Key Laboratory of the Ministry of Education for Medicinal Resources and Natural Pharmaceutical Chemistry (W.Z., Z.W., S.H., Y.G., Y.L., H.S., X.D., Y.F., Y.Y.), National Engineering Laboratory for Resource Development of Endangered Crude Drugs in Northwest of China, College of Life Sciences, Shaanxi Normal University, Xi'an; Department of Neurology (W.Z.), Tianjin Neurological Institute, Tianjin Medical University General Hospital; and Department of Neurology (Z.W.), Xuanwu Hospital, Capital Medical University, Beijing, China.
Neurol Neuroimmunol Neuroinflamm. 2019 Apr 4;6(3):e561. doi: 10.1212/NXI.0000000000000561. eCollection 2019 May.
Using phage display, we sought to screen single-chain variable fragments (scFvs) against complement C5 to treat neuromyelitis optica spectrum disorder (NMOSD).
After 5 rounds of phage display, we isolated individual clones and identified phage clones specifically binding to C5 using ELISA. Using aquaporin-4 (AQP4)-transfected cells in vitro, we confirmed whether these scFvs prevented complement-dependent cytotoxicity (CDC) caused by the serum of patients with NMOSD and human complement (hC). We selected an NMOSD mouse model, in which intracerebral NMOSD immunoglobulin G (IgG) and hC injections induce NMOSD-like lesions in vivo.
We obtained scFvs to test specificity and blocking efficiency. The scFv C5B3 neutralized C5 in the complement activation pathway, which prevented AQP4-IgG-mediated CDC in AQP4-transfected cells. In an NMOSD mouse model, C5B3 prevented AQP4 and astrocyte loss, decreased demyelination, and reduced inflammatory infiltration and membrane attack complex formation in lesions.
We used phage display to screen C5B3 against C5, which was effective in inhibiting cytotoxicity in vitro and preventing CNS pathology in vivo.
通过噬菌体展示技术，筛选针对补体 C5 的单链可变片段（scFv），以治疗视神经脊髓炎谱系疾病（NMOSD）。
经过 5 轮噬菌体展示，我们分离了单个克隆，并通过 ELISA 鉴定出与 C5 特异性结合的噬菌体克隆。我们使用体外转染水通道蛋白-4（AQP4）的细胞，证实这些 scFv 是否能阻止 NMOSD 患者血清和人补体（hC）引起的补体依赖性细胞毒性（CDC）。我们选择了 NMOSD 小鼠模型，在该模型中，脑内 NMOSD IgG 和 hC 注射可在体内诱导 NMOSD 样病变。
我们获得了 scFv 以测试其特异性和阻断效率。scFv C5B3 中和了补体激活途径中的 C5，阻止了 AQP4-IgG 介导的转染 AQP4 细胞中的 CDC。在 NMOSD 小鼠模型中，C5B3 可防止 AQP4 和星形胶质细胞丢失，减少脱髓鞘，减少病变中的炎症浸润和膜攻击复合物形成。
我们使用噬菌体展示技术筛选出针对 C5 的 C5B3，该 scFv 可有效抑制体外细胞毒性，并预防体内中枢神经系统病理。
