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 . A method for producing glycoproteins having carbohydrate structures similar to those produced by human cells in a lower eukaryote comprising providing a unicellular or multicellular fungal host, which does not express one or more enzymes involved in production of high mannose structures, and introducing into the host one or more enzymes for production of a carbohydrate structure selected from the group consisting of Man 5 GlcNAc 2 , Man 8 GlcNAc 2  and Man 9 GlcNAc 2 , wherein the enzymes are selected to have optimal activity at the pH of the location in the host where the carbohydrate structure is produced or which are targeted to a subcellular location in the host where enzyme will have optimal activity to produce the carbohydrate structure. 
     
     
         2 . The method of  claim 1  wherein the host is deficient in the activity of one or more enzymes selected from the group consisting of mannosyltransferases and phosphomannosyltransferases. 
     
     
         3 . The method of  claim 2  wherein the host does not express an enzyme selected from the group consisting of 1,6 mannosyltransferase, 1,3 mannosyltransferase, and 1,2 mannosyltransferase. 
     
     
         4 . The method of  claim 1  wherein the host 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.    
     
     
         5 . The method of  claim 2  wherein the host is an OCH1 mutant of  P. pastoris.    
     
     
         6 . The method of  claim 1  comprising introducing into the host a nucleotide molecule encoding one or more mannosidases involved in the production of Man 5 GlcNAc 2  from Man 8 GlcNAc 2  or Man 9 GlcNAc 2 . 
     
     
         7 . The method of  claim 6  where the at least one mannosidase has a pH optimum within 1.4 pH units of the average pH optimum of other representative enzymes in the organelle in which the mannosidase is localized, or having optimal activity at a pH between 5.1 and 8.0. 
     
     
         8 .- 9 . (canceled) 
     
     
         10 . The method of  claim 1  comprising providing a host that is able to form Man 5 GlcNAc 2  structures, displaying GnT I activity and having UDP-Gn transporter activity. 
     
     
         11 . The method of  claim 1  comprising providing a host which has a UDP specific diphosphatase activity. 
     
     
         12 . The method of  claim 1  comprising introducing into the host one or more enzymes selected from the group consisting of mannosidases, glycosyltransferases and glycosidases, wherein the enzymes are targeted to the endoplasmic reticulum, the early, medial, late Golgi or the trans Golgi network. 
     
     
         13 . The method of  claim 12  wherein the mannosidase enzyme is predominantly localized in the Golgi apparatus or the endoplasmic reticulum. 
     
     
         14 . The method of  claim 12  wherein the enzymes are localized by forming a fusion protein between a catalytic domain of the enzyme and a chimeric localization region encoded by at least one genetic construct formed by the in-frame ligation of a DNA fragment encoding a cellular targeting signal peptide with a DNA fragment encoding a glycosylation enzyme or catalytically active fragment thereof. 
     
     
         15 .- 18 . (canceled) 
     
     
         19 . The method of  claim 1  wherein the glycoprotein includes N-glycans of which greater than 27 mole percent comprise fewer than six mannose residues. 
     
     
         20 . The method of  claim 1  wherein the glycoprotein comprises one or more sugars selected from the group consisting of galactose, sialic acid, and fucose. 
     
     
         21 . The method of  claim 1  wherein the glycoprotein comprises at least one oligosaccharide branch comprising the structure NeuNAc-Gal-GlcNAc-Man. 
     
     
         22 . The method of  claim 1  wherein the glycoprotein comprises N-glycans having fewer than four mannose residues. 
     
     
         23 . The method of  claim 1  wherein subsequent to isolation from the host, the glycoprotein is subjected to at least one further glycosylation or carboxylation reaction in vitro. 
     
     
         24 . The method of  claim 1  comprising the steps of
 (a) providing a DNA library comprising at least two genes encoding exogenous glycosylation enzymes; 
 (b) transforming the host with the library to produce a genetically mixed population expressing at least two distinct exogenous glycosylation enzymes; and 
 (c) selecting from the population a host producing the desired glycosylation phenotype. 
 
     
     
         25 .- 31 . (canceled) 
     
     
         32 . The host produced by the method of  claim 1 . 
     
     
         33 .- 34 . (canceled)