Publications

2024

# Reference PDF

0540

Vukovic, D., Winkelvoß, D., Kapp, J. N., Hänny, A.-C., Bürgisser, H., Riermeier, L., Udovcic, A., Tiefenboeck, P. and Plückthun, A. (2024). Protein degradation kinetics measured by microinjection and live-cell fluorescence microscopy. Sci. Rep. 14, 27153.
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Abstract

We have developed a method combining microinjection and automated fluorescence microscopy to continuously assess the degradation rate, subcellular localization and intracellular concentration of protein analytes at the single-cell level. Cells are unperturbed and grown in unaltered environmental conditions and show high viability. The injection of analytes at defined ratios and concentrations allows for a clearly defined starting point of degradation, without the entanglement of biosynthesis/uptake, often encountered in existing methods. The possibility to evaluate, add, or remove post-translational modifications prior to injection represents a powerful tool to assess minute protein degradation rate changes with high precision and allowed us to determine the absolute degradation rates caused by N-degron pathway engagement, with a focus on the role of acetylation. The low degradation rate of eGFP was found to be caused by inefficient N-terminal proteasomal unfolding. We moreover quantified the surprisingly strong influences of commonly used peptide tags and detected high variation between fluorescent proteins with regard to both protein degradation and subcellular localization. Furthermore, we have validated the use of chemically coupled dyes as robust reporters for protein degradation, and elucidated the significance of their membrane-permeability, thereby extending the applicability of our method to any protein of interest.

DOI: 10.1038/s41598-024-76224-0 | Read all as PDF | SUPPL: 0540 Video1 0540 Video2 0540 Explanation videos 540 | Close Abstract
0539

Zhou, Z., Nguyen, T. L., Li, X, Poujol, C., Berlinska, E., Michelina, S. V., Kapp, J. N., Plückthun, A., Winslow, M. M., Ambrogio, C., Shan, Y., Santamaría, D., Westover, K. D. (2024). Experimental variables determine the outcome of RAS-RAS interactions. J. Biol. Chem. 300, 107859.

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Abstract

RAS clustering at the cell membrane is critical to activate signaling in cells, but whether this clustering is mediated exclusively by its c-terminal hypervariable region, receives contributions from the G-domain of RAS, and/or is influenced by secondary effectors has been intensely debated. Reports that G-domain mutations do not modulate RAS-RAS interactions, have led some to question the validity of previous experiments that indicate the G-domain plays a role in RAS clustering/interactions. Here we reconcile these findings by clarifying the impact of experimental variables, such as protein expression levels, cellular context, RAS zygosity, and secondary effector interactions on RAS clustering. Lack of control over these variables impact the results using G-domain mutations across various assay systems and can lead to unsound conclusions.

DOI: 10.1016/j.jbc.2024.107859 | Read all as PDF | SUPPL: 0539 | Close Abstract
0538

Mahran, R., Kapp, J. N., Valtonen, S., Champagne, A., Ning, J., Gillette, W., Stephen, A. G., Hao, F., Plückthun, A., Härmä, H., Pantsar, T., and Kopra, K. (2924). Beyond KRAS(G12C): biochemical and computational characterization of sotorasib and adagrasib binding specificity and the critical role of H95 and Y96. ACS Chem. Biol. 19, 2152−2164.
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Abstract

Mutated KRAS proteins are frequently expressed in some of the most lethal human cancers and thus have been a target of intensive drug discovery efforts for decades. Lately, KRAS(G12C) switch-II pocket (SII-P)-targeting covalent small molecule inhibitors have finally reached clinical practice. Sotorasib (AMG-510) was the first FDA-approved covalent inhibitor to treat KRAS(G12C)-positive nonsmall cell lung cancer (NSCLC), followed soon by adagrasib (MRTX849). Both drugs target the GDP-bound state of KRAS(G12C), exploiting the strong nucleophilicity of acquired cysteine. Here, we evaluate the similarities and differences between sotorasib and adagrasib in their RAS SII-P binding by applying biochemical, cellular, and computational methods. Exact knowledge of SII-P engagement can enable targeting this site by reversible inhibitors for KRAS mutants beyond G12C. We show that adagrasib is strictly KRAS- but not KRAS(G12C)-specific due to its strong and unreplaceable interaction with H95. Unlike adagrasib, sotorasib is less dependent on H95 for its binding, making it a RAS isoform-agnostic compound, having a similar functionality also with NRAS and HRAS G12C mutants. Our results emphasize the accessibility of SII-P beyond oncogenic G12C and aid in understanding the molecular mechanism behind the clinically observed drug resistance, associated especially with secondary mutations on KRAS H95 and Y96.

DOI: 10.1021/acschembio.4c00315 | Read all as PDF | SUPPL: 0538 | Close Abstract
0537

Freitag, P. C., Kolibius, J., Wieboldt, R., Weber, R., Hartmann, K. P., van Gogh, M., Brücher, D., Läubli, H., and Plückthun, A. (2024). DARPin-fused T cell engager for adenovirus-mediated cancer therapy. Mol. Therapy Oncology 32, 200821.
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Abstract

Abstract: Bispecific T cell engagers are a promising class of therapeutic proteins for cancer therapy. Their potency and small size often come with systemic toxicity and short half-life, making intravenous administration cumbersome. These limitations can be overcome by tumor-specific in situ expression, allowing high local accumulation while reducing systemic concentrations. However, encoding T cell engagers in viral or non-viral vectors and expressing them in situ ablates all forms of quality control performed during recombinant protein production. It is therefore vital to design constructs that feature minimal domain mispairing, and increased homogeneity of the therapeutic product. Here, we report a T cell engager architecture specifically designed for vector-mediated immunotherapy. It is based on a fusion of a designed ankyrin repeat protein (DARPin) to a CD3-targeting single-chain antibody fragment, termed DATE (DARPin-fused T cell Engager). The DATE induces potent T cell-mediated killing of HER2+ cancer cells, both as recombinantly produced therapeutic protein and as in situ expressed payload from a HER2+-retargeted high-capacity adenoviral vector (HC-AdV). We report remarkable tumor remission, DATE accumulation, and T cell infiltration through in situ expression mediated by a HER2+-retargeted HC-AdV in vivo. Our results support further investigations and developments of DATEs as payloads for vector-mediated immunotherapy.

DOI: 10.1016/j.omton.2024.200821 | Read all as PDF | SUPPL: 0537 | Close Abstract
0536

Stark, Y., Menard, F., Jeliazkov, J. R., Ernst, P., Chembath, A., Ashraf, M., Hine, A. V., and Plückthun, A. (2024). Modular binder technology by NGS-aided, high-resolution selection in yeast of designed armadillo modules. Proc. Natl. Acad. Sci. USA 121, e2318198121
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Abstract

Establishing modular binders as diagnostic detection agents represents a cost- and time-efficient alternative to the commonly used binders that are generated one molecule at a time. In contrast to these conventional approaches, a modular binder can be designed in silico from individual modules to, in principle, recognize any desired linear epitope without going through a selection and hit-validation process, given a set of preexisting, amino acid–specific modules. Designed armadillo repeat proteins (dArmRP) have been developed as modular binder scaffolds, and we report here the generation of highly specific dArmRP modules by yeast surface display selection, performed on a rationally designed dArmRP library. A selection strategy was developed to distinguish the binding difference resulting from a single amino acid mutation in the target peptide. Our reverse-competitor strategy introduced here employs the designated target as a competitor to increase the sensitivity when separating specific from cross-reactive binders that show similar affinities for the target peptide. With this switch in selection focus from affinity to specificity, we found that the enrichment during this specificity sort is indicative of the desired phenotype, regardless of the binder abundance. Hence, deep sequencing of the selection pools allows retrieval of phenotypic hits with only 0.1% abundance in the selectivity sort pool from the next-generation sequencing data alone. In a proof-of-principle study, a binder was created by replacing all corresponding wild-type modules with a newly selected module, yielding a binder with very high affinity for the designated target that has been successfully validated as a detection agent in western blot analysis.

DOI: 10.1073/pnas.2318198121 | Read all as PDF | SUPPL: 0536 | Close Abstract
0535

Dederer, V., Sanz Murillo, M., Karasmanis, E. P., Hatch, K. S., Chatterjee, D., Preuss, F., Abdul Azeez, K. .R, Vu Nguyen, L., Galicia, C., Dreier, B., Plückthun, A., Versees, W., Mathea, S., Leschziner, A. E., Reck-Peterson, S. L., and Knapp, S. (2024). A designed ankyrin-repeat protein that targets Parkinson’s disease-associated LRRK2. J. Biol. Chem. 300, 107469.
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Abstract

Leucine rich repeat kinase 2 (LRRK2) is a large multidomain protein containing two catalytic domains, a kinase and a GTPase, as well as protein interactions domains, including a WD40 domain. The association of increased LRRK2 kinase activity with both the familial and sporadic forms of Parkinson’s disease (PD) has led to intense interest in determining its cellular function. However, small molecule probes that can bind to LRRK2 and report on or affect its cellular activity are needed. Here, we report the identification and characterization of the first high-affinity LRRK2-binding designed ankyrin-repeat protein (DARPin), named E11. Using cryo-EM, we show that DARPin E11 binds to the LRRK2 WD40 domain. LRRK2 bound to DARPin E11 showed improved behavior on cryo-EM grids, resulting in higher resolution LRRK2 structures. DARPin E11 did not affect the catalytic activity of a truncated form of LRRK2 in vitro but decreased the phosphorylation of Rab8A, a LRRK2 substrate, in cells. We also found that DARPin E11 disrupts the formation of microtubule-associated LRRK2 filaments in cells, which are known to require WD40-based dimerization. Thus, DARPin E11 is a new tool to explore the function and dysfunction of LRRK2 and guide the development of LRRK2 kinase inhibitors that target the WD40 domain instead of the kinase.

DOI: 10.1016/ j.jbc.2024.107469 | Read all as PDF | SUPPL: 0535 | Close Abstract
0534

Cui, Z., Ayva, C. E., Liew, Y. J., Guo, Z., Mutschler, R., Dreier, B., Fiorito, M. M., Walden, P., Howard, C. B., Ely, F., Plückthun, A., Pretorius, C., Ungerer, J. P., Buckle, A. M., and Alexandrov, K. (2024). mRNA display pipeline for protein biosensor construction. ACS Sens. 9, 2846−2857.

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Abstract

Despite the significant potential of protein biosensors, their construction remains a trial-and-error process. The most obvious approach for addressing this is to utilize modular biosensor architectures where specificity-conferring modalities can be readily generated to recognize new targets. Toward this goal, we established a workflow that uses mRNA display-based selection of hyper-stable monobody domains for the target of choice or ribosome display to select equally stable DARPins. These binders were integrated into a two-component allosteric biosensor architecture based on a calmodulin-reporter chimera. This workflow was tested by developing biosensors for liver toxicity markers such as cytosolic aspartate aminotransferase, mitochondrial aspartate aminotransferase, and alanine aminotransferase 1. We demonstrate that our pipeline consistently produced >10^3 unique binders for each target within a week. Our analysis revealed that the affinity of the binders for their targets was not a direct predictor of the binder's performance in a biosensor context. The interactions between the binding domains and the reporter module affect the biosensor activity and the dynamic range. We conclude that following binding domain selection, the multiplexed biosensor assembly and prototyping appear to be the most promising approach for identifying biosensors with the desired properties.

DOI: 10.1021/acssensors.3c02471 | Read all as PDF | SUPPL: 0534 | Close Abstract
0533

Auger, S. A., Venkatachalapathy, S., Suazo, K. F. G., Wang, Y., Sarkis, A. W., Bernhagen, K., Justyna, K., Schaefer, J. V., Wollack, J. W., Plückthun, A., Li, L., and Distefano, M. D. (2024). Broadening the utility of farnesyltransferase-catalyzed protein labeling using norbornene–tetrazine click chemistry. Bioconjugate Chem. 35, 922-933.
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Abstract

Bioorthogonal chemistry has gained widespread use in the study of many biological systems of interest, including protein prenylation. Prenylation is a post-translational modification, in which one or two 15- or 20-carbon isoprenoid chains are transferred onto cysteine residues near the C-terminus of a target protein. The three main enzymes─protein farnesyltransferase (FTase), geranylgeranyl transferase I (GGTase I), and geranylgeranyl transferase II (GGTase II)─that catalyze this process have been shown to tolerate numerous structural modifications in the isoprenoid substrate. This feature has previously been exploited to transfer an array of farnesyl diphosphate analogues with a range of functionalities, including an alkyne-containing analogue for copper-catalyzed bioconjugation reactions. Reported here is the synthesis of an analogue of the isoprenoid substrate embedded with norbornene functionality (C10NorOPP) that can be used for an array of applications, ranging from metabolic labeling to selective protein modification. The probe was synthesized in seven steps with an overall yield of 7% and underwent an inverse electron demand Diels–Alder (IEDDA) reaction with tetrazine-containing tags, allowing for copper-free labeling of proteins. The use of C10NorOPP for the study of prenylation was explored in the metabolic labeling of prenylated proteins in HeLa, COS-7, and astrocyte cells. Furthermore, in HeLa cells, these modified prenylated proteins were identified and quantified using label-free quantification (LFQ) proteomics with 25 enriched prenylated proteins. Additionally, the unique chemistry of C10NorOPP was utilized for the construction of a multiprotein–polymer conjugate for the targeted labeling of cancer cells. That construct was prepared using a combination of norbornene–tetrazine conjugation and azide–alkyne cycloaddition, highlighting the utility of the additional degree of orthogonality for the facile assembly of new protein conjugates with novel structures and functions.

DOI: 10.1021/acs.bioconjchem.4c00072 | Read all as PDF | SUPPL: 0533 | Close Abstract
0532

Ivanova, J. R., Benk, A. S., Schaefer, J. V., Dreier, B., Hermann, L. O., Plückthun, A., Missirlis, D., and Spatz, J. P. (2024). Designed ankyrin repeat proteins as actin labels of distinct cytoskeletal structures in living cells. ACS Nano 18, 8919-8933.
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Abstract

The orchestrated assembly of actin and actin-binding proteins into cytoskeletal structures coordinates cell morphology changes during migration, cytokinesis, and adaptation to external stimuli. The accurate and unbiased visualization of the diverse actin assemblies within cells is an ongoing challenge. We describe here the identification and use of designed ankyrin repeat proteins (DARPins) as synthetic actin binders. Actin-binding DARPins were identified through ribosome display and validated biochemically. When introduced or expressed inside living cells, fluorescently labeled DARPins accumulated at actin filaments, validated through phalloidin colocalization on fixed cells. Nevertheless, different DARPins displayed different actin labeling patterns: some DARPins labeled efficiently dynamic structures, such as filopodia, lamellipodia, and blebs, while others accumulated primarily in stress fibers. This differential intracellular distribution correlated with DARPin–actin binding kinetics, as measured by fluorescence recovery after photobleaching experiments. Moreover, the rapid arrest of actin dynamics induced by pharmacological treatment led to the fast relocalization of DARPins. Our data support the hypothesis that the localization of actin probes depends on the inherent dynamic movement of the actin cytoskeleton. Compared to the widely used LifeAct probe, one DARPin exhibited enhanced signal-to-background ratio while retaining a similar ability to label stress fibers. In summary, we propose DARPins as promising actin-binding proteins for labeling or manipulation in living cells.

DOI: 10.1021/acsnano.3c12265 | Read all as PDF | SUPPL: 0532 Movie S1 0532 Movie S2 0532 Movie S3 0532 Movie S4 0532 Movie S5 0532 Movie S6 0532 | Close Abstract
0531

Thalmann, L., Martin-Gonzalez, N., Brücher, D., Plückthun, A., de Pablo, P., Suomalainen, M., and Greber, U. (2024). Gutless helper-dependent and first generation HAdV5 vectors have similar mechanical properties and common transduction mechanisms. Hum. Gene Ther. 35, 163-176.
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Abstract

Delivering vectorized information into cells with the help of viruses has been of high interest to fundamental and applied science, and bears significant therapeutic promise. Human adenoviruses (HAdVs) have been at the forefront of gene delivery for many years, and the subject of intensive development resulting in several generations of agents, including replication-competent, -defective or retargeted vectors, and recently also helper-dependent (HD), so-called gutless vectors lacking any viral protein coding information. While it is possible to produce HD-AdVs in significant amounts, physical properties of these virus-like particles and their efficiency of transduction have not been addressed. Here we used single-cell and single virus particle assays to probe the effect of genome length on HadV-C5 vector transduction. Our results demonstrate that first generation C5 vectors lacking the E1/E3 regions of the viral genome as well as HD-AdV-C5 particles with a wild type ~36 kbp or an undersized double-strand DNA genome are similar to HAdV-C5 wild-type regarding attachment to human lung epithelial cells, endocytic uptake, endosome penetration and dependency on the E3 RING ubiquitin ligase Mind Bomb 1 for DNA uncoating at the nuclear pore complex. Atomic force microscopy measurements of single virus particles indicated that small changes in the genome length from 94-103 percent of HAdV-C5 have no major impact on physical and mechanical features of AdV vectors. In contrast, an HD-AdV-C5 with ~30 kbp genome was slightly stiffer and less heat-resistant than the other particles, despite comparable entry and transduction efficiencies in tissue culture cell lines, including murine alveolar macrophage-like MPI-2 cells. Together, our in vitro studies reinforce the use of HD-AdV vectors for effective single round gene delivery. The results illustrate how physical properties and cell entry behaviour of single virus particles can provide functional information for anticipated therapeutic vector applications.

DOI: 10.1089/hum.2023.221 | Read all as PDF | Close Abstract
0530

Abdul-Ridha, A., de Zhang, L. A., Betrie, A. H., Deluigi, M., Vaid, T. M., Whitehead, A., Zhang, Y., Davis, B., Harris, R., Simmonite, H., Hubbard, R. E., Gooley, P. R., Plückthun, A., Bathgate, R. A. D., Chalmers, D. K., and Scott, D. J. (2024). Identification of a novel subtype-selective α 1B -adrenoceptor antagonist. ACS Chem. Neuroscience 15, 671–684.
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Abstract

α1A-, α1B-, and α1D-adrenoceptors (α1-ARs) are members of the adrenoceptor G protein-coupled receptor family that are activated by adrenaline (epinephrine) and noradrenaline. α1-ARs are clinically targeted using antagonists that have minimal subtype selectivity, such as prazosin and tamsulosin, to treat hypertension and benign prostatic hyperplasia, respectively. Abundant expression of α1-ARs in the heart and central nervous system (CNS) makes these receptors potential targets for the treatment of cardiovascular and CNS disorders, such as heart failure, epilepsy, and Alzheimer’s disease. Our understanding of the precise physiological roles of α1-ARs, however, and their involvement in disease has been hindered by the lack of sufficiently subtype-selective tool compounds, especially for α1B-AR. Here, we report the discovery of 4-[(2-hydroxyethyl)amino]-6-methyl-2H-chromen-2-one (Cpd1), as an α1B-AR antagonist that has 10–15-fold selectivity over α1A-AR and α1D-AR. Through computational and site-directed mutagenesis studies, we have identified the binding site of Cpd1 in α1B-AR and propose the molecular basis of α1B-AR selectivity, where the nonconserved V19745.52 residue plays a major role, with contributions from L3146.55 within the α1B-AR pocket. By exploring the structure–activity relationships of Cpd1 at α1B-AR, we have also identified 3-[(cyclohexylamino)methyl]-6-methylquinolin-2(1H)-one (Cpd24), which has a stronger binding affinity than Cpd1, albeit with reduced selectivity for α1B-AR. Cpd1 and Cpd24 represent potential leads for α1B-AR-selective drug discovery and novel tool molecules to further study the physiology of α1-ARs.

DOI: 10.1021/acschemneuro.3c00767 | Read all as PDF | SUPPL: 0530 | Close Abstract

2023

# Reference PDF

0529

Leka, O., Wu, Y., Zanetti, G., Furler, F., Reinberg, T., Marinho, J., Schaefer, J. V., Plückthun, A., Li, X., Pirazzini, M., and Kammerer, R. A. (2023). A DARPin promotes faster onset of botulinum neurotoxin A1 action. Nature Commun. 14, 8317.
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Abstract

In this study, we characterize Designed Ankyrin Repeat Proteins (DARPins) as investigative tools to probe botulinum neurotoxin A1 (BoNT/A1) structure and function. We identify DARPin-F5 that completely blocks SNAP25 substrate cleavage by BoNT/A1 in vitro. X-ray crystallography reveals that DARPin-F5 inhibits BoNT/A1 activity by interacting with a substrate-binding region between the α- and β-exosite. This DARPin does not block substrate cleavage of BoNT/A3, indicating that DARPin-F5 is a subtype-specific inhibitor. BoNT/A1 Glu-171 plays a critical role in the interaction with DARPin-F5 and its mutation to Asp, the residue found in BoNT/A3, results in a loss of inhibition of substrate cleavage. In contrast to the in vitro results, DARPin-F5 promotes faster substrate cleavage of BoNT/A1 in primary neurons and muscle tissue by increasing toxin translocation. Our findings could have important implications for the application of BoNT/A1 in therapeutic areas requiring faster onset of toxin action combined with long persistence.

DOI: 10.1038/s41467-023-44102-4 | Read all as PDF | SUPPL: 0529 | Close Abstract
0528

Cucuzza, S., Sitnik, M., Jurt, S., Michel, E., Dai, W., Müntener, T., Ernst, P., Häussinger, D., Plückthun, A., and Zerbe, O. (2023). Unexpected dynamics in femtomolar complexes of binding proteins with peptides. Nature Commun. 14, 7823.
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Abstract

Ultra-tight binding is usually observed for proteins associating with rigidified molecules. Previously, we demonstrated that femtomolar binders derived from the Armadillo repeat proteins (ArmRPs) can be designed to interact very tightly with fully flexible peptides. Here we show for ArmRPs with four and seven sequence-identical internal repeats that the peptide-ArmRP complexes display conformational dynamics. These dynamics stem from transient breakages of individual protein-residue contacts that are unrelated to overall unbinding. The labile contacts involve electrostatic interactions. We speculate that these dynamics allow attaining very high binding affinities, since they reduce entropic losses. Importantly, only NMR techniques can pick up these local events by directly detecting conformational exchange processes without complications from changes in solvent entropy. Furthermore, we demonstrate that the interaction surface of the repeat protein regularizes upon peptide binding to become more compatible with the peptide geometry. These results provide novel design principles for ultra-tight binders.

DOI: 10.1038/s41467-023-43596-2 | Read all as PDF | SUPPL: 0528 | Close Abstract
0527

Strubel, A., Münick, P., Hartmann, O., Chaikuad, A., Dreier, B., Schaefer, J. V., Gebel, J., Osterburg, C., Tuppi, M., Schäfer, B., Buck, V., Rosenfeldt, M., Knapp, S., Plückthun, A., Diefenbacher, M. E., and Dötsch, V. (2023). DARPins detect the formation of hetero-tetramers of p63 and p73 in epithelial tissues and in squamous cell carcinoma. Cell Death Dis. 14, 674.
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Abstract

The two p53 homologues p63 and p73 regulate transcriptional programs in epithelial tissues and several cell types in these tissues express both proteins. All members of the p53 family form tetramers in their active state through a dedicated oligomerization domain that structurally assembles as a dimer of dimers. The oligomerization domain of p63 and p73 share a high sequence identity, but the p53 oligomerization domain is more divergent and it lacks a functionally important C-terminal helix present in the other two family members. Based on these structural differences, p53 does not hetero-oligomerize with p63 or p73. In contrast, p63 and p73 form hetero-oligomers of all possible stoichiometries, with the hetero-tetramer built from a p63 dimer and a p73 dimer being thermodynamically more stable than the two homo-tetramers. This predicts that in cells expressing both proteins a p632/p732 hetero-tetramer is formed. So far, the tools to investigate the biological function of this hetero-tetramer have been missing. Here we report the generation and characterization of Designed Ankyrin Repeat Proteins (DARPins) that bind with high affinity and selectivity to the p632/p732 hetero-tetramer. Using these DARPins we were able to confirm experimentally the existence of this hetero-tetramer in epithelial mouse and human tissues and show that its level increases in squamous cell carcinoma.

DOI: 10.1038/s41419-023-06213-0 | Read all as PDF | SUPPL: 0527 | Close Abstract
0526

Baumann, C., Chiang, W. C., Valsecchi, R., Jurt, S., Deluigi, M., Schuster, M., Rosengren, K. J,, Plückthun, A., and Zerbe, O. (2023). Side-chain dynamics of the α_1B -adrenergic receptor determined by NMR via methyl relaxation. Protein Sci. 32, e4801.
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Abstract

G protein-coupled receptors (GPCRs) are medically important membrane proteins that sample inactive, intermediate, and active conformational states characterized by relatively slow interconversions (~μs-ms). On a faster timescale (~ps-ns), the conformational landscape of GPCRs is governed by the rapid dynamics of amino acid side chains. Such dynamics are essential for protein functions such as ligand recognition and allostery. Unfortunately, technical challenges have almost entirely precluded the study of side-chain dynamics for GPCRs. Here, we investigate the rapid side-chain dynamics of a thermostabilized α1B -adrenergic receptor (α1B -AR) as probed by methyl relaxation. We determined order parameters for Ile, Leu, and Val methyl groups in the presence of inverse agonists that bind orthosterically (prazosin, tamsulosin) or allosterically (conopeptide ρ-TIA). Despite the differences in the ligands, the receptor's overall side-chain dynamics are very similar, including those of the apo form. However, ρ-TIA increases the flexibility of Ile1764x56 and possibly of Ile2145x49 , adjacent to Pro2155x50 of the highly conserved P5x50 I3x40 F6x44 motif crucial for receptor activation, suggesting differences in the mechanisms for orthosteric and allosteric receptor inactivation. Overall, increased Ile side-chain rigidity was found for residues closer to the center of the membrane bilayer, correlating with denser packing and lower protein surface exposure. In contrast to two microbial membrane proteins, in α1B -AR Leu exhibited higher flexibility than Ile side chains on average, correlating with the presence of Leu in less densely packed areas and with higher protein-surface exposure than Ile. Our findings demonstrate the feasibility of studying receptor-wide side-chain dynamics in GPCRs to gain functional insights.

DOI: 10.1002/pro.4801 | Read all as PDF | SUPPL: 0526 A 0526 B | Close Abstract
0525

Maliqi, L., Friedrich, N., Glögl, M., Schmutz, S., Schmidt, D., Rusert, P., Schanz, M., Zaheri, M., Pasin, C., Niklaus, C., Foulkes, C., Reinberg, T., Dreier, B., Abela, I., Peterhoff, D., Hauser, A., Kouyos, R. D., Günthard, H. F., van Gils, M. J., Sanders, R. W., Wagner, R., Plückthun, A., and Trkola, A. (2023). Assessing immunogenicity barriers of the HIV-1 envelope trimer. npj Vaccines 8, 148.
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Abstract

Understanding the balance between epitope shielding and accessibility on HIV-1 envelope (Env) trimers is essential to guide immunogen selection for broadly neutralizing antibody (bnAb) based vaccines. To investigate the antigenic space of Env immunogens, we created a strategy based on synthetic, high diversity, Designed Ankyrin Repeat Protein (DARPin) libraries. We show that DARPin Antigenicity Analysis (DANA), a purely in vitro screening tool, has the capability to extrapolate relevant information of antigenic properties of Env immunogens. DANA screens of stabilized, soluble Env trimers revealed that stronger trimer stabilization led to the selection of highly mutated DARPins with length variations and framework mutations mirroring observations made for bnAbs. By mimicking heterotypic prime-boost immunization regimens, DANA may be used to select immunogen combinations that favor the selection of trimer-reactive binders. This positions DANA as a versatile strategy for distilling fundamental antigenic features of immunogens, complementary to preclinical immunogenicity testing.

DOI: 10.1038/s41541-023-00746-3 | Read all as PDF | SUPPL: 0525 | Close Abstract
0524

Houvast, R. D., Badr, N., March, T., de Muynck, L. D. A. N., Sier, V. Q., Schomann, T., Bhairosingh, S., Baart, V. M., Peeters, J. A. H. M., van Westen, G. J. P., Plückthun, A., Burggraaf, J., Kuppen, P. J. K., Vahrmeijer, A. L., and Sier, C. F. M. (2024). Preclinical evaluation of EpCAM-binding designed ankyrin repeat proteins (DARPins) as targeting moieties for bimodal near-infrared fluorescence and photoacoustic imaging of cancer. Eur. J. Nucl. Med. Mol. Imaging 51, 2179–2192

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Abstract

Purpose: Fluorescence-guided surgery (FGS) can play a key role in improving radical resection rates by assisting surgeons to gain adequate visualization of malignant tissue intraoperatively. Designed ankyrin repeat proteins (DARPins) possess optimal pharmacokinetic and other properties for in vivo imaging. This study aims to evaluate the preclinical potential of epithelial cell adhesion molecule (EpCAM)-binding DARPins as targeting moieties for near-infrared fluorescence (NIRF) and photoacoustic (PA) imaging of cancer.
Methods: EpCAM-binding DARPins Ac2, Ec4.1, and non-binding control DARPin Off7 were conjugated to IRDye 800CW and their binding efficacy was evaluated on EpCAM-positive HT-29 and EpCAM-negative COLO-320 human colon cancer cell lines. Thereafter, NIRF and PA imaging of all three conjugates were performed in HT-29_luc2 tumor-bearing mice. At 24 h post-injection, tumors and organs were resected and tracer biodistributions were analyzed.
Results: Ac2-800CW and Ec4.1-800CW specifically bound to HT-29 cells, but not to COLO-320 cells. Next, 6 nmol and 24 h were established as the optimal in vivo dose and imaging time point for both DARPin tracers. At 24 h post-injection, mean tumor-to-background ratios of 2.60 ± 0.3 and 3.1 ± 0.3 were observed for Ac2-800CW and Ec4.1-800CW, respectively, allowing clear tumor delineation using the clinical Artemis NIRF imager. Biodistribution analyses in non-neoplastic tissue solely showed high fluorescence signal in the liver and kidney, which reflects the clearance of the DARPin tracers.
Conclusion: Our encouraging results show that EpCAM-binding DARPins are a promising class of targeting moieties for pan-carcinoma targeting, providing clear tumor delineation at 24 h post-injection. The work described provides the preclinical foundation for DARPin-based bimodal NIRF/PA imaging of cancer.

DOI: 10.1007/s00259-023-06407-w | Read all as PDF | Close Abstract
0523

Hartmann, K. P., van Gogh, M., Freitag, P. C., Kast, F., Nagy-Davidescu, G., Borsig, L., and Plückthun, A. (2023). FAP-retargeted Ad5 enables in vivo gene delivery to stromal cells in the tumor microenvironment, Mol. Therapy 31, 2914-2928.
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Abstract

Fibroblast activation protein (FAP) is a cell surface serine protease that is highly expressed on reactive stromal fibroblasts, such as cancer-associated fibroblasts (CAFs), and generally absent in healthy adult tissues. FAP expression in the tumor stroma has been detected in more than 90% of all carcinomas, rendering CAFs excellent target cells for a tumor site-specific adenoviral delivery of cancer therapeutics. Here, we present a tropism-modified human adenovirus 5 (Ad5) vector that targets FAP through trivalent, designed ankyrin repeat protein (DARPin)-based retargeting adapters. We describe the development and validation of these adapters via cell-based screening assays and demonstrate adapter-mediated Ad5 retargeting to FAP fibroblasts in vitro and in vivo. We further show efficient in vivo delivery and in-situ production of a therapeutic payload by CAFs in the tumor microenvironment (TME), resulting in attenuated tumor growth. We thus propose using our FAP-Ad5 vector to convert CAFs into a ‘biofactory’, secreting encoded cancer therapeutics into the TME to enable a safe and effective cancer treatment.

DOI: 10.1016/j.ymthe.2023.08.018. | Read all as PDF | SUPPL: 0523 | Close Abstract
0522

Glögl, M., Friedrich, M., Cerutti, G., Lemmin, T., Kwon, Y. D., Gorman, J., Maliqi, L., Mittl, P. R. E., Hesselman, M. C., Schmidt, D., Weber, J., Foulkes, C., Dingens, A. S., Bylund, T., Olia, A. S. Verardi, R., Reinberg, T., Baumann, N. S., Rusert, P., Dreier, B., Shapiro, L., Kwong, P. D., Plückthun, A., and Trkola, A. (2023). Trapping the HIV-1 V3 loop in a helical conformation enables broad neutralization. Nature Struct. Mol. Biol. 30, 1323–1336.
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Abstract

The third variable (V3) loop on the human immunodeficiency virus 1 (HIV-1) envelope glycoprotein trimer is indispensable for virus cell entry. Conformational masking of V3 within the trimer allows efficient neutralization via V3 only by rare, broadly neutralizing glycan-dependent antibodies targeting the closed prefusion trimer but not by abundant antibodies that access the V3 crown on open trimers after CD4 attachment. Here, we report on a distinct category of V3-specific inhibitors based on designed ankyrin repeat protein (DARPin) technology that reinstitute the CD4-bound state as a key neutralization target with up to >90% breadth. Broadly neutralizing DARPins (bnDs) bound V3 solely on open envelope and recognized a four-turn amphipathic α-helix in the carboxy-terminal half of V3 (amino acids 314-324), which we termed 'αV3C'. The bnD contact surface on αV3C was as conserved as the CD4 binding site. Molecular dynamics and escape mutation analyses underscored the functional relevance of αV3C, highlighting the potential of αV3C-based inhibitors and, more generally, of postattachment inhibition of HIV-1.

DOI: 10.1038/s41594-023-01062-z | Read all as PDF | SUPPL: 0522 | Close Abstract
0521

Klinnert, S., Schenkel, C. D., Freitag, P. C., Günthard, H. F., Plückthun, A., and Metzner, K. J. (2024). Targeted shock-and-kill HIV-1 gene therapy approach combining CRISPR activation, suicide gene tBid and retargeted adenovirus delivery. Gene Therapy, 74–84.
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Abstract

Infections with the human immunodeficiency virus type 1 (HIV-1) are incurable due the long-lasting, latent viral reservoir. The shock-and-kill cure approach aims to activate latent proviruses in HIV-1 infected cells and subsequently kill these cells with strategies such as therapeutic vaccines or immune enhancement. Here, we combined the dCas9-VPR CRISPR activation (CRISPRa) system with gRNA-V, the truncated Bid (tBid)-based suicide gene strategy and CD3-retargeted adenovirus (Ad) delivery vectors, in an all-in-one targeted shock-and-kill gene therapy approach to achieve specific elimination of latently HIV-1 infected cells. Simultaneous transduction of latently HIV-1 infected J-Lat 10.6 cells with a CD3-retargeted Ad-CRISPRa-V and Ad-tBid led to a 57.7 ± 17.0% reduction of productively HIV-1 infected cells and 2.4-fold ± 0.25 increase in cell death. The effective activation of latent HIV-1 provirus by Ad-CRISPRa-V was similar to the activation control TNF-α. The strictly HIV-1 dependent and non-leaky killing by tBid could be demonstrated. Furthermore, the high transduction efficiencies of up to 70.8 ± 0.4% by the CD3-retargeting technology in HIV-1 latently infected cell lines was the basis of successful shock-and-kill. This novel targeted shock-and-kill all-in-one gene therapy approach has the potential to safely and effectively eliminate HIV-1 infected cells in a highly HIV-1 and T cell specific manner.

DOI: 10.1038/s41434-023-00413-1 | Read all as PDF | SUPPL: 0521 | Close Abstract
0520

Shi, J., Hauschulte, K., Mikicic, I., Maharjan, S., Arz, V., Strauch, T., Heidelberger, J. B., Schaefer, J. V., Dreier, B., Plückthun, A., Beli, P., Ulrich, H. D., and Wollscheid. H.-P. (2023). Nuclear myosin VI maintains replication fork stability. Nature Commun. 14, 3787.
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Abstract

The actin cytoskeleton is of fundamental importance for cellular structure and plasticity. However, abundance and function of filamentous actin in the nucleus are still controversial. Here we show that the actin-based molecular motor myosin VI contributes to the stabilization of stalled or reversed replication forks. In response to DNA replication stress, myosin VI associates with stalled replication intermediates and cooperates with the AAA ATPase Werner helicase interacting protein 1 (WRNIP1) in protecting these structures from DNA2-mediated nucleolytic attack. Using functionalized affinity probes to manipulate myosin VI levels in a compartment-specific manner, we provide evidence for the direct involvement of myosin VI in the nucleus and against a contribution of the abundant cytoplasmic pool during the replication stress response.

DOI: 10.1038/s41467-023-39517-y | Read all as PDF | SUPPL: 0520 | Close Abstract
0519

Gut, M., Dreier, B., Furler, S., Sobek, J., Plückthun, A., and Holland, J. P. (2023) Designed ankyrin repeat proteins for detecting prostate-specific antigen expression in vivo. RSC Chem. Biol. 4, 494-505.
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Abstract

Late-stage prostate cancer often acquires resistance to conventional chemotherapies and transforms into a hormone-refractory, drug-resistant, and non-curative disease. Developing non-invasive tools to detect the biochemical changes that correlate with drug efficacy and reveal the onset of drug resistance would have important ramifications in managing the treatment regimen for individual patients. Here, we report the selection of new Designed Ankyrin Repeat Proteins (DARPins) that show high affinity toward prostate-specific antigen (PSA), a biomarker used in clinical monitoring of prostate cancer. Ribosome display and in vitro screening tools were used to select PSA-binding DARPins based on their binding affinity, selectivity, and chemical constitution. Surface plasmon resonance measurements demonstrated that the four lead candidates bind to PSA with nanomolar affinity. DARPins were site-specifically functionalised at a unique C-terminal cysteine with a hexadentate aza-nonamacrocyclic chelate (NODAGA) for subsequent radiolabelling with the positron-emitting radionuclide 68Ga. [68Ga]GaNODAGA-DARPins showed high stability toward transchelation and were stable in human serum for >2 h. Radioactive binding assays using streptavidin-loaded magnetic beads confirmed that the functionalisation and radiolabelling did not compromise the specificity of [68Ga]GaNODAGA-DARPins toward PSA. Biodistribution experiments in athymic nude mice bearing subcutaneous prostate cancer xenografts derived from the LNCaP cell line revealed that three of the four [68Ga]GaNODAGA-DARPins displayed specific tumour-binding in vivo. For DARPin-6, tumour-uptake in the normal group reached 4.16 ± 0.58% ID g−1 (n = 3; 2 h post-administration) and was reduced by ∼50% by competitive binding with a low molar activity formulation (blocking group: 2.47 ± 0.42% ID g−1; n = 3; P value = 0.018). Collectively, the experimental results support the future development of new PSA-specific imaging agents for potential use in monitoring the efficacy of androgen receptor (AR)-targeted therapies.

DOI: 10.1039/D3CB00010A | Read all as PDF | SUPPL: 0519 | Close Abstract
0518

Blanc, M., Lettl, C., Guérin, J., Vieille, A., Furler, S., Briand-Schumacher, S., Dreier, B., Bergé, C., Plückthun, A., Vadon-Le Goff, S., Fronzes, R., Rousselle, P., Fischer, W., and Terradot, L. (2923) Designed Ankyrin Repeat Proteins provide insights into the structure and function of CagI and are potent inhibitors of CagA translocation by the Helicobacter pylori type IV secretion system. PLoS Pathog. 19, e1011368.
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Abstract

The bacterial human pathogen Helicobacter pylori produces a type IV secretion system (cagT4SS) to inject the oncoprotein CagA into gastric cells. The cagT4SS external pilus mediates attachment of the apparatus to the target cell and the delivery of CagA. While the composition of the pilus is unclear, CagI is present at the surface of the bacterium and required for pilus formation. Here, we have investigated the properties of CagI by an integrative structural biology approach. Using Alpha Fold 2 and Small Angle X-ray scattering, it was found that CagI forms elongated dimers mediated by rod-shape N-terminal domains (CagIN) prolonged by globular C-terminal domains (CagIC). Three Designed Ankyrin Repeat Proteins (DARPins) K2, K5 and K8 selected against CagI interacted with CagIC with subnanomolar affinities. The crystal structures of the CagI:K2 and CagI:K5 complexes were solved and identified the interfaces between the molecules, thereby providing a structural explanation for the difference in affinity between the two binders. Purified CagI and CagIC were found to interact with adenocarcinoma gastric (AGS) cells, induced cell spreading and the interaction was inhibited by K2. The same DARPin inhibited CagA translocation by up to 65% in AGS cells while inhibition levels were 40% and 30% with K8 and K5, respectively. Our study suggests that CagIC plays a key role in cagT4SS-mediated CagA translocation and that DARPins targeting CagI represent potent inhibitors of the cagT4SS, a crucial risk factor for gastric cancer development.

DOI: 10.1371/journal.ppat.1011368 | Read all as PDF | Close Abstract
0517

Becker, L., and Plückthun, A. (2023). DARPins bind their cytosolic targets after having been translocated through the protective antigen pore of anthrax toxin. Sci. Rep. 13, 8048.
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Abstract

Intracellular protein–protein interactions in aberrant signaling pathways have emerged as a prime target in several diseases, particularly cancer. Since many protein–protein interactions are mediated by rather flat surfaces, they can typically not be interrupted by small molecules as they require cavities for binding. Therefore, protein drugs might be developed to compete with undesired interactions. However, proteins in general are not able to translocate from the extracellular side to the cytosolic target site by themselves, and thus an efficient protein translocation system, ideally combining efficient translocation with receptor specificity, is in high demand. Anthrax toxin, the tripartite holotoxin of Bacillus anthracis, is one of the best studied bacterial protein toxins and has proven to be a suitable candidate for cell-specific translocation of cargoes in vitro and in vivo. Our group recently developed a retargeted protective antigen (PA) variant fused to different Designed Ankyrin Repeat Proteins (DARPins) to achieve receptor specificity, and we incorporated a receptor domain to stabilize the prepore and prevent cell lysis. This strategy had been shown to deliver high amounts of cargo DARPins fused behind the N-terminal 254 amino acids of Lethal Factor (LFN). Here, we established a cytosolic binding assay, demonstrating the ability of DARPins to refold in the cytosol and bind their target after been translocated by PA.

DOI: 10.1038/s41598-023-34647-1 | Read all as PDF | SUPPL: 0517 | Close Abstract
0516

Schubert, R., Bae, T., Simic, B., Smith, S. N., Park, S. H., Nagy-Davidescu, G., Gradinaru, V., Plückthun, A., and Hur, J. K. (2023). CRISPR-clear imaging of melanin-rich B16-derived solid tumors. Commun. Biol. 6, 370.
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Abstract

Tissue clearing combined with deep imaging has emerged as a powerful technology to expand classical histological techniques. Current techniques have been optimized for imaging sparsely pigmented organs such as the mammalian brain. In contrast, melanin-rich pigmented tissue, of great interest in the investigation of melanomas, remains challenging. To address this challenge, we have developed a CRISPR-based gene editing approach that is easily incorporated into existing tissue-clearing workflows such the PACT clearing method. We term this method CRISPR-Clear. We demonstrate its applicability to highly melanin-rich B16-derived solid tumors, including one made transgenic for HER2, constituting one of very few syngeneic mouse tumors that can be used in immunocompetent models. We demonstrate the utility in detailed tumor characterization by staining for targeting antibodies and nanoparticles, as well as expressed fluorescent proteins. With CRISPR-Clear we have unprecedented access to optical interrogation in considerable portions of intact melanoma tissue for stained surface markers, expressed fluorescent proteins, of subcellular compartments, and of the vasculature.

DOI: 10.1038/s42003-023-04614-7 | Read all as PDF | SUPPL: 0516 Explanation videos 0516 Video1 0516 Video2 0516 Video3 0516 Video4 0516 | Close Abstract
0515

Klenk, C., Scrivens, M., Niederer, A., Shi, S., Mueller, L., Gersz, E., Zauderer, M., Smith, E. S., Strohner, R., and Plückthun, A. (2023). A Vaccinia-based system for directed evolution of GPCRs in mammalian cells. Nature Commun. 14, 1770.
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Abstract

Directed evolution in bacterial or yeast display systems has been successfully used to improve stability and expression of G protein-coupled receptors for structural and biophysical studies. Yet, several receptors cannot be tackled in microbial systems due to their complex molecular composition or unfavorable ligand properties. Here, we report an approach to evolve G protein-coupled receptors in mammalian cells. To achieve clonality and uniform expression, we develop a viral transduction system based on Vaccinia virus. By rational design of synthetic DNA libraries, we first evolve neurotensin receptor 1 for high stability and expression. Second, we demonstrate that receptors with complex molecular architectures and large ligands, such as the parathyroid hormone 1 receptor, can be readily evolved. Importantly, functional receptor properties can now be evolved in the presence of the mammalian signaling environment, resulting in receptor variants exhibiting increased allosteric coupling between the ligand binding site and the G protein interface. Our approach thus provides insights into the intricate molecular interplay required for GPCR activation.

DOI: 10.1038/s41467-023-37191-8 | Read all as PDF | SUPPL: 0515 | Close Abstract
0514

Freitag, P. C., Kaulfuss, M., Flühler, L., Mietz, J., Weiss, F., Brücher, D., Kolibius, K., Hartmann, K. P., Smith, S. N.,Münz, C., Chijioke, O., and Plückthun, A. (2023). Targeted adenovirus-mediated transduction of human T cells in vitro and in vivo. Mol. Therapy Meth. Clin. Dev. 29, 120-132.
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Abstract

Clinical success in T cell therapy has stimulated widespread efforts to increase safety, potency and extend this technology to solid tumors. Yet progress in cell therapy remains restricted by the limited payload-capacity, specificity of target cell transduction and transgenic gene expression efficiency of applied viral vectors. This renders complex reprogramming or direct in vivo applications difficult. Here, we developed a synergistic combination of trimeric adapter constructs enabling T cell-directed transduction by the human adenoviral vector serotype C5 in vitro and in vivo. Rationally chosen binding partners showed receptor-specific transduction of otherwise non-susceptible human T cells by exploiting activation stimuli. This platform remains compatible with high-capacity vectors for up to 37 kb DNA delivery, increasing payload capacity and safety due to the removal of all viral genes. Together, these findings provide a tool for targeted delivery of large payloads in T cells as potential avenue to overcome current limitations of T cell therapy.

DOI: 10.1016/j.omtm.2023.02.012 | Read all as PDF | Close Abstract
0513

Schellhorn, S., Brücher, D., Wolff, N., Schröer, K., Sallard, E., Mese, K., Zhang, W., Ehrke-Schulz, E., Thevenod, F., Plückthun, A., and Ehrhardt, A. (2023). Targeting oncolytic adenoviruses to cancer cells using a designed ankyrin repeat protein (DARPin) lipocalin-2 (LCN2) fusion protein. Hum. Gene Ther. 34, 203-216.
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Abstract

Oncolytic viruses are a promising technology to attack cancer cells and to recruit immune cells to the tumor site. Since the Lipocalin-2 receptor (LCN2R) is expressed on most cancer cells, we used its ligand LCN2 to target oncolytic adenoviruses (Ads) to cancer cells. Therefore, we fused a DARPin adapter binding the knob of Ad type 5 (knob5) to LCN2 to retarget the virus towards LCN2R with the aim of analyzing the basic characteristics of this novel targeting approach. The adapter was tested in vitro with Chinese Hamster Ovary (CHO) cells stably expressing the LCN2R and on 20 cancer cell lines (CCLs) using an Ad5 vector encoding luciferase and green fluorescent protein (GFP). Luciferase assays with the LCN2 adapter (LA) showed 10-fold higher infection compared to blocking adapter (BA) in CHO cells expressing LCN2R and in cells not expressing the LCN2R. Most CCLs showed an increased viral uptake of LA-bound virus compared to BA-bound virus and for five CCLs viral uptake was comparable to unmodified Ad5. Flow cytometry and hexon immunostainings also revealed increased uptake of LA-bound Ads compared with BA-bound Ads in most tested CCLs. Virus spread was studied in 3D cell culture models and nine CCLs showed increased and earlier fluorescence signals for LA-bound virus compared to BA-bound virus. Mechanistically we show that the LCN2 adapter increases viral uptake only in the absence of its ligand Enterobactin (Ent) and independently of iron. Together, we characterized a novel DARPin-based system resulting in enhanced uptake demonstrating potential for future oncolytic virotherapy.

DOI: 10.1089/hum.2022.215 | Read all as PDF | SUPPL: 0513 HeLaAd5BA HeLaAd5LA HeLaAd5only HeLaNoVirus | Close Abstract

2022

# Reference PDF

0512

Kiss, C., Gall, F. M., Dreier, B., Adams, M., Riedl, R., Plückthun, A., and Mittl, P. R. E. (2022). Structure of a hydrophobic leucinostatin derivative determined by host lattice display. Acta Cryst. D78, 1439-1450.
| Abstract + further info

Abstract

Peptides comprising many hydrophobic amino acids are almost insoluble under physiological buffer conditions, which complicates their structural analysis. To investigate the three-dimensional structure of the hydrophobic leucinostatin derivative ZHAWOC6027, the previously developed host lattice display technology was applied. Two designed ankyrin-repeat proteins (DARPins) recognizing a biotinylated ZHAWOC6027 derivative were selected from a diverse library by ribosome display under aqueous buffer conditions. ZHAWOC6027 was immobilized by means of the DARPin in the host lattice and the structure of the complex was determined by X-ray diffraction. ZHAWOC6027 adopts a distorted α-helical conformation. Comparison with the structures of related compounds that have been determined in organic solvents reveals elevated flexibility of the termini, which might be functionally important.

DOI: 10.1107/S2059798322010762 | Read all as PDF | SUPPL: 0512 | Close Abstract
0511

Campbell, B. F. N., Dittmann, A., Dreier, B., Plückthun, A., and Tyagarajan, S. K. (2022) A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology. eLife 11, e80895
| Abstract + further info

Abstract

Neuroscience currently requires the use of antibodies to study synaptic proteins, where antibody binding is used as a correlate to define the presence, plasticity, and regulation of synapses. Gephyrin is an inhibitory synaptic scaffolding protein used to mark GABAergic and glycinergic postsynaptic sites. Despite the importance of gephyrin in modulating inhibitory transmission, its study is currently limited by the tractability of available reagents. Designed Ankyrin Repeat Proteins (DARPins) are a class of synthetic protein binder derived from diverse libraries by in vitro selection and tested by high-throughput screening to produce specific binders. In order to generate a functionally diverse toolset for studying inhibitory synapses, we screened a DARPin library against gephyrin mutants representing both phosphorylated and dephosphorylated states. We validated the robust use of anti-gephyrin DARPin clones for morphological identification of gephyrin clusters in rat neuron culture and mouse brain tissue, discovering previously overlooked clusters. This DARPin-based toolset includes clones with heterogenous gephyrin binding modes that allowed for identification of the most extensive gephyrin interactome to date and defined novel classes of putative interactors, creating a framework for understanding gephyrin’s nonsynaptic functions. This study demonstrates anti-gephyrin DARPins as a versatile platform for studying inhibitory synapses in an unprecedented manner.

DOI: 10.7554/eLife.80895 | Read all as PDF | SUPPL: 0511 1 0511 2 | Close Abstract
0510

Philippi, M., Richter, C. P., Kappen, M., Watrinet, I., Miao, Y., Runge, M., Jorde, L., Korneev, S., Holtmannspötter, M., Kurre, R., Holthuis, J. C. M., Garcia, K. C., Plückthun, A., Steinhart, M., Piehler, J., and You, C. (2022) Biofunctional nanodot arrays in living cells uncover synergistic co-condensation of Wnt signalodroplets. Small 18, 2203723.
| Abstract + further info

Abstract

Qualitative and quantitative analysis of transient signaling platforms in the plasma membrane has remained a key experimental challenge. Here, biofunctional nanodot arrays (bNDAs) are developed to spatially control dimerization and clustering of cell surface receptors at the nanoscale. High-contrast bNDAs with spot diameters of ≈300 nm are obtained by capillary nanostamping of bovine serum albumin bioconjugates, which are subsequently biofunctionalized by reaction with tandem anti-green fluorescence protein (GFP) clamp fusions. Spatially controlled assembly of active Wnt signalosomes is achieved at the nanoscale in the plasma membrane of live cells by capturing the co-receptor Lrp6 into bNDAs via an extracellular GFP tag. Strikingly, co-recruitment is observed of co-receptor Frizzled-8 as well as the cytosolic scaffold proteins Axin-1 and Disheveled-2 into Lrp6 nanodots in the absence of ligand. Density variation and the high dynamics of effector proteins uncover highly cooperative liquid?liquid phase separation (LLPS)-driven assembly of Wnt ?signalodroplets? at the plasma membrane, pinpointing the synergistic effects of LLPS for Wnt signaling amplification. These insights highlight the potential of bNDAs for systematically interrogating nanoscale signaling platforms and condensation at the plasma membrane of live cells.

DOI: 10.1002/smll.202203723 | Read all as PDF | SUPPL: 0510 smll videos1 smll videos2 smll videos3 smll videos4 smll videos5 smll videos6 smll videos7 | Close Abstract
0509

Zhang, Y., Wang, Y., Uslu, S., Venkatachalapathy, S., Rashidian, M., Schaefer, J. V., Plückthun, A. and Distefano, M. D. (2022) Enzymatic construction of DARPin-based targeted delivery systems using protein farnesyltransferase and a capture and release strategy. Int. J. Mol. Sci. 23, 11537.
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Abstract

Protein-based conjugates have been extensively utilized in various biotechnological and therapeutic applications. In order to prepare homogeneous conjugates, site-specific modification methods and efficient purification strategies are both critical factors to be considered. The development of general and facile conjugation and purification strategies is therefore highly desirable. Here, we apply a capture and release strategy to create protein conjugates based on Designed Ankyrin Repeat Proteins (DARPins), which are engineered antigen-binding proteins with prominent affinity and selectivity. In this case, DARPins that target the epithelial cell adhesion molecule (EpCAM), a diagnostic cell surface marker for many types of cancer, were employed. The DARPins were first genetically modified with a C-terminal CVIA sequence to install an enzyme recognition site and then labeled with an aldehyde functional group employing protein farnesyltransferase. Using a capture and release strategy, conjugation of the labeled DARPins to a TAMRA fluorophore was achieved with either purified proteins or directly from crude E. coli lysate and used in subsequent flow cytometry and confocal imaging analysis. DARPin-MMAE conjugates were also prepared yielding a construct manifesting an IC50 of 1.3 nM for cell killing of EpCAM positive MCF-7 cells. The method described here is broadly applicable to enable the streamlined one-step preparation of protein-based conjugates.

DOI: 10.3390/ijms231911537 | Read all as PDF | Close Abstract
0508

Freitag, P. C., Brandl, F., Brücher, D., Weiss, F., Dreier, B., and Plückthun, A. (2022) Modular adapters utilizing binders of different molecular types expand cell-targeting options for adenovirus gene delivery. Bioconjug. Chem. 33, 1595−1601.
| Abstract + further info

Abstract

Efficient and cell-specific delivery of DNA is essential for the effective and safe use of gene delivery technologies. Consequently, a large variety of technologies have been developed and applied in a wide range of ex vivo and in vivo applications, including multiple approaches based on viral vectors. However, widespread success of a technology is largely determined by the versatility of the method and the ease of use. The rationally designed adapter technology previously developed redirects widely used human adenovirus serotype 5 (HAdV-C5) to a defined cell population, by binding and blocking the adenoviral knob tropism while simultaneously allowing fusions of an N-terminal retargeting module. Here we expand modularity, and thus applicability of this adapter technology, by extending the nature of the cell-binding portion. We report successful receptor-specific transduction mediated by a retargeting module consisting of either a DARPin, a single-chain variable fragment (scFv) of an antibody, a peptide, or a small molecule ligand. Furthermore, we show that an adapter can be engineered to carry more than one specificity, allowing dual targeting. Specific HAdV-C5 retargeting was thus demonstrated to human epidermal growth factor receptor 2 (HER2), human folate receptor α, and neurotensin receptor 1, effective at vector concentrations as low as a multiplicity of infection of 2.5. Therefore, we report a modular design which allows plug-and-play combinations of different binding modules, leading to efficient and specific mono- or dual-targeting while circumventing tedious optimization procedures. This extends the technology to combinational applications of cell-specific binding, supporting research in gene therapy, synthetic biology, and biotechnology.

DOI: 10.1021/acs.bioconjchem.2c00346 | Read all as PDF | SUPPL: 0508 | Close Abstract
0507

Strittmatter, T., Wang, Y., Bertschi, A., Scheller, L., Freitag, P. C., Ray, P. G., Stuecheli, P., Schaefer, J. V., Reinberg, T., Tsakiris, D., Plückthun, A., Ye, H., and Fussenegger, M. (2022) Programmable DARPin-based receptors for the detection of thrombotic markers. Nature Chem. Biol. 18, 1125–1134.
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Abstract

Cellular therapies remain constrained by the limited availability of sensors for disease markers. Here we present an integrated target-to-receptor pipeline for constructing a customizable advanced modular bispecific extracellular receptor (AMBER) that combines our generalized extracellular molecule sensor (GEMS) system with a high-throughput platform for generating designed ankyrin repeat proteins (DARPins). For proof of concept, we chose human fibrin degradation products (FDPs) as markers with high clinical relevance and screened a DARPin library for FDP binders. We built AMBERs equipped with 19 different DARPins selected from 160 hits, and found 4 of them to be functional as heterodimers with a known single-chain variable fragments binder. Tandem receptors consisting of combinations of the validated DARPins are also functional. We demonstrate applications of these AMBER receptors in vitro and in vivo by constructing designer cell lines that detect pathological concentrations of FDPs and respond with the production of a reporter and a therapeutic anti-thrombotic protein.

DOI: 10.1038/s41589-022-01095-3 | Read all as PDF | SUPPL: 0507 | Close Abstract
0506

Strubel, A., Münick, P., Chaikuad, A., Dreier, B., Schaefer, J., Gebel, J., Osterburg, C., Tuppi, M., Schäfer, B., Knapp, S., Plückthun, A., and Dötsch, V. (2022) Designed ankyrin repeat proteins as a tool box for analyzing p63. Cell Death Differ. 29, 2445–2458.
| Abstract + further info

Abstract

The function of the p53 transcription factor family is dependent on several folded domains. In addition to a DNA-binding domain, members of this family contain an oligomerization domain. p63 and p73 also contain a C-terminal Sterile α-motif domain. Inhibition of most transcription factors is difficult as most of them lack deep pockets that can be targeted by small organic molecules. Genetic knock-out procedures are powerful in identifying the overall function of a protein, but they do not easily allow one to investigate roles of individual domains. Here we describe the characterization of Designed Ankyrin Repeat Proteins (DARPins) that were selected as tight binders against all folded domains of p63. We determine binding affinities as well as specificities within the p53 protein family and show that DARPins can be used as intracellular inhibitors for the modulation of transcriptional activity. By selectively inhibiting DNA binding of the ΔNp63α isoform that competes with p53 for the same promoter sites, we show that p53 can be reactivated. We further show that inhibiting the DNA binding activity stabilizes p63, thus providing evidence for a transcriptionally regulated negative feedback loop. Furthermore, the ability of DARPins to bind to the DNA-binding domain and the Sterile α-motif domain within the dimeric-only and DNA-binding incompetent conformation of TAp63α suggests a high structural plasticity within this special conformation. In addition, the developed DARPins can also be used to specifically detect p63 in cell culture and in primary tissue and thus constitute a very versatile research tool for studying the function of p63.

DOI: 10.1038/s41418-022-01030-y | Read all as PDF | SUPPL: 0506 | Close Abstract
0505

Michel, E., Cucuzza, S., Mittl, P. R. E., Zerbe, O., and Plückthun, A. (2023) Improved repeat protein stability by combined consensus and computational protein design. Biochemistry 62, 318-329.
| Abstract + further info

Abstract

High protein stability is an important feature for proteins used as therapeutics, as diagnostics, and in basic research. We have previously employed consensus design to engineer optimized Armadillo repeat proteins (ArmRPs) for sequence-specific recognition of linear epitopes with a modular binding mode. These designed ArmRPs (dArmRPs) feature high stability and are composed of M-type internal repeats that are flanked by N- and C-terminal capping repeats that protect the hydrophobic core from solvent exposure. While the overall stability of the designed ArmRPs is remarkably high, subsequent biochemical and biophysical experiments revealed that the N-capping repeat assumes a partially unfolded, solvent-accessible conformation for a small fraction of time that renders it vulnerable to proteolysis and aggregation. To overcome this problem, we have designed new N-caps starting from an M-type internal repeat using the Rosetta software. The superior stability of the computationally refined models was experimentally verified by circular dichroism and nuclear magnetic resonance spectroscopy. A crystal structure of a dArmRP containing the novel N-cap revealed that the enhanced stability correlates with an improved packing of this N-cap onto the hydrophobic core of the dArmRP. Hydrogen exchange experiments further show that the level of local unfolding of the N-cap is reduced by several orders of magnitude, resulting in increased resistance to proteolysis and weakened aggregation. As a first application of the novel N-cap, we determined the solution structure of a dArmRP with four internal repeats, which was previously impeded by the instability of the original N-cap.

DOI: 10.1021/acs.biochem.2c00083 | Read all as PDF | SUPPL: 0505 | Close Abstract
0504

Fay, R., Törő. I., Schinke, A. L., Simic, B,, Schaefer, J. V., Dreier, B., Plückthun. A., and Holland, J. P. (2022) Sortase-mediated site-specific conjugation and 89Zr-radiolabeling of designed ankyrin repeat proteins for PET. Mol. Pharm. 19, 3576-3585.
| Abstract + further info

Abstract

Designed ankyrin repeat proteins (DARPins) are genetically engineered proteins that exhibit high specificity and affinity toward specific targets. Here, the G3-DARPin, which binds the HER2/neu receptor, was site-specifically modified with enzymatic methods and 89Zr-radiolabeled for applications in positron emission tomography (PET). Sortase A transpeptidation was used to install a desferrioxamine B (DFO) chelate bearing a reactive triglycine group to the C-terminal sortase tag of the G3-DARPin, and 89Zr-radiolabeling produced a novel 89ZrDFO-G3-DARPin radiotracer that can detect HER2/neu-positive tumors. The triglycine probe, DFO-Gly3 (1), was synthesized in 29% overall yield. After sortase A transpeptidation and purification from the nonfunctionalized protein component, the DFO-G3-DARPin product was radiolabeled to give 89ZrDFO-G3-DARPin. Binding specificity was assessed in HER2/neu-expressing BT-474 and SK-OV-3 cellular assays. The pharmacokinetics, tumor uptake, and specificity of 89ZrDFO-G3-DARPin were measured in vivo by PET imaging and confirmed by final time point (24 h) biodistribution experiments in female athymic nude mice bearing BT-474 xenografts. Sortase A transpeptidation afforded the site-specific and stoichiometrically precise functionalization of DFO-G3-DARPin with one chelate per protein. The modified DFO-G3-DARPin was purified from the nonfunctionalized DARPin by using Ni-NTA affinity chromatography. 89ZrDFO-G3-DARPin was obtained with a radiochemical purity of >95% measured by radio-size-exclusion chromatography. BT-474 tumor uptake at 24 h postadministration reached 4.41 ± 0.67 %ID/g (n = 3) with an approximate ∼70% reduction in tumor-associated activity in the blocking group (1.26 ± 0.29 %ID/g; 24 h postadministration, n = 5, P-value of <0.001). Overall, the site-specific, enzyme-mediated functionalization and characterization of 89ZrDFO-G3-DARPin in HER2/neu positive BT-474 xenografts demonstrate that DARPins are an attractive platform for generating a new class of protein-based radiotracers for PET. The specific uptake and retention of 89ZrDFO-G3-DARPin in tumors and clearance from most background tissues produced PET images with high tumor-to-background contrast.

DOI: 10.1021/acs.molpharmaceut.2c00136 | Read all as PDF | SUPPL: 0504 | Close Abstract
0503

Waltenspühl, Y., Ehrenmann, J., Vacca, S., Thom, C., Medalia, O., and Plückthun, A. (2022) Structural basis for the activation and ligand recognition of the human oxytocin receptor. Nature Comm. 13, 4154
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Abstract

The small cyclic neuropeptide hormone oxytocin (OT) and its cognate receptor play a central role in the regulation of social behaviour and sexual reproduction. Here we report the single-particle cryo-electron microscopy structure of the active oxytocin receptor (OTR) in complex with its cognate ligand oxytocin. Our structure provides high-resolution insights into the OT binding mode, the OTR activation mechanism as well as the subtype specificity within the oxytocin/vasopressin receptor family.

DOI: 10.1038/s41467-022-31325-0 | Read all as PDF | SUPPL: 0503 | Close Abstract
0502

Kirchhammer, N., Trefny, M. P., Natoli, M., Brücher, D., Smith, S. N., Werner, F., Koch, V., Schreiner, D., Bartoszek, E., Buchi, M., Schmid, M., Breu, D., Hartmann, K. P., Zaytseva, P., Thommen, D. S., Läubli, H., Böttcher, J. P., Stanczak, M. A., Kashyap, A. S., Plückthun, A., and Zippelius, A. (2022) NK cells with tissue-resident traits shape response to immunotherapy by inducing adaptive antitumor immunity. Science Transl. Med. 14, eabm9043
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Abstract

T cell–directed cancer immunotherapy often fails to generate lasting tumor control. Harnessing additional effectors of the immune response against tumors may strengthen the clinical benefit of immunotherapies. Here, we demonstrate that therapeutic targeting of the interferon-γ (IFN-γ)–interleukin-12 (IL-12) pathway relies on the ability of a population of natural killer (NK) cells with tissue-resident traits to orchestrate an antitumor microenvironment. In particular, we used an engineered adenoviral platform as a tool for intratumoral IL-12 immunotherapy (AdV5–IL-12) to generate adaptive antitumor immunity. Mechanistically, we demonstrate that AdV5–IL-12 is capable of inducing the expression of CC-chemokine ligand 5 (CCL5) in CD49a+ NK cells both in tumor mouse models and tumor specimens from patients with cancer. AdV5–IL-12 imposed CCL5-induced type I conventional dendritic cell (cDC1) infiltration and thus increased DC-CD8 T cell interactions. A similar observation was made for other IFN-γ–inducing therapies such as Programmed cell death 1 (PD-1) blockade. Conversely, failure to respond to IL-12 and PD-1 blockade in tumor models with low CD49a+ CXCR6+ NK cell infiltration could be overcome by intratumoral delivery of CCL5. Thus, therapeutic efficacy depends on the abundance of NK cells with tissue-resident traits and, specifically, their capacity to produce the DC chemoattractant CCL5. Our findings reveal a barrier for T cell–focused therapies and offer mechanistic insights into how T cell–NK cell–DC cross-talk can be enhanced to promote antitumor immunity and overcome resistance. A population of CCL5-producing NK cells with tissue-resident traits enhances T cell–DC cross-talk for successful cancer IL-12 immunotherapy. Interleukin-12 (IL-12) has been a successful form of cancer immunotherapy in preclinical studies, but its translation has been lacking in early patient clinical trials. To investigate this dichotomy, Kirchhammer et al. used an adenovirus platform to treat patient-derived xenografts intratumorally with IL-12 immunotherapy. They saw that the efficacy of IL-12 treatment was dependent on a population of tissue-associated natural killer (NK) cells that produced the chemoattractant CCL5. They saw that treatment failure could be overcome with an intratumoral delivery of CCL5, suggesting that a potential combination treatment could be useful in patients and warrants further study.

DOI: 10.1126/scitranslmed.abm9043 | Read all as PDF | SUPPL: 0502 | Close Abstract
0501

Kopra, K., Valtonen. S., Mahran, R., Kapp, J. N., Hassan, N., Gillette, W., Dennis, B., Li, L., Westover, K. D., Plückthun, A., and Härmä, H. (2022) Thermal shift assay for small GTPase stability screening: evaluation and suitability. Int. J. Mol. Sci. 23, 7095.
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Abstract

Thermal unfolding methods are commonly used as a predictive technique by tracking the protein's physical properties. Inherent protein thermal stability and unfolding profiles of biotherapeutics can help to screen or study potential drugs and to find stabilizing or destabilizing conditions. Differential scanning calorimetry (DSC) is a 'Gold Standard' for thermal stability assays (TSA), but there are also a multitude of other methodologies, such as differential scanning fluorimetry (DSF). The use of an external probe increases the assay throughput, making it more suitable for screening studies, but the current methodologies suffer from relatively low sensitivity. While DSF is an effective tool for screening, interpretation and comparison of the results is often complicated. To overcome these challenges, we compared three thermal stability probes in small GTPase stability studies: SYPRO Orange, 8-anilino-1-naphthalenesulfonic acid (ANS), and the Protein-Probe. We studied mainly KRAS, as a proof of principle to obtain biochemical knowledge through TSA profiles. We showed that the Protein-Probe can work at lower concentration than the other dyes, and its sensitivity enables effective studies with non-covalent and covalent drugs at the nanomolar level. Using examples, we describe the parameters, which must be taken into account when characterizing the effect of drug candidates, of both small molecules and Designed Ankyrin Repeat Proteins.

DOI: 10.3390/ijms23137095 | Read all as PDF | SUPPL: 0501 | Close Abstract
0500

Guimaraes Koch, S. S., Thorpe, R., Kawasaki, N., Lefranc, M. P., Malan, S., Martin, A. C. R., Mignot, G., Plückthun, A., Rizzi, M., Shubat, S., Weisser, K., and Balocco, R. (2022) International nonproprietary names for monoclonal antibodies: an evolving nomenclature system. MAbs 14, 2075078.
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Abstract

Appropriate nomenclature for all pharmaceutical substances is important for clinical development, licensing, prescribing, pharmacovigilance, and identification of counterfeits. Nonproprietary names that are unique and globally recognized for all pharmaceutical substances are assigned by the International Nonproprietary Names (INN) Programme of the World Health Organization (WHO). In 1991, the INN Programme implemented the first nomenclature scheme for monoclonal antibodies. To accompany biotechnological development, this nomenclature scheme has evolved over the years; however, since the scheme was introduced, all pharmacological substances that contained an immunoglobulin variable domain were coined with the stem -mab. To date, there are 879 INN with the stem -mab. Owing to this high number of names ending in -mab, devising new and distinguishable INN has become a challenge. The WHO INN Expert Group therefore decided to revise the system to ease this situation. The revised system was approved and adopted by the WHO at the 73rd INN Consultation held in October 2021, and the radical decision was made to discontinue the use of the well-known stem -mab in naming new antibody-based drugs and going forward, to replace it with four new stems: -tug, -bart, -mig, and -ment.

DOI: 10.1080/19420862.2022.2075078 | Read all as PDF | Close Abstract
0499

Wang, L., Moreira, E. A., Kempf, G., Miyake, Y., Oliveira Esteves, B. I., Fahmi, A., Schaefer, J. V., Dreier, B., Yamauchi, Y., Alves, M. P., Plückthun, A., and Matthias, P. (2022) Disrupting the HDAC6-ubiquitin interaction impairs infection by influenza and Zika virus and cellular stress pathways. Cell Reports 39, 110736
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Abstract

The deacetylase HDAC6 has tandem catalytic domains and a zinc finger domain (ZnF) binding ubiquitin (Ub). While the catalytic domain has an antiviral effect, the ZnF facilitates influenza A virus (IAV) infection and cellular stress responses. By recruiting Ub via the ZnF, HDAC6 promotes the formation of aggresomes and stress granules (SGs), dynamic structures associated with pathologies such as neurodegeneration. IAV subverts the aggresome/HDAC6 pathway to facilitate capsid uncoating during early infection. To target this pathway, we generate designed ankyrin repeat proteins (DARPins) binding the ZnF; one of these prevents interaction with Ub in vitro and in cells. Crystallographic analysis shows that it blocks the ZnF pocket where Ub engages. Conditional expression of this DARPin reversibly impairs infection by IAV and Zika virus; moreover, SGs and aggresomes are downregulated. These results validate the HDAC6 ZnF as an attractive target for drug discovery.

DOI: 10.1016/j.celrep.2022.110736 | Read all as PDF | SUPPL: 0499 Data1 0499 Data2 0499 | Close Abstract
0498

Gisdon, F. J., Kynast, J. P., Ayyildiz, M., Hine, A. V., Plückthun, A. and Höcker, B. (2022) Modular peptide binders – development of a predictive technology as alternative for reagent antibodies. Biol. Chem. 403, 535-543.
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Abstract

Current biomedical research and diagnostics critically depend on detection agents for specific recognition and quantification of protein molecules. Monoclonal antibodies have been used for this purpose over decades and facilitated numerous biological and biomedical investigations. Recently, however, it has become apparent that manycommercial reagent antibodies lack specificity or do not recognize their target at all. Thus, synthetic alternatives are needed whose complex designs are facilitated by multidisciplinary approaches incorporating experimental protein engineering with computational modeling. Here, we review the status of such an engineering endeavor based on the modular armadillo repeat protein scaffold and discuss challenges in its implementation.

DOI: https://doi.org/10.1515/hsz-2021-0384 | Read all as PDF | Close Abstract
0497

Brücher, D., Freitag, P. C., und Plückthun, A. (2022). Zellspezifische Produktion von multiplen Therapeutika im Körper. Biospektrum 28, 155-158.
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Abstract

Many conventional cancer therapies suffer from side effects and low efficacy, due to poor tumor localization of systemically injected therapeutics. Utilizing the adenoviral SHREAD platform, therapeutic genes were delivered specifically to cancer cells in vivo that express a defined, freely selectable cell receptor. Antibodies secreted by infected cells were visualized using a cell-clearing technology, revealing a high local concentration of the antibody within the tumor, with minimal concentration in peripheral healthy tissues.

DOI: 10.1007/s12268-022-1714-9 | Read all as PDF | Close Abstract
0496

Qi, C., Lavriha, P., Mehta, V., Khanppnavar, B., Mohammed, I., Li, Y., Lazaratos, M., Schaefer, J. V., Dreier, B., Plückthun, A., Bondar, A.-N., Dessauer, C. W., and Korkhov, V. M. (2022). Structural basis of adenylyl cyclase 9 activation. Nature Commun. 13, 1045.
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Abstract

Adenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts ATP into cAMP. The enzyme is weakly activated by forskolin, fully activated by the G protein Gαs subunit and is autoinhibited by the AC9 C-terminus. Although our recent structural studies of the AC9-Gαs complex provided the framework for understanding AC9 autoinhibition, the conformational changes that AC9 undergoes in response to activator binding remains poorly understood. Here, we present the cryo-EM structures of AC9 in several distinct states: (i) AC9 bound to a nucleotide inhibitor MANT-GTP, (ii) bound to an artificial activator (DARPin C4) and MANT-GTP, (iii) bound to DARPin C4 and a nucleotide analogue ATPαS, (iv) bound to Gαs and MANT-GTP. The artificial activator DARPin C4 partially activates AC9 by binding at a site that overlaps with the Gαs binding site. Together with the previously observed occluded and forskolin-bound conformations, structural comparisons of AC9 in the four conformations described here show that secondary structure rearrangements in the region surrounding the forskolin binding site are essential for AC9 activation.

DOI: 10.1038/s41467-022-28685-y | Read all as PDF | SUPPL: 0496 Movie explanation 0496 Movie1 0496 Movie2 0496 Movie3 0496 | Close Abstract
0495

Schöppe, J., Ehrenmann, J., Waltenspühl, Y., and Plückthun, A. (2022). Universal platform for the generation of thermostabilized GPCRs that crystallize in LCP. Nature Protoc. 17, 698–726.
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Abstract

Structural studies of G-protein-coupled receptors (GPCRs) are often limited by difficulties in obtaining well-diffracting crystals suitable for high-resolution structure determination. During the past decade, crystallization in lipidic cubic phase (LCP) has become the most successful and widely used technique for obtaining such crystals. Despite often intense efforts, many GPCRs remain refractory to crystallization, even if receptors can be purified in sufficient amounts. To address this issue, we have developed a highly efficient screening and stabilization strategy for GPCRs, based on a fluorescence thermal stability assay readout, which seems to correlate particularly well with those GPCR constructs that remain native during incorporation into the LCP. Detailed protocols are provided for rapid and cost-efficient mutant and construct generation using sequence- and ligation-independent cloning, high-throughput magnetic bead-based protein purification from small-scale expressions in mammalian cells, the screening and optimal combination of mutations for increased receptor thermostability and the rapid identification of suitable chimeric fusion protein constructs for successful crystallization in LCP. We exemplify the method on three receptors from two different classes: the neurokinin 1 receptor, the oxytocin receptor and the parathyroid hormone 1 receptor.

DOI: 10.1038/s41594-018-0151-4 | Read all as PDF | Close Abstract
0494

Deluigi, M., Morstein, L., Schuster, M., Klenk, C., Merklinger, L., Cridge, C. R., de Zhang, L. A., Klipp, A., Vacca, S., Vaid, T. M., Mittl, P. R. E., Egloff, P., Eberle, S. A., Zerbe, O., Chalmers, D. K., Scott, D. J., and Plückthun, A. (2022). Crystal structure of the α1B-adrenergic receptor reveals molecular determinants of selective ligand recognition. Nature Commun. 13, 382.
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Abstract

α-adrenergic receptors (αARs) are G protein-coupled receptors that regulate vital functions of the cardiovascular and nervous systems. The therapeutic potential of αARs, however, is largely unexploited and hampered by the scarcity of subtype-selective ligands. Moreover, several aminergic drugs either show off-target binding to αARs or fail to interact with the desired subtype. Here, we report the crystal structure of human α1BAR bound to the inverse agonist (+)-cyclazosin, enabled by the fusion to a DARPin crystallization chaperone. The α1BAR structure allows the identification of two unique secondary binding pockets. By structural comparison of α1BAR with α2ARs, and by constructing α1BAR-α2CAR chimeras, we identify residues 3.29 and 6.55 as key determinants of ligand selectivity. Our findings provide a basis for discovery of α1BAR-selective ligands and may guide the optimization of aminergic drugs to prevent off-target binding to αARs, or to elicit a selective interaction with the desired subtype.

DOI: 10.1038/s41467-021-27911-3 | Read all as PDF | SUPPL: 0494 Movie explanation 0494 Movie 0494 | Close Abstract

2021

# Reference PDF

0493

Bieri, M., Hendrickx, R., Bauer, M., Yu, B., Jetzer, T., Dreier, B., Mittl, P. R. E., Sobek, J., Plückthun, A., Greber, U. F., and Hemmi S. (2921). The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection. PLoS Pathog. 17, e1010083.
| Abstract + further info

Abstract

Mammalian adenoviruses (AdVs) comprise more than ~350 types including over 100 human (HAdVs) and just three mouse AdVs (MAdVs). While most HAdVs initiate infection by high affinity/avidity binding of their fiber knob (FK) protein to either coxsackievirus AdV receptor (CAR), CD46 or desmoglein (DSG)-2, MAdV-1 (M1) infection requires arginine-glycine-aspartate (RGD) binding integrins. To identify the receptors mediating MAdV infection we generated five novel reporter viruses for MAdV-1/-2/-3 (M1, M2, M3) transducing permissive murine (m) CMT-93 cells, but not B16 mouse melanoma cells expressing mCAR, human (h) CD46 or hDSG-2. Recombinant M1 or M3 FKs cross-blocked M1 and M3 but not M2 infections. Profiling of murine and human cells expressing RGD-binding integrins suggested that αvβ6 and αvβ8 heterodimers are associated with M1 and M3 infections. Ectopic expression of mβ6 in B16 cells strongly enhanced M1 and M3 binding, infection, and progeny production comparable with mαvβ6-positive CMT-93 cells, whereas mβ8 expressing cells were more permissive to M1 than M3. Anti-integrin antibodies potently blocked M1 and M3 binding and infection of CMT-93 cells and hαvβ8-positive M000216 cells. Soluble integrin αvβ6, and synthetic peptides containing the RGDLXXL sequence derived from FK-M1, FK-M3 and foot and mouth disease virus coat protein strongly interfered with M1/M3 infections, in agreement with high affinity interactions of FK-M1/FK-M3 with αvβ6/αvβ8, determined by surface plasmon resonance measurements. Molecular docking simulations of ternary complexes revealed a bent conformation of RGDLXXL-containing FK-M3 peptides on the subunit interface of αvβ6/β8, where the distal leucine residue dips into a hydrophobic pocket of β6/8, the arginine residue ionically engages αv aspartate215, and the aspartate residue coordinates a divalent cation in αvβ6/β8. Together, the RGDLXXL-bearing FKs are part of an essential mechanism for M1/M3 infection engaging murine and human αvβ6/8 integrins. These integrins are highly conserved in other mammals, and may favour cross-species virus transmission.

DOI: 10.1371/journal.ppat.1010083 | Read all as PDF | Close Abstract
0492

Thom, C., Ehrenmann, J., Vacca, S., Waltenspühl, Y., Schöppe, J., Medalia, O., and Plückthun A. (2021) Structures of neurokinin 1 receptor in complex with Gq and Gs proteins reveal substance P binding mode and unique activation features. Science Adv. 7, eabk2872.
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Abstract

The neurokinin 1 receptor (NK1R) is involved in inflammation and pain transmission. This pathophysiologically important G protein–coupled receptor is predominantly activated by its cognate agonist substance P (SP) but also by the closely related neurokinins A and B. Here, we report cryo–electron microscopy structures of SP-bound NK1R in complex with its primary downstream signal mediators, Gq and Gs. Our structures reveal how a polar network at the extracellular, solvent-exposed receptor surface shapes the orthosteric pocket and that NK1R adopts a noncanonical active-state conformation with an interface for G protein binding, which is distinct from previously reported structures. Detailed comparisons with antagonist-bound NK1R crystal structures reveal that insurmountable antagonists induce a distinct and long-lasting receptor conformation that sterically blocks SP binding. Together, our structures provide important structural insights into ligand and G protein promiscuity, the lack of basal signaling, and agonist- and antagonist-induced conformations in the neurokinin receptor family.

DOI: 10.1126/sciadv.abk2872 | Read all as PDF | SUPPL: 0492 | Close Abstract
0491

Friedrich, N., Stiegeler, E., Glögl, M., Lemmin, T., Hansen, S., Kadelka, C., Wu, Y., Ernst, P., Maliqi, L., Foulkes, C., Morin, M., Eroglu, M., Liechti, T., Ivan, B., Reinberg, T., Schaefer, J. V., Karakus, U., Ursprung, S., Mann, A., Rusert, P., Kouyos, R. D., Robinson, J. A., Günthard, H. F., Plückthun, A., and Trkola, A. (2021) Distinct conformations of the HIV-1 V3 loop crown are targetable for broad neutralization. Nature Commun. 12, 6705.
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Abstract

The V3 loop of the HIV-1 envelope (Env) protein elicits a vigorous, but largely non-neutralizing antibody response directed to the V3-crown, whereas rare broadly neutralizing antibodies (bnAbs) target the V3-base. Challenging this view, we present V3-crown directed broadly neutralizing Designed Ankyrin Repeat Proteins (bnDs) matching the breadth of V3-base bnAbs. While most bnAbs target prefusion Env, V3-crown bnDs bind open Env conformations triggered by CD4 engagement. BnDs achieve breadth by focusing on highly conserved residues that are accessible in two distinct V3 conformations, one of which resembles CCR5-bound V3. We further show that these V3-crown conformations can, in principle, be attacked by antibodies. Supporting this conclusion, analysis of antibody binding activity in the Swiss 4.5 K HIV-1 cohort (n = 4,281) revealed a co-evolution of V3-crown reactivities and neutralization breadth. Our results indicate a role of V3-crown responses and its conformational preferences in bnAb development to be considered in preventive and therapeutic approaches.

DOI: 10.1038/s41467-021-27075-0 | Read all as PDF | SUPPL: 0491 | Close Abstract
0490

Matsuda, S., Schaefer, J. V., Mii, Y., Hori, Y., Bieli, D., Taira, M., Plückthun, A., and Affolter, M. (2021). Asymmetric requirement of Dpp/BMP morphogen dispersal in the Drosophila wing disc. Nature Commun. 12, 6435.
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Abstract

How morphogen gradients control patterning and growth in developing tissues remains largely unknown due to lack of tools manipulating morphogen gradients. Here, we generate two membrane-tethered protein binders that manipulate different aspects of Decapentaplegic (Dpp), a morphogen required for overall patterning and growth of the Drosophila wing. One is "HA trap" based on a single-chain variable fragment (scFv) against the HA tag that traps HA-Dpp to mainly block its dispersal, the other is "Dpp trap" based on a Designed Ankyrin Repeat Protein (DARPin) against Dpp that traps Dpp to block both its dispersal and signaling. Using these tools, we found that, while posterior patterning and growth require Dpp dispersal, anterior patterning and growth largely proceed without Dpp dispersal. We show that dpp transcriptional refinement from an initially uniform to a localized expression and persistent signaling in transient dpp source cells render the anterior compartment robust against the absence of Dpp dispersal. Furthermore, despite a critical requirement of dpp for the overall wing growth, neither Dpp dispersal nor direct signaling is critical for lateral wing growth after wing pouch specification. These results challenge the long-standing dogma that Dpp dispersal is strictly required to control and coordinate overall wing patterning and growth.

DOI: 10.1038/s41467-021-26726-6 | Read all as PDF | SUPPL: 0490 | Close Abstract
0489

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.

DOI: 10.1038/s41594-021-00667-6 | Read all as PDF | SUPPL: 0489 Video0489 1 Video0489 2 Video0489 3 NewsViews0489 | Close Abstract
0488

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.

DOI: 10.1007/s10858-021-00376-8 | Read all as PDF | SUPPL: 0488 Video S16 | Close Abstract
0487

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.

DOI: 10.1021/acssynbio.1c00357 | Read all as PDF | SUPPL: 0487 | Close Abstract
0486

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.

DOI: 10.3390/pharmaceutics13081285 | Read all as PDF | SUPPL: 0486 | Close Abstract
0485

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.

DOI: 10.1016/j.ultrasmedbio.2021.04.027 | Read all as PDF | SUPPL: 0485 | Close Abstract
0484

Nemergut, M., Škrabana, R., Berta, M., Plückthun, A., Sedlák, E. (2021). Purification of MBP fusion proteins using engineered DARPin affinity matrix. Int. J. Biol. Macromol. 187, 105-112
| Abstract + further info

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.

DOI: 10.1016/j.ijbiomac.2021.07.117 | Read all as PDF | SUPPL: 0484 | Close Abstract
0483

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.

DOI: 10.1016/j.ejpb.2021.07.011 | Read all as PDF | SUPPL: 0483 | Close Abstract
0482

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.

DOI: 10.1080/19420862.2021.1950265 | Read all as PDF | Close Abstract
0481

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.

DOI: 10.1107/S2053230X21006002 | Read all as PDF | SUPPL: 0481 | Close Abstract
0480

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.

DOI: 10.1038/s42003-021-02253-4 | Read all as PDF | SUPPL: 0480 Commun Biol movie5 Commun Biol movie6 Commun Biol movie7 Commun Biol movie8 Commun Biol movie9 Commun Biol movie10 Commun Biol movie11 | Close Abstract
0479

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 Commun. 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.

DOI: 10.1038/s41467-021-23948-6 | Read all as PDF | SUPPL: 0479 | Close Abstract
0478

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.

DOI: 10.1073/pnas.2015037118 | Read all as PDF | SUPPL: 0478 | Close Abstract
0477

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.

DOI: 10.1073/pnas.2017925118 | Read all as PDF | SUPPL: 0477 PNAS Explanation movies PNAS movie5 PNAS movie1 PNAS movie2 PNAS movie3 PNAS movie4 | Close Abstract
0476

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.

DOI: 10.1021/acssensors.0c02480 | Read all as PDF | Close Abstract
0475

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 receptor. Sci. 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.

DOI: 10.1038/s41598-021-87793-9 | Read all as PDF | SUPPL: 0475 | Close Abstract
0474

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.

DOI: 10.3390/molecules26051465 | Read all as PDF | Close Abstract
0473

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.
| Abstract + further info

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.

DOI: 10.1038/s41594-020-00554-6 | Read all as PDF | SUPPL: 0473 | Close Abstract
0472

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.

DOI: 10.1016/j.omtm.2021.01.002 | Read all as PDF | Close Abstract
0471

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. 7, eabe5504.
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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.

DOI: 10.1126/sciadv.abe5504 | Read all as PDF | SUPPL: 0471 corr | Close Abstract
0470

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.

DOI: 10.1002/cbic.202000541 | Read all as PDF | SUPPL: 0470 | Close Abstract
0469

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.

DOI: 10.15252/embj.2020104416 | Read all as PDF | Close Abstract

2020

# Reference PDF

0468

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.
| Abstract + further info

Abstract

No abstract

DOI: 10.1038/s41587-020-0687-9 | Read all as PDF | Close Abstract
0467

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.

| Abstract + further info

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.

DOI: 10.1038/s41467-020-18981-w | Read all as PDF | SUPPL: 0467 1 0467 2 0467 3 0467 4 0467 5 0467 6 0467 7 | Close Abstract
0466

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.
| Abstract + further info

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.

DOI: 10.1186/s12915-020-00827-y | Read all as PDF | SUPPL: 0466 | Close Abstract
0465

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.
| Abstract + further info

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.

DOI: 10.1016/j.jconrel.2020.08.004 | Read all as PDF | SUPPL: 0465 | Close Abstract
0464

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
| Abstract + further info

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.

DOI: 10.1080/19420862.2020.1792084 | Read all as PDF | SUPPL: 0464 1 0464 2 0464 3 0464 4 0464 5 0464 6 | Close Abstract
0463

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
| Abstract + further info

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.

DOI: 10.1080/19420862.2020.1794421 | Read all as PDF | Close Abstract
0462

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. Nature Methods 17, 755-756.
| Abstract + further info

Abstract

No abstract

DOI: 10.1038/s41592-020-0906-9 | Read all as PDF | Close Abstract
0461

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.

DOI: 10.1126/sciadv.abb5419 | Read all as PDF | SUPPL: 0461 | Close Abstract
0460

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.
| Abstract + further info

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.

DOI: 10.1038/s42003-020-1072-4 | Read all as PDF | SUPPL: 0460 1 0460 2 | Close Abstract
0459

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.
| Abstract + further info

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.

DOI: 10.1128/ JVI.00542-20 | Read all as PDF | Close Abstract
0458

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.
| Abstract + further info

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.

DOI: 10.1016/j.bbamem.2020.183354 | Read all as PDF | SUPPL: 0458 | Close Abstract
0457

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 .
| Abstract + further info

Abstract

No abstract

DOI: 10.1038/d41586-020-01474-7 | Read all as PDF | Close Abstract
0456

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.
| Abstract + further info

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.

DOI: 10.1158/1535-7163.MCT-19-0633 | Read all as PDF | Close Abstract
0455

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.
| Abstract + further info

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.

DOI: 10.1038/s41598-020-60861-2 | Read all as PDF | SUPPL: 0455 | Close Abstract
0454

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.
| Abstract + further info

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.

DOI: 10.1021/acschembio.9b00928 | Read all as PDF | SUPPL: 0454 | Close Abstract
0453

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

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.

DOI: 10.1016/j.sbi.2019.12.009 | Read all as PDF | Close Abstract

2019

# Reference PDF

0452

Stüber, J. C. and Plückthun, A. (2019) Labeling surface proteins with high specificity: Intrinsic limitations of phosphopantetheinyl transferase systems. PLoS One 14, e0226579.
| Abstract + further info

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.

DOI: 10.1371/journal.pone.0226579 | Read all as PDF | Close Abstract
0451

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.
| Abstract + further info

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.

DOI: 10.1111/febs.15115 | Read all as PDF | Close Abstract
0450

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.
| Abstract + further info

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.

DOI: 10.1158/1078-0432.CCR-19-1479 | Read all as PDF | SUPPL: 0450 | Close Abstract
0449

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.
| Abstract + further info

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.

DOI: 10.1038/s41598-019-54939-9 | Read all as PDF | SUPPL: 0449 | Close Abstract
0448

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.
| Abstract + further info

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.

DOI: 10.1038/s41598-019-52121-9 | Read all as PDF | SUPPL: 0448 | Close Abstract
0447

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.
| Abstract + further info

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.

DOI: 10.1016/j.celrep.2019.09.062 | Read all as PDF | SUPPL: 0447 1 0447 2 0447 3 0447 4 0447 5 0447 6 0447 7 0447 8 0447 9 | Close Abstract
0446

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.
| Abstract + further info

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.

DOI: 10.1038/s41598-019-51017-y | Read all as PDF | Close Abstract
0445

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.
| Abstract + further info

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.

DOI: 10.1073/pnas.1912705116 | Read all as PDF | SUPPL: 0445 | Close Abstract
0444

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.
| Abstract + further info

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.

DOI: 10.1177/2472555219837344 | Read all as PDF | SUPPL: 0444 | Close Abstract
0443

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.
| Abstract + further info

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.

DOI: 10.1016/j.jconrel.2019.06.030 | Read all as PDF | SUPPL: 0443 | Close Abstract
0442

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.

DOI: 10.1007/978-1-4939-9654-4_17 | Read all as PDF | Close Abstract
0441

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.
| Abstract + further info

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.

DOI: 10.1007/978-1-4939-9654-4_14 | Read all as PDF | Close Abstract
0440

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.

DOI: 10.1021/acschembio.9b00016 | Read all as PDF | SUPPL: 0440 | Close Abstract
0439

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.

DOI: 10.1016/j.jmb.2019.05.005 | Read all as PDF | SUPPL: 0439 | Close Abstract
0438

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.
| Abstract + further info

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.

DOI: 10.1016/j.ejpb.2019.05.015 | Read all as PDF | SUPPL: 0438 | Close Abstract
0437

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.
| Abstract + further info

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.

DOI: 10.1073/pnas.1813559116 | Read all as PDF | SUPPL: 0437 | Close Abstract
0436

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 Commun. 10, 1830.
| Abstract + further info

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.

DOI: 10.1038/s41467-019-09854-y | Read all as PDF | SUPPL: 0436 | Close Abstract
0435

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.
| Abstract + further info

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.

DOI: 10.1016/j.jmb.2019.03.024 | Read all as PDF | SUPPL: 0435 | Close Abstract
0434

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.
| Abstract + further info

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.

DOI: 10.1021/acs.bioconjchem.9b00112 | Read all as PDF | Close Abstract
0433

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.
| Abstract + further info

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.

DOI: 10.1038/s41598-019-39492-9 | Read all as PDF | SUPPL: 0433 1 0433 2 | Close Abstract
0432

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

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.

DOI: 10.1515/hsz-2018-0355 | Read all as PDF | SUPPL: 0432 | Close Abstract
0431

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.
| Abstract + further info

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.

DOI: 10.1126/scisignal.aau2875 | Read all as PDF | SUPPL: 0431 1 0431 2 0431 3 0431 4 | Close Abstract
0430

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.
| Abstract + further info

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.

DOI: 10.1371/journal.pone.0210131 | Read all as PDF | Close Abstract
0429

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 Commun. 10, 17.
| Abstract + further info

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.

DOI: 10.1038/s41467-018-07939-8 | Read all as PDF | SUPPL: 0429 | Close Abstract

2018

# Reference PDF

0428

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.
| Abstract + further info

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.

DOI: 10.1038/s41594-018-0151-4 | Read all as PDF | SUPPL: 0428 | Close Abstract
0427

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.
| Abstract + further info

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.

DOI: 10.1242/bio.036749 | Read all as PDF | SUPPL: 0427 | Close Abstract
0426

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.
| Abstract + further info

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.

DOI: 10.1021/acssynbio.8b00224 | Read all as PDF | SUPPL: 0426 | Close Abstract
0425

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.
| Abstract + further info

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.

DOI: 10.1080/19420862.2018.1450126 | Read all as PDF | SUPPL: 0425 | Close Abstract
0424

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

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.

DOI: 10.1007/978-1-4939-8648-4_13 | Read all as PDF | SUPPL: 0424 | Close Abstract
0423

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.
| Abstract + further info

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.

DOI: 10.1007/s10858-018-0175-4 | Read all as PDF | SUPPL: 0423 | Close Abstract
0422

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.

DOI: 10.1016/j.omtm.2018.07.001 | Read all as PDF | SUPPL: Correction 0422 | Close Abstract
0421

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.

DOI: 10.1038/s41586-018-0325-6 | Read all as PDF | SUPPL: 0421 1 0421 2 0421 3 0421 4 0421 5 | Close Abstract
0420

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.

