new insight into SLD function, suggesting that a major effect of these phosphorylatable proteins is the programmed release of sequestered tubulin for microtubule assembly at the specific cellular locations of members of the stathmin family.

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  • Alfarano, P., Varadamsetty, G., Ewald, C., Parmeggiani, F., Pellarin, R., Zerbe, O., Plückthun, A. and Caflisch, A. (2012) Optimization of designed armadillo repeat proteins by molecular dynamics simulations and NMR spectroscopy. Protein Sci. 21, 1298-1314.

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    Abstract

    A multidisciplinary approach based on molecular dynamics (MD) simulations using homology models, NMR spectroscopy, and a variety of biophysical techniques was used to efficiently improve the thermodynamic stability of armadillo repeat proteins (ArmRPs). ArmRPs can form the basis of modular peptide recognition and the ArmRP version on which synthetic libraries are based must be as stable as possible. The 42-residue internal Arm repeats had been designed previously using a sequence-consensus method. Heteronuclear NMR revealed unfavorable interactions present at neutral but absent at high pH. Two lysines per repeat were involved in repulsive interactions, and stability was increased by mutating both to glutamine. Five point mutations in the capping repeats were suggested by the analysis of positional fluctuations and configurational entropy along multiple MD simulations. The most stabilizing single C-cap mutation Q240L was inferred from explicit solvent MD simulations, in which water penetrated the ArmRP. All mutants were characterized by temperature- and denaturant-unfolding studies and the improved mutants were established as monomeric species with cooperative folding and increased stability against heat and denaturant. Importantly, the mutations tested resulted in a cumulative decrease of flexibility of the folded state in silico and a cumulative increase of thermodynamic stability in vitro. The final construct has a melting temperature of about 85 degrees C, 14.5 degrees higher than the starting sequence. This work indicates that in silico studies in combination with heteronuclear NMR and other biophysical tools may provide a basis for successfully selecting mutations that rapidly improve biophysical properties of the target proteins.

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  • Kummer, L., Parizek, P., Rube, P., Millgramm, B., Prinz, A., Mittl, P. R., Kaufholz, M., Zimmermann, B., Herberg, F. W. and Plückthun, A. (2012) Structural and functional analysis of phosphorylation-specific binders of the kinase ERK from designed ankyrin repeat protein libraries. Proc. Natl. Acad. Sci. U. S. A. 109, E2248-2257.

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    Abstract

    We have selected designed ankyrin repeat proteins (DARPins) from a synthetic library by using ribosome display that selectively bind to the mitogen-activated protein kinase ERK2 (extracellular signal-regulated kinase 2) in either its nonphosphorylated (inactive) or doubly phosphorylated (active) form. They do not bind to other kinases tested. Crystal structures of complexes with two DARPins, each specific for one of the kinase forms, were obtained. The two DARPins bind to essentially the same region of the kinase, but recognize the conformational change within the activation loop and an adjacent area, which is the key structural difference that occurs upon activation. Whereas the rigid phosphorylated activation loop remains in the same form when bound by the DARPin, the more mobile unphosphorylated loop is pushed to a new position. The DARPins can be used to selectively precipitate the cognate form of the kinases from cell lysates. They can also specifically recognize the modification status of the kinase inside the cell. By fusing the kinase with Renilla luciferase and the DARPin to GFP, an energy transfer from luciferase to GFP can be observed in COS-7 cells upon intracellular complex formation. Phosphorylated ERK2 is seen to increase by incubation of the COS-7 cells with FBS and to decrease upon adding the ERK pathway inhibitor PD98509. Furthermore, the anti-ERK2 DARPin is seen to inhibit ERK phosphorylation as it blocks the target inside the cell. This strategy of creating activation-state-specific sensors and kinase-specific inhibitors may add to the repertoire to investigate intracellular signaling in real time.

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  • Parizek, P., Kummer, L., Rube, P., Prinz, A., Herberg, F. W. and Plückthun, A. (2012) Designed ankyrin repeat proteins (DARPins) as novel isoform-specific intracellular inhibitors of c-Jun N-terminal kinases. ACS Chem. Biol. 7, 1356-1366.

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    Abstract

    The c-Jun N-terminal kinases (JNKs) are involved in many biological processes such as proliferation, differentiation, apoptosis, and inflammation and occur in highly similar isoforms in eukaryotic cells. Isoform-specific functions and diseases have been reported for individual JNK isoforms mainly from gene-knockout studies in mice. There is, however, a high demand for intracellular inhibitors with high selectivity to improve the understanding of isoform-specific mechanisms and for use as therapeutic tools. The commonly used JNK inhibitors are based on small molecules or peptides that often target the conserved ATP binding site or docking sites and thus show only moderate selectivity. To target novel binding epitopes, we used designed ankyrin repeat proteins (DARPins) to generate alternative intracellular JNK inhibitors that discriminate two very similar isoforms, JNK1 and JNK2. DARPins are small binding proteins that are well expressed, stable, and cysteine-free, which makes them ideal candidates for applications in the reducing intracellular environment. We performed ribosome display selections against JNK1alpha1 and JNK2alpha1 using highly diverse combinatorial libraries of DARPins. The selected binders specifically recognize either JNK1 or JNK2 or both isoforms in vitro and in mammalian cells. All analyzed DARPins show affinities in the low nanomolar range and isoform-specific inhibition of JNK activation in vitro at physiological ATP concentrations. Importantly, DARPins that selectively inhibit JNK activation in human cells were also identified. These results emphasize the great potential of DARPins as a novel class of highly specific intracellular inhibitors of distinct enzyme isoforms for use in biological studies and as possible therapeutic leads.

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  • Schlinkmann, K. M., Hillenbrand, M., Rittner, A., Künz, M., Strohner, R. and Plückthun, A. (2012) Maximizing detergent stability and functional expression of a GPCR by exhaustive recombination and evolution. J. Mol. Biol. 422, 414-428.

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    Abstract

    To identify structural features in a G-protein-coupled receptor (GPCR) crucial for biosynthesis, stability in the membrane and stability in detergent micelles, we developed an evolutionary approach using expression in the inner membrane of Escherichia coli. From the analysis of 800,000 sequences of the rat neurotensin receptor 1, in which every amino acid had been varied to all 64 codons, we uncovered several ""shift"" positions, where the selected population focuses on a residue different from wild type. Here, we employed in vitro DNA recombination and a comprehensive synthetic binary library made by the Slonomics(R) technology, allowing us to uncover additive and synergistic effects in the structure that maximize both detergent stability and functional expression. We identified variants with >25,000 functional molecules per E. coli cell, a 50-fold increase over wild type, and observed strong coevolution of detergent stability. We arrived at receptor variants highly stable in short-chain detergents, much more so than those found by alanine scanning on the same receptor. These evolved GPCRs continue to be able to signal through the G-protein. We discuss the structural reasons for these improvements achieved through directed evolution.

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  • Pecqueur, L., Duellberg, C., Dreier, B., Jiang, Q., Wang, C., Plückthun, A., Surrey, T., Gigant, B. and Knossow, M. (2012) A designed ankyrin repeat protein selected to bind to tubulin caps the microtubule plus end. Proc. Natl. Acad. Sci. U. S. A. 109, 12011-12016.

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    Abstract

    Microtubules are cytoskeleton filaments consisting of alphabeta-tubulin heterodimers. They switch between phases of growth and shrinkage. The underlying mechanism of this property, called dynamic instability, is not fully understood. Here, we identified a designed ankyrin repeat protein (DARPin) that interferes with microtubule assembly in a unique manner. The X-ray structure of its complex with GTP-tubulin shows that it binds to the beta-tubulin surface exposed at microtubule (+) ends. The details of the structure provide insight into the role of GTP in microtubule polymerization and the conformational state of tubulin at the very microtubule end. They show in particular that GTP facilitates the tubulin structural switch that accompanies microtubule assembly but does not trigger it in unpolymerized tubulin. Total internal reflection fluorescence microscopy revealed that the DARPin specifically blocks growth at the microtubule (+) end by a selective end-capping mechanism, ultimately favoring microtubule disassembly from that end. DARPins promise to become designable tools for the dissection of microtubule dynamic properties selective for either of their two different ends.

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  • Schlinkmann, K. M., Honegger, A., Tureci, E., Robison, K. E., Lipovsek, D. and Plückthun, A. (2012) Critical features for biosynthesis, stability, and functionality of a G protein-coupled receptor uncovered by all-versus-all mutations. Proc. Natl. Acad. Sci. U. S. A. 109, 9810-9815.

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    Abstract

    The structural features determining efficient biosynthesis, stability in the membrane and, after solubilization, in detergents are not well understood for integral membrane proteins such as G protein-coupled receptors (GPCRs). Starting from the rat neurotensin receptor 1, a class A GPCR, we generated a separate library comprising all 64 codons for each amino acid position. By combining a previously developed FACS-based selection system for functional expression [Sarkar C, et al. (2009) Proc Natl Acad Sci USA 105:14808-14813] with ultradeep 454 sequencing, we determined the amino acid preference in every position and identified several positions in the natural sequence that restrict functional expression. A strong accumulation of shifts, i.e., a residue preference different from wild type, is detected for helix 1, suggesting a key role in receptor biosynthesis. Furthermore, under selective pressure we observe a shift of the most conserved residues of the N-terminal helices. This unique data set allows us to compare the in vitro evolution of a GPCR to the natural evolution of the GPCR family and to observe how selective pressure shapes the sequence space covered by functional molecules. Under the applied selective pressure, several positions shift away from the wild-type sequence, and these improve the biophysical properties. We discuss possible structural reasons for conserved and shifted residues.

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  • Madhurantakam, C., Varadamsetty, G., Grütter, M. G., Plückthun, A. and Mittl, P. R. (2012) Structure-based optimization of designed armadillo-repeat proteins. Protein Sci. 21, 1015-1028.

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    Abstract

    The armadillo domain is a right-handed super-helix of repeating units composed of three alpha-helices each. Armadillo repeat proteins (ArmRPs) are frequently involved in protein-protein interactions, and because of their modular recognition of extended peptide regions they can serve as templates for the design of artificial peptide binding scaffolds. On the basis of sequential and structural analyses, different consensus-designed ArmRPs were synthesized and show high thermodynamic stabilities, compared to naturally occurring ArmRPs. We determined the crystal structures of four full-consensus ArmRPs with three or four identical internal repeats and two different designs for the N- and C-caps. The crystal structures were refined at resolutions ranging from 1.80 to 2.50 A for the above mentioned designs. A redesign of our initial caps was required to obtain well diffracting crystals. However, the structures with the redesigned caps caused domain swapping events between the N-caps. To prevent this domain swap, 9 and 6 point mutations were introduced in the N- and C-caps, respectively. Structural and biophysical analysis showed that this subsequent redesign of the N-cap prevented domain swapping and improved the thermodynamic stability of the proteins. We systematically investigated the best cap combinations. We conclude that designed ArmRPs with optimized caps are intrinsically stable and well-expressed monomeric proteins and that the high-resolution structures provide excellent structural templates for the continuation of the design of sequence-specific modular peptide recognition units based on armadillo repeats.

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  • Simon, M., Zangemeister-Wittke, U. and Plückthun, A. (2012) Facile double-functionalization of designed ankyrin repeat proteins using click and thiol chemistries. Bioconjug. Chem. 23, 279-286.

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    Abstract

    Click chemistry is a powerful technology for the functionalization of therapeutic proteins with effector moieties, because of its potential for bio-orthogonal, regio-selective, and high-yielding conjugation under mild conditions. Designed Ankyrin Repeat Proteins (DARPins), a novel class of highly stable binding proteins, are particularly well suited for the introduction of clickable methionine surrogates such as azidohomoalanine (Aha) or homopropargylglycine (Hpg), since the DARPin scaffold can be made methionine-free by an M34L mutation in the N-cap which fully maintains the biophysical properties of the protein. A single N-terminal azidohomoalanine, replacing the initiator Met, is incorporated in high yield, and allows preparation of ""clickable"" DARPins at about 30 mg per liter E. coli culture, fully retaining stability, specificity, and affinity. For a second modification, we introduced a cysteine at the C-terminus. Such DARPins could be conveniently site-specifically linked to two moieties, polyethylene glycol (PEG) to the N-terminus and the fluorophore Alexa488 to the C-terminus. We present a DARPin selected against the epithelial cell adhesion molecule (EpCAM) with excellent properties for tumor targeting as an example. We used these doubly modified molecules to measure binding kinetics on tumor cells and found that PEGylation has no effect on dissociation rate, but slightly decreases the association rate and the maximal number of cell-bound DARPins, fully consistent with our previous model of PEG action obtained in vitro. Our data demonstrate the benefit of click chemistry for site-specific modification of binding proteins like DARPins to conveniently add several functional moieties simultaneously for various biomedical applications.

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  • Schaefer, J. V. and Plückthun, A. (2012) Engineering aggregation resistance in IgG by two independent mechanisms: lessons from comparison of Pichia pastoris and mammalian cell expression. J. Mol. Biol. 417, 309-335.

