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.

The solubility and Immunogenicity of protein significantly impacted the production efficiency and medical application of Nbs. The soluble and humanized framework was rationally designed through bioinformatics analysis. The phagemids carrying genetic diversity of Nb were electro-transformed into TG1 cells for library construction and isolated the binding TNF-α function of Nb. Protein products of TNF-α specific Nb were prepared with E.coli expression system and its bioactivity was verified by cytotoxicity assay. [Display omitted] • Nanobodies are the variable domains of camelid heavy chain antibodies (VHH). • A new method was built to design humanized framework with high solubility using bioinformatics tools. • The constructed library was of high quality and used to screen anti-TNF-a Nanobodies. • NT-3 showed a good expression in supernatant and significant inhibitory effect for L929 cells apoptosis mediated by TNF-α. 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. [ABSTRACT FROM AUTHOR]