title: Selection of scFv Antibody Fragments Binding to Human Blood versus Lymphatic Endothelial Surface Antigens by Direct Cell Phage Display.
abstract: The identification of marker molecules specific for blood and lymphatic endothelium may provide new diagnostic tools and identify new targets for therapy of immune, microvascular and cancerous diseases. Here, we used a phage display library expressing human randomized single-chain Fv (scFv) antibodies for direct panning against live cultures of blood (BECs) and lymphatic (LECs) endothelial cells in solution. After six panning rounds, out of 944 sequenced antibody clones, we retrieved 166 unique/diverse scFv fragments, as indicated by the V-region sequences. Specificities of these phage clone antibodies for respective compartments were individually tested by direct cell ELISA, indicating that mainly pan-endothelial cell (EC) binders had been selected, but also revealing a subset of BEC-specific scFv antibodies. The specific staining pattern was recapitulated by twelve phage-independently expressed scFv antibodies. Binding capacity to BECs and LECs and differential staining of BEC versus LEC by a subset of eight scFv antibodies was confirmed by immunofluorescence staining. As one antigen, CD146 was identified by immunoprecipitation with phage-independent scFv fragment. This antibody, B6-11, specifically bound to recombinant CD146, and to native CD146 expressed by BECs, melanoma cells and blood vessels. Further, binding capacity of B6-11 to CD146 was fully retained after fusion to a mouse Fc portion, which enabled eukaryotic cell expression. Beyond visualization and diagnosis, this antibody might be used as a functional tool. Overall, our approach provided a method to select antibodies specific for endothelial surface determinants in their native configuration. We successfully selected antibodies that bind to antigens expressed on the human endothelial cell surfaces in situ, showing that BECs and LECs share a majority of surface antigens, which is complemented by cell-type specific, unique markers.
authors.affiliation: Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Keller
authors.affiliation: Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria.
authors.forename: Romana
authors.initials: R
authors.lastname: Kalt
authors.affiliation: Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Raab
authors.affiliation: Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria.
authors.forename: Helga
authors.initials: H
authors.lastname: Schachner
authors.affiliation: Institute for Animal Breeding and Genetics, University of Veterinary Medicine, Vienna, Austria.
authors.forename: Corina
authors.initials: C
authors.lastname: Mayrhofer
authors.affiliation: Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria.
authors.forename: Dontscho
authors.initials: D
authors.lastname: Kerjaschki
authors.affiliation: Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Hantusch
mesh_terms: D000818:Animals; D000918:Antibody Specificity / Q000276:immunology; D000941:Antigens / Q000378:metabolism; D000954:Antigens, Surface / Q000378:metabolism*; D001773:Blood Cells / Q000378:metabolism*; D051929:CD146 Antigen / Q000378:metabolism; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D002999:Clone Cells; D019295:Computational Biology; D042783:Endothelial Cells / Q000378:metabolism*; D004729:Endothelium, Lymphatic / Q000166:cytology*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D009928:Organ Specificity; D019151:Peptide Library; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020539:Sequence Analysis, Protein; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000378:metabolism*; D012995:Solubility
chemical_list: D000941:Antigens; D000954:Antigens, Surface; D051929:CD146 Antigen; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies