Establishment of FUT8 knockout Chinese hamster ovary cells: an ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity.
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.
Yamane-Ohnuki
Naoko
N
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6 Asahi-machi, Machida-shi, Tokyo 194-8533, Japan.
Kinoshita
Satoko
S
Inoue-Urakubo
Miho
M
Kusunoki
Machi
M
Iida
Shigeru
S
Nakano
Ryosuke
R
Wakitani
Masako
M
Niwa
Rinpei
R
Sakurada
Mikiko
M
Uchida
Kazuhisa
K
Shitara
Kenya
K
Satoh
Mitsuo
M
D000483:Alleles; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D000920:Antibody-Dependent Cell Cytotoxicity; D018951:Antigens, CD20 / Q000235:genetics / Q000276:immunology; D016466:CHO Cells; D002240:Carbohydrate Sequence; D006224:Cricetinae; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay; D005647:Fucosyltransferases / Q000235:genetics*; D018390:Gene Targeting; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis; D009844:Oligosaccharides / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D018951:Antigens, CD20; D007074:Immunoglobulin G; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; D005647:Fucosyltransferases; C020559:Glycoprotein 6-alpha-L-fucosyltransferase