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

Abstract

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.

High-throughput retrieval of physical DNA for NGS-identifiable clones in phage display library

“…Also, it was reported that clones with high binding activity to a target antigen can exist in a rare population in a library, due to the bias that results from the biopanning 8 . This was shown in our previous research, where certain rare clones in a library were retrieved and confirmed to have a binding activity to a target antigen 9 . Thus, to retrieve these rare clones with high developability and exploit them, in-depth analysis of a library is essential.…”

“…Conventionally, in vitro screening has been used to identify and retrieve clones in a phage display library 10 . Next-generation sequencing (NGS) technology was introduced to improve the screening process in terms of sampling depth 9,11,12 . However, the two existing methodologies, the in vitro screening and the NGS-based process, still have several technical limitations in identifying and retrieving rare clones in the library in high throughput.…”

Integrating high‐throughput screening and sequencing for monoclonal antibody discovery and engineering

“…Besides directly mining natural B‐cell‐derived antibody repertoires of animals and humans, Ig‐seq can also be used to interrogate a diverse set of recombinant antibody libraries like naive, focused, immune, semi‐synthetic and fully synthetic libraries . These repertoires are compatible with in vitro screening platforms such as phage, yeast or ribosome display .…”

“…In order to more fully exploit the potential of various antibody libraries, several studies reported the combined use of traditional recombinant library screening and Ig‐seq analysis . In one of the first examples by Ravn et al ,.…”

“…However, only clones that were previously identified by traditional ELISA screening gave a positive signal using crude supernatants or periplasmic extracts, so providing a plausible explanation as to why the other variants may have been missed by conventional screening. Likewise, when focused immune libraries from hens were explored by a combination of successive rounds of screening and high‐throughput sequencing, specific antibodies isolated by conventional panning as well as many more were identified by clustering and frequency analysis of Ig‐seq data . In an attempt to improve the affinity of a humanized anti‐ErbB2 monoclonal antibody, a diversified CDR library was subjected to panning and Ig‐seq, resulting in the isolation of nearly 40 mutants with improved affinity .…”