Methods for producing modified glycoproteins

Abstract

Cell lines having genetically modified glycosylation pathways that allow them to carry out a sequence of enzymatic reactions, which mimic the processing of glycoproteins I humans, have been developed. Recombinant proteins expressed in these engineered hosts yield glycoproteins more similar, if not substantially identical, to their human counterparts. The lower eukaryotes, which ordinarily produce high-mannose containing N-glycans such as Man 5 GlcNA C2 or other structures along human glycosylation pathways. This is achieved using a combination of engineering and/or selection of strains which: do not express certain enzymes which create the undesirable complex structures characteristic of the fungal glycoproteins, which express exogenous enzymes selected either to have optimal activity under the conditions present in the fungi where activity is desired, or which are targeted to an organelle where optimal activity is achieved, and combinations thereof wherein the genetically engineered eukaryote expresses multiple exogenous enzymes required to produce “human-like” glycoproteins.

1 . A methylotrophic yeast host cell that does not display an alpha-1,6 mannosyltransferase activity with respect to an N-glycan on a glycoprotein comprising a nucleic acid encoding an α-1,2-mannosidase enzyme or catalytic domain thereof wherein the host cell is capable of producing a glycoprotein having a Man 5 GlcNAc 2  oligosaccharide structure. 
     
     
         2 . The host cell of  claim 1 , wherein the α-1,2-mannosidase enzyme is targeted to the early, medial, late Golgi or the trans Golgi network of the host cell. 
     
     
         3 . The host cell of  claim 1 , wherein the host cell further comprises a nucleic acid encoding one or more additional polypeptides selected from the group consisting of glycosidases, glycosyltransferases, nucleotide sugar transporters and nucleotide diphosphatases. 
     
     
         4 . The host cell of  claim 3 , wherein the glycosyltransferase is selected from the group consisting of GnT I, GnT II, GnT III, GnT IV, GnT V, GnT VI, GalT, Fucosyltransferase, and ST. 
     
     
         5 . The host cell of  claim 3 , wherein the nucleotide transporter is selected from the group consisting of UDP-GlcNAc transporter, UDP-galactose transporter, GDP-fucose transporter and CMP-sialic acid transporter. 
     
     
         6 . The host cell of  claim 1 , wherein the host cell is capable of producing glycoproteins comprising one or more sugars selected from the group consisting of N-acetylglucosamine, galactose, sialic acid, and fucose. 
     
     
         7 . The host cell of  claim 1 , wherein the host cell is capable of producing glycoproteins comprising at least one oligosaccharide branch comprising the structure NeuNAc-Gal-GlcNAc-Man. 
     
     
         8 . The host cell of  claim 1 , wherein the host cell lacks the activity of one or more enzymes selected from the group consisting of mannosyltransferases and phosphomannosyltransferases. 
     
     
         9 . The host cell of  claim 8 , wherein the host cell lacks an enzyme activity with respect to the N-glycan on a glycoprotein, the deficient activity selected from the group consisting of 1,6 mannosyltransferase; 1,3 mannosyltransferase; and 1,2 mannosyltransferase. 
     
     
         10 . A methylotrophic yeast host cell that does not display an alpha-1,6 mannosyltransferase activity with respect to an N-glycan on a glycoprotein comprising a nucleic acid encoding an α-1,2-mannosidase enzyme or catalytic domain thereof and a nucleic acid encoding a GnT I enzyme or catalytic domain thereof. 
     
     
         11 . A method for producing a recombinant glycoprotein comprising an N-glycan structure that comprises a Man5GlcNAc2 glycoform in a methylotrophic host cell that does not display alpha-1,6 mannosyltransferase activity with respect to the N-glycan on a glycoprotein, the method comprising the step of introducing into the host cell a nucleic acid encoding an α-1,2 mannosidase enzyme, the enzyme comprising: (a) an alpha-1,2 mannosidase catalytic domain fused to (b) a cellular targeting signal peptide not normally associated with the catalytic domain selected to target the mannosidase enzyme to the ER or Golgi apparatus of the host cell; whereby, upon passage of the recombinant glycoprotein through the ER or Golgi apparatus of the host cell, the N-glycan structures attached thereto have predominantly a Man 5 GlcNAc 2  glycoform. 
     
     
         12 . The method of  claim 11 , wherein the α-1,2-mannosidase enzyme is targeted to the early, medial, late Golgi or the trans Golgi network of the host cell. 
     
     
         13 . The method of  claim 11 , further comprising the step of introducing into the host cell one or more additional nucleic acids encoding one or more additional enzymes selected from the group consisting of mannosidases, glycosyltransferases and glycosidases. 
     
     
         14 . The method of  claim 11 , wherein the recombinant glycoprotein comprising the N-glycan is further modified to comprise one or more sugars selected from the group consisting of N-acetylglucosamine, galactose, sialic acid and fucose. 
     
     
         15 . The method of  claim 11 , wherein the recombinant glycoprotein comprising the N-glycan is further modified to comprise at least one oligosaccharide branch comprising the structure NeuNAc-Gal-GLcNAc-Man. 
     
     
         16 . The method of  claim 11 , wherein the host further lacks the activity of one or more enzymes selected from the group consisting of mannosyltransferases and phosphomannosyltransferases. 
     
     
         17 . The method of  claim 16 , wherein the host lacks an enzyme activity with respect to the N-glycan on a glycoprotein, the activity selected from the group consisting of 1,6 mannosyltransferase; 1,3 mannosyltransferase; and 1,2 mannosyltransferase. 
     
     
         18 . The method of  claim 11 , wherein the host cell further comprises a nucleic acid encoding one or more additional polypeptides selected from the group consisting of glycosidases, glycosyltransferases, nucleotide sugar transporters and nucleotide diphosphatases. 
     
     
         19 . The method of  claim 18 , wherein the glycosyltransferase is selected from the group consisting of GnT I, GnT II, GnT III, GnT IV, GnT V, GnT VI, GalT, Fucosyltransferase, and ST. 
     
     
         20 . The method of  claim 18 , wherein the nucleotide transporter is selected from the group consisting of UDP-GlcNAc transporter, UDP-galactose transporter, GDP-fucose transporter and CMP-sialic acid transporter.