title: Generation of glyco-engineered Nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like N-glycan structure.
abstract: A common argument against using plants as a production system for therapeutic proteins is their inability to perform authentic human N-glycosylation (i.e. the presence of beta1,2-xylosylation and core alpha1,3-fucosylation). In this study, RNA interference (RNAi) technology was used to obtain a targeted down-regulation of the endogenous beta1,2-xylosyltransferase (XylT) and alpha1,3-fucosyltransferase (FucT) genes in Nicotiana benthamiana, a tobacco-related plant species widely used for recombinant protein expression. Three glyco-engineered lines with significantly reduced xylosylated and/or core alpha1,3-fucosylated glycan structures were generated. The human anti HIV monoclonal antibody 2G12 was transiently expressed in these glycosylation mutants as well as in wild-type plants. Four glycoforms of 2G12 differing in the presence/absence of xylose and core alpha1,3-fucose residues in their N-glycans were produced. Notably, 2G12 produced in XylT/FucT-RNAi plants was found to contain an almost homogeneous N-glycan species without detectable xylose and alpha1,3-fucose residues. Plant-derived glycoforms were indistinguishable from Chinese hamster ovary (CHO)-derived 2G12 with respect to electrophoretic properties, and exhibited functional properties (i.e. antigen binding and HIV neutralization activity) at least equivalent to those of the CHO counterpart. The generated RNAi lines were stable, viable and did not show any obvious phenotype, thus providing a robust tool for the production of therapeutically relevant glycoproteins in plants with a humanized N-glycan structure.
authors.affiliation: Institute of Applied Genetics and Cell Biology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Richard
authors.initials: R
authors.lastname: Strasser
authors.forename: Johannes
authors.initials: J
authors.lastname: Stadlmann
authors.forename: Matthias
authors.initials: M
authors.lastname: Schähs
authors.forename: Gabriela
authors.initials: G
authors.lastname: Stiegler
authors.forename: Heribert
authors.initials: H
authors.lastname: Quendler
authors.forename: Lukas
authors.initials: L
authors.lastname: Mach
authors.forename: Josef
authors.initials: J
authors.lastname: Glössl
authors.forename: Koen
authors.initials: K
authors.lastname: Weterings
authors.forename: Martin
authors.initials: M
authors.lastname: Pabst
authors.forename: Herta
authors.initials: H
authors.lastname: Steinkellner
mesh_terms: D000911:Antibodies, Monoclonal / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D005818:Genetic Engineering; D006031:Glycosylation; D015483:HIV Antibodies / Q000378:metabolism*; D015488:HIV Antigens / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D009154:Mutation; D011134:Polysaccharides / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding; D034622:RNA Interference; D014026:Nicotiana / Q000235:genetics*
chemical_list: D000911:Antibodies, Monoclonal; D015483:HIV Antibodies; D015488:HIV Antigens; D007074:Immunoglobulin G; D011134:Polysaccharides