Next-generation sequencing enables the discovery of more diverse positive clones from a phage-displayed antibody library

Phage display technology provides a powerful tool to screen a library for a binding molecule via an enrichment process. It has been adopted as a critical technology in the development of therapeutic antibodies. However, a major drawback of phage display technology is that because the degree of the enrichment cannot be controlled during the bio-panning process, it frequently results in a limited number of clones. In this study, we applied next-generation sequencing (NGS) to screen clones from a library and determine whether a greater number of clones can be identified using NGS than using conventional methods. Three chicken immune single-chain variable fragment (scFv) libraries were subjected to bio-panning on prostate-specific antigen (PSA). Phagemid DNA prepared from the original libraries as well as from the Escherichia coli pool after each round of bio-panning was analyzed using NGS, and the heavy chain complementarity-determining region 3 (HCDR3) sequences of the scFv clones were determined. Subsequently, through two-step linker PCR and cloning, the entire scFv gene was retrieved and analyzed for its reactivity to PSA in a phage enzyme immunoassay. After four rounds of bio-panning, the conventional colony screening method was performed for comparison. The scFv clones retrieved from NGS analysis included all clones identified by the conventional colony screening method as well as many additional clones. The enrichment of the HCDR3 sequence throughout the bio-panning process was a positive predictive factor for the selection of PSA-reactive scFv clones. Next-generation sequencing can identify a greater diversity of potential antibody clones than conventional colony screening methods. Most therapeutic antibodies today are found using a technique called phage display, in which researchers used bacterio-phages that infect bacteria to express antibody fragments that might bind to antigen of interest. A method known as bio-panning then helps scientists to enrich cancidate antibody clones with high binding reactivity. After bio-panning, however, the retrieval of the antibody clone has been inefficient. A team led by Junho Chung from Seoul National University, South Korea, compared a Next-generation sequencing based analysis of antibody clones with standard colony formation method using immunized chickens antibody libraries. They found that Next-generation sequencing based analysis of antibody clones retrieved not only all the antibody clones from the conventional colony formation method but also many more. Hence, this new method could improve antibody selection and better clones for development.