Generation of glyco-engineered Nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like N-glycan structure

Richard Strasser, Johannes Stadlmann, Matthias Schähs, Gabriela Stiegler, Heribert Quendler, Lukas Mach, Josef Glössl, Koen Weterings, Martin Pabst, Herta Steinkellner*

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

353 Citations (Scopus)

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 β1,2-xylosylation and core α1,3-fucosylation). In this study, RNA interference (RNAi) technology was used to obtain a targeted down-regulation of the endogenous β1,2- xylosyltransferase (XylT) and α1,3-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 α1,3-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 α1,3-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 α1,3-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.

Original languageEnglish
Pages (from-to)392-402
Number of pages11
JournalPlant Biotechnology Journal
Volume6
Issue number4
DOIs
Publication statusPublished - 1 May 2008

Keywords

  • 2G12 anti-HIV antibody
  • Nicotiana benthamiana
  • Plant N-glycosylation
  • Recombinant proteins
  • RNAi glyco-engineering

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