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    Abstract

    Aggregation is an important concern for therapeutic antibodies, since it can lead to reduced bioactivity and increase the risk of immunogenicity. In our analysis of immunoglobulin G (IgG) molecules of identical amino acid sequence but produced either in mammalian cells (HEK293) or in the yeast Pichia pastoris (PP), dramatic differences in their aggregation susceptibilities were encountered. The antibodies produced in Pichia were much more resistant to aggregation under many conditions, a phenomenon found to be mainly caused by two factors. First, the mannose-rich glycan of the IgG from Pichia, while slightly thermally destabilizing the IgG, strongly inhibited its aggregation susceptibility, compared to the complex mammalian glycan. Second, on the Pichia-produced IgGs, amino acids belonging to the alpha-factor pre-pro sequence were left at the N-termini of both chains. These additional residues proved to considerably increase the temperature of the onset of aggregation and reduced the aggregate formation after extended incubation at elevated temperatures. The attachment of these residues to IgGs produced in cell culture confirmed their beneficial effect on the aggregation resistance. Secretion of IgGs with native N-termini in the yeast system became possible after systematic engineering of the precursor proteins and the processing site. Taken together, the present results will be useful for the successful production of full-length IgGs in Pichia, give indications on how to engineer aggregation-resistant IgGs and shed new light on potential biophysical effects of tag sequences in general.

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  • Tamaskovic, R., Simon, M., Stefan, N., Schwill, M. and Plückthun, A. (2012) Designed ankyrin repeat proteins (DARPins): from research to therapy. Methods Enzymol. 503, 101-134.

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    Abstract

    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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  • Dreier, B. and Plückthun, A. (2012) Rapid selection of high-affinity binders using ribosome display. Methods Mol. Biol. 805, 261-286.

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    Abstract

    Ribosome display has proven to be a powerful in vitro selection and evolution method for generating high-affinity binders from libraries of folded proteins. It has been successfully applied to single-chain Fv fragments of antibodies and alternative scaffolds, such as Designed Ankyrin Repeat Proteins (DARPins). High-affinity binders with new target specificity can be obtained from highly diverse DARPin libraries in only a few selection rounds. In this protocol, the selection from the library and the process of affinity maturation and off-rate selection are explained in detail.

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  • Plückthun, A. (2012) Ribosome display: a perspective. Methods Mol. Biol. 805, 3-28.

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    Abstract

    Ribosome display is an in vitro evolution technology for proteins. It is based on in vitro translation, but prevents the newly synthesized protein and the mRNA encoding it from leaving the ribosome. It thereby couples phenotype and genotype. Since no cells need to be transformed, very large libraries can be used directly in selections, and the in vitro amplification provides a very convenient integration of random mutagenesis that can be incorporated into the procedure. This review highlights concepts, mechanisms, and different variations of ribosome display and compares it to related methods. Applications of ribosome display are summarized, e.g., the directed evolution of proteins for higher binding affinity, for higher stability or other improved biophysical parameters and enzymatic properties. Ribosome display has developed into a robust technology used in academia and industry alike, and it has made the cell-free Darwinian evolution of proteins over multiple generations a reality.

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2011

# Reference PDF
  • Boersma, Y. L. and Plückthun, A. (2011) DARPins and other repeat protein scaffolds: advances in engineering and applications. Curr. Opin. Biotechnol. 22, 849-857.

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    Abstract

    Antibodies have long been regarded as the only class of binding proteins. With the emergence of protein engineering techniques, new binding proteins based on alternative scaffolds have been designed. Additionally, modern technologies for selection and evolution from libraries are independent of the antibody scaffold and could thus be readily used for obtaining specific binding proteins. One important group of alternative scaffolds is based on repeat proteins. Nature is widely using these proteins to modulate protein-protein interactions, and even in the adaptive immune system of jawless vertebrates; the step to their application as an alternative to antibodies seems therefore logical. In this review, progress on DARPins and other repeat protein scaffolds will be discussed. Advances in their design as well as novel applications will be highlighted.

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  • Boersma, Y. L., Chao, G., Steiner, D., Wittrup, K. D. and Plückthun, A. (2011) Bispecific Designed Ankyrin Repeat Proteins (DARPins) Targeting Epidermal Growth Factor Receptor Inhibit A431 Cell Proliferation and Receptor Recycling. J. Biol. Chem. 286, 41273-41285.

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    Abstract

    The EGF receptor (EGFR) has been implicated in the development and progression of many tumors. Although monoclonal antibodies directed against EGFR have been approved for the treatment of cancer in combination with chemotherapy, there are limitations in their clinical efficacy, necessitating the search for robust targeting molecules that can be equipped with new effector functions or show a new mechanism of action. Designed ankyrin repeat proteins (DARPins) may provide the targeting component for such novel reagents. Previously, four DARPins were selected against EGFR with (sub)nanomolar affinity. As any targeting module should preferably be able to inhibit EGFR-mediated signaling, their effect on A431 cells overexpressing EGFR was examined: three of them were shown to inhibit proliferation by inducing G(1) arrest, as seen for the Food and Drug Administration-approved antibody cetuximab. To understand this inhibitory mechanism, we mapped the epitopes of the DARPins using yeast surface display. The epitopes for the biologically active DARPins overlapped with the EGF-binding site, whereas the fourth DARPin bound to a different domain, explaining the lack of a biological effect. To optimize the biological activity of the DARPins, we combined two DARPins binding to different epitopes with a flexible linker or with a leucine zipper, leading to a homodimer. The latter DARPin was able to reduce surface EGFR by inhibiting receptor recycling, leading to a dramatic decrease in cell viability. These results indicate that multispecific EGFR-specific DARPins are superior to cetuximab and may form the basis of new opportunities in tumor targeting and tumor therapy.

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  • Stefan, N., Martin-Killias, P., Wyss-Stoeckle, S., Honegger, A., Zangemeister-Wittke, U. and Plückthun, A. (2011) DARPins recognizing the tumor-associated antigen EpCAM selected by phage and ribosome display and engineered for multivalency. J. Mol. Biol. 413, 826-843.

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    Abstract

    Designed Ankyrin Repeat Proteins (DARPins) represent a novel class of binding molecules. Their favorable biophysical properties such as high affinity, stability and expression yields make them ideal candidates for tumor targeting. Here, we describe the selection of DARPins specific for the tumor-associated antigen epithelial cell adhesion molecule (EpCAM), an approved therapeutic target on solid tumors. We selected DARPins from combinatorial libraries by both phage display and ribosome display and compared their binding on tumor cells. By further rounds of random mutagenesis and ribosome display selection, binders with picomolar affinity were obtained that were entirely monomeric and could be expressed at high yields in the cytoplasm of Escherichia coli. One of the binders, denoted Ec1, bound to EpCAM with picomolar affinity (K(d)=68 pM), and another selected DARPin (Ac2) recognized a different epitope on EpCAM. Through the use of a variety of bivalent and tetravalent arrangements with these DARPins, the off-rate on cells was further improved by up to 47-fold. All EpCAM-specific DARPins were efficiently internalized by receptor-mediated endocytosis, which is essential for intracellular delivery of anticancer agents to tumor cells. Thus, using EpCAM as a target, we provide evidence that DARPins can be conveniently selected and rationally engineered to high-affinity binders of various formats for tumor targeting.

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  • Dodevski, I. and Plückthun, A. (2011) Engineering integral membrane proteins for expression and stability in: Production of Membrane Proteins – Strategies for Expression and Isolation (Skaja Robinson, A., ed) pp. 277-296. Wiley-VCH Verlag & Co. KGaA., Weinheim, Germany

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    Abstract

    No abstract

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  • Stevens, G. B., Krüger, M., Latychevskaia, T., Lindner, P., Plückthun, A. and Fink, H. W. (2011) Individual filamentous phage imaged by electron holography. Eur. Biophys. J. 40, 1197-201.

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    Abstract

    An in-line electron hologram of an individual f1.K phage was recorded with a purpose-built low energy electron point source (LEEPS) microscope. Cryo-microscopic methods were employed to prepare the specimen so that a single phage could be presented to the coherent low energy electrons: An aqueous phage suspension was applied to a thin carbon membrane with micro-machined slits. The membrane was rapidly cooled to freeze the remaining water as an amorphous ice sheet, which was then sublimated at low temperatures and pressures to leave individual free-standing phages suspended across slits. An image of a phage particle, depicted as the amplitude of the object wave, was reconstructed numerically from a digitized record of the hologram, obtained using 88 eV coherent electrons. The reconstructed image shows a single phage suspended across a slit in a supporting carbon membrane, magnified by a factor of 100,000. The width and shape in the reconstructed image compared well with a TEM image of the same filament. It is thus possible to record and reconstruct electron holograms of an individual phage. The challenge now is to improve the resolution of reconstructed images obtained by this method and to extend these structural studies to other biological molecules.

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  • Dodevski, I. and Plückthun, A. (2011) Evolution of three human GPCRs for higher expression and stability. J. Mol. Biol. 408, 599-615.

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    Abstract

    We recently developed a display method for the directed evolution of integral membrane proteins in the inner membrane of Escherichia coli for higher expression and stability. For the neurotensin receptor 1, a G-protein-coupled receptor (GPCR), we had evolved a mutant with a 10-fold increase in functional expression that largely retains wild-type binding and signaling properties and shows higher stability in detergent-solubilized form. We have now evolved three additional human GPCRs. Unmodified wild-type receptor cDNA was subjected to successive cycles of mutagenesis and fluorescence-activated cell sorting, and functional expression could be increased for all three GPCR targets. We also present a new stability screening method in a 96-well assay format to quickly identify evolved receptors showing increased thermal stability in detergent-solubilized form and rapidly evaluate them quantitatively. Combining the two methods turned out to be very powerful; even for the most challenging GPCR target-the tachykinin receptor NK(1), which is hardly expressed in E. coli and cannot be functionally solubilized-receptor mutants that are functionally expressed at 1 mg/l levels in E. coli and are stable in detergent solution could be quickly evolved. The improvements result from cumulative small changes in the receptor sequence. This combinatorial approach does not require preconceived notions for designing mutations. Our results suggest that this method is generally applicable to GPCRs. Existing roadblocks in structural and biophysical studies can now be removed by providing sufficient quantities of correctly folded and stable receptor protein.

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  • Münch, R. C., Mühlebach, M. D., Schaser, T., Kneissl, S., Jost, C., Plückthun, A., Cichutek, K. and Buchholz, C. J. (2011) DARPins: an efficient targeting domain for lentiviral vectors. Mol. Ther. 19, 686-693.

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    Abstract

    We have recently developed a retargeting system for lentiviral vectors (LVs) that relies on the pseudotyping of LVs with engineered measles virus (MV) glycoproteins (hemagglutinin (H) and fusion protein (F)). Specificity is provided through display of a single-chain antibody (scFv) as targeting domain by fusion to the MV-H protein. As an alternative to scFv, designed ankyrin repeat proteins (DARPins) can be selected to become high-affinity binders to any kind of target molecule. In this study six HER2/neu-specific DARPins exhibiting different affinities and binding to different HER2/neu epitopes were applied as targeting domains. All H-DARPin fusion proteins were efficiently expressed on the cell surface. Upon coexpression with F, syncytia formation was observed in HER2/neu positive cells only and correlated directly with the HER2/neu receptor density. All H-DARPin proteins incorporated into LVs, albeit at different levels. The vectors only transduced HER2/neu-positive cells, while HER2/neu-negative cells remained untransduced. Highest titers were observed with one particular DARPin binding to the membrane distal domain of HER2/neu with medium affinity. When applied in vivo systemically, HER2/neu-targeted LVs showed exclusive gene expression in HER2/neu positive tumor tissue, while vesicular stomatitis virus-glycoprotein (VSV-G) pseudotyped vectors mainly transduced cells in spleen and liver. Thus, DARPins are a promising alternative to scFvs for retargeting of LVs.

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  • Plückthun, A. (2011) Evolution im Reagenzglas in: Darwins langer Arm – Evolutionstheorie heute (Reyer, H.-U., Schmid-Hempel P., eds.) pp. 89-105. vdf Hochschulverlag AG an der ETH Zürich, Zürich

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    Abstract

    No abstract

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  • Martin-Killias, P., Stefan, N., Rothschild, S., Plückthun, A. and Zangemeister-Wittke, U. (2011) A novel fusion toxin derived from an EpCAM-specific designed ankyrin repeat protein has potent antitumor activity. Clin. Cancer Res. 17, 100-110.

