US2015079633A1PendingUtilityA1

Production of modified glycoproteins having multiple antennary structures

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Assignee: GLYCOFI INCPriority: Jun 28, 2000Filed: Jul 25, 2014Published: Mar 19, 2015
Est. expiryJun 28, 2020(expired)· nominal 20-yr term from priority
Inventors:Piotr BobrowiczStephen HamiltonTillman U. GerngrossStefan WildtByung-Kwon ChoiJuergen Hermann NettRobert C. Davidson
C12P 21/005C12N 9/1051
64
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Claims

Abstract

The present invention relates to lower eukaryotic host cells having modified oligosaccharides which may be modified further by heterologous expression of a set of glycosyltransferases, sugar and sugar nucleotide transporters to become host-strains for the production of mammalian, e.g., human therapeutic glycoproteins. The process provides an engineered host cell which can be used to express and target any desirable gene(s) involved in glycosylation. Host cells with modified lipid-linked oligosaccharides are created or selected. N-glycans made in the engineered host cells exhibit GnTIV, GnTV, GnT VI or GnTIX activity, which produce multiantennary N-glycan structures and may be modified further by heterologous expression of one or more enzymes, e.g., glycosyltransferases, sugar, sugar nucleotide transporters, to yield human-like glycoproteins. For the production of therapeutic proteins, this method may be adapted to engineer cell lines in which any desired glycosylation structure may be obtained.

Claims

exact text as granted — not AI-modified
1 . A process for making a glycoprotein in a lower eukaryotic host cell comprising the step of introducing into the cell an N-acetylglucosaminyltransferase activity selected from the group consisting of: N-acetylglucosaminyltransferase IV activity, an N-acetylglucosaminyltransferase V activity, an N-acetylglucosaminyltransferase VI activity; and an N-acetylglucosaminyltransferase IX activity wherein the glycoprotein comprises at least three antennae on the trimannose core. 
     
     
         2 . A process for making a glycoprotein in a lower eukaryotic host cell comprising the step of expressing in the cell one or more enzymatic activities that produce multiple antennary N-glycans comprising GlcNAcβ1,2-Manα1,6 (GlcNAcβ1,4 GlcNAcβ1,2 Manα1,3) Man β1,4-GlcNAcβ1,4-GlcNAcβ1,4-Asn; GlcNAcβ1,4 GlcNAcβ1,2-Manα1,6 (GlcNAcβ1,4 GlcNAcβ1,2 Manα1,3) Man β1,4-GlcNAcβ1,4-GlcNAcβ1,4-Asn; and GlcNAcβ1,6 GlcNAcβ1,4 GlcNAcβ1,2-Manα1,6 (GlcNAcβ1,4 GlcNAcβ1,2 Manα1,3) Man β1,4-GlcNAcβ1,4-GlcNAcβ1,4-Asn structures. 
     
     
         3 . The process of  claim 1  or  2 , wherein the activity is substantially intracellular. 
     
     
         4 . The process of  claim 1  or  2 , further comprising the step of isolating the glycoprotein from the host cell. 
     
     
         5 . The process of  claim 1  or  2 , wherein the host cell is selected from the group consisting of  Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minutia, Ogataea minuta, Pichia lindneri, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia  sp.,  Saccharomyces cerevisiae, Saccharomyces  sp.,  Hansenula polymorpha, Kluyveromyces  sp.,  Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium  sp.,  Fusarium gramineum, Fusarium venenatum , and  Neurospora crassa.    
     
     
         6 . The process of  claim 5 , wherein the host cell is selected from the group consisting of  Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minutia, Ogataea minuta, Pichia lindneri, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica , and  Pichia  sp. 
     
     
         7 . The process of  claim 6 , wherein the host cell is  Pichia pastoris.    
     
     
         8 . The process of  claim 1  or  2  wherein the glycoprotein is a therapeutic protein. 
     
     
         9 . The process of  claim 8 , wherein the therapeutic protein is selected from the group consisting of kringle domains of human plasminogen, erythropoietin, cytokines, coagulation factors, soluble IgE receptor α-chain, IgG, IgG fragments, IgM, interleukins, urokinase, chymase, urea trypsin inhibitor, IGF binding protein, epidermal growth factor, growth hormone-releasing factor, annexin V fusion protein, angiostatin, vascular endothelial growth factor-2, myeloid progenitor inhibitory factor-1, osteoprotegerin, a-1 antitrypsin, DNase II, α-feto proteins, FSH and peptide hormones. 
     
     
         10 . A lower eukaryotic host cell comprising an N-acetylglucosaminyltransferase IV, V, VI, or IX activity. 
     
     
         11 . A lower eukaryotic host cell comprising an N-acetylglucosaminyltransferase IV activity and an N-acetylglucosaminyltransferase V activity. 
     
     
         12 . The host cell of  claim 10  or  11 , wherein the activity is substantially intracellular. 
     
     
         13 . The host cell of  claim 10  or  11 , wherein the cell produces N-glycans comprising GlcNAc2Man3GlcNAc2 structures that are capable of reacting with GnTIV, GnTV or GnT VI activity. 
     
     
         14 . A lower eukaryotic host cell comprising an N-glycan that comprises at least three GlcNAcs on a Man3GlcNAc2 oligosaccharide. 
     
     
         15 . The host cell of  claim 14 , wherein the N-glycan contains at least 50, 60, 70, 80, 90 mole % or greater triantennary glycans. 
     
     
         16 . The host cell of  claim 14 , wherein the N-glycan contains at least 70, 80, 90 mole % or greater tetraantennary glycans. 
     
     
         17 . A lower eukaryotic host cell comprising multiple antennary N-glycans including GlcNAcβ1,2-Manα1,6 (GlcNAcβ1,4 GlcNAcβ1,2 Manα1,3) Man β1,4-GlcNAcβ1,4-GlcNAcβ1,4-Asn; GlcNAcβ1,4 GlcNAcβ1,2-Manα1,6 (GlcNAcβ1,4 GlcNAcβ1,2 Manα1,3) Man β1,4-GlcNAcpβ1,4-GlcNAcβ1,4-Asn; and GlcNAcβ1,6 GlcNAcβ1,4 GlcNAcβ1,2-Manα1,6 (GlcNAcβ1,4 GlcNAcβ1,2 Manα1,3) Man β1,4-GlcNAcβ1,4-GlcNAcβ1,4-Asn structures. 
     
     
         18 . The host cell of  claim 10 ,  11 ,  14 , or  17 , wherein the host cell is selected from the group consisting of  Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minutia, Ogataea minuta, Pichia lindneri, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia  sp.,  Saccharomyces cerevisiae, Saccharomyces  sp.,  Hansenula polymorpha, Kluyveromyces  sp.,  Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium  sp.,  Fusarium gramineum, Fusarium venenatum , and  Neurospora crassa.    
     
     
         19 . The host cell of  claim 18 , wherein the host cell is selected from the group consisting of  Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minutia, Ogataea minuta, Pichia lindneri, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica , and  Pichia  sp. 
     
     
         20 . The host cell of  claim 19 , wherein the host cell is  Pichia pastoris.    
     
     
         21 .- 51 . (canceled)

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