The currently used anti-cytokine therapeutic antibodies cannot selectively neutralize pathogenic cytokine signaling that cause collateral damage to protective signaling cascades carrying the potential for unwanted side effects. The variable domains of heavy-chain only antibodies (HCAbs) discovered in Camelidae are stable and display fully functional in antigen-binding against variable targets, which has been seemed as attractive candidates for the next-generation biologic drug study. The purpose of our study was to establish a simple prokaryotic expression system for large-scale expression, purification, and refolding of the recombinant anti-TNF-α fusion protein (FVH1-1) from inclusion bodies. Over 95% purity of the recombinant anti-TNF-α fusion proteins were obtained by just one purification step in our developed prokaryotic expression system, while the results of surface plasmon resonance (SPR) established the high-efficiency potent binding ability of FVH1-1 to human TNF-α. The counteraction of TNF-α cytotoxic effect experiment on the mouse fibroblast fibrosarcoma cell line (L929) confirmed that the expressed FVH1-1 were able to selectively and highly combine with hTNF-α in vitro. Western blot results showed that FVH1-1 can inhibit the activation of caspase-9 and PARP, which are the apoptotic signaling pathway proteins activated by hTNF-α. Meanwhile, lysosome autophagy signaling pathways stimulated by hTNF-α were inhibited by FVH1-1, which down-regulated the expression of LC3II/LC3I and up-regulated the expression of P62, indicating that the autophagy linked with TNF-α-induced apoptosis in response to rheumatoid arthritis. The results of the AIA rat experiment presented that FVH1-1 can reduce the degree of joint swelling and inflammatory factors to a certain extent in vivo.

中文翻译：

---

重组抗TNF-α融合蛋白通过自噬的保护作用改善类风湿性关节炎。


目前使用的抗细胞因子治疗抗体不能选择性地中和致病性细胞因子信号传导，这些信号传导会对保护性信号级联造成附带损害，从而可能产生不良副作用。在骆驼科动物中发现的纯重链抗体 (HCAb) 的可变结构域是稳定的，并且在针对可变靶标的抗原结合中显示出完整的功能，这似乎是下一代生物药物研究的有吸引力的候选者。我们研究的目的是建立一个简单的原核表达系统，用于从包涵体中大规模表达、纯化和重折叠重组抗TNF-α融合蛋白（FVH1-1）。在我们开发的原核表达系统中，仅通过一步纯化即可获得纯度超过 95% 的重组抗 TNF-α 融合蛋白，而表面等离子共振 (SPR) 结果证实了 FVH1-1 的高效强效结合能力人类 TNF-α。 TNF-α对小鼠成纤维细胞纤维肉瘤细胞系（L929）的细胞毒作用实验证实，表达的FVH1-1能够在体外选择性地与hTNF-α高度结合。 Western blot结果显示，FVH1-1可以抑制caspase-9和PARP的激活，这两种蛋白是hTNF-α激活的凋亡信号通路蛋白。同时，hTNF-α刺激的溶酶体自噬信号通路被FVH1-1抑制，下调LC3II/LC3I的表达，上调P62的表达，表明自噬与TNF-α诱导的细胞凋亡有关。对类风湿性关节炎的反应。 AIA大鼠实验结果表明，FVH1-1在体内可以一定程度减轻关节肿胀程度和炎症因子。