Production of modified glycoproteins having multiple antennary structures

Abstract

The present invention relates to eukaryotic host cells, especially 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 GnTIII, GnTIV, GnTV, GnT VI or GnTIX activity, which produce bisected and/or 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.

1 . A process for making a glycoprotein in a lower eukaryotic host cell engineered to produce glycoproteins having GlcNAc 2 Man 3 GlcNAc 2  N-glycans comprising the step of introducing into the cell a nucleic acid encoding 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, whereby a glycoprotein comprising at least three antennae on a trimannose core is produced. 
     
     
         2 . A process for making a glycoprotein in a lower eukaryotic host cell engineered to produce glycoproteins having GlcNAc 2 Man 3 GlcNAc 2  N-glycans comprising the step of expressing in the cell one or more nucleic acids each nucleic acid encoding an enzymatic activity that produces multiple antennary N-glycan structures selected from the group consisting of GlcNAc β 1,2-Manα1,6 (GlcNAc β1,4 GlcNAc β 1,2 Manα1,3) Man β1,4-GlcNAc β 1,4-GlcNAcβ 1,4-Asn; GlcNAc β1,4 GlcNAc β 1,2-Manα1,6 (GlcNAc β1,4 GlcNAc β 1,2 Manα1,3) Man β1,4-GlcNAc β 1,4-GlcNAcβ 1,4-Asn; and GlcNAc β1,6 GlcNAc β1,4 GlcNAc β 1,2-Manα1,6 (GlcNAc β1,4 GlcNAc β 1,2 Manα1,3) Man β1,4-GlcNAc β P1,4-GlcNAcβ 1,4-Asn. 
     
     
         3 . The process of  claim 1 , wherein the activity is intracellular. 
     
     
         4 . The process of  claim 1 , further comprising the step of isolating the glycoprotein from the host cell. 
     
     
         5 . The process of  claim 1 , 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 . (canceled) 
     
     
         7 . (canceled) 
     
     
         8 . The process of  claim 1  wherein the glycoprotein is a therapeutic protein. 
     
     
         9 . The process of  claim 8 , wherein the therapeutic protein is selected from the group consisting 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, α-1 antitrypsin, DNase II, α-feto proteins, FSH and peptide hormones. 
     
     
         10 . A lower eukaryotic host cell comprising a nucleic acid encoding an N-acetylglucosaminyltransferase (GnT) Iv, V, VI, or IX activity. 
     
     
         11 . A lower eukaryotic host cell comprising a nucleic acid encoding an N-acetylglucosaminyltransferase IV activity and a nucleic acid encoding an N-acetylglucosaminyltransferase V activity. 
     
     
         12 . The host cell of  claim 10 , wherein the activity is intracellular. 
     
     
         13 . The host cell of  claim 10 , wherein the cell has been engineered to produce glycoproteins comprising N-glycans comprising GlcNAc 2 Man 3 GlcNAc 2  structures that are capable of reacting with the GnT IV, GnT V, or GnT VI activity. 
     
     
         14 . A lower eukaryotic host cell capable of producing a glycoprotein comprising an N-glycan that comprises at least three GlcNAcs on a Man 3 GlcNAc 2  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 . (canceled) 
     
     
         17 . A lower eukaryotic host cell capable of producing a glycoprotein comprising multiple antennary N-glycan structures selected from the group consisting of GlcNAc β 1,2-Manα1,6 (GlcNAc β1,4 GlcNAc β 1,2 Manα1,3) Man β1,4-GlcNAc β 1,4-GlcNAcβ 1,4-Asn; GlcNAc β1,4 GlcNAc β 1,2-Manα1,6 (GlcNAc β1,4 GlcNAc β 1,2 Manα1,3) Man β1,4-GlcNAc β 1,4-GlcNAcβ 1,4-Asn; and GlcNAc β1,6 GlcNAc β1,4 GlcNAc β 1,2-Manα1,6 (GlcNAc β1,4 GlcNAc β 1,2 Manα1,3) Man ⊕1,4-GlcNAc β 1,4-GlcNAcβ 1,4-Asn. 
     
     
         18 . The host cell of  claim 10 , 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 . (canceled) 
     
     
         20 . (canceled) 
     
     
         21 . (canceled) 
     
     
         22 . (canceled) 
     
     
         23 . (canceled) 
     
     
         24 . A lower eukaryotic host cell comprising a nucleic acid encoding an N-acetylglucosaminyltransferase I activity, a nucleic acid encoding an N-acetylglucosaminyltransferase II activity and a nucleic acid encoding an N-acetylglucosaminyltransferase IV activity. 
     
     
         25 . The host cell of  claim 24 , wherein the activities are substantially intracellular. 
     
     
         26 . The host cell of  claim 24 , wherein the cell is capable of producing a glycoprotein comprising N-glycans comprising GlcNAc 2 Man 3 GlcNAc 2  structures that are capable of reacting with GnTV, GnTVI or GnTIX activity. 
     
     
         27 . The host cell of  claim 24 , wherein the N-acetylglucosaminyltransferase activities produce a glycoprotein comprising a multiple antennary glycan. 
     
     
         28 . A lower eukaryotic host cell comprising a nucleic acid encoding a GnTIV, V, VI or IX activity and a nucleic acid encoding a mannosidase II activity. 
     
