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

The new EASeL method also maximized the sequence diversity of CDR3 represented in the phage library to increase the selection of high-affinity binders. A previous study produced a shark phage library with 10 7 diversity using the conventional cloning method. Our library has 10 10 diversity and contains all known four types of V NAR single domains. The majority of the sequences are Type II and Type I V NAR as shown in Figure 2C . Interestingly, Type IV V NAR sequences are significantly under-represented as shown in Figure 2C . The shark V NAR sequences in the NCBI database are mostly derived from bamboo shark, dogfish shark, wobbegong shark, and other types of smaller sharks [12] [13] [14] [15] [16] [17] . The percentage of V NAR types can be significantly different between various species of sharks. Interestingly, our deep sequencing analysis showed that 56 174 of the nurse shark V NAR sequences in our library were not categorized in any one of the known V NAR types (Types I-IV). Whether these novel types of V NAR s possess unique confirmations and biophysical properties should be analyzed structurally and functionally when binders for cancer or viral antigens are found from these novel types of V NAR s in the future. Since the method we used to assemble the unique V NAR sequences for analysis is highly accurate and the number of the unique sequences analyzed is large, we used unbiased methods to analyze all the sequences for cysteine number and CDR3 length without further subdividing them into the four known V NAR types. A significant percentage of the sequences are not classical V NAR as defined in the known four types. Analyzing the data sequences altogether would provide more comprehensive picture of the sequence patterns in the naïve nurse shark without having to fit the sequences into a defined V NAR type. The extra cysteines in both Type I and Type II V NAR s are important for stabilizing the antigen-binding regions [11] . Extra disulfide bonds formed by these cysteines are essential for forming diverse antigen-binding surfaces. It may be one strategy for increasing antigen-binding region diversity in heavy chain-only antibodies.