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    Abstract

    PURPOSE: Designed ankyrin repeat proteins (DARPins) hold great promise as a new class of binding molecules to overcome the limitations of antibodies for biomedical applications. Here, we assessed the potential of an epithelial cell adhesion molecule (EpCAM)-specific DARPin (Ec4) for tumor targeting as a fusion toxin with Pseudomonas aeruginosa exotoxin A. Experimental design: DARPin Ec4 was genetically fused to a truncated form of Pseudomonas aeruginosa exotoxin A (ETA'') and expressed in Escherichia coli. The cytotoxicity of Ec4-ETA'' was measured against tumor cell lines of various histotypes in vitro. Tumor localization and antitumor activity were determined in mice bearing 2 different EpCAM-positive tumor xenografts. RESULTS: Ec4-ETA'' expressed very well in soluble form in the cytoplasm of E. coli and yielded up to 40 mg after purification per liter of culture. The protein was monomeric and the disulfides of ETA'' formed spontaneously. Ec4-ETA'' bound to EpCAM with low nanomolar affinity, similar to free Ec4. Furthermore, it was highly cytotoxic against various EpCAM-positive tumor cell lines in vitro with IC(50) values less than 0.005 pmol/L. This effect was competed by free Ec4, but not by unspecific DARPins. Upon systemic administration in athymic mice, Ec4-ETA'' efficiently localized to EpCAM-positive tumors to achieve maximum accumulation 48 to 72 hours after injection, whereas an irrelevant control fusion toxin did not accumulate. Tumor targeting with Ec4-ETA'' resulted in a strong antitumor response including complete regressions in some animals. CONCLUSIONS: Our data show for the first time the potential of DARPins for the generation of protein therapeutics for tumor targeting, and that Ec4-ETA'' deserves attention for clinical development. Clin Cancer Res; 17(1); 100-10. (c)2010 AACR.

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  • Dreier, B., Mikheeva, G., Belousova, N., Parizek, P., Boczek, E., Jelesarov, I., Forrer, P., Plückthun, A. and Krasnykh, V. (2011) Her2-specific multivalent adapters confer designed tropism to adenovirus for gene targeting. J. Mol. Biol. 405, 410-426.

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    Abstract

    Adenoviruses (Ads) hold great promise as gene vectors for diagnostic or therapeutic applications. The native tropism of Ads must be modified to achieve disease site-specific gene delivery by Ad vectors and this should be done in a programmable way and with technology that can realistically be scaled up. To this end, we applied the technologies of designed ankyrin repeat proteins (DARPins) and ribosome display to develop a DARPin that binds the knob domain of the Ad fiber protein with low nanomolar affinity (K(D) 1.35 nM) and fused this protein with a DARPin specific for Her2, an established cell-surface biomarker of human cancers. The stability of the complex formed by this bispecific targeting adapter and the Ad virion resulted in insufficient gene transfer and was subsequently improved by increasing the valency of adapter-virus binding. In particular, we designed adapters that chelated the knob in a bivalent or trivalent fashion and showed that the efficacy of gene transfer by the adapter-Ad complex increased with the functional affinity of these molecules. This enabled efficient transduction at low stoichiometric adapter-to-fiber ratios. We confirmed the Her2 specificity of this transduction and its dependence on the Her2-binding DARPin component of the adapters. Even the adapter molecules with four fused DARPins could be produced and purified from Escherichia coli at very high levels. In principle, DARPins can be generated against any target and this adapter approach provides a versatile strategy for developing a broad range of disease-specific gene vectors.

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  • Dreier, B. and Plückthun, A. (2011) Ribosome display: a technology for selecting and evolving proteins from large libraries. Methods Mol. Biol. 687, 283-306.

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    Abstract

    The selection and concomitant affinity maturation of proteins to bind to user-defined target molecules have become a key technology in biochemical research, diagnostics, and therapy. One of the most potent selection technologies for such applications is ribosome display. It works entirely in vitro, and this has two important consequences. First, since no transformation of any cells is required, libraries with much greater diversity can be handled than with most other techniques. Second, since a library does not have to be cloned and transformed, it is very convenient to introduce random errors in the library by PCR-based methods and select improved binders. Thus, a true directed evolution, an iteration between randomization and selection over several generations, can be conveniently carried out, e.g., for affinity maturation. Ribosome display has been used successfully for the selection of antibody fragments and other binding proteins, such as Designed Ankyrin Repeat Proteins (DARPins).

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2010

# Reference PDF
  • Kramer, M. A., Wetzel, S. K., Plückthun, A., Mittl, P. R. and Grütter, M. G. (2010) Structural determinants for improved stability of designed ankyrin repeat proteins with a redesigned C-capping module. J. Mol. Biol. 404, 381-391.

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    Abstract

    Designed ankyrin repeat proteins (DARPins) that specifically bind to almost any target can be obtained by ribosome display or phage display from combinatorial libraries. Although DARPins are already very stable molecules, molecular dynamics simulations, equilibrium denaturation experiments, structural studies, and recent NMR experiments suggested that the unfolding of the original C-terminal capping repeat (C-cap), taken from a natural ankyrin repeat protein, limits the stability of the initial DARPin design. Several point mutations had been introduced to optimize the C-cap and were shown to indeed further increase the stability of DARPins. We now determined crystal structures of DARPins with one or three full-consensus internal repeats (NI(1)C or NI(3)C) between an N-terminal capping repeat and mutants of the C-cap. An NI(1)C mutant, in which the C-cap was only extended by three additional helix-forming residues, showed no structural change but reduced B-factors in the C-cap. An NI(3)C C-cap mutant carrying five additional mutations in the interface to the preceding repeat, previously designed by using the consensus sequence as a guide, showed a rigid-body movement of the C-cap towards the internal repeat. This movement results in an increased buried surface area and a superior surface complementarity and explains the improved stability in equilibrium unfolding, compared to the original C-cap. A C-cap mutant with three additional mutations introducing suitably spaced charged residues did not show formation of salt bridges, explaining why its stability was not increased further. These structural studies underline the importance of repeat coupling for stability and help in the further design of this protein family.

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  • Plückthun, A. (2010) HIV: Antibodies with a split personality. Nature 467, 537-538.

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    Abstract

    No abstract

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  • Theurillat, J. P., Dreier, B., Nagy-Davidescu, G., Seifert, B., Behnke, S., Zürrer-Härdi, U., Ingold, F., Plückthun, A. and Moch, H. (2010) Designed ankyrin repeat proteins: a novel tool for testing epidermal growth factor receptor 2 expression in breast cancer. Mod. Pathol. 23, 1289-1297.

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    Abstract

    Designed ankyrin repeat proteins are a novel class of specific binding molecules, which display increased thermodynamic stability, smaller size and at least equal target affinity compared to immunoglobulins, making them potentially powerful tools in diagnostic pathology and therapeutic oncology. Here, we investigated whether designed ankyrin repeat proteins can reliably identify the amplification status of the epidermal growth factor receptor 2 in breast cancer. Designed ankyrin repeat proteins specific for epidermal growth factor receptor 2 were tested in paraffin-embedded tissue sections. Detection using enzymatic biotinylation proved to be most specific and sensitive. The affinity of the designed ankyrin repeat proteins was found crucial, but for a picomolar binder no further gain was found by making it multivalent. The best designed ankyrin repeat protein, G3 (K(D) 90 pM) was compared on breast cancer tissue microarrays (n=792) to an FDA-approved rabbit monoclonal antibody against epidermal growth factor receptor 2 (clone 4B5; Ventana Medical Systems) and correlated with corresponding epidermal growth factor receptor 2 amplification status measured by fluorescent in situ hybridization. Amplification status and epidermal growth factor receptor 2 expression measured by designed ankyrin repeat protein and antibody correlated strongly with each other (P<0.0001 each), the correlation between designed ankyrin repeat protein and amplification status being the strongest (0.87 compared to 0.77 for the antibody, Kendall's tau-beta). Using a modified scoring system for the designed ankyrin repeat protein, we show that the designed ankyrin repeat protein detects a positive epidermal growth factor receptor 2 amplification status with similar sensitivity and significantly higher specificity than the antibody (P=0.0005). This study suggests that designed ankyrin repeat proteins provide a valuable alternative to antibodies for the detection of epidermal growth factor receptor 2 expression in breast cancer and adds further compelling evidence for the use of designed ankyrin repeat proteins in diagnostic pathology and therapeutic oncology.

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  • Wetzel, S. K., Ewald, C., Settanni, G., Jurt, S., Plückthun, A. and Zerbe, O. (2010) Residue-resolved stability of full-consensus ankyrin repeat proteins probed by NMR. J. Mol. Biol. 402, 241-258.

      |  

    Abstract

    We investigated the stability determinants and the unfolding characteristics of full-consensus designed ankyrin repeat proteins (DARPins) by NMR. Despite the repeating sequence motifs, the resonances could be fully assigned using (2)H,(15)N,(13)C triple-labeled proteins. To remove further ambiguities, we attached paramagnetic spin labels to either end of these elongated proteins, which attenuate the resonances of the spatially closest residues. Deuterium exchange experiments of DARPins with two and three internal repeats between N- and C-terminal capping repeats (NI(2)C, NI(3)C) and NI(3)C_Mut5, where the C-cap had been reengineered, indicate that the stability of the full-consensus ankyrin repeat proteins is strongly dependent on the coupling between repeats, as the stabilized cap decreases the exchange rate throughout the whole protein. Some amide protons require more than a year to exchange at 37 degrees C, highlighting the extraordinary stability of the proteins. Denaturant-induced unfolding, followed by deuterium exchange, chemical shift change, and heteronuclear nuclear Overhauser effects, is consistent with an Ising-type description of equilibrium folding for NI(3)C_Mut5, while for native-state deuterium exchange, we postulate local fluctuations to dominate exchange as unfolding events are too slow in these very stable proteins. The location of extraordinarily slowly exchanging protons indicates a very stable core structure in the DARPins that combines hydrophobic shielding with favorable electrostatic interactions. These investigations help the understanding of repeat protein architecture and the further design of DARPins for biomedical applications where high stability is required.

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  • Zahnd, C., Sarkar, C. A. and Plückthun, A. (2010) Computational analysis of off-rate selection experiments to optimize affinity maturation by directed evolution. Protein Eng. Des. Sel. 23, 175-184.

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    Abstract

    Directed evolution is a powerful approach for isolating high-affinity binders from complex libraries. In affinity maturation experiments, binders with the highest affinities in the library are typically isolated through selections for decreased off rate using a suitable selection platform (e.g. phage display or ribosome display). In such experiments, the library is initially exposed to biotinylated antigen and the binding reaction is allowed to proceed. A large excess of unbiotinylated antigen is then added as a competitor to capture the vast majority of rapidly dissociating molecules; the slowly dissociating library members can subsequently be rescued by capturing the biotin-carrying complexes. To optimize the parameters for such affinity maturation experiments, we performed both deterministic and stochastic simulations of off-rate selection experiments using different input libraries. Our results suggest that the most critical parameters for achieving the lowest off rates after selection are the ratio of competitor antigen to selectable antigen and the selection time. Furthermore, the selection time has an optimum that depends on the experimental setup and the nature of the library. Notably, if selections are carried out for times much longer than the optimum, equilibrium is reached and the selection pressure is weakened or lost. Comparison of different selection strategies revealed that sequential selection rounds with lower stringency are favored over high-stringency selection experiments due to enhanced diversity in the selected pools. Such simulations may be helpful in optimizing affinity maturation strategies and off-rate selection experiments.

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  • Brient-Litzler, E., Plückthun, A. and Bedouelle, H. (2010) Knowledge-based design of reagentless fluorescent biosensors from a designed ankyrin repeat protein. Protein Eng. Des. Sel. 23, 229-241.

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    Abstract

    Designed ankyrin repeat proteins (DARPins) can be selected from combinatorial libraries to bind any target antigen. They show high levels of recombinant expression, solubility and stability, and contain no cysteine residue. The possibility of obtaining, from any DARPin and at high yields, fluorescent conjugates which respond to the binding of the antigen by a variation of fluorescence, would have numerous applications in micro- and nano-analytical sciences. This possibility was explored with Off7, a DARPin directed against the maltose binding protein (MalE) from Escherichia coli, with known crystal structure of the complex. Eight residues of Off7, whose solvent accessible surface area varies on association with the antigen but which are not in direct contact with the antigen, were individually mutated into cysteine and then chemically coupled with a fluorophore. The conjugates were ranked according to their relative sensitivities. All of them showed an increase in their fluorescence intensity on antigen binding by >1.7-fold. The best conjugate retained the same affinity as the parental DARPin. Its signal increased linearly and specifically with the concentration of antigen, up to 15-fold in buffer and 3-fold in serum when fully saturated, the difference being mainly due to the absorption of light by serum. Its lower limit of detection was equal to 0.3 nM with a standard spectrofluorometer. Titrations with potassium iodide indicated that the fluorescence variation was due to a shielding of the fluorescent group from the solvent by the antigen. These results suggest rules for the design of reagentless fluorescent biosensors from any DARPin.

