Compositions and methods for humanization and optimization of n-glycans in plants

Abstract

Methods for altering the N-glycosylation pattern of proteins in higher plants are provided. The methods comprise introducing into the plant a recombinant construct that provides for the inhibition of expression of α1,3-fucosyltransferase (FucT) and β1,2-xylosyltransferase (XylT) in a plant. Use of these constructs to inhibit or suppress expression of both of these enzymes, and isoforms thereof, advantageously provides for the production of endogenous and heterologous proteins having a “humanized” N-glycosylation pattern without impacting plant growth and development. Stably transformed higher plants having this protein N-glycosylation pattern are provided. Glycoprotein compositions, including monoclonal antibody compositions, having substantially homogeneous glycosylation profiles, and which are substantially homogeneous for the G0 glycoform, are also provided.

1 . A composition comprising a glycoprotein produced by a duckweed plant or duckweed plant cell or nodule that expresses glycoproteins having an altered N-glycosylation pattern, wherein said altered N-glycosylation pattern is characterized by a reduction in the attachment of α1,3-fucose residues, β1,2-xylose residues, or both α1,3-fucose residues and β1,2-xylose residues to N-glycans of said glycoproteins. 
     
     
         2 . The composition of  claim 1 , wherein said N-glycans are devoid of said fucose and xylose residues. 
     
     
         3 . The composition of  claim 2 , wherein said glycoprotein has an N-glycosylation profile characterized by the presence of a single predominant peak corresponding to the G0 glycan species. 
     
     
         4 . The composition of  claim 3 , wherein said glycoprotein is a monoclonal antibody of interest. 
     
     
         5 . A composition comprising a heterologous glycoprotein that has been produced in a higher plant, wherein said higher plant comprises a nucleotide construct comprising a fusion polynucleotide that is capable of inhibiting expression or function of an α1,3-fucosyltransferase (FucT) and a β1,2-xylosyltransferase (XylT) in said plant, wherein said fusion polynucleotide is operably linked to a promoter that is functional in a plant cell. 
     
     
         6 . The composition of  claim 5 , wherein said higher plant is a member of the Lemnaceae. 
     
     
         7 . The composition of  claim 5 , wherein said fusion polynucleotide comprises in the 5′-to-3′ orientation and operably linked:
 (a) a chimeric forward fragment, said chimeric forward fragment comprising:
 (i) a first fragment comprising about 500 to about 650 contiguous nucleotides having at least 90% sequence identity to a nucleotide sequence of about 500 to about 650 contiguous nucleotides of a polynucleotide encoding said FucT; and 
 (ii) a second fragment comprising about 500 to about 650 contiguous nucleotides having at least 90% sequence identity to a nucleotide sequence of about 500 to about 650 contiguous nucleotides of a polynucleotide encoding said XylT; 
 
 (b) a spacer sequence comprising about 200 to about 700 nucleotides; 
 (c) and a reverse fragment, said reverse fragment having sufficient length and sufficient complementarity to said chimeric forward fragment such that said fusion polynucleotide is transcribed as an RNA molecule capable of forming a hairpin RNA structure. 
 
     
     
         8 . The composition of  claim 5 , wherein said glycoprotein has an N-glycosylation profile characterized by the presence of a single predominant peak corresponding to the G0 glycan species. 
     
     
         9 . The composition of  claim 8 , wherein said glycoprotein is a monoclonal antibody of interest.