A rapid phage-free platform for antigen-specific VNAR screening with BATCH system.

Shark-derived variable new antigen receptors (VNARs) are promising single-domain antibodies due to their compact size, high stability, and cryptic epitope access. While conventional screening via phage display is highly effective, complementary approaches can further expand the methodological toolkit. We developed a phage-free bacterial two-hybrid (BATCH) platform for rapid antigen-specific VNAR discovery from immunized Chiloscyllium plagiosum. A Cre recombinase-immunized spleen cDNA library (~108 diversity) was cloned into pUT18c, screened against pKT25c-Cre in BTH101 cells using dual lacZ and CmR readouts enhanced by triple lacO sites and cAMP feedback, yielding two unique binders (VNAR1 and VNAR2) in 16 h. AlphaFold3 modeling highlighted CDR3 dominance in the Cre paratope, with C84-C90 disulfide stabilizing the loop; mutagenesis halved enzyme-linked immunosorbent assay affinity and disrupted BATCH function but retained transcriptional activation in 293 T cells (~27% EGFP+). This 1- to 2-day screening phase provides a streamlined alternative to conventional methods, facilitating the rapid identification of stable VNARs. By leveraging a simplified in vivo selection process, this BATCH-based platform offers a practical tool for VNAR discovery in various therapeutics and diagnostics applications.