DK2508612T3 - Glykosylering af molekyler - Google Patents

Glykosylering af molekyler Download PDF

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DK2508612T3
DK2508612T3 DK12000268.8T DK12000268T DK2508612T3 DK 2508612 T3 DK2508612 T3 DK 2508612T3 DK 12000268 T DK12000268 T DK 12000268T DK 2508612 T3 DK2508612 T3 DK 2508612T3
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cell
protein
glycosylation
cells
polypeptide
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DK12000268.8T
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Nico Luc Marc Callewaert
Wouter Vervecken
Pourq Karen Jacqueline Marcel De
Steven Christian Jozef Geysens
Mouna Guerfal
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Oxyrane Uk Ltd
Vib Vzw
Univ Gent
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    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
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    • C12N15/09—Recombinant DNA-technology
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    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2465—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)
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    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
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    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
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    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01045—Glucosylceramidase (3.2.1.45), i.e. beta-glucocerebrosidase
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Claims (17)

1. Fremgangsmåde til fremstilling af en ændret N-glykosyleringsform af et målprotein eller -polypeptid, hvilken fremgangsmåde omfatter: a) at tilvejebringe en celle, der er gensplejset til at omfatte mindst to modificerede N-glykosyleringsaktiviteter i forhold til en ikke-gensplejset celle af samme art og indføre i cellen en nukleinsyre, der koder for målproteinet eller -polypeptidet, hvor cellen producerer målproteinet eller -polypeptidet i en ændret N-glykosyleringsform, hvilken celle er en Yarrowia lipolytica-celle, en Arxula adeninivorans eller en celle fra en relateret art af dimorf gær; eller b) at bringe målproteinet eller -polypeptidet i kontakt med et cellelysat fremstillet ud fra en celle, der er gensplejset til at omfatte mindst to modificerede N-glykosyleringsaktiviteter i forhold til en ikke-gensplejset celle af samme art, hvilken celle er en Yarrowia lipolytica-celle, en Arxula adeninivorans eller en relateret art af dimorf gær; hvor de mindst to modificerede N-glykosyleringsaktiviteter omfatter en mangel på OCH1-aktivitet og en ekspression af et alpha-1,2-mannosidase-polypeptid, der er målrettet til det endoplasmatiske reticulum, hvor målproteinet eller -polypeptidet er et farmaceutisk egnet protein eller polypeptid, og hvor mindst 90 % af N-glykanstrukturerne af målproteinet er Man5GlcNAc2.
2. Fremgangsmåde ifølge krav 1, hvor alpha-mannosidasen er MNS1.
3. Fremgangsmåde ifølge krav 1, hvor alpha-mannosidasen er Saccharomy-ces cerews/ae-MNS1-polypeptidet eller S. cerews/ae-MNS1-polypeptidet, der omfatter en eller flere aminosyresubstitutioner udvalgt fra gruppen bestående af R273L, R269S og S272G.
4. Fremgangsmåde ifølge krav 1, hvor alpha-mannosidasen er en Trichoderma reesei-alpha-1,2-mannosidase.
5. Fremgangsmåde ifølge krav 1, hvor mannosidase-polypeptidet omfatter, fusioneret dermed, et FIDEL-endoplasmatisk reticulum-retentionspeptid.
6. Fremgangsmåde ifølge krav 1, hvor mannosidase-polypeptidet er et fusionsprotein, der omfatter en alpha-mannosidase og et FIDEL-endoplasmatisk reticulum-retentionspeptid.
7. Fremgangsmåde ifølge krav 1, hvor alpha-mannosidasen er en Trichoderma reese/-alpha-1,2-mannosidase fusioneret med et HDEL-endoplasmatisk reticulum-retentionspeptid.
8. Fremgangsmåde ifølge et hvilket som helst af kravene 1-7, hvor målproteinet eller -polypeptidet er heterologt til cellen.
9. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvor målproteinet eller -polypeptidet er et humant protein eller polypeptid.
10. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, der yderligere omfatter at tilvejebringe en farmaceutisk sammensætning, der omfatter en terapeutisk virksom mængde af det farmaceutisk egnede protein eller polypeptid og en eller flere adjuvanser, excipienser, bærere og/eller fortyndingsmidler.
11. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvor målproteinet eller -polypeptidet er egnet til behandling af en lysosomal lagringssygdom, er et antistof eller et antigenbindende fragment deraf, er et patogent protein, er en vækstfaktor, er et cytokin, er et kemokin eller er et fusionsprotein.
12. Fremgangsmåde ifølge krav 10 eller 11, hvor målproteinet eller -polypeptidet er et, der er egnet til behandling af en lysosomal lagringssygdom og er udvalgt fra gruppen bestående af glucocerebrosidase, galactoce-rebrosidase, alpha-L-iduronidase, beta-D-galactosidase, beta-glucosidase, beta-hexosaminidase, beta-D-mannosidase, alpha-L-fucosidase, arylsulfata-se B, arylsulfatase A, alpha-N-acteylgalactosaminidase, aspartylglucosa-minidase, iduronat-2-sulfatase, alpha-glucosaminid-N-acetyltransferase, be-ta-D-glucoronidase, hyaluronidase, alpha-L-mannosidase, alpha-neuraminidase, phosphotransferase, syrelipase, syre-ceramidase, sphinogmyelinase, thioesterase, cathepsin K og lipoproteinlipase; eller er et fusionsprotein omfattende et hvilket som helst af de førnævnte.
13. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvor cellen er en gensplejset Yarrowia lipolytica-ce\\e.
14. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, hvor cellen er en gensplejset Arxula adeninivorans-celle.
15. Fremgangsmåde ifølge et hvilket som helst af de foregående krav, yderligere omfattende at isolere målproteinet eller -polypeptidet.
16. Celle, der er gensplejset til at producere et målprotein eller -polypeptid i ændret N-glykosyleringsform, hvor cellen er en Yarrowia lipolytica-celle, en Arxula adeninivorans-ce\\e eller en celle fra en relateret art af dimorf gær, og cellen er gensplejset til at omfatte mindst to modificerede N-glykosyleringsaktiviteter i forhold til en ikke-gensplejset celle af den samme art, hvilke mindst to modificerede N-glykosyleringsaktiviteter omfatter en mangel på OCH1-aktivitet og en ekspression af et alpha-1,2-mannosidase-polypeptid, der er målrettet til det endoplasmatiske reticulum, hvilken celle yderligere omfatter en nukleinsyre, der koder for målproteinet eller -polypeptidet, således at cellen producerer målproteinet eller -polypeptidet i en ændret N-glykosyleringsform, hvor mindst 90 % af N-glykanstrukturerne af målproteinet er Man5GlcNAc2.
17. I det væsentlige ren kultur af Yarrowia lipolytica-celler, eller af Arxula adeninivorans-celler eller af celler fra en anden art af en celle fra en relateret art af dimorf gær, hvor et væsentligt antal af cellerne er celler ifølge krav 16.
DK12000268.8T 2007-04-03 2008-04-03 Glykosylering af molekyler DK2508612T3 (da)

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US8202523B2 (en) * 2005-09-22 2012-06-19 ProSci, Inc. Glycosylated polypeptides produced in yeast mutants and methods of use thereof
US8911964B2 (en) 2006-09-13 2014-12-16 Abbvie Inc. Fed-batch method of making human anti-TNF-alpha antibody
CA2910619A1 (en) 2006-09-13 2008-03-20 Abbvie Inc. Cell culture improvements
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