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  • Schaefer, J. V. and Plückthun, A. (2010) Improving expression of scFv fragments by co-expression of periplasmic chaperones in: Antibody Engineering 2nd ed. (Kontermann, R., and Dübel, S., eds) Vol. 2 pp. 345-361. Springer Verlag, Berlin, Heidelberg, Germany

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    Abstract

    For more than 20 years now, periplasmic expression in Escherichia coli has become the standard technology for preparing functional antibody fragments in a rapid and

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  • Schaefer, J. V., Lindner, P. and Plückthun, A. (2010) Miniantibodies in: Antibody Engineering 2nd ed. (Kontermann, R., and Dübel, S., eds) Vol. 2 pp. 85-99. Springer Verlag, Berlin, Heidelberg, Germany

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    Abstract

    The usual motivation to create a multivalent molecule is to increase its functional affinity (avidity) to a corresponding multimeric antigen structure, which can be a

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  • Schaefer, J. V., Honegger, A. and Plückthun, A. (2010) Construction of scFv fragments from hybridoma or spleen cells by PCR assembly in: Antibody Engineering 2nd ed. (Kontermann, R., and Dübel, S., eds) Vol. 1 pp. 21-44. Springer Verlag, Berlin, Heidelberg, Germany

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    Abstract

    Today, antibodies can be obtained from naive repertoires (Winter et al. 1994; Vaughan et al. 1996) or libraries of fully synthetic genes (Knappik et al. 2000),

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  • Zahnd, C., Kawe, M., Stumpp, M. T., de Pasquale, C., Tamaskovic, R., Nagy-Davidescu, G., Dreier, B., Schibli, R., Binz, H. K., Waibel, R. and Plückthun, A. (2010) Efficient tumor targeting with high-affinity designed ankyrin repeat proteins: effects of affinity and molecular size. Cancer Res. 70, 1595-1605.

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    Abstract

    Slow-clearing, tumor-targeting proteins such as monoclonal antibodies typically exhibit high tumor accumulation but low tissue contrast, whereas intermediate-sized proteins such as scFvs show faster clearance but only moderate tumor accumulation. For both, tumor targeting does not seem to improve further above an optimal affinity. We show here that with very small high-affinity proteins such as designed ankyrin repeat proteins (DARPins), these limits can be overcome. We have systematically investigated the influence of molecular mass and affinity on tumor accumulation with DARPins with specificity for HER2 in SK-OV-3.ip nude mouse xenografts. DARPins with a mass of 14.5 kDa and affinities between 270 nmol/L and 90 pmol/L showed a strong correlation of tumor accumulation with affinity to HER2, with the highest affinity DARPin reaching 8% ID/g after 24 hours and 6.5% ID/g after 48 hours (tumor-to-blood ratio >60). Tumor autoradiographs showed good penetration throughout the tumor mass. Genetic fusion of two DARPins (30 kDa) resulted in significantly lower tumor accumulation, similar to values observed for scFvs, whereas valency had no influence on accumulation. PEGylation of the DARPins increased the circulation half-life, leading to higher tumor accumulation (13.4% ID/g after 24 hours) but lower tumor-to-blood ratios. Affinity was less important for tumor uptake of the PEGylated constructs. We conclude that two regimes exist for delivering high levels of drug to a tumor: small proteins with very high affinity, such as unmodified DARPins, and large proteins with extended half-life, such as PEGylated DARPins, in which the importance of affinity is less pronounced.

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2009

# Reference PDF
  • Plückthun, A. (2009) Alternative scaffolds: Expanding the options of antibodies in: Recombinant Antibodies for Immunotherapy (Little, M., ed) pp. 243-271. Cambridge University Press, New York

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    Abstract

    In the language of modern biotechnology, monoclonal antibodies (Köhler & Milstein, 1975) were the first ""library"" of proteins that was available, and the immune system was the first ""selection"" technology by which a specific binder could be obtained. However, only the subsequent introduction of molecular biology into this field allowed a true control over the molecules (reviewed, e.g., in Plückthun & Moroney, 2005; Weiner & Carter, 2003). This development of technologies was largely driven by the desire to use antibodies therapeutically, since the extraordinarily strong immune response to a nonhuman antibody in humans had put an end to essentially all of these endeavors. As will be illustrated in the following paragraphs, technological developments intended to solve this problem made not only the use of an animal immune system, but, ironically, also the antibody molecule itself dispensable.

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  • Veesler, D., Dreier, B., Blangy, S., Lichière, J., Tremblay, D., Moineau, S., Spinelli, S., Tegoni, M., Plückthun, A., Campanacci, V. and Cambillau, C. (2009) Crystal structure and function of a DARPin neutralizing inhibitor of lactococcal phage TP901-1: comparison of DARPin and camelid VHH binding mode. J. Biol. Chem. 284, 30718-30726.

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    Abstract

    Combinatorial libraries of designed ankyrin repeat proteins (DARPins) have been proven to be a valuable source of specific binding proteins, as they can be expressed at very high levels and are very stable. We report here the selection of DARPins directed against a macromolecular multiprotein complex, the baseplate BppUxBppL complex of the lactococcal phage TP901-1. Using ribosome display, we selected several DARPins that bound specifically to the tip of the receptor-binding protein (RBP, the BppL trimer). The three selected DARPins display high specificity and affinity in the low nanomolar range and bind with a stoichiometry of one DARPin per BppL trimer. The crystal structure of a DARPin complexed with the RBP was solved at 2.1 A resolution. The DARPinxRBP interface is of the concave (DARPin)-convex (RBP) type, typical of other DARPin protein complexes and different from what is observed with a camelid VHH domain, which penetrates the phage p2 RBP inter-monomer interface. Finally, phage infection assays demonstrated that TP901-1 infection of Lactococcus lactis cells was inhibited by each of the three selected DARPins. This study provides proof of concept for the possible use of DARPins to circumvent viral infection. It also provides support for the use of DARPins in co-crystallization, due to their rigidity and their ability to provide multiple crystal contacts.

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  • Winkler, J., Martin-Killias, P., Plückthun, A. and Zangemeister-Wittke, U. (2009) EpCAM-targeted delivery of nanocomplexed siRNA to tumor cells with designed ankyrin repeat proteins. Mol. Cancer Ther. 9, 2674-2683.

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    Abstract

    Specific delivery to tumors and efficient cellular uptake of nucleic acids remain major challenges for gene-targeted cancer therapies. Here we report the use of a designed ankyrin repeat protein (DARPin) specific for the epithelial cell adhesion molecule (EpCAM) as a carrier for small interfering RNA (siRNA) complementary to the bcl-2 mRNA. For charge complexation of the siRNA, the DARPin was fused to a truncated human protamine-1 sequence. To increase the cell binding affinity and the amount of siRNA delivered into cells, DARPin dimers were generated and used as fusion proteins with protamine. All proteins expressed well in Escherichia coli in soluble form, yet, to remove tightly bound bacterial nucleic acids, they were purified under denaturing conditions by immobilized metal ion affinity chromatography, followed by refolding. The fusion proteins were capable of complexing four to five siRNA molecules per protamine, and fully retained the binding specificity for EpCAM as shown on MCF-7 breast carcinoma cells. In contrast to unspecific LipofectAMINE transfection, down-regulation of antiapoptotic bcl-2 using fusion protein complexed siRNA was strictly dependent on EpCAM binding and internalization. Inhibition of bcl-2 expression facilitated tumor cell apoptosis as shown by increased sensitivity to the anticancer agent doxorubicin. (Mol Cancer Ther 2009;8(9):OF1-10).

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  • Milovnik, P., Ferrari, D., Sarkar, C. A. and Plückthun, A. (2009) Selection and characterization of DARPins specific for the neurotensin receptor 1. Protein Eng. Des. Sel. 22, 357-366.

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    Abstract

    We describe here the selection and characterization of designed ankyrin repeat proteins (DARPins) that bind specifically to the rat neurotensin receptor 1 (NTR1), a G-protein coupled receptor (GPCR). The selection procedure using ribosome display and the initial clone analysis required <10 microg of detergent-solubilized, purified NTR1. Complex formation with solubilized GPCR was demonstrated by ELISA and size-exclusion chromatography; additionally, the GPCR could be detected in native membranes of mammalian cells using fluorescence microscopy. The main binding epitope in the GPCR lies within the 33 amino acids following the seventh transmembrane segment, which comprise the putative helix 8, and additional binding interactions are possibly contributed by the cytoplasmic loop 3, thus constituting a discontinuous epitope. Since the selected binders recognize the GPCR both in detergent-solubilized and in membrane-embedded forms, they will be potentially useful both in co-crystallization trials and for signal transduction experiments.

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  • Kawe, M., Horn, U. and Plückthun, A. (2009) Facile promoter deletion in Escherichia coli in response to leaky expression of very robust and benign proteins from common expression vectors. Microb. Cell Fact. 8, 8.

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    Abstract

    ABSTRACT: BACKGROUND: Overexpression of proteins in Escherichia coli is considered routine today, at least when the protein is soluble and not otherwise toxic for the host. We report here that the massive overproduction of even such ""benign"" proteins can cause surprisingly efficient promoter deletions in the expression plasmid, leading to the growth of only non-producers, when expression is not well repressed in the newly transformed bacterial cell. Because deletion is so facile, it might impact on high-throughput protein production, e.g. for structural genomics, where not every expression parameter will be monitored. RESULTS: We studied the high-level expression of several robust non-toxic proteins using a T5 promoter under lac operator control. Full induction leads to no significant growth retardation. We compared expression from almost identical plasmids with or without the lacI gene together in strains expressing different levels of LacI. Any combination without net overexpression of LacI led to an efficient promoter deletion in the plasmid, although the number of growing colonies and even the plasmid size - all antibiotic-resistant non-producers - was almost normal, and thus the problem not immediately recognizable. However, by assuring sufficient repression during the initial establishment phase of the plasmid, deletion was completely prevented. CONCLUSION: The deletions in the insufficiently repressed system are caused entirely by the burden of high-level translation. Since the E. coli Dps protein, known to protect DNA against stress in the stationary phase, is accumulated in the deletion mutants, the mutation may have taken place during a transient stationary phase. The cause of the deletion is thus distinct from the well known interference of high-level transcription with plasmid replication. The deletion can be entirely prevented by overexpressing LacI, a useful precaution even without any signs of stress caused by the protein.

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  • Kügler, M., Stein, C., Schwenkert, M., Saul, D., Vockentanz, L., Huber, T., Wetzel, S. K., Scholz, O., Plückthun, A., Honegger, A. and Fey, G. H. (2009) Stabilization and humanization of a single-chain Fv antibody fragment specific for human lymphocyte antigen CD19 by designed point mutations and CDR-grafting onto a human framework. Protein Eng. Des. Sel. 22, 135-147.

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    Abstract

    A single-chain Fv (scFv) fragment derived from the murine antibody 4G7, specific for human lymphocyte CD19, was engineered for stability and expression in Escherichia coli in view of future use as a therapeutic protein. We compared two orthogonal knowledge-based procedures. In one approach, we designed a mutant with 14 single amino-acid substitutions predicted to correct destabilizing residues in the 4G7-wt sequence to create 4G7-mut. In the second variant, the murine CDRs were grafted to the human acceptor framework huVkappa3-huV(H)3, with 11 additional point mutations introduced to obtain a better match between CDR graft and acceptor framework, to arrive at 4G7-graft. Compared to 4G7-wt, 4G7-mut showed greater thermodynamic stability in guanidinium chloride-induced equilibrium denaturation experiments and somewhat greater stability in human serum. The loop graft maintained the comparatively high stability of the murine loop donor, but did not improve it further. Our analysis indicates that this is due to subtle strain introduced between CDRs and framework, mitigating the otherwise highly favorable properties of the human acceptor framework. This slight strain in the loop graft is also reflected in the binding affinities for CD19 on leukemic cells of 8.4 nM for 4G7-wt, 16.4 nM for 4G7-mut and 30.0 nM for 4G7-graft. This comparison of knowledge-based mutation and loop-grafting-based approaches will be important, when moving molecules forward to therapeutic applications.

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  • Honegger, A., Malebranche, A. D., Röthlisberger, D. and Plückthun, A. (2009) The influence of the framework core residues on the biophysical properties of immunoglobulin heavy chain variable domains. Protein Eng. Des. Sel. 22, 121-134.

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    Abstract

    Antibody variable domains differ considerably in stability. Single-chain Fv (scFv) fragments derived from natural repertoires frequently lack the high stability needed for therapeutic application, necessitating reengineering not only to humanize their sequence, but also to improve their biophysical properties. The human V(H)3 domain has been identified as having the best biophysical properties among human subtypes. However, complementarity determining region (CDR) grafts from highly divergent V(H) domains to huV(H)3 frequently fail to reach its superior stability. In previous experiments involving a CDR graft from a murine V(H)9 domain of very poor stability to huV(H)3, a hybrid V(H) framework was obtained which combines the lower core residues of muV(H)9 with the surface residues of huV(H)3. It resulted in a scFv with far better biophysical properties than the corresponding grafts to the consensus huV(H)3 framework. To better understand the origin of the superior properties of the hybrid framework, we constructed further hybrids, but now in the context of the consensus CDR-H1 and -H2 of the original human V(H)3 domain. The new hybrids included elements from either murine V(H)9, human V(H)1 or human V(H)5 domains. From guanidinium chloride-induced equilibrium denaturation measurements, kinetic denaturation experiments, measurements of heat-induced aggregation and comparison of soluble expression yield in Escherichia coli, we conclude that the optimal V(H) framework is CDR-dependent. The present work pinpoints structural features responsible for this dependency and helps to explain why the immune system uses more than one framework with different structural subtypes in framework 1 to optimally support widely different CDRs.

