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.

Purpose: Recent studies have revealed that fucosylated therapeutic IgG1s need high concentrations to compensate for FcgRIIIa-competitive inhibition of antibody-dependent cellular cytotoxicity (ADCC) by endogenous human plasma IgG. Here, we investigated whether ADCC of nonfucosylated therapeutic IgG1 is also influenced by plasma IgG in the same way as fucosylated IgG1s. Experimental Design: Ex vivo ADCC upon CD20 + human B cells was induced by incubation of human whole blood with nonfucosylated and/or fucosylated anti-CD20 IgG1s rituximab, and quantified by measuring the remaining CD19 + human B cells using flow cytometry. Results: Nonfucosylated anti-CD20 showed markedly higher (over 100-fold based on EC 50 ) ex vivo B-cell depletion activity than its fucosylated counterpart in the presence of plasma IgG. The efficacy of fucosylated anti-CD20 was greatly diminished in plasma, resulting in the need for a high concentration (over 1.0 Ag/mL) to achieve saturated efficacy. In contrast, nonfucosylated anti-CD20 reached saturated ADCC at lower concentrations (0.01-0.1 Ag/mL) with much higher efficacy than fucosylated anti-CD20 in all nine donors through improved FcgRIIIa binding. Noteworthy, the high efficacy of nonfucosylated anti-CD20 was inhibited by addition of fucosylated anti-CD20. Thus, the efficacy of a 1:9 mixture (10 Ag/mL) of nonfucosylated and fucosylated anti-CD20s was inferior to that of a 1,000-fold dilution (0.01 Ag/mL) of nonfucosylated anti-CD20 alone. Conclusions: Our data showed that nonfucosylated IgG1, not including fucosylated counterparts, can evade the inhibitory effect of plasma IgG on ADCC through its high FcgRIIIa binding. Hence, nonfucosylated IgG1exhibits strong therapeutic potential through dramatically enhanced ADCC at low doses in humans in vivo.

We explored the possibility of converting established antibody-producing cells to cells producing high antibody-dependent cellular cytotoxicity (ADCC) antibodies. The conversion was made by constitutive expression of small interfering RNA (siRNA) against alpha1,6 fucosyltransferase (FUT8). We found two effective siRNAs, which reduce FUT8 mRNA expression to 20% when introduced into Chinese hamster ovary (CHO)/DG44 cells. Selection for Lens culinaris agglutinin (LCA)-resistant clones after introduction of the FUT8 siRNA expression plasmids yields clones producing highly defucosylated (approximately 60%) antibody with over 100-fold higher ADCC compared to antibody produced by the parental cells (approximately 10% defucosylated). Moreover, the selected clones remain stable, producing defucosylated antibody even in serum-free fed-batch culture. Our results demonstrate that constitutive FUT8 siRNA expression can control the oligosaccharide structure of recombinant antibody produced by CHO cells to yield antibodies with dramatically enhanced ADCC.

The clinical success of therapeutic antibodies is demonstrated by the number of antibody therapeutics that have been brought to market and the increasing number of therapeutic antibodies in development. Recombinant antibodies are molecular-targeted therapeutic agents and represent a major new class of drugs. However, it is still very important to optimize and maximize the clinical efficacy of therapeutic antibodies, in part to help lower the cost of therapeutic antibodies by potentially reducing the dose or the duration of treatment. Clinical trials using therapeutic antibodies fully lacking core fucose residue in the Fc oligosaccharides are currently underway, and their remarkable physiological activities in humans in vivo have attracted attention as next-generation therapeutic antibody approaches with improved efficacy. Thus, an industrially applicable antibody production process that provides consistent yields of fully nonfucosylated antibody therapeutics with fixed quality has become a key goal in the successful development of next-generation therapeutic agents. In this article, we review the current technologies for production of therapeutic antibodies with control of fucosylation of the Fc N-glycans.