Publications by authors named "Eva Bosse-Doenecke"

11 Publications

  • Page 1 of 1

Mabfilin and Fabfilin - New antibody-scaffold fusion formats for multispecific targeting concepts.

Protein Expr Purif 2018 09 25;149:51-65. Epub 2018 Apr 25.

Navigo Proteins GmbH, Formerly Scil Proteins GmbH, Heinrich-Damerow-Straße 1, D-06120 Halle Saale, Germany. Electronic address:

Protein based binding molecules have a broad applicability from therapeutic to technical use. Monoclonal antibodies represent the major class of this type of agents complemented by innovative approaches using scaffold proteins with tailor-made properties. Various concepts for new formats combining antibody chains or antibody fragments and fusions with other entities have been developed recently. This strategy opens up options to design molecules with biophysical, biochemical and pharmacological characteristics in a broad range while simultaneously addressing several targets or epitopes. The demand for such compounds is still growing as reflected by the literature and further new ideas are expected. In this context we developed so called Mabfilin and Fabfilin molecules. The formats synergistically bring together the classical antibody or fragments thereof supplemented with additional binding moieties, the Affilin molecules. These are based on the scaffold ubiquitin endowed with novel targeting properties by local randomization and selection from combinatorial libraries. Mab-/Fabfilin variants show advantageous biochemical properties and open a new route for the development of multispecific compounds for flexible applications.
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http://dx.doi.org/10.1016/j.pep.2018.04.013DOI Listing
September 2018

Construction and Selection of Affilin Phage Display Libraries.

Methods Mol Biol 2018 ;1701:205-238

Navigo Proteins GmbH, Heinrich-Damerow-Straße 1, 06120, Halle (Saale), Germany.

Affilin molecules represent a new class of so-called scaffold proteins. The concept of scaffold proteins is to use stable and versatile protein structures which can be endowed with de novo binding properties and specificities by introducing mutations in surface exposed amino acid residues. Complex variations and combinations are generated by genetic methods of randomization resulting in large cDNA libraries. The selection for candidates binding to a desired target can be executed by display methods, especially the very robust and flexible phage display. Here, we describe the construction of ubiquitin based Affilin phage display libraries and their use in biopanning experiments for the identification of novel protein ligands.
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http://dx.doi.org/10.1007/978-1-4939-7447-4_11DOI Listing
July 2018

Ubiquitin is a versatile scaffold protein for the generation of molecules with de novo binding and advantageous drug-like properties.

FEBS Open Bio 2015 10;5:579-93. Epub 2015 Jul 10.

Scil Proteins GmbH, Heinrich-Damerow-Straße 1, D-06120 Halle (Saale), Germany.

In the search for effective therapeutic strategies, protein-based biologicals are under intense development. While monoclonal antibodies represent the majority of these drugs, other innovative approaches are exploring the use of scaffold proteins for the creation of binding molecules with tailor-made properties. Ubiquitin is especially suited for this strategy due to several key characteristics. Ubiquitin is a natural serum protein, 100% conserved across the mammalian class and possesses high thermal, structural and proteolytic stability. Because of its small size and lack of posttranslational modifications, it can be easily produced in Escherichia coli. In this work we provide evidence that ubiquitin is safe as tested experimentally in vivo. In contrast to previously published results, we show that, in our hands, ubiquitin does not act as a functional ligand of the chemokine receptor CXCR4. Cellular assays based on different signaling pathways of the receptor were conducted with the natural agonist SDF-1 as a benchmark. In none of the assays could a response to ubiquitin treatment be elicited. Furthermore, intravenous application to mice at high concentrations did not induce any detectable effect on cytokine levels or hematological parameters.
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http://dx.doi.org/10.1016/j.fob.2015.07.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4522466PMC
August 2015

Novel ubiquitin-derived high affinity binding proteins with tumor targeting properties.

J Biol Chem 2014 Mar 28;289(12):8493-507. Epub 2014 Jan 28.

From Scil Proteins GmbH, Heinrich-Damerow-Strasse 1, 06120 Halle (Saale), Germany.

