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...

Plants have attracted attention as bio-drug production platforms because of their economical and safety benefits. The preliminary efficacy of ZMapp, a cocktail of antibodies produced in N. benthamiana (Nicotiana benthamiana L.), suggested plants may serve as a platform for antibody production. However, because the amino acid sequences of the Fab fragment are diverse and differences in post-transcriptional processes between animals and plants remain to be elucidated, it is necessary to confirm functional equivalence of plant-produced antibodies to the original antibody. In this study, Obinutuzumab, a third generation anti-CD20 antibody, was produced in N. benthamiana leaves (plant-obinutuzumab) and compared to the original antibody produced in glyco-engineered Chinese hamster ovary (CHO) cells (CHO-obinutuzumab). Two forms (with or without an HDEL tag) were generated and antibody yields were compared. The HDEL-tagged form was more highly expressed than the non-HDEL-tagged form which was cleaved in the N-terminus. To determine the equivalence in functions of the Fab region between the two forms, we compared the CD20 binding affinities and direct binding induced cell death of a CD20-positive B cells. Both forms showed similar CD20 binding affinities and direct cell death of B cell. The results suggested that plant-obinutuzumab was equivalent to CHO-obinutuzumab in CD20 binding, cell aggregation, and direct cell death via binding. Therefore, our findings suggest that Obinutuzumab is a promising biosimilar candidate that can be produced efficiently in plants.

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Background The induction of pyroptosis holds great promise as a strategy for improving the tumor immune microenvironment. Previous pyroptosis inducers have faced limitations, including drug resistance, toxic side effects, and a lack of targeting capabilities. As a result, there is a growing demand for tumor therapeutic molecules that can overcome these obstacles. With this in mind, the objective of this study is to develop a multifunctional nanospheres that addresses these challenges by enabling high-precision targeting of tumor cells and effective pyroptosis induction. Results We prepared a mannose-modified MOF called mannose-doped Fe3O4@NH2-MIL-100, referred to as M-FNM. M-FNM has the ability to enter CAL27 cells through MR-mediated endocytosis, which results in a significant increase in intracellular ROS levels. This increase subsequently triggers endoplasmic reticulum stress (ER stress) and activates the PERK-eIF2α-ATF4-CHOP signaling pathway. CHOP then mediates the downstream cascade of Caspase-1, inducing pyroptosis. In in vivo experiments, M-FNM demonstrates excellent targeting ability and exhibits anti-tumor effects. Additionally, M-FNM reshapes the immune microenvironment by promoting the infiltration of anti-tumor immune cells, primarily T lymphocytes. Conclusions M-FNM significantly decreased tumor growth. This novel approach of inducing pyroptosis in tumor cells using M-FNM may offer new avenues for the development of effective immunotherapies for cancer treatment.

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