Publications by authors named "Manja Gloser"

2 Publications

  • Page 1 of 1

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

Recombinant production of bioactive human TNF-alpha by SUMO-fusion system--high yields from shake-flask culture.

Protein Expr Purif 2010 Aug 2;72(2):238-43. Epub 2010 Apr 2.

NWG Künstliche Bindeproteine, Institut für Biochemie und Biotechnologie, Technische Biochemie, Martin-Luther-Universität Halle-Wittenberg, Heinrich-Damerow-Str. 4, 06120 Halle, Germany.

Tumor necrosis factor (TNF-alpha) inhibitors, used for the treatment of common inflammatory diseases, currently belong among the most important biotechnologically produced pharmaceuticals. So far four TNF-alpha antagonists have been approved by regulatory authorities for defined subsets of applications. Furthermore, numerous approaches are being taken to develop new protein-based pharmaceuticals and to broaden their application areas in the treatment of TNF-alpha -related diseases. Both the fundamental understanding of disease-related TNF-alpha activity and the subsequent development of corresponding drug candidates demand the availability of large amounts of TNF-alpha as a bioactive protein. We have therefore established a protocol for the rapid high-level synthesis of recombinant human TNF-alpha in Escherichia coli shake-flask cultures and the subsequent purification of the mature protein. Using the advantages of SUMO-fusion technology we were able to produce protein with an authentic N-terminus in high yield. Two immobilized metal ion-affinity chromatography steps with a protease cleavage step in between and subsequent size-exclusion chromatography were utilized to purify the protein. The protein was obtained from the last chromatography step as a trimer, while purity was at least 96% as estimated by SDS-PAGE. The identity of the protein was confirmed by MALDI-TOF mass spectrometry. Recombinant mature TNF-alpha was correctly folded as assessed by CD spectroscopy and its biological activity was confirmed by an L929 cell assay.
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http://dx.doi.org/10.1016/j.pep.2010.03.022DOI Listing
August 2010