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2008

# Reference PDF
  • Sarkar, C. A., Dodevski, I., Kenig, M., Dudli, S., Mohr, A., Hermans, E. and Plückthun, A. (2008) Directed evolution of a G protein-coupled receptor for expression, stability, and binding selectivity. Proc. Natl. Acad. Sci. U. S. A. 105, 14808-14813.

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    Abstract

    We outline a powerful method for the directed evolution of integral membrane proteins in the inner membrane of Escherichia coli. For a mammalian G protein-coupled receptor, we arrived at a sequence with an order-of-magnitude increase in functional expression that still retains the biochemical properties of wild type. This mutant also shows enhanced heterologous expression in eukaryotes (12-fold in Pichia pastoris and 3-fold in HEK293T cells) and greater stability when solubilized and purified, indicating that the biophysical properties of the protein had been under the pressure of selection. These improvements arise from multiple small contributions, which would be difficult to assemble by rational design. In a second screen, we rapidly pinpointed a single amino acid substitution in wild type that abolishes antagonist binding while retaining agonist-binding affinity. These approaches may alleviate existing bottlenecks in structural studies of these targets by providing sufficient quantities of stable variants in defined conformational states.

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  • Steiner, D., Forrer, P. and Plückthun, A. (2008) Efficient selection of DARPins with sub-nanomolar affinities using SRP phage display. J. Mol. Biol. 382, 1211-1227.

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    Abstract

    There is an ever-increasing demand to select specific, high-affinity binding molecules against targets of biomedical interest. The success of such selections depends strongly on the design and functional diversity of the library of binding molecules employed, and on the performance of the selection strategy. We recently developed SRP phage display that employs the cotranslational signal recognition particle (SRP) pathway for the translocation of proteins to the periplasm. This system allows efficient filamentous phage display of highly stable and fast-folding proteins, such as designed ankyrin repeat proteins (DARPins) that are virtually refractory to conventional phage display employing the post-translational Sec pathway. DARPins comprise a novel class of binding molecules suitable to complement or even replace antibodies in many biotechnological or biomedical applications. So far, all DARPins have been selected by ribosome display. Here, we harnessed SRP phage display to generate a phage DARPin library containing more than 10(10) individual members. We were able to select well behaved and highly specific DARPins against a broad range of target proteins having affinities as low as 100 pM directly from this library, without affinity maturation. We describe efficient selection on the Fc domain of human IgG, TNFalpha, ErbB1 (EGFR), ErbB2 (HER2) and ErbB4 (HER4) as examples. Thus, SRP phage display makes filamentous phage display accessible for DARPins, allowing, for example, selection under harsh conditions or on whole cells. We envision that the use of SRP phage display will be beneficial for other libraries of stable and fast-folding proteins.

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  • Frey, D., Huber, T., Plückthun, A. and Grütter, M. G. (2008) Structure of the recombinant antibody Fab fragment f3p4. Acta Crystallogr. D Biol. Crystallogr. 64, 636-643.

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    Abstract

    The structure of the antibody Fab fragment f3p4, which was selected from a subset of the synthetic HuCAL antibody library to bind the sodium citrate symporter CitS, is described at 1.92 angstrom resolution. Comparison with computational models revealed deviations in a few framework positions and in the binding loops. The crystals belong to space group P2(1)2(1)2 and contain four molecules in the asymmetric unit, with unit-cell parameters a = 102.77, b = 185.92, c = 102.97 angstrom. These particular unit-cell parameters allowed pseudo-merohedral twinning; interestingly, the twinning law relates a twofold screw axis to a twofold axis.

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  • Parmeggiani, F., Pellarin, R., Larsen, A. P., Varadamsetty, G., Stumpp, M. T., Zerbe, O., Caflisch, A. and Plückthun, A. (2008) Designed armadillo repeat proteins as general peptide-binding scaffolds: consensus design and computational optimization of the hydrophobic core. J. Mol. Biol. 376, 1282-1304.

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    Abstract

    Armadillo repeat proteins are abundant eukaryotic proteins involved in several cellular processes, including signaling, transport, and cytoskeletal regulation. They are characterized by an armadillo domain, composed of tandem armadillo repeats of approximately 42 amino acids, which mediates interactions with peptides or parts of proteins in extended conformation. The conserved binding mode of the peptide in extended form, observed for different targets, makes armadillo repeat proteins attractive candidates for the generation of modular peptide-binding scaffolds. Taking advantage of the large number of repeat sequences available, a consensus-based approach combined with a force field-based optimization of the hydrophobic core was used to derive soluble, highly expressed, stable, monomeric designed proteins with improved characteristics compared to natural armadillo proteins. These sequences constitute the starting point for the generation of designed armadillo repeat protein libraries for the selection of peptide binders, exploiting their modular structure and their conserved binding mode.

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  • Wetzel, S. K., Settanni, G., Kenig, M., Binz, H. K. and Plückthun, A. (2008) Folding and unfolding mechanism of highly stable full-consensus ankyrin repeat proteins. J. Mol. Biol. 376, 241-257.

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    Abstract

    Full-consensus designed ankyrin repeat proteins were designed with one to six identical repeats flanked by capping repeats. These proteins express well in Escherichia coli as soluble monomers. Compared to our previously described designed ankyrin repeat protein library, randomized positions have now been fixed according to sequence statistics and structural considerations. Their stability increases with length and is even higher than that of library members, and those with more than three internal repeats are resistant to denaturation by boiling or guanidine hydrochloride. Full denaturation requires their heating in 5 M guanidine hydrochloride. The folding and unfolding kinetics of the proteins with up to three internal repeats were analyzed, as the other proteins could not be denatured. Folding is monophasic, with a rate that is nearly identical for all proteins ( approximately 400-800 s(-1)), indicating that essentially the same transition state must be crossed, possibly the folding of a single repeat. In contrast, the unfolding rate decreases by a factor of about 10(4) with increasing repeat number, directly reflecting thermodynamic stability in these extraordinarily slow denaturation rates. The number of unfolding phases also increases with repeat number. We analyzed the folding thermodynamics and kinetics both by classical two-state and three-state cooperative models and by an Ising-like model, where repeats are considered as two-state folding units that can be stabilized by interacting with their folded nearest neighbors. This Ising model globally describes both equilibrium and kinetic data very well and allows for a detailed explanation of the ankyrin repeat protein folding mechanism.

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  • Interlandi, G., Wetzel, S. K., Settanni, G., Plückthun, A. and Caflisch, A. (2008) Characterization and further stabilization of designed ankyrin repeat proteins by combining molecular dynamics simulations and experiments. J. Mol. Biol. 375, 837-854.

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    Abstract

    Multiple molecular dynamics simulations with explicit solvent at room temperature and at 400 K were carried out to characterize designed ankyrin repeat (AR) proteins with full-consensus repeats. Using proteins with one to five repeats, the stability of the native structure was found to increase with the number of repeats. The C-terminal capping repeat, originating from the natural guanine-adenine-binding protein, was observed to denature first in almost all high-temperature simulations. Notably, a stable intermediate is found in experimental equilibrium unfolding studies of one of the simulated consensus proteins. On the basis of simulation results, this intermediate is interpreted to represent a conformation with a denatured C-terminal repeat. To validate this interpretation, constructs without C-terminal capping repeat were prepared and did not show this intermediate in equilibrium unfolding experiments. Conversely, the capping repeats were found to be essential for efficient folding in the cell and for avoiding aggregation, presumably because of their highly charged surface. To design a capping repeat conferring similar solubility properties yet even higher stability, eight point mutations adapting the C-cap to the consensus AR and adding a three-residue extension at the C-terminus were predicted in silico and validated experimentally. The in vitro full-consensus proteins were also compared with a previously published designed AR protein, E3_5, whose internal repeats show 80% identity in primary sequence. A detailed analysis of the simulations suggests that networks of salt bridges between beta-hairpins, as well as additional interrepeat hydrogen bonds, contribute to the extraordinary stability of the full consensus.

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  • Merz, T., Wetzel, S. K., Firbank, S., Plückthun, A., Grütter, M. G. and Mittl, P. R. (2008) Stabilizing ionic interactions in a full-consensus ankyrin repeat protein. J. Mol. Biol. 376, 232-240.

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    Abstract

    Full-consensus designed ankyrin repeat proteins (DARPins), in which randomized positions of the previously described DARPin library have been fixed, are characterized. They show exceptionally high thermodynamic stabilities, even when compared to members of consensus DARPin libraries and even more so when compared to naturally occurring ankyrin repeat proteins. We determined the crystal structure of a full-consensus DARPin, containing an N-capping repeat, three identical internal repeats and a C-capping repeat at 2.05 A resolution, and compared its structure with that of the related DARPin library members E3_5 and E3_19. This structural comparison suggests that primarily salt bridges on the surface, which arrange in a network with almost crystal-like regularity, increase thermostability in the full-consensus NI(3)C DARPin to make it resistant to boiling. In the crystal structure, three sulfate ions complement this network. Thermal denaturation experiments in guanidine hydrochloride directly indicate a contribution of sulfate binding to the stability, providing further evidence for the stabilizing effect of surface-exposed electrostatic interactions and regular charge networks. The charged residues at the place of randomized residues in the DARPin libraries were selected based on sequence statistics and suggested that the charge interaction network is a hidden design feature of this protein family. Ankyrins can therefore use design principles from proteins of thermophilic organisms and reach at least similar stabilities.

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2007

# Reference PDF
  • Hussain, S., Plückthun, A., Allen, T. M. and Zangemeister-Wittke, U. (2007) Antitumor activity of an epithelial cell adhesion molecule targeted nanovesicular drug delivery system. Mol. Cancer Ther. 6, 3019-3027.

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    Abstract

    Site-specific delivery of anticancer agents to tumors represents a promising therapeutic strategy because it increases efficacy and reduces toxicity to normal tissues compared with untargeted drugs. Sterically stabilized immunoliposomes (SIL), guided by antibodies that specifically bind to well internalizing antigens on the tumor cell surface, are effective nanoscale delivery systems capable of accumulating large quantities of anticancer agents at the tumor site. The epithelial cell adhesion molecule (EpCAM) holds major promise as a target for antibody-based cancer therapy due to its abundant expression in many solid tumors and its limited distribution in normal tissues. We generated EpCAM-directed immunoliposomes by covalently coupling the humanized single-chain Fv antibody fragment 4D5MOCB to the surface of sterically stabilized liposomes loaded with the anticancer agent doxorubicin. In vitro, the doxorubicin-loaded immunoliposomes (SIL-Dox) showed efficient cell binding and internalization and were significantly more cytotoxic against EpCAM-positive tumor cells than nontargeted liposomes (SL-Dox). In athymic mice bearing established human tumor xenografts, pharmacokinetic and biodistribution analysis of SIL-Dox revealed long circulation times in the blood with a half-life of 11 h and effective time-dependent tumor localization, resulting in up to 15% injected dose per gram tissue. These favorable pharmacokinetic properties translated into potent antitumor activity, which resulted in significant growth inhibition (compared with control mice), and was more pronounced than that of doxorubicin alone and nontargeted SL-Dox at low, nontoxic doses. Our data show the promise of EpCAM-directed nanovesicular drug delivery for targeted therapy of solid tumors.

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  • Morfill, J., Blank, K., Zahnd, C., Luginbühl, B., Kuhner, F., Gottschalk, K. E., Plückthun, A. and Gaub, H. E. (2007) Affinity-matured recombinant antibody fragments analyzed by single-molecule force spectroscopy. Biophys. J. 93, 3583-90.

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    Abstract

    For many applications, antibodies need to be engineered toward maximum affinity. Strategies are in demand to especially optimize this process toward slower dissociation rates, which correlate with the (un)binding forces. Using single-molecule force spectroscopy, we have characterized three variants of a recombinant antibody single-chain Fv fragment. These variants were taken from different steps of an affinity maturation process. Therefore, they are closely related and differ from each other by a few mutations only. The dissociation rates determined with the atomic force microscope differ by one order of magnitude and agree well with the values obtained from surface plasmon resonance measurements. However, the effective potential width of the binding complexes, which was derived from the dynamic force spectroscopy measurements, was found to be the same for the different mutants. The large potential width of 0.9 nm indicates that both the binding pocket and the peptide deform significantly during the unbinding process.

