Abstract
BackgroundFilamentous phage display has become an ordinary tool to engineer antibody fragments. Several capsid proteins have been applied for displaying antibodies, of which gene III (p3) protein is used the most followed by experiments with gene IX (p9) protein. Despite the popularity, there are no library scale studies to objectively compare differences in the selection performance of the libraries, when displayed via different capsid proteins.ResultsIn this study, an identical antibody repertoire was displayed as Fab fragments on p9, p3 and truncated p3 (p3Δ). In addition, the library clones were displayed as ScFv fragments on p3Δ and the Fab-p3 display valency was modulated by hyperphage and VCS-M13 superinfections. The selection performances of the libraries were followed in repeated parallel panning reactions against streptavidin (STR) and digoxigenin (DIG). Selection was successful with all display formats, but the enrichment of specific clones from Fab-p9 library was clearly less efficient than from the other libraries. The most diverse outputs were obtained from p3Δ display and the highest affinity anti-DIG antibodies from the ScFv repertoire. Unfortunately, the number of retrieved specific clones was too low for explicit analysis of the differences in the number of obtained unique clones from each library. However, severe reduction in sequence diversity was observed in p3-Fab libraries prior to panning, which in turn, materialized as a low number of unique specific clones. Oligovalent display by hyperphage resulted in a higher number of unique clones, but the same highest affinity anti-DIG Fab was recovered also by VCS-M13 superinfection.ConclusionsThe compromised enrichment of the target-specific clones from the Fab repertoire as a fusion to p9 capsid protein in our experiments, the significant loss of functional diversity in Fab-p3 library after single phage packing cycle and the retrieval of higher affinity anti-digoxigenin clones as ScFv molecules than as Fab molecules from the same source repertoire indicate that the chosen display format may have a significant impact on the selection outcome. This study demonstrates that in addition to library content, also display related issues, should be taken into consideration when planning directed evolution experiments.
Highlights
Filamentous phage display has become an ordinary tool to engineer antibody fragments
Two, six, two and four positions were randomized in the complementary determining regions (CDRs)-L1, CDR-L3, CDR-H1 and CDR-H2 loops, respectively, with the majority of diversity introduced at the CDR-H3 loop containing variating loop lengths from 5 to 12 residues (IMGT definition)
We have shown earlier that ScFv-p9 display truly is a working concept [24], but as the enrichment of binding clones in ScFv-format seemed to be faster by p3Δ display, our latest ScFv libraries are displayed as p3Δ fusions [14]
Summary
Filamentous phage display has become an ordinary tool to engineer antibody fragments. Several capsid proteins have been applied for displaying antibodies, of which gene III (p3) protein is used the most followed by experiments with gene IX (p9) protein. According to the timeline analysis, hybridoma technology was intensively researched in the 80s and early 90s, but since year 2000 phage display has been cited more often in scientific articles. A review of 100 accessed original research papers, covering 1/4 of all “phage display”-citing articles published in year 2009 (Sep–Dec), confirms that antibody fragments were displayed in 50% of the studies, followed by peptides with a 30% share. Sorting the same sample pile of 100 articles by phage type demonstrated that filamentous phage is still by far the most popular choice for in vitro evolution studies (91/100) followed by T7 phage display (4/100)
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