The Absence of Fucose but Not the Presence of Galactose or Bisecting N-Acetylglucosamine of Human IgG1 Complex-type Oligosaccharides Shows the Critical Role of Enhancing Antibody-dependent Cellular Cytotoxicity

Abstract

An anti-human interleukin 5 receptor (hIL-5R) humanized immunoglobulin G1 (IgG1) and an anti-CD20 chimeric IgG1 produced by rat hybridoma YB2/0 cell lines showed more than 50-fold higher antibody-dependent cellular cytotoxicity (ADCC) using purified human peripheral blood mononuclear cells as effector than those produced by Chinese hamster ovary (CHO) cell lines. Monosaccharide composition and oligosaccharide profiling analysis showed that low fucose (Fuc) content of complex-type oligosaccharides was characteristic in YB2/0-produced IgG1s compared with high Fuc content of CHO-produced IgG1s. YB2/0-produced anti-hIL-5R IgG1 was subjected to Lens culinaris aggulutin affinity column and fractionated based on the contents of Fuc. The lower Fuc IgG1 had higher ADCC than the IgG1 before separation. In contrast, the content of bisecting GlcNAc of the IgG1 affected ADCC much less than that of Fuc. In addition, the correlation between Gal and ADCC was not observed. When the combined effect of Fuc and bisecting GlcNAc was examined in anti-CD20 IgG1, only a severalfold increase of ADCC was observed by the addition of GlcNAc to highly fucosylated IgG1. Quantitative PCR analysis indicated that YB2/0 cells had lower expression level of FUT8 mRNA, which codes ␣1,6-fucosyltransferase, than CHO cells. Overexpression of FUT8 mRNA in YB2/0 cells led to an increase of fucosylated oligosaccharides and decrease of ADCC of the IgG1. These results indicate that the lack of fucosylation of IgG1 has the most critical role in enhancement of ADCC, although several reports have suggested the importance of Gal or bisecting GlcNAc and provide important information to produce the effective therapeutic antibody. Antibody-dependent cellular cytotoxicity (ADCC),1 a lytic attack on antibody-targeted cells, is triggered upon binding of lymphocyte receptors (Fc␥Rs) to the constant region (Fc) of the antibodies. ADCC is considered to be a major function of some of the therapeutic antibodies, although antibodies have multiple therapeutic functions (e.g. antigen binding, induction of apoptosis, and complement-dependent cellular cytotoxicity) (1, 2).One IgG molecule contains two N-linked oligosaccharide sites in its Fc region (3). The general structure of N-linked oligosaccharide on IgG is complex-type, characterized by a mannosyl-chitobiose core (Man3GlcNAc2-Asn) with or without bisecting GlcNAc/L-fucose (Fuc) and other chain variants including the presence or absence of Gal and sialic acid. In addition, oligosaccharides may contain zero (G0), one (G1), or two (G2) Gal.Recent studies have shown that engineering the oligosaccharides of IgGs may yield optimized ADCC. ADCC requires the presence of oligosaccharides covalently attached at the conserved Asn 297 in the Fc region and is sensitive to change in the oligosaccharide structure. In the oligosaccharide, sialic acid of IgG has no effect on ADCC (4). The relationship between the Gal residue and ADCC is controversial. Boyd et al. (4) have shown that obvious change was not found in ADCC after removal of ...

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

“…Similar results were obtained when two other antibodies were produced in CHO cells and YB2/0 cells. Glycan analysis showed that lower level of core fucose in YB2/0 cells-produced antibodies was the main reason for the enhanced ADCC 15 . Analysis showed that YB2/0 cells have significantly lower levels of the Fut8 mRNA than CHO cells.…”

“…Shinkawa et al. subsequently reported that the absence of fucose, but not the presence of galactose or bisecting GlcNAc, is critical for enhancing ADCC 15 . Another study also suggested that the removal of core fucose from antibodies was sufficient to achieve maximal ADCC activity 62 .…”

“…employed rat hybridoma YB2/0 cells to produce humanized anti-human interleukin-5 receptor (IL-5R) IgG1 antibody (KM8399) and compared it against the same antibody produced in CHO cells (KM8404) 15 . Although both antibodies showed similar levels of antigen binding, the ADCC activity of YB2/0-produced KM8399 was 50-fold higher than CHO-produced KM8404.…”

“…These include engineering the Fc region through amino acid mutations 12 and glycoengineering the Fc N -glycan to reduce core fucose. 13-15 It is now widely recognized that removal of the core fucose from Fc N -glycans represents the most effective approach to enhance ADCC activity 14 15 A high-throughput study of the IgG glycome of three isolated human populations showed that most of the human plasma IgG antibodies are core fucosylated with levels of afucosylated IgG ranging from 1.3% to 19.3%, underlying the difference in ADCC efficacy of naturally occurring antibodies to protect against diseases 16 .…”

“…13-15 It is now widely recognized that removal of the core fucose from Fc N -glycans represents the most effective approach to enhance ADCC activity 14 15 A high-throughput study of the IgG glycome of three isolated human populations showed that most of the human plasma IgG antibodies are core fucosylated with levels of afucosylated IgG ranging from 1.3% to 19.3%, underlying the difference in ADCC efficacy of naturally occurring antibodies to protect against diseases 16 . Dramatic shifts in IgG glycan profile towards reduced galactosylation and fucosylation have been observed in human immunodeficiency virus (HIV)-specific antibodies and are associated with improved antiviral activity and HIV control 17 …”