High reproducible ADCC analysis revealed a competitive relation between ADCC and CDC and differences between FcγRllla polymorphism

The anti-CD20 chimeric monoclonal antibody rituximab mediates cytotoxicity in malignant B cells via multiple mechanisms, including complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and direct induction of apoptosis. To optimize treatment of non-Hodgkin's lymphoma, a fuller understanding of these mechanisms and their relative contributions to clinical efficacy is required. Here, we report the characteristics of the mutual impact between ADCC and CDC, the two major effector functions through the Fc receptors. To compare ADCC induced under various conditions, we developed a highly reproducible method of estimating ADCC activity using immortalized effector cells. The set of the effector cells that we established was able to calculate net ADCC with high reproducibility by comparing the cytotoxicity of effector cells expressing exogeneous FcγRIIIa to those of mock effector cells. In addition, the different property of effector cells of two FcγRIIIa single-nucleotide polymorphisms (SNP) could be also evaluated in exactly identical background. ADCC assessment in the presence of human serum directly provided the evidence of the competitive interaction of ADCC and CDC. The inhibition of ADCC of effector cells having low affinity SNP of FcγRIIIa by active complement was more potent than those having high-affinity SNP at the rituximab-concentration comparable to the serum level obtained in patients. These findings could have a profound impact on optimization of the regimen of therapeutic antibodies and on the development of antibodies that will enhance effector function.