Expression of GnTIII in a recombinant anti‐CD20 CHO production cell line: Expression of antibodies with altered glycoforms leads to an increase in ADCC through higher affinity for FCγRIII

Abstract

The gene encoding the rat glycosylation enzyme beta1-4-N-acetylglucosaminyltransferase III (GnTIII) was cloned and coexpressed in a recombinant production Chinese hamster ovary (CHO) cell line expressing a chimeric mouse/human anti-CD20 IgG1 antibody. The new cell lines expressed high levels of antibody and have growth kinetics similar to that of the parent. Relative QPCR showed the cell lines to express varying levels of mRNA. High-performance liquid chromatography (HPLC) analysis showed the enzyme to have added bisecting N-acetylglucosamine (GlcNAc) residues in most (48% to 71%) of the N-linked oligosaccharides isolated from antibody preparations purified from the cell lines. In an ADCC assay the new antibody preparations promoted killing of CD20-positive target cells at approximately 10- to 20-fold lower concentrations than the parent. This activity was blocked using an anti-Fc gamma RIII antibody, supporting the role of Fc gamma RIII binding in this increase. In addition, cell binding assays showed the modified antibody bound better to Fc gamma RIII-expressing cells. The increase in ADCC activity is therefore likely due to an increased affinity of the modified antibody for the Fc gamma RIII receptor.

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

“…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…”

Using cell engineering and omic tools for the improvement of cell culture processes

“…Cell engineering of glycosylation patterns (Davies et al 2001;Natsume et al 2006;Shields et al 2002;Shinkawa et al 2003;Umañ a et al 1999;Warner 1999;Weikert et al 1999) and in vitro glycosylation (Butler 2005;Raju et al 2001) has provided new possibilities for biopharmaceutical design and optimization thus lowering immunogenicity effects, increasing stability and enhancing functional activity. Weikert et al (1999) engineered CHO cells that secreted either tissue necrosis factor receptor-IgG1 fusion protein (TNFR-IgG) or tissue plasminogen activator (TNK-tPA) to over-express a2,3-sialyltransferase and the resultant glycoproteins had a considerable longer pharmacokinetic mean residence time in rabbit models.…”

“…Bisected oligosaccharides have been implicated in enhanced antibody dependant cellular cytotoxicity (ADCC) activity. When CHO cells were engineered to over express b1,4 N-acetyl glucosaminoyltransferase (GnTIII), the bisected oligosaccharide glycoforms, ADCC activity was increased 20-100 fold (Davies et al 2001;Umañ a et al 1999). A silencing approach has also been used in CHO cells to increase the antibody effector function of ADCC by introducing siRNA targeting a1,6 fucosyltransferase (FUT8) mRNA (Mori et al 2004).…”

Application of a Reversible Immortalization System for the Generation of Proliferation-Controlled Cell Lines

“…The contents of the dead and lysed cells are released to the cell culture supernatant, leading to contamination of the product. Most of the presently popular mammalian producer cell lines have been optimized with respect to technological requirements by adaptation and maintenance for extended periods of time under the appropriate culture conditions, while adaptation of the cellular physiology by genetic engineering is still in its infancy (Grabenhorst et al 1995;Monaco et al 1996;Irani et al 1999Irani et al , 2002Uman˜a et al 1999;Davies et al 2001;Fogolin et al 2004; for a review see Grabenhorst et al 1999). However, due to comparatively high costs and low productivities of cultured mammalian cells, there is a constant pressure to further improve the producer cell lines as well as the protein production processes.…”