COMPOSITIONS AND METHODS FOR HUMANIZATION AND OPTIMIZATION OF N-GLYCANS IN PLANTS
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.