补体调节因子靶向治疗重症肌无力的研究

Myasthenia gravis (MG) is a typical organ-specific auto-immune disease. Autoantibody-induced complement activation, which causes disruption of the postsynaptic membrane, is recognized as a key pathogenic factor in MG. Our previous work demonstrated that specific targeting of complement inhibitors to the site of complement activation to pretect the AchR and complement regulator protein (CRP) at the cell level, however its mechanics and effect to the conduction of normal neuromuscular junction need to be further discussed. This study tends to construct scFv-CRP targeting fusion protein based on AChR with different subunit, and its antibody affinity and inhibitory capacity of complement attack will be measured. In order to confirm the mechanics of scFv-CRP and screen the scFv-CRP with safety and high efficiency, the protection ability and effect of scFv-CRP to AChR are compered in this study from melecular, cell, tissue, and animal levels by immunohistochemistry, flow cytometry, and radioimmunoassay based on TE671 cell line and experimental autoimmune myasthenia gravis in Lewis rat. This study may lay a solid foundation for the specific immunotherapy of MG, and provide a novel idea for the treatment of other autoimmune disease.

重症肌无力（myasthenia gravis，MG）是典型的器官特异性自身免疫性疾病。其发病与自身抗体攻击乙酰胆碱受体（AChR）引起补体介导细胞膜溶解和免疫调变作用相关。我们前期试验证实针对AChR 的单链抗体（scFv）与补体调节因子（DAF）的靶向融合蛋白能在细胞水平保护AChR免受自身抗体攻击，但其作用机制及对正常神经肌肉接头传导的影响尚待进一步探讨。本研究拟构建针对AChR不同亚基的scFvs-DAF靶向融合蛋白，并检测其抗体亲和力、补体攻击保护力。通过TE671细胞系和Lewis大鼠重症肌无力模型，采用免疫组化、流式细胞、放射免疫等手段，从分子、细胞、组织及动物整体水平等多层次比较scFvs-DAF对AChR的保护及影响，进一步明确scFvs-DAF 的作用机制并筛选出高效安全的scFvs-DAF，为MG的特异性免疫治疗奠定基础，并为其他自身免疫病的治疗提供一个新的思路。

项目背景：重症肌无力（MG）是一种乙酰胆碱受体抗体（AChR Abs）介导、细胞免疫依赖、补体参与的自身免疫性疾病。将针对AChR的单链抗体与补体调节因子偶联（scFv-CRP），通过阻止二价的病理性自身抗体与AChR的结合及补体系统激活来保护AChR及突触后膜免受补体系统的活化所致的免疫损伤是一种可能的靶向治疗策略。 研究内容：筛选出对乙酰胆碱受体AChR有较高亲和力的单链抗体scFv，并将其通过连接序列(G4S1)3与促衰变因子（DAF）在基因水平偶联。用原核表达系统表达融合蛋白scFv-DAF，经过优化表达及纯化、复性后得到正确折叠的可溶性scFv-DAF。并在分子和细胞水平检测scFv-DAF是否保留了两个组分scFv和DAF的功能。重要结果：实验筛选出有较高AChR结合能力的单链抗体，并成功将其与DAF在基因水平偶联。用原核表达系统对融合蛋白进行了表达纯化和重折叠，得到可溶性scFv-DAF。融合蛋白scFv-DAF不仅在分子水平保留了特异性结合AChR和补体抑制的活性，而且在细胞水平成功将DAF靶向到AChR处，并抑制了经典途径补体活化，scFv靶向的DAF对补体攻击破坏的保护作用要优于单纯可溶性DAF。关键数据及其科学意义：融合蛋白scFv-DAF的成功构建、表达及其功能鉴定，不仅为MG的特异性免疫治疗奠定基础，并且为其他自身免疫病的治疗提供新的思路和实验依据，具有重要应用前景和意义。