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

Abstract

Obinutuzumab is thought to exert its effects through its high antibodydependent 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 bindinginduction 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.STIM1 increases obinutuzumab affinity to CD20 © 2020 British Society for Immunology, Clinical and Experimental Immunology, 200: 260-271 261Ca 2+ via B cell receptor activation [19,20]. Additionally, Ca 2+ influx of CD20 is related to resistance to rituximab treatment [21]. The relationship between Ca 2+ and obinutuzumab binding is not clearly understood. The fact that cell death occurs only by obinutuzumab binding leads us to expect that excessive Ca 2+ influx through CD20 is the underlying cell death mechanism. However, reports of intracellular calcium responses after binding of obinutuzumab remain controversial, and there are no reports supporting the hypothesis that Ca 2+ influx is a key cell death mechanism. Therefore, in-depth understanding of the CD20 channel features, its exact biological role and interactions with obinutuzumab binding and cell death are essential to unveil the pharmacological mechanism of obinutuzumab.Rituximab binding to CD20 causes CD20 aggregation in the raft region, which is one of the typical characteristics of Type I categorized CD20 antibodies [16]. This enhances the efficient establishment of a mega pore by activating the complement cascade which, in turn, causes complement-dependent cytotoxicity [1,22,23]. In contrast, Type II antibodies such as obinutuzumab do not cause CD20 aggregation in the raft region, and thus show low complement-dependent cell death and instead exhibit high ADCC [3,24]. The epitope region is bound by rituximab and obinutuzumab overlap [25]; however, only obinutuzumab causes binding-induced cell death [3,8,9]. Structural analysis has revealed that the binding mode and plasma membrane localization of rituximab and obinutuzumab diff...

Deregulated calcium signaling in blood cancer: Underlying mechanisms and therapeutic potential

“…Monoclonal anti-CD20 antibodies such as rituximab and obinutuzumab activate Ca 2+ influx in patient CLL cells and cell lines such as SUDHL-4, BL2, Ramos, BL60, Raji, Daudi, and normal B-cells ( 202 – 206 ). Using genetically encoded Ca 2+ indicator GCaMP-CD20 as a precise method to measure Ca 2+ concentration changes around CD20, it was determined that anti-CD20 antibodies do not cause Ca 2+ influx through or near CD20 ( 207 ). Instead, obinutuzumab activates intracellular Ca 2+ efflux from either lysosomes or the ER into the cytosol ( 206 ) (see Figure 4A and the corresponding legend for molecular details).…”

“…Instead, obinutuzumab activates intracellular Ca 2+ efflux from either lysosomes or the ER into the cytosol ( 206 ) (see Figure 4A and the corresponding legend for molecular details). Inhibition of this intracellular Ca 2+ movement reduces obinutuzumab-induced cell death ( 206 , 207 ). Binding of rituximab to CD20 induces co-clustering of CD20 with Orai1 and STIM1 in SUDHL-4 cells, leading to extracellular Ca 2+ influx and internal Ca 2+ store release ( 205 ).…”

“…Binding of rituximab to CD20 induces co-clustering of CD20 with Orai1 and STIM1 in SUDHL-4 cells, leading to extracellular Ca 2+ influx and internal Ca 2+ store release ( 205 ). CD20 overexpression in HEK293 cells increases Ca 2+ influx, which is abolished when Orai1 and STIM1 are knocked down ( 207 ). CD20 strongly interacts with STIM1 but only when Orai1 is present ( 207 ).…”

“…CD20 overexpression in HEK293 cells increases Ca 2+ influx, which is abolished when Orai1 and STIM1 are knocked down ( 207 ). CD20 strongly interacts with STIM1 but only when Orai1 is present ( 207 ). Influx of Ca 2+ induced by rituximab or obinutuzumab is significantly reduced in Orai1 knockdown cells ( 205 , 206 ).…”