Highly efficient deletion of FUT8 in CHO cell lines using zinc‐finger nucleases yields cells that produce completely nonfucosylated antibodies

Abstract

IgG1 antibodies produced in Chinese hamster ovary (CHO) cells are heavily a1,6-fucosylated, a modification that reduces antibody-dependent cellular cytotoxicity (ADCC) and can inhibit therapeutic antibody function in vivo. Addition of fucose is catalyzed by Fut8, a a1,6-fucosyltransferase. FUT8À/À CHO cell lines produce completely nonfucosylated antibodies, but the difficulty of recapitulating the knockout in protein-production cell lines has prevented the widespread adoption of FUT8 À/À cells as hosts for antibody production. We have created zinc-finger nucleases (ZFNs) that cleave the FUT8 gene in a region encoding the catalytic core of the enzyme, allowing the functional disruption of FUT8 in any CHO cell line. These reagents produce FUT8 À/À CHO cells in 3 weeks at a frequency of 5% in the absence of any selection. Alternately, populations of ZFN-treated cells can be directly selected to give FUT8 À/À cell pools in as few as 3 days. To demonstrate the utility of this method in bioprocess, FUT8 was disrupted in a CHO cell line used for stable protein production. ZFNderived FUT8 À/À cell lines were as transfectable as wild-type, had similar or better growth profiles, and produced equivalent amounts of antibody during transient transfection. Antibodies made in these lines completely lacked core fucosylation but had an otherwise normal glycosylation pattern. Cell lines stably expressing a model antibody were made from wild-type and ZFN-generated FUT8 À/À cells. Clones from both lines had equivalent titer, specific productivity distributions, and integrated viable cell counts. Antibody titer in the best ZFN-generated FUT8 À/À cell lines was fourfold higher than in the best-producing clones of FUT8 À/À cells made by standard homologous recombination in a different CHO subtype. These data demonstrate the straightforward, ZFN-mediated transfer of the Fut8À phenotype to a production CHO cell line without adverse phenotypic effects. This process will speed the production of highly active, completely nonfucosylated therapeutic antibodies.

Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases

“…Alternately, the small deletion of one base may have happened by coincidence or by a biased DNA repair process at this locus. The type of mutations recovered are similar to previously observed ZFN-created alleles and include deletions and insertions (9,10,(25)(26)(27)(28). These small mutations (−7 bp to +4 bp) (Fig.…”

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

“…The FUT8 −/− cell line also demonstrated similar growth kinetics and productivity compared to the parental cell line when cultured in 1 L bioreactors. The FUT8 gene has also been targeted for inactivation using the zinc finger nuclease platform 78 . This also led to the production of completely afucosylated antibodies.…”

The GalNAc-type O-Glycoproteome of CHO Cells Characterized by the SimpleCell Strategy

“…Given the accomplishment of whole genome sequencing of several CHO lines and the original Chinese hamster (2,3), CHO has entered a new era with respect to genetic engineering to improve desirable features. The first successful design engineering of glycosylation in CHO was performed by two rounds of highly laborious and time consuming homologous recombinations to knockout FUT8 (37), but with the emerging precise gene editing technologies the efforts required have been drastically reduced as demonstrated by fast knock out of FUT8 with ZFN-mediated gene targeting (38). More recently, MGAT1 was knocked out in CHO GS cells to design a CHO platform for production of N-glycoproteins with homogeneous high mannose N-glycosylation, which is desirable for crystallization studies (39).…”