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

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Line graph representing population shifts in HCDR3 sequences through bio-panning rounds. (a) scFv library 1, (b) scFv library 2 and (c) scFv library 3.
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fig2: Line graph representing population shifts in HCDR3 sequences through bio-panning rounds. (a) scFv library 1, (b) scFv library 2 and (c) scFv library 3.

Mentions: The diversity of the antibody clones is represented by the number of HCDR3 sequences that belong to each cluster (Figure 1). The abundance of the HCDR3 sequences in each cluster is represented by heat map color; high and low populations are indicated in red and blue, respectively. HCDR3 sequences in cluster 1 were highly abundant before bio-panning and up to the second bio-panning round. However, there was a sudden impoverishment in rounds 3 and 4 of bio-panning. In contrast, HCDR3 sequences that belonged to clusters 2 and 3 (including cluster 4 of library 1) showed the opposite pattern. Their populations were very low before bio-panning, remained low after the second round of bio-panning, and started to enrich from the third round of bio-panning. The increase continued in the fourth round of bio-panning. This population shift of HCDR3 sequences throughout bio-panning is represented in Figure 2. All 22 HCDR3 sequences in clones found via the conventional colony screening method existed among the HCDR3 sequences obtained from NGS analysis of phagemid DNA prepared after the fourth round of bio-panning (Table 1). Two out of the 22 unique HCDR3 sequences belonged to cluster 1, and the other 20 HCDR3 sequences belonged to clusters 2, 3 or 4.


Next-generation sequencing enables the discovery of more diverse positive clones from a phage-displayed antibody library
Line graph representing population shifts in HCDR3 sequences through bio-panning rounds. (a) scFv library 1, (b) scFv library 2 and (c) scFv library 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5382563&req=5

fig2: Line graph representing population shifts in HCDR3 sequences through bio-panning rounds. (a) scFv library 1, (b) scFv library 2 and (c) scFv library 3.
Mentions: The diversity of the antibody clones is represented by the number of HCDR3 sequences that belong to each cluster (Figure 1). The abundance of the HCDR3 sequences in each cluster is represented by heat map color; high and low populations are indicated in red and blue, respectively. HCDR3 sequences in cluster 1 were highly abundant before bio-panning and up to the second bio-panning round. However, there was a sudden impoverishment in rounds 3 and 4 of bio-panning. In contrast, HCDR3 sequences that belonged to clusters 2 and 3 (including cluster 4 of library 1) showed the opposite pattern. Their populations were very low before bio-panning, remained low after the second round of bio-panning, and started to enrich from the third round of bio-panning. The increase continued in the fourth round of bio-panning. This population shift of HCDR3 sequences throughout bio-panning is represented in Figure 2. All 22 HCDR3 sequences in clones found via the conventional colony screening method existed among the HCDR3 sequences obtained from NGS analysis of phagemid DNA prepared after the fourth round of bio-panning (Table 1). Two out of the 22 unique HCDR3 sequences belonged to cluster 1, and the other 20 HCDR3 sequences belonged to clusters 2, 3 or 4.

View Article: PubMed Central - PubMed

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.

No MeSH data available.