Antibody Fab display and selection through fusion to the pIX coat protein of filamentous phage

Tornetta, Mark A.; Baker, Scott I.; Whitaker, Brian; Lu, Jinhua; Chen, Qiang; Pisors, Eileen; Shi, Lei; Luo, Jinquan; Sweet, Raymond W.; Tsui, Ping

doi:10.1016/j.jim.2010.06.001

Cited by 18 publications

(14 citation statements)

References 20 publications

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“…In the other studies on Fab-p9 display, conclusions were withdrawn by meta-analysis. P9 display was concluded to be a comparable platform to p3 display, because similar affinities and numbers of unique clones were obtained as reported by others using p3 display [28, 29]. We have also shown earlier that ScFv-p9 display truly is a working concept [24], but as the enrichment of binding clones also in ScFv-format seemed to be faster by p3Δ display, our latest ScFv libraries are displayed as p3Δ fusions [14].…”

Section: Resultssupporting

confidence: 67%

“…In contrast to this, there are reports in which the performance of p9 displayed ScFv [27] or Fab [28, 29] libraries have been esteemed to be equal or even superior to p3 display. However, a closer look at these references reveals that valid side-by-side comparison between p3 and p9 were not carried out.…”

Section: Resultsmentioning

confidence: 95%

“…Naturally, it cannot be excluded that the sequence of the linker has an effect on the selection properties of the p9 repertoire. However, in the other p9 library initiatives the fusion gene constructions have been similar, such as PelB-ScFv-Flag-G 4 S 1 -p9 [27], PelB-Fd-TSG 4 S 1 -p9 [29] and PelB-ScFv-AAAGSKDIR-p9 [30]. Myc-tag is also used in p3 display libraries without reported effect on the display efficiency [31].…”

Section: Resultsmentioning

confidence: 99%

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The selection performance of an antibody library displayed on filamentous phage coat proteins p9, p3 and truncated p3

et al. 2014

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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.

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Section: Resultssupporting

confidence: 67%

Section: Resultsmentioning

confidence: 95%

Section: Resultsmentioning

confidence: 99%

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The selection performance of an antibody library displayed on filamentous phage coat proteins p9, p3 and truncated p3

et al. 2014

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“…Selection efficiency from the library was found to exceed that normally seen with pIII display. The use of pIX as antibody fragment display scaffold has also been independently reported by others, confirming that type 9 + 9 display is an effective display avenue for antibody discovery and affinity maturation [89,90].…”

Section: The Resurrection Of Pvii and Pix As Alternative Ff Display Ssupporting

confidence: 66%

Next generation phage display by use of pVII and pIX as display scaffolds

Løset

Sandlie

2012

Methods

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“…The pIII protein is responsible for phage infection of Escherichia coli [3] and thus proteins fused to pIII can interfere with phage infection. Additionally, display of antibody fragments can also be achieved by fusion to the smaller pIX protein at the opposite end of the virion [3,4]. Because it is not involved in the infection process, display on pIX allows for efficient recovery of phage by direct incubation with E. coli cells.…”

Section: Introductionmentioning

confidence: 99%