Yeast strain for the production of proteins with terminal alpha-1,3-linked galactose

Abstract

Lower eukaryotic host cells have been engineered to produce glycoprotein having at least one terminal α-galactosyl epitope. The glycoproteins are useful for the production of highly antigenic glycoprotein compositions with advantages for the production of vaccines.

1 . A yeast or filamentous fungus host cell that can produce recombinant glycoproteins having N-glycans that have at least one terminal α-galactosyl epitope. 
     
     
         2 . The host cell of  claim 1  wherein said host cell is impaired in initiating α-1,6 mannosyltransferase activity with respect to the glycan on the glycoprotein. 
     
     
         3 . The host cell of  claim 1  wherein the host cell is diminished or depleted in dolichy-P-Man:Man5GlcNAc2-PP-dolichyl α-1,3 mannosyltransferase activity. 
     
     
         4 . The host cell of  claim 1  wherein said host cell expresses a mannosidase activity selected from the group consisting of an α-1,2 mannosidase I activity, mannosidase II activity, mannosidase IIx activity and class III mannosidase activity. 
     
     
         5 . The host cell of  claim 1  wherein said host cell expresses an N-acetyl glucosamine transferase I (GnT) activity a β-galactosyl transferase (β-GalT) activity, and a UDP-galactose-4 epimerase activity. 
     
     
         6 . The host cell 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 minuta  ( Ogataea minuts, 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 luchnowense, Fusarium  sp.,  Fursarium gramineum, Fusarium venenatum, Physcomitrella patens  and  Neurospora crassa.    
     
     
         7 . The host of  claim 1  wherein the host expresses a fusion protein comprising a catalytic domain of an α-galactosyltransferase linked to a targeting peptide that targets the fusion protein to the ER or Golgi of the host cell. 
     
     
         8 . The host cell of  claim 1 , wherein the host cell produce glycoproteins having a N-glycan comprising a terminal α-galactosyl epitope, wherein the predominant N-glycan has a structure selected from the group consisting of:
 (α-Gal)(β-Gal)GlcNAcMan5GlcNAc2; 
 (α-Gal)(β-Gal)2GlcNAc2Man3GlcNAc2; and 
 (α-Gal)2(β-Gal)2GlcNAc2Man3GlcNAc2. 
 
     
     
         9 . The host cell of  claim 1 , wherein the host cell has been engineered to produce a vaccine protein. 
     
     
         10 . The host cell of  claim 9 , wherein the vaccine protein is selected from the group consisting of an anti-viral vaccine glycoprotein, an anti-bacterial vaccine glycoprotein and an anti-cancer vaccine glycoprotein. 
     
     
         11 . A method for producing a host cell capable of producing N-glycans that have terminal α-galactosyl residues, comprising:
 (a) providing a recombinant yeast or filamentous fungus host cell capable of producing N-glycans that have at least one terminal α-galactose on the non-reducing end of the N-glycan; and 
 (b) introducing a nucleic acid encoding an fusion protein comprising the catalytic domain of an α-galactosyltransferase linked to a targeting peptide that targets the fusion protein to the ER or Golgi of the host cell into the host cell to provide the host cell producing N-glycans that have terminal α-galactosyl residues. 
 
     
     
         12 . (canceled) 
     
     
         13 . The host cell of  claim 1 , wherein the host cell produces human-like glycoproteins, the host cell comprising a nucleic acid encoding a fusion protein capable of transferring an α-galactose residue onto a terminal β-galactose residue of an N-linked oligosaccharide branch of an N-glycan having a trimannose core of a glycoprotein produced by the host cell, wherein the N-linked oligosaccharide branch has a structure selected from the group consisting of:
 Galβ1,4-GlcNAcβ1,2-Manα1,3; 
 Galβ1,4-GlcNAcβ1,4-Manα1,3; 
 Galβ1,4-GlcNAcβ1,2-Manα1,6; 
 Galβ1,4-GlcNAcβ1,4 Manα1,6; and 
 Galβ1,4-GlcNacβ1,6-Manα1,6 
 
     
     
         14 . A recombinant glycoprotein produced by a yeast or filamentous fungal host cell, wherein the N-glycans of said glycoprotein comprises a predominant N-glycan comprising a terminal α-galactosyl epitope. 
     
     
         15 . The recombinant glycoprotein of  claim 14 , wherein the predominant N-glycan has a structure selected from the group consisting of
 (α-Gal)(β-Gal)GlcNAcMan5GlcNAc2;   (α-Gal)(β-Gal)2GlcNAc2Man3GlcNAc2; and   (α-Gal)2(β-Gal)2GlcNAc2Man3GlcNAc2.   
     
     
         16 . A vaccine composition comprising the glycoprotein of  claim 14 . 
     
     
         17 . The vaccine composition of  claim 16 , wherein the predominant N-glycan is selected from the group consisting of
 (α-Gal)(β-Gal)GlcNAcMan5GlcNAc2;   (α-Gal)(β-Gal)2GlcNAc2Man3GlcNAc2; and   (α-Gal)2(β-Gal)2GlcNAc2Man3GlcNAc2.