Ab-dependent cellular cytotoxicity (ADCC) is usually considered an important mechanism of action for immunotherapy with human IgG1 but not IgG2 Abs. The epidermal growth factor receptor (EGF-R) Ab panitumumab represents the only human IgG2 Ab approved for immunotherapy and inhibition of EGF-R signaling has been described as its principal mechanism of action. In this study, we investigated effector mechanisms of panitumumab compared with zalutumumab, an EGF-R Ab of the human IgG1 isotype. Notably, panitumumab was as effective as zalutumumab in recruiting ADCC by myeloid effector cells (i.e., neutrophils and monocytes) in contrast to NK cell-mediated ADCC, which was only induced by the IgG1 Ab. Neutrophil-mediated tumor cell killing could be stimulated by myeloid growth factors and was triggered via FcγRIIa. Panitumumab-mediated ADCC was significantly affected by the functional FcγRIIa-R131H polymorphism and was induced more effectively by neutrophils from FcγRIIa-131H homozygous donors than from -131R individuals. This polymorphism did not affect neutrophil ADCC induced by the IgG1 Ab zalutumumab. The in vivo activity of both Abs was assessed in two animal models: a high-dose model, in which signaling inhibition is a dominant mechanism of action, and a low-dose model, in which effector cell recruitment plays a prominent role. Zalutumumab was more effective than panitumumab in the high-dose model, reflecting its stronger ability to induce EGF-R downmodulation and growth inhibition. In the low-dose model, zalutumumab and panitumumab similarly prevented tumor growth. Thus, our results identify myeloid cell-mediated ADCC as a potent and additional mechanism of action for EGF-R–directed immunotherapy.

Glycosylation of the antibody Fc fragment is essential for Fc receptormediated activity. Carbohydrate heterogeneity is known to modulate the activity of effector cells in the blood, in which fucosylation particularly affects NK cellmediated killing. Here, we investigated how the glycosylation profile of 2F8, a human IgG 1 monoclonal antibody against epidermal growth factor receptor in clinical development, impacted effector function. Various 2F8 batches differing in fucosylation, galactosylation, and sialylation of the complex-type oligosaccharides in the Fc fragment were investigated. Our results confirmed that low fucose levels enhance mononuclear cell-mediated antibody-mediated cellular cytotoxicity (ADCC). In contrast, polymorphonuclear cells were found to preferentially kill via high-fucosylated antibody. Whole blood ADCC assays, containing both types of effector cells, revealed little differences in tumor cell killing between both batches. Significantly, however, high-fucose antibody induced superior ADCC in blood from granulocyte colony-stimulating factor-primed donors containing higher numbers of activated polymorphonuclear cells. In conclusion, our data demonstrated for the first time that lack of fucose does not generally increase the ADCC activity of therapeutic antibodies and that the impact of Fc glycosylation on ADCC is critically dependent on the recruited effector cell type. (Blood. 2008; 112:2390-2399) IntroductionMonoclonal antibodies constitute a growing class of therapeutics, with major indications in oncology, infectious diseases, and autoimmunity. 1 In oncology, antibody-mediated cellular cytotoxicity (ADCC) is considered a particularly relevant mechanism of action for therapeutic antibodies. 2 Evidence for this is mainly derived from studies with the CD20 antibody rituximab, the most intensively investigated antibody in this regard. For example, rituximab lost most of its therapeutic efficacy against xenotransplanted human tumors in mice lacking activating Fc receptors by knockout of the common FcR␥-chain. 3 Syngeneic B-cell depletion by murine CD20 antibodies has furthermore been correlated with antibody isotypes and with their respective binding to activating Fc␥ receptors, compared with inhibitory Fc␥ receptors. 4,5 In patients, rituximab's therapeutic efficacy has been correlated with well-defined FcR polymorphisms affecting binding of human IgG and the ability to induce ADCC in vitro. 6,7 These and other observations stimulated studies exploring opportunities to improve antibodies' capacity to trigger ADCC. 8,9 This can be achieved by increasing antibody binding to activating cellular Fc receptors, such as NK cell-expressed Fc␥RIIIa, and by decreasing binding to the inhibitory Fc␥RIIb isoform. At least 2 different methodologies have been established: one modifying the protein structure of the antibody Fc region by mutating the respective cDNAs 10,11 and the other based on technologies altering the glycosylation profile of antibodies. [12][13][14][15] Posttranslational modifications, s...

Therapeutic monoclonal antibodies against the epidermal growth factor receptor (EGFR) have advanced the treatment of colon and head and neck cancer, and show great promise for the development of treatments for other solid cancers. Antibodies against EGFR have been shown to act via inhibition of receptor signaling and induction of antibody-dependent cellular cytoxicity. However, complement-dependent cytotoxicity, which is considered one of the most powerful cell killing mechanisms of antibodies, seems inactive for such antibodies. Here, we show a remarkable synergy for EGFR antibodies. Combinations of antibodies against EGFR were identified, which resulted in potent complement activation via the classic pathway and effective lysis of tumor cells. Studies on a large panel of antibodies indicated that the observed synergy is a general mechanism, which can be activated by combining human IgG1 antibodies recognizing different, nonoverlapping epitopes. Our findings show an unexpected quality of therapeutic EGFR antibodies, which may be exploited to develop novel and more effective treatments for solid cancers. [Cancer Res 2008;68(13):4998-5003]

During the menstrual cycle, ovarian estradiol and progesterone stimulate the ordered growth and differentiation of endometrial tissue compartments. In the human, this includes synchronous growth and coiling of the spiral arteries, secretory transformation of glandular epithelium, migration of bone marrow-derived cells, and decidualization of the stroma, which is thought to be essential for blastocyst implantation and subsequent formation of a hemochorial placenta. Decidualization of human endometrial stromal (ES)

IgA is the most abundantly produced Ab isotype in humans, but its potential as immunotherapeutic reagent has hardly been explored. In this study, we describe anti-tumor mechanisms of mouse/human chimeric IgA Abs against the epidermal growth factor receptor (EGF-R). EGF-R Abs of IgG isotype are currently approved for the treatment of colon or head and neck cancers. As expected, the human IgG1, IgA1, and IgA2 variants of the 225 Ab demonstrated similar binding to EGF-R. Furthermore, IgA Abs were as effective as IgG in mediating direct effector mechanisms such as blockade of EGF binding, inhibition of EGF-R phosphorylation, and induction of growth inhibition. None of the three variants induced complement-mediated lysis. Human IgG1 effectively recruited MNC for ADCC, but activated PMN only weakly, whereas both IgA isoforms proved to be effective in triggering neutrophils. Interestingly, the IgA2 isoform was significantly superior to its IgA1 counterpart in recruiting PMN as effector cells. Because neutrophils constitute the most abundant effector cell population in human blood, this enhanced neutrophil recruitment lead to increased killing of EGF-R expressing tumor cells in whole blood assays. This killing was further enhanced when blood from G-CSF-primed donors was compared with healthy donor blood. Together, these data suggest EGF-R Abs of human IgA isotype to bear promise for therapeutic use in cancer.