Publications by authors named "Uli Binder"

8 Publications

  • Page 1 of 1

PASylated Thymosin α1: A Long-Acting Immunostimulatory Peptide for Applications in Oncology and Virology.

Int J Mol Sci 2020 Dec 24;22(1). Epub 2020 Dec 24.

Lehrstuhl für Biologische Chemie, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354 Freising, Germany.

Thymosin α1 (Tα1) is an immunostimulatory peptide for the treatment of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections and used as an immune enhancer, which also offers prospects in the context of COVID-19 infections and cancer. Manufacturing of this N-terminally acetylated 28-residue peptide is demanding, and its short plasma half-life limits in vivo efficacy and requires frequent dosing. Here, we combined the PASylation technology with enzymatic in situ N-acetylation by RimJ to produce a long-acting version of Tα1 in at high yield. ESI-MS analysis of the purified fusion protein indicated the expected composition without any signs of proteolysis. SEC analysis revealed a 10-fold expanded hydrodynamic volume resulting from the fusion with a conformationally disordered Pro/Ala/Ser (PAS) polypeptide of 600 residues. This size effect led to a plasma half-life in rats extended by more than a factor 8 compared to the original synthetic peptide due to retarded kidney filtration. Our study provides the basis for therapeutic development of a next generation thymosin α1 with prolonged circulation. Generally, the strategy of producing an N-terminally protected PASylated peptide solves three major problems of peptide drugs: (i) instability in the expression host, (ii) rapid degradation by serum exopeptidases, and (iii) low bioactivity because of fast renal clearance.
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http://dx.doi.org/10.3390/ijms22010124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7795856PMC
December 2020

PASylation of IL-1 receptor antagonist (IL-1Ra) retains IL-1 blockade and extends its duration in mouse urate crystal-induced peritonitis.

J Biol Chem 2020 01 9;295(3):868-882. Epub 2019 Dec 9.

Department of Medicine, University of Colorado, Aurora, Colorado 80045.

Interleukin-1 (IL-1) is a key mediator of inflammation and immunity. Naturally-occurring IL-1 receptor antagonist (IL-1Ra) binds and blocks the IL-1 receptor-1 (IL-1R1), preventing signaling. Anakinra, a recombinant form of IL-1Ra, is used to treat a spectrum of inflammatory diseases. However, anakinra is rapidly cleared from the body and requires daily administration. To create a longer-lasting alternative, PASylated IL-1Ra (PAS-IL-1Ra) has been generated by in-frame fusion of a long, defined-length, N-terminal Pro/Ala/Ser (PAS) random-coil polypeptide with IL-1Ra. Here, we compared the efficacy of two PAS-IL-1Ra molecules, PAS600-IL-1Ra and PAS800-IL-1Ra (carrying 600 and 800 PAS residues, respectively), with that of anakinra in mice. PAS600-IL-1Ra displayed markedly extended blood plasma levels 3 days post-administration, whereas anakinra was undetectable after 24 h. We also studied PAS600-IL-1Ra and PAS800-IL-1Ra for efficacy in monosodium urate (MSU) crystal-induced peritonitis. 5 days post-administration, PAS800-IL-1Ra significantly reduced leukocyte influx and inflammatory markers in MSU-induced peritonitis, whereas equimolar anakinra administered 24 h before MSU challenge was ineffective. The 6-h pretreatment with equimolar anakinra or PAS800-IL-1Ra before MSU challenge similarly reduced inflammatory markers. In cultured A549 lung carcinoma cells, anakinra, PAS600-IL-1Ra, and PAS800-IL-Ra reduced IL-1α-induced IL-6 and IL-8 levels with comparable potency. In human peripheral blood mononuclear cells, these molecules suppressed -induced production of the cancer-promoting cytokine IL-22. Surface plasmon resonance analyses revealed significant binding between PAS-IL-1Ra and IL-1R1, although with a slightly lower affinity than anakinra. These results validate PAS-IL-1Ra as an active IL-1 antagonist with marked potency and a significantly extended half-life compared with anakinra.
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http://dx.doi.org/10.1074/jbc.RA119.010340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6970921PMC
January 2020

Half-life extension and non-human primate pharmacokinetic safety studies of i-body AD-114 targeting human CXCR4.

MAbs 2019 10 23;11(7):1331-1340. Epub 2019 Aug 23.

The Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University , Bundoora , Melbourne , Australia.

