Humanized synthetic nanobody library for antivenom development

Humanized synthetic nanobodies are increasingly important in biomedical research and therapeutic antibody development owing to their high specificity, small size, superior tissue penetration, and reduced immunogenicity. In this study, we constructed a humanized synthetic nanobody library by combining eight sub-libraries. Each sub-library comprised four framework regions derived from human immunoglobulin framework sequences, whereas the complementarity-determining regions (CDRs) were designed based on camelid nanobody sequences. The resulting combined library reached a titer of 1.8 × 10⁸ CFU/mL, with approximately 80% of clones containing correct inserts. To evaluate the functionality of the library, a synthetic 15-mer peptide mimic (Asp49-41) corresponding to an enzymatic region of Asp49 phospholipase A₂ (PLA₂) from snake (Crotalus atrox) venom was synthesized and used as the target antigen. Four rounds of phage display bio-panning were conducted against Asp49-41, followed by phage Enzyme-linked Immunosorbent Assay (ELISA) screening of 1344 bio-panning-derived clones. This screening identified 80 positive clones, from which 30 exhibiting stronger binding signals were selected for subsequent ELISA analysis, resulting in the identification of 16 high-affinity clones. The four clones demonstrating the strongest binding capacity were further evaluated for their ability to inhibit the enzymatic activity of Asp49 PLA₂, and all four nanobodies exhibited inhibitory effects. This nanobody library holds significant potential for broad applications in biomedical research and therapeutic antivenom development.