DOI: 10.1371/journal.pcbi.1006182 | Read all as PDF | SUPPL: 0420 | Close Abstract
0419

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.
| Abstract + further info

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.

DOI: 10.1016/j.jmb.2017.10.032 | Read all as PDF | SUPPL: 0419 | Close Abstract
0418

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.
| Abstract + further info

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.

DOI: 10.1021/acschembio.8b00191 | Read all as PDF | SUPPL: 0418 | Close Abstract
0417

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.

DOI: 10.1002/anie.201713377 | Read all as PDF | SUPPL: 0417 | Close Abstract
0416

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.
| Abstract + further info

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.

DOI: 10.1371/journal.ppat.1006914 | Read all as PDF | Close Abstract
0415

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.
| Abstract + further info

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.

DOI: 10.1016/j.jsb.2017.08.009 | Read all as PDF | SUPPL: 0415 | Close Abstract
0414

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 Commun. 9, 450.
| Abstract + further info

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.

DOI: 10.1038/s41467-017-02707-6 | Read all as PDF | SUPPL: 0414 | Close Abstract

2017

# Reference PDF

0413

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.
| Abstract + further info

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.

DOI: 10.1038/s41598-017-15711-z | Read all as PDF | SUPPL: 0413 | Close Abstract
0412

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.
| Abstract + further info

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.

DOI: 10.1002/pro.3278 | Read all as PDF | SUPPL: 0412 | Close Abstract
0411

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.
| Abstract + further info

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.

DOI: 10.1038/s41598-017-13469-y | Read all as PDF | SUPPL: 0411 | Close Abstract
0410

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.
| Abstract + further info

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.

DOI: 10.1002/pro.3229 | Read all as PDF | Close Abstract
0409

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.
| Abstract + further info

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.

DOI: 10.1038/s41598-017-11472-x | Read all as PDF | SUPPL: 0409 | Close Abstract
0408

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.
| Abstract + further info

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.

DOI: 10.1002/pro.3216 | Read all as PDF | SUPPL: 0408 | Close Abstract
0407

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.
| Abstract + further info

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.

DOI: 10.1080/19420862.2017.1341029 | Read all as PDF | Close Abstract
0406

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 Commun. 8, 45.
| Abstract + further info

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.

DOI: 10.1038/s41467-017-00062-0 | Read all as PDF | SUPPL: 0406 | Close Abstract
0405

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.
| Abstract + further info

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.

DOI: 10.1371/journal.pone.0175842 | Read all as PDF | SUPPL: 0405 1 0405 3 0405 3 0405 4 0405 5 0405 6 0405 7 0405 8 0405 9 0405 10 | Close Abstract
0404

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.
| Abstract + further info

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.

DOI: 10.1021/acschembio.6b00646 | Read all as PDF | SUPPL: 0404 | Close Abstract
0403

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.
| Abstract + further info

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.

DOI: 10.1007/978-1-4939-6857-2_14 | Read all as PDF | Close Abstract
0402

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.
| Abstract + further info

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.

DOI: 10.1007/s00216-016-0127-3 | Read all as PDF | SUPPL: 0402 | Close Abstract
0401

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.
| Abstract + further info

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.

DOI: 10.1038/srep39756 | Read all as PDF | SUPPL: 0401 | Close Abstract
0400

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.
| Abstract + further info

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.

DOI: 10.1038/nmeth.4079 | Read all as PDF | SUPPL: 0400 | Close Abstract
0399

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

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.

DOI: 10.1515/hsz-2016-0233 | Read all as PDF | Close Abstract

2016

# Reference PDF

0398

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.
| Abstract + further info

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.

DOI: 10.1016/j.jmb.2016.09.012 | Read all as PDF | SUPPL: 0398 | Close Abstract
0397

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.
| Abstract + further info

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.

DOI: 10.1038/srep28922 | Read all as PDF | SUPPL: 0397 | Close Abstract
0396

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 + further info

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.

DOI: 10.1073/pnas.1604125113 | Read all as PDF | SUPPL: 0396 | Close Abstract
0395

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.
| Abstract + further info

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.

DOI: 10.1371/journal.ppat.1005641 | Read all as PDF | SUPPL: 0395 | Close Abstract
0394

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 Commun. 7, 11672.
| Abstract + further info

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

DOI: 10.1038/ncomms11672 | Read all as PDF | SUPPL: 0394 | Close Abstract
0393

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.
| Abstract + further info

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.

DOI: 10.1016/j.jmb.2016.03.002 | Read all as PDF | SUPPL: 0393 | Close Abstract
0392

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.
| Abstract + further info

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.

DOI: 10.1016/j.jmb.2016.01.017 | Read all as PDF | SUPPL: 0392 | Close Abstract
0391

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.
| Abstract + further info

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.

DOI: 10.1021/jacs.6b00099 | Read all as PDF | SUPPL: 0391 | Close Abstract
0390

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.
| Abstract + further info

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.

DOI: 10.1038/srep21508. | Read all as PDF | SUPPL: 0390 | Close Abstract
0389

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.
| Abstract + further info

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.

DOI: 10.1038/mto.2016.3 | Read all as PDF | SUPPL: 0389 | Close Abstract
0388

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.
| Abstract + further info

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.

DOI: 10.1038/srep21294 | Read all as PDF | SUPPL: 0388 | Close Abstract
0387

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.
| Abstract + further info

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.

DOI: 10.1080/19420862.2015.1114320 | Read all as PDF | Close Abstract
0386

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.
| Abstract + further info

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

0385

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.
| Abstract + further info

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.

DOI: 10.1038/nmeth.3475 | Read all as PDF | SUPPL: 0385 | Close Abstract
0384

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.
| Abstract + further info

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.

DOI: 10.1021/acs.bioconjchem.5b00260 | Read all as PDF | Close Abstract
0383

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.
| Abstract + further info

Abstract

No abstract

DOI: 10.1093/protein/gzv051 | Read all as PDF | Close Abstract
0382

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.
| Abstract + further info

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.

DOI: 10.1002/pro.2691 | Read all as PDF | SUPPL: 0382 | Close Abstract
0381

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.
| Abstract + further info

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.

DOI: 10.1016/j.jmb.2015.02.022 | Read all as PDF | SUPPL: 0381 0381 RealTimeSignalingVideo | Close Abstract
0380

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.
| Abstract + further info

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.

DOI: 10.1093/protein/gzv005 | Read all as PDF | SUPPL: 0380 | Close Abstract
0379

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.

DOI: 10.1073/pnas.1417573112 | Read all as PDF | SUPPL: 0379 | Close Abstract
0378

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.
| Abstract + further info

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.

DOI: 10.1016/j.pep.2014.10.006 | Read all as PDF | SUPPL: 0378 | Close Abstract
0377

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.
| Abstract + further info

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.

DOI: 10.1016/j.jconrel.2015.01.027 | Read all as PDF | SUPPL: 0377 | Close Abstract
0376

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 Commun. 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.

DOI: 10.1038/ncomms7246 | Read all as PDF | SUPPL: 0376 | Close Abstract
0375

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.
| Abstract + further info

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.

DOI: 10.1007/s00259-014-2940-2 | Read all as PDF | SUPPL: 0375 | Close Abstract
0374

Bradbury, A. and Plückthun, A. (2015) Standardize antibodies used in research. Nature 518, 27-29.
| Abstract + further info

Abstract

No abstract

Read all as PDF | SUPPL: 0374 | Close Abstract
0373

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.

DOI: 10.1002/anie.201409791 | Read all as PDF | SUPPL: 0373 | Close Abstract
0372

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

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.

DOI: 10.1146/annurev-pharmtox-010611-134654 | Read all as PDF | Close Abstract
0371

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.
| Abstract + further info

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.

DOI: 10.1016/j.jconrel.2014.12.019 | Read all as PDF | SUPPL: 0371 | Close Abstract

2014

# Reference PDF

0370

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.
| Abstract + further info

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.

DOI: 10.1021/bc500468s | Read all as PDF | SUPPL: 0370 | Close Abstract
0369

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.
| Abstract + further info

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.

DOI: 10.1242/bio.201410041 | Read all as PDF | Close Abstract
0368

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.
| Abstract + further info

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.

DOI: 10.1002/pro.2535 | Read all as PDF | SUPPL: 0368 | Close Abstract
0367

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.
| Abstract + further info

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.

DOI: 10.1074/jbc.M114.564013 | Read all as PDF | Close Abstract
0366

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.
| Abstract + further info

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.

DOI: 10.1371/journal.pone.0108061 | Read all as PDF | SUPPL: 0366 | Close Abstract
0365

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.
| Abstract + further info

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.

DOI: 10.1126/scisignal.2005157 | Read all as PDF | SUPPL: 0365 0365 MovieS1 0365 MovieS2 0365 MovieS3 0365 MovieS4 | Close Abstract
0364

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.
| Abstract + further info

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.

DOI: 10.1093/nar/gku571 | Read all as PDF | SUPPL: 0364 | Close Abstract
0363

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.
| Abstract + further info

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.

DOI: 10.1016/j.bbamem.2014.07.015 | Read all as PDF | Close Abstract
0362

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.
| Abstract + further info

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.

DOI: 10.1016/j.sbi.2014.05.011 | Read all as PDF | Close Abstract
0361

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.
| Abstract + further info

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.

DOI: 10.1016/j.str.2014.05.002 | Read all as PDF | Close Abstract
0360

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.

DOI: 10.1016/j.jmb.2014.04.010 | Read all as PDF | SUPPL: 0360 | Close Abstract
0359

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.

DOI: 10.1073/pnas.1317903111 | Read all as PDF | Close Abstract
0358

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.

DOI: 10.1016/j.jsb.2013.07.012 | Read all as PDF | Close Abstract
0357

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.

DOI: 10.1158/1535-7163.MCT-13-0523 | Read all as PDF | SUPPL: 0357 | Close Abstract
0356

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.

DOI: 10.1016/j.jmb.2013.10.026 | Read all as PDF | SUPPL: 0356 | Close Abstract

2013

# Reference PDF

0355

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.

DOI: 10.1021/bc4004102 | Read all as PDF | SUPPL: 0355 | Close Abstract
0354

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.

DOI: 10.1016/j.str.2013.08.020 | Read all as PDF | SUPPL: 0354 | Close Abstract
0353

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.

DOI: 10.1038/nsmb.2624 | Read all as PDF | SUPPL: 0353 1 0353 2 | Close Abstract
0352

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.

DOI: 10.1016/j.chembiol.2013.04.016 | Read all as PDF | SUPPL: 0352 0352 RealTimeSignalingVideo1 0352 RealTimeSignalingVideo2 | Close Abstract
0351

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.

DOI: 10.1016/j.cbpa.2013.04.002 | Read all as PDF | Close Abstract
0350

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.

DOI: 10.1128/JVI.00152-13 | Read all as PDF | SUPPL: 0350 | Close Abstract
0349

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.

DOI: 10.1371/journal.pone.0059163 | Read all as PDF | SUPPL: 0349 | Close Abstract
0348

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.

DOI: 10.1038/mt.2013.16 | Read all as PDF | Close Abstract
0347

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.

DOI: 10.1517/17425247.2013.759938 | Read all as PDF | Close Abstract
0346

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.

DOI: 10.1073/pnas.1213653110 | Read all as PDF | Close Abstract
0345

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.

DOI: 10.1016/j.jmb.2012.11.015 | Read all as PDF | Close Abstract
0344

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

DOI: 10.1016/B978-0-12-391861-1.00004-6 | Read all as PDF | Close Abstract

2012

# Reference PDF

0343

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.
| Abstract + further info

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.

DOI: 10.1016/j.nbt.2012.05.005 | Read all as PDF | Close Abstract
0342

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.

DOI: 10.1016/j.jmb.2012.08.029 | Read all as PDF | SUPPL: 0342 | Close Abstract
0341

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.
| Abstract + further info

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.

DOI: 10.1038/nbt.2354 | Read all as PDF | SUPPL: 0341 1 0341 2 0341 3 0341 4 0341 5 0341 6 0341 7 0341 8 0341 9 | Close Abstract
0340

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.

DOI: 10.1093/protein/gzs039 | Read all as PDF | SUPPL: 0340 | Close Abstract
0339

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.

DOI: 10.1074/jbc.M112.383869 | Read all as PDF | Close Abstract
0338

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.

DOI: 10.1002/pro.2117 | Read all as PDF | SUPPL: 0338 | Close Abstract
0337

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.

DOI: 10.1073/pnas.1205399109 | Read all as PDF | Close Abstract
0336

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.

DOI: 10.1021/cb3001167 | Read all as PDF | SUPPL: 0336 | Close Abstract
0335

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.

DOI: 10.1016/j.jmb.2012.05.039 | Read all as PDF | SUPPL: 0335 | Close Abstract
0334

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.

DOI: 10.1073/pnas.1204129109 | Read all as PDF | Close Abstract
0333

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.

DOI: 10.1073/pnas.1202107109 | Read all as PDF | SUPPL: 0333 | Close Abstract
0332

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.

DOI: 10.1002/pro.2085 | Read all as PDF | SUPPL: 0332 | Close Abstract
0331

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.

DOI: 10.1021/bc200591x | Read all as PDF | SUPPL: 0331 | Close Abstract
0330

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.

DOI: 10.1016/j.jmb.2012.01.027 | Read all as PDF | SUPPL: 0330 | Close Abstract
0329

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.

DOI: 10.1016/B978-0-12-396962-0.00005-7 | Read all as PDF | Close Abstract
0328

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.

DOI: 10.1007/978-1-61779-379-0_15 | Read all as PDF | Close Abstract
0327

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.

DOI: 10.1007/978-1-61779-379-0_1 | Read all as PDF | Close Abstract

2011

# Reference PDF

0326

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.

DOI: 10.1016/j.copbio.2011.06.004 | Read all as PDF | Close Abstract
0325

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.

DOI: 10.1074/jbc.M111.293266 | Read all as PDF | SUPPL: 0325 | Close Abstract
0324

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.

DOI: 10.1016/j.jmb.2011.09.016 | Read all as PDF | SUPPL: 0324 | Close Abstract
0323

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

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0322

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.

DOI: 10.1007/s00249-011-0743-y | Read all as PDF | Close Abstract
0321

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.

DOI: 10.1016/j.jmb.2011.02.051 | Read all as PDF | SUPPL: 0321 | Close Abstract
0320

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.
| Abstract + further info

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.

DOI: 10.1038/mt.2010.298 | Read all as PDF | Close Abstract
0319

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

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0318

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.

DOI: 10.1158/1078-0432.CCR-10-1303 | Read all as PDF | SUPPL: 0318 | Close Abstract
0317

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.

DOI: 10.1016/j.jmb.2010.10.040 | Read all as PDF | SUPPL: 0317 | Close Abstract
0316

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).

DOI: 10.1007/978-1-60761-944-4_21 | Read all as PDF | Close Abstract

2010

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0315

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.

DOI: 10.1016/j.jmb.2010.09.023 | Read all as PDF | Close Abstract
0314

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

No abstract

DOI: 10.1038/467537a | Read all as PDF | Close Abstract
0313

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.

DOI: 10.1038/modpathol.2010.103 | Read all as PDF | SUPPL: 0313 1 0313 2 0313 3 | Close Abstract
0312

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.
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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.

DOI: 10.1016/j.jmb.2010.07.031 | Read all as PDF | SUPPL: 0312 | Close Abstract
0311

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.

DOI: 10.1093/protein/gzp087 | Read all as PDF | Close Abstract
0310

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.

DOI: 10.1093/protein/gzp074 | Read all as PDF | Close Abstract
0309

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

DOI: 10.1007/978-3-642-01147-4_27 | Read all as PDF | Close Abstract
0308

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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0307

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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0306

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.

DOI: 10.1158/0008-5472.CAN-09-2724 | Read all as PDF | SUPPL: 0306 | Close Abstract

2009

# Reference PDF

0305

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
| Abstract + further info

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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0304

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.
| Abstract + further info

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.

DOI: 10.1074/jbc.M109.037812 | Read all as PDF | Close Abstract
0303

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).

DOI: 10.1158/1535-7163.MCT-09-0402 | Read all as PDF | Close Abstract
0302

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.

DOI: 10.1093/protein/gzp011 | Read all as PDF | Close Abstract
0301

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.

DOI: 10.1186/1475-2859-8-8 | Read all as PDF | SUPPL: 0301 | Close Abstract
0300

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.

DOI: 10.1093/protein/gzn079 | Read all as PDF | SUPPL: 0300 | Close Abstract
0299

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.

DOI: 10.1093/protein/gzn077 | Read all as PDF | SUPPL: 0299 | Close Abstract

2008

# Reference PDF

0298

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.

DOI: 10.1073/pnas.0803103105 | Read all as PDF | SUPPL: 0298 | Close Abstract
0297

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.
| Abstract + further info

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.

DOI: 10.1016/J.Jmb.2008.07.085 | Read all as PDF | SUPPL: 0297 | Close Abstract
0296

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.
| Abstract + further info

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.

DOI: 10.1107/S0907444908007282 | Read all as PDF | Close Abstract
0295

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.

DOI: 10.1016/j.jmb.2007.12.014 | Read all as PDF | SUPPL: 0295 | Close Abstract
0294

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.

DOI: 10.1016/j.jmb.2007.11.046 | Read all as PDF | SUPPL: 0294 | Close Abstract
0293

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.
| Abstract + further info

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.

DOI: 10.1016/j.jmb.2007.09.042 | Read all as PDF | SUPPL: 0293 | Close Abstract
0292

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.
| Abstract + further info

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.

DOI: 10.1016/j.jmb.2007.11.047 | Read all as PDF | Close Abstract

2007

# Reference PDF

0291

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.
| Abstract + further info

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.

DOI: 10.1158/1535-7163.MCT-07-0615 | Read all as PDF | Close Abstract
0290

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.
| Abstract + further info

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.

DOI: 10.1529/biophysj.107.112532 | Read all as PDF | Close Abstract
0289

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 + further info

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.

DOI: 10.1016/j.jsb.2007.01.013 | Read all as PDF | SUPPL: 0289 | Close Abstract
0288

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 + further info

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.

DOI: 10.1016/j.jmb.2007.03.028 | Read all as PDF | SUPPL: 0288 | Close Abstract
0287

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 + further info

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.

DOI: 10.1016/j.str.2007.03.014 | Read all as PDF | SUPPL: 0287 | Close Abstract
0286

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 + further info

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.

DOI: 10.1038/nmeth1003 | Read all as PDF | SUPPL: 0286 | Close Abstract
0285

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 + further info

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.

DOI: 10.1093/protein/gzl058 | Read all as PDF | Close Abstract
0284

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 + further info

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.

DOI: 10.1038/nmeth0107-13 | Read all as PDF | SUPPL: 0284 | Close Abstract

2006

# Reference PDF

0283

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.
| Abstract + further info

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.

DOI: 10.1074/jbc.M602506200 | Read all as PDF | SUPPL: 0283 | Close Abstract
0282

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 + further info

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.

DOI: 10.1158/1535-7163.MCT-06-0412 | Read all as PDF | Close Abstract
0281

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 + further info

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.

DOI: 10.1074/jbc.M604127200 | Read all as PDF | Close Abstract
0280

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 + further info

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.

DOI: 10.1074/jbc.M602547200 | Read all as PDF | SUPPL: 0280 | Close Abstract
0279

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 + further info

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.

DOI: 10.1002/prot.20991 | Read all as PDF | Close Abstract
0278

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.
| Abstract + further info

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.

DOI: 10.1002/prot.20930 | Read all as PDF | Close Abstract
0277

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.
| Abstract + further info

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.

DOI: 10.1016/j.jmb.2006.07.027 | Read all as PDF | Close Abstract
0276

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.
| Abstract + further info

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.

DOI: 10.1038/nbt1218 | Read all as PDF | SUPPL: 0276 1 0276 2 0276 3 0276 4 | Close Abstract
0275

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.
| Abstract + further info

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.

DOI: 10.1016/j.bbrc.2006.02.126 | Read all as PDF | Close Abstract
0274

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
| Abstract + further info

Abstract

No abstract

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0273

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.
| Abstract + further info

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.

DOI: 10.1093/protein/gzl004 | Read all as PDF | SUPPL: 0273 | Close Abstract
0272

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.

DOI: 10.1016/j.jmb.2005.12.043 | Read all as PDF | Close Abstract
0271

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.
| Abstract + further info

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.

DOI: 10.1016/j.jmb.2005.12.005 | Read all as PDF | SUPPL: 0271 | Close Abstract
0270

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.
| Abstract + further info

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

0269

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
| Abstract + further info

Abstract

No abstract

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0268

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.
| Abstract + further info

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.

DOI: 10.1124/mol.105.014910 | Read all as PDF | SUPPL: 0268 | Close Abstract
0267

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
| Abstract + further info

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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0266

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

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.

DOI: 10.1038/nbt1127 | Read all as PDF | SUPPL: 0266 | Close Abstract
0265

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.
| Abstract + further info

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.

DOI: 10.1073/pnas.0504917102 | Read all as PDF | Close Abstract
0264

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.
| Abstract + further info

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.

DOI: 10.1110/ps.051520605 | Read all as PDF | Close Abstract
0263

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.
| Abstract + further info

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.

DOI: 10.1016/j.jmb.2005.06.073 | Read all as PDF | SUPPL: 0263 | Close Abstract
0262

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.
| Abstract + further info

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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0261

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

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.

DOI: 10.1016/j.copbio.2005.06.005 | Read all as PDF | Close Abstract
0260

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.
| Abstract + further info

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.

DOI: 10.1074/jbc.M501746200 | Read all as PDF | Close Abstract
0259

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

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.

DOI: 10.1093/protein/gzi030 | Read all as PDF | Close Abstract
0258

Iwai, H., Forrer, P., Plückthun, A. and Güntert, P. (2005) NMR solution structure of the monomeric form of the bacteriophage lambda capsid stabilizing protein gpD. J. Biomol. NMR 31, 351-356.
| Abstract + further info

Abstract

No abstract

DOI: 10.1007/s10858-005-0945-7 | Read all as PDF | Close Abstract
0257

Ott, D., Neldner, Y., Cebe, R., Dodevski, I. and Plückthun, A. (2005) Engineering and functional immobilization of opioid receptors. Protein Eng. Des. Sel. 18, 153-160.
| Abstract + further info

Abstract

Opioid receptors, like many G protein-coupled receptors (GPCRs), are notoriously unstable in detergents. We have now developed a more stable variant of the mu-opioid receptor (MOR) and also a method for the immobilization of solubilized, functional opioid receptors on a solid phase (magnetic beads). Starting with the intrinsically more stable kappa-opioid receptor (KOR), we optimized the conditions (i.e. detergents and stabilizing ligands) for receptor extraction from lipid bilayers of HEK293T cells to obtain maximal amounts of functional, immobilized receptor. After immobilization, the ligand binding profile remains the same as observed for the membrane-embedded receptor. For the immobilized wild-type mu-opioid receptor, however, no conditions were found under which ligand binding capacity was retained. To solve this problem, we engineered the receptor chimera KKM where the N-terminus and the first transmembrane helix (TM1) of wild-type MOR is exchanged for the homologous receptor parts of the wild-type KOR. This hybrid receptor behaves exactly as the wild-type MOR in functional assays. Interestingly, the modified MOR is expressed at six times higher levels than wild-type MOR and is similarly stable as wild-type KOR after immobilization. Hence the immobilized MOR, represented by the chimera KKM, is now also amenable for biophysical characterization. These results are encouraging for future stability engineering of GPCRs.

DOI: 10.1093/protein/gzi012 | Read all as PDF | Close Abstract
0256

Schimmele, B. and Plückthun, A. (2005) Engineering Proteins for Stability and Efficient Folding in: Protein Folding Handbook (Buchner, J., and Kiefhaber, T., eds) Vol. 5 pp. 1281-1333. Wiley Verlag GmbH & Co. KGaA., Weinheim, Germany
| Abstract + further info

Abstract

The industrial, biotechnological, and medical applications of proteins are often limited by an insufficient protein stability or related problems. Such applications commonly require that proteins be produced on a large scale and remain stable enough to fulfill their functions for a reasonable length of time, often under harsh conditions. However, natural proteins are typically only marginally stable, and it is thus a major challenge for protein engineers to optimize stability and folding efficiency. The approaches that have been successfully employed to achieve this goal are rational design, semi-rational strategies based on sequence comparisons, and the methods of directed protein evolution. Of course, these methods are not mutually exclusive and can be combined to solve practical problems. All studies employing these methods have revealed important rules for protein engineering and at the same time shed light on the principles and mechanisms responsible for the folding and stability of proteins. Recent advances in stability engineering have demonstrated that merely small changes in a given protein sequence can have profound effects on its biophysical properties. The major challenge is therefore to correctly identify and remedy these shortcomings. It is the goal of this chapter to summarize the biophysical principles and technological approaches useful in improving the biophysical properties of proteins through sequence modification. Considering the enormous array of technologies involved in this endeavor, ranging from computer algorithms to selection technologies, it is not possible to give detailed experimental protocols in this chapter; instead, we will guide the reader to the cited literature.

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0255

Röthlisberger, D., Honegger, A. and Plückthun, A. (2005) Domain interactions in the Fab fragment: A comparative evaluation of the single-chain Fv and Fab format engineered with variable domains of different stability. J. Mol. Biol. 347, 773-789.
| Abstract + further info

Abstract

Recombinant antibody fragments, most notably Fab and scFv, have become important tools in research, diagnostics and therapy. Since different recombinant antibody formats exist, it is crucial to understand the difference in their respective biophysical properties. We assessed the potential stability benefits of changing the scFv into the Fab format, the influence of the variable domains on the stability of the Fab fragment, and the influence of the interchain disulfide bond in the Fab fragment. To analyze domain interactions, the Fab fragment was broken down into its individual domains, several two-domain assemblies and one three-domain assembly. The equilibrium denaturation properties of these constructs were then compared to those of the Fab fragment. It was found that mutual stabilization occurred across the V(H)/V(L) and the C(H)1/C(L) interface, whereas the direct interaction between the V(L) and the C(L) domain had no influence on the stability of either domain. This observation can be explained by the different interfaces used for interaction. In contrast, the whole C(H)1C(L) and V(H)V(L) unit showed significant mutual stabilization, indicating a high degree of cooperation between the V(H)/V(L) and C(H)1/C(L) interface. The interchain disulfide bond in the Fab fragment plays an essential role in this stabilization. In addition to the effects of domain association on the thermodynamic (equilibrium) stability, Fab fragments differ from scFv fragments of similar equilibrium stability by having a very slow unfolding rate. This kinetic stabilization may increase significantly the resistance of Fab fragments against short time exposure to adverse conditions.

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2004

# Reference PDF

0254

Chang, C., Plückthun, A. and Wlodawer, A. (2004) Crystal structure of a truncated version of the phage lambda protein gpD. Proteins 57, 866-868.
| Abstract + further info

Abstract

No abstract.

DOI: 10.1002/prot.20254 | Read all as PDF | Close Abstract
0253

Forrer, P., Chang, C., Ott, D., Wlodawer, A. and Plückthun, A. (2004) Kinetic stability and crystal structure of the viral capsid protein SHP. J. Mol. Biol. 344, 179-193.
| Abstract + further info

Abstract

SHP, the capsid-stabilizing protein of lambdoid phage 21, is highly resistant against denaturant-induced unfolding. We demonstrate that this high functional stability of SHP is due to a high kinetic stability with a half-life for unfolding of 25 days at zero denaturant, while the thermodynamic stability is not unusually high. Unfolding experiments demonstrated that the trimeric state (also observed in crystals and present on the phage capsid) of SHP is kinetically stable in solution, while the monomer intermediate unfolds very rapidly. We also determined the crystal structure of trimeric SHP at 1.5A resolution, which was compared to that of its functional homolog gpD. This explains how a tight network of H-bonds rigidifies crucial interpenetrating residues, leading to the observed extremely slow trimer dissociation or denaturation. Taken as a whole, our results provide molecular-level insights into natural strategies to achieve kinetic stability by taking advantage of protein oligomerization. Kinetic stability may be especially needed in phage capsids to allow survival in harsh environments.

DOI: 10.1016/j.jmb.2004.09.030 | Read all as PDF | Close Abstract
0252

Devi, V. S., Binz, H. K., Stumpp, M. T., Plückthun, A., Bosshard, H. R. and Jelesarov, I. (2004) Folding of a designed simple ankyrin repeat protein. Protein Sci. 13, 2864-2870.
| Abstract + further info

Abstract

Ankyrin repeats (AR) are 33-residue motifs containing a beta-turn, followed by two alpha-helices connected by a loop. AR occur in tandem arrangements and stack side-by-side to form elongated domains involved in very different cellular tasks. Recently, consensus libraries of AR repeats were constructed. Protein E1_5 represents a member of the shortest library, and consists of only a single consensus repeat flanked by designed N- and C-terminal capping repeats. Here we present a biophysical characterization of this AR domain. The protein is compactly folded, as judged from the heat capacity of the native state and from the specific unfolding enthalpy and entropy. From spectroscopic data, thermal and urea-induced unfolding can be modeled by a two-state transition. However, scanning calorimetry experiments reveal a deviation from the two-state behavior at elevated temperatures. Folding and unfolding at 5 degrees C both follow monoexponential kinetics with k(folding) = 28 sec(-1) and k(unfolding) = 0.9 sec(-1). Kinetic and equilibrium unfolding parameters at 5 degrees C agree very well. We conclude that E1_5 folds in a simple two-state manner at low temperatures while equilibrium intermediates become populated at higher temperatures. A chevron-plot analysis indicates that the protein traverses a very compact transition state along the folding/unfolding pathway. This work demonstrates that a designed minimal ankyrin repeat protein has the thermodynamic and kinetic properties of a compactly folded protein, and explains the favorable properties of the consensus framework.

DOI: 10.1110/ps.04935704 | Read all as PDF | Close Abstract
0251

Ewert, S., Honegger, A. and Plückthun, A. (2004) Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable frameworks and structure-based framework engineering. Methods 34, 184-99.
| Abstract + further info

Abstract

By combining the knowledge gained from an analysis of the biophysical properties of natural antibody variable domains, the effects of mutations obtained in directed evolution experiments, and the detailed structural comparison of antibodies, it has now become possible to engineer antibodies for higher thermodynamic stability and more efficient folding. This is particularly important when antibodies are to be used under conditions where the disulfide bonds cannot form, i.e., in intracellular applications (as ""intrabodies""). We describe in detail two methods for the knowledge-based improvement of antibody stability and folding efficiency. While CDR grafting from a non-human to the most closely related human antibody framework is an established technique to reduce the immunogenicity of a therapeutic antibody, CDR grafting for stabilization implies the use of a more distantly related acceptor framework with superior biophysical characteristics. The use of such dissimilar frameworks requires particular attention to antigen contact residues outside the classical CDR definition and to residues capable of indirectly affecting the conformation of the antigen binding site. As a second alternative, the stability of a suboptimal framework can be improved by the introduction of point mutations designed to optimize key residue interactions. We describe the analysis methods used to identify such point mutations, which can be introduced all at once, while maintaining the framework features necessary for antigen binding. These rational approaches render the continued ""rediscovery"" of certain mutations by directed evolution unnecessary, but they can also be used in conjunction with such methods to discover even better molecules.

DOI: 10.1016/j.ymeth.2004.04.007 | Read all as PDF | Close Abstract
0250

Lipovsek, D. and Plückthun, A. (2004) In-vitro protein evolution by ribosome display and mRNA display. J. Immunol. Methods 290, 51-67.
| Abstract + further info

Abstract

In-vitro display technologies combine two important advantages for identifying and optimizing ligands by evolutionary strategies. First, by obviating the need to transform cells in order to generate and select libraries, they allow a much higher library diversity. Second, by including PCR as an integral step in the procedure, they make PCR-based mutagenesis strategies convenient. The resulting iteration between diversification and selection allows true Darwinian protein evolution to occur in vitro. We describe two such selection methods, ribosome display and mRNA display. In ribosome display, the translated protein remains connected to the ribosome and to its encoding mRNA; the resulting ternary complex is used for selection. In mRNA display, mRNA is first translated and then covalently bonded to the protein it encodes, using puromycin as an adaptor molecule. The covalent mRNA-protein adduct is purified from the ribosome and used for selection. Successful examples of high-affinity, specific target-binding molecules selected by in-vitro display methods include peptides, antibodies, enzymes, and engineered scaffolds, such as fibronectin type III domains and synthetic ankyrins, which can mimic antibody function.

DOI: 10.1016/j.jim.2004.04.008 | Read all as PDF | Close Abstract
0249

Binz, H. K., Amstutz, P., Kohl, A., Stumpp, M. T., Briand, C., Forrer, P., Grütter, M. G. and Plückthun, A. (2004) High-affinity binders selected from designed ankyrin repeat protein libraries. Nature Biotechnol. 22, 575-582.
| Abstract + further info

Abstract

We report here the evolution of ankyrin repeat (AR) proteins in vitro for specific, high-affinity target binding. Using a consensus design strategy, we generated combinatorial libraries of AR proteins of varying repeat numbers with diversified binding surfaces. Libraries of two and three repeats, flanked by 'capping repeats,' were used in ribosome-display selections against maltose binding protein (MBP) and two eukaryotic kinases. We rapidly enriched target-specific binders with affinities in the low nanomolar range and determined the crystal structure of one of the selected AR proteins in complex with MBP at 2.3 Å resolution. The interaction relies on the randomized positions of the designed AR protein and is comparable to natural, heterodimeric protein-protein interactions. Thus, our AR protein libraries are valuable sources for binding molecules and, because of the very favorable biophysical properties of the designed AR proteins, an attractive alternative to antibody libraries.

DOI: 10.1038/nbt962 | Read all as PDF | SUPPL: 0249 1 0249 2 0249 3 0249 4 | Close Abstract
0248

Zahnd, C., Spinelli, S., Luginbühl, B., Amstutz, P., Cambillau, C. and Plückthun, A. (2004) Directed in vitro evolution and crystallographic analysis of a peptide binding scFv antibody with low picomolar affinity. J. Biol. Chem. 279, 18870-18877.
| Abstract + further info

Abstract

We generated a single-chain antibody fragment (scFv) with a binding affinity of about 5 pM to a short peptide by applying rigorous directed evolution. Starting from a high affinity peptide binder, originally obtained by ribosome display from a murine library, we generated libraries of mutants with the error-prone polymerase chain reaction (PCR) and DNA shuffling and applied off-rate selection using ribosome display. Crystallographic analysis of the scFv in its antigen-bound and free state showed that only few mutations, which do not make direct contact to the antigen, lead to a 500-fold affinity improvement over its potential germ line precursor. These results suggest that the affinity optimization of very high affinity binders is achieved by modulating existing interactions via subtle changes in the framework rather than by introducing new contacts. Off-rate selection in combination with ribosome display can evolve binders to the low picomolar affinity range, even for peptide targets.

DOI: 10.1074/jbc.M309169200 | Read all as PDF | Close Abstract
0247

Röthlisberger, D., Pos, K. M. and Plückthun, A. (2004) An antibody library for stabilizing and crystallizing membrane proteins selecting binders to the citrate carrier CitS. FEBS Lett. 564, 340-348.
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Abstract

Abstract Co-crystallization of membrane proteins with antibody

DOI: 10.1016/S0014-5793(04)00359-X | Read all as PDF | Close Abstract
0246

Hu, K. F., Plückthun, A. and Pervushin, K. (2004) Backbone H-N, N, C_, C› and C_ chemical shift assignments and secondary structure of FkpA, a 245-residue peptidyl-prolyl cis/trans isomerase with chaperone activity. J. Biomol. NMR 28, 405-406.
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Abstract

No abstract

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0245

Forrer, P., Binz, H. K., Stumpp, M. T. and Plückthun, A. (2004) Consensus design of repeat proteins. ChemBioChem 5, 183-189.
| Abstract + further info

Abstract

Consensus design is a valuable protein-engineering method that is based on statistical information derived from sequence alignments of homologous proteins. Recently, consensus design was adapted to repeat proteins. We discuss the potential of this novel repeat-based approach for the design of consensus repeat proteins and repeat protein libraries and summarize recent results from such experiments.

DOI: 10.1002/cbic.200300762 | Read all as PDF | Close Abstract
0244

Matsuura, T., Ernst, A., Zechel, D. L. and Plückthun, A. (2004) Combinatorial approaches to novel proteins. ChemBioChem 5, 177-182.
| Abstract + further info

Abstract

No Abstract

DOI: 10.1002/cbic.200300755 | Read all as PDF | Close Abstract
0243

Iwai, H., Forrer, P., Plückthun, A. and Güntert, P. (2004) Assignments of 1H and 15N resonances of the bacteriophage lambda capsid stabilizing protein gpD. J. Biomol. NMR 28, 89-90.
| Abstract + further info

Abstract

No abstract

DOI: 10.1023/B:JNMR.0000012844.21707.8c | Read all as PDF | Close Abstract
0242

Ott, D., Frischknecht, R. and Plückthun, A. (2004) Construction and characterization of a kappa opioid receptor devoid of all free cysteines. Protein Eng. 17, 37-48.
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Abstract

We have constructed an optimized mutant of the kappa opioid receptor (KOR), which is devoid of its 10 free cysteines. It was necessary to test different amino acid replacements at various positions and we used a structural model and homology with other receptor family members as a guide. This mutant binds ligands and couples to the cognate G-proteins in a very similar fashion to wild-type KOR. The addition of the antagonist naloxone during cell growth greatly enhances heterogeneous expression of the mutant in mammalian cells, such that amounts similar to wild-type could be produced. We showed by fluorescence microscopy that naloxone stabilizes the mutant in the plasma membrane. This mutant, which now permits the insertion of single cysteines, was designed for use in spectroscopic studies of ligand-induced receptor conformational changes as well as to simplify folding studies.

DOI: 10.1093/Protein/Gzh004 | Read all as PDF | Close Abstract
0241

Matsuura, T. and Plückthun, A. (2004) Strategies for selection from protein libraries composed of de novo designed secondary structure modules. Origins Life Evol. Biosphere 34, 151-157.
| Abstract + further info

Abstract

As more and more protein structures are determined, it has become clear that there is only a limited number of protein folds in nature. To explore whether the protein folds found in nature are the only solutions to the protein folding problem, or that a lack of evolutionary pressure causes the paucity of different protein folds found, we set out to construct protein libraries without any restriction of topology. We generated different libraries (all a-helix, all b-strand and a-helix plus b-strand) with average lenght of 100 amino acid residues, composed of designed secondary structure modules (a-helix, b-strand and b-turn) in various proportions, based primarily on the patterning of polar and non-polar residues. From the analysis of proteins chosen randomly from the libraries, we found that a substantial portion of pure a-helical proteins show properties similar to native proteins. Using these libraries as a starting point, we aim to establish a selection system which allows us to enrich proteins with favorable folding properties (non aggregating, compactly folded) from the libraries. We have developed such a method based on ribosome display. This selection is based on two concepts: (1) misfolded proteins are more sensitive to proteolysis, (2) misfolded and/or aggregated proteins are mor hydrophobic. We show that by applying each of these selection criteria proteins that are compactly folded and soluble can be enriched over insoluble and random coil proteins.

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2003

# Reference PDF

0240

Deyev, S. M., Waibel, R., Lebedenko, E. N., Schubiger, A. P. and Plückthun, A. (2003) Design of multivalent complexes using the barnase*barstar module. Nature Biotechnol. 21, 1486-1492.
| Abstract + further info

Abstract

The ribonuclease barnase (12 kDa) and its inhibitor barstar (10 kDa) form a very tight complex in which all N and C termini are accessible for fusion. Here we exploit this system to create modular targeting molecules based on antibody scFv fragment fusions to barnase, to two barnase molecules in series and to barstar. We describe the construction, production and purification of defined dimeric and trimeric complexes. Immobilized barnase fusions are used to capture barstar fusions from crude extracts to yield homogeneous, heterodimeric fusion proteins. These proteins are stable, soluble and resistant to proteolysis. Using fusions with anti-p185(HER2-ECD) 4D5 scFv, we show that the anticipated gain in avidity from monomer to dimer to trimer is obtained and that favorable tumor targeting properties are achieved. Many permutations of engineered multispecific fusion proteins become accessible with this technology of quasi-covalent heterodimers.

DOI: 10.1038/nbt916 | Read all as PDF | SUPPL: 0240 1 0240 2 | Close Abstract
0239

Binz, H. K., Stumpp, M. T., Forrer, P., Amstutz, P. and Plückthun, A. (2003) Designing repeat proteins: well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins. J. Mol. Biol. 332, 489-503.
| Abstract + further info

Abstract

We describe an efficient way to generate combinatorial libraries of stable, soluble and well-expressed ankyrin repeat (AR) proteins. Using a combination of sequence and structure consensus analyses, we designed a 33 amino acid residue AR module with seven randomized positions having a theoretical diversity of 7.2x10(7). Different numbers of this module were cloned between N and C-terminal capping repeats, i.e. ARs designed to shield the hydrophobic core of stacked AR modules. In this manner, combinatorial libraries of designed AR proteins consisting of four to six repeats were generated, thereby potentiating the theoretical diversity. All randomly chosen library members were expressed in soluble form in the cytoplasm of Escherichia coli in amounts up to 200 mg per 1 l of shake-flask culture. Virtually pure proteins were obtained in a single purification step. The designed AR proteins are monomeric and display CD spectra identical with those of natural AR proteins. At the same time, our AR proteins are highly thermostable, with T(m) values ranging from 66 degrees C to well above 85 degrees C. Thus, our combinatorial library members possess the properties required for biotechnological applications. Moreover, the favorable biophysical properties and the modularity of the AR fold may account, partly, for the abundance of natural AR proteins.

DOI: 10.1016/s0022-2836(03)00896-9 | Read all as PDF | Close Abstract
0238

Stumpp, M. T., Forrer, P., Binz, H. K. and Plückthun, A. (2003) Designing repeat proteins: modular leucine-rich repeat protein libraries based on the mammalian ribonuclease inhibitor family. J. Mol. Biol. 332, 471-487.
| Abstract + further info

Abstract

We present a novel approach to design repeat proteins of the leucine-rich repeat (LRR) family for the generation of libraries of intracellular binding molecules. From an analysis of naturally occurring LRR proteins, we derived the concept to assemble repeat proteins with randomized surface positions from libraries of consensus repeat modules. As a guiding principle, we used the mammalian ribonuclease inhibitor (RI) family, which comprises cytosolic LRR proteins known for their extraordinary affinities to many RNases. By aligning the amino acid sequences of the internal repeats of human, pig, rat, and mouse RI, we derived a first consensus sequence for the characteristic alternating 28 and 29 amino acid residue A-type and B-type repeats. Structural considerations were used to replace all conserved cysteine residues, to define less conserved positions, and to decide where to introduce randomized amino acid residues. The so devised consensus RI repeat library was generated at the DNA level and assembled by stepwise ligation to give libraries of 2-12 repeats. Terminal capping repeats, known to shield the continuous hydrophobic core of the LRR domain from the surrounding solvent, were adapted from human RI. In this way, designed LRR protein libraries of 4-14 LRRs (equivalent to 130-415 amino acid residues) were obtained. The biophysical analysis of randomly chosen library members showed high levels of soluble expression in the Escherichia coli cytosol, monomeric behavior as characterized by gel-filtration, and alpha-helical CD spectra, confirming the success of our design approach.

DOI: 10.1016/s0022-2836(03)00897-0 | Read all as PDF | Close Abstract
0237

Di Paolo, C., Willuda, J., Kubetzko, S., Lauffer, I., Tschudi, D., Waibel, R., Plückthun, A., Stahel, R. A. and Zangemeister-Wittke, U. (2003) A recombinant immunotoxin derived from a humanized epithelial cell adhesion molecule-specific single-chain antibody fragment has potent and selective antitumor activity. Clin. Cancer Res. 9, 2837-2848.
| Abstract + further info

Abstract

PURPOSE: Epithelial cell adhesion molecule (Ep-CAM) is a tumor-associated antigen overexpressed in many solid tumors but shows limited expression in normal epithelial tissues. To exploit this favorable expression pattern for targeted cancer therapy, an Ep-CAM-specific recombinant immunotoxin was developed and its antitumor activity investigated. Experimental Design: The immunotoxin 4D5MOCB-ETA was developed by genetically fusing a truncated form of Pseudomonas aeruginosa exotoxin A (ETA) (ETA(252-608)KDEL) to the highly stable humanized single-chain antibody fragment (scFv) 4D5MOCB. Cytotoxicity of 4D5MOCB-ETA was measured in cell growth and leucine incorporation assays in vitro. Tumor localization and antitumor activity were assessed in athymic mice bearing established human tumor xenografts. RESULTS: Fusion of the toxin moiety to the scFv did neither affect its thermal stability nor its antigen-binding affinity. In vitro, 4D5MOCB-ETA potently and specifically inhibited protein synthesis and reduced the viability of Ep-CAM-positive carcinoma cells of diverse histological origins with IC(50)s ranging from 0.005 to 0.2 pM. Upon systemic administration in mice, 4D5MOCB-ETA showed similar organ distribution as the scFv 4D5MOCB and preferentially localized to Ep-CAM-positive tumor xenografts with a tumor:blood ratio of 5.4. The potent antitumor activity of 4D5MOCB-ETA was demonstrated by its ability to strongly inhibit the growth and induce regression of relatively large tumor xenografts derived from lung, colon, or squamous cell carcinomas. CONCLUSIONS: We describe for the first time the development of a fully recombinant Ep-CAM-specific immunotoxin and demonstrate its potent activity against solid tumors of various histological origins. 4D5MOCB-ETA is currently being evaluated in a Phase I study in patients with refractory squamous cell carcinoma of the head and neck.

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0236

Cesaro-Tadic, S., Lagos, D., Honegger, A., Rickard, J. H., Partridge, L. J., Blackburn, G. M. and Plückthun, A. (2003) Turnover-based in vitro selection and evolution of biocatalysts from a fully synthetic antibody library. Nature Biotechnol. 21, 679-685.
| Abstract + further info

Abstract

This report describes the selection of highly efficient antibody catalysts by combining chemical selection from a synthetic library with directed in vitro protein evolution. Evolution started from a naive antibody library displayed on phage made from fully synthetic, antibody-encoding genes (the Human Combinatorial Antibody Library; HuCAL-scFv). HuCAL-scFv was screened by direct selection for catalytic antibodies exhibiting phosphatase turnover. The substrate used was an aryl phosphate, which is spontaneously transformed into an electrophilic trapping reagent after cleavage. Chemical selection identified an efficient biocatalyst that then served as a template for error-prone PCR (epPCR) to generate randomized repertoires that were subjected to further selection cycles. The resulting superior catalysts displayed cumulative mutations throughout the protein sequence; the ten-fold improvement of their catalytic proficiencies (>10(10) M(-1)) resulted from increased k(cat) values, thus demonstrating direct selection for turnover. The strategy described here makes the search for new catalysts independent of the immune system and the antibody framework.

DOI: 10.1038/nbt828 | Read all as PDF | Close Abstract
0235

Ciatto, C., Capitani, G., Tissot, A. C., Pécorari, F., Plückthun, A. and Grütter, M. G. (2003) Structural analysis of mycobacterial and murine hsp60 epitopes in complex with the class I MHC molecule H-2Db. FEBS Lett. 543, 11-15.
| Abstract + further info

Abstract

The decameric peptide SALQNAASIA from the Mycobacterium bovis heat shock protein (hsp) 60 is recognized by the murine T-cell receptor UZ-3-4 in complex with the murine class I major histocompatibility complex molecule H-2D(b). This T-cell receptor cross-reacts with the H-2D(b)-bound non-homologous decameric peptide KDIGNIISDA from the murine hsp60, but does not recognize the nonameric mycobacterial peptide SALQNAASI. Cross-recognition of the KDIGNIISDA/H-2D(b) complex induces autoimmune pathology in immunodeficient mice. We solved the X-ray crystal structure of the SALQNAASIA/H-2D(b) complex at 3.0 A resolution, and we modelled the KDIGNIISDA and SALQNAASI peptides in the H-2D(b) binding site. The structural analysis of the H-2D(b)-bound hsp60 epitopes offers insight into T-cell receptor cross-reactivity.

