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

Interestingly, the sequence variation mostly lies in CDR3 with minimal variation in CDR1, HV2, and HV4. Previous literature suggested there are sequence variability in HV2 and HV4 during in vivo affinity maturation [24] . It was also shown that somatic mutations within HV2/4 can contribute to antigen binding [21] . However, NGS results in the present study showed there is minimal sequence variability in CDR1, HV2, and HV4 in our naïve shark library. It is possible that these naïve sharks used in our study have been kept in captivity for a long time, therefore do not have as much sequences diversity in Antibody Therapeutics, 2019 9 CDR1, HV2, and HV4. Another major type of heavy chain-only domain antibody V H H from camels also has highly diversified CDR3. The lengths of V H H CDR3 are much longer than conventional VH from camel IgG. The median CDR3 length of V H H is 20 amino acids (similar with shark V NAR ), whereas median length for camel VH CDR3 is only 15 amino acids [32] . However, there is no comprehensive study on the cysteine numbers and locations in camel V H H sequences for comparison with shark V NAR .