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  • Huber, T., Steiner, D., Röthlisberger, D. and Plückthun, A. (2007) In vitro selection and characterization of DARPins and Fab fragments for the co-crystallization of membrane proteins: The Na(+)-citrate symporter CitS as an example. J. Struct. Biol. 159, 206-221.

      |  

    Abstract

    The determination of 3D structures of membrane proteins is still extremely difficult. The co-crystallization with specific binding proteins may be an important aid in this process, as these proteins provide rigid, hydrophilic surfaces for stable protein-protein contacts. Also, the conformational homogeneity of the membrane protein may be increased to obtain crystals suitable for high resolution structures. Here, we describe the efficient generation and characterization of Designed Ankyrin Repeat Proteins (DARPins) as specific binding molecules for membrane proteins. We used both phage display and ribosome display to select DARPins in vitro that are specific for the detergent-solubilized Na(+)-citrate symporter CitS of Klebsiella pneumoniae. Compared to classical hybridoma technology, the in vitro selection systems allow a much better control of the structural integrity of the target protein and allow the use of other protein classes in addition to recombinant antibodies. We also compared the selected DARPins to a Fab fragment previously selected by phage display and demonstrate that different epitopes are recognized, unique to each class of binding molecules. Therefore, the use of several classes of binding molecules will make suitable crystal formation and the determination of their 3D structure more likely.

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  • Zahnd, C., Wyler, E., Schwenk, J. M., Steiner, D., Lawrence, M. C., McKern, N. M., Pecorari, F., Ward, C. W., Joos, T. O. and Plückthun, A. (2007) A designed ankyrin repeat protein evolved to picomolar affinity to Her2. J. Mol. Biol. 369, 1015-28.

      |  

    Abstract

    Designed ankyrin repeat proteins (DARPins) are a novel class of binding molecules, which can be selected to recognize specifically a wide variety of target proteins. DARPins were previously selected against human epidermal growth factor receptor 2 (Her2) with low nanomolar affinities. We describe here their affinity maturation by error-prone PCR and ribosome display yielding clones with zero to seven (average 2.5) amino acid substitutions in framework positions. The DARPin with highest affinity (90 pM) carried four mutations at framework positions, leading to a 3000-fold affinity increase compared to the consensus framework variant, mainly coming from a 500-fold increase of the on-rate. This DARPin was found to be highly sensitive in detecting Her2 in human carcinoma extracts. We have determined the crystal structure of this DARPin at 1.7 A, and found that a His to Tyr mutation at the framework position 52 alters the inter-repeat H-bonding pattern and causes a significant conformational change in the relative disposition of the repeat subdomains. These changes are thought to be the reason for the enhanced on-rate of the mutated DARPin. The DARPin not bearing the residue 52 mutation has an unusually slow on-rate, suggesting that binding occurred via conformational selection of a relatively rare state, which was stabilized by this His52Tyr mutation, increasing the on-rate again to typical values. An analysis of the structural location of the framework mutations suggests that randomization of some framework residues either by error-prone PCR or by design in a future library could increase affinities and the target binding spectrum.

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  • Schweizer, A., Roschitzki-Voser, H., Amstutz, P., Briand, C., Gulotti-Georgieva, M., Prenosil, E., Binz, H. K., Capitani, G., Baici, A., Plückthun, A. and Grütter, M. G. (2007) Inhibition of caspase-2 by a designed ankyrin repeat protein: specificity, structure, and inhibition mechanism. Structure 15, 625-636.

      |  

    Abstract

    Specific and potent caspase inhibitors are indispensable for the dissection of the intricate pathways leading to apoptosis. We selected a designed ankyrin repeat protein (DARPin) from a combinatorial library that inhibits caspase-2 in vitro with a subnanomolar inhibition constant and, in contrast to the peptidic caspase inhibitors, with very high specificity for this particular caspase. The crystal structure of this inhibitor (AR_F8) in complex with caspase-2 reveals the molecular basis for the specificity and, together with kinetic analyses, the allosteric mechanism of inhibition. The structure also shows a conformation of the active site that can be exploited for the design of inhibitory compounds. AR_F8 is a specific inhibitor of an initiator caspase and has the potential to help identify the function of caspase-2 in the complex biological apoptotic signaling network.

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  • Zahnd, C., Amstutz, P. and Plückthun, A. (2007) Ribosome display: selecting and evolving proteins in vitro that specifically bind to a target. Nature Methods 4, 269-79.

      |  

    Abstract

    Ribosome display is an in vitro selection and evolution technology for proteins and peptides from large libraries. As it is performed entirely in vitro, there are two main advantages over other selection technologies. First, the diversity of the library is not limited by the transformation efficiency of bacterial cells, but only by the number of ribosomes and different mRNA molecules present in the test tube. Second, random mutations can be introduced easily after each selection round, as no library must be transformed after any diversification step. This allows facile directed evolution of binding proteins over several generations. A prerequisite for the selection of proteins from libraries is the coupling of genotype (RNA, DNA) and phenotype (protein). In ribosome display, this link is accomplished during in vitro translation by stabilizing the complex consisting of the ribosome, the mRNA and the nascent, correctly folded polypeptide. The DNA library coding for a particular library of binding proteins is genetically fused to a spacer sequence lacking a stop codon. This spacer sequence, when translated, is still attached to the peptidyl tRNA and occupies the ribosomal tunnel, and thus allows the protein of interest to protrude out of the ribosome and fold. The ribosomal complexes are allowed to bind to surface-immobilized target. Whereas non-bound complexes are washed away, mRNA of the complexes displaying a binding polypeptide can be recovered, and thus, the genetic information of the binding polypeptides is available for analysis. Here we describe a step-by-step procedure to perform ribosome display selection using an Escherichia coli S30 extract for in vitro translation, based on the work originally described and further refined in our laboratory. A protocol that makes use of eukaryotic in vitro translation systems for ribosome display is also included in this issue.

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  • Kolly, R., Thiel, M. A., Herrmann, T. and Plückthun, A. (2007) Monovalent antibody scFv fragments selected to modulate T-cell activation by inhibition of CD86-CD28 interaction. Protein Eng. Des. Sel. 20, 91-98.

      |  

    Abstract

    Beside the interaction of the antigen-presenting major histocompatibility complex with the T-cell receptor, a co-stimulatory signal is required for T-cell activation in an immune response. To reduce immune-mediated graft rejection in corneal transplantation, where topical application of drugs in ointments or eye-drops may be possible, we selected single-chain antibody fragments (scFv) with binding affinity to rat CD86 (B7.2) that inhibit the co-stimulatory signal. We produced the IgV-like domain of rat CD86 as a fusion protein in Escherichia coli by refolding from inclusion bodies. This protein was used as a target for phage display selection of scFv from HuCAL-1((R)), a fully artificial human antibody library. Selected binding molecules were shown to specifically bind to rat CD86 and inhibit the interaction of CD86 with CD28 and CTLA4 (CD152) in flow cytometry experiments. In an assay for CD86-dependent co-stimulation, the selected scFv fragment successfully inhibited the proliferation of T-cells induced by CD86-expressing P815 cells.

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  • Taussig, M. J., Stoevesandt, O., Borrebaeck, C. A., Bradbury, A. R., Cahill, D., Cambillau, C., de Daruvar, A., Dubel, S., Eichler, J., Frank, R., Gibson, T. J., Gloriam, D., Gold, L., Herberg, F. W., Hermjakob, H., Hoheisel, J. D., Joos, T. O., Kallioniemi, O., Koegl, M., Konthur, Z., Korn, B., Kremmer, E., Krobitsch, S., Landegren, U., van der Maarel, S., McCafferty, J., Muyldermans, S., Nygren, P. A., Palcy, S., Plückthun, A., Polic, B., Przybylski, M., Saviranta, P., Sawyer, A., Sherman, D. J., Skerra, A., Templin, M., Ueffing, M. and Uhlen, M. (2007) ProteomeBinders: planning a European resource of affinity reagents for analysis of the human proteome. Nature Methods 4, 13-7.

      |  

    Abstract

    ProteomeBinders is a new European consortium aiming to establish a comprehensive resource of well-characterized affinity reagents, including but not limited to antibodies, for analysis of the human proteome. Given the huge diversity of the proteome, the scale of the project is potentially immense but nevertheless feasible in the context of a pan-European or even worldwide coordination.

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2006

# Reference PDF
  • Kawe, M., Forrer, P., Amstutz, P. and Plückthun, A. (2006) Isolation of intracellular proteinase inhibitors derived from designed ankyrin repeat proteins by genetic screening. J. Biol. Chem. 281, 40252-63.

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    Abstract

    The specific intracellular inhibition of protein activity at the protein level is a highly valuable tool for the validation or modulation of cellular processes. We demonstrate here the use of designed ankyrin repeat proteins (DARPins) as tailor-made intracellular proteinase inhibitors. Site-specific proteolytic processing plays a critical role in the regulation of many biological processes, ranging from basic cellular functions to the propagation of viruses. The NIa(pro) proteinase of tobacco etch virus, a major plant pathogen, can be functionally expressed in Escherichia coli without harming the bacterium. To identify inhibitors of this proteinase, we first selected binders to it from combinatorial libraries of DARPins and tested this pool with a novel in vivo screen for proteinase inhibition. For this purpose, a hybrid protein consisting of the omega subunit of E. coli RNA polymerase was covalently fused to a DNA-binding protein, the lambdacI repressor, containing an NIa(pro) cleavage site in the linker between the two proteins. Thus, this transcriptional activator is inactivated by site-specific proteolytic cleavage, and inhibitors of this cleavage can be identified by the reconstitution of transcription of a reporter gene. Following this two-step approach of selection and screening, we could rapidly isolate NIa(pro) proteinase inhibitors active inside the cell from highly diverse combinatorial DARPin libraries. These findings underline the great potential of DARPins for modulation of protein functionality in the intracellular space. In addition, our novel genetic screen can help to select and identify tailor-made proteinase inhibitors based on other protein scaffolds or even on low molecular weight compounds.

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  • Hussain, S., Plückthun, A., Allen, T. M. and Zangemeister-Wittke, U. (2006) Chemosensitization of carcinoma cells using epithelial cell adhesion molecule-targeted liposomal antisense against bcl-2/bcl-xL. Mol. Cancer Ther. 5, 3170-3180.

      |  

    Abstract

    Nanoscale drug delivery systems, such as sterically stabilized immunoliposomes binding to internalizing tumor-associated antigens, can increase therapeutic efficacy and reduce toxicity to normal tissues compared with nontargeted liposomes. The epithelial cell adhesion molecule (EpCAM) is of interest as a ligand for targeted drug delivery because it is abundantly expressed in solid tumors but shows limited distribution in normal tissues. To generate EpCAM-specific immunoliposomes for targeted cancer therapy, the humanized single-chain Fv antibody fragment 4D5MOCB was covalently linked to the exterior of coated cationic liposomes. As anticancer agent, we encapsulated the previously described antisense oligonucleotide 4625 specific for both bcl-2 and bcl-xL. The EpCAM-targeted immunoliposomes (SIL25) showed specific binding to EpCAM-overexpressing tumor cells, with a 10- to 20-fold increase in binding compared with nontargeted control liposomes. No enhanced binding was observed on EpCAM-negative control cells. On cell binding, SIL25 was efficiently internalized by receptor-mediated endocytosis, ultimately leading to down-regulation of both bcl-2 and bcl-xL expression on both the mRNA and protein level, which resulted in enhanced tumor cell apoptosis. In combination experiments, the use of SIL25 led to a 2- to 5-fold sensitization of EpCAM-positive tumor cells of diverse origin to death induction by doxorubicin. Our data show the promise of EpCAM-specific drug delivery systems, such as antisense-loaded immunoliposomes, for targeted cancer therapy.

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  • Kubetzko, S., Balic, E., Waibel, R., Zangemeister-Wittke, U. and Plückthun, A. (2006) PEGylation and multimerization of the anti-p185HER-2 single chain Fv fragment 4D5: effects on tumor targeting. J. Biol. Chem. 281, 35186-35201.

      |  

    Abstract

    A major goal in antibody design for cancer therapy is to tailor the pharmacokinetic properties of the molecule according to specific treatment requirements. Key parameters determining the pharmacokinetics of therapeutic antibodies are target specificity, affinity, stability, and size. Using the p185HER-2 (HER-2)-specific scFv 4D5 as model system, we analyzed how changes in molecular weight and valency independently affect antigen binding and tumor localization. By employing multimerization and PEGylation, four different antibody formats were generated and compared with the scFv 4D5. First, dimeric and tetrameric miniantibodies were constructed by fusion of self-associating, disulfide-linked peptides to the scFv 4D5. Second, we attached a 20-kDa PEG moiety to the monovalent scFv and to the divalent miniantibody at the respective C terminus. In all formats, serum stability and full binding reactivity of the scFv 4D5 were retained. Functional affinity, however, did change. An avidity increase was achieved by multimerization, whereas PEGylation resulted in a 5-fold decreased affinity. Nevertheless, the PEGylated monomer showed an 8.5-fold, and the PEGylated dimer even a 14.5-fold higher tumor accumulation than the corresponding scFv, 48 h post-injection, because of a significantly longer serum half-life. In comparison, the non-PEGylated bivalent and tetravalent miniantibodies showed only a moderate increase in tumor localization compared with the scFv, which correlated with the degree of multimerization. However, these non-PEGylated formats resulted in higher tumor-to-blood ratios. Both multimerization and PEGylation represent thus useful strategies to tailor the pharmacokinetic properties of therapeutic antibodies and their combined use can additively improve tumor targeting.

