Establishment of <i>FUT8</i> knockout Chinese hamster ovary cells: An ideal host cell line for producing completely defucosylated antibodies with enhanced antibody‐dependent cellular cytotoxicity

Yamane-Ohnuki, Naoko; Kinoshita, Shigeyoshi; Inoue-Urakubo, Miho; Kusunoki, Machi; Iida, Shigeru; Nakano, Ryoko; Wakitani, Masako; Niwa, Rinpei; Sakurada, Mikiko; Uchida, Kazuaki; Shitara, Kenya; Satoh, Mitsuo

doi:10.1002/bit.20151

Cited by 518 publications

(406 citation statements)

References 46 publications

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“…Most of the presently available glycoengineering techniques address and alter the processing machinery by knocking down, inhibiting, or overexpressing glycosylation enzymes (22,25,30,32,55,56) and thus are designed to modify simultaneously all the glycans carried by a given protein. Combining site-directed glycoprotein mutagenesis and classic glycoengineering techniques toward the limiting enzymes, as determined by flux analysis, can yield specific and targeted distributions of glycan on a single site.…”

Section: Discussionmentioning

confidence: 99%

Influence of protein/glycan interaction on site‐specific glycan heterogeneity

Losfeld¹,

Scibona²,

Lin³

et al. 2017

FASEB j.

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To study how the interaction between N-linked glycans and the surrounding amino acids influences oligosaccharide processing, we used protein disulfide isomerase (PDI), a glycoprotein bearing 5 N-glycosylation sites, as a model system and expressed it transiently in a Chinese hamster ovary (CHO)-S cell line. PDI was produced as both secreted Sec-PDI and endoplasmic reticulum-retained glycoprotein (ER)-PDI, to study glycan processing by ER and Golgi resident enzymes. Quantitative site-specific glycosylation profiles were obtained, and flux analysis enabled modeling site-specific glycan processing. By altering the primary sequence of PDI, we changed the glycan/ protein interaction and thus the site-specific glycoprofile because of the improved enzymatic fluxes at enzymatic bottlenecks. Our results highlight the importance of direct interactions between N-glycans and surface-exposed amino acids of glycoproteins on processing in the ER and the Golgi and the possibility of changing a site-specific N-glycan profile by modulating such interactions and thus the associated enzymatic fluxes. Altering the primary protein sequence can therefore be used to glycoengineer recombinant proteins.-Losfeld, M.-E., Scibona, E., Lin, C.-W., Villiger, T. K., Gauss, R., Morbidelli, M., Aebi, M. Influence of protein/glycan interaction on site-specific glycan heterogeneity. FASEB J. 31, 4623-4635 (2017). www.fasebj.org KEY WORDS: glycoengineering • glycobiology • glycoprotein maturation • Golgi processing • enzymatic flux N-glycosylation is a major posttranslational modification of proteins that modulates folding, maturation, trafficking, and secretion, as well as specific protein functions (1-3). Contrary to many other posttranslational modifications, N-glycans are composed of many monosaccharide units, creating a large diversity of glycan structures (4, 5). In eukaryotic cells, the N-glycosylation occurs in the endoplasmic reticulum (ER) after a conserved pathway. The oligosaccharide GlcNAc 2 Man 9 Glc 3 is assembled on the lipid dolicholpyrophosphate at the membrane of the ER and then is covalently linked to the side-chain amide of the asparagine residue in the sequon N-X-S/T by oligosaccharyltransferase (OST) (1, 6, 7). Multiple sites on one glycoprotein can be modified by the OST complex and with the increasing complexity of multicellular organism, an increasing number of N-glycosylation sites in the glycoproteome are observed. It is estimated that up to 6000 different sites are modified by OST in mammalian cells (8). In the second phase of the N-glycosylation pathway, the protein-linked glycan is trimmed by glucosidases and mannosidases (1, 9) and is subsequently modified by different glycosyltransferases spatially distributed along the secretory pathway. N-glycan processing enzymes have defined substrate specificities and locations in the secretory pathway (9, 10), and their expression level can be regulated upon internal and external signals.Glycan maturation in the secretory pathway is not a template-driven process. The...

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Section: Discussionmentioning

confidence: 99%

Influence of protein/glycan interaction on site‐specific glycan heterogeneity

Losfeld¹,

Scibona²,

Lin³

et al. 2017

FASEB j.

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“…In the study by Yamane-Ohnuki et al. , the FUT8 gene in an anti-CD20 antibody-producing CHO DG44 cell line was targeted for disruption using sequential homologous recombination 77 . In the resultant cell line, both FUT8 alleles were knocked out from the FUT8 genomic region.…”

Section: Strategies To Produce Afucosylated Antibodiesmentioning

confidence: 99%

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

Pereira

Chan

Lin

et al. 2018

mAbs

253

211

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Therapeutic monoclonal antibodies are the fastest growing class of biological therapeutics for the treatment of various cancers and inflammatory disorders. In cancer immunotherapy, some IgG1 antibodies rely on the Fc-mediated immune effector function, antibody-dependent cellular cytotoxicity (ADCC), as the major mode of action to deplete tumor cells. It is well-known that this effector function is modulated by the N-linked glycosylation in the Fc region of the antibody. In particular, absence of core fucose on the Fc N-glycan has been shown to increase IgG1 Fc binding affinity to the FcγRIIIa present on immune effector cells such as natural killer cells and lead to enhanced ADCC activity. As such, various strategies have focused on producing afucosylated antibodies to improve therapeutic efficacy. This review discusses the relevance of antibody core fucosylation to ADCC, different strategies to produce afucosylated antibodies, and an update of afucosylated antibody drugs currently undergoing clinical trials as well as those that have been approved.

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“…Similarly, glycosylation pathway engineering has been developed to improve the biological function and reduce the heterogenecity of therapeutic antibodies (23,24). Of these methods, the most practical way to acquire homogeneous glycoproteins is based on the strategy of glycoprotein remodeling, a strategy first reported in 1997…”

mentioning

confidence: 99%

A common glycan structure on immunoglobulin G for enhancement of effector functions

Lin

Tsai²,

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

183

178

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Antibodies have been developed as therapeutic agents for the treatment of cancer, infection, and inflammation. In addition to binding activity toward the target, antibodies also exhibit effector-mediated activities through the interaction of the Fc glycan and the Fc receptors on immune cells. To identify the optimal glycan structures for individual antibodies with desired activity, we have developed an effective method to modify the Fc-glycan structures to a homogeneous glycoform. In this study, it was found that the biantennary N-glycan structure with two terminal alpha-2,6-linked sialic acids is a common and optimized structure for the enhancement of antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and antiinflammatory activities.endoglycosidase | Fc glycosylation | glycoengineered antibodies | homogeneous antibodies | sugar oxazoline

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Establishment of FUT8 knockout Chinese hamster ovary cells: An ideal host cell line for producing completely defucosylated antibodies with enhanced antibody‐dependent cellular cytotoxicity

Cited by 518 publications

References 46 publications

Influence of protein/glycan interaction on site‐specific glycan heterogeneity

Influence of protein/glycan interaction on site‐specific glycan heterogeneity

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

A common glycan structure on immunoglobulin G for enhancement of effector functions

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