title: An efficient strategy for cell-based antibody library selection using an integrated vector system.
abstract: BACKGROUND
  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. 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.
  
  
  RESULTS
  A 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.
  
  
  CONCLUSIONS
  This 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.
authors.affiliation: Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology, 111 Gwahangno, Yuseong-gu, Daejon 305-806, Republic of Korea.
authors.forename: Hyerim
authors.initials: H
authors.lastname: Yoon
authors.forename: Jin Myung
authors.initials: JM
authors.lastname: Song
authors.forename: Chun Jeih
authors.initials: CJ
authors.lastname: Ryu
authors.forename: Yeon-Gu
authors.initials: YG
authors.lastname: Kim
authors.forename: Eun Kyo
authors.initials: EK
authors.lastname: Lee
authors.forename: Sunghyun
authors.initials: S
authors.lastname: Kang
authors.forename: Sang Jick
authors.initials: SJ
authors.lastname: Kim
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D000939:Epitopes / Q000276:immunology; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D019151:Peptide Library; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D060245:Tetraspanin 29 / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D014162:Transfection
chemical_list: D000939:Epitopes; D019151:Peptide Library; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D060245:Tetraspanin 29