# A high diversity naïve variable new antigen receptor, vNAR, phage library for rapid nanobody discovery across diverse antigens

## TL;DR

This paper describes the creation of a highly diverse vNAR phage library from a shark, which can rapidly discover nanobodies that bind to a wide range of antigens with high affinity.

## Contribution

The study introduces a high-diversity vNAR phage library with 100% hit rate for diverse antigens and provides structural insights into class IV vNARs.

## Key findings

- The library achieved a 100% hit rate for isolating potent nanobodies with micro to nanomolar affinity.
- The library encompasses a broad range of classical functional vNAR types, as confirmed by next-generation sequencing.
- X-ray crystallography revealed structural details of underexplored class IV vNARs from bamboo sharks.

## Abstract

Conventional antibodies are among the most frequently used and effective biological tools explored for therapeutic and diagnostic applications. However, they face significant limitations when it comes to challenges that demand specialized attributes such as rapid tissue penetration, the ability to bind to concealed epitopes, and stability in nonphysiological environments. In recent years, shark-derived immunoglobulin variable new antigen receptor (vNAR) has emerged as a promising alternative to overcome these limitations. In this study, we constructed a naïve vNAR phage display library from a white-spotted bamboo shark (Chiloscyllium plagiosum), with a library diversity size of ∼3 × 1011 clones. Next generation sequencing analysis revealed the high diversity of the library, allowing it to encompass a broad range of classical functional vNAR types. To confirm the usability of the library for the successful isolation of positive clones, we screened the library against wide range of antigens (n = 9;) from different origin that includes viral, cancer, autoimmune, toxins, parasite, algae, and plant antigens. We achieved a hit rate of ∼100%, of potent binders with micro to nanomolar range affinity. The total number of unique binder’s clones varied from 30%-100%, depending on the antigens and screening strategy. Furthermore, we provide an in-depth structural analysis by using X-ray crystallography of class IV vNARs from bamboo sharks, which remain underexplored. Our study represents a significant step forward in the field of single-domain antibody research and development.