Biopharmaceutical Lab, College of Life Science, Northeast Agricultural University, Harbin, China, 150030.
Curr Pharm Biotechnol. 2014;14(12):1048-61. doi: 10.2174/1389201015666140113111711.
IL-1β and TNF-α play key roles in the inflammatory response. Their abnormal expression may cause the occurrence of various diseases, such as RA. Recently, medicines of target TNF-α and IL-1β have become popular in the clinical practice. Although these biological agents can get mostly good results, they are not effective in all patients. The reason for this result may be that these biological agents could not fully inhibit a variety of inflammatory cytokines in the inflammatory response. In the present study, a fusion protein gene which encoded human interleukin-1β scfv and soluble TNF receptor I (sTNFRI) was cloned. A number of in vitro assays demonstrated that anti-IL-1β scfv/TNFRI simultaneously bound to both targets. The bioactivity assay showed that the fusion protein could inhibit both the cytotoxicity of hTNF-α on L929 cells and hIL-1β-induced proliferation of L929 cells, indicating that the fusion protein has the ability to neutralize both hTNF-α and hIL-1β. In this study, we established the chicken type II collagen-induced rheumatoid arthritis model in Kunming mice, and evaluated the pharmacological effect of the fusion protein in vivo. Model mice were randomly divided into 8 groups (n=8): CIA model control group, DEX treatment group (1 mg/kg), intraperitoneal treatment group (highdose: 5 mg/kg; medium-dose: 2 mg/kg; low-dose: 0.8 mg/kg), subcutaneous treatment group (high-dose: 5 mg/kg; medium- dose: 2 mg/kg; low-dose: 0.8 mg/kg), and healthy mice as control. The control group received the same volume of saline. The mice were administrated once every 2 days. Arthritis index, anti-CII antibody titers, cytokine levels, histopathological changes were examined. The results showed that anti-IL-1β scfv/TNFRI fusion protein could reduce the degree of joint swelling, inflammatory cell infiltration, synovial cell proliferation and the level of CII antibody in the sera. The Real-time PCR analysis showed that anti-IL-1β scfv/TNFRI had the ability to reduce the expression of IL-1β, TNF-α, IL-17A, MMP-3, IL-6 and improve the expression of IL-10 in a dose-dependent manner, suggesting that the fusion protein is the mediator for IL-1β and TNF-α involved in the RA process. Compared with DEX positive medicine control, anti-IL-1β scfv/TNFRI appeared more beneficial in treatment of CIA mice. The therapeutic effect of the anti-IL-1β scfv/TNFRI at 5mg/kg was significantly better than that of DEX treatment. So the anti-IL-1β scfv/TNFRI can become a candidate for treatment of RA.
IL-1β 和 TNF-α 在炎症反应中发挥关键作用。它们的异常表达可能导致各种疾病的发生，如 RA。最近，靶向 TNF-α 和 IL-1β 的药物在临床实践中变得流行。尽管这些生物制剂大多能取得良好的效果，但并非对所有患者都有效。造成这种结果的原因可能是这些生物制剂不能完全抑制炎症反应中多种炎症细胞因子的产生。在本研究中，克隆了编码人白细胞介素-1β scfv 和可溶性 TNF 受体 I（sTNFRI）的融合蛋白基因。多项体外检测表明，抗 IL-1β scfv/TNFRI 同时与两个靶标结合。生物活性检测表明，融合蛋白能抑制 hTNF-α 对 L929 细胞的细胞毒性和 hIL-1β 诱导的 L929 细胞增殖，表明该融合蛋白具有中和 hTNF-α 和 hIL-1β 的能力。在本研究中，我们建立了昆明小鼠 II 型胶原诱导的类风湿关节炎模型，并在体内评估了融合蛋白的药理学作用。模型小鼠随机分为 8 组（n=8）：CIA 模型对照组、DEX 治疗组（1mg/kg）、腹腔内治疗组（高剂量：5mg/kg；中剂量：2mg/kg；低剂量：0.8mg/kg）、皮下治疗组（高剂量：5mg/kg；中剂量：2mg/kg；低剂量：0.8mg/kg）和健康小鼠作为对照组。对照组给予相同体积的生理盐水。每 2 天给药一次。检查关节炎指数、抗 CII 抗体滴度、细胞因子水平、组织病理学变化。结果表明，抗 IL-1β scfv/TNFRI 融合蛋白可降低关节肿胀程度、炎性细胞浸润、滑膜细胞增殖和血清中 CII 抗体水平。实时 PCR 分析表明，抗 IL-1β scfv/TNFRI 能降低 IL-1β、TNF-α、IL-17A、MMP-3、IL-6 的表达，并能改善 IL-10 的表达，呈剂量依赖性，提示该融合蛋白是 RA 过程中 IL-1β 和 TNF-α 参与的介质。与阳性药物 DEX 对照组相比，抗 IL-1β scfv/TNFRI 对 CIA 小鼠的治疗效果更有益。抗 IL-1β scfv/TNFRI 5mg/kg 的治疗效果明显优于 DEX 治疗。因此，抗 IL-1β scfv/TNFRI 可成为治疗 RA 的候选药物。
