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WO2008151845A2 - Procédés de production de l'antitrypsine alpha-1 humaine glycosylée (a1at) dans des cellules hôtes de mammifère - Google Patents

Procédés de production de l'antitrypsine alpha-1 humaine glycosylée (a1at) dans des cellules hôtes de mammifère Download PDF

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Publication number
WO2008151845A2
WO2008151845A2 PCT/EP2008/004844 EP2008004844W WO2008151845A2 WO 2008151845 A2 WO2008151845 A2 WO 2008151845A2 EP 2008004844 W EP2008004844 W EP 2008004844W WO 2008151845 A2 WO2008151845 A2 WO 2008151845A2
Authority
WO
WIPO (PCT)
Prior art keywords
alat
glycosylation
antitrypsin
mammalian host
gene sequence
Prior art date
Application number
PCT/EP2008/004844
Other languages
English (en)
Other versions
WO2008151845A3 (fr
Inventor
Markus Berger
Uwe Marx
Volker Sandig
René BRECHT
Susann Koch
Richard Ammer
Silke Rieck
Werner Reutter
Veronique Blanchard
Matthias Kaup
Rudolf Tauber
Stefan Zietze
Original Assignee
Charite - Universitätsmedizin Berlin
Probiogen Ag
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Charite - Universitätsmedizin Berlin, Probiogen Ag filed Critical Charite - Universitätsmedizin Berlin
Priority to EP08773472A priority Critical patent/EP2167539A2/fr
Publication of WO2008151845A2 publication Critical patent/WO2008151845A2/fr
Publication of WO2008151845A3 publication Critical patent/WO2008151845A3/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8125—Alpha-1-antitrypsin