     
         29 . The host cell of  claim 28 , further comprising a nucleic acid encoding an N-acetylglucosaminyltransferase I activity. 
     
     
         30 . The host cell of  claim 29 , further comprising a nucleic acid encoding an N-acetylglucosaminyltransferase II activity. 
     
     
         31 . The host cell of  claim 28 , further comprising a nucleic acid encoding an N-acetylglucosaminyltransferase I activity and a nucleic acid encoding an N-acetylglucosaminyltransferase II activity. 
     
     
         32 . (canceled) 
     
     
         33 . (canceled) 
     
     
         34 . (canceled) 
     
     
         35 . (canceled) 
     
     
         36 . The glycoprotein made by the process of  claim 1 . 
     
     
         37 . The glycoprotein made by the process of  claim 2 . 
     
     
         38 . (canceled) 
     
     
         39 . (canceled) 
     
     
         40 . (canceled) 
     
     
         41 . (canceled) 
     
     
         42 . (canceled) 
     
     
         43 . A glycoprotein comprising at least three GlcNAcβ1,4 residues or at least three GlcNAcβ1,6 residues on a GlcNAc 2 Man 3 GlcNAc 2  core structure, wherein said glycoprotein is produced in a lower eukaryotic host cell. 
     
     
         44 . A glycoprotein comprising a GlcNAcβ1,4 residue attached to either the Manα1,3 or Manα1,6 arm of a substrate selected from the group consisting of GlcNAc 2 Man 3 GlcNAc 2 , GlcNAc 3 Man 3 GlcNAc 2 , GlcNAcMan 3 GlcNAc 2 , and Man 3 GlcNAc 2 , wherein said glycoprotein is produced in a lower eukaryotic host cell. 
     
     
         45 . (canceled) 
     
     
         46 . The glycoprotein of  claim 44 , wherein greater than 50 mole % of the core structures are modified by GnTV. 
     
     
         47 . (canceled) 
     
     
         48 . A vector capable of expressing GnTIV, GnTV, GnTVI, or GnTIX activity in a lower eukaryotic host. 
     
     
         49 . A lower eukaryotic host cell capable of producing a glycoprotein comprising N-glycans having at least two GlcNAc residues on either the Manα1,3 or Manα1,6 arm of the trimannose core oligosaccharide intermediate. 
     
     
         50 . (canceled) 
     
     
         51 . (canceled) 
     
     
         52 . A process for making a glycoprotein in a yeast or filamentous fungal host cell engineered to produce a glycoproteins having GlcNAc 2 Man 3 GlcNAc 2  N-glycans comprising the step of introducing into the cell a nucleic acid encoding 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, whereby a glycoprotein comprising at least three antennae on a trimannose core is produced. 
     
     
         53 . The process of  claim 52  wherein the activity is intracellular. 
     
     
         54 . The process of  claim 52 , further comprising the step of isolating the glycoprotein from the host cell. 
     
     
         55 . The process of  claim 52 , 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.    
     
     
         56 . The process of  claim 52  wherein the glycoprotein is a therapeutic protein. 
     
     
         57 . The process of  claim 56 , wherein the therapeutic protein is selected from the group consisting 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, α-1 antitrypsin, DNase II, α-feto proteins, FSH and peptide hormones. 
     
     
         58 . A yeast or filamentous fungal host cell comprising a nucleic acid encoding an N-acetylglucosaminyltransferase (GnT) IV, V, VI, or IX activity. 
     
     
         59 . The host cell of  claim 58 , wherein the activity is intracellular. 
     
     
         60 . The host cell of  claim 58 , wherein the cell has been engineered to produce glycoproteins comprising N-glycans comprising GlcNAc 2 Man 3 GlcNAc 2  structures that are capable of reacting with GnT IV, GnT V, or GnT VI activity. 
     
     
         61 . The host cell of  claim 58  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.    
     
     
         62 . A process for making a glycoprotein in a  Pichia pastoris  host cell engineered to produce a glycoproteins having GlcNAc 2 Man 3 GlcNAc 2  N-glycans comprising the step of introducing into the cell a nucleic acid encoding 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, whereby a glycoprotein comprising at least three antennae on a trimannose core is produced. 
     
     
         63 . The process of  claim 62  wherein the activity is intracellular. 
     
     
         64 . The process of  claim 62 , further comprising the step of isolating the glycoprotein from the host cell. 
     
     
         65 . The process of  claim 62  wherein the glycoprotein is a therapeutic protein. 
     
     
         66 . The process of  claim 65 , wherein the therapeutic protein is selected from the group consisting 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, α-1 antitrypsin, DNase II, α-feto proteins, FSH and peptide hormones. 
     
     
         67 . A  Pichia pastoris  host cell comprising a nucleic acid encoding an N-acetylglucosaminyltransferase (GnT) IV, V, VI, or IX activity. 
     
     
         68 . The host cell of  claim 67 , wherein the activity is intracellular. 
     
     
         69 . The host cell of  claim 58 , wherein the cell has been engineered to produce glycoproteins comprising N-glycans comprising GlcNAc 2 Man 3 GlcNAc 2  structures that are capable of reacting with GnT IV, GnT V, or GnT VI activity.