Development of Immunotherapy of Refractory Periodontitis with Anti-Fc Receptor Bispecific Antibodies

在获得知情同意后，通过研究IgG和IgA介导的牙龈卟啉单胞菌清除，分析了21例牙周炎患者龈沟液（GCF）和外周血（PB）中性粒细胞（PMN）IgG和伊加PcR（FcγR和FcαR）的表达和功能。与PB-PMN相比，GCF-PMN表现出FcαR水平升高和FcγR水平降低。功能研究显示，与PB-PMN相比，GCF-PMN表现出吞噬和杀死IgG 1调理的牙龈卟啉单胞菌的能力降低。然而，IgA 1介导的吞噬和杀伤能力在GCF和PB-PMN之间是相当的。结果表明，FcαR可作为牙周炎免疫治疗的候选靶点。重组牙龈卟啉单胞菌40 kDa外膜蛋白（rOMP-40）经超声波纯化后获得。通过注射rOMP-40免疫产生人免疫球蛋白的小鼠KM小鼠<TM>。收集脾细胞，然后与小鼠骨髓瘤细胞系融合。用酶联免疫吸附试验筛选出102株抗rOMP-40人源单克隆抗体。IgG的数量为99，而IgM为3。未建立伊加克隆。测量显示与rOMP-40的高反应性的hMAb克隆的调理吞噬活性。我们的结果表明hMAb克隆的调理吞噬活性显著高于人多克隆抗体。这些结果提供了局部应用hMAb将有助于PMN依赖性消除牙龈卟啉单胞菌的可能性。

After informed consent was obtained, gingival crevicular fluid (GCF)- and peripheral blood (PB)-neutrophils (PMN) from 21 adult periodohtitis patients were analyzed for their IgG and IgA PcR (FcγR and FcαR) expression and function by studying IgG- and IgA-mediated elimination of Porphyromonas gingivalis. GCF-PMN exhibited increased FcαR levels and decreased FcγR levels compared to PB-PMN. Functional studies revealed GCF-PMN to exhibit a decreased capacity to phagocytose and kill IgG1-opsonized P.gingivalis compared to PB-PMN. IgA1-mediated phagocytosis and killing capacity was, however, comparable between GCF- and PB-PMN. These results suggest FcαR to represent a candidate target for FcR-directed immunotherapy of periodontitis.Recombinant 40-kDa outer membrane protein (rOMP-40) of P. gingivalis was obtained by purification with homogeneity from the cell sonicate. Human immunoglobulin producing mice, KM mouse^<TM>, were immunized by injection of rOMP-40. The spleen cells were collected, and then fused with the mouse myeloma cell line. Total 102 clones of human mononlonal antibodies (hMAbs) specific for rOMP-40 were obtained and screened by enzyme-linked immunosorbent assay. The number of IgG was 99, whereas three were IgM. IgA clone was not established. The opsonophagocytic activity of hMAb clones showing high reactivity with rOMP-40 was measured. Our results indicated hMAb clones to be significantly higher in opsonophagocytic activity than human polyclonal antibodies. These results provided the possibility that topical application of hMAbs would be aid in PMN-dependent elimination of P. gingivalis.