RANKL-TNF样区双靶点人源化抗体对RA模型鼠的保护作用和机制研究

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by progressive synovitis, cartilage erosion and bone destruction. Raised levels of TNF-α in sera and synovial fluid suggest that TNF-α plays an important role in the pathogenesis of RA through triggering inflammatory cytokine cascades in patients with RA. In addition, osteoclasts derived from the myeloid haemopoietic lineage are the principal cells involved in bone resorption, while receptor activator of NF-κB ligand (RANKL) is crucial regulator of osteoclast differentiation, formation, activation and apoptosis. Reducing inflammatory and preventing bone resorption are the key point to successfully cure RA, such as blockade of TNF-α activity with either anti-TNF-α monoclonal antibodies or soluble chimeric TNF-α receptors, and inhibition the binding of RANKL to RANK. In clinic, TNF-α antagonist could significantly ameliorate inflammation, and RANKL monoclonal antibody could delay bone injury. Combined blockade of TNF-α and RANKL had more prominent therapeutic effects in arthritic models. However, their expensive price limited their application. TNF-α and RANKL, as two members of TNF superfamily, share some similarities in structures and pathological functions, which were considered as attractive therapeutic targets for inhibiting inflammation and bone resorption. We previously found a single agent with the dual functions of mitigating inflammation and bone resorption. High titers of antibodies against both human TNF-α and RANKL were generated with the immunization of RANKL-TNF-like core fusion protein (RTFP). Moreover, RTFP also prevented the synovial inflammation and focal bone erosion in collagen-induced arthritis (CIA). The monoclonal antibody would be more useful than the vaccine. A positive anti-RANKL/TNF homologous domains cell line (ARTHC) was screened. This hybridoma secreted a dual functional monoclonal antibody (mAb), named 8G12, targeting TNF-α and RANKL. This mAb 8G12 neutralized the bioactivity of TNF-α and RANKL and increased bone mass and reduced inflammation due to binding to the free forms of TNF-α and RANKL locally. In this funding, a new humanized antibody simultaneously targeting RANKL and TNF-α will be produced, and the mechanisms and dual protective functions of inhibiting inflammation and bone destruction in RA will also be evaluated. The novel humanized antibody will be a good candidate to treat inflammatory bone diseases, such as RA.

滑膜炎和骨损伤是类风湿关节炎（RA）最显著且相关联的两个病理学特征。TNF-α是介导RA炎症的重要因子，并诱发骨破坏；RANKL是骨损伤的必需因子。有效抑制炎症、并从疾病早期预防骨破坏是成功治疗RA的关键。临床数据表明，TNF-α拮抗剂可显著改善炎症，抗RANKL单抗能延缓骨损伤，但使用两种药物费用昂贵。已知RANKL与TNF-α同为TNFSF成员，TNF样区氨基酸序列高度相似，这为应用一种制剂同时抑制RANKL和TNF-α的致病作用成为可能。基于以上设想，本课题组前期开展了双靶点多肽疫苗研究，对RA动物模型有明显保护作用。但与疫苗相比，抗体治疗更及时可控，且更具应用价值。本课题拟以RANKL中与TNF-α高相似区作为靶标制备一新型双靶点RANKL-TNF样区人源化改形抗体，在RA动物模型中评估其抑制炎症和骨破坏的双重疗效，并探讨其作用机制。鉴于该抗体的特点和优势应具有应用前景。

本项目主要研究内容为针对滑膜炎症和骨破坏这两个类风湿关节炎（RA）的重要病理学特征，以其诱发的关键致病因子TNF和RANKL为靶点，用人RANKL-TNF样区肽类抗原免疫小鼠，研究制备鼠源性单克隆抗体，并改造为人源化抗体，观察其对RA动物模型—CIA小鼠的双功能保护作用及其机制。在本项目的研究中还探讨了与抑制RA滑膜炎症和骨破坏相关的其他分子的作用。.研究取得以下成果：研制出的鼠源性RANKL-TNF样区单克隆抗体能有效缓解模型鼠的炎症和骨损伤；通过CDR替换技术，将能同时拮抗TNF-α和RANKL的鼠源性单抗改造成人源化抗体；人源化抗体对DBA/1小鼠单关节炎保护作用的研究显示，其可有效抑制TNF-α引起的炎症反应和拮抗RANKL引起的骨破坏；人源化抗体对CIA小鼠保护作用的研究表明，其能明显抑制RA动物模型的滑膜炎症反应及骨破坏；与该人源化抗体作用相似的IL-17RC的研究显示，其通过中和IL-17A和IL-17F可明显降低TNF-α、RANKL等RA致病相关因子的分泌，缓解RA模型鼠的滑膜炎症和骨破坏，以后可进行其与人源化抗体作用的比较研究；RA骨破坏的新靶点研究显示，芳香烃受体（Ahr)与RA小鼠的骨破坏成正相关关系，因此认为抑制Ahr也可作为阻止RA骨破坏的新靶点，为抑制RA骨破坏的新型生物制剂的研究奠定了基础。