Construction and next-generation sequencing analysis of a large phage-displayed V(NAR) single-domain antibody library from six naïve nurse sharks

Feng, Mingqian; Bian, Hejiao; Wu, Xiaolin; Fu, Tianyun; Fu, Ying; Hong, Jessica; Fleming, Bryan D; Flajnik, Martin F; Ho, Mitchell

Phage display technology has been used to isolate shark V NAR antibodies. In one study, two shark V NAR libraries with a size of 10 7 clones were constructed from both naïve spiny dogfish (Squalus acanthias) and smooth dogfish (Mustelus canis) sharks [20] . Phage antibodies were isolated from immunized sharks for specific antigens such as hen egg white lysozyme [21] [22] [23] [24] . Synthetic shark V NAR singledomain libraries were also pursued [25] [26] [27] [28] [29] . However, none of these approaches has generated a large shark V NAR single-domain library that covers the wide range of diversity required (>10 10 ) commonly for therapeutic antibody discovery. In this study, we developed a PCR-Extension Assembly and Self-Ligation-based method (named EASeL) to make a large phage-displayed V NAR single-domain library from six nurse sharks. Nurse sharks were chosen to maximize the diversity of the V NAR library because they exclusively have been reported to contain Type I V NAR domains. To assess its diversity and analyze the V NAR sequences on the largest scale in the field, we conducted next-generation sequencing (NGS) analysis on 1.19 million unique full-length V NAR s. The unique sequences were then analyzed using unbiased methods to investigate their cysteine numbers and CDR3 lengths for all V NAR sequences together as well as Type I and Type II/III V NAR s separately. To validate the potential of this library as a new platform for therapeutic antibody discovery, we conducted phage panning to identify shark Antibody Therapeutics, 2019 3 V NAR binders to a panel of tumor and viral antigens. These included antigens associated with liver and breast cancers, as well as antigens against viral proteins associated with SARS and MERS. These selected binders including Type I and II V NAR s were produced successfully in E. coli as soluble proteins for antigen-binding validation. This work validated the large diversity of the nurse shark V NAR library and the utility of the shark V NAR library as a platform for therapeutic antibody discovery.