In vitro selection of VNAR antibodies using bioinformatics-based library design and cDNA display

Variable domains of new antigen receptors (VNARs) derived from shark immunoglobulin new antigen receptors (IgNARs) have gained attention as promising single-domain antibody modalities owing to their compact structures, structural stability, and unique binding capabilities. This study aimed to demonstrate the feasibility of screening a synthetic library based on VNARs derived from the brownbanded bamboo shark ( Chiloscyllium punctatum ) using a complementary DNA (cDNA) display system. A synthetic VNAR library was constructed by randomizing the CDR1 and CDR3 loops, resulting in a genetic diversity of approximately 10 12 sequences in the cDNA display format. Using fluorescent proteins as model antigens, in vitro selection was performed, followed by next-generation sequencing and biochemical validation. VNAR-E1 and VNAR-N1 were identified as binding to EGFP and mNeonGreen, respectively. This work represents the first demonstration of applying a cDNA display system to VNAR selection.