Construction of a Synthetic Shark-Derived Nanobody Library with Conserved CDR3 Tryptophan and Its Application in...

Shark-derived variable new antigen receptors (VNARs) exhibit broad biomedical application prospects owing to their small size, exceptional thermal stability, and resistance to extreme pH conditions. Synthetic library construction enables the selection of specific VNARs without shark immunization, while employing stable universal scaffolds with rationally designed complementarity-determining region 3 (CDR3) length and amino acid composition. Here, a synthetic phage display library was constructed using a highly expressed and stable scaffold, guided by a systematic analysis of existing VNAR sequences. Its framework regions were retained, while the CDR1 and CDR3 were randomized with the conserved tryptophan (W) in CDR3 preserved. Using this library, VNARs specifically targeting the urinary tumor biomarkers hyaluronidase-1 (Hyal-1), Engrailed-2 (EN2), and prostate-specific antigen (PSA) were successfully identified. The affinity of all selected VNARs reached the micromolar (μM) level, and the expression level can reach 2.4-14.3 mg/mL. In summary, this study established a high-performance synthetic VNAR phage display library and preliminarily explored the role of the conserved tryptophan (W) in the CDR3. The VNARs targeting distinct epitopes of Hyal-1, EN2, and PSA obtained through screening represent promising candidate molecules for the diagnosis and treatment of related cancers.