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

TLDR

Fc N‑linked glycans modulate IgG1 pharmacokinetics and effector functions, but the impact of different core‑fucose‑lacking glycoforms on ADCC and CDC remains unclear, with the nonreducing end influencing CDC and core‑fucose removal enhancing FcγRIIIa binding. The study aimed to generate core‑fucose‑lacking IgG1 antibodies bearing high‑mannose, hybrid, or complex Fc glycans from a single clone and compare their biological activities. Core‑fucose‑lacking IgG1 were produced by culturing FUT8‑knockout CHO cells with or without glycosidase inhibitors (swainsonine or kifunensine) to yield each glycoform without cross‑contamination. High‑mannose and hybrid glycans reduced FcγRIIIa and C1q binding, diminishing ADCC and CDC, whereas the complex glycan preserved activity and exhibited a longer serum half‑life, highlighting the structural influence of the nonreducing end.

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 α-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 (FcγRIIIa) 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 FcγRIIIa 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.