DOI: 10.1016/s0014-5793(03)00325-9 | Read all as PDF | Close Abstract
0234

Matsuura, T. and Plückthun, A. (2003) Selection based on the folding properties of proteins with ribosome display. FEBS Lett. 539, 24-28.
| Abstract + further info

Abstract

Ribosome display is a powerful tool for selecting and evolving protein functions through ligand-binding. Here, this in vitro system was used to perform selection based on the folding properties of proteins, independent of specific ligand-binding. The selection is based on two properties of misfolded proteins: (1) increased sensitivity to proteolysis and (2) greater exposure of hydrophobic area. By targeting these properties, we show that compactly folded and soluble proteins can be enriched over insoluble and random coil proteins. This approach may be especially useful for selection and evolution of folded proteins from random sequence libraries.

DOI: 10.1016/s0014-5793(03)00178-9 | Read all as PDF | Close Abstract
0233

Forrer, P., Stumpp, M. T., Binz, H. K. and Plückthun, A. (2003) A novel strategy to design binding molecules harnessing the modular nature of repeat proteins. FEBS Lett. 539, 2-6.
| Abstract + further info

Abstract

Repeat proteins, such as ankyrin or leucine-rich repeat proteins, are ubiquitous binding molecules, which occur, unlike antibodies, intra- and extracellularly. Their unique modular architecture features repeating structural units (repeats), which stack together to form elongated repeat domains displaying variable and modular target-binding surfaces. Based on this modularity, we developed a novel strategy to generate combinatorial libraries of polypeptides with highly diversified binding specificities. This strategy includes the consensus design of self-compatible repeats displaying variable surface residues and their random assembly into repeat domains. We envision that such repeat protein libraries will be highly valuable sources for novel binding molecules especially suitable for intracellular applications.

DOI: 10.1016/s0014-5793(03)00177-7 | Read all as PDF | Close Abstract
0232

Ewert, S., Honegger, A. and Plückthun, A. (2003) Structure-based improvement of the biophysical properties of immunoglobulin VH domains with a generalizable approach. Biochemistry 42, 1517-1528.
| Abstract + further info

Abstract

In a systematic study of V gene families carried out with consensus V(H) and V(L) domains alone and in combinations in the scFv format, we found comparatively low expression yields and lower cooperativity in equilibrium unfolding in antibody fragments containing V(H) domains of human germline families 2, 4, and 6. From an analysis of the packing of the hydrophobic core, the completeness of charge clusters, the occurrence of unsatisfied hydrogen bonds, and residues with low beta-sheet propensities, positive Phi angles, and exposed hydrophobic side chains, we pinpointed residues potentially responsible for the unsatisfactory properties of these germline-encoded sequences. Several of those are in common between the domains of the even-numbered subgroups, but do not occur in the odd-numbered ones. In this study, we have systematically exchanged those residues alone and in combination in two different scFvs using the V(H)6 framework, and we describe their effect on equilibrium stability and folding yield. We improved the stability by 20.9 kJ/mol and the expression yield by a factor of 4 and can now use these data to rationally engineer antibodies derived from this and similar germline families for better biophysical properties. Furthermore, we provide an improved design for libraries exploiting the significant additional diversity provided by these frameworks. Both antibodies studied here completely retain their binding affinity, demonstrating that the CDR conformations were not affected.

DOI: 10.1021/bi026448p | Read all as PDF | Close Abstract
0231

Kohl, A., Binz, H. K., Forrer, P., Stumpp, M. T., Plückthun, A. and Grütter, M. G. (2003) Designed to be stable: Crystal structure of a consensus ankyrin repeat protein. Proc. Natl. Acad. Sci. U. S. A. 100, 1700-1705.
| Abstract + further info

Abstract

Ankyrin repeat (AR) proteins mediate innumerable protein-protein interactions in virtually all phyla. This finding suggested the use of AR proteins as designed binding molecules. Based on sequence and structural analyses, we designed a consensus AR with fixed framework and randomized interacting residues. We generated several combinatorial libraries of AR proteins consisting of defined numbers of this repeat. Randomly chosen library members are expressed in soluble form in the cytoplasm of Escherichia coli constituting up to 30% of total cellular protein and show high thermodynamic stability. We determined the crystal structure of one of those library members to 2.0-A resolution, providing insight into the consensus AR fold. Besides the highly complementary hydrophobic repeat-repeat interfaces and the absence of structural irregularities in the consensus AR protein, the regular and extended hydrogen bond networks in the beta-turn and loop regions are noteworthy. Furthermore, all residues found in the turn region of the Ramachandran plot are glycines. Many of these features also occur in natural AR proteins, but not in this rigorous and standardized fashion. We conclude that the AR domain fold is an intrinsically very stable and well-expressed scaffold, able to display randomized interacting residues. This scaffold represents an excellent basis for the design of novel binding molecules.

DOI: 10.1073/pnas.0337680100 | Read all as PDF | SUPPL: 0231 1 | Close Abstract
0230

Ewert, S., Huber, T., Honegger, A. and Plückthun, A. (2003) Biophysical properties of human antibody variable domains. J. Mol. Biol. 325, 531-553.
| Abstract + further info

Abstract

There are great demands on the stability, expression yield and resistance to aggregation of antibody fragments. To untangle intrinsic domain effects from domain interactions, we present first a systematic evaluation of the isolated human immunoglobulin variable heavy (V(H)) and light (V(L)) germline family consensus domains and then a systematic series of V(H)-V(L) combinations in the scFv format. The constructs were evaluated in terms of their expression behavior, oligomeric state in solution and denaturant-induced unfolding equilibria under non-reducing conditions. The seven V(H) and seven V(L) domains represent the consensus sequences of the major human germline subclasses, derived from the Human Combinatorial Antibody Library (HuCAL((R))). The isolated V(H) and V(L) domains with the highest thermodynamic stability and yield of soluble protein were V(H)3 and V(kappa)3, respectively. Similar measurements on all domain combinations in scFv fragments allowed the scFv fragments to be classified according to thermodynamic stability and in vivo folding yield. The scFv fragments containing the variable domain combinations H3kappa3, H1bkappa3, H5kappa3 and H3kappa1 show superior properties concerning yield and stability. Domain interactions diminish the intrinsic differences of the domains. ScFv fragments containing V(lambda) domains show high levels of stability, even though V(lambda) domains are surprisingly unstable by themselves. This is due to a strong interaction with the V(H) domain and depends on the amino acid sequence of the CDR-L3. On the basis of these analyses and model structures, we suggest possibilities for further improvement of the biophysical properties of individual frameworks and give recommendations for library design.

DOI: 10.1021/bi026448p | Read all as PDF | Close Abstract

2002

# Reference PDF

0229

Lindner, P., Blank, K., Diefenbach, B. and Plückthun, A. (2002) Chitin binding domains for immobilizing antibody fragments in immunaffinity chromatography in: EUCHIS’02, 5th International Conference of the European Chitin Society (Vårum, K. M., Domard, A., and Smidsrød, O., eds) Vol. VI pp. 261-262. NTNU Trondheim, Norway, Trondheim, Norway
| Abstract + further info

Abstract

No abstract

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0228

Auf Der Maur, A., Zahnd, C., Fischer, F., Spinelli, S., Honegger, A., Cambillau, C., Escher, D., Plückthun, A. and Barberis, A. (2002) Direct in vivo screening of intrabody libraries constructed on a highly stable single-chain framework. J. Biol. Chem. 277, 45075-45086.
| Abstract + further info

Abstract

Single-chain Fv antibody fragments (scFv) represent a convenient antibody format for intracellular expression in eukaryotic or prokaryotic cells. These so-called intrabodies have a great potential in functional genomics as a tool to study the function of newly identified proteins in vivo, for example by binding-induced modulation of their activity or by blocking interactions with other proteins. However, the intracellular expression and activity of many scFvs are limited by their instability and folding efficiency in the reducing intracellular environment, where the highly conserved intrachain disulfide bonds do not form. In the present work, we used an in vivo selection system to isolate novel antigen-binding intrabodies. We screened two intrabody libraries carrying a randomized third hypervariable loop (CDR-H3) onto the heavy chain (VH) of a stable framework, which had been further optimized by random mutagenesis for better behaviour in the selection system, and we biophysically characterized the selected variants to interpret the outcome of the selection. Our results show that single-framework intrabody libraries can be directly screened in vivo to rapidly select antigen-specific intrabodies.

DOI: 10.1074/jbc.M205264200 | Read all as PDF | Close Abstract
0227

Matsuura, T., Ernst, A. and Plückthun, A. (2002) Construction and characterization of protein libraries composed of secondary structure modules. Protein Sci. 11, 2631-2643.
| Abstract + further info

Abstract

Only a minute fraction of all possible protein sequences can exist in the genomes of all life forms. To explore whether physicochemical constraints or a lack of need causes the paucity of different protein folds, we set out to construct protein libraries without any restriction of topology. We generated different libraries (all alpha-helix, all beta-strand, and alpha-helix plus beta-strand) with an average length of 100 amino acid residues, composed of designed secondary structure modules (alpha-helix, beta-strand, and beta-turn) in various proportions, based primarily on the patterning of polar and nonpolar residues. We wished to explore that part of sequence space that is rich in secondary structure. The analysis of randomly chosen clones from each of the libraries showed that, despite the low sequence homology to known protein sequences, a substantial proportion of the library members containing alpha-helix modules were indeed helical, possess a defined oligomerization state, and showed cooperative chemical unfolding behavior. On the other hand, proteins composed of mainly beta-strand modules tended to form amyloid-like fibrils and were among the least soluble proteins ever reported. We found that a large fraction of members in non-beta-strand-containing protein libraries that are distant from natural proteins in sequence space possess unexpectedly favorable properties. These results reinforce the efficacy of applying binary patterning to design proteins with native-like properties despite lack of restriction in topology. Because of the intrinsic tendency of beta-strand modules to aggregate, their presence requires precise topologic arrangement to prevent fibril formation.

DOI: 10.1110/ps.0215102 | Read all as PDF | Close Abstract
0226

Valle, F., DeRose, J. A., Dietler, G., Kawe, M., Plückthun, A. and Semenza, G. (2002) AFM structural study of the molecular chaperone GroEL and its two-dimensional crystals: an ideal «»living»» calibration sample. Ultramicroscopy 93, 83-89.
| Abstract + further info

Abstract

Supramolecular complexes, such as chaperonins, are suitable samples for atomic force microscope structural studies because they have a very well defined shape. High-resolution images can be made using tapping mode in liquid under native conditions. Details about the two-dimensional structures formed onto the surface upon adsorption and of the single protein can be observed. Dissection of the upper ring of the supramolecular complex as a result of the applied lateral force through scanning tip is observed. Finally, the combination of lateral convolution and tip penetration into the cavity of chaperonins offers a direct evaluation of the tip convolution effect on images of macromolecular samples.

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0225

Arndt, K., Pelletier, J., Muller, K., Plückthun, A. and Alber, T. (2002) Comparison of in vivo selection and rational design of heterodimeric coiled coils. Structure 10, 1235-1248.
| Abstract + further info

Abstract

To investigate how electrostatic interactions restrict the associations of coiled coils, we improved a heterodimeric coiled coil (WinZip-A1B1) by in vivo selection and, alternatively, by rational design. Selection from libraries encoding variable edge (g and e) residues enriched g/e' ion pairs, but the optimum selected heterodimers unexpectedly retained two predicted repulsive g/e' pairs. The best genetically selected heterodimer displayed similar thermodynamic stability and specificity as a rationally designed dimer with predicted ion pairs at all edge positions. This rationally designed pair, however, was less effective than the best genetically selected pair in mediating dimerization in vivo. Thus, the effects of predicted charge pairs depend on sequence context, and complementary charges at the edge positions rationalize only a fraction of the sequences that form stable, specific coiled coils.

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0224

Hofmann, A., Iwai, H., Hess, S., Plückthun, A. and Wlodawer, A. (2002) Structure of cyclized green fluorescent protein. Acta Crystallogr. D Biol. Crystallogr. 58, 1400-1406.
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Abstract

Crystals of cyclic green fluorescent protein (cGFP) engineered by the previously reported split intein technology [Iwai et al. (2001), J. Biol. Chem. 276, 16548-16554] were obtained and the structure was solved using molecular replacement. Although the core of the protein can unambiguously be fitted from the first to the last residue of the genuine sequence, the electron density in the region of the linker peptide is rather poor owing to the high water content of the crystals. Therefore, it is concluded that this part of the protein is highly disordered in the present structure and is very flexible. This is supported by the absence of crystal contacts in the linker-peptide region and the fact that the core of the protein exhibits a very similar conformation to that known from other GFP structures, thereby not implicating any constraints arising from the presence of the artificial linker. Nevertheless, the density is consistent with the loop being intact, as confirmed by mass spectroscopy of dissolved crystals. The present structure contains an antiparallel cGFP dimer where the dimer interface is clearly different from other crystal structures featuring two GFP molecules. This adds further support to the fact that the cylinder surface of GFP is rather versatile and can employ various polar and non-polar patches in protein-protein interactions.

DOI: 10.1107/S0907444902010454 | Read all as PDF | Close Abstract
0223

Amstutz, P., Pelletier, J. N., Guggisberg, A., Jermutus, L., Cesaro-Tadic, S., Zahnd, C. and Plückthun, A. (2002) In vitro selection for catalytic activity with ribosome display. J. Am. Chem. Soc. 124, 9396-9403.
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Abstract

We report what is, to our knowledge, the first in vitro selection for catalytic activity based on catalytic turnover by using ribosome display, a method which does not involve living cells at any step. RTEM-beta-lactamase was functionally displayed on ribosomes as a complex with its encoding mRNA. We designed and synthesized a mechanism-based inhibitor of beta-lactamase, biotinylated ampicillin sulfone, appropriate for selection of catalytic activity of the ribosome-displayed beta-lactamase. This derivative of ampicillin inactivated beta-lactamase in a specific and irreversible manner. Under appropriate selection conditions, active RTEM-beta-lactamase was enriched relative to an inactive point mutant over 100-fold per ribosome display selection cycle. Selection for binding, carried out with beta-lactamase inhibitory protein (BLIP), gave results similar to selection with the suicide inhibitor, indicating that ribosome display is similarly efficient in catalytic activity and affinity selections. In the future, the capacity to select directly for enzymatic activity using an entirely in vitro process may allow for a significant increase in the explorable sequence space relative to existing strategies.

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0222

Hoyer, W., Ramm, K. and Plückthun, A. (2002) A kinetic trap is an intrinsic feature in the folding pathway of single-chain Fv fragments. Biophys. Chem. 96, 273-284.
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Abstract

We have studied the equilibrium unfolding and the kinetics of folding and unfolding of an antibody scFv fragment devoid of cis-prolines. An anti-GCN4 scFv fragment carrying a V(L) lambda domain, obtained by ribosome display, served as the model system together with an engineered destabilized mutant in V(H) carrying the R66K exchange. Kinetic and equilibrium unfolding experiments indicate that the V(H) mutation also affects V(L) unfolding, possibly by partially destabilizing the interface provided by V(H), even though the mutation is distant from the interface. Upon folding of the scFv fragment, a kinetic trap is populated whose escape rate is much faster with the more stable V(H) domain. The formation of the trap can be avoided if refolding is carried out stepwise, with V(H) folding first. These results show that antibody scFv fragments do not fold by the much faster independent domain folding, but instead form a kinetically trapped off-pathway intermediate, which slows down folding under native conditions. This intermediate is characterized by premature interaction of the unfolded domains, and particularly involving unfolded V(H), independent of proline cis-trans isomerization in V(L). This work also implies that V(H) should be a prime target in engineering well behaving antibody fragments.

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0221

Jermutus, L., Kolly, R., Földes-Papp, Z., Hanes, J., Rigler, R. and Plückthun, A. (2002) Ligand binding of a ribosome-displayed protein detected in solution at the single molecule level by fluorescence correlation spectroscopy. Eur. Biophys. J. 31, 179-184.
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Abstract

Interaction of a single-chain antibody fragment (scFv) with its cognate antigen while still attached to the ribosome was studied by fluorescence correlation spectroscopy (FCS). In experiments with purified scFv, FCS was capable of resolving the difference in diffusion time between free and antibody-bound labelled antigen. Ribosome-displayed antibody fragments generated by in vitro translation, in which neither the protein nor the mRNA leaves the ribosome owing to the absence of a stop codon and stabilizing buffer conditions, could be shown to specifically bind the antigen. The antibody-antigen interaction was specific, as shown by inhibition or displacement with unlabelled antigen and by control experiments with a non-cognate antibody fragment.

DOI: 10.1007/s00249-001-0204-0 | Read all as PDF | Close Abstract
0220

Kaufmann, M., Lindner, P., Honegger, A., Blank, K., Tschopp, M., Capitani, G., Plückthun, A. and Grütter, M. G. (2002) Crystal structure of the anti-His tag antibody 3D5 single-chain fragment complexed to its antigen. J. Mol. Biol. 318, 135-147.
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Abstract

The crystal structure of a mutant form of the single-chain fragment (scFv), derived from the monoclonal anti-His tag antibody 3D5, in complex with a hexahistidine peptide has been determined at 2.7 Å resolution. The peptide binds to a deep pocket formed at the interface of the variable domains of the light and the heavy chain, mainly through hydrophobic interaction to aromatic residues and hydrogen bonds to acidic residues. The antibody recognizes the C-terminal carboxylate group of the peptide as well as the main chain of the last four residues and the last three imidazole side-chains. The crystals have a solvent content of 77% (v/v) and form 70 Å-wide channels that would allow the diffusion of peptides or even small proteins. The anti-His scFv crystals could thus act as a framework for the crystallization of His-tagged target proteins. Designed mutations in framework regions of the scFv lead to high-level expression of soluble protein in the periplasm of Escherichia coli. The recombinant anti-His scFv is a convenient detection tool when fused to alkaline phosphatase. When immobilized on a matrix, the antibody can be used for affinity purification of recombinant proteins carrying a very short tag of just three histidine residues, suitable for crystallization. The experimental structure is now the basis for the design of antibodies with even higher stability and affinity.

DOI: 10.1016/S0022-2836(02)00038-4 | Read all as PDF | Close Abstract
0219

Ewert, S., Cambillau, C., Conrath, K. and Plückthun, A. (2002) Biophysical properties of camelid VHH domains compared to those of human VH3 domains. Biochemistry 41, 3628-3636.
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Abstract

Camelidae possess an unusual form of antibodies lacking the light chains. The variable domain of these heavy chain antibodies (VHH) is not paired, while the VH domain of all other antibodies forms a heterodimer with the variable domain of the light chain (VL), held together by a hydrophobic interface. Here, we analyzed the biophysical properties of four camelid VHH fragments (H14, AMD9, RN05, and CA05) and two human consensus VH3 domains with different CDR3 loops to gain insight into factors determining stability and aggregation of immunoglobulin domains. We show by denaturant-induced unfolding equilibria that the free energies of unfolding of VHH fragments are characterized by DGN-U values between 21.1 and 35.0 kJ/mol and thus lie in the upper range of values for VH fragments from murine and human antibodies. Nevertheless, the VHH fragments studied here did not reach the high values between 39.7 and 52.7 kJ/mol of the human consensus VH3 domains with which they share the highest degree of sequence similarity. Temperature-induced unfolding of the VHH fragments that were studied proved to be reversible, and the binding affinity after cooling was fully retained. The melting temperatures were determined to be between 60.1 and 66.7 °C. In contrast, the studied VH3 domains aggregated during temperature-induced denaturation at 63-65 °C. In summary, the camelid VHH fragments are characterized by a favorable but not unusually high stability. Their hallmark is the ability to reversibly melt without aggregation, probably mediated by the surface mutations characterizing the VHH domains, which allow them to regain binding activity after heat renaturation.

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0218

Betley, J. R., Cesaro-Tadic, S., Mekhalfia, A., Rickard, J. H., Denham, H., Partridge, L. J., Plückthun, A. and Blackburn, G. M. (2002) Direct screening for phosphatase activity by turnover-based capture of protein catalysts. Angew. Chem. Int. Ed. Engl. 41, 775-777.
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Abstract

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0217

Blank, K., Lindner, P., Diefenbach, B. and Plückthun, A. (2002) Self-immobilizing recombinant antibody fragments for immunoaffinity chromatography: generic, parallel, and scalable protein purification. Protein Expr. Purif. 24, 313-322.
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Abstract

We present the directed immobilization of recombinant antibody fragments as ligands for general immunoaffinity chromatography methods. It is based on fusion proteins of scFv fragments with several chitin- binding domains which can be immobilized directly from a crude bacterial lysate on inexpensive chitin beads for the purification of proteins without any gradient or detector. It has been used with a positive pressure manifold, allowing the parallel processing of 24 different samples on a milligram scale, as convenient as plasmid isolation. The method is demonstrated with several anti-protein antibodies. In addition, methods are presented of using an anti-His tag antibody either alone or directly coupled to IMAC to obtain very pure protein. As those methods are scalable, they should prove very useful in the parallel purification of natural and recombinant proteins on small scales (for proteomics), medium scales (for crystallography and NMR), and very large scales (for therapeutic proteins).

DOI: 10.1006/prep.2001.1575 | Read all as PDF | Close Abstract

2001

# Reference PDF

0216

Schaffitzel, C., Zahnd, C., Amstutz, P., Luginbühl, B. and Plückthun, A. (2001) In vitro selection and evolution of protein-ligand interactions by ribosome display in: Protein-Protein Interactions, A Molecular Cloning Manual (Golemis, E., ed) pp. 535-567. Cold Spring Harbor Laboratory Press, New York
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Abstract

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0215

Paci, E., Caflisch, A., Plückthun, A. and Karplus, M. (2001) Forces and energetics of hapten-antibody dissociation: a biased molecular dynamics simulation study. J. Mol. Biol. 314, 589-605.
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Abstract

The unbinding of fluorescein from the single-chain Fv fragment of the 4D5Flu antibody is investigated by biased molecular dynamics with an implicit solvation model. To obtain statistically meaningful results, a large number of unbinding trajectories are calculated; they involve a total simulation time of more than 200 ns. Simulations are carried out with a time-dependent perturbation and in the presence of a constant force. The two techniques, which provide complementary information, induce unbinding by favoring an increase in the distance between the ligand and the antibody. This distance is an appropriate progress variable for the dissociation reaction and permits direct comparison of the unbinding forces in the simulations with data from atomic force microscopy (AFM). The time-dependent perturbation generates unfolding pathways that are close to equilibrium and can be used to reconstruct the mean force; i.e. the derivative of the potential of mean force, along the reaction coordinate. This is supported by an analysis of the overall unbinding profile and the magnitude of the mean force, which are similar to those of the unbinding force (i.e. the external force due to the time-dependent perturbation) averaged over several unbinding events.The multiple simulations show that unbinding proceeds along a rather well-defined pathway for a broad range of effective pulling speeds. Initially, there is a distortion of the protein localized in the C-terminal region followed by the fluorescein exit from the binding site. This occurs in steps that involve breaking of specific electrostatic and van der Waals interactions. It appears that the simulations do not explore the same barriers as those measured in the AFM experiments because of the much higher unfolding speed in the former. The dependence of the force on the logarithm of the loading rate is linear and the slope is higher than in the AFM, in agreement with experiment in other systems, where different slopes were observed for different regimes. Based on the unbinding events, mutations in the 4D5Flu antigen binding site are predicted to result in significant changes in the unbinding force. Copyright 2001 Academic Press.

DOI: 10.1006/jmbi.2001.5103 | Read all as PDF | Close Abstract
0214err

Ciatto, C., Tissot, A. C., Tschopp, M., Capitani, G., Pecorari, F., Plückthun, A. and Grütter, M. G. (2001) ERRATUM: Zooming in on the hydrophobic ridge of H-2Db: implications for the conformational variability of bound peptides. J. Mol. Biol. 314, 1257.
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Abstract

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0214

Ciatto, C., Tissot, A. C., Tschopp, M., Capitani, G., Pecorari, F., Plückthun, A. and Grütter, M. G. (2001) Zooming in on the hydrophobic ridge of H-2Db: implications for the conformational variability of bound peptides. J. Mol. Biol. 312, 1059-1071.
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Abstract

Class I major histocompatibility complex (MHC) molecules, which display intracellularly processed peptides on the cell surface for scanning by T-cell receptors (TCRs), are extraordinarily polymorphic. MHC polymorphism is believed to result from natural selection, since individuals heterozygous at the corresponding loci can cope with a larger number of pathogens. Here, we present the crystal structures of the murine MHC molecule H-2D(b) in complex with the peptides gp276 and np396 from the lymphocytic choriomeningitis virus (LCMV), solved at 2.18 A and 2.20 A resolution, respectively. The most prominent feature of H-2D(b) is a hydrophobic ridge that cuts across its antigen-binding site, which is conserved in the L(d)-like family of class I MHC molecules. The comparison with previously solved crystal structures of peptide/H-2D(b) complexes shows that the hydrophobic ridge focuses the conformational variability of the bound peptides in a ""hot-spot"", which could allow optimal TCR interaction and discrimination. This finding suggests a functional reason for the conservation of this structural element. Copyright 2001 Academic Press.

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0213

Arndt, K. M., Müller, K. M. and Plückthun, A. (2001) Helix-stabilized Fv (hsFv) antibody fragments: substituting the constant domains of a Fab fragment for a heterodimeric coiled-coil domain. J. Mol. Biol. 312, 221-228.
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Abstract

Antibody Fv fragments would in principle be useful for a variety of biotechnological applications because of their small size and the possibility to produce them in relatively large amounts in recombinant form; however, their limited stability is a drawback. To solve this problem, both domains are usually fused via a peptide linker to form a single-chain Fv (scFv) fragment, but in some cases this leads to a dimerization. We present an alternative format for stabilizing antibody Fv fragments. The C(H)1 and C(L) domain of the Fab fragment were replaced with a heterodimeric coiled coil (WinZip-A2B1), which had previously been selected using a protein-fragment complementation assay in Escherichia coli. This new antibody format was termed helix-stabilized Fv fragment (hsFv), and was compared to the corresponding Fv, Fab and single-chain Fv format. Bacterial growth and expression of the hsFv was significantly improved compared to the Fab fragment. The hsFv fragment formed a heterodimer of heavy and light chain with the expected molecular mass, also under conditions where the scFv fragment was predominantly dimeric. The hsFv fragment was significantly more stable than the Fv fragment, and nearly as stable as the scFv fragment under the conditions used (80 nM protein concentration). Thus, the format of a helix-stabilized Fv (hsFv) fragment can be a useful alternative to existing recombinant antibody formats, especially in cases where poor expression of Fab fragments or multimerization of scFv fragments is a problem. Copyright 2001 Academic Press.

DOI: 10.1006/jmbi.2001.4915 | Read all as PDF | Close Abstract
0212

Valle, F., Derose, J.A., Dietler, G., Kawe, M., Semenza, G. and Plückthun, A. (2001) Imaging the native structure of the charperone protein GroEL without fixation using atomic force microscopy. J. Microsc. 203, 195-198.
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Abstract

Most sample preparation methods for scanning probe or electron microscopy require that biomolecules, such as proteins, be fixed. Fixation destroys the molecular functionality and can possibly affect the true molecular structure. Here we report sample preparation conditions that allow the imaging of an unfixed protein, GroEL, under in-vivo conditions, by atomic force microscopy. Under these conditions, the protein should maintain its native structure and biological activity. The typical toroidal shape with pore of the GroEL complex was easily visible in the images. Images of a single complex show dimensions that agree well with crystallographic data. Under in-vivo conditions, it should be possible to study the biological activity and function of proteins.

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0211

Schaffitzel, C. and Plückthun, A. (2001) Protein-fold evolution in the test tube. Trends Biochem. Sci. 26, 577-579.
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Abstract

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0210

Lindner, P. and Plückthun, A. (2001) Miniantibodies in: Antibody Engineering (Kontermann, R., and Dübel, S., eds) pp. 637-647. Springer-Verlag, Berlin, Heidelberg
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Abstract

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0209

Bothmann, H. and Plückthun, A. (2001) Improving expression of scFv fragments by coexpression of periplasmic chaperones. in: Antibody Engineering (Kontermann, R., and Dübel, S., eds) pp. 307-317. Springer-Verlag, Berlin, Heidelberg
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Abstract

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0208

Burmester, J. and Plückthun, A. (2001) Construction of scFv fragments from hybridoma or spleen cells by PCR assembly in: Antibody Engineering (Kontermann, R., and Dübel, S., eds) pp. 19-40. Springer-Verlag, Berlin, Heidelberg
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Abstract

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0207

Ramm, K. and Plückthun, A. (2001) High enzymatic activity and chaperone function are mechanistically related features of the dimeric E. coli peptidyl-prolyl-isomerase FkpA. J. Mol. Biol. 310, 485-498.
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Abstract

We have recently described the existence of a chaperone activity for the dimeric peptidyl-prolyl cis/trans isomerase FkpA from the periplasm of Escherichia coli that is independent of its isomerase activity. We have now investigated the molecular mechanism of these two activities in vitro in greater detail. The isomerase activity with a protein substrate (RNaseT1) is characterized by a 100-fold higher k(cat)/K(M) value than with a short tetrapeptide substrate. This enhanced activity with a protein is due to an increased affinity towards the protein substrate mediated by a polypeptide-binding site that is distinct from the active site. The chaperone activity is also mediated by interaction of folding and unfolding intermediates with a binding site that is most likely identical to the polypeptide-binding site which enhances catalysis. Both activities are thus mechanistically related, being based on the transient interaction with this high-affinity polypeptide-binding site. Only the isomerase activity, but not the chaperone activity, with the substrate citrate synthase can be inhibited by FK520. Experiments with the isolated domains of FkpA imply that both the isomerase and the chaperone site are located on the highly conserved FKBP domain. The additional amino-terminal domain mediates the dimerization and thus places the two active sites of the FKBP domains in juxtaposition, such that they can simultaneously interact with a protein, and this is required for full catalytic activity. Copyright 2001 Academic Press.

DOI: 10.1006/jmbi.2001.4747 | Read all as PDF | Close Abstract
0206

Honegger, A. and Plückthun, A. (2001) The influence of the buried glutamine or glutamate residue in position 6 on the structure of immunoglobulin variable domains. J. Mol. Biol. 309, 687-699.
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Abstract

Immunoglobulin V(H) domain frameworks can be grouped into four distinct types, depending on the main-chain conformation of framework 1. Based on the analysis of over 200 X-ray structures representing more than 100 non-redundant V(H) domain sequences, we have come to the conclusion that the marked structural variability of the V(H) framework 1 region is caused by three residues: the buried side-chain of H6, which can be either a glutamate or a glutamine residue, the residue in position H7, which may be proline only if H6 is glutamine, and by H9 (H10 according to a new consensus nomenclature), which has to be either glycine or proline if H6 is a glutamate residue. In natural antibodies, these three residues are encoded in combinations that are compatible with each other and with the rest of the structure and therefore will yield functional molecules. However, the degenerate primer mixtures commonly used for PCR cloning of antibody fragments can and frequently do introduce out-of-context mutations to combinations that can lead to severe reduction of stability, production yield and antigen affinity. Copyright 2001 Academic Press.

DOI: 10.1006/jmbi.2001.4664 | Read all as PDF | Close Abstract
0205

Burmester, J., Spinelli, S., Pugliese, L., Krebber, A., Honegger, A., Jung, S., Schimmele, B., Cambillau, C. and Plückthun, A. (2001) Selection, characterization and X-ray structure of anti-ampicillin single-chain Fv fragments from phage-displayed murine antibody libraries. J. Mol. Biol. 309, 671-685.
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Abstract

Single-chain Fv (scFv) antibody libraries were constructed from mice immunized with an ampicillin-bovine serum albumin conjugate. Several antibodies with specificity for intact ampicillin were selected by phage display and characterized. The antibody scFv fragment aL2 binds to intact ampicillin and shows no detectable cross-reactivity with hydrolyzed ampicillin. We determined the X-ray structures of two crystal forms of w.t. aL2, which differ mainly in the side-chain conformation of Trp H109 (according to a new consensus nomenclature Kabat residue number H95) in the extremely short (three residues) CDR H3 and the presence or absence of a well-resolved molecule of 2-methyl-pentane-2,4-diol in the bottom of the binding pocket. Attempts to co-crystallize aL2 with its antigen or to diffuse ampicillin into the wild-type aL2 crystals were unsuccessful, since crystal contacts obstruct the binding pocket. However, a mutant with two point mutations near the N terminus (Gln H6 replaced by Glu and Ala H10 (Kabat H9) replaced by Gly) crystallized in a form compatible with antigen-binding. Although the mutations affect the conformation of framework I, the conformations of the binding pocket of the uncomplexed wild-type aL2 and of the mutant complex were almost identical. The structure explains the specificity of the antibody for intact ampicillin and the degree of cross-reactivity of aL2 with a wide variety of ampicillin analogs. This antibody system will be very useful as a diagnostic reagent for antibiotics use and abuse, as a model for the effect of expression of antibiotic binding molecules in Escherichia coli, and for directed evolution towards high antibiotic resistance. Copyright 2001 Academic Press.

DOI: 10.1006/jmbi.2001.4663 | Read all as PDF | Close Abstract
0204

Honegger, A. and Plückthun, A. (2001) Yet another numbering scheme for immunoglobulin variable domains: An automatic modeling and analysis tool.. J. Mol. Biol. 309, 657-670.
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Abstract

A common residue numbering scheme for all immunoglobulin variable domains (immunoglobulin light chain lambda (V(lambda)) and kappa (V(kappa)) variable domains, heavy chain variable domains (V(H)) and T-cell receptor alpha (V(alpha)), beta (V(beta)), gamma (V(gamma)) and delta (V(delta)) variable domains) has been devised. Based on the spatial alignment of known three-dimensional structures of immunoglobulin domains, it places the alignment gaps in a way that minimizes the average deviation from the averaged structure of the aligned domains. This residue numbering scheme was applied to the immunoglobulin variable domain structures in the PDB database to automate the extraction of information on structural variations in homologous positions of the different molecules. A number of methods are presented that allow the automated projection of information derived from individual structures or from the comparison of multi-structure alignments onto a graphical representation of the sequence alignment. Copyright 2001 Academic Press.

DOI: 10.1006/Jmbi.2001.4662 | Read all as PDF | Close Abstract
0203

Jung, S., Spinelli, S., Schimmele, B., Honegger, A., Pugliese, L., Cambillau, C. and Plückthun, A. (2001) The importance of framework residue H6, H7 and H10 in antibody heavy chains: Experimental evidence for a new structural subclassification of antibody VH domains. J. Mol. Biol. 309, 701-716.
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Abstract

The N-terminal segment (FR-H1) of the heavy chain (V(H)) of antibodies shows significant conformational variability correlating with the nature of the amino acids H6, H7 and H10 (Kabat H9). In this study, we have established a causal relationship between the local sequence and the structure of this framework region and linked this relationship to important biophysical properties such as affinity, folding yield and stability. We have generated six mutants of the scFv fragment aL2, covering some of the most abundant amino acid combinations in positions H6, H7 and H10 (according to a new consensus nomenclature, Kabat H9). For the aL2 wild-type (w.t.) with the sequence 6(Q)7(P)10(A) and for two of the mutants, the X-ray structures have been determined. The structure of the triple mutant aL2-6(E)7(S)10(G) shows the FR-H1 backbone conformations predicted for this amino acid combination, which is distinctly different from the structure of the w.t, thus supporting our hypothesis that these residues determine the conformation of this segment. The mutant aL2-6(E)7(P)10(G) represents a residue combination not occurring in natural antibody sequences. It shows a completely different, unique structure in the first beta-strand of V(H), not observed in natural Fv fragments and forms a novel type of diabody. Two V(H) domains of the mutant associate by swapping the first beta-strand. Concentration-dependent changes in Trp fluorescence indicate that this dimerization also occurs in solution. The mutations in amino acids H6, H7 and H10 (Kabat H9) influence the dimerization behavior of the scFv and its thermodynamic stability. All the observations reported here have practical implications for the cloning of Fv fragments with degenerate primers, as well as for the design of new antibodies by CDR grafting or synthetic libraries. Copyright 2001 Academic Press.

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0202

Schaffitzel, C., Berger, I., Postberg, J., Hanes, J., Lipps, H. J. and Plückthun, A. (2001) In vitro generated antibodies specific for telomeric guanine-quadruplex DNA react with Stylonychia lemnae macronuclei. Proc. Natl. Acad. Sci. U. S. A. 98, 8572-8577.
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Abstract

Most eukaryotic telomeres contain a repeating motif with stretches of guanine residues that form a 3'-terminal overhang extending beyond the telomeric duplex region. The telomeric repeat of hypotrichous ciliates, d(T(4)G(4)), forms a 16-nucleotide 3'-overhang. Such sequences can adopt parallel-stranded as well as antiparallel-stranded quadruplex conformations in vitro. Although it has been proposed that guanine-quadruplex conformations may have important cellular roles including telomere function, recombination, and transcription, evidence for the existence of this DNA structure in vivo has been elusive to date. We have generated high-affinity single-chain antibody fragment (scFv) probes for the guanine-quadruplex formed by the Stylonychia telomeric repeat, by ribosome display from the Human Combinatorial Antibody Library. Of the scFvs selected, one (Sty3) had an affinity of K(d) = 125 pM for the parallel-stranded guanine-quadruplex and could discriminate with at least 1,000-fold specificity between parallel or antiparallel quadruplex conformations formed by the same sequence motif. A second scFv (Sty49) bound both the parallel and antiparallel quadruplex with similar (K(d) = 3--5 nM) affinity. Indirect immunofluorescence studies show that Sty49 reacts specifically with the macronucleus but not the micronucleus of Stylonychia lemnae. The replication band, the region where replication and telomere elongation take place, was also not stained, suggesting that the guanine-quadruplex is resolved during replication. Our results provide experimental evidence that the telomeres of Stylonychia macronuclei adopt in vivo a guanine-quadruplex structure, indicating that this structure may have an important role for telomere functioning.

DOI: 10.1073/pnas.141229498 | Read all as PDF | Close Abstract
0201

Mössner, E., Koch, H. and Plückthun, A. (2001) Fast selection of antibodies without antigen purification: Adaption of the protein fragment complementation assay to select antigen-antibody pairs.. J. Mol. Biol. 308, 115-122.
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Abstract

We have adapted the protein fragment complementation assay (PCA) to the screening and selection of antibodies in the single-chain Fv (scFv) format. In this assay, two interacting proteins (target and antibody) are genetically fused to the two halves of the dissected enzyme dihydrofolate reductase. Binding of the two partners reassembles this enzyme and reconstitutes its activity, thus allowing growth on minimal medium. We have optimized this system with regard to linker length and orientation, and can reach an efficiency for antigen/antibody interactions similar to that with fused leucine zippers. Using several model antibodies specific for peptides and proteins, we show that cognate interactions give rise to about seven orders of magnitude more colonies than non-specific interactions. When transforming mixtures of plasmids encoding different antigens and/or antibodies, all colonies tested contained plasmids encoding cognate pairs. We believe that this system will be very powerful as a routine system for generating antibodies, especially in functional genomics, since it does not require purification and immobilization of the antigen. The identification of an antibody specific for a cDNA or EST-encoded protein will require only cloning, transformation and plating of bacteria. Copyright 2001 Academic Press.

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0200

Chang, Ch., Mooser, A., Plückthun, A. and Wlodawer, A. (2001) Crystal structure of the dimeric C-terminal domain of TonB reveals a novel fold. J. Biol. Chem. 276, 27535-27540.
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Abstract

The TonB-dependent complex of Gram-negative bacteria couples the inner membrane proton motive force to the active transport of iron.siderophore and vitamin B(12) across the outer membrane. The structural basis of that process has not been described so far in full detail. The crystal structure of the C-terminal domain of TonB from Escherichia coli has now been solved by multiwavelength anomalous diffraction and refined at 1.55-A resolution, providing the first evidence that this region of TonB (residues 164-239) dimerizes. Moreover, the structure shows a novel architecture that has no structural homologs among any known proteins. The dimer of the C-terminal domain of TonB is cylinder-shaped with a length of 65 A and a diameter of 25 A. Each monomer contains three beta strands and a single alpha helix. The two monomers are intertwined with each other, and all six beta-strands of the dimer make a large antiparallel beta-sheet. We propose a plausible model of binding of TonB to FhuA and FepA, two TonB-dependent outer-membrane receptors.

DOI: 10.1074/jbc.M102778200 | Read all as PDF | Close Abstract
0199

Amstutz, P., Forrer, P., Zahnd, C. and Plückthun, A. (2001) In vitro display technologies: Novel developments and applications.. Curr. Opin. Biotechnol. 12, 400-405.
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Abstract

In vitro display techniques are powerful tools to select polypeptide binders against various target molecules. Novel applications include maturation of protein affinity and stability, selection for enzymatic activity, and the display of cDNA and random polypeptide libraries. Taken together, these display techniques have great potential for biotechnological, medical and proteomic applications.

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0198

Mössner, E. and Plückthun, A. (2001) Directed evolution with fast and efficient selection technologies.. Chimia 55, 325-329.
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Abstract

Directed molecular evolution has proven to be a very powerful concept for the generation of proteins with improved properties, such as increased activity, binding affinity, folding efficiency or enhanced chemical and/or thermodynamic stability. We review here advances in the selection of proteins carrying desired mutations from pools of proteins that mostly carry unfavourable alterations. A short overview of the concept of directed evolution with a discussion of randomisation strategies is given first. Two technologies for the selection of proteins, each with its own advantages, are then discussed: In Ribosome Display, all steps are carried out in a cell-free system, which allows one to create very large libraries (diversity > 1011), rapidly introduce mutations and thus obtain an iterative evolution. Examples with antibodies evolved for affinity or stability are discussed. In the Protein Fragment Complementation Assay, a library-versus-library selection is possible, that is, a simultaneous selection of binders against many targets. Examples with peptide and antibody libraries are discussed.

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0197

Jäger, M., Gehrig, P. and Plückthun, A. (2001) The scFv fragment of the antibody hu4D5-8: Evidence for early premature domain interaction in refolding.. J. Mol. Biol. 305, 1111-1129.
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Abstract

Fluorescence spectroscopy and 1H/2H-exchange techniques have been applied to characterize the folding of an scFv fragment, derived from the humanized anti-HER2 antibody hu4D5-8. A stable intermediate, consisting of a native VL domain and an unfolded VH domain, is populated under equilibrium unfolding conditions. A partially structured intermediate, with 1H/2H-exchange protection significantly less than that of the two isolated domains together, is detectable upon refolding the equilibrium-denatured scFv fragment. This means that the domains in the heterodimer do not fold independently. Rather, they associate prematurely before full 1H/2H-exchange protection can be gained. The formation of the native heterodimer from the non-native intermediate is a slow, cooperative process, which is rate-limited by proline cis/trans-isomerization. Unproductive domain association is also detectable after short-term denaturation, i.e. with the proline residues in native conformation. Only a fraction of the short-term denatured protein folds into the native protein in a fast, proline-independent reaction, because of spontaneous proline cis/trans-reisomerization in the early non-native intermediate. The comparison with the previously studied antibody McPC603 has now allowed us to delineate similarities in the refolding pathway of scFv fragments.

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0196

Iwai, H., Lingel, A. and Plückthun, A. (2001) Cyclic green fluoroscent protein produced in vivo using and artifically Split PI-PfuI intein from Pyrococcus furiosus. J. Biol. Chem. 276, 16548-16554.
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Abstract

A cyclic protein was produced in vivo using the intein from Pyrococcus furiosus PI-PfuI in a novel approach to create a circular permutation of the precursor protein by introducing new termini in the intein domain. Green fluorescent protein (GFP) was cyclized with this method in vivo on milligram scales. There was no by-product of linear or polymerized species isolated, unlike with other in vitro or in vivo cyclization methods utilizing inteins. Cyclized GFP unfolded at half the rate of the linear form upon chemical denaturation and required >2 days in 7 m guanidine hydrochloride until a residual fast folding phase (consistent with a persistent cis-proline) had disappeared. Cyclic GFP might become a novel tool for studying the role of termini and backbone topology in various biological processes such as protein degradation and translocation in vivo as well as in vitro.

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0195

Willuda, J., Kubetzko, S., Waibel, R., Schubiger, P. A., Zangemeister-Wittke, U. and Plückthun, A. (2001) Tumor targeting of mono-, di- and tetravalent Anti-p185HER-2 miniantibodies multimerized by self-associating peptides.. J. Biol. Chem. 276, 14385-14392.
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Abstract

Multimerization of antibody fragments increases the valency and the molecular weight, both identified as key features in the design of the optimal targeting molecule. Here, we report the construction of mono-, di-, and tetrameric variants of the anti-tumor p185(HER-2) single chain Fv fragment 4D5 by fusion of self-associating peptides to the carboxyl terminus. Dimeric miniantibodies with a synthetic helix-turn-helix domain and tetrameric ones with the multimerization domain of the human p53 protein were produced in functional form in the periplasm of Escherichia coli. We have directly compared these molecules and the single-chain Fv fragment in the targeting of SK-OV-3 xenografts. Tetramerization of the 4D5 antibody fragment resulted in increased serum persistence, significantly reduced off-rate, due to the avidity effect, both in surface plasmon resonance measurements on purified p185(HER-2) and on SK-OV-3 cells. The (99m)technetium-tricarbonyl-labeled tetrameric 4D5-p53 miniantibody localized with the highest dose at the tumor and remained stably bound for at least 72 h. The highest total dose was 4.3% injected dose/g after 24 h, whereas the highest tumor-to-blood ratio was found to be 13.5:1 after 48 h, with a total dose of 3.2% injected dose/g. The tetramer shows no higher avidity than the dimer, presumably since the simultaneous binding to more than two antigen molecules on the surface of cells is not possible, and the improvement in performance over the dimer must at least be due in part to the molecular weight. These results demonstrate that multimerization by self-associating peptides can be used for the development of more effective targeting molecules for medical diagnostics and therapy.

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0194

Wörn, A. and Plückthun, A. (2001) Stability engineering of antibody single-chain Fv fragments. J. Mol. Biol. 305, 989-1010.
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Abstract

The application of single-chain Fv fragments (scFv) in medicine and biotechnology places great demands on their stability. Only recently has attention been given to the production of highly stable scFvs, and in a number of examples it was found that such fragments indeed perform better during practical applications. The structural parameters influencing scFv stability are now beginning to be elucidated. This review summarizes progress in rational and evolutionary engineering methods, the structural implications of these results, as well as some examples where stability engineering has been successfully applied.

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0193

Jermutus, L., Honegger, A., Schwesinger, F., Hanes, J. and Plückthun, A. (2001) Tailoring in vitro evolution for protein affinity or stability. Proc. Natl. Acad. Sci. U. S. A. 98, 75-80.
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Abstract

We describe a rapid and general technology working entirely in vitro to evolve either the affinity or the stability of ligand-binding proteins, depending on the chosen selection pressure. Tailored in vitro selection strategies based on ribosome display were combined with in vitro diversification by DNA shuffling to evolve either the off-rate or thermodynamic stability of single-chain Fv antibody fragments (scFvs). To demonstrate the potential of this method, we chose to optimize two proteins already possessing favorable properties. A scFv with an initial affinity of 1.1 nM (k(off) at 4 degrees C of 10(-4) s(-1)) was improved 30-fold by the use of off-rate selections over a period of several days. As a second example, a generic selection strategy for improved stability exploited the property of ribosome display that the conditions can be altered under which the folding of the displayed protein occurs. We used decreasing redox potentials in the selection step to select for molecules stable in the absence of disulfide bonds. They could be functionally expressed in the reducing cytoplasm, and, when allowed to form disulfides again, their stability had increased to 54 kJ/mol from an initial value of 24 kJ/mol. Sequencing revealed that the evolved mutant proteins had used different strategies of residue changes to adapt to the selection pressure. Therefore, by a combination of randomization and appropriate selection strategies, an in vitro evolution of protein properties in a predictable direction is possible.

