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

寻常型天疱疮(PV)是一种以循环抗桥粒蛋白3(Dsg3)致病抗体为特征的自身免疫性水疱病。本研究的目的是开发抗Dsg3抗体的杂交瘤细胞株(5H10、12A2)作为B细胞靶向的体外模型系统。Dsg3-GFG是一种包含整个Dsg3胞外区的杆状蛋白，与绿色荧光蛋白融合，通过杂交瘤细胞的表面免疫球蛋白受体以抗原特异性的方式识别和靶向杂交瘤细胞。这些单抗的表位被映射到EC1的氨基末端和EC2的一部分，这是一个被认为在钙粘附素中具有重要功能的区域。在细菌(Dsg3_N1-PF4O-KDEL和PE37-Dsg3_N1-KDEL)中产生含有作图区域(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试验。这些嵌合毒素，而不仅仅是Dsg3_N1，表现出剂量依赖的毒性活性，杂交瘤细胞数量减少到毒素阴性对照培养的40%-60%，而对抗Dsg3阴性杂交瘤细胞几乎没有影响。此外，这些毒素对Dsg3N1免疫的小鼠产生抗Dsg3Ig G的B细胞有毒性作用，与Dsg3N1单独免疫相比，细胞数量减少了60%。因此，在PV中证明了抗原特异性B细胞的特异性识别和靶向，这一策略有望成为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.