Tumor necrosis factor (TNF-α) is an important clinically tested cytokine that could induce autoimmune diseases and inflammation. Therefore, the anti-TNF-α therapy strategy was developed and used therapeutically in various diseases, especially in the cytokine storm associated chimeric antigen receptor (CAR) T-cell therapy and antiviral therapy. Compare with other anti-TNF-α inhibitors, anti-TNF-α Nb (nanobody) has many unique advantages. Herein, we reported a novel humanized scaffold for library construction, which could be soluble and expressed in Escherichia coli (E.coli), and the efficiency capacity could reach as high as 2.01 × 10⁹. Meanwhile, an anti-TNF-α Nb was selected for further study after 4 rounds of screening, NT-3, as the optimal Nb could effectively inhibit TNF-mediated cytotoxicity. The IC50 of NT-3 was determined as 0.804 μM, and its apoptosis inhibition rate was 62.47 % in L929 cells. Furthermore, the molecular docking results showed that complementarity-determining regions (CDRs) of NT-3 could connect to TNF for blocking function through strong hydrogen bonds and salt bridges. In general, our study not only provided a good Nb screening platform in vitro without animal immunization, but also generated a series of novel humanized anti-TNF-α Nb candidates with potential applications.

肿瘤坏死因子（TNF-α）是一种经过临床测试的重要细胞因子，可诱导自身免疫性疾病和炎症。因此，开发了抗TNF-α疗法，并将其用于各种疾病，特别是在细胞因子风暴相关的嵌合抗原受体（CAR）T细胞疗法和抗病毒疗法中。与其他抗TNF-α抑制剂相比，抗TNF-αNb（纳米抗体）具有许多独特的优势。在本文中，我们报道了一种新型的人源化的用于文库构建的支架，该支架可以在大肠杆菌（E.coli）中溶解并表达，效率最高可以达到2.01×10 ^9。。同时，经过4轮筛选，选择抗TNF-αNb进行进一步研究，NT-3，因为最佳的Nb可以有效抑制TNF介导的细胞毒性。NT-3的IC50确定为0.804μM，在L929细胞中其凋亡抑制率为62.47％。此外，分子对接结果表明NT-3的互补决定区（CDRs）可以通过强大的氢键和盐桥与TNF连接，以发挥阻断作用。总的来说，我们的研究不仅在没有动物免疫的情况下提供了良好的体外Nb筛选平台，而且还产生了一系列具有潜在应用前景的新型人源化抗TNF-αNb候选物。