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2000

# Reference PDF

0192

Plückthun, A., Schaffitzel, C., Hanes, J. and Jermutus, L. (2000) In vitro selection and evolution of proteins. Adv. Protein Chem. 55, 367-403.
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Abstract

No abstract

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0191

Hanes, J., Schaffitzel, C., Knappik, A. and Plückthun, A. (2000) Picomolar affinity antibodies from a fully synthetic naive library selected and evolved by ribosome display. Nature Biotechnol. 18, 1287-1292.
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Abstract

Here we applied ribosome display to in vitro selection and evolution of single-chain antibody fragments (scFvs) from a large synthetic library (Human Combinatorial Antibody Library; HuCAL) against bovine insulin. In three independent ribosome display experiments different clusters of closely related scFvs were selected, all of which bound the antigen with high affinity and specificity. All selected scFvs had affinity-matured up to 40-fold compared to their HuCAL progenitors, by accumulating point mutations during the ribosome display cycles. The dissociation constants of the isolated scFvs were as low as 82 pM, which validates the design of the naive library and the power of this evolutionary method. We have thus mimicked the process of antibody generation and affinity maturation with a synthetic library in a cell-free system in just a few days, obtaining molecules with higher affinities than most natural antibodies.

DOI: 10.1038/82407 | Read all as PDF | Close Abstract
0190

Hanes, J., Jermutus, L. and Plückthun, A. (2000) Selecting and evolving functional proteins in vitro by ribosome display. Methods Enzymol. 328, 404-430.
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Abstract

No abstract

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0189

Arndt, K. M., Jung, S., Krebber, C. and Plückthun, A. (2000) Selectively infective phage technology. Methods Enzymol. 328, 364-388.
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Abstract

No abstract

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0188err

Tissot, A. C., Ciatto, C., Mittl, P. R. E., Grütter, M. G. and Plückthun, A. (2000) ERRATUM: Viral escape at the molecular level explained by quantitative T-cell receptor/peptide/MHC interactions and the crystal structure of a peptide/MHC complex.. J. Mol. Biol. 304, 683.
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Abstract

No abstract

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0188

Tissot, A. C., Ciatto, C., Mittl, P. R. E., Grütter, M. G. and Plückthun, A. (2000) Viral escape at the molecular level explained by quantitative T-cell receptor/peptide/MHC interactions and the crystal structure of a peptide/MHC complex.. J. Mol. Biol. 302, 873-885.
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Abstract

Viral escape, first characterized for the lymphocytic choriomeningitis virus (LCMV) in a mouse transgenic for the P14 T cell-receptor (TCR), can be due to mutations in T-cell epitopes. We have measured the affinity between the H-2D(b) containing the wild-type and two of its ""viral escape"" epitopes, as well as other altered peptide ligands (APL), by using BIACORE analysis, and solved the crystal structure of H-2D(b) in complex with the wild-type peptide at 2.75 A resolution. We show that viral escape is due to a 50 to 100-fold reduction in the level of affinity between the P14 TCR and the binary complexes of the MHC molecule with the different peptides. Structurally, one of the mutations alters a TCR contact residue, while the effect of the other on the binding of the TCR must be indirect through structural rearrangements. The former is a null ligand, while the latter still leads to some central tolerance. This work defines the structural and energetic threshold for viral escape. Copyright 2000 Academic Press.

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0186

Bothmann, H. and Plückthun, A. (2000) The periplasmic Escherichia coli peptidylprolyl cis,trans-isomerase FkpA: I. Increased functional expression of antibody fragments with and without cis-prolines. J. Biol. Chem. 275, 17100-17105.
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Abstract

The production of recombinant proteins in the periplasm of Escherichia coli can be limited by folding problems, leading to periplasmic aggregates. We used a selection system for periplasmic chaperones based on the coexpression of an E. coli library with a poorly expressing antibody single-chain Fv (scFv) fragment displayed on filamentous phage (Bothmann, H., and Pluckthun, A. (1998) Nature Biotechnol. 16, 376-380). By selection for a functional antibody, the protein Skp had been enriched previously and shown to improve periplasmic expression of a wide range of scFv fragments. This selection strategy was now repeated with a library constructed from the genomic DNA of an skp-deficient strain, leading to enrichment of the periplasmic peptidylprolyl cis,trans-isomerase (PPIase) FkpA. Coexpression of FkpA increased the amount of fusion protein displayed on the phage and dramatically improved functional periplasmic expression even of scFv fragments not containing cis-prolines. In contrast, the coexpression of the periplasmic PPIases PpiA and SurA showed no increase in the functional scFv fragment level in the periplasm or displayed on phage. Together with the in vitro data in the accompanying paper (Ramm, K., and Pluckthun, A. (2000) J. Biol. Chem. 275, 17106-17113), we conclude that the effect of FkpA is independent of its PPIase activity.

DOI: 10.1074/Jbc.M910233199 | Read all as PDF | Close Abstract
0185

Ramm, K. and Plückthun, A. (2000) The periplasmic Escherichia coli peptidylprolyl cis,trans-isomerase FkpA: II. Isomerase-independent chaperone activity in vitro. J. Biol. Chem. 275, 17106-17113.
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Abstract

We recently identified FkpA by selecting for the increased yield of antibody single-chain Fv (scFv) fragments in phage display, even of those not containing cis-prolines. We have now investigated the properties of FkpA in vitro. The peptidylprolyl cis-trans-isomerase activity of FkpA was found to be among the highest of any such enzyme with a protein substrate, yet FkpA is not able to enhance the proline-limited refolding rate of the disulfide-free hu4D5-8 scFv fragment, probably due to inaccessibility of Pro-L95. Nevertheless, the yield of the soluble and functional scFv fragment was dramatically increased in vitro in the presence of FkpA. Similar effects were observed for an scFv fragment devoid of cis-prolines. We are thus forced to conclude that the observed folding-assisting function is independent of the isomerase activity of the protein. The beneficial effect of FkpA was found to be due to two components. First, FkpA interacts with early folding intermediates, thus preventing their aggregation. Additionally, it has the ability to reactivate inactive protein, possibly also by binding to a partially unfolded species that may exist in equilibrium with the aggregated form, which may thus be released on a productive pathway. These in vitro measurements therefore fully reflect the in vivo results from periplasmic overexpression of FkpA.

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0184

Jäger, M. and Plückthun, A. (2000) Direct evidence by H/D exchange and ESI-MS for transient unproductive domain interaction in the refolding of an antibody scFv fragment. Protein Sci. 9, 552-563.
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Abstract

The refolding kinetics of a single-chain Fv (scFv) fragment, derived from a stabilized mutant of the phosphorylcholine binding antibody McPC603, was investigated by H/D exchange and ESI-MS and compared with the folding kinetics of its constituting domains V(H) and V(L). Both V(H) and V(L) adopt essentially native-like exchange protection within the dead time of the manual-mixing H/D exchange experiment (10 s) and in the case of V(L), which contains two cis-prolines in the native conformation, this fast protection is independent of proline cis/trans isomerization. At the earliest time point resolvable by manual mixing, fewer deuterons are protected in the scFv fragment than in the two isolated domains together, despite the fact that the scFv fragment is significantly more stable than V(L) and V(H). Full H/D exchange protection in the scFv fragment is gained on a time scale of minutes. This means that the domains in the scFv fragment do not refold independently. Rather, they associate prematurely and in nonnative form, a kinetic trap. Unproductive domain association is observed both after equilibrium- and short-term denaturation. For the equilibrium-denatured scFv fragment, whose native structure formation is dependent on a cis conformation of an interface proline in V(L), this cis/trans isomerization reaction proceeds about one order in magnitude more slowly than the escape from the trap to a conformation where full H/D exchange protection is already achieved. We interpret these data in terms of a general kinetic scheme involving intermediates with and without domain association.

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0183

Tissot, A. C., Pecorari, F. and Plückthun, A. (2000) Characterizing the functionality of recombinant T-cell receptors in vitro: A pMHC tetramer based approach. J. Immunol. Methods 236, 147-165.
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Abstract

The very low affinity of the T-cell receptor (TCR) for the peptide-major histocompatibility complex (pMHC) has made it very challenging to design assays for testing the functionality of these molecules on small scales, which in turn has severely hampered the progress in developing expression and refolding methodologies for the TCR. We have now developed an ELISA assay for detecting pMHC binding to functional recombinant TCRs. It uses tetramers of biotinylated pMHCs bound to a neutravidin-horseradish peroxidase conjugate and detects the presence of functional TCR, bound in a productive orientation to an immobilized anti-Cbeta antibody. Specificity can be stringently demonstrated by inhibition with monomeric pMHCs. The assay is very sensitive and specific, and requires only very small amounts of protein. It has allowed us to study the unstable recombinant TCR P14, which we expressed and refolded from Escherichia coli. The TCR P14 is directed against the most abundant epitope of LCMV. We have confirmed the specificity of the interaction by BIAcore, and were able to determine the dissociation constant of the interaction of the P14 TCR and of the gp33-pMHC as 6 microM. This affinity ranks it among the tighter ones of TCR-pMHC interactions, and unusually low affinity thus does not seem to be the cause of the modest protective power of these T-cells, compared to others elicited in the anti-LCMV response. This strategy of multimerizing one partner and immobilizing the other in both a native form and productive orientation should be generally useful for characterizing the weak interactions of cell-surface molecules.

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0182

Linke, R. P., Schäeffer, J., Gielow, P., Lindner, P., Lottspeich, F., Plückthun, A. and Weiss, E. H. (2000) Production of recombinant human _2-microglobulin for scintigraphic diagnosis of amyloidosis in uremia and hemodialysis. Eur. J. Biochem. 267, 627-633.
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Abstract

Amyloid of beta2-microglobulin (beta2m) origin can be diagnosed using 131I-radiolabelled-beta2m scintigraphy in patients with uremia and hemodialysis treatment. As the tracer beta2m is isolated from another patient, it carries the common risks, including viral infections such as Hepatitis B, C and HIV, which are associated with human plasma products. In order to exclude these risks we have produced recombinant human beta2m (rhbeta2m) in Escherichia coli. The expression vector pASK40DeltaLbeta2m(His)5 contains a C-terminal (His)5-tag for purification via immobilized metal ion affinity chromatography (IMAC). Size exclusion chromatography on a Superose 12 column represents the second step of purification. The isolated rhbeta2mH5 reacted in an immunochemically identical manner to native human beta2m, and showed a single band of approximately 11.8 kDa in Western blot analysis and revealed a single spot in two-dimensional gel electrophoresis. Mass spectrometry analysis revealed a single peak at the expected molecular mass of 12 415.8 Da. Uniformity was further proven by crystallization and N-terminal amino-acid sequence analysis. The rhbeta2mH5 protein was then produced under conditions that allow the intravenous use in humans. Intraveneously applied indium-111-labelled rhbeta2mH5 was monitored in hemodialysed patients with and without known beta2m-amyloidosis. The tracer was localized specifically to particular areas known to contain amyloid. Thus, this rhbeta2mH5 preparation is suitable for detecting amyloid-containing organs of the beta2m-class in vivo and fulfils the requirements of a tracer for common use. Finally, the use of indium-111 instead of iodine-131 has reduced the radioactive load and resulted in higher resolution.

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0181

Yang, F., Forrer, P., Dauter, Z., Conway, J. F., Cheng, N., Cerritelli, M. E., Steven, A. C., Plückthun, A. and Wlodawer, A. (2000) Novel fold and capsid-binding properties of the _-phage display platform protein gpD. Nature Struct. Biol. 7, 230-237.
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Abstract

The crystal structure of gpD, the capsid-stabilizing protein of bacteriophage lambda, was solved at 1.1 A resolution. Data were obtained from twinned crystals in space group P21 and refined with anisotropic temperature factors to an R-factor of 0.098 (Rfree = 0. 132). GpD (109 residues) has a novel fold with an unusually low content of regular secondary structure. Noncrystallographic trimers with substantial intersubunit interfaces were observed. The C-termini are well ordered and located on one side of the trimer, relatively far from its three-fold axis. The N-termini are disordered up to Ser 15, which is close to the three-fold axis and on the same side as the C-termini. A density map of the icosahedral viral capsid at 15 A resolution, obtained by cryo-electron microscopy and image reconstruction, reveals gpD trimers, seemingly indistinguishable from the ones seen in the crystals, at all three-fold sites. The map further reveals that the side of the trimer that binds to the capsid is the side on which both termini reside. Despite this orientation of the gpD trimer, fusion proteins connected by linker peptides to either terminus bind to the capsid, allowing protein and peptide display.

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0180

Knappik, A., Ge, L., Honegger, A., Pack, P., Fischer, M., Wellnhofer, G., Hoess, A., Wölle, J., Plückthun, A. and Virnekäs, B. (2000) Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J. Mol. Biol. 296, 57-86.
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Abstract

By analyzing the human antibody repertoire in terms of structure, amino acid sequence diversity and germline usage, we found that seven V(H) and seven V(L) (four Vkappa and three Vlambda) germline families cover more than 95 % of the human antibody diversity used. A consensus sequence was derived for each family and optimized for expression in Escherichia coli. In order to make all six complementarity determining regions (CDRs) accessible for diversification, the synthetic genes were designed to be modular and mutually compatible by introducing unique restriction endonuclease sites flanking the CDRs. Molecular modeling verified that all canonical classes were present. We could show that all master genes are expressed as soluble proteins in the periplasm of E. coli. A first set of antibody phage display libraries totalling 2x10(9) members was created after cloning the genes in all 49 combinations into a phagemid vector, itself devoid of the restriction sites in question. Diversity was created by replacing the V(H) and V(L) CDR3 regions of the master genes by CDR3 library cassettes, generated from mixed trinucleotides and biased towards natural human antibody CDR3 sequences. The sequencing of 257 members of the unselected libraries indicated that the frequency of correct and thus potentially functional sequences was 61 %. Selection experiments against many antigens yielded a diverse set of binders with high affinities. Due to the modular design of all master genes, either single binders or even pools of binders can now be rapidly optimized without knowledge of the particular sequence, using pre-built CDR cassette libraries. The small number of 49 master genes will allow future improvements to be incorporated quickly, and the separation of the frameworks may help in analyzing why nature has evolved these distinct subfamilies of antibody germline genes. Copyright 2000 Academic Press.

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0179

Arndt, K. M., Pelletier, J. N., Müller, K. M., Alber, T., Michnick, S. W. and Plückthun, A. (2000) A heterodimeric coiled-coil peptide pair selected in vivo from a designed library-versus-library ensemble. J. Mol. Biol. 295, 627-639.
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Abstract

Novel heterodimeric coiled-coil pairs were selected simultaneously from two DNA libraries using an in vivo protein-fragment complementation assay with dihydrofolate reductase, and the best pair was biophysically characterized. We randomized the interface-flanking e and g positions to Gln, Glu, Arg or Lys, and the core a position to Asn or Val in both helices simultaneously, using trinucleotide codons in DNA synthesis. Selection cycles with three different stringencies yielded sets of coiled-coil pairs, of which 80 clones were statistically analyzed. Thereby, properties most crucial for successful heterodimerization could be distinguished from those mediating more subtle optimization. A strong bias towards an Asn pair in the core a position indicated selection for structural uniqueness, and a reduction of charge repulsions at the e/g positions indicated selection for stability. Increased stringency led to additional selection for heterospecificity by destabilizing the respective homodimers. Interestingly, the best heterodimers did not contain exclusively complementary charges. The dominant pair, WinZip-A1B1, proved to be at least as stable in vitro as naturally occurring coiled coils, and was shown to be dimeric and highly heterospecific with a K(D) of approximately 24 nM. As a result of having been selected in vivo it possesses all characteristics required for a general in vivo heterodimerization module. The combination of rational library design and in vivo selection presented here is a very powerful strategy for protein design, and it can reveal new structural relationships. Copyright 1999 Academic Press.

DOI: 10.1006/jmbi.1999.3352 | Read all as PDF | Close Abstract
0178

Wörn, A., Auf der Maur, A., Escher, D., Honegger, A., Barberis, A. and Plückthun, A. (2000) Correlation between in vitro stability and in vivo performance of anti-GCN4 intrabodies as cytoplasmic inhibitors. J. Biol. Chem. 275, 2795-2803.
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Abstract

A cellular assay system for measuring the activity of cytoplasmically expressed anti-GCN4 scFv fragments directed against the Gcn4p dimerization domain was established in the budding yeast Saccharomyces cerevisiae. The inhibitory potential of different constitutively expressed anti-GCN4 scFv intrabodies was monitored by measuring the activity of beta-galactosidase expressed from a GCN4-dependent reporter gene. The in vivo performance of these scFv intrabodies in specifically decreasing reporter gene activity was related to their in vitro stability, measured by denaturant-induced equilibrium unfolding. A framework-engineered stabilized version showed significantly improved activity, while a destabilized point mutant of the anti-GCN4 wild-type showed decreased effects in vivo. These results indicate that stability engineering can result in improved performance of scFv fragments as intrabodies. Increasing the thermodynamic stability appears to be an essential factor for improving the yield of functional scFv in the reducing environment of the cytoplasm, where the conserved intradomain disulfides of antibody fragments cannot form.

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1999

# Reference PDF

0177

Pelletier, J. N., Arndt, K. M., Plückthun, A. and Michnick, S. W. (1999) An in vivo library-versus-library selection of optimized protein-protein interactions. Nature Biotechnol. 17, 683-690.
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Abstract

We describe a rapid and efficient in vivo library-versus-library screening strategy for identifying optimally interacting pairs of heterodimerizing polypeptides. Two leucine zipper libraries, semi-randomized at the positions adjacent to the hydrophobic core, were genetically fused to either one of two designed fragments of the enzyme murine dihydrofolate reductase (mDHFR), and cotransformed into Escherichia coli. Interaction between the library polypeptides reconstituted enzymatic activity of mDHFR, allowing bacterial growth. Analysis of the resulting colonies revealed important biases in the zipper sequences relative to the original libraries, which are consistent with selection for stable, heterodimerizing pairs. Using more weakly associating mDHFR fragments, we increased the stringency of selection. We enriched the best-performing leucine zipper pairs by multiple passaging of the pooled, selected colonies in liquid culture, as the best pairs allowed for better bacterial propagation. This competitive growth allowed small differences among the pairs to be amplified, and different sequence positions were enriched at different rates. We applied these selection processes to a library-versus-library sample of 2.0 x 10(6) combinations and selected a novel leucine zipper pair that may be appropriate for use in further in vivo heterodimerization strategies.

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0176

Jung, S., Honegger, A. and Plückthun, A. (1999) Selection for improved protein stability by phage display. J. Mol. Biol. 294, 163-180.
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Abstract

A library of mutants of a single-chain Fv fragment (scFv) was generated by a combination of directed and random mutagenesis, using oligonucleotides randomized at defined positions and two rounds of DNA shuffling. The library was based on the already well folding and stable scFv fragment 4D5Flu. In order to further improve this framework and test the efficiency of various selection strategies, phage display selection was carried out under different selective pressures for higher thermodynamic stability. Incubation of the display phages at elevated temperatures was compared to exposure of the phages to high concentrations of guanidinium chloride. Temperature stress-guided selection yielded the most stable scFv mutant after two rounds of mutagenesis and selection, due to the irreversibility of the unfolding process. It possessed only two mutations (His(L27d)Asn and Phe(L55)Val) and showed a thermodynamic stability improved by roughly 4 kcal/mol, threefold better expression yields in Escherichia coli as well as a 20-fold better binding constant than the 4D5Flu wild-type. The selection results obtained in this study delineate the advantages, disadvantages and limitations of different stability stress selection methods in phage display. Copyright 1999 Academic Press.

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0175

Schaffitzel, C., Hanes, J., Jermutus, L. and Plückthun, A. (1999) Ribosome display: an in vitro method for selection and evolution of antibodies from libraries. J. Immunol. Methods 231, 119-135.
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Abstract

Combinatorial approaches in biology require appropriate screening methods for very large libraries. The library size, however, is almost always limited by the initial transformation steps following its assembly and ligation, as other all screening methods use cells or phages and viruses derived from them. Ribosome display is the first method for screening and selection of functional proteins performed completely in vitro and thus circumventing many drawbacks of in vivo systems. We review here the principle and applications of ribosome display for generating high-affinity antibodies from complex libraries. In ribosome display, the physical link between genotype and phenotype is accomplished by a mRNA-ribosome-protein complex that is used for selection. As this complex is stable for several days under appropriate conditions, very stringent selections can be performed. Ribosome display allows protein evolution through a built-in diversification of the initial library during selection cycles. Thus, the initial library size no longer limits the sequence space sampled. By this method, scFv fragments of antibodies with affinities in the low picomolar range have been obtained. As all steps of ribosome display are carried out entirely in vitro, reaction conditions of individual steps can be tailored to the requirements of the protein species investigated and the objectives of the selection or evolution experiment. [References: 89]

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0174

Jung, S., Arndt, K. M., Müller, K. M. and Plückthun, A. (1999) Selectively infective phage (SIP) technology: scope and limitations. J. Immunol. Methods 231, 93-104.
| Abstract + further info

Abstract

We review here the selectively infective phage (SIP) technology, a powerful tool for the rapid selection of protein-ligand and peptide-ligand pairs with very high affinities. SIP is highly suitable for discriminating between molecules with subtle stability and folding differences. We discuss the preferred types of applications for this technology and some pitfalls inherent in the in vivo SIP method that have become apparent in its application with highly randomized libraries, as well as some precautions that should be taken in successfully applying this technology. [References: 38]

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0173

Willuda, J., Honegger, A., Waibel, R., Schubiger, P. A., Stahel, R., Zangemeister-Wittke, U. and Plückthun, A. (1999) High thermal stability is essential for tumor targeting of antibody fragments: Engineering of a humanized anti-epithelial glycoprotein-2 (epithelial cell adhesion molecule) single-chain Fv fragment. Cancer Res. 59, 5758-5767.
| Abstract + further info

Abstract

The epithelial glycoprotein-2 is abundantly expressed on many solid tumors and is a suitable target for antibody-based therapy. In the present study, an antiepithelial glycoprotein-2 single-chain Fv (scFv) was derived from the hybridoma MOC31 by phage display. Despite its high affinity (KD = 3.9 x 10(-9) M), however, this antibody fragment failed to significantly enrich at lung tumor xenografts in mice, mostly because of its insufficient thermal stability. To overcome this limitation, the antigen-binding residues of the MOC31 scFv fragment were grafted onto the framework of the highly stable and well-folding anti-c-erbB2 scFv 4D5. Further modification of the resulting 4D5 MOC-A, which was performed by transferring eight additional residues of the heavy chain variable domain core of the parent MOC31 antibody, produced 4D5 MOC-B, resulting in increased serum stability at 37 degrees C and also significantly improved expression behavior while retaining the antigen specificity and affinity of the parent MOC31 scFv. In mice, the scFv 4D5 MOC-B, which was radiolabeled with 99mtechnetium using a new histidine-tag specific labeling method (Waibel et al., Nature Biotechnol., 17: 897-901, 1999), showed favorable blood clearance and efficient enriches at lung tumor xenografts, with a tumor:blood ratio of 5.25 and a total dose of 1.47% injected dose per gram after 24 h. Biophysical properties such as high thermal stability are thus decisive for whether these molecules are useful in vivo, and our approach may provide a general strategy to solve this problem. This is also the first report of using a humanized anti-EGP-2 scFv in vivo for targeting solid tumors, which is a promising targeting moiety for the diagnostics and therapy of EGP-2-positive tumors in patients.

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0172

Jäger, M. and Plückthun, A. (1999) Domain interactions in antibody Fv and scFv fragments: effects on unfolding kinetics and equilibria. FEBS Lett. 462, 307-312.
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Abstract

The equilibrium denaturation and unfolding kinetics of the domains V(L) and V(H) have been compared with those of the Fv and single-chain Fv (scFv) fragment of an engineered variant of the antibody McPC603 in the presence and absence of the antigen phosphorylcholine. The scFv fragment is significantly more stable than the isolated constituting domains. Antigen binding stabilizes the heterodimeric assembly even further. Domain dissociation and domain unfolding are coupled processes, giving rise to a highly cooperative unfolding transition. For the Fv fragment, cooperative unfolding is only observed in the presence of antigen. At low protein concentrations and in the absence of antigen, the Fv fragment is significantly destabilized, leading to quantitative domain dissociation before significant domain unfolding takes place. The kinetic unfolding of V(H), V(L) and the scFv fragment is monophasic. Unfolding of the scFv fragment is much slower, when extrapolated to zero denaturant, than either of the isolated domains, suggesting that the higher thermodynamic stability of the scFv fragment is at least partially due to a high-energy transition state for unfolding. These studies emphasize the enormous importance of mutual domain stabilization in engineering stable antibodies.

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0171

zur Mühlen, E., Koschinski, P., Gehring, S., Ros, R., Tiefenauer, L., Haltner, E., Lehr, C.-M., Hartmann, U., Schwesinger, F. and Plückthun, A. (1999) Force microscopy of cells to measure bioadhesion in: Bioadhesive Drug Delivery Systems. Drugs and the Pharmaceutical Sciences (Mathiowitz, E., Chickering III, D. E., and Lehr, C.-M., eds) Vol. 98 pp. 197-221. Marcel Dekker Inc., New York
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Abstract

No abstract

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0170

Iwai, H. and Plückthun, A. (1999) Circular b-lactamase: Stability enhancement by cyclizing the backbone. FEBS Lett. 459, 166-172.
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Abstract

We have cyclized the polypeptide backbone of beta-lactamase with a short peptide loop as a novel method for protein stabilization, using intein-mediated protein ligation. Successful cyclization was proven by mass spectrometry and subsequent re-linearization by proteolytic cleavage, as well as by resistance against carboxypeptidase. Under the conditions of the experiment, no disulfide bond is present. The circular form of beta-lactamase was found to be significantly more stable against irreversible aggregation upon heating than the linear form. The circular form could be purified from the linear one either by this heat treatment or by a his-tag which became exopeptidase-resistant by cyclization. The increased stability of the circular form is probably due to the decreased conformational entropy in the unfolded state and in the intermediate states. While the introduction of additional disulfide bonds for protein stabilization follows the same rationale, the cyclization strategy may disturb the structure less and thus constitute a general method for stabilizing those proteins with N- and C-termini in close proximity.

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0169

Langedijk, A. C., Spinelli, S., Anguille, C., Hermans, P., Nederlof, J., Butenandt, J., Honegger, A., Cambillau, C. and Plückthun, A. (1999) Insight into odorant perception: The crystal structure and binding characteristics of antibody fragments directed against the musk odorant traseolide. J. Mol. Biol. 292, 855-869.
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Abstract

Monoclonal antibodies were elicited against the small hydrophobic hapten traseolide, a commercially available musk fragrance. Antibody variable region sequences were found to belong to different sequence groups, and the binding characteristics of the corresponding antibody fragments were investigated. The antibodies M02/01/01 and M02/05/01 are highly homologous and differ in the binding pocket only at position H93. M02/05/01 (H93 Val) binds the hapten traseolide about 75-fold better than M02/01/01 (H93 Ala). A traseolide analog, missing only one methyl group, does not have the characteristic musk odorant fragrance. The antibody M02/05/01 binds this hapten analog about tenfold less tightly than the original traseolide hapten, and mimics the odorant receptor in this respect, while the antibody M02/01/01 does not distinguish between the analog and traseolide. To elucidate the structural basis for the fine specificity of binding, we determined the crystal structure of the Fab fragment of M02/05/01 complexed with the hapten at 2.6 A resolution. The crystal structure showed that only van der Waals interactions are involved in binding. The somatic Ala H93 Val mutation in M02/05/01 fills up an empty cavity in the binding pocket. This leads to an increase in binding energy and to the ability to discriminate between the hapten traseolide and its derivatives. The structural understanding of odorant specificity in an antibody gives insight in the physical principles on how specificity for such hydrophobic molecules may be achieved.

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0168

Waibel, R., Alberto, R., Willuda, J., Finnern, R., Schibli, R., Stichelberger, A., Egli, A., Abram, U., Mach, J. P., Plückthun, A. and Schubiger, P. A. (1999) Stable one-step technetium-99m labeling of His-tagged recombinant proteins with a novel Tc(I)-carbonyl complex. Nature Biotechnol. 17, 897-901.
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Abstract

We have developed a technetium labeling technology based on a new organometallic chemistry, which involves simple mixing of the novel reagent, a 99m Tc(I)-carbonyl compound, with a His-tagged recombinant protein. This method obviates the labeling of unpaired engineered cysteines, which frequently create problems in large-scale expression and storage of disulfide-containing proteins. In this study, we labeled antibody single-chain Fv fragments to high specific activities (90 mCi/mg), and the label was very stable to serum and all other challenges tested. The pharmacokinetic characteristics were indistinguishable from iodinated scFv fragments, and thus scFV fragments labeled by the new method will be suitable for biodistribution studies. This novel labeling method should be applicable not only to diagnostic imaging with 99mTc, but also to radioimmunotherapy approaches with 186/188 Re, and its use can be easily extended to almost any recombinant protein or synthetic peptide.

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0167

Wall, J. G. and Plückthun, A. (1999) The hierarchy of mutations influencing the folding of antibody domains in Escherichia coli. Protein Eng. 12, 605-611.
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Abstract

In a systematic study of the periplasmic folding of antibody fragments in Escherichia coli, we have analysed the expression of an aggregation-prone and previously non-functional anti-phosphorylcholine antibody, T15, as a model system and converted it to a functional molecule. Introduction of heavy chain framework mutations previously found to improve the folding of a related antibody led to improved folding of T15 fragments and improved physiology of the host E.coli cells. Manipulation of the complementarity determining regions (CDR) of the framework-mutated forms of T15 further improved folding and bacterial host physiology, but no improvement was seen in the wild type, suggesting the existence of a hierarchy in sequence positions leading to aggregation. Rational mutagenesis of the T15 light chain led to the production of functional T15 fragments for the first time, with increased levels of functional protein produced from V(H) manipulated constructs. We propose that a hierarchical analysis of the primary amino acid sequence, as we have described, provides guidelines on how correctly folding, functional antibodies might be achieved and will allow further delineation of the decisive structural factors and pathways favouring protein aggregation.

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0166

Ramm, K., Gehrig, P. and Plückthun, A. (1999) Removal of the conserved disulfide bridges from the scFv fragment of an antibody: Effects on folding kinetics and aggregation. J. Mol. Biol. 290, 535-546.
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Abstract

Fluorescence measurements and H/2H exchange experiments monitored by mass spectrometry have been applied to investigate the influence of the conserved disulfide bridges on the folding behavior and in vitro aggregation properties of the scFv fragment of the antibody hu4D5-8. A set of four proteins, carrying none, one, or both of the disulfide bridges have been compared regarding their stabilities, folding kinetics and tendency to aggregate. The results show that refolding of all four scFvs is ultimately limited by a slow proline isomerization in the VLdomain, since the native cis -conformation of proline L95 seems to be a prerequisite for formation of the native interface. Starting from short-term denatured protein, with the proline residues in their native conformation, a kinetically trapped intermediate is populated depending on the conditions, whose rate of conversion is slower than that of the fast-folding molecules. According to deuteron protection patterns determined by mass spectrometry, those domains retaining the disulfide bridge are able to form stable native-like structure, independent of native interface formation. The disulfide-free domains, in contrast, require the native interface for sufficient stabilization. The resistance of the scFvs towards aggregation seems to be critically dependent on the presence of the disulfide bridge in the VHdomain, and thus on the ability of the VHdomain to form stable structure prior to interaction with the VLdomain. The presence of a stable VLdomain in combination with a disulfide-free VHdomain appears to further promote aggregation, indicating the involvement of structured domains in the aggregates. Copyright 1999 Academic Press.

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0165

Forrer, P., Jung, S. and Plückthun, A. (1999) Beyond binding: using phage display to select for structure, folding and enzymatic activity in proteins. Curr. Opin. Struct. Biol. 9, 514-520.
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Abstract

Phage display of a wide range of polypeptides has been increasingly used to identify novel molecules with useful binding properties for research, medical and industrial applications. Recent developments include methods for the selection of stabilized variants of a protein, the selection of regulatable enzymes and promising strategies for the selection and evolution of protein catalysts. [References: 55]

DOI: 10.1016/S0959-440x(99)80073-6 | Read all as PDF | Close Abstract
0164

Wörn, A. and Plückthun, A. (1999) Different equilibrium stability behavior of scFv fragments: Identification, classification, and improvement by protein engineering. Biochemistry 38, 8739-8750.
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Abstract

A classification of scFv fragments concerning their unfolding/refolding equilibria is proposed. It is based on the analysis of different mutants of the levan-binding A48 scFv fragment and the HER-2 binding 4D5 scFv fragment as well as a ""hybrid"" scFv carrying the VL domain of 4D5 and the VH domain of an A48 mutant. The denaturant-induced unfolding curves of the corresponding scFv fragments were measured and, if necessary for the classification, compared with the denaturation of the isolated domains. Depending on the relative intrinsic stabilities of the domains and the stability of the interface, the different scFv fragments were grouped into different classes. We also demonstrate with several examples how such a classification can be used to improve the stability of a given scFv fragment, by concentrating engineering efforts on the ""weak part"" of the particular molecule, which may either be the intrinsic stability of VL, of VH, or the stability of the interface. One of the scFv fragments obtained by this kind of approach is extremely stable, starting denaturation only at about 7 M urea. We believe that such extremely stable frameworks may be very suitable recipients in CDR grafting experiments. In addition, the thermodynamic equilibrium stabilities of seven related A48 scFv mutants covering a broad range of stabilities in urea unfolding were shown to be well correlated with thermal aggregation properties measured by light scattering and analytical gel filtration.

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0163

Hanes, J. and Plückthun, A. (1999) In vitro selection methods for screening of peptide and protein libraries in: Combinatorial Chemistry in Biology. Current Topics in Microbiology and Immunology (Famulok, M., Winnacker, E.-L., and Wong, C.-H., eds) Vol. 243 pp. 107-122. Springer Verlag, Berlin, Heidelberg
| Abstract + further info

Abstract

No abstract

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0162

Lubkowski, J., Hennecke, F., Plückthun, A. and Wlodawer, A. (1999) Filamentous phage infection: crystal structure of g3p in complex with its coreceptor, the C-terminal domain of TolA. Structure 7, 711-722.
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Abstract

BACKGROUND: Infection of male Escherichia coli cells by filamentous Ff bacteriophages (M13, fd, and f1) involves interaction of the phage minor coat gene 3 protein (g3p) with the bacterial F pilus (primary receptor), and subsequently with the integral membrane protein TolA (coreceptor). G3p consists of three domains (N1, N2, and CT). The N2 domain interacts with the F pilus, whereas the N1 domain--connected to N2 by a flexible glycine-rich linker and tightly interacting with it on the phage--forms a complex with the C-terminal domain of TolA at later stages of the infection process. RESULTS: The crystal structure of the complex between g3p N1 and TolA D3 was obtained by fusing these domains with a long flexible linker, which was not visible in the structure, indicating its very high disorder and presumably a lack of interference with the formation of the complex. The interface between both domains, corresponding to approximately 1768 A2 of buried molecular surface, is clearly defined. Despite the lack of topological similarity between TolA D3 and g3p N2, both domains interact with the same region of the g3p N1 domain. The fold of TolA D3 is not similar to any previously known protein motifs. CONCLUSIONS: The structure of the fusion protein presented here clearly shows that, during the infection process, the g3p N2 domain is displaced by the TolA D3 domain. The folds of g3p N2 and TolA D3 are entirely different, leading to distinctive interdomain contacts observed in their complexes with g3p N1. We can now also explain how the interactions between the g3p N2 domain and the F pilus enable the g3p N1 domain to form a complex with TolA.

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0161

Berger, C., Weber-Bornhauser, S., Eggenberger, J., Hanes, J., Plückthun, A. and Bosshard, H. R. (1999) Antigen recognition by conformational selection. FEBS Lett. 450, 149-153.
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Abstract

Conformational adaptation between antigen and antibody can modulate the antibody specificity. The phenomenon has often been proposed to result from an 'induced fit', which implies that the binding reaction induces a conformational change in the antigen and the antibody. Thus, an 'induced fit' requires initial complex formation followed by a conformational change in the complex. However, an antibody may select those antigen molecules that happen to be in a fitting conformational state. This leads to the same end result as an induced fit. Here, we demonstrate conformational selection by a single chain antibody fragment, raised against a random coil variant of the leucine zipper domain of transcription factor GCN4, when it cross-reacts with the wild-type dimeric leucine zipper. Kinetic and equilibrium data show that the single chain antibody fragment fragment selects monomeric peptides from the population in equilibrium with the leucine zipper dimer.

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0160

Hanes, J., Jermutus, L., Schaffitzel, C. and Plückthun, A. (1999) Comparison of Escherichia coli and rabbit reticulocyte ribosome display systems. FEBS Lett. 450, 105-110.
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Abstract

Ribosome display is a technology for library selection and simultaneous molecular evolution in vitro. We present here a comparison between an optimized Escherichia coli system and different rabbit reticulocyte ribosome display systems, optimized in a number of parameters, as a coupled eukaryotic system had been suggested to result in high enrichment factors [He and Taussig (1997) Nucleic Acids Res. 25, 5132-5134]. With all systems, antibody scFv fragments, complexed to the ribosomes and the corresponding mRNA, were enriched by binding to their cognate antigen and enrichment was always dependent on the absence of a stop codon and the presence of cognate antigen. However, the efficiency of the E. coli ribosome display system was 100-fold higher than an optimized uncoupled rabbit reticulocyte ribosome display system, with separate in vitro transcription and translation, which was in turn several-fold more efficient than the reported coupled system. Neither the E. coli nor the rabbit reticulocyte ribosome display system was dependent on the orientation of the domains of an antibody scFv fragment or on the spacer sequence. In summary, we could not detect any intrinsic advantage of using a eukaryotic translation system for ribosome display.

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0159

Pecorari, F., Tissot, A. C. and Plückthun, A. (1999) Folding, heterodimeric association and specific peptide recognition of a murine ab T-cell receptor expressed in Escherichia coli. J. Mol. Biol. 285, 1831-1843.
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Abstract

In a systematic study of the murine T-cell receptor UZ3-4, expressed and refolded from inclusion bodies in Escherichia coli, it was found that functional molecules can be obtained only under a very narrow set of conditions. The refolded T-cell receptor UZ3-4 specifically recognizes its cognate peptide (from mycobacterial Hsp60) in the context of H-2Db, but not another peptide bound to H-2Db, and the dissociation constant was determined by BIAcore as 10(-4) M. Using T-cell receptor constructs comprising all extracellular domains (ValphaCalpha and VbetaCbeta), found to be necessary for stability of the final product, significant amounts of native molecules were obtained only if the intermolecular Calpha-Cbeta disulfide bridge bond was deleted, even though the interaction between the complete alpha and beta-chain was determined to be very weak and fully reversible (KD approximately 10(-7) to 10(-6) M). Fusion of Jun and Fos to the constant domains also decreased the folding yield, because of premature association of intermediates leading to aggregation. Furthermore, only in a very narrow set of concentrations of oxidized and reduced glutathione, native disulfide bonds dominated. This shows that T-cell receptor domains are very prone to aggregation and misassociation during folding, compounded by incorrect disulfide bond formation. Once folded, however, the heterodimeric molecule is very stable and could be concentrated to millimolar concentration. Copyright 1999 Academic Press.

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0158

Jäger, M. and Plückthun, A. (1999) Folding and assembly of an antibody Fv fragment, a heterodimer stabilized by antigen. J. Mol. Biol. 285, 2005-2019.
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Abstract

The folding and assembly of the Fv fragment of the phosphorylcholine binding antibody McPC603, a non-covalent heterodimer of the variable domains VH and VL, was investigated. Since both domains, each engineered for stability and folding efficiency, could now be obtained in native and soluble form by themselves, fluorescence spectra of VH and VL in unfolded, folded and associated states can be reported. VH and VL only associate when they are native, and the stability of the heterodimer is strongly increased in the presence of antigen. VH rapidly folds into an hyperfluorescent intermediate, and the native state is reached in two parallel, proline-independent reactions. VL displays two fast refolding reactions, which are followed by two slower phases, limited by proline cis/trans-isomerization. The rate-limiting step for both the Fv and the scFv (single-chain Fv) fragment is the formation of the native VH-VL interface, which depends on ProL95 being in cis. The folding of the Fv fragment is fast after short-term denaturation or in the presence of proline cis/trans-isomerase catalysis, but the scFv fragment falls into a kinetic trap, observed by the persistence of the slow phases under all conditions. Furthermore, the scFv fragment, but not the Fv fragment, gives rise to premature interface formation, indicated by the fluorescence spectra and a much higher transient binding of 8-anilino-1-naphthalene sulfonate. The analysis of the folding pathway of the domains VH and VL in isolation and in non-covalent and covalent assemblies should provide helpful insights into the folding of multimeric proteins in general, and for the further engineering of stable and well-folding antibody fragments in particular. Copyright 1999 Academic Press.

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1998

# Reference PDF

0157

Spada, S., Honegger, A. and Plückthun, A. (1998) Reproducing the natural evolution of protein structural features with the selectively infective phage (SIP) technology. The kink in the first strand of antibody kappa domains. J. Mol. Biol. 283, 395-407.
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Abstract

The beta-sandwich structure of immunoglobulin variable domains is characterized by a typical kink in the first strand, which allows the first part of the strand to hydrogen bond to the outer beta-sheet (away from the VH-VL interface) and the second part to the inner beta-sheet. This kink differs in length and sequence between the Vkappa, Vlambda and VH domains and yet is involved in several almost perfectly conserved interactions with framework residues. We have used the selectively infective phage (SIP) system to select the optimal kink region from several defined libraries, using an anti-hemagglutinin single-chain Fv (scFv) fragment as a model system. Both for the kink with the Vkappa domain length and that with the Vlambda length, a sequence distribution was selected that coincides remarkably well with the sequence distribution of natural antibodies. The selected scFv fragments were purified and characterized, and thermodynamic stability was found to be the prime factor responsible for selection. These data show that the SIP technology can be used for optimizing protein structural features by evolutionary approaches. Copyright 1998 Academic Press.

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0156

Langedijk, A. C., Honegger, A., Maat, J., Planta, R. J., van Schaik, R. C. and Plückthun, A. (1998) The nature of antibody heavy chain residue H6 strongly influences the stability of a VH domain lacking the disulfide bridge. J. Mol. Biol. 283, 95-110.
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Abstract

Monoclonal antibody mAb 03/01/01, directed against the musk odorant traseolide, carries a serine residue instead of the conserved Cys H92 in the heavy chain variable domain, and is thus lacking the highly conserved disulfide bridge. We investigated the energetic consequence of restoring the disulfide bond and the nature of residue H6 (Glu or Gln), which is poised to interact with Ser H92 in the recombinant scFv fragment obtained from this antibody. In the scFv fragment derived from this antibody, the stabilizing effect of Gln H6 over Glu was found to be as large as the effect of reintroducing the disulfide bond. We have analyzed the conformation and hydrogen bond pattern of Gln H6 and Glu H6 in antibodies carrying these residues and suggest mechanisms by which this residue could contribute to VH domain stability. We also show that the unpaired cysteine H22 is buried, and conforms to the expected VH structure. The antibody appears to have acquired two somatic mutations (Ser H52 and Arg H66), which had been previously characterized as having a positive effect on VH stability. The overall domain stability is the decisive factor for generating functional, disulfide-free antibody domains, and several key residues play dominant roles. Copyright 1998 Academic Press.

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0155

Plückthun, A. (1998) Studying protein structure and function by directed evolution in: Protein Dynamics, Function and Design. NATO ASI Series, Series A: Life Sciences (Jardetzky, O., and Lefèvre, J. F., eds) Vol. 301 pp. 37-57. Plenum Press, New York
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Abstract

Directed molecular evolution is a powerful strategy for investigating the structure and function of proteins. When a function, such as ligand binding, can only be carried out by the native state of the protein, the biological selection for this function can be used to improve structural properties of the protein. Thus, thermodynamic stability and folding efficiency, which is the ability to avoid aggregation during folding, can be optimized. Three methods of selection are reviewed: phage display, selectively infective phages (SIP) and ribosome display, a cell-free method. Examples for optimizing antibody stability are discussed. In one case, antibodies have been generated under evolutionary pressure, which are stable in the absence of any disulfide bond, in the other case, a kink in the first strand of the beta-sandwich of kappa domains has been optimized.

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0154

Hanes, J., Jermutus, L., Weber-Bornhauser, S., Bosshard, H. R. and Plückthun, A. (1998) Ribosome display efficiently selects and evolves high-affinity antibodies in vitro from immune libraries. Proc. Natl. Acad. Sci. U. S. A. 95, 14130-14135.
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Abstract

Ribosome display was applied for affinity selection of antibody single-chain fragments (scFv) from a diverse library generated from mice immunized with a variant peptide of the transcription factor GCN4 dimerization domain. After three rounds of ribosome display, positive scFvs were isolated and characterized. Several different scFvs were selected, but those in the largest group were closely related to each other and differed in 0 to 5 amino acid residues with respect to their consensus sequence, the likely common progenitor. The best scFv had a dissociation constant of (4 +/- 1) x 10(-11) M, measured in solution. One amino acid residue in complementarity determining region L1 was found to be responsible for a 65-fold higher affinity than the likely progenitor. It appears that this high-affinity scFv was selected from the mutations occurring during ribosome display in vitro, and that this constitutes an affinity maturation inherent in this method. The in vitro-selected scFvs could be functionally expressed in the Escherichia coli periplasm with good yields or prepared by in vitro refolding. Thus, ribosome display can be a powerful methodology for in vitro library screening and simultaneous sequence evolution.

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0153

Sieber, V., Plückthun, A. and Schmid, F. X. (1998) Selecting proteins with improved stability by a phage-based method. Nature Biotechnol. 16, 955-960.
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Abstract

We describe a method for the stabilization of proteins that links the protease resistance of stabilized variants of a protein with the infectivity of a filamentous phage. A repertoire of variants of the protein to be stabilized is inserted between two domains (N2 and CT) of the gene-3-protein of the fd phage. The infectivity of fd phage is lost when the three domains are disconnected by the proteolytic cleavage of unstable protein inserts. Rounds of in vitro proteolysis, infection, and propagation can thus be performed to enrich those phage containing the most stable variants of the protein insert. This strategy discriminates between variants of a model protein (ribonuclease T1) differing in conformational stability and selects from a large repertoire variants that are only marginally more stable than others. Because fd phage are exceptionally stable and the proteolysis in the selection step takes place in vitro a wide range of solvent conditions can be used, tailored for the protein to be stabilized.

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0152

Jermutus, L., Ryabova, L. A. and Plückthun, A. (1998) Recent advances in producing and selecting functional proteins by using cell-free translation. Curr. Opin. Biotechnol. 9, 534-548.
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Abstract

Prokaryotic and eukaryotic in vitro translation systems have recently become the focus of increasing interest for tackling fundamental problems in biochemistry. Cell-free systems can now be used to study the in vitro assembly of membrane proteins and viral particles, rapidly produce and analyze protein mutants, and enlarge the genetic code by incorporating unnatural amino acids. Using in vitro translation systems, display techniques of great potential have been developed for protein selection and evolution. Furthermore, progress has been made to efficiently produce proteins in batch or continuous cell-free translation systems and to elucidate the molecular causes of low yield and find possible solutions for this problem. [References: 120]

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0151

Wörn, A. and Plückthun, A. (1998) Mutual stabilization of VL and VH in single-chain antibody fragments, investigated with mutants engineered for stability. Biochemistry 37, 13120-13127.
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Abstract

A set of six mutants of the levan binding single-chain Fv (scFv) fragment A48 (ABPC48), which have the identical light chain but differ gradually in the stability of the heavy chain, was generated. This was achieved by introducing one or both of the stabilizing mutations H-K66R and H-N52S into the VH domain of the A48 wild-type protein, which is naturally missing the conserved disulfide bridge in VH, and into the cysteine-restored variant A48cys scFv. The stabilizing effects of these two mutations in VH, which had been selected in the context of a disulfide-free derivative of this scFv fragment [Proba, K., et al. (1998) J. Mol. Biol. 275, 245-253], were found to be additive and transferable to the cysteine-restored variant of the A48 scFv, thereby generating extremely stable VH domains. The equilibrium denaturation of these scFv fragments was compared with the corresponding isolated VL domain and two of the different isolated VH domains. In the scFv fragment, the VL domain was found to be stabilized by a more stable VH domain, and, conversely, the VH domain was stabilized by a more stable VL domain. A folding intermediate with nativelike VH and denatured VL was found at equilibrium, if VH was significantly more stable than VL. In all other cases, a cooperative unfolding of the scFv was observed. We explain this observation with different contributions of intrinsic domain stability and extrinsic stabilization provided by the partner domain in the single-chain antibodies.

