靶向于TNF-α和RANKL的人源化抗体对DBA/1小鼠单关节炎的保护作用  

The humanized monoclonal antibody h8G12 prevented arthritis in targeting both TNF-α and RANKL in DBA/1 mono-arthritis mouse model

作　　者：娄苇苇 杜晓楠[1] 袁慧慧[1] 窦云鹏[1] 赵文明[1] 李慎涛[1] 

机构地区：[1]首都医科大学基础医学院免疫学系风湿免疫研究室,北京100069

出　　处：《微生物学免疫学进展》2017年第2期8-16,共9页Progress In Microbiology and Immunology

基　　金：国家自然科学基金(31370936)

摘　　要：目的制备同时拮抗肿瘤坏死因子α(tumor necrosis factorα,TNF-α)和核因子κB受体活化因子配体(re-ceptor activator of NF-κB ligand,RANKL)的人源化单克隆抗体(h8G12),通过单关节炎小鼠模型,评估h8G12在抑制TNF-α引起的炎症反应和拮抗RANKL引起的骨破坏中的保护作用。方法培养稳定表达h8G12的CHO细胞株,观察细胞生长状态,用间接ELISA检测细胞培养上清中h8G12的表达水平。纯化的h8G12经SDS-PAGE、West-ern blot分析及鉴定,用间接ELISA观察其亲和力。通过向DBA/1小鼠膝关节腔注射人TNF-α重组蛋白和人RANKL重组蛋白,建立单关节炎小鼠模型。用组织学评估观察不同给药方案[阴性对照组、阳性对照组、阿达木单抗(adalimumab,ADA)组和h8G12组]对小鼠单关节炎引起的炎症浸润、软骨侵蚀、膝关节腔内破骨细胞分化的保护作用。结果 CHO细胞株培养96 h时细胞生长状态良好,可稳定分泌h8G12;纯化的h8G12纯度可达90%,Western blot可见特异性条带,且可与rh TNF-α和rh RANKL结合。在单关节炎小鼠模型中,苏木精-伊红染色显示,h8G12组关节腔、周围软组织及骨髓腔内炎性细胞浸润明显少于阳性对照组,炎症评分降低了50%,治疗效果与ADA相似。甲苯胺蓝染色显示,h8G12组病理评分出现显著下降,h8G12治疗后小鼠关节结构相对完好,关节软骨厚度接近正常水平。抗酒石酸酸性磷酸酶染色显示,h8G12组关节腔内破骨细胞明显减少。结论 h8G12可有效抑制TNF-α引起的炎症反应和拮抗RANKL引起的骨破坏,为类风湿关节炎的治疗提供了新方法。Objective To prepare humanized bispecific antibody h8G12 targeting both human tumor necrosis factor-α （TNF-α） and receptor activator for nuclear factor-κB Ligand （RANKL） and to evaluate its therapeutic effects on reducing TNF-α-induced inflammation and RANKL-mediated bone destruction in a mouse model with mnnoarthritis. Methods The Chinese hamster ovary （CHO） cells expressing h8G12 were cultured. The growth status of the CHO cells was observed, and the expression level of h8G12 in supernatants was measured by indirect ELISA. The purified h8G12 antibody was identified by SDS-PAGE and Western blot, and affinity was detected by indirect ELISA. The monoarthritic mouse model was prepared by intra-articular injection of rhTNF-α and rhRANKL in DBA/1 mice. The protective effects of h8G12 were evaluated by histological observation on inflammatory infiltration, cartilage erosion and the osteoclast differentiation in knee joint cavity resulted from different dose regime （including neagtive control, positive control, ADA and hSG12 group）. Results The CHO cells expressing h8G12 grew well and antibody concentration reached peak and kept at a steady state in supernatant, after cultured 96 h. The purity of the purified h8G12 reached 90% and specific bands were observed by Western blot analysis. The purified h8G12 could bind to rhTNF-α and rhRANKL. In the mouse model, HE staining showed that hgG12 significantly inhibited infiltration from inflammatory cells in the joint cavity, peripheral soft tissue and bone marrow cavity. Inflammatory infihration scores in h8G12-treated mice decreased by more than 50% compared with those in positive control group, and the therapeutic effect of h8G12 was similar to that of ADA. TB staining showed that the pathological scores of h8G12-treated mice were significantly lower than that in positive control group. The joint structure of articular cartilage of the mice in treated group was relative intact and the thickness of the articular cartilage was nearly to normal level. The

关 键 词：类风湿关节炎 肿瘤坏死因子Α 核因子ΚB受体活化因子配体 人源化单克隆抗体(h8G12) 

分 类 号：R593.22[医药卫生—内科学]