Development of a simple and rapid method for producing non‐fucosylated oligomannose containing antibodies with increased effector function

Abstract

Glycosylation in the Fc region of antibodies has been shown to play an important role in antibody function. In the current study, glycosylation of human monoclonal antibodies was metabolically modulated using a potent alpha-mannosidase I inhibitor, kifunensine, resulting in the production of antibodies with oligomannose-type N-glycans. Growing Chinese hamster ovary cells for 11 days in batch culture with a single treatment of kifunensine was sufficient to elicit this effect without any significant impact on cell viability or antibody production. Antibodies expressed in the presence of kifunensine at a concentration as low as 60 ng/mL contained mainly oligomannose-type glycans and demonstrated increased ADCC activity and affinity for FcgammaRIIIA, but reduced C1q binding. Although the kifunensine-mediated shift to oligomannose-type glycans could, in theory, result in rapid clearance of the antibody through increased mannose receptor binding, the serum levels of antibody in mice were not significantly altered up to 168 h following injection. The use of kifunensine provides a simple and rapid method for the production of antibodies with increased ADCC without the time-consuming need to re-engineer either the antibody molecule or the host cell line.

Molecular modeling of antibodies for the treatment of TNFα‐related immunological diseases

“…Both cell lines deleted for the fucosyl transferase gene and cell lines treated with fucosyl transferase inhibitors express IgG1 afucosylated or poorly fucosylated (Zhou et al. 2008; Ferrara et al. 2011).…”

Tailoring recombinant protein quality by rational media design

“…For example, IgG antibody expressing hybridoma cells generated exclusively oligomannose structures (Man9 to Man5) if the cells were exposed to 80-100 ng/mL kifunensine throughout the culture. At intermediate kifunensine concentrations (40-60 ng/mL), oligomannose abundance settled in the range of 69 to 90% and decreased to about 18% at 20 ng/mL of kifunensine 91 . It is not surprising that they concluded that kifunensine is effective in producing antibodies with oligomannose-type glycans from CHO cells, as no impact on both titer and viability but only on maximum viable cell density was observed.…”

“…As a result, selective inhibition of sialic acid addition by adding a fluorinated sialic acid analog increases fucosylation 112 . Due to its effective inhibition of steps occurring prior to fucose attachment, kifunensine effectively retains oligomannose residues and hence allows to express non fucosylated proteins at concentrations beyond 60 ng/mL 91 . It is important to take into account the existence of a mannosyl-glycoprotein-N-acetylglucosaminyltransferase I-independent (GnT I) fucosylation pathway, as described by various authors when modulating the glycosylation levels, since inhibition of GnT does not necessarily prevent further processing of the oligosaccharide chain including fucosylation.…”

“…A significant amount of research has been performed to produce monoclonal antibodies with increased effector functions including antibody-dependent cell-mediated cytotoxicity (ADCC) 91 and cell-dependent cytotoxicity (CDC) 200 . It has been shown that the N-linked glycan affected the FcγIIIa receptor binding and ADCC activity of the antibodies 201 .…”

“…Cultures supplemented with N-acetylglucosamine (GlcNAc) at the millimolar level resulted in reduced complexity of glycan profiles, hence favoring the G0 glycoform 204 . Media supplementation with kifunensine, a potent α-mannosidase I inhibitor entailed an increase of oligomannose containing monoclonal antibodies (mAb) 91 . That study exhibited increased ADCC activity due to the increase of high mannose (HM) glycans; in particular mannose 8 and 9 entities.…”