DOI: 10.1021/bi980712q | Read all as PDF | Close Abstract
0150

Shusta, E. V., Raines, R. T., Plückthun, A. and Wittrup, K. D. (1998) Increasing the secretory capacity of Saccharomyces cerevisiae for production of single-chain antibody fragments. Nature Biotechnol. 16, 773-777.
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Abstract

We have produced single-chain antibody fragments (scFv) in Saccharomyces cerevisiae at levels up to 20 mg/L in shake flask culture by a combination of expression level tuning and overexpression of folding assistants. Overexpression of the chaperone BiP or protein disulfide isomerase (PDI) increases secretion titers 2-8 fold for five scFvs. The increases occur for scFv expression levels ranging from low copy to ER-saturating overexpression. The disulfide isomerase activity of PDI, rather than its chaperone activity, is responsible for the secretion increases. A synergistic increase in scFv production occurs upon cooverexpression of BiP and PDI.

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0149

Arndt, K. M., Müller, K. M. and Plückthun, A. (1998) Factors influencing the dimer to monomer transition of an antibody single-chain Fv fragment. Biochemistry 37, 12918-12926.
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Abstract

Antibody single-chain Fv (scFv) fragments are able to form dimers under certain conditions, and the extent of dimerization appears to depend on linker length, antibody sequence, and external factors. We analyzed the factors influencing dimer-monomer equilibrium as well as the rate of interconversion, using the scFv McPC603 as a model system. In this molecule, the stability of the VH-VL interaction can be conveniently varied by adjusting the ionic strength (because of its influence on the hydrophobic effect), by pH (presumably because of the presence of titratable groups in the interface), and by the presence or absence of the antigen phosphorylcholine, which can be rapidly removed due to its very fast off-rate. It was found that the monomer is the thermodynamically stable form with linkers of 15 and 25 amino acids length under all conditions tested (35 &mgr;M or less). The dimer is initially formed in periplasmic expression, presumably by domain swapping, and can be trapped by all factors which stabilize the VH-VL interface, such as the presence of the antigen, high ionic strength, and pH below 7.5. Under all other conditions, it converts to the monomer. Predominantly monomer is obtained during in vitro folding. Monomer is stabilized against dimerization at very high concentrations by the same factors which stabilize the VH-VL interaction. These results should be helpful in producing molecules with defined oligomerization states.

DOI: 10.1021/bi9810407 | Read all as PDF | Close Abstract
0148

Müller, K. M., Arndt, K. M. and Plückthun, A. (1998) Model and simulation of multivalent binding to fixed ligands. Anal. Biochem. 261, 149-158.
| Abstract + further info

Abstract

A model to quantitate the principal aspects of multivalent binding was developed. It describes the random distribution of an immobilized component (the ligand) taking into account local densities. The binding of a bivalent molecule (the analyte) to the ligand is described as occurring in two steps, the second of which is driven by the local concentration of neighboring ligands. The model was used to simulate the kinetics of bivalent binding in surface plasmon resonance biosensors such as BIAcore. The simulations are compared with measured data. The simulation quantitates the influence of bivalent binding on the sensor signal, as a function of ligand density, analyte concentration, and binding site distance. Such simulations will be helpful for understanding and designing experiments to assess avidity effects as well as for developing molecules with high avidity. Furthermore, they help to analyze the inherent complexity in seemingly simple sensorgrams. Copyright 1998 Academic Press.

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0147

Gervasoni, P., Staudenmann, W., James, P. and Plückthun, A. (1998) Identification of the binding surface on _-lactamase for GroEL by limited proteolysis and MALDI-mass spectrometry. Biochemistry 37, 11660-11669.
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Abstract

Escherichia coli beta-lactamase, alone or as a complex with GroEL at 48 degreesC, was partially digested with trypsin, endoproteinase Glu-C, or thermolysin. Peptides were analyzed by matrix-assisted laser desorption and ionization mass spectrometry and aligned with the known sequence. From the protease cleavage sites which become protected upon binding and those which become newly accessible, a model of the complex is proposed in which the carboxy-terminal helix has melted, two loops form the binding interface and the large beta-sheet become partially uncovered by the slight dislocation of other structural elements. This explains how hydrophobic surface on the substrate protein can become accessible while scarcely disrupting the hydrogen bond network of the native structure. An analysis of the GroEL-bound peptides bound after digestion of the beta-lactamase showed no obvious sequence motifs, indicating that binding is provided by hydrophobic patches in the three-dimensional structure.

DOI: 10.1021/bi980258q | Read all as PDF | Close Abstract
0146

Müller, K. M., Arndt, K. M. and Plückthun, A. (1998) A dimeric bispecific miniantibody combines two specificities with avidity. FEBS Lett. 432, 45-49.
| Abstract + further info

Abstract

Bispecific antibodies extend the capabilities of nature and might be applied in immunotherapy and biotechnology. By fusing the gene of a single-chain Fv (scFv) fragment to a helical dimerization domain, followed by a second scFv fragment of different specificity, we were able to express a functional protein in E. coli, which is bispecific and has two valencies for each specificity. The dimeric bispecific (DiBi) miniantibody preserves the natural avidity of antibodies in a very small-sized molecule of only 120 kDa. The generality of the principle was shown with a scFv fragment binding the EGF-receptor (named scFv 425) in three combinations with scFv fragments either directed against CD2 (ACID2.M1), phosphorylcholine (McPC603) or fluorescein (FITC-E2). Binding was analyzed by sandwich surface plasmon resonance biosensor (BIAcore) measurements.

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0145

Ros, R., Schwesinger, F., Anselmetti, D., Kubon, M., Schäfer, R., Plückthun, A. and Tiefenauer, L. (1998) Antigen binding forces of individually addressed single-chain Fv antibody molecules. Proc. Natl. Acad. Sci. U. S. A. 95, 7402-7405.
| Abstract + further info

Abstract

Antibody single-chain Fv fragment (scFv) molecules that are specific for fluorescein have been engineered with a C-terminal cysteine for a directed immobilization on a flat gold surface. Individual scFv molecules can be identified by atomic force microscopy. For selected molecules the antigen binding forces are then determined by using a tip modified with covalently immobilized antigen. An scFv mutant of 12% lower free energy for ligand binding exhibits a statistically significant 20% lower binding force. This strategy of covalent immobilization and measuring well separated single molecules allows the characterization of ligand binding forces in molecular repertoires at the single molecule level and will provide a deeper insight into biorecognition processes.

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0144

Vinckier, A., Gervasoni, P., Zaugg, F., Ziegler, U., Lindner, P., Groscurth, P., Plückthun, A. and Semenza, G. (1998) Atomic force microscopy detects changes in the interaction forces between GroEL and substrate proteins. Biophys. J. 74, 3256-3263.
| Abstract + further info

Abstract

The structure of the Escherichia coli chaperonin GroEL has been investigated by tapping-mode atomic force microscopy (AFM) under liquid. High-resolution images can be obtained, which show the up-right position of GroEL adsorbed on mica with the substrate-binding site on top. Because of this orientation, the interaction between GroEL and two substrate proteins, citrate synthase from Saccharomyces cerevisiae with a destabilizing Gly-->Ala mutation and RTEM beta-lactamase from Escherichia coli with two Cys-->Ala mutations, could be studied by force spectroscopy under different conditions. The results show that the interaction force decreases in the presence of ATP (but not of ATPgammaS) and that the force is smaller for native-like proteins than for the fully denatured ones. It also demonstrates that the interaction energy with GroEL increases with increasing molecular weight. By measuring the interaction force changes between the chaperonin and the two different substrate proteins, we could specifically detect GroEL conformational changes upon nucleotide binding.

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0143

Hennecke, F., Krebber, C. and Plückthun, A. (1998) Non-repetitive single-chain Fv linkers selected by selectively infective phage (SIP) technology. Protein Eng. 11, 405-410.
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Abstract

By using the selectively infective phage (SIP) technology, we selected non-repetitive linkers for a single-chain Fv fragment to have genes more robust against deletions in PCR-based gene assembly and directed evolution experiments than is the case for the classical (Gly4Ser)3 linker. We designed linkers encoding turns at both ends and random positions in the middle where glycines and polar and charged residues were allowed to occur. After only a single round of SIP, all clones obtained were fully functional. Properties such as antigen binding constants, urea denaturation curves and expression of soluble scFv fragments were identical with those of the parental fragment with the (Gly4Ser)3 linker. This demonstrates that SIP is a very fast and powerful technique to remove rapidly sequences of poor functionality, exclusively yielding sequences of the desired overall property in a single round.

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0142

Gallimore, A., Glithero, A., Godkin, A., Tissot, A. C., Plückthun, A., Elliott, T., Hengartner, H. and Zinkernagel, R. (1998) Induction and exhaustion of lymphocytic choriomeningitis virus-specific cytotoxic T lymphocytes visualized using soluble tetrameric major histocompatibility complex class I-peptide complexes. J. Exp. Med. 187, 1383-1393.
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Abstract

This study describes the construction of soluble major histocompatibility complexes consisting of the mouse class I molecule, H-2Db, chemically biotinylated beta2 microglobulin and a peptide epitope derived from the glycoprotein (GP; amino acids 33-41) of lymphocytic choriomeningitis virus (LCMV). Tetrameric class I complexes, which were produced by mixing the class I complexes with phycoerythrin-labeled neutravidin, permitted direct analysis of virus-specific cytotoxic T lymphocytes (CTLs) by flow cytometry. This technique was validated by (a) staining CD8+ cells in the spleens of transgenic mice that express a T cell receptor (TCR) specific for H-2Db in association with peptide GP33-41, and (b) by staining virus-specific CTLs in the cerebrospinal fluid of C57BL/6 (B6) mice that had been infected intracranially with LCMV-DOCILE. Staining of spleen cells isolated from B6 mice revealed that up to 40% of CD8(+) T cells were GP33 tetramer+ during the initial phase of LCMV infection. In contrast, GP33 tetramers did not stain CD8+ T cells isolated from the spleens of B6 mice that had been infected 2 mo previously with LCMV above the background levels found in naive mice. The fate of virus-specific CTLs was analyzed during the acute phase of infection in mice challenged both intracranially and intravenously with a high or low dose of LCMV-DOCILE. The results of the study show that the outcome of infection by LCMV is determined by antigen load alone. Furthermore, the data indicate that deletion of virus-specific CTLs in the presence of excessive antigen is preceded by TCR downregulation and is dependent upon perforin.

DOI: 10.1084/Jem.187.9.1383 | Read all as PDF | Close Abstract
0141

Müller, K. M., Arndt, K. M., Bauer, K. and Plückthun, A. (1998) Tandem immobilized metal-ion affinity chromatography/immunoaffinity purification of His-tagged proteins – evaluation of two anti-His-tag monoclonal antibodies. Anal. Biochem. 259, 54-61.
| Abstract + further info

Abstract

A tag comprising four to six histidines genetically fused to the protein of interest (His-tag) has been widely used to purify proteins by immobilized metal-ion affinity chromatography (IMAC). Here we report the utilization of the same tag twice in series, first for IMAC and subsequently for immunoaffinity purification. Both steps are based on completely different physical principles and can therefore remove different contaminants. Two anti-His-tag antibodies (3D5 and PentaHis) were characterized for their binding and elution properties using the BIAcore surface plasmon resonance biosensor. The dissociation constant of the PentaHis antibody was determined to be 1 x 10(-8) M and for the 3D5 antibody 3.4 x 10(-7) M at pH 7.4. Imidazole in the sample did interfere with binding, whereas chelating agents such as EDTA and high salt did not. The antibody 3D5 was coupled to a column matrix and used for a coupled two-step purification, in which the IMAC column is eluted with EDTA and the eluent is loaded directly on the immunoaffinity column. This method may constitute a very general procedure to purify proteins to near homogeneity without the need to tailor conditions individually, and it may thus be very attractive for high-throughput screening programs and for developing general protocols for clinical grade material.

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0140

Stegmann, R., Manakova, E., Rössle, M., Heumann, H., Nieba-Axmann, S. E., Plückthun, A., Hermann, T., May, R. P. and Wiedenmann, A. (1998) Structural changes of the Escherichia coli GroEL-GroES chaperonins upon complex formation in solution: A neutron small angle scattering study. J. Struct. Biol. 121, 30-40.
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Abstract

We applied neutron scattering in conjunction with deuterium (D-) labeling in order to obtain information about the domain structure of GroEL and GroES isolated and in the complex. Each subunit of the heptameric GroES consists of two domains, a core domain (Met1 to Lys15 and Lys34 to Ala97) and an intervening loop region (Glu16 to Ala33). Neutron scattering shows that both regions change their conformation upon GroEL/GroES complex formation. The interdomain angle between the core regions of the heptameric GroES increases from 120 to 140 degrees, leading to a less dome-like shape of GroES, and the loop regions turn inwards by 75 degrees. The 23 C-terminal amino acids of the 14 GroEL subunits (Lys526 to Met548), which are unresolved in the crystal structure, are located either at the bottom of the cavity formed by the seven-membered GroEL ring or at the inner wall of the cavity. Upon complex formation the apical domains of GroEL move outwards, which facilitates binding of GroES at a Gro-EL-GroES center-to-center distance of (87 +/- 8) A. These structural changes may be important for the dissociation of the unfolded protein bound to the central cavity upon GroES binding. The overall structure determined by neutron scattering in solution tallies with the crystallographic model published after completion of this study. Differences in the conformation of GroES observed in the complex by the two methods support the view that the chaperonin complex is a flexible molecule which might switch in solution between different conformations.

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0139

Wörn, A. and Plückthun, A. (1998) An intrinsically stable antibody scFv fragment can tolerate the loss of both disulfide bonds and fold correctly. FEBS Lett. 427, 357-361.
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Abstract

A fully functional cysteine-free derivative of the intrinsically stable anti-HER2 scFv fragment hu4D5-8 was generated by replacing the disulfide forming cysteine residues in VH and VL with the amino acid combination valine-alanine in both domains. The antigen binding properties, determined by ELISA and BIAcore measurements, were not affected by removal of the disulfide bonds. The thermodynamic stability of the disulfide-containing scFv of 8.1 kcal/mol is decreased upon complete reduction of both disulfides to 2.7 kcal/mol, while that of the valine-alanine variant is somewhat higher (about 3.8 kcal/ mol). Our results suggest that, in principle, a disulfide-free fully functional derivative of any scFv can be obtained, as long as the corresponding disulfide-containing scFv has a high enough thermodynamic stability.

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0138

Bothmann, H. and Plückthun, A. (1998) Selection for a periplasmic factor improving phage display and functional periplasmic expression. Nature Biotechnol. 16, 376-380.
| Abstract + further info

Abstract

The efficiency of both phage display in Escherichia coli and periplasmic expression of recombinant proteins may be limited by the same periplasmic folding steps. To search for E. coli factors that improve the efficiency of both procedures, a library of E. coli proteins was coexpressed in a phagemid vector that contained a poorly folding single-chain Fv antibody (scFv) fragment fused to g3p. We enriched, by panning for antigen binding, those phagemids in which the amount of displayed scFv is highest. We thus identified the periplasmic protein Skp/OmpH/HlpA as improving phage display of a wide range of scFv fragments. This occurs as a result of an increase in the amount of hybrid protein displayed on the phage. Coexpression of skp also increases the functional yield of scFv fragments when expressed by secretion to the periplasm.

DOI: 10.1038/nbt0498-376 | Read all as PDF | Close Abstract
0137

Müller, K. M., Arndt, K. M., Strittmatter, W. and Plückthun, A. (1998) The first constant domain (CH1 and CL) of an antibody used as heterodimerization domain for bispecific miniantibodies. FEBS Lett. 422, 259-264.
| Abstract + further info

Abstract

Bispecific miniantibodies were constructed by genetically fusing the C(H)1 domain of an IgG1 to the C-terminus of a single-chain Fv fragment (scFv-425), specific for the EGF receptor, and fusing the C(L) domain of a kappa light chain to the C-terminus of a scFv specific for CD2 (scFv-M1). An efficient dicistronic gene arrangement for functional expression in Escherichia coli was constructed. Immunoblots demonstrated correct domain assembly and the formation of the natural C(H)1-C(L) disulfide bridge. Gel filtration confirmed the correct size, sandwich ELISAs demonstrated bispecific functionality, and SPR biosensor measurements determined binding to EGF-R in comparison to bivalent constructs. Bispecific anti-EGF-R/anti-CD2 miniantibodies are candidates for the immunotherapy of cancer.

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0136

Gervasoni, P., Gehrig, P. and Plückthun, A. (1998) Two conformational states of _-lactamase bound to GroEL: A biophysical characterization. J. Mol. Biol. 275, 663-675.
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Abstract

Escherichia coli RTEM beta-lactamase, in which both cysteine residues which form the single disulfide bond have been mutated to alanine residues, can form stable reversible complexes with GroEL under two different sets of conditions. Starting with the GdmCl-denatured enzyme, it is bound to GroEL in a state where no protons are protected against exchange with 2H2O, as determined by electrospray ionization mass spectrometry (ESI-MS). In contrast, when native protein is destabilized at high temperature and added to GroEL, a conformation is bound with 18 protected protons after two hours of exchange. While the high-temperature complex can form both with the wild-type enzyme (with intact disulfide bond) and the Cys-Ala double mutant, only the latter protein can form a complex starting from GdmCl denatured states. Thus, two different sets of conformations of the same protein can be bound, depending both on the conditions used to form the complex and on the intrinsic stability of the intermediate recognized by GroEL, and we have characterized the properties of both complexes.

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0135

Proba, K., Wörn, A., Honegger, A. and Plückthun, A. (1998) Antibody scFv fragments without disulfide bonds made by molecular evolution. J. Mol. Biol. 275, 245-253.
| Abstract + further info

Abstract

We generated stable and functional cysteine-free antibody single-chain fragments (scFv) lacking the conserved disulfide bonds in both VH and VL. This was achieved by molecular evolution, starting from the scFv fragment of the levan binding antibody ABPC48, which is naturally missing one of the conserved cysteine residues, by using DNA shuffling and phage display. Several of the selected sequences were expressed and the resulting scFv proteins characterized by equilibrium urea denaturation. Three of the characterized proteins exhibit thermodynamic stability similar to the wild-type protein, and these cysteine-free mutant proteins can now be expressed in functional form in the Escherichia coli cytoplasm. We believe that such molecules are of great utility for use as intrabodies, can be produced by simpler expression strategies and may give further insight into the folding and stability of the immunoglobulin fold.

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0134

Lubkowski, J., Hennecke, F., Plückthun, A. and Wlodawer, A. (1998) The structural basis of phage display elucidated by the crystal structure of the N-terminal domains of g3p. Nature Struct. Biol. 5, 140-147.
| Abstract + further info

Abstract

The structure of the two N-terminal domains of the gene 3 protein of filamentous phages (residues 1-217) has been solved by multiwavelength anomalous diffraction and refined at 1.46 A resolution. Each domain consists of either five or eight beta-strands and a single alpha-helix. Despite missing sequence homology, their cores superimposed with a root-mean-square deviation of 2 A. The domains are engaged in extensive interactions, resulting in a horseshoe shape with aliphatic amino acids and threonines lining the inside, delineating the likely binding site for the F-pilus. The glycine-rich linker connecting the domains is invisible in the otherwise highly ordered structure and may confer flexibility between the domains required during the infection process.

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0133

Apostolakis, J., Plückthun, A. and Caflisch, A. (1998) Docking small ligands in flexible binding sites. J. Comput. Chem. 19, 21-37.
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Abstract

A novel procedure for docking ligands in a flexible binding site is presented. It relies on conjugate gradient minimization, during which nonbonded interactions are gradually switched on. Short Monte Carlo minimization runs are performed on the most promising candidates. Solvation is implicitly taken into account in the evaluation of structures with a continuum model. It is shown that the method is very accurate and can model induced fit in the ligand and the binding site. The docking procedure has been successfully applied to three systems. The first two are the binding of progesterone and 5 b-androstane-3,17-dione to the antigen binding fragment of a steroid binding antibody. A comparison of the crystal structures of the free and the two complexed forms reveals that any attempt to model binding must take protein rearrangements into account. Furthermore, the two ligands bind in two different orientations, posing an additional challenge. The third test case is the docking of a . N - 2-naphthyl-sulfonyl-glycyl -D-para-amidino-phenyl-alanyl-piperidine . NAPAP to human a-thrombin. In contrast to steroids, NAPAP is a very flexible ligand, and no information of its conformation in the binding site is used. All docking calculations are started from X-ray conformations of proteins with the uncomplexed binding site. For all three systems the best minima in terms of free energy have a root mean square deviation from the X-ray structure ° smaller than 1.5 A for the ligand atoms.

DOI: 10.1002/(Sici)1096-987x(19980115)19:1<21::Aid-Jcc2>3.0.Co;2-0 | Read all as PDF | Close Abstract

1997

# Reference PDF

0132

Freund, C., Gehrig, P., Baici, A., Holak, T. A. and Plückthun, A. (1997) Parallel pathways in the folding of a short-term denatured scFv fragment of an antibody. Folding and Design 3, 39-49.
| Abstract + further info

Abstract

BACKGROUND: Antibodies are prototypes of multimeric proteins and consist of structurally similar domains. The two variable domains of an antibody (VH and VL) interact through a large hydrophobic interface and can be expressed as covalently linked single-chain Fv (scFv) fragments. The in vitro folding of scFv fragments after long-term denaturation in guanidinium chloride is known to be slow. In order to delineate the nature of the rate-limiting step, the folding of the scFv fragment of an antibody after short-term denaturation has been investigated. RESULTS: Secondary structure formation, measured by H/D-exchange protection, of a mutant scFv fragment of an antibody after short incubation in 6 M guanidinium chloride was shown to be multiphasic. NMR analysis shows that an intermediate with significant proton protection is observed within the dead time of the manual mixing experiments. Subsequently, the folding reaction proceeds via a biphasic reaction and mass spectrometry analyses of the exchange experiments confirm the existence of two parallel pathways. In the presence of cyclophilin, however, the faster of the two phases vanishes (when followed by intrinsic tryptophan fluorescence), while the slower phase is not significantly enhanced by equimolar cyclophilin. CONCLUSIONS: The formation of an early intermediate, which shows amide-proton exchange protection, is independent of proline isomerization. Subsequently, a proline cis-trans isomerization reaction in the rapidly formed intermediate, producing 'non-native' isomers, competes with the fast formation of native species. Interface formation in a folding intermediate of the scFv fragment is proposed to prevent the back-isomerization of these prolines from being efficiently catalyzed by cyclophilin.

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0131

Jäger, M. and Plückthun, A. (1997) The rate-limiting steps for the folding of an antibody scFv fragment. FEBS Lett. 418, 106-110.
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Abstract

The refolding kinetics of a single-chain Fv (scFv) fragment, derived from the phosphorylcholine binding antibody McPC603, was investigated. Both prolyl-peptide bonds which are cis in the native state affect the refolding kinetics of long-term denatured protein. The rate-limiting step is the trans --> cis isomerization at the ProL95-peptide bond, which is catalyzed by peptidyl-prolyl-cis/trans-isomerase (PPIase), and is the prerequisite for correct V(H)/V(L) domain association. Refolding of short-term denatured protein resulted in complex refolding kinetics, too. This kinetic heterogeneity could be ascribed to cis --> trans re-isomerization at the ProL95-peptide bond to the wrong conformation in a folding intermediate. PPIase was shown to increase the fraction of slowly folding species, thereby competing with the fast folding of short-term denatured scFv, having native proline conformations. A trapped intermediate is rapidly populated, and the return from this state becomes rate-limiting.

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0130

Jung, S. and Plückthun, A. (1997) Improving in vivo folding and stability of a single-chain Fv antibody fragment by loop grafting. Protein Eng. 10, 959-966.
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Abstract

The complementary determining regions (CDRs) from the fluorescein-binding antibody 4-4-20, which yields almost no soluble protein in periplasmic expression in Escherichia coli, were transplanted to the framework of the humanized antibody 4D5. The resulting single-chain Fv fragment (scFv) 4D5Flu showed both a dramatic improvement in soluble expression, even at 37 degrees C, and an improved thermodynamic stability. Antigen affinity was maintained upon this engineering by paying attention to crucial framework-CDR contacts. This demonstrates that the use of superior frameworks is a robust strategy to improve the physical properties of scFv fragments. We also report that the grafted version was selected in phage display over several competing variants of the same antibody with identical binding constant but poorer folding or stability properties. The selection required four panning rounds and a temperature of 37 degrees C and we show that the underlying reason for this selection is a higher fraction of phages carrying functional scFv molecules.

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0129

Nieba, L., Nieba-Axmann, S. E., Persson, A., Hämäläinen, M., Edebratt, F., Hansson, A., Lidholm, J., Magnusson, K., Karlsson, A. F. and Plückthun, A. (1997) BIACORE analysis of histidine-tagged proteins using a chelating NTA sensor chip. Anal. Biochem. 252, 217-228.
| Abstract + further info

Abstract

While BIACORE instruments are routinely used for kinetic measurements and for the determination of binding constants, the immobilization of a ligand onto the sensor chip surface has to be individually optimized for every system. We show here that the histidine (His) tag, routinely used in protein purification and in detection is an ideal tag for immobilization, despite the intrinsically low affinity between an immobilized metal ion and the His tag. This is due to strong rebinding effects caused by the high surface density of immobilized Ni2+-nitrilotriacetic acid (NTA) on the chips used here. The immobilization of the ligand can be adjusted to a low level using the same chip, such that mass transport limitation and rebinding of the analyte to the immobilized ligand is minimal. Nine different proteins with different numbers of His tags were tested for stable binding to the Ni2+-NTA surface. Most proteins with one His tag dissociate very rapidly from the Ni2+-NTA surface, and the KD for the interaction between His tag and Ni2+-NTA was estimated to about 10(-6) m at neutral pH. In contrast, two His tags are usually found to be sufficient for stable binding. The kinetics of the chaperonin system of Escherichia coli GroEL and GroES were analyzed as a model using this system and found to be very similar to those obtained with covalently immobilized ligands. The sensor chip can be reused many times, because of the powerful regeneration methods. The ligand can be freshly immobilized after each cycle, thus eliminating potential denaturation upon regeneration as a source of error. Copyright 1997 Academic Press.

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0128

Pedrazzi, G., Schwesinger, F., Honegger, A., Krebber, C. and Plückthun, A. (1997) Affinity and folding properties both influence the selection of antibodies with the selectively infective phage (SIP) methodology. FEBS Lett. 415, 289-293.
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Abstract

We investigated which molecules are selected from a model library by the selectively infective phage (SIP) methodology. As a model system, we used the fluorescein binding single-chain Fv fragment FITC-E2, and from a 3D-model, we identified 11 residues potentially involved in hapten binding and mutated them individually to alanines. The binding constant of each mutant was determined by fluorescence titration, and each mutant was tested individually as well as in competitive SIP experiments for infectivity. After three rounds of SIP, only molecules with KD values within a factor of 2 of the tightest binder remain, and among those, a mutant no longer carrying an unnecessary exposed tryptophan residue is preferentially selected. SIP is shown to select for the best overall properties of the displayed molecules, including folding behavior, stability and affinity.

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0127

Nieba-Axmann, S. E., Ottiger, M., Wüthrich, K. and Plückthun, A. (1997) Multiple cycles of global unfolding of GroEL-bound cyclophilin A evidenced by NMR. J. Mol. Biol. 271, 803-818.
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Abstract

GroE, the chaperonin system of Escherichia coli, prevents the aggregation of partially folded or misfolded proteins by complexing them in a form competent for subsequent folding to the native state. We examined the exchange of amide protons of cyclophilin A (CypA) interacting with GroEL, using NMR spectroscopy. We have applied labeling pulses in H2O to the deuterated GroEL-CypA-complex. When ATP and GroES were added after the labeling pulse, refolding of CypA could be accelerated to rates comparable to the amide proton exchange. This allowed the calculation of protection factors (PF) for the backbone amide protons in the GroEL-bound substrate protein. A set of highly protected protons in the native state (PF 10(5) to 10(7)) was observed to be much less protected (PF 10(2) to 10(4)) in complex with GroEL and, in contrast to the native structure, the protection factors were found to be quite uniform along the sequence suggesting that CypA with native-like structure undergoes multiple cycles of unfolding while bound to GroEL, which are faster than unfolding in free solution. Because of the small sequence dependence of the protection factors, unfolding must be global, and in this way the chaperone appears to resolve off-pathway intermediates and to support protein folding by annealing. Although in the complex with GroEL native-like states still predominate over globally unfolded states, this equilibrium is shifted 10(2) to 10(4)-fold toward the unfolded state when compared to CypA in free solution. Repeated global unfolding may be a key step in achieving a high yield of correctly folded proteins. Copyright 1997 Academic Press Limited.

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0126

Nieba, S.E. and Plückthun, A. (1997) Chaperone-mediated protein folding. BIOforum International 1, 20-24.
| Abstract + further info

Abstract

Molecular chaperones of the chaperonin (Hsp60) and Hsp70 families are basic constituents of the cellular machinery that mediates protein folding. The Escherichia coli chaperonin system GroEL/ GroES is well characterized in terms of structure and function. GroEL prevents the aggregation of partially folded or misfolded proteins by complexing them in a form competent for subsequent folding to the native state. Although many detailed biochemical studies have been performed in order to elucidate the mechanism of GroEL function, very little is known about the conformation of GroEL-bound substrate proteins. Here, we summarize amide proton exchange studies in combination with nuclear magnetic resonance spectroscopy or mass spectrometry as a powerful tool to investigate the conformational properties of proteins bound to the chaperonin. From different studies and model proteins a wide range of folding states has been proposed.

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0125

Spada, S., Krebber, C. and Plückthun, A. (1997) Selectively infective phages (SIP). Biol. Chem. 378, 445-456.
| Abstract + further info

Abstract

We review here advances in the selectively infective phage (SIP) technology, a novel method for the in vivo selection of interacting protein-ligand pairs. A 'selectively infective phage' consists of two components, a filamentous phage particle made non-infective by replacing its N-terminal domains of gene3 protein (g3p) with a ligand-binding protein, and an 'adapter' molecule in which the ligand is linked to those N-terminal domains of g3p which are missing from the phage particle. Infectivity is restored when the displayed protein binds the ligand and thereby attaches the missing N-terminal domains of g3p to the phage particle. Phage propagation becomes strictly dependent on the protein-ligand interaction. This method shows promise both in the area of library screening and in the optimization of peptides or proteins. [References: 58]

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0124

Spada, S. and Plückthun, A. (1997) Selectively infective phage (SIP) technology: A novel method for in vivo selection of interacting protein-ligand pairs. Nature Med. 3, 694-696.
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Abstract

No abstract

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0123

Plückthun, A. and Pack, P. (1997) New protein engineering approaches to multivalent and bispecific antibody fragments. Immunotechnol. 3, 83-105.
| Abstract + further info

Abstract

Multivalency is one of the hallmarks of antibodies, by which enormous gains in functional affinity, and thereby improved performance in vivo and in a variety of in vitro assays are achieved. Improved in vivo targeting and more selective localization are another consequence of multivalency. We summarize recent progress in engineering multivalency from recombinant antibody fragments by using miniantibodies (scFv fragments linked with hinges and oligomerization domains), spontaneous scFv dimers with short linkers (diabodies), or chemically crosslinked antibody fragments. Directly related to this are efforts of bringing different binding sites together to create bispecific antibodies. For this purpose, chemically linked fragments, diabodies, scFv-scFv tandems and bispecific miniantibodies have been investigated. Progress in E. coli expression technology makes the amounts necessary for clinical studies now available for suitably engineered fragments. We foresee therapeutic advances from a modular, systematic approach to optimizing pharmacokinetics, stability and functional affinity, which should prove possible with the new recombinant molecular designs. [References: 174]

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0122

Hottenrott, S., Schumann, T., Plückthun, A., Fischer, G. and Rahfeld, J. U. (1997) The Escherichia coli SlyD is a metal ion-regulated peptidyl-prolyl cis/trans-isomerase. J. Biol. Chem. 272, 15697-15701.
| Abstract + further info

Abstract

In Escherichia coli as many as nine different genes coding for proteins with significant homology to peptidyl-prolyl cis/trans-isomerases (PPIases) have been found. However, for three of them, the histidine-rich SlyD, the homologous gene product of ORF149, and parvulin-like SurA, it was not known whether these proteins really possess PPIase activity. To gain access to the full set of PPIases in E. coli, SlyD, the N-terminal fragment of SlyD devoid of the histidine-rich region, as well as the protein product of ORF149 of E. coli named SlpA (SlyD-like protein) were cloned, overexpressed, and purified to apparent homogeneity. On the basis of the amino acid sequences, both proteins proved to be of the FK506-binding protein type of PPIases. Only when using trypsin instead of chymotrypsin as helper enzyme in the PPIase assay, the enzymatic activity of full-length SlyD and its N-terminal fragment can be measured. For Suc-Ala-Phe-Pro-Arg-4-nitroanilide as substrate, kcat/Km of 29,600 M-1 s-1 for SlyD and 18,600 M-1 s-1 for the N-terminal fragment were obtained. Surprisingly, the PPIase activity of SlyD is reversibly regulated by binding of three Ni2+ ions to the histidine-rich, C-terminal region. Because the PPIase activity of SlpA could be established as well, we now know eight distinct PPIases with proven enzyme activity in E. coli.

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0121

Slootstra, J. W., Kuperus, D., Plückthun, A. and Meloen, R. H. (1997) Identification of new tag sequences with differential and selective recognition properties for the anti-FLAG monoclonal antibodies M1, M2 and M5. Mol. Divers. 2, 156-164.
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Abstract

The FLAG peptides DYKDDDDK and MDYKDDDDK are widely used affinity tags. Here we describe new variants of the FLAG peptides which, in direct ELISA, showed selective and differential binding to the commercially available anti-FLAG monoclonal antibodies M1, M2 and M5. Variants of the FLAG peptides were synthesized on polymer-grafted plastic pins, and in an ELISA incubated with mAbs M1, M2 and M5. Among the newly identified tag sequences are those that bind only one of the anti-FLAG mAbs and those that bind only two or all three of the anti-FLAG mAbs. Examples of new tag sequences are MDFKDDDDK (which binds mAb M5 and does not bind mAbs M1 and M2) and MDYKAFDNL (which binds mAb M2 and does not bind mAbs M1 and M5). The sensitivity in direct ELISA of some variants was increased, e.g. using mAb M2 it was found that replacing DDDDK in MDYKDDDDK by AFDNL increased the sensitivity in ELISA at least 10-fold. The activity of this peptide was studied in more detail. In different direct ELISAs, in which MDYKAFDNL was synthesized on polyethylene pins, coated onto polystyrene microtiter plates or onto nitrocellulose paper, the activity of this peptide was similar, i.e. increased at least 10-fold over that of MDYKDDDDK. Remarkably, in competitive ELISA the binding activity of soluble MDYKAFDNL was decreased 10-fold over those of soluble MDYKDDDDK or DYKDDDDK. The results seem to suggest that, in solution, the conformation of MDYKAFDNL is more 'unstructured' compared to its conformation when coated or linked to a carrier. We postulate that the newly described tag sequences may be used as affinity tags to separately detect, quantify and purify multiple co-expressed proteins and/or subunits.

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0120

Krebber, C., Spada, S., Desplancq, D., Krebber, A., Ge, L. and Plückthun, A. (1997) Selectively-infective phage (SIP): A mechanistic dissection of a novel in vivo selection for protein-ligand interactions. J. Mol. Biol. 268, 607-618.
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Abstract

Selectively-infective phage (SIP) is a novel methodology for the in vivo selection of interacting protein-ligand pairs. It consists of two components, (1) a phage particle made non-infective by replacing its N-terminal domains of geneIII protein (gIIIp) with a ligand-binding protein, and (2) an ""adapter"" molecule in which the ligand is linked to those N-terminal domains of gIIIp which are missing from the phage particle. Infectivity is restored when the displayed protein binds to the ligand and thereby attaches the missing N-terminal domains of gIIIp to the phage particle. Phage propagation is thus strictly dependent on the protein-ligand interaction. We have shown that the insertion of beta-lactamase into different positions of gIIIp, mimicking the insertion of a protein-ligand pair, led to highly infective phage particles. Any phages lacking the first N-terminal domain were not infective at all. In contrast, those lacking only the second N-terminal domain showed low infectivity irrespective of the presence or absence of the F-pilus on the recipient cell, which could be enhanced by addition of calcium. An anti-fluorescein scFv antibody and its antigen fluorescein were examined as a protein-ligand model system for SIP experiments. Adapter molecules, synthesized by chemical coupling of fluorescein to the purified N-terminal domains, were mixed with non-infective anti-fluorescein scFv-displaying phages. Infection events were strictly dependent on fluorescein being coupled to the N-terminal domains and showed a strong dependence on the adapter concentration. Up to 10(6) antigen-specific events could be obtained from 10(10) input phages, compared to only one antigen-independent event. Since no separation of binders and non-binders is necessary, SIP is promising as a rapid procedure to select for high affinity interactions.

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0119

Freund, C., Gehrig, P., Holak, T. A. and Plückthun, A. (1997) Comparison of the amide proton exchange behavior of the rapidly formed folding intermediate and the native state of an antibody scFv fragment. FEBS Lett. 407, 42-46.
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Abstract

We have investigated the stability of backbone amide protons of the intermediate and the native state of the scFv fragment of an antibody. Stopped flow experiments analyzed by MS and NMR detected the formation of an exchange protected intermediate within the deadtime of the stopped flow apparatus (17 ms). H/D exchange rates of the native protein identified a number of very stable backbone amide protons in the V(L) and the V(H) domains. In the V(L) domain, this slowly exchanging core of the scFv fragment is similar to the folding core of the intermediate, while the V(H) domain possesses a great number of very stable amide protons which are not stabilized to a significant degree in the folding intermediate of the scFv fragment.

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0118

Hanes, J. and Plückthun, A. (1997) In vitro selection and evolution of functional proteins by using ribosome display. Proc. Natl. Acad. Sci. U. S. A. 94, 4937-4942.
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Abstract

We report here a system with which a correctly folded complete protein and its encoding mRNA both remain attached to the ribosome and can be enriched for the ligand-binding properties of the native protein. We have selected a single-chain fragment (scFv) of an antibody 10(8)-fold by five cycles of transcription, translation, antigen-affinity selection, and PCR. The selected scFv fragments all mutated in vitro by acquiring up to four unrelated amino acid exchanges over the five generations, but they remained fully compatible with antigen binding. Libraries of native folded proteins can now be screened and made to evolve in a cell-free system without any transformation or constraints imposed by the host cell.

DOI: 10.1073/Pnas.94.10.4937 | Read all as PDF | Close Abstract
0117

Nieba, L., Honegger, A., Krebber, C. and Plückthun, A. (1997) Disrupting the hydrophobic patches at the antibody variable/constant domain interface: Improved in vivo folding and physical characterization of an engineered scFv fragment. Protein Eng. 10, 435-444.
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Abstract

By constructing Fv and single-chain Fv (scFv) fragments of antibodies, the variable domains are taken out of their natural context in the Fab fragment, where they are associated with the constant domains of the light (CL) and heavy chain (CH1). As a consequence, all residues of the former variable/constant domain interface become solvent exposed. In an analysis of 30 non-redundant Fab structures it was found that at the former variable/constant domain interface of the Fv fragment the frequency of exposed hydrophobic residues is much higher than in the rest of the Fv fragment surface. We investigated the importance of these residues for different properties such as folding in vivo and in vitro, thermodynamic stability, solubility of the native protein and antigen affinity. The experimental model system was the scFv fragment of the anti-fluorescein antibody 4-4-20, of which only 2% is native when expressed in the periplasm of Escherichia coli. To improve its in vivo folding, a mutagenesis study of three newly exposed interfacial residues in various combinations was carried out. The replacement of one of the residues (V84D in VH) led to a 25-fold increase of the functional periplasmic expression yield of the scFv fragment of the antibody 4-4-20. With the purified scFv fragment it was shown that the thermodynamic stability and the antigen binding constant are not influenced by these mutations, but the rate of the thermally induced aggregation reaction is decreased. Only a minor effect on the solubility of the native protein was observed, demonstrating that the mutations prevent aggregation during folding and not of the native protein. Since the construction of all scFv fragments leads to the exposure of these residues at the former variable/constant domain interface, this strategy should be generally applicable for improving the in vivo folding of scFv fragments and, by analogy, also the in vivo folding of other engineered protein domains.

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0116

Ryabova, L. A., Desplancq, D., Spirin, A. S. and Plückthun, A. (1997) Functional antibody production using cell-free translation: Effects of protein disulfide isomerase and chaperones. Nature Biotechnol. 15, 79-84.
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Abstract

To create a rapid system to test the effect of sequence changes on recombinant antibody binding, we have developed a procedure for producing functional scFv fragments in an Escherichia coli cell-free translation system. Functional antibodies with antigen-binding activity are obtained only if disulfide formation and rearrangement is allowed to take place during the translation reaction. The inclusion of protein disulfide isomerase (PDI) leads to a threefold increase in yield over that obtained in the presence of glutathione redox systems. DsbA had no such effect, indicating that disulfide shuffing, and not net formation, is the crucial yield-limiting step. The addition of the molecular chaperones DnaK and DnaJ increased the amount of soluble protein but not the amount of functional scFv, which appears to be limited entirely by correct disulfide formation. None of these factors significantly influenced total protein synthesis. In the presence of PDI, chaperones, reduced glutathione and oxidized glutathione, 50% of the scFv produced (about 8 micrograms/ml in only 15 min) could be recovered from immobilized antigen.

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0115

Lindner, P., Bauer, K., Krebber, A., Nieba, L., Kremmer, E., Krebber, C., Honegger, A., Klinger, B., Mocikat, R. and Plückthun, A. (1997) Specific detection of His-tagged proteins with recombinant anti-His tag scFv-phosphatase or scFv-phage fusions. Biotechniques 22, 140-149.
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Abstract

Using a cell-bound immunogen, we have generated a monoclonal antibody, 3D5, that recognizes carboxy-terminal oligo-histidine tags (His tags) on a wide variety of proteins. From this monoclonal antibody, we have generated a single-chain fragment of the variable domains (scFv), a dimeric scFv-alkaline phosphatase fusion and an oligovalent scFv-display phage. The antibody in its various formats is an effective tool used in fluorescence-activated cell sorting analysis, the BIAcore method, Western blots and enzyme-linked immunosorbent assay (ELISA). Western blots and ELISAs can be developed directly by using crude extracts of E.coli cells that produce the scFv-alkaline phosphatase fusion, thus providing an inexhaustable and convenient supply of detection reagent. Alternatively, oligovalent scFv-displaying phage can be used directly from culture supernatants for this purpose. The dissociation constants, KD of the peptide KGGHHHHH (KD = 4 x 10(-7) M) and of imidazole (KD = 4 x 10(-4) M) were determined. Molecular modeling of the Fv fragment suggests the occurrence of two salt bridges between the protonated histidine side chains of the peptide and the acidic groups in the antibody, explaining why the antibody or the substrate may be eluted under mildly basic conditions.

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0114

Proba, K., Honegger, A. and Plückthun, A. (1997) A natural antibody missing a cysteine in VH: Consequences for thermodynamic stability and folding. J. Mol. Biol. 265, 161-172.
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Abstract

While the disulfide bridge is highly conserved within the immunoglobulin fold, a few antibody variable domains lack one of the essential cysteine residues. In the levan binding antibody ABPC48 one of the essential cysteine residues (Cys H92) of the heavy chain variable domain is replaced by tyrosine. We expressed scFv fragments with the ABPC48 sequence and a mutant in which the VH disulfide bond has been restored in Escherichia coli, purified both proteins by antigen affinity chromatography and characterized them by equilibrium denaturation. While the ABPC48 protein was found to be significantly less stable than an average scFv molecule, the restored disulfide increased its stability above that of other, unrelated scFv fragments, explaining why it tolerates the disulfide loss. Surprisingly, we observed that under some refolding conditions, the unpaired cysteine residue of functional scFv of ABPC48 is derivatized by glutathione. It is easily accessible to other reagents and thus appears to be solvent-exposed, in contrast to the deeply buried disulfide of ordinary variable domains. This implies a very unusual conformation of stand b containing the unpaired Cys H22, which might be stabilized by interactions with the tyrosine residue in position H92.

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0113

Krebber, A., Bornhauser, S., Burmester, J., Honegger, A., Willuda, J., Bosshard, H. R. and Plückthun, A. (1997) Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J. Immunol. Methods 201, 35-55.
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Abstract

A prerequisite for the use of recombinant antibody technologies starting from hybridomas or immune repertoires is the reliable cloning of functional immunoglobulin genes. For this purpose, a standard phage display system was optimized for robustness, vector stability, tight control of scFv-delta geneIII expression, primer usage for PCR amplification of variable region genes, scFv assembly strategy and subsequent directional cloning using a single rare cutting restriction enzyme. This integrated cloning, screening and selection system allowed us to rapidly obtain antigen binding scFvs derived from spleen-cell repertoires of mice immunized with ampicillin as well as from all hybridoma cell lines tested to date. As representative examples, cloning of monoclonal antibodies against a his tag, leucine zippers, the tumor marker EGP-2 and the insecticide DDT is presented. Several hybridomas whose genes could not be cloned in previous experimental setups, but were successfully obtained with the present system, expressed high amounts of aberrant heavy and light chain mRNAs, which were amplified by PCR and greatly exceeded the amount of binding antibody sequences. These contaminating variable region genes were successfully eliminated by employing the optimized phage display system, thus avoiding time consuming sequencing of non-binding scFv genes. To maximize soluble expression of functional scFvs subsequent to cloning, a compatible vector series to simplify modification, detection, multimerization and rapid purification of recombinant antibody fragments was constructed.

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0112

Gervasoni, P. and Plückthun, A. (1997) Folding intermediates of _-lactamase recognized by GroEL. FEBS Lett. 401, 138-142.
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Abstract

beta-Lactamase, from which the disulfide bond was removed by two Cys-->Ala mutations, forms stable complexes with GroEL only during the first 30 s of folding, while wild-type beta-lactamase forms no stable complex under these conditions. The 3-phasic kinetics of folding are very similar between wild-type and mutant. After 4 s, Trp-210 is already juxtaposed to the disulfide bond, but proline cis-trans isomerization has not yet taken place and almost no enzymatic activity is observed. This shows that GroEL is unable to bind late folding intermediates and also discriminates between the degree of unfolding possible in wild-type disulfide-containing beta-lactamase and the Cys-Ala mutant.

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1996

# Reference PDF

0111

Nieba, L., Krebber, A. and Plückthun, A. (1996) Competition BIAcore for measuring true affinities: Large differences from values determined from binding kinetics. Anal. Biochem. 234, 155-165.
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Abstract

In attempting to use the BIAcore instrument for the determination of binding constants of several haptens or peptides to different antibodies by measuring on- and off-rates, we found that neither the absolute nor the relative values of the binding constants corresponded to the measurements in solution. Even at the lowest coupling densities useful for measurements, rebinding and bivalency effects offset the measurements by a factor of up to 500. We caution therefore about using on- and off-rates for the determination of absolute or even relative binding constants without controlling for rebinding and avidity effects. Instead, we show that binding constants in solution can be reproduced well by using on-rate determinations of antibody preincubated with antigen, and we derive the conditions under which such an approach is valid.

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0110

Gervasoni, P., Staudenmann, W., James, P., Gehrig, P. and Plückthun, A. (1996) _-Lactamase binds to GroEL in a conformation highly protected against hydrogen/deuterium exchange. Proc. Natl. Acad. Sci. U. S. A. 93, 12189-12194.
| Abstract + further info

Abstract

Escherichia coli RTEM beta-lactamase reversibly forms a stable complex with GroEL, devoid of any enzymatic activity, at 48 degrees C. When beta-lactamase is diluted from this complex into denaturant solution, its unfolding rate is identical to that from the native state, while the unfolding rate from the molten globule state is too fast to be measured. Electrospray mass spectrometry shows that the rate of proton exchange in beta-lactamase in the complex at 48 degrees C is slower than in the absence of GroEL at the same temperature, and resembles the exchange of the native state at 25 degrees C. Similarly, the final number of protected deuterons is higher in the presence of GroEL than in its absence. We conclude that, for beta-lactamase, a state with significant native structure is bound to GroEL. Thus, different proteins are recognized by GroEL in very different states, ranging from totally unfolded to native-like, and this recognition may depend on which state can provide sufficient accessible hydrophobic amino acids in a suitably clustered arrangement. Reversible binding of native-like states with hydrophobic patches may be an important property of GroEL to protect the cell from aggregating protein after heat-shock.

