p53四价功能域架构抗环瓜氨酸肽小分子抗体(CCP-TeAb)的构建及其拮抗类风湿关节炎自身免疫炎症作用的研究

Rheumatoid arthritis(RA) is an autoimmune disease characterized by a pathological feature of chronic and progressive synovitis，affecting 0.32-0.38％of Chinese population with high disability and mortality. Anti-cyclic citrullinated peptide(CCP) antibodies has been suggested to be a specific serologic marker in the diagnosis of RA ,and to play an important role in the pathogenesis. Environmental factors induced the citrullination of self-antigen in the genetic susceptibly. Conformational modified citrullinated epitope increased affinity to HLA-DRB1 shared epitope(SE) and were presented to autoreactive helper T cell 17(Th17), the balance of Th17/Treg cells were broken, and then B cells were active to product anti-CCP antibodies. The RA pathological immune response cycle was formed. This study is aimed to construct a tetravalent miniantibody specifically binding to CCP (CCP-TeAb) on p53 tetramerization to improve the molecular weight and avidity of the single chain antibody by the phagemid display techniques. CCP-TeAb will be analyzed whether or not to act as a blocker of CCP to break the pathogenic RA circle with anti-CCP in vitro and vivo. It is of promise for the development of biological agent to inhibit the autoimmune inflammation of RA.

类风湿关节炎（RA）是以慢性进展性滑膜炎为主要病理改变的自身免疫性疾病。我国患病率0.32-0.38％，有较高致残率死亡率。抗环胍氨酸肽（CCP）抗体不仅是RA诊断的高特异性血清学标志，研究表明其可能在RA的发病机制中也有重要作用。RA遗传易感者在环境因素影响下瓜氨酸化自身抗原，使其空间构象表位发生改变，CCP是关键共同表位，与HLA-DRB1共同表位亲和力增加，递呈给自身免疫性Th17细胞，打破Th17/Treg调控平衡，进而活化B细胞产生抗CCP抗体，产生RA免疫应答环。本研究拟用噬菌体展示技术以p53四聚结构域来改造现有单价噬菌体抗CCP-ScFv抗体，构建四价小分子抗体（CCP-TeAb），以增加单链抗体的分子量和亲和力。由体外、体内试验两个方面来验证CCP-TeAb是否能够作为封闭抗体，阻断抗CCP抗体为介质的RA发病机制环路，抑制自身免疫炎症过程。为在生物制药开发奠定试验基础。

本研究拟通过构建p53四聚化抗CCP-ScFv抗体（TeAb-Anti-CCP-ScFv-P53，TeAb）增加亲和力。在离体和在体试验，检验TeAb能否作为暴露表位封闭抗体的治疗作用。构建成功的TeAb亲和常数显著提升（Ka≌8.19×1011M-1）。TeAb处理7天后显著性抑制了上清液抗CCP抗体滴度和CCP抗体分泌性B细胞计数，抑制了CD3+CD8- IL17+ Th17细胞/CD3+CD8-T比率和IL-6、TNFα和IL-17a的分泌，抑制FLS增殖、活化、迁移、侵蚀以及金属蛋白酶分泌和自身抗体及抗原瓜氨酸化酶的产生。TeAb处理后的PBMC+FLS共培养上清液对破骨细胞（OC）分化有明显的抑制作用（TeAb vs Control组OC形成细胞融合指数0.36±0.04 vs 0.51±0.04，T=2.54 P=0.021），抑制率29.11％，平均视野骨片吸收陷窝形成数分别为3.2±0.44 vs 5.3±0.45，T=3.33 P＜0.01），抑制39.62％。1mg/50g TeAb治疗21天后，抗体封闭组（BLK）足垫厚度从治疗前11.03±1.14mm 显著地减低至4.70±0.95 ，T值=12.09 P＜0.01；关节炎指数从治疗前3.83±0.35显著地降至1.37±0.74，T值=8.45 P＜0.01；BLK组滑膜炎病理评分显著改善。BLK组18F-FDG标记的滑膜炎症T/NT从治疗前1.27±0.08显著性降低至0.99±0.10（T值5.89 P＜0.001）。99mTc-3P4-RGD2标记的滑膜血管翳T/NT由11.02±1.34显著地降低至4.70±0.95（T值=12.08，P＜0.001）。BLK组治疗后21天与基线比较：前炎症细胞因子IL-6、TNFα、IL-17a和VEGF水平有显著性减低；Th17/Treg细胞比率的变化值与对照组比较，有显著性的减低；CD19+CD24hiCD38hiBreg百分比有显著性的上升。BLK组与ConA组间CCP分泌性B细胞/CD19+B比值（7.76±3.29 vs 20.56±4.87，T值=-5.45 P＜0.001）差异有显著。综上所述，TeAb-Anti-CCP-ScFv-P53在体外和在体模型动物实验均证实了其针对RA发病机制环节的治疗作用，为在人体研究奠定了基础。