title: Comparison of cell lines for stable production of fucose-negative antibodies with enhanced ADCC.
abstract: Several methods have been described to enhance antibody-dependent cellular cytotoxicity (ADCC) using different host cells that produce antibody with reduced levels of fucose on their carbohydrates. We compared the suitability of these methods for the serum-free fed-batch production of antibody for clinical trials and commercial uses. Recombinant anti-human CD20 chimeric IgG1-producing clones were established from host-cells that have been shown to produce more than 90% fucose-negative antibody. The cell lines were a FUT8 (alpha-1,6-fucosyltransferase) knockout Chinese hamster ovary (CHO) cell line, Ms704, and two Lens culinaris agglutinin (LCA)-resistant cell lines, one derived from a variant CHO line, Lec13 and the other from a rat hybridoma cell line, YB2/0. The amount of fucose-negative antibody produced by Lec13 and YB2/0 significantly decreased with the culture. The increase in fucosylation was due to remaining synthesis of GDP-fucose via de novo pathway for the CHO line and the elevation of FUT8 expression by the YB2/0 cells. In contrast, Ms704 cells stably produced fucose-negative antibody with a consistent carbohydrate structure until the end of the culture. The productivity of the Ms704 cells reached 1.76 g/L with a specific production rate (SPR) of 29 pg/cell/day for 17 days in serum-free fed-batch culture using a 1 L spinner bioreactor. Our results demonstrate that FUT8 knockout has the essential characteristics of host cells for robust manufacture of fucose-negative therapeutic antibodies with enhanced ADCC.
authors.affiliation: Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6 Asahi-machi, Machida-shi, Tokyo 194-8533, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Kanda
authors.forename: Naoko
authors.initials: N
authors.lastname: Yamane-Ohnuki
authors.forename: Naoto
authors.initials: N
authors.lastname: Sakai
authors.forename: Kazuya
authors.initials: K
authors.lastname: Yamano
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Nakano
authors.forename: Miho
authors.initials: M
authors.lastname: Inoue
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Misaka
authors.forename: Shigeru
authors.initials: S
authors.lastname: Iida
authors.forename: Masako
authors.initials: M
authors.lastname: Wakitani
authors.forename: Yoshinobu
authors.initials: Y
authors.lastname: Konno
authors.forename: Keiichi
authors.initials: K
authors.lastname: Yano
authors.forename: Kenya
authors.initials: K
authors.lastname: Shitara
authors.forename: Shinji
authors.initials: S
authors.lastname: Hosoi
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Satoh
mesh_terms: D000818:Animals; D000917:Antibody Formation; D000920:Antibody-Dependent Cell Cytotoxicity; D019149:Bioreactors; D016466:CHO Cells / Q000276:immunology; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D006224:Cricetinae; D016895:Culture Media, Serum-Free; D018076:DNA, Complementary / Q000235:genetics; D005643:Fucose / Q000276:immunology*; D005647:Fucosyltransferases / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D009005:Monosaccharides / Q000737:chemistry / Q000302:isolation & purification; D009844:Oligosaccharides / Q000737:chemistry / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D016895:Culture Media, Serum-Free; D018076:DNA, Complementary; D009005:Monosaccharides; D009844:Oligosaccharides; D005643:Fucose; D005647:Fucosyltransferases