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0109

Freund, C., Honegger, A., Hunziker, P., Holak, T. A. and Plückthun, A. (1996) Folding nuclei of the scFv fragment of an antibody. Biochemistry 35, 8457-8464.
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Abstract

The folding kinetics of the variable domains of the phosphorylcholine-binding antibody McPC603, combined into a scFv fragment [VH-(Gly4Ser)3-VL], were investigated by the use of fluorescence spectroscopy, nuclear magnetic resonance (NMR), and mass spectrometry (MS). All three methods gave evidence for the occurrence of a major kinetic intermediate during the refolding of the denatured, oxidized scFv fragment. This intermediate is formed within the first 30 s of folding and comprises exchange-protected amide protons of hydrophobic and aromatic amino acids, most of which are localized within the inner beta-sheet of the V(L) domain. In the subsequent slow step, most of the amide protons become protected with rate constants that are very similar for residues of both domains. These data are in agreement with the MS results, which indicate a cooperative folding event from the intermediate to the native state of the scFv fragment.

DOI: 10.1021/bi952764a | Read all as PDF | Close Abstract
0108

Plückthun, A., Krebber, A., Krebber, C., Horn, U., Knüpfer, U., Wenderoth, R., Nieba, L., Proba, K. and Riesenberg, D. (1996) Producing antibodies in Escherichia coli: From PCR to fermentation in: Antibody Engineering, A Practical Approach (McCafferty, J., Hoogenboom, H. R., and Chiswell, D. J., eds) pp. 203-252. IRL Press, Oxford
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Abstract

No abstract

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0107

Krebber, A., Burmester, J. and Plückthun, A. (1996) Inclusion of an upstream transcriptional terminator in phage display vectors abolishes background expression of toxic fusions with coat protein g3p. Gene 178, 71-74.
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Abstract

Expression of toxic gene products affects bacterial cell growth and phage display, causing a strong selection against plasmid maintenance and integrity. During phage propagation steps, in particular, phagemid instability can dramatically affect diversity of antibody libraries or even lead to the deletion of antibody genes. We constructed a modified phage display vector by introducing a strong transcriptional terminator upstream of the lac promoter, which together with glucose suppression of its CAP-dependent activation, very efficiently represses product formation before induction.

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0106

Rheinnecker, M., Hardt, C., Ilag, L. L., Kufer, P., Gruber, R., Hoess, A., Lupas, A., Rottenberger, C., Plückthun, A. and Pack, P. (1996) Multivalent antibody fragments with high functional affinity for a tumor-associated carbohydrate antigen. J. Immunol. 157, 2989-2997.
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Abstract

We report in this work a human-derived self-assembling polypeptide based on the tetramerization domain of the human transcription factor p53, which can be fused to single-chain Fv Ab (scFv) fragments via a long and flexible hinge sequence of human origin, allowing exploitation of the functional affinity increase of binding to a ligand or cell surface with multimeric binding sites. We have demonstrated the use of this polypeptide by applying it to the construction of a tetrameric scFv against the tumor-associated carbohydrate Ag Lewis Y (Fuc alpha 1-->2Gal beta 1-->4[Fuc alpha 1-->3] GlcNAc beta 1-->3R). For comparison purposes, the corresponding scFv and dimeric mini-antibody, comprising the scFv fused via a flexible murine hinge to an artificial dimerization domain, were also created. The recombinant mini-antibody proteins were expressed in functional form in Escherichia coli and showed the expected m.w. of a dimer and tetramer, respectively. Analysis of Lewis Y-binding behavior by surface plasmon resonance revealed specific but very weak binding of the scFv fragment. In contrast, both dimeric and tetrameric scFv fusion proteins exhibited an enormous gain in functional affinity that was greatest in the case of the tetrameric mini-antibody.

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0105

Kalinke, U., Krebber, A., Krebber, C., Bucher, E., Plückthun, A., Zinkernagel, R. M. and Hengartner, H. (1996) Monovalent single-chain Fv fragments and bivalent miniantibodies bound to vesicular stomatitis virus protect against lethal infection. Eur. J. Immunol. 26, 2801-2806.
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Abstract

Several antibody-dependent mechanisms have been postulated to mediate neutralization of different animal viruses, including blocking of docking to receptors, induction of conformational changes in the virus coat, and Fc-dependent opsonization. We have studied the molecular requirements for antibody-mediated neutralization of vesicular stomatitis virus (VSV) in vitro and protection against lethal disease in vivo with a single-chain Fv fragment (scFv) and the corresponding bivalent miniantibody (scFv-dHLX) generated from a VSV-neutralizing monoclonal antibody. Both monovalent scFv and bivalent scFv-dHLX miniantibodies were able to neutralize VSV in vitro and to protect interferon-alphabeta receptor-deficient (IFN-alphabeta R-/-) mice against lethal disease after intravenous injection of 50 plaque-forming units (pfu) VSV pre-incubated with the scFv reagents. Similarly, severe-combined immunodeficient (SCID) mice infected with immune complexes of 10(8) pfu VSV and bivalent scFv-dHLX were protected against lethal disease; however, mice infected with immune complexes of 10(8) pfu VSV and monovalent scFv were not. Although repeated scFv-dHLX treatment reduced virus quantities in the blood, neither SCID nor IFN-alphabeta R-/- mice were protected against lethal disease after passive immunization and subsequent VSV infection. This was due to the short half-life of 17 min of scFv-dHLX in the circulation. These data demonstrate that neutralization of VSV and protection against lethal disease do not require Fc-mediated mechanisms and that cross-linking is not crucial for protection against physiologically relevant virus doses in vivo.

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0104

Zahn, R., Lindner, P., Axmann, S. E. and Plückthun, A. (1996) Effect of single point mutations in citrate synthase on binding to GroEL. FEBS Lett. 380, 152-156.
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Abstract

Six single point mutants of yeast citrate synthase were analyzed for binding to the molecular chaperone GroEL. In contrast to the wild-type and G276S, all other G276-mutants were able to displace pre-beta-lactamase from GroEL. The off-rate constant for pre-beta-lactamase must be at least partially rate-limiting, leading to an equilibrium dissociation constant between 10(-10) M and 10(-12)M. Direct evidence for binding of citrate synthase was obtained from gel filtration experiments. The results suggest that thermodynamic rather than structural features of the mutants determine the degree of binding to the chaperone.

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0103

Horn, U., Strittmatter, W., Krebber, A., Knüpfer, U., Kujau, M., Wenderoth, R., Müller, K. M., Matzku, S., Plückthun, A. and Riesenberg, D. (1996) High volumetric yields of functional dimeric miniantibodies in Escherichia coli, using an optimized expression vector and high-cell-density fermentation under non-limited growth conditions. Appl. Microbiol. Biotechnol. 46, 524-532.
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Abstract

Functional bivalent miniantibodies, directed against the epidermal growth factor receptor, accumulated to more than 3 gl-1 in high-cell-density cultures of Escherichia coli RV308(pHKK) on a pilot scale. The miniantibodies consist of scFv fragments with a C-terminal hinge followed by a helix-turn-helix motif, which homodimerizes in vivo. The improved expression vector pHKK is characterized by the hoklsok suicide system, improving plasmid maintenance, and the inducible lac pl o promoter system with the very strong T7g10 Shine-Dalgarno sequence. The expression unit is flanked by terminators. The prototrophic RV308 cells were cultivated in glucose mineral salt medium and reached a cell density of 145 g dry biomass l-1 after 33 h. After induction, growth continued almost unchanged for a further 4 h with concomitant miniantibody formation. In the fedbatch phase, the concentration of glucose was kept almost constant at the physiological level of approximately 1.5 gl-1, using on-line flow injection analysis for control. Surprisingly, E. coli RV308(pHKK) did not accumulate significant amounts of the metabolic by-product acetate under these unlimited aerobic growth conditions.

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1995

# Reference PDF

0102

Wülfing, C. and Plückthun, A. (1995) T-cell receptor signal sequences. Immunol. Today 16, 405-406.
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Abstract

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0101

Proba, K., Ge, L. and Plückthun, A. (1995) Functional antibody single-chain fragments from the cytoplasm of Escherichia coli: Influence of thioredoxin reductase (TrxB). Gene 159, 203-207.
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Abstract

The cytoplasmic expression of a functional antibody (Ab) fragment, containing the correct intradomain disulfide bonds, was investigated in E. coli. We used a single-chain Fv (scFv) fragment of the levan-binding Ab ABPC48, which was shown to be functional only in the presence of the disulfide bonds. Significant amounts of functional, disulfide-containing scFv could be produced in the cytoplasm of E. coli in the absence of thioredoxin reductase (TrxB) activity. The amount of soluble protein remained largely unchanged by this null mutation. A stronger promoter did not result in further improved yields of functional Ab fragment, despite much higher protein production, suggesting that inefficient disulfide formation was still limiting the yield of active scFv. This method of expressing functional Ab fragments in the cytoplasm of E. coli may be important for screening and selection systems.

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0100

Wülfing, C. and Plückthun, A. (1995) Soluble T-cell receptor fragments – guidance of folding and assembly. The Immunologist 3, 59-66.
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Abstract

The TcR is the central recognition molecule in cellular immunity, yet our knowledge about its structure and interactions with the MHC/peptide complex and with its co receptors is sparse. Therefore, the production of soluble TcR fragments is of considerable interest, in particular for biophysical studies. Here, Christoph Wülfing and Andreas Plückthun discuss soluble TcR fragments produced in E.coli and in eukaryotic systems, and analyze the success to date.

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0099

Ge, L., Lupas, A., Peraldi-Roux, S., Spada, S. and Plückthun, A. (1995) A mouse Ig k domain of very unusual framework structure loses function when converted to the consensus. J. Biol. Chem. 270, 12446-12451.
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Abstract

Antibody gene sequences, particularly those of kappa light chains, are very well conserved in the framework region, and the variability is concentrated in the complementarity-determining regions (CDR). We now found that the murine antibody 93-6 (Djavadi-Ohaniance, L., Friguet, B., and Goldberg, M. (1984) Biochemistry 23, 97-104) whose Fab fragment binds the beta-subunit of Escherichia coli tryptophan synthase with high affinity (Kd of 6.7.10(-9) M) has a highly unusual kappa light chain framework, which is crucial for the function of this antibody. It carries an insertion of 8 amino acids in a conserved framework loop that faces the antigen, and its framework region 2 (FR2) which precedes CDR2 is shortened by one amino acid, normally leucine and part of an absolutely conserved beta-bulge preceding CDR2. Removal of the insertion to restore the consensus sequence reduced the binding affinity of 93-6 by a factor 3, while insertion of the missing leucine into FR2 completely abolished binding.

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0098

Haunschild, J., Faro, H.-P., Pack, P. and Plückthun, A. (1995) Pharmacokinetic properties of bivalent miniantibodies and comparison to other immunoglobulin forms. Antibody, Immunoconjugates and Radiopharmaceuticals 8, 111-128.
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Abstract

The pharmacokinetics of bivalent miniantibodies were compared in the mouse, and compared with those of a single-chain Fv (scFv) fragment and a whole antibody. Miniantibodies are based on scfv-fragments, which are fused, via a flexible hinge-region, to association domains consisting of different kinds of amphipathic, associating helices. All molecules in this study had the same binding site for antigen, which was not a murine molecule, and thus the kinetics independent of localization phenomena could be measured. The miniantibodies were found to have plasma elimination half-lives somewhat slower than a scFv fragment but much faster than a whole antibody, to be almost quantitatively excreted (via the kidneys) and to show no sign of breakdown in the plasma. Since miniantibodies can easily be prepared from Escherichia coil and show a greatly increased surface binding compared to monovalent scFv fragments, they should be useful molecules for in vivo diagnostics and therapy.

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0097

Knappik, A. and Plückthun, A. (1995) Engineered turns of a recombinant antibody improve its in vivo folding. Protein Eng. 8, 81-89.
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Abstract

Using recombinant antibodies functionally expressed by secretion to the periplasm in Escherichia coli as a model system, we identified mutations located in turns of the protein which reduce the formation of aggregates during in vivo folding or which influence cell stability during expression. Unexpectedly, the two effects are based on different mutations and could be separated, but both mutations act synergistically in vivo. Neither mutation increases the thermodynamic stability in vitro. However, the in vivo folding mutation correlates with the yield of oxidative folding in vitro, which is limited by the side reaction of aggregation. The in vivo folding data also correlate with the rate and activation entropy of thermally induced aggregation. This analysis shows that it is possible to engineer improved frameworks for semi-synthetic antibody libraries which may be important in maintaining library diversity. Moreover, limitations in recombinant protein expression can be overcome by single amino acid substitutions.

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0096

Wall, J. G. and Plückthun, A. (1995) Effects of overexpressing folding modulators on the in vivo folding of heterologous proteins in Escherichia coli. Curr. Opin. Biotechnol. 6, 507-516.
| Abstract + further info

Abstract

Interest continues to increase in the use of folding modulators to overcome problems with heterologous protein folding in Escherichia coli. Currently, this approach, though highly successful with a number of individual proteins, remains a somewhat hit-and-miss affair. Ongoing research directed at unraveling the precise role and specificity of these folding modulators should generate a clearer understanding of the potential and limitations of overexpressing folding catalysts in vivo. This will facilitate the development, in the not too distant future, of a more structured and rational approach to improving the folding of heterologous gene products in E. coli. [References: 77]

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0095

Harris, J. R., Zahn, R. and Plückthun, A. (1995) Electron microscopy of the GroEL-GroES filament. J. Struct. Biol. 115, 68-77.
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Abstract

Electron microscopy of a filamentous complex of GroEL and GroES has been performed on negatively stained specimens. The filaments have been formed when GroEL and GroES at relatively low molar ratios(e.g., 1:1 to 1:3) are incubated at room temperature in the presence of magnesium and ATP. At higher molar ratio, the symmetrical GroEL-GroES complex predominates. Within the GroEL-GroES filaments, the same structural rearrangements of GroEL were observed as in the ''bulletshaped'' or symmetrical GroEL-GroES complexes. In the absence of GroES, GroEL molecules have no tendency to form linear filaments, The sharing of one GroES by two GroEL molecules might indicate a high plasticity of the GroEL-GroES complex. Preliminary Fourier analysis of filaments of increasing length indicates that they possess an inherent helicity. Using the negative staining-carbon film procedure, two-dimensional (2-D) crystal nucleation induced by ammonium molybdate-polyethylene glycol can create undulatory rows of side-on GroEL molecules, which are able extend as a p2 2-D crystal form, readily distinguishable from the filamentous GroEL-GroES aggregates. Crystallographic image processing indicates that the GroEL 2 x 7-mer itself possesses a dyad screw axis, in accord with existing higher resolution X-ray structure data.

DOI: 10.1006/Jsbi.1995.1031 | Read all as PDF | Close Abstract
0094

Krebber, C., Spada, S., Desplancq, D. and Plückthun, A. (1995) Co-selection of cognate antibody-antigen pairs by selectively-infective phages. FEBS Lett. 377, 227-231.
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Abstract

We have developed a chloramphenicol resistant derivative of fd phage with which cognate pairs of antibodies and antigens can be selected. The phage genome encodes a fusion of single-chain antibody to the C-terminal domain of gIIIp, rendering the phage non-infective. The antigen fused to the N-terminal domains of gIIIp is encoded in the same phage genome. Antigen and antibody fusion interact with each other in the periplasm of the phage-producing cell, restoring infectivity. This system has a very low background and will allow simultaneous randomisation of antibody and antigen.

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0093

Ge, L., Knappik, A., Pack, P., Freund, C. and Plückthun, A. (1995) Expressing antibodies in Escherichia coli in: Antibody Engineering 2nd ed. (Borrebaeck, C. A. K., ed) pp. 229-266. Oxford University Press, Inc., New York
| Abstract + further info

Abstract

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0092

Pack, P., Müller, K. M., Zahn, R. and Plückthun, A. (1995) Tetravalent miniantibodies with high avidity assembling in Escherichia coli. J. Mol. Biol. 246, 28-34.
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Abstract

We have designed tetravelent miniantibodies assembling in the periplasm of Escherichia coli. They are based on single-chain Fv fragments, connected via a flexible hinge to an amphipathic helix which tetramerizes the molecule. The amphipathic helix is derived from the coiled coil helix of the transcription factor GCN4, in which all hydrophobic a positions of every heptad repeat have been exchanged to leucine and all d positions to isoleucine. Gel filtration shows tetramer assembly of the miniantibody even at low concentrations. As expected, the functional affinity (avidity) of the tetravalent miniantibody is higher in ELISA and BIAcore measurements than that of the bivalent construct and the gain is dependent on surface epitope density.

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1994

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0091

Knappik, A. and Plückthun, A. (1994) An improved affinity tag based on the FLAG® peptide for the detection and purification of recombinant antibody fragments. Biotechniques 17, 754-761.
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Abstract

The commercially available monoclonal antibodies M1 and M2 were raised against and bind the FLAG sequence DYKDDDDK with high specificity. Using the calcium-dependent M1 antibody and the FLAG tag attached to the N terminus of various fragments of the antibody McPC603 expressed in Escherichia coli, we found that the M1 antibody binds with almost the same affinity to a much shorter version of this sequence (DYKD). Since most antibody light chains start with an aspartate, the addition of only three additional amino acids to the N terminus is sufficient to detect and quantify the expressed antibody fragments using standard immunological methods. Similarly, the heavy chain can be detected specifically with the sequence DYKD, which requires four additional amino acids since most heavy chains do not start with Asp. The signal sequence of both chains that is necessary for the transport of the chains to the periplasm of E. coli is processed correctly. Furthermore, we investigated the influence of the amino acid at the fifth position of the FLAG sequence on the binding affinity of the M1 antibody and found that a glutamate at this position increased the sensitivity in Western blots sixfold over the original long FLAG sequence containing an aspartate residue at this position. Together, the improved FLAG is a versatile tool for both sensitive detection and one-step purification of recombinant proteins.

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0090

Wülfing, C. and Plückthun, A. (1994) Correctly folded T-cell receptor fragments in the periplasm of Escherichia coli. Influence of folding catalysts. J. Mol. Biol. 242, 655-669.
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Abstract

The T-cell receptor is the central recognition molecule in cellular immunity. Its extracellular domains are homologous with and thought to be structurally similar to an antibody Fab fragment. Despite the biological importance of the TCR and the ease of bacterial expression of antibody fragments, there are only few reports of TCR-fragment expression in E. coli. In order to understand the difficulties of expressing correctly folded TCR fragments in E. coli, we have characterized the expression behavior of single-chain Fv analogs of three different TCRs (scTCR). All of them can be folded into the correct conformation in the periplasm of E. coli, yet the extent of correct folding varies greatly. In order to overcome the folding problems of some of the scTCRs, we have developed a system with enhanced in vivo folding capability based on the simultaneous induction of the heat-shock response and over-expression of the E. coli disulfide isomerase DsbA at low temperature. We present a model describing the folding of the scTCRs in the periplasm of E. coli and possible points of folding assistance. The role of the periplasm as an independent folding compartment is emphasized and the existence of a general periplasmic chaperone is postulated. We have also shown that a bivalent scTCR, dimerized in vivo with helix-turn-helix modules, can be expressed in a correctly folded form.

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0089

Steipe, B., Schiller, B., Plückthun, A. and Steinbacher, S. (1994) Sequence statistics reliably predict stabilizing mutations in a protein domain. J. Mol. Biol. 240, 188-192.
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Abstract

Immunoglobulin variable domains are generally thought of as well conserved platforms providing the base for antigen binding loops of highly varying sequence and structure. However, domain evolution must ensure a balance between optimizing antigen affinity and the requirements of a stable, cooperatively folding domain. Since random mutations can carry a significant penalty for domain stability, constraints are imposed both on the repertoire of germline sequences and on somatic amino acid replacements during affinity maturation. Analyzing these constraints in the conceptual framework of statistical mechanics, we have been able to predict stabilizing mutations in the McPC603 V kappa domain from sequence information alone with better than 60% success rate. The validity of this concept not only has far reaching implications for antibody engineering but may also be generalized to engineer other proteins for higher stability.

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0088

Plückthun, A. (1994) Recombinant antibodies in: Immunochemistry (van Oss, C. J., and van Regenmortel, M. H. V., eds) pp. 201-236. Marcel Dekker Inc., New York
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Abstract

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0087

Wülfing, C., Lombardero, J. and Plückthun, A. (1994) An Escherichia coli protein consisting of a domain homologous to FK506-binding proteins (FKBP) and a new metal binding motif. J. Biol. Chem. 269, 2895-2901.
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Abstract

Initially detected as a persistent contaminant in immobilized metal affinity chromatography of recombinant proteins in Escherichia coli, a 196-amino acid protein was isolated, cloned, overexpressed, and characterized. It consists of two domains, of which the first (146 amino acids) shows some homology to the FK506-binding proteins. The second domain (50 amino acids) is extremely rich in potentially metal-binding amino acids, such as histidine, cysteine, and acidic amino acids. The protein binds Ni2+ and Zn2+ tightly with 1:1 stoichiometry, Cu2+ and Co2+ with lower affinity, and Mn2+, Fe2+, Fe3+, Mg2+, and Ca2+ hardly at all.

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0086

Zahn, R. and Plückthun, A. (1994) Thermodynamic partitioning model for hydrophobic binding of polypeptides by GroEL. II. GroEL recognizes thermally unfolded mature _-lactamase. J. Mol. Biol. 242, 165-174.
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Abstract

By thermal equilibrium measurements we found a three-state folding behavior of mature Escherichia coli beta-lactamase TEM2. The thermodynamically stable intermediate H had no enzymatic activity, but a native-like secondary structure. State H was 9 kcal mol-1 less stable than the native state N and 4 kcal mol-1 more stable than the totally unfolded state U, which is consistent with urea equilibrium measurements of mature beta-lactamase measured under similar conditions. Between 38 degrees C and 50 degrees C there was a decrease in the apparent equilibrium constant for dissociation K'D of the complex between GroEL and mature beta-lactamase, at least partially caused by a decrease in the thermodynamic stability of the native form of mature beta-lactamase. GroEL-bound beta-lactamase was released either after addition of ATP, or in the presence of a competing substrate (i.e. a single-chain antibody), or after lowering the temperature. Whereas at 10 degrees C the folding reaction of mature beta-lactamase was rate limiting, at 37 degrees C the release reaction was the rate-determining step for the regain of beta-lactamase activity, consistent with a decrease of the equilibrium constant for dissociation KD of the complex with temperature. A temperature dependent behavior of GroEL was also observed, when measuring the anilinonaphthalene sulfonic acid (ANS) fluorescence of the chaperone. Similar to all other substrate proteins studied so far, the maximal tryptohan fluorescence of GroEL-bound beta-lactamase was observed at 342 nm. Our results are compatible with a hydrophobic binding pocket of GroEL and confirm the suggested thermodynamic partitioning model for hydrophobic binding of polypeptides by GroEL.

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0085

Zahn, R., Spitzfaden, C., Ottiger, M., Wüthrich, K. and Plückthun, A. (1994) Destabilization of the complete protein secondary structure on binding to the chaperone GroEL. Nature 368, 261-265.
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Abstract

Protein folding in vivo is mediated by helper proteins, the molecular chaperones, of which Hsp60 and its Escherichia coli variant GroEL are some of the best characterized. GroEL is an oligomeric protein with 14 subunits each of M(r) 60K, which possesses weak, co-operative ATPase activity and high plasticity. GroEL seems to interact with non-native proteins, binding one or two molecules per 14-mer in a 'central cavity', but little is known about the conformational state of the bound polypeptides. Here we use nuclear magnetic resonance techniques to show that the interaction of the small protein cyclophilin with GroEL is reversible by temperature changes, and all amide protons in GroEL-bound cyclophilin are exchanged with the solvent, although this exchange does not occur in free cyclophilin. The complete secondary structure of cyclophilin must be disrupted when bound to GroEL.

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0084

Freund, C., Ross, A., Plückthun, A. and Holak, T. A. (1994) Structural and dynamic properties of the Fv fragment and the single-chain Fv fragment of an antibody in solution investigated by heteronuclear three-dimensional NMR spectroscopy. Biochemistry 33, 3296-3303.
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Abstract

Fv fragments, heterodimers of the variable light (VL) and variable heavy chain (VH) domains, are the smallest functional antibody units with molecular masses of approximately 26 kDa. The structural and dynamic properties of the Fv fragment and the corresponding single-chain Fv fragment (scFv: VH-linker-VL, 252 amino acids) of the phosphorylcholine-binding antibody McPC603 in the presence of hapten have been studied in solution by heteronuclear multidimensional NMR spectroscopy. Both 15N TOCSY-HMQC and triple-resonance experiments (HNCA and HN(CA)H, with 15N-13C-labeled protein) gave poor spectra, due to short T2 relaxation times for most of the backbone 1H, 15N, and 13C alpha atoms. The assignment procedure therefore relied upon the combination of amino acid and domain (VL) specifically labeled spectra and the 3D NOESY-HMQC spectrum of the uniformly 15N labeled Fv and scFv fragments. Approximately 80% of the 15N and 1H backbone and 60% of the 1H side-chain resonances have been assigned. Short- and long-range NOEs were used to determine the extent of beta-sheet structure and were compared to the X-ray crystallographic data. The 1H-15N NOE data indicate that the scFv backbone has a well-defined structure of limited conformational flexibility. However, the linker of the scFv fragment exhibits substantial fast internal motion (on the picosecond to nanosecond time scale) compared with the overall rotational correlation time of the whole molecule. Several residues in the CDRs, in turns, or at the C-terminal end of the protein have smaller NOEs, reflecting some degree of rapid motion in the protein backbone.

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0083

Wülfing, C. and Plückthun, A. (1994) Protein folding in the periplasm of Escherichia coli. Mol. Microbiol. 12, 685-692.
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Abstract

With the discovery of molecular chaperones and the development of heterologous gene expression techniques, protein folding in bacteria has come into focus as a potentially limiting factor in expression and as a topic of interest in its own right. Many proteins of importance in biotechnology contain disulphide bonds, which form in the Escherichia coli periplasm, but most work on protein folding in the periplasm of E. coli is very recent and is often speculative. This MicroReview gives a short overview of the possible fates of a periplasmic protein from the moment it is translocated, as well as of the E. coli proteins involved in this process. After an introduction to the specific physiological situation in the periplasm of E. coli, we discuss the proteins that might help other proteins to obtain their correctly folded conformation--disulphide isomerase, rotamase, parts of the translocation apparatus and putative periplasmic chaperones--and briefly cover the guided assembly of multi-subunit structures. Finally, our MicroReview turns to the fate of misfolded proteins: degradation by periplasmic proteases and aggregation phenomena. [References: 96]

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0082

Virnekäs, B., Ge, L., Plückthun, A., Schneider, K. C., Wellnhofer, G. and Moroney, S. E. (1994) Trinucleotide phosphoramidites: Ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis. Nucleic Acids Res. 22, 5600-5607.
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Abstract

Trinucleotide phosphoramidites representing codons for all 20 amino acids have been prepared and used in automated, solid-phase DNA synthesis. In contrast to an earlier report, we show that these substances can be used to introduce entire codons into oligonucleotides in excess of 98% yield, and are ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis.

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0081

Plückthun, A. (1994) Antibodies from Escherichia coli in: The pharmacology of monoclonal antibodies. Handbook of Experimental Pharmacology (Rosenberg, M., and Moore, G. P., eds) Vol. 113 pp. 269-315. Springer Verlag, Berlin
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Abstract

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0080

Zahn, R., Axmann, S. E., Rücknagel, K. P., Jaeger, E., Laminet, A. A. and Plückthun, A. (1994) Thermodynamic partitioning model for hydrophobic binding of polypeptides by GroEL. I. GroEL recognizes the signal sequences of _-lactamase precursor. J. Mol. Biol. 242, 150-164.
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Abstract

From equilibrium measurements with urea we found a three-state thermodynamic and kinetic folding behavior for the precursor and mature form of Escherichia coli beta-lactamase TEM2. The thermodynamic intermediate H of Escherichia coli beta-lactamase and its precursor had no enzymatic activity, and a quenched tryptophan fluorescence intensity, but a native-like wavelength of maximum intensity. State H of mature beta-lactamase was 8.7 kcal mol-1 less stable than the native state N and about 4.2 kcal mol-1 more stable than the unfolded state U, extrapolated to absence of urea. In contrast, state H of precursor beta-lactamase was even more stable than N by about 0.5 kcal mol-1 and about 6.9 kcal mol-1 more stable than U. Native pre-beta-lactamase could be stabilized by lowering the pH value from 7.0 to 5.5, probably by protonating a histidine residue leading to an improved solubility of the signal sequence. Synthetic peptides, containing 23 or 38 N-terminal amino acid residues of pre-beta-lactamase, were unable to compete with pre-beta-lactamase for binding to GroEL. However, GroEL prevented the inactivation of mature beta-lactamase by p38, consistent with competition between GroEL and mature beta-lactamase for binding to p38. The equilibrium constant for dissociation KD of the complex between GroEL and p23, a peptide containing exclusively the signal sequence of pre-beta-lactamase, was measured with the BIAcore instrument to be in the range 10(-7) to 10(-8) M. Our results are consistent with co-operative binding of GroEL to the mature part and to the signal sequence of pre-beta-lactamase. We suggest a thermodynamic partitioning model for hydrophobic binding of polypeptides by GroEL.

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0079

Harris, J. R., Plückthun, A. and Zahn, R. (1994) Transmission electron microscopy of GroEL, GroES, and the symmetrical GroEL/ES complex. J. Struct. Biol. 112, 216-230.
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Abstract

Two new 2-D crystal forms of the Escherichia coli chaperone GroEL (cpn60) 2 x 7-mer have been produced using the negative staining-carbon film (NS-CF) technique. These 2-D crystals, which contain the cylindrical GroEL in side-on and end-on orientations, both possess p21 symmetry, with two molecules in the respective unit cells. The crystallographically averaged images correlate well with those obtained by other authors from single particle analysis of GroEL and our own previous crystallographic analysis. 2-D crystallization of the smaller chaperone GroES (cpn10) 7-mer has also been achieved using the NS-CF technique. Crystallographically averaged images of GroES single particle images indicate considerable variation in molecular shape, which is most likely due to varying molecular orientation on the carbon support film. The quaternary structure of GroES does, nevertheless, approximate to a ring-like shape. The complex formed by GroEL and GroES in the presence of ATP at room temperature has been shown to possess a symmetrical hollow ellipsoidal conformation. This symmetrical complex forms in the presence of a 2:1 or greater molar ratio of GroES:GroEL. At lower molar ratios linear chains of GroEL form, apparently linked by GroES in a 1:1 manner, which provide supportive evidence for the ability of both ends of the GroEL cylinder to interact with GroES. The apparent discrepancy between our data and that of other groups who have described an asymmetrical ""bullet-shaped"" (holo-chaperone) GroEL/ES complex is discussed in detail.

DOI: 10.1006/jsbi.1994.1022 | Read all as PDF | Close Abstract
0078

Plückthun, A. (1994) Escherichia coli producing recombinant antibodies in: Recombinant microbes for industrial and agricultural applications (Murooka, Y., and Imanaka, T., eds) pp. 233-252. Marcel Dekker Inc., New York
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Abstract

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1993

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0077

Plückthun, A. (1993) Stability of engineered antibody fragments in: Stability and Stabilization of Enzymes (van den Tweel, W. J. J., Harder, A., and Buitelaar, R. M., eds) pp. 81-90. Elsevier Science Publishers B.V.
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Abstract

Antibody Fv fragments, consisting of only the variable domains, do bind the antigen with the same thermodynamic affinity as the whole antibody, but may dissociate into their components VH and VL. Methods of covalently linking the domains also increase the stability against irreversible denaturation, most dramatically by engineering disulfide bonds. New framework positions have been identified in which a majority of antibody fragments can probably be linked, as judged from the structural fit of the disulfides. Surprisingly, almost all antibody Fv fragments contain cavities, albeit in individual positions.

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0076

Zahn, R., Harris, J. R., Pfeifer, G., Plückthun, A. and Baumeister, W. (1993) Two-dimensional crystals of the molecular chaperone GroEL reveal structural plasticity. J. Mol. Biol. 229, 579-584.
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Abstract

For two-dimensional (2-D) crystallization we have purified the molecular chaperone GroEL from Escherichia coli to homogeneity. The final and important step for crystallization in the purification procedure was an ATP-agarose column, on which the spacer between ATP and agarose was attached to C8 of adenine. Using the mica spreading ""negative staining-carbon film"" procedure and polyethylene glycol as a precipitant, we obtained four different 2-D periodic arrays. Two of them turned out to be true crystals. One crystal has P2 symmetry and lattice constants of a = 24.3 nm and b = 16.9 nm, the other has essentially P4 symmetry and shows smoothly varying local changes in the lattice parameters (a = b = 23 (+/- 1.3) nm). Very striking in the P4 crystal is the departure within each individual GroEL particle from the GroEL-typical seven-fold symmetry, which seems to be required for GroEL to accommodate to a crystal symmetry.

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0075

Pack, P., Kujau, M., Schroeckh, V., Knüpfer, U., Wenderoth, R., Riesenberg, D. and Plückthun, A. (1993) Improved bivalent miniantibodies, with identical avidity as whole antibodies, produced by high cell density fermentation of Escherichia coli. Bio/Technology 11, 1271-1277.
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Abstract

The combination of single-chain Fv-fragments (scFv) with a C-terminal, flexible linking region followed by a designed or natural dimerization domain provides a versatile system for targeted association of functional fragments in the periplasmic space of Escherichia coli. For homodimerization in vivo, two scFv fragments with a C-terminal hinge followed by a helix-turn-helix motif form ""miniantibodies"" with significantly higher avidity than in the case of leucine zipper containing constructs. The favorable design probably results in an antiparallel four-helix bundle and brings the homodimer to the same avidity as the whole IgA antibody, from which the binding site was taken. The molecular weight of the bivalent miniantibody is almost the same as that of a monovalent Fab fragment. We report here a high-cell density fermentation of E. coli producing these miniantibodies and a work-up procedure suitable for large scale production. Without any need of subsequent chemical coupling in vitro, approximately 200 mg/l of functional dimeric miniantibodies can be directly obtained from the E. coli culture.

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0074

Knappik, A., Krebber, C. and Plückthun, A. (1993) The effect of folding catalysts on the in vivo folding process of different antibody fragments expressed in Escherichia coli. Bio/Technology 11, 77-83.
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Abstract

The Fv and Fab fragment and both orientations of the single-chain Fv fragment (VH-linker-VL and VL-linker-VH) of an antibody can be expressed in functional form in the periplasm of Escherichia coli, but the yield of these correctly assembled proteins is limited by the periplasmic folding process. While the periplasmic E. coli disulfide isomerase DsbA is required for this assembly, its functional over-expression does not significantly change the folding limit. Similarly, the functionally over-expressed E. coli proline cis-trans isomerase does not change the amount of all but one of the antibody fragments, not even if DsbA is over-expressed as well. Therefore, aggregation steps in the periplasm appear to compete with periplasmic folding, and they may occur before disulfide formation and/or proline cis-trans isomerization takes place and be independent of their extent.

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0073

Brecht, M., Kellermann, J. and Plückthun, A. (1993) Cloning and sequencing of glutamate mutase component E from Clostridium tetanomorphum. FEBS Lett. 319, 84-89.
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Abstract

The nucleotide sequence of the large subunit E of glutamate mutase of Clostridium tetanomorphum was determined. The protein consists of 483 amino acids and is not made in a precursor form, thus excluding the possibility of subunit E being a pyruvoyl enzyme. It shows no homology to any other protein in the database, and while binding coenzyme B12, a conspicuous B12 binding motif, shared amongst other proteins, is not detectable at the sequence level.

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0072

Wülfing, C. and Plückthun, A. (1993) A versatile and highly repressible Escherichia coli expression system based on invertible promoters: Expression of a gene encoding a toxic product.. Gene 136, 199-203.
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Abstract

A very flexible and tightly regulatable expression system has been constructed. It uses the principle of invertible promoters [Podhajska et al., Gene 40 (1985) 163-168]. Here, we describe the construction of a plasmid that provides the integrase, which causes promoter inversion in a tightly regulated fashion, as well as modified plasmids carrying the invertible module. The way the integrase is provided on a separate plasmid closely mimicks expression of the integrase from a lambda lysogen. Thus, the flexibility of the original system is considerably extended by making it strain-independent without compromising the tight regulation. We present the expression of a single-chain T-cell receptor fragment as an example of application, in order to illustrate the properties of this expression system.

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0071

Freund, C., Ross, A., Guth, B., Plückthun, A. and Holak, T. A. (1993) Characterization of the linker peptide of the single-chain Fv fragment of an antibody by NMR spectroscopy. FEBS Lett. 320, 97-100.
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Abstract

A comparison of the single-chain Fv fragment of the antibody McPC603 (scFv) with its corresponding unlinked Fv fragment has been carried out with 15N-edited NMR spectroscopy. The two Fv fragments adopt the same structure, indicating that the linker does not perturb the folding of the domains. This also directly demonstrates that folding in vivo (Fv fragment) and in vitro (scFv fragment) leads to the same structure. The main differences in the spectra of the uniformly 15N-labeled scFv and Fv fragments are due to signals of Gly and Ser from the linker peptide of the scFv fragment. The linker peptide has been mapped with NMR spectra of 15N-glycine- and 15N-glycine/15N-serine-labeled scFv fragments. The 15N T2 relaxation data indicate that the linker peptide is more flexible than the rest of the molecule.

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0070

Plückthun, A. (1993) Antibody Engineering to study protein-ligand interactions and catalysis: The phosphorylcholine binding antibodies in: Bioorganic Chemistry Frontiers (Dugas, H., and Schmidtchen, F. P., eds) Vol. 3 pp. 25-66. Springer Verlag, Berlin
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Abstract

Antibodies binding the small hapten phosphorylcholine are an ideal model system for investigating the structural logic of the selection of only a few genes in the immune response to this antigen, as well as the structural and energetic consequences of point mutations. Using bacterial expression technology, originally developed with this model system, a large number of mutations have been analyzed with Fv and Fab fragments of these antibodies. While the hapten is very small and only few amino acids are in direct contact, residues up to 20 Å away can have a dramatic effect on antigen binding. This is probably due to indirect conformational effects on the residues that do make direct contacts, giving these interactions improved geometries. These antibodies are able to catalyze the hydrolysis of choline carbonate esters, and the structure-function relationship of this reaction can be studied, because of the known crystal structure of one of the antibodies.

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0069

Plückthun, A., Pack, P., Knappik, A., Freund, C. and Holak, T. (1993) Monovalent single-chain antibodies and bivalent miniantibodies from E. coli: Structure and properties. Ann. Biol. Clin. 51, 553.
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Abstract

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0068

Plückthun, A. (1993) Catalytic antibodies in: Methods of Immunological Analysis (Masseyeff, R. F., Albert, W. H., and Staines, N. A., eds) Vol. 2 pp. 413-430. VCH Verlagsgesellschaft mbH, Weinheim
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Abstract

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1992

# Reference PDF

0067

Zahn, R. and Plückthun, A. (1992) GroE prevents the accumulation of early folding intermediates of pre-_-lactamase without changing the folding pathway. Biochemistry 31, 3249-3255.
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Abstract

In folding studies of pre-beta-lactamase in the presence of GroE, we investigated the pH dependence of the folding reaction. Two critical intermediates in the folding pathway were defined kinetically. I1 is an early folding intermediate recognized by GroE; the misfolding of I1 leads to aggregation, and this is prevented by GroE. A second intermediate I2 is released from GroE after ATP hydrolysis. Its pH-dependent misfolding to a nonnative form, which is not an aggregate, is not prevented by GroE. From these results, a model is proposed, in which the crucial role of GroE consists of allowing the change from I1 to I2 to take place in the complex. Fluorescence spectra of the pre-beta-lactamase complexed to GroE are very similar to those of the native state. The pathway of pre-beta-lactamase folding is not changed by GroE as evidenced by the same half-time and pH dependence of the folding reaction. GroE probably recognizes the signal sequence and some portion of the mature protein since mature beta-lactamase does not interact with GroE even under conditions of slow folding.

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0066

Pack, P. and Plückthun, A. (1992) Miniantibodies: Use of amphipathic helices to produce functional, flexibly linked dimeric Fv fragments with high avidity in Escherichia coli. Biochemistry 31, 1579-1584.
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Abstract

We have designed dimeric antibody fragments that assemble in Escherichia coli. They are based on single-chain FV fragments, with a flexible hinge region from mouse IgG3 and an amphiphilic helix fused to the C-terminus of the antibody fragment. The sequence of the helix was taken either from that of a previously reported four-helix bundle design or from a leucine zipper, optionally extended with a short cysteine-containing peptide. The bivalent fragments associate in vivo, either with covalent linkage or with a monomer-dimer equilibrium, and results from ultracentrifugation sedimentation studies and SDS-PAGE are consistent with dimers. All constructs are able to bind to surface-bound antigen under conditions in which only bivalent but not monovalent antibody fragments bind. The covalent bundle helix construct shows binding characteristics nearly identical to those of the much larger whole mouse antibody, resulting in substantially more stable immunoglobulin-antigen complexes than in the case of monovalent fragments. This modular design of natural and engineered protein domains directly leads to a boost of avidity, and it allows the construction of bispecific antibody fragments in functional form in E. coli.

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0065

Pack, P., Knappik, A., Krebber, C. and Plückthun, A. (1992) Mono- and bivalent antibody fragments produced in E.coli: Binding properties and folding in vivo in: Harnessing Biotechnology for the 21st Century (Ladisch, M. R., and Bose, A., eds) pp. 10-13. American Chemical Society, Washington, DC
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Abstract

We have designed dimeric antibody fragments of minimal size that assemble in E. coli and show an increase in avidity approaching a whole antibody. They are based on single-chain Fv fragments with a flexible hinge region from mouse IgG3 and an amphiphilic helix fused to the C-terminus of the antibody fragment. The sequence of the helix was taken either from that of a four-helix bundle design or a leucine zipper, optionally extended with a short cysteine containing peptide. To investigate the folding and assembly process of antibody fragments in E. coli, co-expression experiments with proline cis-trans-isomerase and disulfide isomerase were carried out. These folding steps do not appear to be limiting the folding process in E. coli.

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0064

Steipe, B., Plückthun, A. and Huber, R. (1992) Refined crystal structure of a recombinant immunoglobulin domain and a complementarity-determining region 1-grafted mutant. J. Mol. Biol. 225, 739-753.
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Abstract

We report the solution of the crystal structure of a mutant of the immunoglobulin VL domain of the antibody McPC603, in which the complementarity-determining region 1 segment is replaced with that of a different antibody. The wild-type and mutant crystal structures have been refined to a crystallographic R-factor of 14.9% at a nominal resolution of 1.97 A. A detailed description of the structures is given. Crystal packing results in a dimeric association of domains, in a fashion closely resembling that of an Fv fragment. The comparison of this VL domain with the same domain in the Fab fragment of McPC603 shows that the structure of an immunoglobulin VL domain is largely independent of its mode of association, even in places where the inter-subunit contacts are not conserved between VL and VH. In all three complementarity-determining regions we observe conformations that would not have been predicted by the canonical structure hypothesis. Significant differences between the VL domain dimer and the Fab fragment in the third complementarity-determining region show that knowledge of the structure of the dimerization partner and its exact mode of association may be needed to predict the precise conformation of antigen-binding loops.

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0063

Lindner, P., Guth, B., Wülfing, C., Krebber, C., Steipe, B., Müller, F. and Plückthun, A. (1992) Purification of native proteins from the cytoplasm and periplasm of Escherichia coli using IMAC and histidine tails: A Comparison of proteins and protocols. Methods 4, 41-56.
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Abstract

Several proteins were purified in a single step from Escherichia coli in native form using fused histidine tails and immobilized metal affinity chromatography (IMAC). Procedures are reported and compared: Antibody fragments (single-chain Fv fragments and VL domains) carrying a C-terminal (his)5 tail purified from the periplasm, the E. coli disulfide isomerase (DsbA) carrying a C-terminal (his)6 tail purified from the periplasm, and yeast citrate synthase carrying both a N-terminal and a C-terminal (his)5 tail purified from the cytoplasm. Because of blocks in the periplasmic folding process, the investigated murine single-chain T-cell receptor could only be purified in denatured form, despite being soluble, demonstrating the sensitivity of the native purification to the folding state. A comparison of columns and metal ions showed that Zn2+-imino-diacetic acid and Ni2+-nitrilo-triacetic acid gave equally good one-step purifications of VL domains, but only in this combination of metal and chelator. The buffer is of secondary importance, and the preferred method of elution depends on the stability of the protein. In a crystal structure determination of the VL domain carrying the (metal-free) histidine tail, no significant density beyond the first two histidine residues was detected, implying a disordered structure of the unliganded affinity tail. In using IMAC under denaturing conditions, an E. coli protease can be co-purified and refolded, which can digest sensitive proteins during their in vitro refolding.

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0062

Plückthun, A. (1992) Mono- and bivalent antibody fragments produced in Escherichia coli: Engineering, folding and antigen binding. Immunol. Rev. 130, 151-188.
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Abstract

No abstract

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0061

Glockshuber, R., Schmidt, T. and Plückthun, A. (1992) The disulfide bonds in antibody variable domains: Effects on stability, folding in vitro, and functional expression in Escherichia coli. Biochemistry 31, 1270-1279.
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Abstract

The formation of the disulfide bonds in the variable domains VH and VL of the antibody McPC603 was found to be essential for the stability of all antigen binding fragments investigated. Exposure of the Fv fragment to reducing conditions in vitro resulted in irreversible denaturation of both VH and VL. In vitro refolding of the reduced Fv fragment was only possible when the disulfide bonds were allowed to form under oxidizing conditions. The analysis of a series of mutants of the Fv fragment, the Fab fragment and the single-chain Fv fragment, all secreted into the periplasm of Escherichia coli, in which each of the cysteine residues of the variable domains was replaced by a series of other amino acids, showed that functional antigen binding fragments required the presence of both the disulfide bond in VH and the one in VL. These results were also used to devise an alternative expression system based on the production of insoluble fusion proteins consisting of truncated beta-galactosidase and antibody domains, enzymatic cleavage, and refolding and assembly in vitro. This strategy should be useful for providing access to unstable antibody domains and fragments.

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1991

# Reference PDF

0060

Plückthun, A. (1991) Chemie mit Genen und Computern?. Chemie Heute, Fonds der chemischen Industrie (Frankfurt) , 60-64.
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Abstract

Weitsichtig sah Emil Fischer anlässlich seiner Nobelpreisverleihung im Jahre 1902 die Zeit voraus, ""wo die Chemie nicht allein die natürlichen Enzyme in ausgedehntem Masse als Agenzien verwendet, sondern wo sie sich aus künstliche Fermente für ihre Zwecke bereitet"". Heute arbeiten zahlreiche Chemiker, Biochemiker und Biotechnologen daran, die Worte des Chemie-Genies in die Tat umzusetzen. Sie bemühen sich, künstliche Enzyme -- früher Fermente genannt -- zu schaffen, die als wirksame Katalysatorn in chemischen Reaktionen eigesetzt werden können. Manche gehen sogar schon einen Schritt weiter: Sie wollen Amtikörper - enzym-ähnliche ""Abwehrspezialisten"" des Immunsystems -- als Katalysatoren einsetzen. Ein spannender Forschungsbericht von Privatdozent Dr. Andreas Plückthun vom Max-Planck-Institut für Biochemie in Martinsried.

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0059

Köhler, J., Kikuno, R. and Plückthun, A. (1991) Non-covalent interactions in biologically relevant complexes. Importance of electrostatics and the atomic charge concept in: Molecules in natural science and medicine. An Encomium for Linus Pauling (Maksic, Z. B., and Eckert-Maksic, M., eds) pp. 441-465. Ellis Horwood, New York
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Abstract

No abstract

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0058

Plückthun, A. (1991) Antibody Engineering. Curr. Opin. Biotechnol. 2, 238-246.
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Abstract

Antibody engineering has received a boost from the development of an Escherichia coli expression system that now allows the screening of libraries with bacteria or phages. These random selection techniques can be applied using knowledge obtained from new X-ray structures of recombinant antibody domains, and anti-peptide antibodies. The first crystal structure of an anti-idiotype complex has also been solved. Additionally, the engineering of binding sites for metals and haptens, and the design of new immunotoxins have been reported.

