title: Selection of single chain antibody fragments binding to the extracellular domain of 4-1BB receptor by phage display technology.
abstract: The 4-1BB is a surface glycoprotein that pertains to the tumor necrosis factor-receptor family. There is compelling evidence suggesting important roles for 4-1BB in the immune response, including cell activation and proliferation and also cytokine induction. Because of encouraging results of different agonistic monoclonal antibodies against 4-1BB in the treatment of cancer, infectious, and autoimmune diseases, 4-1BB has been suggested as an attractive target for immunotherapy. In this study, single chain variable fragment phage display libraries, Tomlinson I+J, were screened against specific synthetic oligopeptides (peptides I and II) designed from 4-1BB extracellular domain. Five rounds of panning led to selection of four 4-1BB specific single chain variable fragments (PI.12, PI.42, PII.16, and PII.29) which showed specific reaction to relevant peptides in phage enzyme-linked immunosorbent assay. The selected clones were successfully expressed in Escherichia coli Rosetta-gami 2, and their expression was confirmed by western blot analysis. Enzyme-linked immunosorbent assay experiments indicated that these antibodies were able to specifically recognize 4-1BB without any cross-reactivity with other antigens. Flow cytometry analysis demonstrated an acceptable specific binding of the single chain variable fragments to 4-1BB expressed on CCRF-CEM cells, while no binding was observed with an irrelevant antibody. Anti-4-1BB single chain variable fragments enhanced surface CD69 expression and interleukin-2 production in stimulated CCRF-CEM cells which confirmed the agonistic effect of the selected single chain variable fragments. The data from this study have provided a rationale for further experiments involving the biological functions of anti-4-1BB single chain variable fragments in future studies.
authors.affiliation: 1 Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.|2 Department of Immunology, School of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.|3 Hybridoma Laboratory, Department of Immunology, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Salman
authors.initials: S
authors.lastname: Bagheri
authors.affiliation: 1 Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.|2 Department of Immunology, School of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Yousefi
authors.affiliation: 1 Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.|2 Department of Immunology, School of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.|3 Hybridoma Laboratory, Department of Immunology, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Elmira
authors.initials: E
authors.lastname: Safaie Qamsari
authors.affiliation: 4 Department of Immunology, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Farhad
authors.initials: F
authors.lastname: Riazi-Rad
authors.affiliation: 3 Hybridoma Laboratory, Department of Immunology, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Mohsen
authors.initials: M
authors.lastname: Abolhassani
authors.affiliation: 5 Pishtaz Teb Zaman Diagnostics, Tehran, Iran.
authors.forename: Vahid
authors.initials: V
authors.lastname: Younesi
authors.affiliation: 6 Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.
authors.forename: Ruhollah
authors.initials: R
authors.lastname: Dorostkar
authors.affiliation: 2 Department of Immunology, School of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.
authors.forename: Ali Akbar
authors.initials: AA
authors.lastname: Movassaghpour
authors.affiliation: 3 Hybridoma Laboratory, Department of Immunology, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Zahra
authors.initials: Z
authors.lastname: Sharifzadeh
mesh_terms: D000888:Antibodies, Anti-Idiotypic / Q000276:immunology* / Q000627:therapeutic use; D015703:Antigens, CD / Q000096:biosynthesis; D000945:Antigens, Differentiation, T-Lymphocyte / Q000096:biosynthesis; D000951:Antigens, Neoplasm / Q000276:immunology; D045744:Cell Line, Tumor; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D006801:Humans; D007113:Immunity, Innate; D007167:Immunotherapy; D007376:Interleukin-2 / Q000096:biosynthesis; D037181:Lectins, C-Type / Q000096:biosynthesis; D007938:Leukemia / Q000276:immunology* / Q000628:therapy; D019151:Peptide Library; D010455:Peptides / Q000276:immunology / Q000627:therapeutic use; D000072417:Protein Domains / Q000276:immunology; D057127:Single-Chain Antibodies / Q000276:immunology* / Q000302:isolation & purification / Q000627:therapeutic use; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9 / Q000276:immunology* / Q000302:isolation & purification / Q000627:therapeutic use; D000099192:CD69 Antigens
chemical_list: D000888:Antibodies, Anti-Idiotypic; D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D000951:Antigens, Neoplasm; D007376:Interleukin-2; D037181:Lectins, C-Type; D019151:Peptide Library; D010455:Peptides; D057127:Single-Chain Antibodies; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9; D000099192:CD69 Antigens; C505714:TNFRSF9 protein, human
keywords: 4-1BB; T cell; cancer; immunotherapy; phage display; single chain variable fragment