Designing and constructing a phage display synthesized single domain antibodies library based on camel VHHs frame for screening and identifying humanized TNF-α-specific nanobody

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.