Targeting effector molecules to tumor cells is a promising mode of action for cancer therapy and diagnostics. Binding proteins with high affinity and specificity for a tumor target that carry effector molecules such as toxins, cytokines, or radiolabels to their intended site of action are required for these applications. In order to yield high tumor accumulation while maintaining low levels in healthy tissues and blood, the half-life of such conjugates needs to be in an optimal range. Scaffold-based binding molecules are small proteins with high affinity and short systemic circulation. Due to their low molecular complexity, they are well suited for combination with effector molecules as well as half-life extension technologies yielding therapeutics with half-lives adapted to the specific therapy. We have identified ubiquitin as an ideal scaffold protein due to its outstanding biophysical and biochemical properties. Based on a dimeric ubiquitin library, high affinity and specific binding molecules, so-called Affilin® molecules, have been selected against the extradomain B of fibronectin, a target almost exclusively expressed in tumor tissues. Extradomain B-binding molecules feature high thermal and serum stability as well as strong in vitro target binding and in vivo tumor accumulation. Application of several half-life extension technologies results in molecules of largely unaffected affinity but significantly prolonged in vivo half-life and tumor retention. Our results demonstrate the utility of ubiquitin as a scaffold for the generation of high affinity binders in a modular fashion, which can be combined with effector molecules and half-life extension technologies.
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http://dx.doi.org/10.1074/jbc.M113.519884DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3961674PMC
March 2014

Binding specificity of the ectodomain of the parathyroid hormone receptor.

Biophys Chem 2011 Mar 14;154(2-3):66-72. Epub 2011 Jan 14.

Institute of Biochemistry/Biotechnology, Faculty of Science I, Martin-Luther-University Halle-Wittenberg, Germany.

The parathyroid hormone (PTH)1 receptor is a member of the class B G protein-coupled receptor (GPCR) family and regulates bone and mineral metabolism of vertebrates. A truncated highly active parathyroid hormone fragment PTH (1-34) exerts stimulatory effects on the receptor and is used for treatment of osteoporosis. To study the interacting amino acids of the natural peptide ligand PTH (1-84) with the ectodomain of its receptor we used peptide micro arrays on solid cellulose membranes. The amino acids Arg20 and Trp23 within the identified core binding stretch PTH (20-26) were found to be most important for affinity to the ectodomain of PTH1R. Isothermal titration calorimetry and NMR spectroscopy allowed peptide binding studies in solution and verified peptide positions required for high affinity. With this combination of biochemical and biophysical methods we extend former findings on this essential interaction and can now provide a strategy to screen for optimized therapeutic peptides.
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http://dx.doi.org/10.1016/j.bpc.2011.01.002DOI Listing
March 2011

Recombinant expression, in vitro refolding, and biophysical characterization of the human glucagon-like peptide-1 receptor.

Biochemistry 2010 Sep;49(36):7956-65

Institute of Biochemistry and Biotechnology, Martin-Luther-University, Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120 Halle/Saale, Germany.

Activation of the glucagon-like peptide-1 receptor (GLP-1R) upon ligand binding leads to the release of insulin from pancreatic cells. This strictly glucose-dependent process renders the receptor and its ligands useful in the treatment of type II diabetes mellitus. To enable a biophysical characterization in vitro, we expressed the human full-length GLP-1R in the cytosol of Escherichia coli as inclusion bodies. After purification, refolding of the SDS-solubilized receptor was achieved by the exchange of SDS against the detergent Brij78 using an artificial chaperone system. Far-UV circular dichroism spectroscopic studies revealed that the receptor adopts a characteristic alpha-helical structure in Brij78 micelles. Ligand binding of the renatured protein was quantified by fluorescence quenching and surface plasmon resonance spectroscopy. In the presence of Brij micelles, the refolded receptor binds the agonist exendin-4 with an apparent dissociation constant of approximately 100 nM in a reversible one-step mechanism. To demonstrate that the detected ligand binding activity is not only due to an autonomously functional N-terminal domain (nGLP-1R) but also due to additional contacts with the juxtamembrane part, we separately expressed and refolded the extracellular domain relying on identical protocols established for the full-length GLP-1R. In support of the suggested multidomain binding mode, the nGLP-1R binds exendin-4 with a lower affinity (K(app) in the micromolar range) and a different kinetic mechanism. The lower ligand affinity of the nGLP-1R results entirely from a decreased kinetic stability of the receptor-ligand complex, dissociation of which is approximately 40-fold faster in the case of the nGLP-1R compared to the full-length GLP-1R. In summary, a framework was developed to produce functional human full-length GLP-1R by recombinant expression in E. coli as a prerequisite for eventual structure determination and a rigorous biophysical characterization including protein variants.
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http://dx.doi.org/10.1021/bi101159sDOI Listing
September 2010

