Generation of glyco‐engineered <i>Nicotiana benthamiana</i> for the production of monoclonal antibodies with a homogeneous human‐like <i>N</i>‐glycan structure

TLDR

Plants are often rejected as therapeutic protein production systems because they cannot perform authentic human N‑glycosylation, producing beta1,2‑xylose and core alpha1,3‑fucose residues. This study used RNA interference to down‑regulate the endogenous beta1,2‑xylosyltransferase and alpha1,3‑fucosyltransferase genes in *Nicotiana benthamiana*, a widely used recombinant protein host. The authors generated three glyco‑engineered lines with reduced xylosylation and/or core fucosylation and transiently expressed the human anti‑HIV monoclonal antibody 2G12 in these mutants and wild‑type plants. The engineered RNAi lines produced a human anti‑HIV monoclonal antibody 2G12 with an almost homogeneous N‑glycan lacking xylose and core fucose, matching CHO‑derived antibody in electrophoretic profile and functional activity, and the lines were stable, viable, and phenotypically normal, demonstrating a robust platform for human‑like glycoprotein production in plants.

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.