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0057

Plückthun, A. (1991) Jahresrückblick Biochemie und Molekularbiologie; Proteinforschung. Nachr. Chem. Tech. Lab. 39, 178-182.
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Abstract

No abstract

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0056

Plückthun, A. and Ge, L. (1991) Wie rational ist Zufalls-Screening? – Effiziente Methoden der Selektion von Peptid- und Oligonucleotid-Liganden. Angew. Chem. 103, 301-303.
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Abstract

No abstract

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0055

Plückthun, A. (1991) Biotechnological aspects of antibody production in E. coli. Acta Biotechnol. 11, 449-456.
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Abstract

The production of genetically engineered antibodies in Escherichia coli is now possible. The resulting fragments are completely functional and have antigen binding constants indistinguishable from the natural antibody. This article summarizes the biochemical basis of this newly developed technology and the properties of the resulting fragments. It is likely that this technology will have an important role in antibody production for technical, medical and research uses. Screening of E. coli libraries may mount a challenge to traditional antibody production methods.

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0054

Plückthun, A. (1991) Antibody engineering: Advances from the use of Escherichia coli expression systems. Bio/Technology 9, 545-551.
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Abstract

Synthesis in Escherichia coli of correctly folded antibody fragments that bind antigen with the same affinity as the whole antibody is now possible. Here I review the techniques for achieving this and the physical properties of the various fragments produced. This technology not only facilitates antibody engineering but is also the basis of screening libraries for binding activity. Although the immunization of animals has not been made unnecessary in the production of monoclonal antibodies, steps toward this goal are now feasible. [References: 68]

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0053

Plückthun, A. (1991) Strategies for the expression of antibody fragments in Escherichia coli. Methods 2, 88-96.
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Abstract

This article summarizes the techniques for the expression of various antibody fragments in Escherichia coli. The properties of various antigen binding fragments are discussed and several strategies for expression are compared. Emphasis is placed on the secretory approach, as it leads directly to functional fragments and thus forms the basis for all screening approaches, be it with cells or phages. Purification procedures of the fragments from E. coli are also discussed.

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0052

Plückthun, A. and Pfitzinger, I. (1991) Comparison of the Fv fragments of different phosphorylcholine binding antibodies expressed in Escherichia coli. Ann. N. Y. Acad. Sci. 646, 115-124.
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Abstract

The development of general methods to express functional antibody fragments in E. coli greatly facilitates the engineering of antibodies. Some of the essential features of the technology are summarized. As a model system, phosphorylcholine binding antibodies are used. The immune response against this antigen results in three classes of antibodies, exemplified by the myeloma proteins McPC603, TEPC15, and MOPC167. Fv fragments of these antibodies can now be conveniently prepared in E. coli to aid in understanding the structural logic of this well-characterized immune response.

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0051

Skerra, A., Pfitzinger, I. and Plückthun, A. (1991) The functional expression of antibody Fv fragments in Escherichia coli: Improved vectors and a generally applicable purification technique. Bio/Technology 9, 273-278.
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Abstract

We have previously demonstrated that the expression of fully functional Fv and Fab fragments in E. coli is possible by the simultaneous secretion of both chains to the periplasm. To increase production levels and facilitate engineering and random mutagenesis, we improved our previous vectors by introducing a resident repressor gene and a filamentous phage origin. We also developed a new purification strategy based on immobilized metal ion chromatography, with which a single-chain Fv fragment can be purified to homogeneity in a single step. We investigated the most efficient tail constructions and found that only a minimal structural change of three additional C-terminal amino acids is necessary. This modification has no deleterious effect on in vivo transport and folding or antigen affinity.

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0050

Skerra, A. and Plückthun, A. (1991) Secretion and in vivo folding of the Fab fragment of the antibody McPC603 in Escherichia coli: Influence of disulphides and cis-prolines.. Protein Eng. 4, 971-979.
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Abstract

Using the well-characterized antibody McPC603 as a model, we had found that the Fv fragment can be isolated from Escherichia coli as a functional protein in good yields, whereas the amount of the correctly folded Fab fragment of the same antibody produced under identical conditions is significantly lower. In this paper, we analyse the reasons for this difference. We found that a variety of signal sequences function in the secretion of the isolated chains of the Fab fragment or in the co-secretion of both chains in E.coli. The low yield of functional Fab fragment is not caused by inefficient expression or secretion in E.coli, but by inefficient folding and/or assembly in the periplasm. We compared the folding yields for the Fv and the Fab fragment in the periplasm under various conditions. Several diagnostic framework variants were constructed and their folding yields measured. The results show that substitutions affecting cis-proline residues and those affecting various disulphide bonds in the protein are by themselves insufficient to dramatically change the partitioning of the folding pathway to the native structure, and the cause must lie in a facile aggregation of folding intermediates common to all structural variants. However, all structural variants could be obtained in native form, demonstrating the general utility of the secretory expression strategy.

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0049

Glockshuber, R., Stadlmüller, J. and Plückthun, A. (1991) Mapping and modification of an antibody hapten binding site: A site-directed mutagenesis study of McPC603.. Biochemistry 30, 3049-3054.
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Abstract

The quantitative contributions of various amino acid residues to hapten binding in the Fv fragment of the antibody McPC603 were investigated by site-directed mutagenesis. The three-dimensional structure of the Fab' fragment of McPC603 is known to atomic resolution. The haptens phosphocholine, choline sulfate, 3-(trimethylammonium)propane-1-sulfonate, 4-(trimethylammonium)butyric acid, and 4-(trimethyl-ammonium)butyric acid methyl ester were tested for binding. It was found that the phosphate group but not the sulfate and sulfonate groups, interacts with the hydroxyl group of Tyr33(h). The required positive charge for the binding of the phosphate must be contributed by Arg52(h); a lysine at this position or an additional positive charge at position 33(h) abolishes the binding to a phosphocholine affinity column. The interaction between Tyr100(l) and Glu35(h) was found to be essential and could not be functionally replaced by any other pair of residues tested. Binding of the quaternary ammonium ion needs a negative charge; it can reside in either Asp97(l) or Asp101(h), but both together prevent binding to the affinity column. These data may serve as the basis for the development of quantitative treatments of antigen-antibody interactions.

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0048

Plückthun, A. and Stadlmüller, J. (1991) Catalytic antibodies: Contributions from engineering and expression in Escherichia coli. Ciba Foundation Symposium 159, 103-112; discussion 112-117.
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Abstract

Antibodies have been raised against the transition state of many reactions and shown to catalyse the relevant reaction. Their moderate catalytic efficiencies can be increased by protein engineering, if ways can be found to express the engineered antibody. We have developed a system by which fully functional Fv and Fab fragments can be expressed in Escherichia coli. The Fv fragment dissociates at low concentrations; we therefore devised methods to stabilize the fragment. We showed that the Fv fragment of the antibody McPC603, a phosphorylcholine-binding immunoglobulin A, binds the antigen with the same affinity as does the intact antibody isolated from mouse ascites. Phosphorylcholine is an analogue of the transition state for the hydrolysis of choline carboxylate ester. The Fv fragment of McPC603 catalysed this hydrolysis. Mutational analysis of the residues in the binding site of the antibody has shown which are essential for binding and for catalysis, and the importance of charged residues in certain positions. The E. coli expression system combined with protein engineering and screening methods will facilitate understanding of enzyme catalysis and the development of new catalytic antibodies. [References: 51]

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0047

Laminet, A. A., Kumamoto, C. A. and Plückthun, A. (1991) Folding in vitro and transport in vivo of pre-b-lactamase are SecB independent. Mol. Microbiol. 5, 117-122.
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Abstract

The rate of folding of the precursor of beta-lactamase is not influenced by the presence of SecB under conditions in which GroEL/ES retards the folding. Wild-type beta-lactamase and several mutants in the signal or the mature protein, affecting either transport or enzyme kinetics and probably folding, were examined for total expression, total enzymatic activity, and transported beta-lactamase (in vivo resistance) in secB- and secB+ strains. We conclude that there is no indication of any relevant interaction between SecB and pre-beta-lactamase in vitro, nor did the secB- mutation affect the transport of wild-type beta-lactamase or any of the mutant in vivo. Thus, putative Escherichia coli ""folding modulators' must be of limited specificity.

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1990

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0046

Plückthun, A. and Köhler, J. (1990) Protein-Struktur und Funktion: Die Möglichkeiten und Probleme der Computersimulation aus der Sicht des Experimentators in: Forum ’90 Wissenschaft und Technik. Informatik Fachberichte (Friemel, H.-J., Müller-Schönberger, G., and Schütt, A., eds) Vol. 259 pp. 258-274. Springer-Verlag, Trier
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Abstract

Die drei-dimensionale Struktur eines Proteins ist die Grundlage seiner spezifischen Funktion. Obwohl die Gentechnologie heute erlaubt, Proteine beliebiger Aminosäure-Sequenz herzustellen, ist die Vorhersage von Struktur und Eigenschaften eines Proteins heute noch nicht möglich, und das enorme Potential der neuen experimentellen Methoden kann deshalb noch nicht voll technisch ausgenutzt werden. Es ist zunächst notwendig, Methoden zu entwickeln, die in verläßlicher Weise die sehr geringen Beträge von Wechselwirkungsenergien vohersagen, die bei biologischen Prozessen wie Proteinfaltung und Protein-Liganden- echselwirkungen die entscheidende Rolle spielen. Dafür sind, um auch nur zu einer qualitativ korrekten Aussage über freie Energien zu kommen, sehr genaue Berechnungen nötig. Viele solcher Prozesse verlaufen nicht nach rein enthalpischen Prinzipien, sondern der Entropieterm kann die entscheidende Rolle spielen, und die Entropie ist nur über die statistische Thermodynamik in aufwendigen Simulationen zugänglich. Der Artikel diskutiert Fortschritte der Molekulardynamik mit empirischen Kraftfeldern und Fallbeispiele an einem Antikörper-Antigen-Komplex, der mit dieser Methode und verschiedenen experimentellen Ansätzen analysiert wurde.

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0045

Plückthun, A. (1990) Engineering and heterologous expression of antibodies in Escherichia coli in: 6th International Symposium on genetics of industrial microorganisms (GIM 90) (Heslot, H., Davies, J., Florent, J., Bobichon, L., Durand, G., and Penasse, L.) pp. 17-23. Société Française de Microbiologie
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Abstract

No abstract

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0044

Plückthun, A. (1990) Antigen-bindende Fragmente eines Antikörpers, produziert in Escherichia coli in: 40 Jahre Fonds der Chemischen Industrie. Wissenschaftliche Beiträge von Dozenten-Stipendiaten pp. 187-196. Fonds der Chemischen Industrie
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Abstract

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0043

Plückthun, A. (1990) Towards new enzymes: Protein engineering and catalytic antibodies. Kontakte (Darmstadt) 1990, 40-59.
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Abstract

The article first discusses the principle factors contributing to rate accelerations in enzymes. Then, the chances and the problems associated with four strategies to new enzymatic activities are scrutinized: the screening of microorganisms, random mutagenesis of a cloned enzyme, protein engeneering and the generation of catalytic antibody. Each of these topics are illustrated by several examples from the literature (80 refs.).

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0042

Plückthun, A. (1990) Antibodies from Escherichia coli. Nature 347, 497-498.
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Abstract

Use of Escherichia coli as an expression host has opened up new possibilities in antibody research and its applications. It greatly facilitates rational engineering and random mutagenesis.

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0041

Plückthun, A. (1990) Recombinant antibodies produced in E.coli: Prospects for catalysis. Fresenius J. Anal. Chem. 337, 13.
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Abstract

No abstract

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0040

Plückthun, A., Glockshuber, R., Skerra, A. and Stadlmüller, J. (1990) Engineering of Fv and Fab fragments of the antibody McPC603 expressed in E. coli. ICSU Short Rep. 10, 94-95.
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Abstract

No abstract

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0039

Plückthun, A. (1990) Jahresrückblick Physikal.Chemie/Biochemie und Molekularbiologie. Nachrichten aus Chemie Technik und Laboratorium 38, 216-222.
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Abstract

No abstract

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0038

Ganter, C. and Plückthun, A. (1990) Glycine to alanine substitutions in helices of glyceraldehyde-3-phosphate dehydrogenase: Effects on stability. Biochemistry 29, 9395-9402.
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Abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from chicken was expressed in and purified from Escherichia coli. To investigate the physical basis of possible protein stabilization strategies, the effect of substitutions of glycine residues by alanine in helical regions was determined. One Gly to Ala substitution (G316A) located in the central core of the subunit was found to strongly stabilize the protein, while the other mutations are neutral or destabilize the protein. The effect seen for the stabilizing mutant in irreversible heat denaturation correlates with the first transition in folding equilibrium experiments that is observable by fluorescence, but not with the one detected by circular dichroism measurements or in dilution-induced dissociation experiments. The stabilizing effect of a Gly to Ala substitution therefore does not seem to be caused by an entropic effect on the unfolded state. Rather, an internal cavity is filled by the substitution G316A, probably stabilizing the native state. In large oligomeric proteins, imperfect packing may be a frequent cause of limited stability.

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0037

Skerra, A., Glockshuber, R. and Plückthun, A. (1990) Structural features of the McPC603 Fab fragment not defined in the X-ray structure. FEBS Lett. 271, 203-206.
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Abstract

The proteolytic Fab fragment of the well characterized antibody McPC603 was compared to the recombinant Fab fragment, which was obtained in functional form from an Escherichia coli expression system [(1989) Methods Enzymol. 178, 497-515]. We found evidence that the proteolytic fragment is glycosylated at Asn H160 in the CH1 domain, where additional electron density had been observed in the crystal structure [J. Mol. Biol. 190, 593-604]. In addition, its heavy chain is about 30 amino acids longer than visible in the electron density and thus contains the complete hinge region. These structural differences between the recombinant Fab fragment, which had been designed exactly according to the defined electron density, and the proteolytic Fab fragment of McPC603 had no effect on the hapten binding properties of these antigen binding fragments. Yet, it may be important to be aware of these structural features of McPC603 in folding studies and some comparative analyses of antibody structures.

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0036

Glockshuber, R., Steipe, B., Huber, R. and Plückthun, A. (1990) Crystallization and preliminary X-ray studies of the VL domain of the antibody McPC603 produced in Escherichia coli. J. Mol. Biol. 213, 613-615.
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Abstract

The VL domain, obtained from a recombinant Fv fragment of the antibody McPC603 expressed in Escherichia coli, has been crystallized as a dimer from 2 M-(NH4)2SO4 (pH 4.0). The crystals are hexagonal, space group P6(1)22. The cell dimensions are a = b = 86.48 A, c = 76.64 A, with a VL monomer as the asymmetric unit. The crystals diffract to 2.0 A. The structure was solved by Patterson search using the VL domain of the Fab fragment of McPC603 and the VL dimer REI.

DOI: 10.1016/S0022-2836(05)80247-5 | Read all as PDF | Close Abstract
0035

Laminet, A. A., Ziegelhoffer, T., Georgopoulos, C. and Plückthun, A. (1990) The Escherichia coli heat shock proteins GroEL and GroES modulate the folding of the _-lactamase precursor. EMBO J. 9, 2315-2319.
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Abstract

One of the fundamental problems in biochemistry is the role of accessory proteins in the process of protein folding. The Escherichia coli heat shock protein complex GroEL/ES has been suggested to be a 'chaperonin' and be involved in both oligomer assembly as well as protein transport through the membrane. We show here that the folding of the purified precursor of beta-lactamase is inhibited by purified GroEL or the GroEL/ES complex with a stoichiometry of one particle per molecule of pre-beta-lactamase. Purified GroES alone has no effect on folding. After Mg2+ ATP addition folding resumes and the yield of active enzyme is higher than in the absence of GroEL or GroEL/ES. Unexpectedly, GroEL or GroEL/ES, when added to folded pre-beta-lactamase, lead to an apparent net 'unfolding', probably to a collapsed state of the protein, which can be reversed by the addition of Mg2+ ATP. The reversible and Mg2+ ATP-dependent association of GroEL/ES with non-native proteins might explain its postulated role in both protein transport and oligomer assembly.

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0034

Glockshuber, R., Malia, M., Pfitzinger, I. and Plückthun, A. (1990) A comparison of strategies to stabilize immunoglobulin Fv-fragments. Biochemistry 29, 1362-1367.
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Abstract

Fv-Fragments of antibodies may dissociate at low protein concentrations and are too unstable for many applications at physiological temperatures. To stabilize Fv-fragments against dissociation, we have tested and compared three different strategies on the Fv-fragment of the well-characterized phosphocholine binding antibody McPC603 expressed and secreted in Escherichia coli: chemical cross-linking of the variable domains, introduction of an intermolecular disulfide bond, and construction of a peptide linker to produce a ""single-chain"" Fv-fragment. All the linked fragments show hapten affinities nearly identical with that of the whole antibody independent of protein concentration and are significantly (up to 60-fold) stabilized against irreversible thermal denaturation. All genetically engineered linked Fv-fragments can be obtained in native conformation in E. coli. The reported strategies for generating Fv-fragments with improved physicochemical properties may extend their usefulness in biotechnology as well as in therapeutic and diagnostic applications.

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0033

Plückthun, A., Glockshuber, R., Skerra, A. and Stadlmüller, J. (1990) Properties of Fv and Fab fragments of the antibody McPC603 expressed in E. coli. Behring Institute Mitt. 87, 48-55.
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Abstract

The FV and Fab fragments of the phosphorylcholine binding antibody McPC603 were functionally expressed in E. coli. This was achieved by the co-expression and co-secretion of both chains to the periplasm, where correct processing, folding and assembly occurred. Interestingly, the fraction of correctly folded Fab fragment is smaller than that of the Fv fragment in E. coli. The intrinsic hapten binding affinity was shown to be identical for the recombinant FV or Fab fragment, the whole antibody and the Fab fragment obtained by proteolysis from the mouse antibody. Fluorescence and crosslinking analyses showed that the FV fragment dissociates at high dilution, but that it is stabilized by hapten binding. The recombinant FV fragment was shown to have catalytic activity to hydrolyze choline-p-nitrophenyl carbonate and constitutes therefore a promising model system with which the structural requirements of catalytic antibodies can be studied by altering the protein itself.

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0032

Plückthun, A. (1990) Wege zu neuen Enzymen: Protein Engineering und Katalytische Antikörper. Chemie in unserer Zeit 24, 182-198.
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Abstract

Bevor die zahlreichen und noch lange nicht alle überwundenen Probleme auf den Wegen zu neuen Enzymen diskutiert werden, erläutert der Autor, wie ein Enzym funktioniert, genauer: welche Faktoren -- denn es sind viele -- dafür verantwortlich sind, dass eine enzymatische Reaktion im Extremfall um bis zu 20 Zehnerpotenzen schneller abläuft als eine unkatalysierte Reaktion.

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0030

Holland, I. B., Kenny, B., Steipe, B. and Plückthun, A. (1990) Secretion of heterologous proteins in Escherichia coli. Methods Enzymol. 182, 132-143.
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Abstract

No abstract

DOI: 10.1016/0076-6879(90)82013-R | Read all as PDF | Close Abstract

1989

# Reference PDF

0031

Hörz, W., Kröger, M., Kuschmitz, D. and Plückthun, A. (1989) Recent developments in molecular biology – an overview in: Biotechnology: A comprehensive treatise (Rehm, H.-J., and Reed, G., eds) Vol. 7b pp. 530-552. VCH Verlagsgesellschaft, Weinheim
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Abstract

No abstract

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0029

Plückthun, A., Stadlmüller, J., Skerra, A., Glockshuber, R. and Steipe, B. (1989) Bacterial expression and characterization of antigen binding fragments of the antibody McPC603. J. Cell. Biochem. 13A, 92.
| Abstract + further info

Abstract

The phosphorylcholine binding antibody McPC603 is particularly well studied and the crystal structure of its Fab fragment with hapten bound is known. Thus, the Fv and Fab fragment of this antibody are convenient model systems for quantitatively investigating binding interactions and transition state stabilization (i.e. catalysis) by systematic modification of the antigen binding site and the hapten. We have developed an espression system with which fully functional Fv or Fab fragments can be expressed in E. coli. Both chains are co-expressed and co-secreted into the periplasm of E. coli, with correct signal-processing, disulfide formation, and chain association. The Fv and Fab fragment can be purified to homogeneity in a single step by hapten affinity chromatography. The binding constant of the hapten to the Fv fragment was found to be identical to that of the whole antibody. Also, the variable domains were expressed as fusion proteins with b-galactosidase, precisely cleaved with the protease factor Xa, and refolded in vitro to give a functional Fv fragment. The association constant between the VH and VL domains was determined by crosslinking and fluorescence experiments. Furthermore, we could show that VL dimerizes with itself with an association constant similar to that of the heterodimer, but VH does not. The binding of the hapten stabilizes the Fv fragment considerably. We also showed that the recombinant Fv fragment of McPC603 possesses catalytic activity toward the hydrolysis of suitable carboxylic acid derivatives. This constitutes the first catalytic antibody for which a three-dimensional structure and a convenient expression and mutagenesis system is available.

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0028

Ellinger, S., Glockshuber, R., Jahn, G. and Plückthun, A. (1989) Cleavage of procaryotically expressed human immunodeficiency virus fusion proteins by factor Xa and application in western blot (immunoblot) assays.. J. Clin. Microbiol. 27, 971-976.
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Abstract

The proteins p15 and p24 of the human immunodeficiency virus (HIV) type 1 gag gene were expressed as fusion proteins in Escherichia coli for detecting antibodies against the acquired immunodeficiency virus by Western blot (immunoblot) analysis. These fusion proteins contain amino acids 1 to 375 of the E. coli beta-galactosidase linked to the viral protein(s) by a recognition sequence for the specific protease factor Xa. They are obtained in large amounts in insoluble inclusion bodies. To avoid ambiguous results caused by cross-reaction of sera with bacterial proteins in Western blots, we purified the recombinant fusion proteins and subsequently removed the bacterial part of the fusions by cleavage with factor Xa. The cleavage mixtures were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. The viral proteins obtained by this method did not contain any bacterial proteins or protein fragments. Thus, false-positive results in HIV Western blot analysis with bacterially expressed HIV proteins can be excluded with these purified recombinant viral antigens.

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0027

Laminet, A. A. and Plückthun, A. (1989) The precursor of b-lactamase: Purification, properties and folding kinetics. EMBO J. 8, 1469-1477.
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Abstract

The precursor of Escherichia coli RTEM beta-lactamase was purified to homogeneity on a milligram scale by a procedure independent of the binding properties of the protein and refolded to an active, reduced form. For comparing the folding kinetics, the wild-type enzyme was reduced and a mutant was constructed, in which the two cysteines that form a very stable disulfide bond in the RTEM enzyme were both changed into alanines. The rate of folding was determined by directly measuring the increase in enzymatic activity. The reduced precursor folds at least 15 times more slowly than either the reduced mature enzyme or the mature Cys----Ala double mutant under identical conditions. The wild-type enzyme, the Cys----Ala double mutant and the precursor protein all had similar KM values, demonstrating a very similar native state. The slow folding of the precursor compared with the mature form may be an essential and general feature to secure a transport competent conformation necessary for the translocation through a membrane in protein export. This folding assay of a precursor by directly following its enzymatic activity may facilitate the characterization of putative folding modulators in bacterial membrane transport.

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0026

Plückthun, A. and Skerra, A. (1989) Expression of functional antibody Fv and Fab fragments in Escherichia coli. Methods Enzymol. 178, 497-515.
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Abstract

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1988

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0025

Plückthun, A., Skerra, A., Glockshuber, R. and Stadlmüller, J. (1988) Synthetic antibodies with a known three-dimensional structure in: Protein structure and protein engineering. 39th Colloquium Mosbach (Winnacker, E.-L., and Huber, R., eds) pp. 123-131. Springer Verlag, Berlin, Heidelberg
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Abstract

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0024

Plückthun, A., Skerra, A., Glockshuber, R., Stadlmüller, J. and Pfitzinger, I. (1988) Synthetic antibodies with known three-dimensional structure. Biological Chemistry Hoppe-Seyler 369, 206-207.
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Abstract

The particularly well studied antibody combining site of McPc603 is used by us as a model system for quantitatively investigating factors that contribute to efficient hapten binding, subunit interactions, as well as for the potential of stabilizing a transition state (i.e. chemical catalysis) through the controlled modification of the protein.

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0023

Skerra, A. and Plückthun, A. (1988) Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. Science 240, 1038-1041.
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Abstract

An expression system was developed that allows the production of a completely functional antigen-binding fragment of an antibody in Escherichia coli. The variable domains of the phosphorylcholine-binding antibody McPC603 were secreted together into the periplasmic space, where protein folding as well as heterodimer association occurred correctly. Thus, the assembly pathway for the Fv fragment in E. coli is similar to that of a whole antibody in the eukaryotic cell. The Fv fragment of McPC603 was purified to homogeneity with an antigen-affinity column in a single step. The correct processing of both signal sequences was confirmed by amino-terminal protein sequencing. The functionality of the recombinant Fv fragment was demonstrated by equilibrium dialysis. These experiments showed that the affinity constant of the Fv fragment is identical to that of the native antibody McPC603, that there is one binding site for phosphorylcholine in the Fv fragment, and that there is no inactive protein in the preparation. This expression system should facilitate future protein engineering experiments on antibodies.

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0022

Plückthun, A. and Pfitzinger, I. (1988) Membrane-bound b-lactamase forms in Escherichia coli. J. Biol. Chem. 263, 14315-14322.
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Abstract

Frameshift pseudo-revertants of Escherichia coli RTEM beta-lactamase were obtained by a selection procedure, starting from frameshift mutants at the signal-processing site. These pseudo-revertant proteins, which have a totally altered COOH-terminal part of the signal sequence, are attached to the outer face of the inner membrane. The mutant proteins are enzymatically active in vitro and in vivo, and the membrane localization has no deleterious effect on cell growth. We conclude that initiation of transport across the membrane does not require the COOH-terminal part of the signal, but this part of the sequence determines whether the protein is released to the periplasm either with or without cleavage of the signal, or whether the protein remains anchored to the membrane. Mutants with two signals in series were used to show that a truncated signal is not refractory to transport per se. If neither signal contains a functional cleavage site, the protein is at least partially found on the outer face of the inner membrane. If both signals contain functional cleavage sites, both are removed and the protein is released to the periplasm. If only the first signal contains a cleavage site, a longer fusion protein is transported and released. The results presented here show that a pre-beta-lactamase-like protein can fold properly even as a membrane-bound species.

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1987

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0021

Plückthun, A., Glockshuber, R., Stadlmüller, J. and Skerra, A. (1987) Synthetic antibodies with known 3-D structure. J. Cell. Biochem. 11C, 236.
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Abstract

The genes encoding the variable domains (VH and VL) of the phosphorylcholine binding antibody McPc603 were obtained by DNA synthesis. In addition, we constructed genes encoding the variable and the appropriate constant domains of each chain in order to directly express the exact Fab fragment whose crystal structure is known. The design of the synthetic genes took into consideration the facile replacement of gene fragments (e.g. the hypervariable loops) as well as current knowledge about efficient expression. We have investigated purifications of the cloned gene products from bacterial expression systems and are comparing their efficiency in obtaining large amounts of protein. The essence of antibody architecture is a framework of fairly constant residues and hypervariable loops (complementary determining region, CDR) that contain the antigen recognition sequences to a great variety of antigens. The particularly well studied antibody combining site of McPc603 is used by us as a model system for quantitatively investigating factors that contribute to efficient hapten bindiing, subunit interactions, as well as for the potential of stabilizing a transition state through the controled modification of the protein.

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0020

Plückthun, A., Glockshuber, R., Pfitzinger, I., Skerra, A. and Stadlmüller, J. (1987) Engineering of antibodies with a known three-dimensional structure. Cold Spring Harbor Symp. Quant. Biol. 52, 105-112.
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Abstract

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0019

Plückthun, A. and Knowles, J. R. (1987) The consequences of stepwise deletions from the signal-processing site of _-lactamase. J. Biol. Chem. 262, 3951-3957.
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Abstract

Amino acids have been deleted from the processing site of pre-beta-lactamase, either into the signal sequence or into the mature protein. Whereas the loss of more than 2 amino acid residues from the C-terminal end of the signal sequence prevents the translocation of the protein into the periplasm, the removal of two or more amino acids from the beginning of the mature protein has no effect on the translocation of the truncated protein. The insertion of an additional one to three amino acids at the processing site has no detectable phenotypic consequence either. It appears that many sequences for the first few residues of the mature protein allow successful translocation and processing. In sharp contrast, the removal of one (but not both) of the amino acids that flank the processing site results in a severe growth defect in the host cell and very low expression of the protein. Yet removal of two amino acids from either side of the processing site, or removal of both the flanking residues of the processing site, results in normal secretion and signal cleavage. These results illustrate the limits on the amino acid sequence around the processing junction and suggest that interference with the signal cleavage step can lead not only to aborted secretion but also to pleiotropic consequences for the growth of the host organism.

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1986

# Reference PDF

0018

Glockshuber, R., Mertz, R., Stadlmüller, J. and Plückthun, A. (1986) Engineering of antibody variable domains with known structure. Biological Chemistry Hoppe-Seyler 367 Suppl., 164.
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Abstract

The genes encoding the variable domains (VH and VL) of the phosphorylcholine binding antibody McPc603, whose crystal structure with and without antigene bound is known, were obtained by DNA synthesis. This antibody combining site is used by us as a model system to elucidate factors contributing to efficient hapten binding as well as the potential for stabilizing a transition state. The synthetic genes were designed for facile complete or partial replacement of hypervariable loops as well as for incorporating current knowledge about efficient expression. Bacterial expression systems are described and their effectiveness in obtaining large amounts of correctly folded protein is compared.

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0017

Plückthun, A., DeBony, J., Fanni, T. and Dennis, E. A. (1986) Conformation of fatty acyl chains in _- and _-phosphatidylcholine and phosphatidylethanolamine derivatives in sonicated vesicles. Biochim. Biophys. Acta 856, 144-154.
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Abstract

Mono- and dimethylated derivatives constitute important intermediates in the conversion of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) in eucaryote membranes. 1H-NMR techniques were utilized to examine the conformation of the region of the fatty acyl chains that is close to the polar group in the series of alpha-phospholipids: PE, N-methyl-PE, N,N-dimethyl-PE, and PC. The same series of polar groups, but on phospholipid containing sn-1 and/or sn-3 fatty acyl chains (beta-phospholipids) were also examined. All of the phospholipids were in the form of small sonicated vesicles which are widely utilized as membrane models. The alpha-methylene group of the sn-1 and sn-2 fatty acyl chains of the alpha-phospholipids give rise to separate signals due to the non-equivalency of these chains with respect to the glycerol phosphate backbone on all alpha-phospholipids tested. Additionally, differences in the environment of the PC molecules as well as N-methyl-PE, and N,N-dimethyl-PE, but not PE itself on the inside and outside of the vesicles are reflected in the chemical shift of the alpha-methylene protons. On the other hand, all of the beta-phospholipids (including beta-PE) were found to reflect the inside/outside packing differences in their alpha-methylene groups. The bilayer packing does not induce any nonequivalence in the chemically equivalent acyl chains. In mixed micelles with detergents, beta-phospholipids showed one alpha-CH2 signal for all phospholipids. These results are consistent with a common conformational arrangement for the fatty acyl chains in all alpha-phospholipids that have been investigated no matter what aggregated form. The conformational arrangement in the beta-phospholipids is different, but again is similar for all of the compounds tested in various aggregated forms.

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0016

Dennis, E. A. and Plückthun, A. (1986) Mechanism of interaction of phospholipase A2 with phospholipid substrates and activators in: Enzymes of lipid metabolism II (Freysz, L., Dreyfus, H., Massarelli, R., and Gatt, S., eds) pp. 121-132. Plenum Press, New York
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Abstract

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0015

Lombardo, D., Fanni, T., Plückthun, A. and Dennis, E. A. (1986) Rate-determining step in phospholipase A2 mechanism. 18O isotope exchange determined by 13C NMR. J. Biol. Chem. 261, 11663-11666.
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Abstract

H2(18)O isotope exchange into specifically 13C-labeled substrate was used to obtain information on the rate-limiting step in the action of the phospholipase A2 from the venom of the Indian cobra (Naja naja naja). Incorporation of 18O was detected by the effect of 18O on 13C chemical shifts in 13C NMR. The enzymatic hydrolysis of a micellar phosphatidylcholine analogue of platelet-activating factor 1-alkyl-2-[1-13C]lauroyl-sn-glycero-3-phosphorylcholine proceeds by an O-acyl cleavage of the sn-2 ester bond. The reaction was examined for simultaneous 18O incorporation into the substrate. No exchange was found, suggesting that the hydrolytic step is not followed by a higher energy transition state and that it or a step before it appears to be rate-limiting. Previous experiments on phosphatidylethanolamine activation indicate that kcat is altered but that the km remains the same upon activation, suggesting that the binding steps occurring before the hydrolytic step are not affected. This strongly suggests that the hydrolytic step is in fact the rate-limiting step under these conditions. The 13C, 18O NMR technique should be generally applicable to mechanistic questions of this type.

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0014

Fanni, T., Lombardo, D., Plückthun, A. and Dennis, E. A. (1986) Rate determining step in phospholipase A2 mechanism: 18O Isotope exchange determined by 13C NMR. Fed. Proc. 45, 1557.
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Abstract

H218O isotope exchange into specifically 13C-labeled substrate was used to obtain information on the rate-limiting step in the action of the phospholipase A2 from the venom of the Indian cobra (Naja naja naja). Incorporation of 18O was detected by the effect of 18O on 13C chemical shifts in 13C NMR. The enzymatic hydrolysis of a micellar phosphatidylcholine analogue of platelet-activating factor 1-alkyl-2-[1-13C]lauroyl-sn-glycero-3-phosphorylcholine proceeds by an O-acyl cleavage of the sn-2 ester bond. The reaction was examined for simultaneous 18O incorporation into the substrate. No exchange was found, suggesting that the hydrolytic step is not followed by a higher energy transition state and that it or a step before it appears to be rate-limiting. Previous experiments on phosphatidylethanolamine activation indicate that kcat is altered but that the km remains the same upon activation, suggesting that the binding steps occurring before the hydrolytic step are not affected. This strongly suggests that the hydrolytic step is in fact the rate-limiting step under these conditions. The 13C, 18O NMR technique should be generally applicable to mechanistic questions of this type.

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1985

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0013

Plückthun, A. and Dennis, E. A. (1985) Activation, aggregation, and product inhibition of cobra venom phospholipase A2 and comparison with other phospholipases. J. Biol. Chem. 260, 11099-11106.
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Abstract

The kinetics of phospholipid hydrolysis by cobra venom phospholipase A2 were examined and compared to those of phospholipase A2 from porcine pancreas, Crotalus adamanteus (rattlesnake) venom, and bee venom. Only the enzyme from Naja naja naja (cobra) venom was found to be activated significantly by phosphorylcholine-containing compounds when hydrolyzing phosphatidylethanolamine. The cobra venom enzyme was also the only one in which these activators induced protein aggregation. The parallel specificity for activators and aggregators suggests that these two phenomena are linked. Product effects were also shown to vary between these four phospholipases. These effects manifest themselves in nonlinear time courses, in changes in steady state velocity, and in the differential effects of serum albumin on reaction rates. Different effects were even seen for the same enzyme when acting on different substrates. A model is presented to account for these observations; its main features are enzyme activation by an activator molecule, whose specificity depends on the enzyme, and an activator-induced aggregation of the enzyme.

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0012

Kadonaga, J. T., Plückthun, A. and Knowles, J. R. (1985) Signal sequence mutants of beta-lactamase. J. Biol. Chem. 260, 16192-16199.
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Abstract

The function of the NH2-terminal signal peptide in the translocation of beta-lactamase across the inner membrane of Escherichia coli has been studied by characterization of 15 signal sequence mutants. Three amino acid substitutions (Pro 20 to Ser, Pro 20 to Phe, and Cys 18 to Tyr) in the 23-amino acid signal sequence each cause, to varying degrees, a defect in the proteolytic processing of pre-beta-lactamase, abnormal growth of the host strain, and a severe reduction in the expression of beta-lactamase in vivo but not in vitro. The results are consistent with a model for protein secretion in E. coli that parallels the pathway proposed for translocation across the endoplasmic reticulum in eucaryotic cells.

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0011

Plückthun, A., Rohlfs, R., Davidson, F. F. and Dennis, E. A. (1985) Short-chain phosphatidylethanolamines: Physical properties and susceptibility of the monomers to phospholipase A2 action.. Biochemistry 24, 4201-4208.
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Abstract

The homologous series of optically active short-chain phosphatidylethanolamines (PE) from dibutyryl-PE to dioctanoyl-PE was synthesized. In addition, two monomeric short-chain phospholipid analogues that are not degraded by phospholipase A2 (1,2-bis[(butylcarbamyl)oxy]-sn-glycero-3-phosphocholine and the corresponding ethanolamine derivative) were synthesized. In contrast to the short-chain phosphatidylcholines (PC), short-chain PE's have defined solubilities in water. No break below the solubility limit was found in surface tension plots, suggesting that these compounds exist as monomers in aqueous solution. Only when a significant fraction of the molecules is negatively charged can they form micelles by themselves. Cobra venom phospholipase A2 hydrolyzes monomeric short-chain PE's at about the same rate as short-chain PC's but hydrolyzes long-chain PC's much more rapidly than long-chain PE's. The hydrolysis of short-chain PE's is found to be activated by phosphocholine-containing compounds only in the presence of an interface; in its absence phosphocholine-containing compounds can act as competitive inhibitors. Possible explanations for this phenomenon are considered.

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1984

# Reference PDF

0010

Dennis, E. A. and Plückthun, A. (1984) Phosphorus-31 NMR of phospholipids in micelles in: Phosphorus-31 NMR, Principles and Applications (Gorenstein, D. G., ed) pp. 423-447. Academic Press Inc.
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Abstract

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1983

# Reference PDF

0009

Vogel, C. W., Plückthun, A., Podack, E. R., Dennis, E. A. and Müller-Eberhard, H. J. (1983) The membrane attack complex of complement and its precursor proteins lack phospholipase activity. Mol. Immunol. 20, 377-382.
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Abstract

The membrane attack complex of human complement and its highly purified precursor proteins have been analyzed for phospholipase activity. Using three different sensitive assays, phospholipase A1, A2, C or D activity could not be detected. Based on the sensitivity of the assays employed, these results indicate the complement-mediated membrane damage is not enhanced by covalent breakdown of membrane phospholipids, but is entirely caused by physical action of the membrane attack complex. The results also imply that the putative serine esterase sites of C6 and C7 are not acting on phospholipids.

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1982

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0008

Plückthun, A. and Dennis, E. A. (1982) Role of monomeric activators in cobra venom phospholipase A2 action. Biochemistry 21, 1750-1756.
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Abstract

Phospholipase A2 from cobra venom (Naja naja naja), which acts poorly on phosphatidylethanolamine (PE) in mixed micelles, is activated toward PE by the monomeric phospholipid dibutyrylphosphatidylcholine (dibutyryl-PC) which is an, even poorer substrate. Phosphorus-31 nuclear magnetic resonance spectroscopy was employed to show that only PE is hydrolyzed in mixtures of PE and dibutyryl-PC of various concentrations. The activation shows saturation behavior, and the fully activated enzyme hydrolyzes PE at a rate similar to its optimal substrate PC containing long chain fatty acid groups. Because dibutyryl-PC is not incorporated into the micelles, these results are consistent with a mechanism of direct activation of the enzymes by dibutyryl-PC rather than a change in the properties of the interface being responsible for the activation of phospholipase A2. Furthermore, if either PC pr PE as substrate is dispersed in mixed micelles, increasing amounts of the detergent Triton X-100 decrease the hydrolysis rate. The same detergent effect occurs if PE hydrolysis is activated by sphingomyelin (SPH). However, if the enzyme is activated by the monomeric dibutyryl-PC, this detergent effect can be overcome at high enough dibutyryl-PC concentrations. The hydrolysis of the monomeric dibutyryl-PC can also be stimulated by SPH in mixed micelles. This reaction shows no effect of detergent. Several models are considered to explain these observations, and it is suggested that the enzyme has two types of functional sites: an activator site and a catalytic site.

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0007

Plückthun, A. and Dennis, E. A. (1982) Acyl and phosphoryl migration in lysophospholipids: Importance in phospholipid synthesis and phospholipase specificity. Biochemistry 21, 1743-1750.
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Abstract

Three isomeric lysophosphatidylcholines (1-palmitoyl-sn-glycero-3-phosphorylcholine, 2-palmitoyl-sn-glycero-3-phosphorylcholine, and 3-palmitoyl-sn-glycero-2-phosphorylcholine) have been prepared by the action of phospholipase A2 or lipase on 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine or phospholypase A2 on 1,3-dipalmitoyl-sn-glycero-2-phosphorylcholine. The structures of the lyso compounds have been confirmed by a complete assignment of the polar head groups using 1H NMR spectroscopy. The product of phospholipase A2 action on phosphatidylcholine is 1-acyl-sn-glycero-3-phosphorylcholine. Acyl migration between this compound and the 2-acyl isomer and phosphoryl migration between this compound and the 2-phosphoryl isomer were followed by 31P NMR. The acyl migration was found to be first order in both lysophospholipid and acid or base with a base-catalyzed second-order rate constant of about 4 x 10-4 M-1 s-1. At alkaline pHs, the equilibrium mixture contains about 90% of the 1-acyl and about 10% of the 2-acyl isomer. A slow acyl migration also occurs in organic solvents, most notably in the presents of basic catalysts used in common acylation procedures for the synthesis of phospholipids from lysophospholipids. At alkaline pHs, no phosphoryl migration was detected in the time scale of acyl migration and hydrolysis. 31P NMR could also directly demonstrate the positional specificity of phospholipase A2 and lipase, which acts as a phospholipase A1, by the direct observation of the products formed under conditions where migration was slow. While it is well-known that phospholipase A2 is specific for the sn-2 position of phospholipids in micelles and bilayer membranes, it was demonstrated by this technique that this specificity also holds for the monomeric phopholipid dibutyrylphospatidylcholine.

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0006

Plückthun, A., deBony, J. and Dennis, E. A. (1982) 1H-NMR Investigation of the conformation of charged and zwitterionic _- and _-phospholipids in sonicated vesicles. Biophys. J. 37, 13a.
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Abstract

The two fatty acyl chains of a-phospholipids are conformationally distinct when the phospholipids are in aggregated structures such as micelles. These differences can also be detected in sonicated vesicles by 1H-NMR spectroscopy (360 MHz) of the a-methylene group using resolution enhancement techniques (J. deBony and E. A. Dennis, Biochemistry 20, 5256-5260 (1981)). In vesicles with anionic phospholipids or PE at high pH, the spectra of the a-methylene region can be well resolved and fully accounted for. Zwitterionic a-PC, however, shows a more complicated pattern. On the other hand, b-phospholipids such as b-PE, N-methyl-b-PE, and N,N-dimethyl-b-PE give rise to two signals in sonicated vesicles (although both acyl chains are equivalent by symmetry) but only one signal in mixed micelles with detergent. Zwitterionic b-PC, however, shows only one peak in either case. Since this behavior is paralleled by the signals of the N-methyl groups, it probably derives from inside-outside differences of the b-phospholipids in the vesicle. The unusual spectra of both a- and b-PC compared to their charged analogues will be discussed in terms of packing differences within the bilayer.

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1981

# Reference PDF

0005

Vogel, C. W., Plückthun, A., Müller-Eberhard, H. J. and Dennis, E. A. (1981) Hemolytic assay for venom phospholipase A2. Anal. Biochem. 118, 262-268.
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Abstract

A rapid and sensitive spectrophotometric assay for venom phospholipase A2 based on the hemolysis of guinea pig erythrocytes in the presence of decomplemented serum and cardiotoxin (direct lytic factor) is described. This assay is particularly useful for rapid multisample analyses, such as those used in monitoring chromatography fractions, and is specific for phospholipase A2 in the presence of other potentially hemolytic venom components. The hemolytic mechanism is shown to be a combination of the action of lysophospholipids liberated from lipoproteins in the serum and the synergistic action of phospholipase A2 and cardiotoxin on the erythrocyte membrane.

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0004

Dennis, E. A., Darke, P. L., Deems, R. A., Kensil, C. R. and Plückthun, A. (1981) Cobra venom phospholipase A2: A review of its action toward lipid/water interfaces. Mol. Cell. Biochem. 36, 37-45.
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Abstract

This review focuses on the mechanism of action of phospholipase A2 from cobra venom (Naja naja naja) toward the lipid/water interface. Particular points of interest include dramatic changes in the enzyme activity if the physical state of its substrate is altered and the activation of the enzyme by phosphorylcholine containing lipids. The experimental findings include the following: Micellar substrates are hydrolyzed faster by the enzyme than various bilayer forms of substrate aggregation. The activity of the enzyme toward short chain phospholipids increases suddenly above their critical micelle concentrations. An abrupt change in susceptibility to the enzyme is observed at the thermotropic phase transition of phospholipid vesicles. The enzyme shows the kinetic phenomena of surface dilution and activation by certain lipids, which suggest a two-step mechanism of action. A model is discussed which accommodates the present data both for the action of this enzyme at various lipid/water interfaces as well as its interaction with synthetic monomeric ligands and substrates.

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0003

Plückthun, A. and Dennis, E. A. (1981) Phospholipase A2 hydrolysis of phospholipids: Use of 31P NMR to study the hydrolysis, acyl migration, regiospecific synthesis, and solubilization of phospholipids. Phosphorus Chemistry, ACS Symposium Series 171, 591-594.
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Abstract

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0002

Plückthun, A. and Dennis, E. A. (1981) Phospholipid activation of phospholipase A2 from cobra venom. Fed. Proc. 40, 1805.
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Abstract

The enzymatic hydrolysis of phosphatidylethanolamine (PE) in mixed micelles, which is a very poor substrate alone, can be greatly increased by addition of dibutyrylphosphatidylcholine (DiC4PC), which is an even poorer substrate. 31P-NMR studies have demonstated that in these mixtures only PE is hydrolyzed and that DiC4PC is not incorporated into the Triton X-100/PE mixed micelles that form the substrate. The activation by DiC4PC shows saturation behavior with a maximal velocity similar to the optimal substrate phosphatidylcholine (PC). The activator only increases Vmax but does not change Km. If increasing amounts of Triton X-100 are added, the rate of hydrolysis decreases if the substate is PC, PE or PE activated by sphingomyelin, which is in the micelle, but cannot be hydrolyzed. With sufficient amounts of monomeric DiC4PC as an activator, however, no decrease in activity with higher detergent concentrations is observed. If monomeric DiC4PC is the substrate and micellar sphingomyelin the activator, a detergent dependence is not observed. These findings are explained in terms of surface dilution kinetics and two functional sites on the enzyme: an activator site and a catalytic site. Those functional sites may be on different subunits of an enzyme dimer or constitute separate functional domains on a monomeric enzyme.

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0001

Plückthun, A. and Dennis, E. A. (1981) 31P Nuclear magnetic resonance study on the incorporation of monomeric phospholipids into nonionic detergent micelles. J. Phys. Chem. 85, 678-683.
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Abstract

31P nuclear magnetic resonance chemical shifts of monomeric and micellar phospholipids are reported. Micellization of dihexanoylphosphatidylcholine as a function of concentration was followed, and the critical micelle concentration determined. Chemical-shift differences and gel permeation chromatography were employed to follow the solubilization of monomeric phospholipids by the nonionic surfactant Triton X-100 to form mixed micelles. For dihexanoylphosphatidylcholine, a partition coefficient between Triton X-100 micelles and free solution was calculated as a function of the Triton X-100 concentration. Dibutyrylphosphatidylcholine was only sparingly incorporated into the surfactant micelles in contrast to phosphatidylcholine containing long-chain fatty acyl groups, which is completely solubilized.

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