Impact of antibody Fc carbohydrate fucosylation on antibody-dependent cellular cytotoxicity by HuMax-EGFr —A fully human epidermal growth factor receptor antibody

Abstract

2543 Background: HuMax-EGFr - a human IgG1 EGF-R antibody in phase II clinical trials - was demonstrated to block EGF binding, to interfere with cellular signaling and to efficiently recruit effector cells for ADCC. Triggering of ADCC requires antibody binding to cellular Fc receptors which can be modulated by antibody glycosylation. Here, we investigated whether the glycosylation profile of HuMax-EGFr altered effector functions, and compared the contribution of mononuclear (MNC) and polymorphonuclear (PMN) cells for ADCC in human blood. Methods: Varying concentrations of two HuMax-EGFr batches, differing in the level of fucosylation of the complex-type oligosaccharides present in the Fc fragment, were compared in their capacity to kill EGF-R expressing tumor cells. Antibody binding and inhibition of proliferation was analyzed by flow cytometry and measuring vital cell mass, respectively. ADCC by isolated PMN, MNC and human whole blood was analyzed by chromium release assays. Results: Both batches of HuMax-EGFr bound similarly to target cells, and inhibited their proliferation to a similar extent. Low-fucosylated HuMax-EGFr demonstrated higher affinity for recombinant FcγRIIIa than fully fucosylated antibody. As expected, low-fucosylated antibody was significantly better in mediating ADCC by MNC (EC50 obtained at 10-fold lower antibody concentrations, p<0.05). In contrast, PMN-mediated ADCC was unaffected by the antibody fucosylation level. Importantly, whole blood ADCC assays also revealed little differences between both batches. ADCC was significantly enhanced by blood from G-CSF-primed patients, but again both high- and low-fucosylated antibody performed similarly. Conclusions: Both HuMax-EGFr batches were equally potent in inhibiting cell proliferation, and were highly active in triggering ADCC by MNC and PMN. Differences in fucosylation of Fc carbohydrate affected MNC-mediated lysis, whereas PMN- and whole blood-mediated cell killing were only minimally affected. Thus, fucosylation of antibody Fc may only prove critical for therapeutic antibodies which predominantly recruit NK cells for ADCC, while it may be less relevant for PMN-recruiting antibodies. [Table: see text]