Identification of a disulfide bridge essential for transport function of the human proton-coupled amino acid transporter hPAT1.

J Biol Chem 2009 Aug 23;284(33):22123-32. Epub 2009 Jun 23.

Institute of Biochemistry/Biotechnology, Faculty of Science I, Biozentrum, Martin-Luther-University Halle-Wittenberg, Halle, Germany.

The proton-coupled amino acid transporter 1 (PAT1, SLC36A1) mediates the uptake of small neutral amino acids at the apical membrane of intestinal epithelial cells after protein digestion. The transporter is currently under intense investigation, because it is a possible vehicle for oral drug delivery. Structural features of the protein such as the number of transmembrane domains, the substrate binding site, or essential amino acids are still unknown. In the present study we use mutagenesis experiments and biochemical approaches to determine the role of the three putative extracellular cysteine residues on transport function and their possible involvement in the formation of a disulfide bridge. As treatment with the reducing reagent dithiothreitol impaired transport function of hPAT1 wild type protein, substitution of putative extracellular cysteine residues Cys-180, Cys-329, and Cys-473 by alanine or serine was performed. Replacement of the two highly conserved cysteine residues Cys-180 and Cys-329 abolished the transport function of hPAT1 in Xenopus laevis oocytes. Studies of wild type and mutant transporters expressed in human retinal pigment epithelial (HRPE) cells suggested that the binding of the substrate was inhibited in these mutants. Substitution of the third putative extracellular nonconserved cysteine residue Cys-473 did not affect transport function. All mutants were expressed at the plasma membrane. Biotinylation of free sulfhydryl groups using maleimide-PEG(11)-biotin and SDS-PAGE analysis under reducing and nonreducing conditions provided direct evidence for the existence of an essential disulfide bond between Cys-180 and Cys-329. This disulfide bridge is very likely involved in forming or stabilizing the substrate binding site.
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http://dx.doi.org/10.1074/jbc.M109.023713DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2755936PMC
August 2009

The role of N-glycosylation in transport function and surface targeting of the human solute carrier PAT1.

FEBS Lett 2009 May 3;583(10):1631-6. Epub 2009 May 3.

Institute of Biochemistry/Biotechnology, Faculty of Science I, Martin-Luther-University Halle-Wittenberg, Halle, Germany.

In the present study we show in the Xenopus laevis expression system that the proton-coupled amino acid transporter 1 (PAT1, SLC36A1) is glycosylated at asparagine residues N174, N183 and N470. To determine the functional role of N-glycosylation, glycosylation-deficient mutants were analyzed by two-electrode voltage-clamp measurements after expression in X. laevis oocytes. Single replacements of asparagine residues had no effect on transport activity. However, multiple substitutions resulted in a decreased transport rate, leaving K(t) unchanged. Immunofluorescence localisation revealed a diminished plasma membrane expression of glycosylation-defective mutants. This indicates that N-glycans are not required for transport function, but are important for membrane targeting.
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http://dx.doi.org/10.1016/j.febslet.2009.04.037DOI Listing
May 2009

Pharmaceutical and pharmacological importance of peptide transporters.

J Pharm Pharmacol 2008 May;60(5):543-85

Membrane Transport Group, Biozentrum of the Martin-Luther-University Halle-Wittenberg, D-06120 Halle, Germany.

