In Vivo Glycan Engineering via the Mannosidase I Inhibitor (Kifunensine) Improves Efficacy of Rituximab Manufactured in Nicotiana benthamiana Plants

Int J Mol Sci. 2019 Jan 7;20(1):194. doi: 10.3390/ijms20010194.


N-glycosylation has been shown to affect the pharmacokinetic properties of several classes of biologics, including monoclonal antibodies, blood factors, and lysosomal enzymes. In the last two decades, N-glycan engineering has been employed to achieve a N-glycosylation profile that is either more consistent or aligned with a specific improved activity (i.e., effector function or serum half-life). In particular, attention has focused on engineering processes in vivo or in vitro to alter the structure of the N-glycosylation of the Fc region of anti-cancer monoclonal antibodies in order to increase antibody-dependent cell-mediated cytotoxicity (ADCC). Here, we applied the mannosidase I inhibitor kifunensine to the Nicotiana benthamiana transient expression platform to produce an afucosylated anti-CD20 antibody (rituximab). We determined the optimal concentration of kifunensine used in the infiltration solution, 0.375 µM, which was sufficient to produce exclusively oligomannose glycoforms, at a concentration 14 times lower than previously published levels. The resulting afucosylated rituximab revealed a 14-fold increase in ADCC activity targeting the lymphoma cell line Wil2-S when compared with rituximab produced in the absence of kifunensine. When applied to the cost-effective and scalable N. benthamiana transient expression platform, the use of kifunensine allows simple in-process glycan engineering without the need for transgenic hosts.

Keywords: ADCC; glycosylation; kifunensine; monoclonal antibody; plant made pharmaceuticals.

MeSH terms

  • Alkaloids / pharmacology*
  • Antibody-Dependent Cell Cytotoxicity / drug effects
  • Antigens, CD20 / metabolism
  • Fucose / metabolism
  • Glycosylation / drug effects
  • Mannose / metabolism
  • Mannosidases / antagonists & inhibitors
  • Mannosidases / metabolism
  • Metabolic Engineering / methods*
  • Polysaccharides / metabolism*
  • Rituximab / pharmacology*
  • Tobacco / drug effects
  • Tobacco / metabolism*


  • Alkaloids
  • Antigens, CD20
  • Polysaccharides
  • kifunensine
  • Fucose
  • Rituximab
  • Mannosidases
  • mannosyl-oligosaccharide 1,2-alpha-mannosidase
  • Mannose