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 in 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, including unicellular and multicellular fungi are modified to produce N-glycans such as Man 5 GlcNAc 2 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 - 34 . (canceled) 
     
     
         35 . A host cell that is a unicellular or filamentous fungus that does not display alpha-1,6 mannosyltransferase activity with respect to the N-glycan on a glycoprotein and which provides N-glycans having terminal galactose residues in the trans Golgi or TGN, having in its endoplasmic reticulum (ER) or Golgi apparatus a hybrid enzyme selected to have optimal activity in the ER or Golgi of said host cell, the hybrid enzyme comprising:
 a sialyltransferase catalytic domain fused to a cellular targeting signal peptide not normally associated with the catalytic domain, wherein said cellular targeting signal peptide targets said exogenous sialyltransferase catalytic domain to said ER or Golgi apparatus;   said host cell further comprising a Golgi CMP-sialic acid transporter.   
     
     
         36 . The host cell of claim  1 , wherein said sialyltransferase is an α2,3-sialyltransferase or α2,6-sialyltransferase. 
     
     
         37 . The host cell of any one of claims  1 , wherein the cellular targeting signal peptide is selected from KTR1, MNN1 ( S. cerevisiae ), MNT1 ( S. cerevisiae ), Kre2/Mnt1 ( S. cerevisiae ), Kre2 ( P. pastoris ), Ktr1 ( S. cerevisiae ), Ktr1 ( P. pastoris ), and MNN1 ( S. cerevisiae ). 
     
     
         38 . The host cell of claim  1 , wherein the host cell further includes at least one fusion protein comprising a catalytic domain of a glycosyltransferase selected from the group consisting of GnT III, GnT IV, GnT V, GnT VI fused to a cellular targeting signal peptide not normally associated with the catalytic domain that targets it to the endoplasmic reticulum, the early, medial or late Golgi, or the trans Golgi network of the host cell. 
     
     
         39 . The host cell of claim  1 , further expressing one or more exogenous enzymes selected from the group consisting of UDP-GlcNAc transferase and nucleotide diphosphatases. 
     
     
         40 . The host cell of claim  1 , further expressing one or more exogenous enzymes selected from: a UDP-specific diphosphatase and a GDP-specific diphosphatase. 
     
     
         41 . The host cell of claim  1 , obtained by introducing nucleic acid molecules encoding said enzymes. 
     
     
         42 . The host cell of claim  31 , wherein at least one of the nucleic acid molecules encoding said enzymes is integrated into the host cell chromosome. 
     
     
         43 . The host cell of claim  1 , which is selected from the group consisting of  Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, 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,  and  Trichoderma reesei.    
     
     
         44 . The host cell of claim  1 , which is additionally deficient in the activity of one or more enzymes selected from the group consisting of mannosyltransferases and phosphomannosyltransferases. 
     
     
         45 . The host cell of claim  34 , which does additionally not express an enzyme selected from the group consisting of 1,6 mannosyltransferase; 1,3 mannosyltransferase; and 1,2 mannosyltransferase with respect to the N-glycan. 
     
     
         46 . The host cell of claim  1 , wherein the host is an OCH1 mutant of  P. pastoris.    
     
     
         47 . A method for producing a glycoprotein comprising expressing the glycoprotein in a host cell of claim  1 . 
     
     
         48 . The method of  claim 37 , wherein said glycoprotein comprises N-glycans having a structure selected from the group consisting of NANA (1-4) Gal (1-4) GlcNAc (1-4) Man 3 GlcNAc 2 . 
     
     
         49 . The method of any one of  claims 37 , wherein said glycoprotein is selected from the group consisting of erythropoietin, interferon-α, interferon-β, interferon-γ, interferon-ω, granulocyte-CSF, factor VIII, factor IX, human protein C soluble IgE receptor α-chain, IgG, IgM; 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 and osteoprotegerin. 
     
     
         50 . The method of any one of  claims 37 , further comprising the step of isolating the glycoprotein from the host. 
     
     
         51 . The method of  claim 40 , further comprising the step of subjecting the isolated glycoprotein to at least one further glycosylation reaction in vitro, subsequent to its isolation from the host.