Definitions

  • AlAT deficiency is one of the most common hereditary disorders. A deficiency of AlAT has been observed in a number of patients with chronic emphysema of the lungs. A proportion of individuals with serum deficiency of AlAT may progress to cirrhosis and liver failure (Wu et al., 1991).
  • AlAT As an elastase inhibitor, and because of the prevalence of genetic diseases resulting in deficient serum levels of AAT, there has been an active interest in recombinant synthesis of AAT, for human therapeutic use.
  • the AlAT produced by the above methods is either unglycosylated or has a glycosylation pattern different to that of human AlAT.
  • Chang et al. 2003 disclose the production of sialylated human AlAT in insect cells by introducing human/mammalian N-glycosyliation machinery into the insect cells.
  • Chang et al. describe, that because asialylated glycoproteins have a shorter half-life in blood circulation, it was investigated if sialylated therapeutic glycoprotein can be produced from insect cells by enhancing the N-glycosylation machinery of the cells.
  • h ⁇ lAT human ⁇ l -antitrypsin
  • GnT2 GIcNAc transferase II
  • ⁇ l4GT ⁇ l,4-galactosyltransferase
  • ⁇ 26ST ⁇ 2,6-sialyltransferase
  • human AlAT can be produced in the milk of transgenic animals.
  • Archibald et al., 1990 examined the potential of transgenic animals as an alternative means of producing human AlAT.
  • a hybrid gene constructed by using sequences from an ovine milk protein fused to an AlAT "minigene" was used to generate transgenic mice.
  • Human AlAT was secreted into the milk of the mice.
  • AlAT from transgenic mouse milk was similar in size to human plasma-derived AlAT and showed a similar capacity to inhibit trypsin.
  • WO 00/63403 A3 (Introgene B.V., Bout et al.) and US 2007/0054394 Al (Bout et al.) disclose a method for stably expressing human recombinant proteins, such as glycoproteins, in the human cell line PER.C6TM, which originates from embryonic retina cells (ECACC number 96022940). As an example, the production of human erythropoetin is described.
  • WO 99/61650, US 5,705,364 and US 2004/0259205 Al disclose processes for preparing glycoproteins by mammalian cell culture wherein the sialic acid content of the glycoprotein produced is controlled by manipulating the cell culture environment.
  • process parameters such as osmolality, temperature, addition of organic acids and salts thereof, transcription enhancers, addition of copper ions to the cell culture medium, on the cell specific productivity and the sialic acid content of the glycoprotein is described.
  • the production of TNFRl -IgG-subtyp 1 is shown.
  • the problem is solved by the present invention by providing a method by providing a method for modulating the glycosylation status of human alpha-1 antitrypsin (AlAT), i.e. a method for modulating the glycosylation of human alpha-1 antitrypsin (AlAT) .
  • AlAT human alpha-1 antitrypsin
  • the method for modulating the glycosylation status of human alpha- 1 antitrypsin (AlAT) of the present invention is characterized in that the glycosylation of AlAT is modulated or controlled.
  • loop A amino acids N83-H93
  • loop C3 loop Cl (amino acids S121-V127)
  • a "human glycosylation status or pattern" of AlAT means a glycosylation at the three original glycosylation sites and a glycosylation of a complex type, as e.g. described in Kolarich et al., 2006, which is incorporated herein by reference.
  • the three original iV-glycosylation sites of AlAT contain diantennary JV-glycans but also triantennary and even traces of tetraantennary structures leading to the typical IEF pattern observed for AlAT (see Kolarich et al., 2006).
  • modifying the AlAT gene sequence comprises one or more of the following:
  • glycosylation sites, that are inserted into the AlAT gene sequence are inserted at one or more positions that are different to the original glycosylation sites within the AlAT gene sequence.
  • positions that are different to the original glycosylation sites within the AlAT gene sequence are preferably selected from positions in the loop regions, preferably loop A, loop B, loop C, loop D and loop E, as defined above. These positions can preferably be identified using a mutation strategy. Further positions can be identified by a person of skill in the art by applying the teaching of the present invention.
  • one or more original glycosylation sites within the AlAT gene sequence are deleted, wherein the original glycosylation sites are selected from N46, N83 and N247. These positions can preferably be identified using a mutation strategy. In some embodiments this deletion can occur while glycosylation sites are inserted, see below.
  • the one or more glycosylation site that is inserted is selected from the group comprising N48, N81, N90, N 108, N 123, N201, N249 and N323.
  • the following primers can be used for introducing these glycosylation sites into AlAT:
  • modifying the AlAT gene sequence comprises the insertion of a glycosylation tag.
  • the glycosylation tag comprises one or more glycosylation sites or one or more glycosylation domains of another glycoprotein or the glycosylation tag comprises one or more synthetic glycosylation domains, i.e. not based on a known glycoprotein.
  • the other glycoprotein is a highly glycosylated glycoprotein, preferably a glycoprotein with highly glycosylated glycosylation domain(s).
  • Preferred highly glycosylated glycoproteins are alphal-acid glycoprotein (AGP) (Higai et al., 2005) and erythropoietin (EPO) (Takeuchi et al., 1991).
  • the sugars or sugar derivatives are selected from N-acetyl mannosamine and derivatives thereof, e.g. 2-desoxy-2-N-propanoylamino-D-mannose (ManNProp), 2-desoxy-2- N-Cyclopropyl-acetyl- amino-D-mannose (ManNcyProp), 2-desoxy-2-N-pentanoylamino-D- mannose (ManNPent) (see also Figures 3 and 4).
  • ManNProp 2-desoxy-2-N-propanoylamino-D-mannose
  • ManNcyProp 2-desoxy-2- N-Cyclopropyl-acetyl- amino-D-mannose
  • ManNPent 2-desoxy-2-N-pentanoylamino-D- mannose
  • the functional variant of the heterologous regulator protein is preferably a fusion protein, preferably said fusion protein comprising at least one first domain comprising an adenovirus PIX regulator protein and at least one second domain that modulates or expands the activity or the subcellular distribution of the adenovirus pIX; and/or said at least one second domain comprises a protein or peptide acting as a transcription modulator, preferably said transcription modulator is a transcription factor including the retinoic acid receptor alpha, is a marker protein, preferably said marker protein is a fluorescence marker including GFP, DsRed and its variants or is an enzyme including LacZ, or is a transit peptide including a NLS.
  • the disease to be treated is selected from the group of AlAT deficiency, deficiency emphysema, neonatal cholestasis, chronic hepatic cirrhosis and cystic fibrosis.
  • the disease is AlAT deficiency.
  • the person of skill in the art will be able to determine further treatment and/or prevention methods of diseases and for human therapy.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention porte sur des procédés de modulation de l'état de glycosylation de l'antitrypsine alpha-1 humaine (A1AT) dans des cellules hôtes humaines et sur des procédés de production de l'A1AT humaine glycosylée, sur l'antitrypsine alpha-1 humaine obtenue par ces procédés et sur les utilisations de celle-ci. Les procédés de l'invention ont pour résultat une A1AT qui a un motif de glycosylation humain ou qui a un motif de glycosylation modulé. Ainsi, la présente invention permet la production de A1AT avec un état de glycosylation « personnalisé » qui convient pour différentes utilisations, de préférence, en thérapie humaine.
PCT/EP2008/004844 2007-06-15 2008-06-16 Procédés de production de l'antitrypsine alpha-1 humaine glycosylée (a1at) dans des cellules hôtes de mammifère WO2008151845A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP08773472A EP2167539A2 (fr) 2007-06-15 2008-06-16 Procédés de production de l'antitrypsine alpha-1 humaine glycosylée (a1at) dans des cellules hôtes de mammifère

