STIM1 knock‐down decreases the affinity of obinutuzumab for CD20 by altering CD20 localization to Triton‐soluble membrane

Summary Obinutuzumab is thought to exert its effects through its high antibody‐dependent cellular cytotoxicity (ADCC) via glyco‐engineering of the Fc region. In addition, obinutuzumab causes direct binding‐induced cell death (DCD) only by specifically binding to its target CD20, a Ca 2+ channel. However, the specific features of CD20 related to obinutuzumab binding‐induction of cell death are not clearly understood. In this study, we evaluated the relationship between the Ca 2+ channel features of CD20 as a store‐operated Ca 2+ channel (SOC) and obinutuzumab binding‐induced cell death. Ca 2+ channel function and biochemical analysis revealed that CD20 is an Orai1‐ and stromal interaction molecule (STIM1)‐dependent Ca 2+ pore. However, binding of obinutuzumab on CD20 did not have any effect on Ca 2+ influx activity of CD20; the direct cell death rate mediated by obinutuzumab binding was almost equivalent with or without the extracellular Ca 2+ condition. Given the apparent interaction between STIM1 and CD20, we observed Triton‐X solubilized obinutuzumab‐bound CD20 accompanied by STIM1. Subsequently, obinutuzumab binding and cell death were decreased by STIM1 knock‐down in Ramos B cells. Thus, STIM1 directly contributes to cell death by increasing the affinity of cells for obinutuzumab by transferring CD20 to the Triton‐soluble membrane region.