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

To evaluate the library's potential for therapeutic development, we chose a variety of human tumor biomarkers and virus antigen proteins as selection targets. These include glypican-3 (GPC3), HER2 and PD1, the spike proteins of the MERS and SARS viruses, and Pseudomonas exotoxin (PE38). After four rounds of panning, specific binders to the listed targets were identified by monoclonal phage ELISA (Fig. 6A ). The binders were assigned the following names based on their targets and well numbers, which include GPC3-F1, HER2-A6, PD1-A1, MERS-A3, MERS-A7, MERS-A8, MERS-B4, MERS-B5, SARS-01, and PE38-B6. Only one binder was isolated for all antigens, except MERS spike protein. Five binders were isolated for MERS spike protein. Sequence analysis showed that most of these binders were Type II V NAR except for one Type I and two undefined types. One Type II binder (PE38-B6) targeting PE38 fragment was produced in E. coli HB2151 strain as a single-domain protein. It had 10.1 nM K d binding affinity for its antigen as measured by Octet kinetic assay ( Fig. 6B and C) . The affinity is high as a monomeric single-domain soluble protein isolated from a naïve shark library without immunization. We also produced the Type I binder (MERS A8) and Type II binder MERS A7 in E. coli. The protein yield varied based on protein sequences. PE38-B6 has a relatively low yield (3 mg from 2 L E. coli culture). MERS A8 binder yielded 3.1 mg and MERS A7 yielded 8.7 mg of single-domain soluble protein from 600 ml E. coli culture. Nickel-charged HisTrap columns (GE Healthcare, Chicago, IL) were used to purify these proteins from the polymyxin B lysed bacteria pellet supernatants. The elution profile showed the protein elution in Figure 7A and D. sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the elution peaks in Figure 7B and E showed the later peak eluted by higher imidazole concentrations had over 90% purity of the target soluble V NAR s. Both MERS A8 and A7 had specific binding to the phage panning antigen MERS spike protein in Figure 7C and F. The Type I binder (MERS A8) had higher signals. TSK size exclusion column purification for the Type II binder PE38-B6-his soluble single-domain protein showed this protein is monomeric. The 16.8 kDa protein was eluted with one peak that is slightly earlier than 13.7 kDa control protein peak ( Fig. 7G and H) . Taken together, binders to tumor and viral antigens, including Type I and Type II single domains containing multiple cysteine residues, can be selected from the library and produced as functional single-domain proteins in E. coli.