单克隆抗体Tocilizumab模拟表位的筛选和鉴定

Rheumatoid arthritis (RA) is a symmetric polyarticular arthritis that primarily affects the small diarthrodial joints of the hands and feet. In addition to inflammation in the synovium, which is the joint lining, the aggressive front of tissue called pannus invades and destroys local articular structures..The success of biologic DMARDs that target the pro-inflammatory cytokine tumour-necrosis factor-α (TNF-α) in the treatment of RA - as well as recognition of their limitations - stimulated extensive research on the involvement of other cytokines in the disease process. Among these is interleukin-6 (IL-6), a pleiotropic cytokine that has important roles in a range of biological processes, including regulation of the immune response and inflammation. Signalling by IL-6 is mediated by a receptor system that consists of the ligand-binding membrane protein IL-6R (mIL-6R) and the signal-transducing membrane protein gp130. Several lines of evidence indicated the involvement of IL-6 in the pathogenesis of RA, including increased expression of IL-6 in the synovium of patients with RA and a correlation between elevated IL-6 levels in serum or synovial fluid and measurements of disease..Tocilizumab is a humanized monoclonal antibody that binds specifically to both sIL-6R and mIL-6R, and inhibits sIL-6R and mIL-6R-mediated signalling. It is the first biologic agent specifically targeting IL‐6. It has demonstrated clinical efficacy in a number of RA populations. It was first granted regulatory approval in 2005 in Japan for the treatment of Castleman's disease, a rare lymphoproliferative disorder, and was approved for RA and juvenile idiopathic arthritis in Japan in 2008. Tocilizumab (Actemra) was approved by the FDA on January 8, 2010 for the treatment of rheumatoid arthritis. However, it became evident that the monoclonal antibody approach had serious practical limitations, like poor efficacy and limiting side effects due to passive application of - then murine - monoclonal antibodies. The mimotope is a minimal structural elements which are required to elicit a specific humoral and cellular immune response and found to be peptides from 6 to 20 amino acids length. Definition of peptide mimics of epitopes, so-called mimotopes, was made feasible by the application of a novel technique: screenings of random peptide phage display libraries. A special advantage of the phage display technique is that the epitope to be mimicked does not need to be defi ned; the only prerequisite is that an antibody recognizes it. Here our aim was to identify peptide mimics of the IL-6 epitope recognized by tocilizumab and to develop a mimotope vaccine. Epitopespecific immunization is feasible for active anti-IL-6 immunotherapy. The in vitro biologic features of mimotopeinduced antibodies are similar to those of the monoclonal antibody Tocilizumab.

近年来，不同的研究和资料显示IL-6在类风湿关节炎的发生和发展过程中起着相当重要的作用。在2010年，经FDA批准的Tocilizumab(ROACTEMRA)抗体是一个新型的靶向IL-6的药物，也是第一个临床试验证明单用治疗类风湿关节炎的疗效要好于MTX的药物。但单克隆抗体在类风湿关节炎的免疫治疗中仍旧存在着许多不足之处，其中反复的被动免疫和昂贵的治疗费用是最主要的问题。因此，能够刺激机体产生主动免疫的模拟表位肽成为了一种新型的免疫治疗方法。模拟表位肽是一种能刺激机体产生特异性免疫的多肽，模拟表位同自然表位可能有不同的序列但在空间结构上类似，从而具有相同的生物学活性。本研究拟通过噬菌体展示技术筛选Tocilizumab的模拟表位，用模拟表位免疫小鼠后，刺激小鼠产生抗体，该抗体不仅能识别模拟表位，并且能识别IL-6R,从而抑制IL-6的活性，产生与Tocilizumab类似的治疗效果。

类风湿关节炎(RA)是一种慢性、系统性、多向性的炎性自身免疫性疾病，主要累及手足小关节，最大的特征就是持续性的滑膜炎，症状包括关节疼痛、关节肿胀以及进展性的关节破坏，最终导致患者的残疾。近年来，研究者发现IL-6在类风湿关节炎的发生和发展过程中起着相当重要的作用。在2010年，经FDA批准的Tocilizumab(ROACTEMRA)抗体是一个新型的靶向IL-6的药物，也是第一个临床试验证明单用治疗类风湿关节炎的疗效要好于MTX的药物。但单克隆抗体在类风湿关节炎的免疫治疗中仍旧存在着许多不足之处，其中反复的被动免疫和昂贵的治疗费用是最主要的问题。由此，能够诱导机体产生主动免疫的模拟表位肽随之出现。模拟表位肽能够诱导机体产生特异性免疫靶向预期靶点，从而与抑制靶点信号通路和生物学功能。本研究拟通过噬菌体展示技术筛选Tocilizumab的模拟表位肽，通过免疫动物观察动物的主动免疫是否被活化从而起到同Tocilizumab相似的生物学活性。我们运用噬菌体文库技术共筛选到4个能特异性结合Tocilizumab的模拟表位肽。通过测序、多肽合成和免疫动物后，我们运用免疫学实验发现小鼠免疫后的抗血清能结合人肿瘤细胞系和RA-FLS表面表达的天然IL-6R，4个模拟肽产生的抗血清对不同细胞系信号通路的影响不同并且能显著杀伤肿瘤细胞系。另外，在延伸研究中发现，EGFR模拟肽能够有效缓解干燥综合症小鼠模型中小鼠腺体中的淋巴细胞浸润和炎症因子的分泌，机制研究同时发现EGFR模拟肽能有效提高淋巴细胞表面FAS蛋白的表达从而促进淋巴细胞凋亡。本研究成功筛选到了Tocilizumab的模拟表位肽，推进了模拟肽研究的不断发展，为研发优于单克隆抗体的药物提供了一个可行的方向，为未来类风湿关节炎的治疗提供了可能的选择。另外，延伸项目的完成也证实了在其他自身免疫病模型中，模拟肽能起到激活免疫靶向治疗的作用。该研究提示了模拟肽不仅能作为一个靶向特异性靶点的多肽药物，而且模拟肽也能作为研究分子信号的工具。