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.

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

“…This has resulted in the application of Lec13 cells as the host cell line for the production of afucosylated antibodies 14 . However studies have shown that single clones isolated from Lec13 cells display a wide variety of fucosylation range, with most clones producing 50–70% fucosylated antibody when cultured to confluence in a static flask 75 . Further analysis revealed low-level expression of GMD at mRNA level as well as the presence of fucosylated oligosaccharides on cell surface using LCA-staining.…”

Advantages of AlaGln as an additive to cell culture medium: use with anti-CD20 chimeric antibody-producing POTELLIGENT™ CHO cell lines

“…Both CHO cell lines are derived from Ms704 which is FUT8 knock out CHO/DG44 cells. Construction method of 1A7-15 clone was described elsewhere (Kanda et al 2006). 12C-5 clone was another cell line screened from same host cell pool as 1A7-15 clone.…”

Anti-RhD-Mediated Immunosuppression: Can Monoclonal Antibodies Imitate the Action of Polyclonal Antibodies?

“…Antibodies with low fucose can be produced by the cell lines with a reduced fucosylation activity such as rat myeloma YB2/0 cells or the CHO variant cell line, Lec13 (Shields et al, 2002;Shinkawa et al, 2003). New host cell lines which produce completely defucosylated antibodies with enhanced effector functions were generated by knockout of the fucosyltransferase gene (Yamane-Ohnuki et al, 2004;Kanda et al, 2006). A number of other approaches to improve the FcγRIIIA affinity are being developed: for example, human IgG1 bearing immature oligomannose-type glycans also display an increased ADCC (Crispin et al, 2009).…”