Peptide transport is currently a prominent topic in membrane research. The transport proteins involved are under intense investigation because of their physiological importance in protein absorption and also because peptide transporters are possible vehicles for drug delivery. Moreover, in many tissues peptide carriers transduce peptidic signals across membranes that are relevant in information processing. The focus of this review is on the pharmaceutical relevance of the human peptide transporters PEPT1 and PEPT2. In addition to their physiological substrates, both carriers transport many beta-lactam antibiotics, valaciclovir and other drugs and prodrugs because of their sterical resemblance to di- and tripeptides. The primary structure, tissue distribution and substrate specificity of PEPT1 and PEPT2 have been well characterized. However, there is a dearth of knowledge on the substrate binding sites and the three-dimensional structure of these proteins. Until this pivotal information becomes available by X-ray crystallography, the development of new drug substrates relies on classical transport studies combined with molecular modelling. In more than thirty years of research, data on the interaction of well over 700 di- and tripeptides, amino acid and peptide derivatives, drugs and prodrugs with peptide transporters have been gathered. The aim of this review is to put the reports on peptide transporter-mediated drug uptake into perspective. We also review the current knowledge on pharmacogenomics and clinical relevance of human peptide transporters. Finally, the reader's attention is drawn to other known or proposed human peptide-transporting proteins.
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http://dx.doi.org/10.1211/jpp.60.5.0002DOI Listing
May 2008

High yield production of recombinant native and modified peptides exemplified by ligands for G-protein coupled receptors.

Protein Expr Purif 2008 Mar;58(1):114-21

Institut für Biochemie und Biotechnologie, Technische Biochemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Str. 3, D-06120 Halle, Germany.

G-protein coupled receptors (GPCRs) comprise a large family of membrane proteins and attract pharmaceutical interest as therapeutic targets. Two examples of class B GPCRs that are involved in metabolic diseases are the Parathyroid hormone receptor 1 (PTHR1) and the Glucagon-like-peptide-1 receptor (GLP-1R) which play central roles in osteoporosis and diabetes mellitus type II, respectively. Class B GPCRs are characterised by a large extracellular N-terminal domain with a typical disulfide bridge pattern. This domain is responsible for the binding of peptide hormone ligands. Here we report the recombinant expression of these ligands in natural and several modified forms for their use in functional assays, NMR analyses or affinity purification of receptor/ligand complexes for crystallisation. Applying the SUMO system, low cost expression of soluble fusion-proteins is achieved. Moreover, via the SUMO cleavage site, the authentic N-terminal sequence which is essential for ligand-receptor interactions can be obtained. Purification of the peptide by RP-HPLC results in >98% pure preparations. The strategy can also be adopted for many other purposes, especially if small peptides are needed at either large amounts or with specific features like isotope, affinity or fluorescent labels. Furthermore, for the growing demand for therapeutic peptides, this method could represent a straightforward production process.
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http://dx.doi.org/10.1016/j.pep.2007.10.012DOI Listing
March 2008

Mutational analysis of histidine residues in the human proton-coupled amino acid transporter PAT1.

Biochim Biophys Acta 2008 Apr 12;1778(4):1042-50. Epub 2008 Jan 12.

Membrane Transport Group, Biozentrum, Martin-Luther-University Halle-Wittenberg, D-06120 Halle, Germany.

The proton-coupled amino acid transporter 1 (PAT1) represents a major route by which small neutral amino acids are absorbed after intestinal protein digestion. The system also serves as a novel route for oral drug delivery. Having shown that H+ affects affinity constants but not maximal velocity of transport, we investigated which histidine residues are obligatory for PAT1 function. Three histidine residues are conserved among the H+-coupled amino acid transporters PAT1 to 4 from different animal species. We individually mutated each of these histidine residues and compared the catalytic function of the mutants with that of the wild type transporter after expression in HRPE cells. His-55 was found to be essential for the catalytic activity of hPAT1 because the corresponding mutants H55A, H55N and H55E had no detectable l-proline transport activity. His-93 and His-135 are less important for transport function since H93N and H135N mutations did not impair transport function. The loss of transport function of His-55 mutants was not due to alterations in protein expression as shown both by cell surface biotinylation immunoblot analyses and by confocal microscopy. We conclude that His-55 might be responsible for binding and translocation of H+ in the course of cellular amino acid uptake by PAT1.
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http://dx.doi.org/10.1016/j.bbamem.2007.12.026DOI Listing
April 2008