Unconjugated, target-cell killing antibodies of the human IgG1 isotype are now established as successful therapeutic agents, as demonstrated by the use of rituximab and trastuzumab for the treatment of B cell malignancies and Her2-overexpressing breast cancer, respectively. While both Fc-dependent and independent mechanisms can contribute to the efficacy of these drugs, it is clear that for both rituximab and trastuzumab, significant in vivo target cell depletion requires the Fc portion of the antibody. In vivo, the Fc region may either engage complement activation and/or interact with Fcγ receptors that are important for cellular immune effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), which can be mediated by various effector cells such as natural killer (NK) cells and macrophages.

Increasing evidence indicates an important role for the interaction of antibodies with FcγRIIIa. In particular, retrospective studies have correlated superior objective response rates and progression-free survival with being homozygous for the higher affinity allele of FcγRIIIa encoding a valine residue at position 158.– Only approximately 15% of the population is homozygous for this form of the receptor. Therefore, it may be valuable to generate therapeutic antibody variants that bind to all forms of this receptor with at least as high affinity as current IgG1 antibodies bind to FcγRIIIa-158V.

Both the polypeptide chain and the oligosaccharide component may be engineered in order to increase affinity for FcγRIII. We have chosen the latter path and first demonstrated that recombinant engineering of the glycosylation pattern of antibodies generates antibody glycosylation variants with increased FcγRIII binding affinity and increased ADCC. As explained in more detail below, this was achieved by overexpression of a glycosyltransferase gene in Chinese hamster ovary (CHO) cells, which are the preferred and established cell host for the commercial production of therapeutic antibodies.