Single domain antibodies that combine antigen specificity with high tissue penetration are an attractive alternative to conventional antibodies. However, rapid clearance from the bloodstream owing to their small size can be a limitation of therapeutic single domain antibodies. Here, we describe and evaluate the conjugation of a single domain i-body, AD-114, which targets CXCR4, to a panel of half-life extension technologies including a human serum albumin-binding peptide, linear and branched PEG, and PASylation (PA600). The conjugates were assessed in murine, rat and cynomolgus monkey pharmacokinetic studies and showed that the branched PEG was most effective at extending circulating half-life in mice; however, manufacturing limitations of PEGylated test material precluded scale-up and assessment in larger animals. PA600, by comparison, was amenable to scale-up and afforded considerable half-life improvements in mice, rats and cynomolgus monkeys. In mice, the circulating half-life of AD-114 was extended from 0.18 h to 7.77 h following conjugation to PA600, and in cynomolgus monkeys, the circulating half-life of AD-114-PA600 was 24.27 h. AD-114-PA600 was well tolerated in cynomolgus monkeys at dose rates up to 100 mg/kg with no mortalities or drug-related clinical signs.
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http://dx.doi.org/10.1080/19420862.2019.1626652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748587PMC
October 2019

Author Correction: Consistent success in life-supporting porcine cardiac xenotransplantation.

Nature 2019 Apr;568(7752):E7

Institute of Molecular Animal Breeding and Biotechnology, Gene Center, LMU Munich, Munich, Germany.

In this Letter, Mayuko Kurome and Valeri Zakhartchenko have been added to the author list (affiliated with Institute of Molecular Animal Breeding and Biotechnology, Gene Center, LMU Munich, Munich, Germany). The author list and 'Author contributions' section have been corrected online; see accompanying Amendment.
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http://dx.doi.org/10.1038/s41586-019-1108-4DOI Listing
April 2019

Consistent success in life-supporting porcine cardiac xenotransplantation.

Nature 2018 12 5;564(7736):430-433. Epub 2018 Dec 5.

Institute of Molecular Animal Breeding and Biotechnology, Gene Center, LMU Munich, Munich, Germany.

Heart transplantation is the only cure for patients with terminal cardiac failure, but the supply of allogeneic donor organs falls far short of the clinical need. Xenotransplantation of genetically modified pig hearts has been discussed as a potential alternative. Genetically multi-modified pig hearts that lack galactose-α1,3-galactose epitopes (α1,3-galactosyltransferase knockout) and express a human membrane cofactor protein (CD46) and human thrombomodulin have survived for up to 945 days after heterotopic abdominal transplantation in baboons. This model demonstrated long-term acceptance of discordant xenografts with safe immunosuppression but did not predict their life-supporting function. Despite 25 years of extensive research, the maximum survival of a baboon after heart replacement with a porcine xenograft was only 57 days and this was achieved, to our knowledge, only once. Here we show that α1,3-galactosyltransferase-knockout pig hearts that express human CD46 and thrombomodulin require non-ischaemic preservation with continuous perfusion and control of post-transplantation growth to ensure long-term orthotopic function of the xenograft in baboons, the most stringent preclinical xenotransplantation model. Consistent life-supporting function of xenografted hearts for up to 195 days is a milestone on the way to clinical cardiac xenotransplantation.
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http://dx.doi.org/10.1038/s41586-018-0765-zDOI Listing
December 2018

PASylation: a biological alternative to PEGylation for extending the plasma half-life of pharmaceutically active proteins.

Protein Eng Des Sel 2013 Aug 10;26(8):489-501. Epub 2013 Jun 10.

Munich Center for Integrated Protein Science CIPS-M & Lehrstuhl für Biologische Chemie, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85350 Freising-Weihenstephan, Germany.

A major limitation of biopharmaceutical proteins is their fast clearance from circulation via kidney filtration, which strongly hampers efficacy both in animal studies and in human therapy. We have developed conformationally disordered polypeptide chains with expanded hydrodynamic volume comprising the small residues Pro, Ala and Ser (PAS). PAS sequences are hydrophilic, uncharged biological polymers with biophysical properties very similar to poly-ethylene glycol (PEG), whose chemical conjugation to drugs is an established method for plasma half-life extension. In contrast, PAS polypeptides offer fusion to a therapeutic protein on the genetic level, permitting Escherichia coli production of fully active proteins and obviating in vitro coupling or modification steps. Furthermore, they are biodegradable, thus avoiding organ accumulation, while showing stability in serum and lacking toxicity or immunogenicity in mice. We demonstrate that PASylation bestows typical biologics, such as interferon, growth hormone or Fab fragments, with considerably prolonged circulation and boosts bioactivity in vivo.
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http://dx.doi.org/10.1093/protein/gzt023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3715784PMC
August 2013

Endoplasmic reticulum stress response promotes cytotoxic phenotype of CD8αβ+ intraepithelial lymphocytes in a mouse model for Crohn's disease-like ileitis.

