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ABSTRACT
The fully synthetic humanized phage antibody library has the advantages including the minimized immunogenicity, which frequently happened in hybridoma cell-based antibody production. In this paper, using the constructed diverse complementarity determining region gene library and the germline gene as the backbone, we constructed eight single-chain antibody libraries and a combinatorial antibody library with a big capacity of 1.41 × 1010. M13EEA helper phage that was engineered from M13KO7 was applied to prepare phage antibody library. The eukaryotic expression of T-cell immune receptor with Ig and ITIM domain (TIGIT) antigen was used as a target antigen for screening. The screening of antigen-specific single-chain Fc-fused protein was performed through evaluation of binding affinity based on ELISA analysis. The IgG antibody was prepared with the screened single-chain protein. Finally, the CB3 antibody was screened out which exhibits the highest binding affinity with TIGIT with the Kd value of 8.155 × 10−10 M.
Graphical abstract
The schematic illustrates the procedure of selection of TIGIT-specific human monoclone antibody with high specificity and binding activity.
Acknowledgments
We gratefully thank Professor J. L. (University of Science and Technology of China) for help with using the Absys antibody database, Wenyi Zou (Anke Biotechnology Co.) for valuable advice on NGS data collection and analysis.
Author contributions
H.L., Y.S., and L.Q. designed the experiments. Y. D., X. T., Z. W., Y. W. L. H., and H. T. performed the experiments. Y. D., Y. S., Z. W., and Y. W. analyzed the data. All the authors contributed to the manuscript writing and editing.
Disclosure statement
No potential conflict of interest was reported by the authors.