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  • Zahnd, C., Pécorari, F., Straumann, N., Wyler, E. and Plückthun, A. (2006) Selection and characterization of Her2 binding-designed ankyrin repeat proteins. J. Biol. Chem. 281, 35167-35175.

      |  

    Abstract

    Designed ankyrin repeat proteins (DARPins) are a novel class of binding proteins that bind their target protein with high affinity and specificity and have very favorable expression and stability properties. We describe here the in vitro selection of DARPins against human epidermal growth factor receptor 2 (Her2), an important target for cancer therapy and diagnosis. Several DARPins bind to the same epitope as trastuzumab (Herceptin), but none were selected that bind to the epitope of pertuzumab (Omnitarg). Some of the selected DARPins bind with low nanomolar affinity (Kd=7.3 nm) to the target. Further analysis revealed that all DARPins are highly specific and do not cross-react with epidermal growth factor receptor I (EGFR1) or any other investigated protein. The selected DARPins specifically bind to strongly Her2-overexpressing cell lines such as SKBR-3 but also recognize small amounts of Her2 on weakly expressing cell lines such as MCF-7. Furthermore, the DARPins also lead to a highly specific and strong staining of plasma membranes of paraffinated sections of human mamma-carcinoma tissue. Thus, the selected DARPins might be used for the development of diagnostic tests for the status of Her2 overexpression in different adenocarcinomas, and they may be further evaluated for their potential in targeted therapy since their favorable expression properties make the construction of fusion proteins very convenient.

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  • Yu, H., Kohl, A., Binz, H. K., Plückthun, A., Grütter, M. G. and van Gunsteren, W. F. (2006) Molecular dynamics study of the stabilities of consensus designed ankyrin repeat proteins. Proteins 65, 285-295.

      |  

    Abstract

    Two designed ankyrin repeat (AR) proteins (E3_5 and E3_19) are high homologous (with about 87% sequence identity) and their crystal structures have a Calpha atom-positional root-mean-square difference of about 0.14 nm. However, it was found that E3_5 is considerably more stable than E3_19 in guanidinium hydrochloride and thermal denaturation experiments. With the goal of providing insights into the various factors contributing to the stabilities of the designed AR proteins and suggesting possible mutations to enhance their stabilities, homology modeling and molecular dynamics (MD) simulations with explicit solvent have been performed. Because the crystal structure of E3_19 was solved later than that of E3_5, a homology model of E3_19 based on the crystal structure of E3_5 was also used in the simulations. E3_5 shows a very stable trajectory in both crystal and solution simulations. In contrast, the C-terminal repeat of E3_19 unfolds in the simulations starting from either the modeled structure or the crystal structure, although it has a sequence identical to that of E3_5. A continuum electrostatic model was used to estimate the effect of single mutations on protein stability and to study the interaction between the internal ARs and the C-terminal capping AR. Mutations involving charged residues were found to have large effects on stability. Due to the difference in charge distribution in the internal ARs of E3_19 and E3_5, their interaction with the C-terminal capping AR is less favorable in E3_19. The simulation trajectories suggest that the stability of the designed AR proteins can be increased by optimizing the electrostatic interactions within and between the different repeats.

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  • Binz, H. K., Kohl, A., Plückthun, A. and Grütter, M. G. (2006) Crystal structure of a consensus-designed ankyrin repeat protein: implications for stability. Proteins 65, 280-284.

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    Abstract

    Consensus-designed ankyrin repeat (AR) proteins are thermodynamically very stable. The structural analysis of the designed AR protein E3_5 revealed that this stability is due to a regular fold with highly conserved structural motifs and H-bonding networks. However, the designed AR protein E3_19 exhibits a significantly lower stability than E3_5 (9.6 vs. 14.8 kcal/mol), despite 88% sequence identity. To investigate the structural correlations of this stability difference between E3_5 and E3_19, we determined the crystal structure of E3_19 at 1.9 A resolution. E3_19 as well has a regular AR domain fold with the characteristic H-bonding patterns. All structural features of the E3_5 and E3_19 molecules appear to be virtually identical (RMSD(Calpha) approximately 0.7 A). However, clear differences are observed in the surface charge distribution of the two AR proteins. E3_19 features clusters of charged residues and more exposed hydrophobic residues than E3_5. The atomic coordinates of E3_19 have been deposited in the Protein Data Bank. PDB ID: 2BKG.

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  • Luginbühl, B., Kanyo, Z., Jones, R. M., Fletterick, R. J., Prusiner, S. B., Cohen, F. E., Williamson, R. A., Burton, D. R. and Plückthun, A. (2006) Directed evolution of an anti-prion protein scFv fragment to an affinity of 1 pM and its structural interpretation. J. Mol. Biol. 363, 75-97.

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    Abstract

    Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative prion disease affecting cattle that is transmissible to humans, manifesting as a variant of Creutzfeldt-Jakob disease (vCJD) likely following the consumption of meat contaminated with BSE prions. High-affinity antibodies are a prerequisite for the development of simple, highly sensitive and non-invasive diagnostic tests that are able to detect even small amounts of the disease-associated PrP conformer (PrP(Sc)). We describe here the affinity maturation of a single-chain Fv antibody fragment with a binding affinity of 1 pM to a peptide derived from the unstructured region of bovine PrP (BoPrP (90-105)). This is the tightest peptide-binding antibody reported to date and may find useful application in diagnostics, especially when PrP(Sc) is pretreated by denaturation and/or proteolysis for peptide-like presentation. Several rounds of directed evolution and off-rate selection with ribosome display were performed using an antibody library generated from a single PrP binder with error-prone PCR and DNA-shuffling. As the correct determinations of affinities in this range are not straightforward, competition biosensor techniques and KinExA methods were both applied and compared. Structural interpretation of the affinity improvement was performed based on the crystal structure of the original prion binder in complex with the BoPrP (95-104) peptide by modeling the corresponding mutations.

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  • Steiner, D., Forrer, P., Stumpp, M. T. and Plückthun, A. (2006) Signal sequences directing cotranslational translocation expand the range of proteins amenable to phage display. Nature Biotechnol. 24, 823-31.

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    Abstract

    Even proteins that fold well in bacteria are frequently displayed poorly on filamentous phages. Low protein presentation on phage might be caused by premature cytoplasmic folding, leading to inefficient translocation into the periplasm. As translocation is an intermediate step in phage assembly, we tested the display levels of a range of proteins using different translocation pathways by employing different signal sequences. Directing proteins to the cotranslational signal recognition particle (SRP) translocation pathway resulted in much higher display levels than directing them to the conventional post-translational Sec translocation pathway. For example, the display levels of designed ankyrin-repeat proteins (DARPins) were improved up to 700-fold by simply exchanging Sec- for SRP-dependent signal sequences. In model experiments this exchange of signal sequences improved phage display from tenfold enrichment to >1,000-fold enrichment per phage display selection round. We named this method 'SRP phage display' and envision broad applicability, especially when displaying cDNA libraries or very stable and fast-folding proteins from libraries of alternative scaffolds.

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  • Bès, C., Troadec, S., Chentouf, M., Breton, H., Lajoix, A. D., Heitz, F., Gross, R., Plückthun, A. and Chardes, T. (2006) PIN-bodies: a new class of antibody-like proteins with CD4 specificity derived from the protein inhibitor of neuronal nitric oxide synthase. Biochem. Biophys. Res. Commun. 343, 334-344.

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    Abstract

    By inserting the CB1 paratope-derived peptide (PDP) from the anti-CD4 13B8.2 antibody binding pocket into each of the three exposed loops of the protein inhibitor of neuronal nitric oxide synthase (PIN), we have combined the anti-CD4 specificity of the selected PDP with the stability, ease of expression/purification, and the known molecular architecture of the phylogenetically well-conserved PIN scaffold protein. Such ""PIN-bodies"" were able to bind CD4 with a better affinity and specificity than the soluble PDP; additionally, in competitive ELISA experiments, CD4-specific PIN-bodies were more potent inhibitors of the binding of the parental recombinant antibody 13B8.2 to CD4 than the soluble PDP. The efficiency of CD4-specific CB1-inserted PIN-bodies was confirmed in biological assays where these constructs showed higher potencies to block antigen presentation by inhibition of IL-2 secretion and to inhibit the one-way and two-way mixed lymphocyte reactions, compared with soluble anti-CD4 PDP CB1. Insertion of the PDP into the first exposed loop (position 33/34) of PIN appeared to be the most promising scaffold. Taken together, our findings demonstrate that the PIN molecule is a suitable scaffold to expose new peptide loops and generate small artificial ligand-binding products with defined specificities.

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  • Amstutz, P., Binz, H. K., Zahnd, C. and Plückthun, A. (2006) Ribosome display: in vitro selection of protein-protein interactions in: Cell Biology – A laboratory handbook, 3rd ed. (Celis, J., ed) Vol. 1 pp. 497-509. Elsevier Academic Press

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    Abstract

    No abstract

    PDF
  • Amstutz, P., Koch, H., Binz, H. K., Deuber, S. A. and Plückthun, A. (2006) Rapid selection of specific MAP kinase-binders from designed ankyrin repeat protein libraries. Protein Eng. Des. Sel. 19, 219-229.

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    Abstract

    We describe here the rapid selection of specific MAP-kinase binders from a combinatorial library of designed ankyrin repeat proteins (DARPins). A combined in vitro/in vivo selection approach, based on ribosome display and the protein fragment complementation assay (PCA), yielded a large number of different binders that are fully functional in the cellular cytoplasm. Ribosome-display selection pools of four successive selection rounds were examined to monitor the enrichment of JNK2-specific DARPins. Surprisingly, only one round of ribosome display with subsequent PCA selection of this pool was necessary to isolate a first specific binder with micromolar affinity. After only two rounds of ribosome-display selection followed by PCA, virtually all DARPins showed JNK2-specific binding, with affinities in the low nanomolar range. The enrichment factor of ribosome display thus approaches 10(5) per round. In a second set of experiments, similar results were obtained with the kinases JNK1 and p38 as targets. Again, almost all investigated DARPins obtained after two rounds of ribosome display showed specific binding to the targets used, JNK1 or p38. In all three selection experiments the identified DARPins possess very high specificity for the target kinase. Taken together, the combination of ribosome display and PCA selections allowed the identification of large pools of binders at unparalleled speed. Furthermore, DARPins are applicable in intracellular selections and immunoprecipitations from the extract of eukaryotic cells.

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  • Koch, H., Grafe, N., Schiess, R. and Plückthun, A. (2006) Direct selection of antibodies from complex libraries with the protein fragment complementation assay. J. Mol. Biol. 357, 427-41.

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    Abstract

    The aim of the present study was to develop the protein fragment complementation assay (PCA) for the intracellular selection of specific binding molecules from the fully synthetic HuCAL antibody library. Here, we describe the first successful selections of specific antibodies by PCA, and we discuss the opportunities and limitations of this approach. First, we enriched an antibody specific for the capsid protein D of bacteriophage lambda (gpD) by ten successive rounds of competitive liquid culture selection. In an independent approach, we selected a specific antibody for the c-Jun N-terminal kinase 2 (JNK2) in a single-step selection setup. In order to obtain specific antibodies in only a single PCA selection round, the selection system was thoroughly investigated and several strategies to reduce the amount of false positives were evaluated. When expressed in the cytoplasm of Escherichia coli, the PCA-selected scFv antibody fragments could be purified as soluble and monomeric proteins. Denaturant-induced unfolding experiments showed that both antibody fragments are stable molecules, even when the disulfide bonds are reduced. Furthermore, antigen-specificity of the PCA-selected antibody fragments is demonstrated by in vivo and in vitro experiments. As antigen binding is retained regardless of the antibody redox state, both PCA-selected antibody fragments can tolerate the loss of disulfide bridge formation. Our results illustrate that it is possible to select well-expressed, stable, antigen-specific, and intracellular functional antibodies by PCA directly.

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  • Kawe, M. and Plückthun, A. (2006) GroEL walks the fine line: the subtle balance of substrate and co-chaperonin binding by GroEL. A combinatorial investigation by design, selection and screening. J. Mol. Biol. 357, 411-26.

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    Abstract

    While support in protein folding by molecular chaperones is extremely efficient for endogenous polypeptides, it often fails for recombinant proteins in a bacterial host, thus constituting a major hurdle for protein research and biotechnology. To understand the reasons for this difference and to answer the question of whether it is feasible to design tailor-made chaperones, we investigated one of the most prominent bacterial chaperones, the GroEL/ES ring complex. On the basis of structural data, we designed and constructed a combinatorial GroEL library, where the substrate-binding site was randomized. Screening and selection experiments with this library demonstrated that substrate binding and release is supported by many variants, but the majority of the library members failed to assist in chaperonin-mediated protein folding under conditions where spontaneous folding is suppressed. These findings revealed a conflict between binding of substrate and binding of the co-chaperonin GroES. As a consequence, the window of mutational freedom in that region of GroEL is very small. In screening experiments, we could identify GroEL variants slightly improved for a given substrate, which were still promiscuous. As the substrate-binding site of the GroEL molecule overlaps strongly with the site of cofactor binding, the outcome of our experiments suggests that maintenance of cofactor binding affinity is more critical for chaperonin-mediated protein folding than energetically optimized substrate recognition.