J Immunol 2012 Aug 2;189(3):1510-20. Epub 2012 Jul 2.

Chair for Biofunctionality, Research Centre for Nutrition and Food Science, Centre for Diet and Disease, Technical University of Munich, 85350 Freising-Weihenstephan, Germany.

Endoplasmic reticulum (ER) unfolded protein responses (UPR) are implicated in the pathogenesis of inflammatory bowel disease. Cytotoxic CD8αβ(+) intraepithelial lymphocytes (IEL) contribute to the development of Crohn's disease-like ileitis in TNF(ΔARE/+) mice. In this study, we characterized the role of ER-UPR mechanisms in contributing to the disease-associated phenotype of cytotoxic IEL under conditions of chronic inflammation. Inflamed TNF(ΔARE/+) mice exhibited increased expression of Grp78, ATF6, ATF4, and spliced XBP1 in CD8αβ(+) IEL but not in CD8αα(+) IEL or in lamina propria lymphocytes. Chromatin immunoprecipitation analysis in CD8αβ(+) T cells showed selective recruitment of ER-UPR transducers to the granzyme B gene promoter. Heterozygous Grp78(-/+) mice exhibited an attenuated granzyme B-dependent cytotoxicity of CD8αβ(+) T cells against intestinal epithelial cells, suggesting a critical activity of this ER-associated chaperone in maintaining a cytotoxic T cell phenotype. Granzyme B-deficient CD8αβ(+) T cells showed a defect in IL-2-mediated proliferation in Grp78(-/+) mice. Adoptively transferred Grp78(-/+) CD8αβ(+) T cells had a decreased frequency of accumulation in the intestine of RAG2(-/-) recipient mice. The tissue pathology in TNF(ΔARE/+) × Grp78(-/+) mice was similar to TNF(ΔARE/+) mice, even though the cytotoxic effector functions of CD8αβ(+) T cells were significantly reduced. In conclusion, ER stress-associated UPR mechanisms promote the development and maintenance of the pathogenic cytotoxic CD8αβ(+) IEL phenotype in the mouse model of Crohn's disease-like ileitis.
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http://dx.doi.org/10.4049/jimmunol.1200166DOI Listing
August 2012

High-throughput sorting of an Anticalin library via EspP-mediated functional display on the Escherichia coli cell surface.

J Mol Biol 2010 Jul 26;400(4):783-802. Epub 2010 May 26.

Munich Center for Integrated Protein Science and Lehrstuhl für Biologische Chemie, Technische Universität München, 85350 Freising-Weihenstephan, Germany.

We demonstrate that small engineered single-chain binding proteins based on the lipocalin scaffold, so-called Anticalins, can be functionally displayed on the Gram-negative bacterial cell envelope. To this end, the beta-domains of five different bacterial autotransporters (the IgA protease from Neisseria gonorrhoeae, the esterase EstA from Pseudomonas aeruginosa, the YpjA autotransporter from E. coli K12, the AIDA-I adhesin from enteropathogenic E. coli O127:H27 strain 2787 and the protease EspP from enterohemorrhagic E. coli O157:H7 strain EDL933) were compared with respect to display level, functional variance, and bacterial cell viability. Use of the EspP autotransporter led to a system with high genetic stability for the display of fully functional Anticalins in high density on the cell surface of E. coli as shown by quantitative flow cytofluorimetry. This system was applied to engineer an immunostimulatory Anticalin that binds and blocks the extracellular region of human CTLA-4 to achieve a slower dissociation rate. A combinatorial library of the original Anticalin was generated by error-prone PCR, subjected to E. coli cell surface display, and applied to repeated cycles of cell sorting after incubation with the fluorescently labelled target protein under competition with the unlabelled extracellular domain of CTLA-4. The resulting Anticalin variants, which were expressed and purified as soluble proteins, showed more than eightfold decelerated target dissociation, as revealed by real time surface plasmon resonance analysis. Hence, the EspP autotransporter-mediated E. coli surface display in combination with high-throughput fluorescence-activated cell sorting (FACS) provides an efficient strategy to select for Anticalins, and possibly other small protein scaffolds, with improved binding properties, which is particularly useful for in vitro affinity maturation but may also serve for the selection of novel target specificity from naive libraries.
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http://dx.doi.org/10.1016/j.jmb.2010.05.049DOI Listing
July 2010
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