title: Phage display-based on-slide selection of tumor-specific antibodies on formalin-fixed paraffin-embedded human tissue biopsies.
abstract: Phage display is an effective method for the generation of target-specific human antibodies. Standard phage display panning use purified proteins, antigen-transfected cells or tumor cell lines as target structure to generate specific antibodies. However, recombinant proteins can be difficult to express and purify in their native conformation and suitable cell lines are not always available. Additionally the antigen expression profile may change during cultivation and thus differ from the malignant cells in patient. Here we describe a method for the selection of specific antibodies from phage display libraries by panning against formalin-fixed paraffin-embedded (FFPE) tissue biopsies immobilized on glass slides, using small cell lung cancer (SCLC) as a case study. The human Tomlinson single-chain variable fragment (scFv) phage libraries I and J were panned against SCLC FFPE tissue slides for positive selection and healthy lung tissue for subtraction. The specificity of the selected scFv antibodies was confirmed in vitro by ELISA on immobilized SCLC cell membranes, by flow cytometry using the SCLC cell lines NCI-H69, NCI-H82 and DMS 273, and ex vivo against tissue microarrays containing 35 different SCLC samples and 20 types of normal organs. We monitored the internalization of three selected scFv antibodies and fused them with Pseudomonas exotoxin A (ETA') to produce immunotoxins whose cytotoxicity was confirmed by cell viability and apoptosis assays on different SCLC cell lines, achieving IC50 values of up to 23nM. The selection of SCLC-specific scFv antibodies by panning against FFPE tissue slides circumvents the challenges of using purified antigens or cell lines for antibody selection.
authors.affiliation: Institute of Pathology, University Hospital, Justus-Liebig-University Giessen, Langhans-Str. 10, 35392 Giessen, Germany.
authors.forename: Andre
authors.initials: A
authors.lastname: Ten Haaf
authors.affiliation: Institute of Pathology, University Hospital, Justus-Liebig-University Giessen, Langhans-Str. 10, 35392 Giessen, Germany.
authors.forename: Sibylle
authors.initials: S
authors.lastname: Pscherer
authors.affiliation: Institute of Pathology, University Hospital, Justus-Liebig-University Giessen, Langhans-Str. 10, 35392 Giessen, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Fries
authors.affiliation: Department of Integrative Biomedical Sciences, Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Anzio Road, Observatory 7925, Cape Town, South Africa.
authors.forename: Stefan
authors.initials: S
authors.lastname: Barth
authors.affiliation: Institute of Pathology, University Hospital, Justus-Liebig-University Giessen, Langhans-Str. 10, 35392 Giessen, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Gattenlöhner
authors.affiliation: Institute of Pathology, University Hospital, Justus-Liebig-University Giessen, Langhans-Str. 10, 35392 Giessen, Germany. Electronic address: mehmet.k.tur@patho.med.uni-giessen.de.
authors.forename: Mehmet Kemal
authors.initials: MK
authors.lastname: Tur
mesh_terms: D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology; D017209:Apoptosis / Q000187:drug effects / Q000276:immunology; D001706:Biopsy; D045744:Cell Line, Tumor; D061505:Cell Surface Display Techniques; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007150:Immunohistochemistry / Q000379:methods; D000922:Immunotoxins / Q000276:immunology / Q000378:metabolism / Q000633:toxicity; D009369:Neoplasms / Q000175:diagnosis* / Q000276:immunology*; D019151:Peptide Library; D011485:Protein Binding; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D000922:Immunotoxins; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies
keywords: Antigen retrieval; Formalin fixed paraffin-embedded (FFPE) biopsy; On-slide phage display selection; Recombinant immunotoxin; Small cell lung cancer (SCLC); Tumor-specific antibody fragment