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  • Li, L., Wetzel, S., Plückthun, A. and Fernandez, J. M. (2006) Stepwise unfolding of ankyrin repeats in a single protein revealed by atomic force microscopy. Biophys. J. 90, L30-2.

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    Abstract

    Using single-molecule atomic force microscopy, we find that a protein consisting of six identical ankyrin repeat units flanked by N- and C-terminal modules (N6C) unfolds in a stepwise, unit-by-unit fashion under a mechanical force. Stretching a N6C molecule results in a sawtooth pattern fingerprint, with as many as six peaks separated by approximately 10 nm and an average unfolding force of 50 +/- 20 pN. Our results demonstrate that a stretching force can unfold multiple repeat units individually in a single protein molecule, despite extensive hydrophobic interactions between adjacent units.

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2005

# Reference PDF
  • Schaffitzel, C., Zahnd, C., Amstutz, P., Luginbühl, B. and Plückthun, A. (2005) In vitro selection and evolution of protein-ligand interactions by ribosome display in: Protein-Protein Interactions A Molecular Cloning Manual, 2nd ed. (Golemis, E., and Adams, P., eds) pp. 517-548. Cold Spring Harbor Laboratory Press, NY, New York

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    Abstract

    No abstract

    PDF
  • Kubetzko, S., Sarkar, C. A. and Plückthun, A. (2005) Protein PEGylation decreases observed target association rates via a dual blocking mechanism. Mol. Pharmacol. 68, 1439-1454.

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    Abstract

    PEGylation is an attractive strategy to enhance the therapeutic efficacy of proteins with a short serum half-life. It can be used to extend the serum persistence and to reduce the immunogenicity of proteins. However, PEGylation can also lead to a decrease in the functional activity of the molecule to which it is applied. We constructed site-specifically PEGylated variants of anti-p185(HER-2) antibody fragments in the format of a monovalent single-chain variable fragment and a divalent miniantibody and characterized the antigen binding properties in detail. Mass-transport limited BIAcore measurements and binding assays on HER-2-overexpressing cells demonstrated that the immunoreactivity of the antibody fragments is fully maintained after PEGylation. Nevertheless, we found that the attachment of a 20-kDa polyethylene glycol (PEG) moiety led to a reduction in apparent affinity of approximately 5-fold, although in both formats, the attachment site was most distal to the antigen binding regions. This decrease in affinity was observed in kinetic BIAcore measurements as well as in equilibrium binding assays on whole cells. By analysis of the binding kinetics, we could pinpoint this reduction exclusively to slower apparent on rates. Through both experimental and computational analyses, we demonstrate that these reduced on-rates do not arise from diffusion limitations. We show that a mathematical model accounting for both intramolecular and intermolecular blocking mechanisms of the PEG moiety can robustly explain the observed binding kinetics. The results suggest that PEGylation can significantly alter the binding-competent fraction of both ligands and receptors and may help to explain some of the beneficial effects of PEGylation in vivo.

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  • Plückthun, A. and Moroney, S. E. (2005) Modern antibody technology: The impact on drug development in: Modern Biopharmaceuticals (Knäblein, J., ed) Vol. 3 pp. 1147-1186. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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    Abstract

    Antibodies are now the mainstream of biopharmaceuticals. By the end of 2003. 17 marketed therapeutic antibodies generated over $5 billion in combined annual sales, with market growth at 30%. Ten years earlier this class of biopharmaceutical drugs was almost written off, based on disappointments experienced with the first generation of murine monoclonal antibodies. This chapter will look at how new technologies have provided solutions to problems that hampered early efforts to develop effective antibody therapeutics and transformed the market for antibody drugs. This includes the generation of fully human antibodies, their affinity maturation and the selection of antibodies to bind to particular epitopes on disease-relevant targets. The chapter will also highlight what distinguishes a therapeutic from a simple binding molecule - different modes of actions of antibodies in different molecular and cellular settings will be compared. Finally, some of the available formats of the antibody and their effect on molecular/pharmacological properties will be discussed.

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  • Binz, H. K., Amstutz, P. and Plückthun, A. (2005) Engineering novel binding proteins from nonimmunoglobulin domains. Nature Biotechnol. 23, 1257-1268.

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    Abstract

    Not all adaptive immune systems use the immunoglobulin fold as the basis for specific recognition molecules: sea lampreys, for example, have evolved an adaptive immune system that is based on leucine-rich repeat proteins. Additionally, many other proteins, not necessarily involved in adaptive immunity, mediate specific high-affinity interactions. Such alternatives to immunoglobulins represent attractive starting points for the design of novel binding molecules for research and clinical applications. Indeed, through progress and increased experience in library design and selection technologies, gained not least from working with synthetic antibody libraries, researchers have now exploited many of these novel scaffolds as tailor-made affinity reagents. Significant progress has been made not only in the basic science of generating specific binding molecules, but also in applications of the selected binders in laboratory procedures, proteomics, diagnostics and therapy. Challenges ahead include identifying applications where these novel proteins can not only be an alternative, but can enable approaches so far deemed technically impossible, and delineate those therapeutic applications commensurate with the molecular properties of the respective proteins.

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  • Backmann, N., Zahnd, C., Huber, F., Bietsch, A., Plückthun, A., Lang, H. P., Güntherodt, H. J., Hegner, M. and Gerber, C. (2005) A label-free immunosensor array using single-chain antibody fragments. Proc. Natl. Acad. Sci. U. S. A. 102, 14587-14592.

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    Abstract

    We report a microcantilever-based immunosensor operated in static deflection mode with a performance comparable with surface plasmon resonance, using single-chain Fv (scFv) antibody fragments as receptor molecules. As a model system scFv fragments with specificity to two different antigens were applied. We introduced a cysteine residue at the C terminus of each scFv construct to allow covalent attachment to gold-coated sensor interfaces in directed orientation. Application of an array enabled simultaneous deflection measurements of sensing and reference cantilevers. The differential deflection signal revealed specific antigen binding and was proportional to the antigen concentration in solution. Using small, oriented scFv fragments as receptor molecules we increased the sensitivity of microcantilevers to approximately 1 nM.

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  • Honegger, A., Spinelli, S., Cambillau, C. and Plückthun, A. (2005) A mutation designed to alter crystal packing permits structural analysis of a tight-binding fluorescein-scFv complex. Protein Sci. 14, 2537-2549.

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    Abstract

    The structure of the scFv fragment FITC-E2, obtained from a naive phage antibody scFv library derived from human donors, was determined at 2.1 A resolution in the free form and at 3.0 A in the complexed form. The wild-type (wt) scFv binds fluorescein with a K(D) of 0.75 nM. The free scFv readily crystallizes by compacting its 18 amino acid-long CDR-H3, partially occluding the binding site and further blocking access by binding to the ""bottom"" of a neighboring scFv molecule with a cluster of exposed aromatic residues within CDR-H3. Only upon mutating one of the residues involved in this dominant crystal contact, an exposed tryptophan in the middle of CDR-H3, crystals of the complex could be obtained. A series of alanine mutants within the putative antigen binding site, covering a range of binding affinities, were used to relate macroscopic thermodynamic and kinetic binding parameters to single-molecule disruption forces measured by AFM. The effects of the mutations on the binding properties, particularly on the fraction of binding-competent molecules within the population, cannot be fully explained by changes in the strength of local interactions. The significant conformational change of CDR-H3 between the free and the liganded form illustrates the plasticity of the binding site. An accompanying study in this issue by Curcio and colleagues presents the molecular dynamics simulation of the forced unbinding experiments and explores possible effects of the mutations on the unbinding pathway of the hapten.

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  • Schimmele, B. and Plückthun, A. (2005) Identification of a functional epitope of the Nogo receptor by a combinatorial approach using ribosome display. J. Mol. Biol. 352, 229-241.

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    Abstract

    The Nogo receptor (NgR) plays a central role in mediating growth-inhibitory activities of myelin-derived proteins, thereby severely limiting axonal regeneration after injury of the adult mammalian central nervous system (CNS). The inhibitory proteins Nogo, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp) all bind to the extracellular leucine-rich repeat (LRR) domain of NgR, which provides a large molecular surface for protein-protein interactions. However, epitopes within the LRR domain of NgR for binding Nogo, MAG and OMgp have not yet been revealed. Here, we report an evolutionary approach based on the ribosome display technology for detecting regions involved in ligand binding. By applying this method of ""affinity fingerprinting"" to the NgR ligand binding domain we were able to detect a distinct region important for binding to Nogo. Several residues defining the structural epitope of NgR involved in interaction with Nogo were subsequently confirmed by alanine scanning mutagenesis.

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  • Kohl, A., Amstutz, P., Parizek, P., Binz, H. K., Briand, C., Capitani, G., Forrer, P., Plückthun, A. and Grütter, M. G. (2005) Allosteric inhibition of aminoglycoside phosphotransferase by a designed ankyrin repeat protein. Structure 13, 1131-1141.

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    Abstract

    Aminoglycoside phosphotransferase (3')-IIIa (APH) is a bacterial kinase that confers antibiotic resistance to many pathogenic bacteria and shares structural homology with eukaryotic protein kinases. We report here the crystal structure of APH, trapped in an inactive conformation by a tailor-made inhibitory ankyrin repeat (AR) protein, at 2.15 A resolution. The inhibitor was selected from a combinatorial library of designed AR proteins. The AR protein binds the C-terminal lobe of APH and thereby stabilizes three alpha helices, which are necessary for substrate binding, in a significantly displaced conformation. BIAcore analysis and kinetic enzyme inhibition experiments are consistent with the proposed allosteric inhibition mechanism. In contrast to most small-molecule kinase inhibitors, the AR proteins are not restricted to active site binding, allowing for higher specificity. Inactive conformations of pharmaceutically relevant enzymes, as can be elucidated with the approach presented here, represent powerful starting points for rational drug design.

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  • Binz, H. K. and Plückthun, A. (2005) Engineered proteins as specific binding reagents. Curr. Opin. Biotechnol. 16, 459-69.

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    Abstract

    Over the past 30 years, monoclonal antibodies have become the standard binding proteins and currently find applications in research, diagnostics and therapy. Yet, monoclonal antibodies now face strong competition from synthetic antibody libraries in combination with powerful library selection technologies. More recently, an increased understanding of other natural binding proteins together with advances in protein engineering, selection and evolution technologies has also triggered the exploration of numerous other protein architectures for the generation of designed binding molecules. Valuable protein-binding scaffolds have been obtained and represent promising alternatives to antibodies for biotechnological and, potentially, clinical applications.

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  • Amstutz, P., Binz, H. K., Parizek, P., Stumpp, M. T., Kohl, A., Grütter, M. G., Forrer, P. and Plückthun, A. (2005) Intracellular kinase inhibitors selected from combinatorial libraries of designed ankyrin repeat proteins. J. Biol. Chem. 280, 24715-24722.

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    Abstract

    The specific intracellular inhibition of protein activity at the protein level allows the determination of protein function in the cellular context. We demonstrate here the use of designed ankyrin repeat proteins as tailor-made intracellular kinase inhibitors. The target was aminoglycoside phosphotransferase (3')-IIIa (APH), which mediates resistance to aminoglycoside antibiotics in pathogenic bacteria and shares structural homology with eukaryotic protein kinases. Combining a selection and screening approach, we isolated 198 potential APH inhibitors from highly diverse combinatorial libraries of designed ankyrin repeat proteins. A detailed analysis of several inhibitors revealed that they bind APH with high specificity and with affinities down to the subnanomolar range. In vitro, the most potent inhibitors showed complete enzyme inhibition, and in vivo, a phenotype comparable with the gene knockout was observed, fully restoring antibiotic sensitivity in resistant bacteria. These results underline the great potential of designed ankyrin repeat proteins for modulation of intracellular protein function.

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  • Schimmele, B., Gräfe, N. and Plückthun, A. (2005) Ribosome display of mammalian receptor domains. Protein Eng. Des. Sel. 18, 285-294.

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    Abstract

    Many mammalian receptor domains, among them a large number of potential therapeutic target proteins, are highly aggregation-prone upon heterologous expression in bacteria. This severely limits functional studies of such receptor domains and also their engineering towards improved properties. One of these proteins is the Nogoreceptor, which plays a central role in mediating the inhibition of axon growth and functional recovery after injury of the adult mammalian central nervous system. We show here that the ligand binding domain of the Nogoreceptor folds to an active conformation in ternary ribosomal complexes, as formed in ribosome display. In these complexes the receptor is still connected, via a C-terminal tether, to the peptidyl tRNA in the ribosome and the mRNA also stays connected. The ribosome prevents aggregation of the protein, which aggregates as soon as the release from the ribosome is triggered. In contrast, no active receptor was observed in phage display, where aggregation appears to prevent incorporation of the protein into the phage coat. This strategy sets the stage for rapidly studying defined mutations of such aggregation-prone receptors in vitro and to improve their properties by in vitro evolution using the ribosome display technology.