In Vitro Complement-Dependent Cytotoxicity and Antibody-Dependent Cellular Cytotoxicity by the Anti-TNF Agents Adalimumab, Etanercept, Infliximab, and Certolizumab Pegol (CDP870)

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Purpose: To determine the ability of the anti-TNF agents adalimumab, etanercept, infliximab, and certolizumab pegol to kill cells expressing membrane TNF by complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). Methods: NS0 cells transfected with human TNF (TNF 6.5 cell line) were used for this study. These cells express high levels of membrane TNF on their cell surface. Directly fluoresceinated reagents were used to determine the level of binding of the anti-TNF agents compared with negative control reagents on the TNF 6.5 cell line by flow cytometry. CDC of the TNF 6.5 cell line by the different reagents was determined by measuring lactate dehydrogenase release from the cells and by measuring propidium iodide (PI) staining using flow cytometry. ADCC was performed on the TNF 6.5 cells using monocyte-depleted peripheral blood mononuclear cells as the effector cells. Cell killing was also quantified using PI staining measured by flow cytometry. Results: All four anti-TNF agents bound to the membrane TNF on the TNF 6.5 cells, although etanercept bound to a lesser degree than the other reagents. However, CDC and ADCC of TNF 6.5 cells were mediated only by adalimumab, etanercept, and infliximab, which all have an active human IgG1 Fc. Certolizumab pegol did not kill cells by either method because it is a PEGylated Fab' fragment that does not have an Fc region. Conclusions: It has been reported that etanercept does not mediate CDC or ADCC. However, in this model system, etanercept was clearly capable of killing TNF 6.5 cells by both methods, similar to infliximab and adalimumab. Owing to its unique structure, certolizumab pegol is the only anti-TNF agent that did not mediate in vitro killing by CDC or ADCC of cells expressing TNF on their membrane.