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

Abstract

Summary 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.

The “less-is-more” in therapeutic antibodies: Afucosylated anti-cancer antibodies with enhanced antibody-dependent cellular cytotoxicity

“…, 93 These sugars are immunogenic to humans, and serum antibodies against core xylose and core α1,3-fucose have been detected in healthy human blood donors 94 . Strategies to overcome this immunogenicity include use of RNAi knockdown of α1,3-fucosyltransferase (FucT) and β1,2-xylosyltransferase (XylT) in plants 95 , 96 and FucT/XylT-knockout lines 97 98 An afucosylated anti-CD30 monoclonal antibody with G0 structure was produced using glycoengineered aquatic plant Lemna minor and shown to have improved ADCC over the same CHO cell-produced antibody 95 .…”

“…, 98 An afucosylated anti-CD30 monoclonal antibody with G0 structure was produced using glycoengineered aquatic plant Lemna minor and shown to have improved ADCC over the same CHO cell-produced antibody 95 . Anti-HIV 2G12 produced in XylT/FucT-knockdown N. benthamiana was found to be homogeneous G0 structures with terminal N -acetylglucosamine and lacking both xylose and α1,3-fucose residues 96 . Further glycoengineering in XylT/FucT knockdown N. benthamiana by expressing a modified human β1,4-galactosyltransferase was reported to produce anti-HIV monoclonal antibodies with fully β1,4-galactosylated N -glycans and improved virus neutralization potency 99 …”

Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant

“…By using a transgenic line of N. benthamiana (ΔXTFT) lacking plant-specific N-glycan residues (14), mAbs with highly homogenous human-like glycoforms can be generated (9,14,15). Comparing the efficacy of h-13F6 produced aglycosylated and glycosylated in two different production systems may help identify the mechanism(s) of action and a preferred manufacturing system for continued development of the EBOV therapeutic.…”

Role of complexN-glycans in plant stress tolerance

“…8 Since then, several mutants and transgenic plants altered in N-glycan maturation in the Golgi apparatus have been reported. [9][10][11][12] Plants with altered N-glycan modification pathways that are devoid of potentially immunogenic complex N-glycans are used for the production of pharmaceutical proteins 12,13 and could serve as potential food crops with reduced allergenicity. Until recently, however, plant complex N-glycans have not been associated with essential biological functions in their host plants due to lack of obvious phenotypes of mutant plants defective in complex N-glycan biosynthesis.…”