Comparison of biological activity among nonfucosylated therapeutic IgG1 antibodies with three different N-linked Fc oligosaccharides: the high-mannose, hybrid, and complex types

Abstract

The structure of asparagine-linked oligosaccharides attached to the antibody constant region (Fc) of human immunoglobulin G1 (IgG1) has been shown to affect the pharmacokinetics and antibody effector functions of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). However, it is still unclear how differences in the N-linked oligosaccharide structures impact the biological activities of antibodies, especially those lacking core fucose. Here, we succeeded in generating core fucose-lacking human IgG1 antibodies with three different N-linked Fc oligosaccharides, namely, a high-mannose, hybrid, and complex type, using the same producing clone, and compared their activities. Cultivation of an alpha-1,6-fucosyltransferase (FUT8) knockout Chinese hamster ovary cell line in the presence or absence of a glycosidase inhibitor (either swainsonine or kifunensine) yielded antibody production of each of the three types without contamination by the others. Two of three types of nonnaturally occurring atypical oligosaccharide IgG1, except the complex type, reduced the affinity for both human lymphocyte receptor IIIa (FcgammaRIIIa) and the C1q component of the complement, resulting in reduction of ADCC and CDC. The bulky structure of the nonreducing end of N-linked Fc oligosaccharides is considered to contribute the CDC change, whereas the structural change in the reducing end, i.e. the removal of core fucose, causes ADCC enhancement through improved FcgammaRIIIa binding. In the pharmacokinetic profile, although no significant difference of human neonatal Fc receptor (FcRn)-binding affinity was observed among the three types, the complex type showed longer serum half-lives than the other types irrespective of core fucosylation in mice, which also suggests the contribution of the nonreducing end structure. The present study provides basic information on the effects of core fucose-lacking N-linked Fc oligosaccharides on antibody biological activities.

The availability of glucose to CHO cells affects the intracellular lipid-linked oligosaccharide distribution, site occupancy and the N-glycosylation profile of a monoclonal antibody

“…Possible reason for specific glycoforms of monoclonal antibodies affect ADCC activity is because that it can interact solely with the Fc-RIIIa receptor of natural killer cells, so that a lack of or deformed glycan structure in immunoglobulin G (IgG) can result in decreased Fcmediated ADCC activities, or even induce an immunogenic response, etc. (Kanda et al, 2006;Nimmerjahn et al, 2007;Shields et al, 2002). Altogether, the production of a biotherapeutic monoclonal antibody needs to be focused not only on the Mab itself, but an appropriate N-glycan structure attachment is also required to maximize therapeutic potential.…”

Lessons for the clinic from rituximab pharmacokinetics and pharmacodynamics

“…Therefore, the Asn297 glycosylation does not affect significantly the half-life of the antibody in vivo. 35 Finally, C1q binding and CDC may be affected by the glycosylation pattern of IgG1 antibodies, but is not significantly modified by defucosylation [38][39][40] Rituximab PK Doses and routes of administration. RTX is usually administered by intravenous (i.v.)…”

Classical galactosaemia: novel insights in IgG N-glycosylation and N-glycan biosynthesis

“…15 Changes in core fucose and sialic acid content of IgG can lead to very substantial functional changes as seen in the antibody-dependent cellular cytotoxicity activity or conversely, may confer anti-inflammatory properties. 16 It is established that before and after initiation of treatment, galactosaemia patients exhibit defects in both assembly and processing of N-glycans. [17][18][19][20][21] These defects resemble those observed in a number of congenital disorders of glycosylation (CDG) type I (N-glycan assembly defects) and II (N-glycan processing defects).…”