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 ...

To generate industrially applicable new host cell lines for antibody production with optimizing antibody-dependent cellular cytotoxicity (ADCC) we disrupted both FUT8 alleles in a Chinese hamster ovary (CHO)/DG44 cell line by sequential homologous recombination. FUT8 encodes an alpha-1,6-fucosyltransferase that catalyzes the transfer of fucose from GDP-fucose to N-acetylglucosamine (GlcNAc) in an alpha-1,6 linkage. FUT8(-/-) cell lines have morphology and growth kinetics similar to those of the parent, and produce completely defucosylated recombinant antibodies. FUT8(-/-)-produced chimeric anti-CD20 IgG1 shows the same level of antigen-binding activity and complement-dependent cytotoxicity (CDC) as the FUT8(+/+)-produced, comparable antibody, Rituxan. In contrast, FUT8(-/-)-produced anti-CD20 IgG1 strongly binds to human Fcgamma-receptor IIIa (FcgammaRIIIa) and dramatically enhances ADCC to approximately 100-fold that of Rituxan. Our results demonstrate that FUT8(-/-) cells are ideal host cell lines to stably produce completely defucosylated high-ADCC antibodies with fixed quality and efficacy for therapeutic use.

Human IgG1 antibodies with low fucose contents in their asparaginelinked oligosaccharides have been shown recently to exhibit potent antibody-dependent cellular cytotoxicity (ADCC) in vitro. To additionally investigate the efficacy of the human IgG1 with enhanced ADCC, we generated the defucosylated chimeric anti-CC chemokine receptor 4 (CCR4) IgG1 antibody KM2760. KM2760 exhibited much higher ADCC using human peripheral blood mononuclear cells (PBMCs) as effector cells compared with the highly fucosylated, but otherwise identical IgG1, KM3060. In addition, KM2760 also exhibited potent ADCC in the presence of lower concentrations of human PBMCs than KM3060. Because CCR4 is a selective marker of T-cell leukemia/lymphoma, the effectiveness of KM2760 for T-cell malignancy was evaluated in several mouse models. First, to compare the antitumor activity of KM2760 and KM3060, we constructed a human PBMC-engrafted mouse model to determine ADCC efficacy with human effector cells. In this model, KM2760 showed significantly higher antitumor efficacy than KM3060, indicating that KM2760 retains its high potency in vivo. Second, KM2760 suppressed tumor growth in both syngeneic and xenograft mouse models in which human PBMCs were not engrafted. Although murine effector cells exhibited marginal ADCC mediated by KM2760 and KM3060, KM2760 unexpectedly showed higher efficacy than KM3060 in a syngeneic mouse model, suggesting that KM2760 functions in murine effector system in vivo via an unknown mechanism that differs from that in human. These results indicate that defucosylated antibodies with enhanced ADCC as well as potent antitumor activity in vivo are promising candidates for the novel antibody-based therapy.

Results: We found KM3065-mediated antibody-dependent cellular cytotoxicity was increased 10 to 100-fold compared with rituximab for each of the 20 donors. In contrast to rituximab, KM3065 antibody-dependent cellular cytotoxicity enhancement was similar for both FCGR3A alleles and thus independent of genotype. In addition, antibody-dependent cellular cytotoxicity of both KM3065 and rituximab requires natural killer cells but not monocytes nor polymorphonuclear cells. The antibody-dependent cellular cytotoxicity (ADCC) of each of the 20 donors correlated with the natural killer cell numbers present in the PBMCs. Importantly, using KM3065, the ADCC mediated by effector cells bearing the lower affinity variant Fc␥RIIIa-158F was significantly increased compared with rituximab-mediated ADCC using effector cells bearing the higher affinity Fc␥RIIIa-158V receptors.Conclusions: The use of low-fucose antibodies might improve the therapeutic effects of anti-CD20 therapy for all patients independent of Fc␥RIIIa phenotype beyond that currently seen with even the most responsive patients.