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US94423207P 2007-06-15 2007-06-15
US60/944,232 2007-06-15

Publications (2)

Publication Number Publication Date
WO2008151845A2 true WO2008151845A2 (fr) 2008-12-18
WO2008151845A3 WO2008151845A3 (fr) 2009-09-03

Family

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Family Applications (1)

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PCT/EP2008/004844 WO2008151845A2 (fr) 2007-06-15 2008-06-16 Procédés de production de l'antitrypsine alpha-1 humaine glycosylée (a1at) dans des cellules hôtes de mammifère

Country Status (2)

Country Link
EP (1) EP2167539A2 (fr)
WO (1) WO2008151845A2 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013039295A1 (fr) 2011-09-15 2013-03-21 (주)알테오젠 Nouveau variant d'alpha 1-antitrypsine, et ses procédés de préparation et d'utilisation
WO2019177982A1 (fr) * 2018-03-12 2019-09-19 Protease Pharmaceuticals Inc. Méthodes et compositions pour des troubles d'une maladie associée à l'alpha-1 antitrypsine
US11624080B2 (en) 2017-11-28 2023-04-11 Danmarks Tekniske Universitet Glycosylation of proteins
CN116115629A (zh) * 2016-08-17 2023-05-16 菲克特生物科学股份有限公司 核酸产品及其施用方法

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
COURTNEY M ET AL: "SYNTHESIS IN E. COLI OF ALPHA1-ANTITRYPSIN VARIANTS OF THERAPEUTIC POTENTIAL FOR EMPHYSEMA AND THROMBOSIS" NATURE, NATURE PUBLISHING GROUP, LONDON, UK, vol. 313, 10 January 1985 (1985-01-10), pages 149-151, XP000919013 ISSN: 0028-0836 *
GRABENHORST E ET AL: "Genetic engineering of recombinant glycoproteins and the glycosylation pathway in mammalian host cells" 19990201, vol. 16, no. 2, 1 February 1999 (1999-02-01), pages 81-97, XP002203702 *
HOSOKAWA N ET AL: "Enhancement of endoplasmic reticulum (ER) degradation of misfolded null Hong Kong [alpha]1-antitrypsin by human ER mannosidase I" JOURNAL OF BIOLOGICAL CHEMISTRY 20030711 AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY INC. US, vol. 278, no. 28, 11 July 2003 (2003-07-11), pages 26287-26294, XP002533548 *
KOLARICH DANIEL ET AL: "Comprehensive glyco-proteomic analysis of human alpha(1)-antitrypsin and its charge isoforms" PROTEOMICS, vol. 6, no. 11, June 2006 (2006-06), pages 3369-3380, XP002502192 ISSN: 1615-9853 *
SAMANDARI TARAZ ET AL: "A study of the effects of altering the sites for N-glycosylation in alpha-1-proteinase inhibitor variants M and S" PROTEIN SCIENCE, vol. 2, no. 9, 1993, pages 1400-1410, XP002502191 ISSN: 0961-8368 *
TOROSSI T ET AL: "Endomannosidase processes oligosaccharides of [alpha]1-antitrypsin and its naturally occurring genetic variants in the Golgi apparatus" CMLS CELLULAR AND MOLECULAR LIFE SCIENCES, BIRKHÄUSER-VERLAG, BA, vol. 63, no. 16, 27 July 2006 (2006-07-27), pages 1923-1932, XP019419226 ISSN: 1420-9071 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013039295A1 (fr) 2011-09-15 2013-03-21 (주)알테오젠 Nouveau variant d'alpha 1-antitrypsine, et ses procédés de préparation et d'utilisation
US20140371160A1 (en) * 2011-09-15 2014-12-18 Alteogen, Inc Novel alpha-1 antitrypsin variant, preparation method thereof and use thereof
US9051395B2 (en) * 2011-09-15 2015-06-09 Alteogen, Inc. Alpha-1 antitrypsin variant, preparation method thereof and use thereof
CN116115629A (zh) * 2016-08-17 2023-05-16 菲克特生物科学股份有限公司 核酸产品及其施用方法
US11624080B2 (en) 2017-11-28 2023-04-11 Danmarks Tekniske Universitet Glycosylation of proteins
WO2019177982A1 (fr) * 2018-03-12 2019-09-19 Protease Pharmaceuticals Inc. Méthodes et compositions pour des troubles d'une maladie associée à l'alpha-1 antitrypsine

Also Published As

Publication number Publication date
EP2167539A2 (fr) 2010-03-31
WO2008151845A3 (fr) 2009-09-03

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