Abstract

BackgroundCell panning of phage-displayed antibody library is a powerful tool for the development of therapeutic and imaging agents since disease-related cell surface proteins in native complex conformation can be directly targeted. Here, we employed a strategy taking advantage of an integrated vector system which allows rapid conversion of scFv-displaying phage into scFv-Fc format for efficient cell-based scFv library selection on a tetraspanin protein, CD9.ResultsA mouse scFv library constructed by using a phagemid vector, pDR-D1 was subjected to cell panning against stable CD9 transfectant, and the scFv repertoire from the enriched phage pool was directly transferred to a mammalian cassette vector, pDR-OriP-Fc1. The resulting constructs enabled transient expression of enough amounts of scFv-Fcs in HEK293E cells, and flow cytometric screening of binders for CD9 transfectant could be performed simply by using the culture supernatants. All three clones selected from the screening showed correct CD9-specificity. They could immunoprecipitate CD9 molecules out of the transfectant cell lysate and correctly stain endogenous CD9 expression on cancer cell membrane. Furthermore, competition assay with a known anti-CD9 monoclonal antibody (mAb) suggested that the binding epitopes of some of them overlap with that of the mAb which resides within the large extracellular loop of CD9.ConclusionsThis study demonstrates that scFv-Fc from mammalian transient expression can be chosen as a reliable format for rapid screening and validation in cell-based scFv library selection, and the strategy described here will be applicable to efficient discovery of antibodies to diverse cell-surface targets.

Highlights

  • Cell panning of phage-displayed antibody library is a powerful tool for the development of therapeutic and imaging agents since disease-related cell surface proteins in native complex conformation can be directly targeted

  • Cell panning procedure that allows selection of phage-displayed antibody library directly on intact cells has been employed to target the antigens in their native conformation at the surface of cells [6,7,8,9,10]

  • In this study, we demonstrated an efficient selection of antibodies recognizing cell surface CD9 from phagedisplayed scFv library by taking advantage of an integrated vector system

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Summary

Introduction

Cell panning of phage-displayed antibody library is a powerful tool for the development of therapeutic and imaging agents since disease-related cell surface proteins in native complex conformation can be directly targeted. For the evaluation of candidate clones, antibody fragments in the form of scFv or Fab can usually be expressed in enough quantity by bacterial cells. Cell panning procedure that allows selection of phage-displayed antibody library directly on intact cells has been employed to target the antigens in their native conformation at the surface of cells [6,7,8,9,10]. Some cell surface proteins cannot be expressed in recombinant forms that retain their native conformation, and antibodies selected using the recombinant proteins may not bind to original proteins on cell surface. The procedure gives chances to target novel epitope space created by disease-related overexpression or modification of cell surface proteins

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