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 Ca2+ 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 Ca2+ channel features of CD20 as a store-operated Ca2+ channel (SOC) and obinutuzumab binding-induced cell death. Ca2+ channel function and biochemical analysis revealed that CD20 is an Orai1- and stromal interaction molecule (STIM1)-dependent Ca2+ pore. However, binding of obinutuzumab on CD20 did not have any effect on Ca2+ influx activity of CD20; the direct cell death rate mediated by obinutuzumab binding was almost equivalent with or without the extracellular Ca2+ 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.

中文翻译：

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STIM1敲低通过改变CD20在Triton可溶性膜上的定位来降低obinutuzumab对CD20的亲和力。

据认为，奥比努单抗通过其Fc区的糖工程改造，通过其高抗体依赖性细胞毒性（ADCC）发挥其作用。此外，obinutuzumab仅通过特异性结合其靶CD20（Ca2 +通道）而导致直接结合诱导的细胞死亡（DCD）。但是，尚不清楚与奥比妥珠单抗结合诱导细胞死亡有关的CD20的具体特征。在这项研究中，我们评估了CD20的Ca2 +通道特征作为存储操作的Ca2 +通道（SOC）与obinutuzumab结合诱导的细胞死亡之间的关系。Ca2 +通道功能和生化分析表明，CD20是Orai1和基质相互作用分子（STIM1）依赖的Ca2 +孔。然而，obinutuzumab与CD20的结合对CD20的Ca2 +内流活性没有任何影响。在有或没有细胞外Ca2 +条件下，由obinutuzumab结合介导的直接细胞死亡率几乎相等。给定STIM1和CD20之间的明显相互作用，我们观察到Triton-X溶解了与obututuzumab结合的CD20，并伴有STIM1。随后，拉莫斯B细胞中的STIM1敲低降低了obinutuzumab的结合和细胞死亡。因此，STIM1通过将CD20转移到Triton可溶性膜区域来增加细胞对obinutuzumab的亲和力，直接导致细胞死亡。