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

Cartilaginous fish (sharks, rays, skates, and chimaeras) are phylogenetically the oldest living organisms that use antibodies as part of their adaptive immune system [3, 4] . They produce three different antibody isotypes that function in their humoral immune responses, immunoglobulin M (IgM), immunoglobulin W (IgW), and immunoglobulin new antigen receptor (IgNAR) [3] [4] [5] [6] . IgNAR antibodies are homodimeric proteins composed of heavy chains with an antigen-binding region at the end of each chain. They serve as a major component in humoral responses [4, 7] . A number of camelid V H H domains are being evaluated in phase I, II, and III clinical trials [8, 9] . Even though the shark V NAR is less known, it has the potential to be used in biological therapeutics based on (i) their small size and ability to penetrate dense tissues inaccessible to IgG [10] , (ii) their ability to bind in protein clefts and buried functional sites (e.g. enzyme pocket sites for substrate) [11] , (iii) their solubility and robustness in harsh conditions [12] , and (iv) their ability for high-affinity (including sub-nanomolar) binding. Additionally, these antibodies have the potential to bind a wide range of antigens, despite the nature of their single-domain architecture [13] .