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 Fc gamma RIII

The gene encoding the rat glycosylation enzyme beta1-4-N-acetylglucosaminyl transferase III (GnTIII) was cloned and coexpressed in a recombinant produc tion Chinese hamster ovary (CHO) cell line expressing a chimeric mouse/huma n anti-CD20 IgG1 antibody. The new cell lines expressed high levels of anti body and have growth kinetics similar to that of the parent. Relative QPCR showed the cell lines to express varying levels of mRNA. High-performance l iquid chromatography (HPLC) analysis showed the enzyme to have added bisect ing N-acetylglucosamine (GlcNAc) residues in most (48% to 71%) of the N-lin ked 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 concen trations than the parent. This activity was blocked using an anti-Fc gamma RIII antibody, supporting the role of Fc gamma RIII binding in this increas e. In addition, cell binding assays showed the modified antibody bound bett er to Fc gamma RIII-expressing cells. The increase in ADCC activity is ther efore likely due to an increased affinity of the modified antibody for the Fc gamma RIII receptor. (C) 2001 John Wiley & Sons, Inc.