Development of antigen-specific B cell elimination by recombinant toxins in autoimmune diseases

寻常天疱疮（PV）是一种自身免疫性水疱性疾病，其特征在于循环的针对桥粒芯糖蛋白3（Dsg 3）的致病性IgG抗体。本研究的目的是开发用于特异性识别和消除PV中自身免疫性B细胞的嵌合分子。开发了产生抗Dsg 3抗体的小鼠杂交瘤细胞系（5 H10，12 A2）作为B细胞靶向的体外模型系统。Dsg 3-GFg是一种杆状病毒蛋白，含有完整的Dsg 3胞外结构域和绿色荧光蛋白，通过其表面的免疫球蛋白受体以抗原特异性的方式识别并靶向杂交瘤细胞。这些单克隆抗体的表位被映射到氨基末端EC 1和EC2的一部分，该区域被认为在钙粘蛋白中具有重要的功能。在细菌（Dsg3_N1-PF 4 O-KDEL，PE 37-Dsg3_N1-KDEL）中产生含有映射区域（Dsg3_N1）和修饰的假单胞菌外毒素（PE）的嵌合毒素分子，并在杂交瘤细胞系上进行体外测试。嵌合毒素，但不是单独的Dsg3_N1，显示剂量依赖性毒性活性，与对抗Dsg 3阴性杂交瘤细胞几乎没有影响或没有影响相比，杂交瘤细胞数量减少至毒素阴性对照培养物的40-60%。此外，这些毒素对来自Dsg3_N1免疫小鼠的产生抗Dsg 3 IgG的B细胞显示毒性作用，与单独的Dsg3_N1相比，细胞数量减少60%。因此，证明了PV中抗原特异性B细胞的特异性识别和靶向，并且这种策略可能有望作为PV和其他自身免疫性疾病的未来治疗选择。

Pemphigus vulgaris (PV) is an autoimmune blistering disease characterized by circulating pathogenic IgG antibodies against desmoglein 3 (Dsg3). The purpose of this study was to develop chimeric molecules for specific recognition and elimination of autoimmune B cells in PV.Mouse hybridoma cell lines producing anti-Dsg3 antibody (5H10, 12A2) were developed as an in vitro model system for B cell targeting. Dsg3-GFg a baculoprotein containing the entire extracellular domain of Dsg3 fused with green fluorescence protein, recognized and targeted the hybridoma cells through their surface immunoglobulin receptors in an antigen-specific way. The epitopes of these monoclonal antibodies were mapped on the amino-terminal EC1 and part of EC2, a region considered functionally important in cadherins. Chimeric toxin molecules containing the mapped region (Dsg3_N1) and modified Pseudomonas exotoxin (PE) were produced in bacteria (Dsg3_N1-PF4O-KDEL, PE37-Dsg3_N1-KDEL) and tested in vitro on hybridoma cell lines. The chimeric toxins, but not Dsg3_N1 alone, showed dose-dependent toxic activity with a reduction in hybridoma cell number to 40-60% of toxin-negative control cultures, compared with little or no effect on anti-Dsg3-negative hybridoma cells. Furthermore, these toxins showed toxic effects on anti-Dsg3 IgG-producing B cells from Dsg3_N1-immunized mice, with a 60% reduction in cell number compared with Dsg3_N1 alone. Thus specific recognition and targeting of antigen-specific B cells in PV was demonstrated and this strategy may hold promise as a future therapeutic option for PV and other autoimmune diseases.