title: Generation and characterization of small single domain antibodies inhibiting human tumor necrosis factor receptor 1.
abstract: The cytokine TNF is a well known drug target for several inflammatory diseases such as Crohn disease. Despite the great success of TNF blockers, therapy could be improved because of high costs and side effects. Selective inhibition of TNF receptor (TNFR) 1 signaling holds the potential to greatly reduce the pro-inflammatory activity of TNF, thereby preserving the advantageous immunomodulatory signals mediated by TNFR2. We generated a selective human TNFR1 inhibitor based on Nanobody (Nb) technology. Two anti-human TNFR1 Nbs were linked with an anti-albumin Nb to generate Nb Alb-70-96 named "TNF Receptor-One Silencer" (TROS). TROS selectively binds and inhibits TNF/TNFR1 and lymphotoxin-α/TNFR1 signaling with good affinity and IC50 values, both of which are in the nanomolar range. Surface plasmon resonance analysis reveals that TROS competes with TNF for binding to human TNFR1. In HEK293T cells, TROS strongly reduces TNF-induced gene expression, like IL8 and TNF, in a dose-dependent manner; and in ex vivo cultured colon biopsies of CD patients, TROS inhibits inflammation. Finally, in liver chimeric humanized mice, TROS antagonizes inflammation in a model of acute TNF-induced liver inflammation, reflected in reduced human IL8 expression in liver and reduced IL6 levels in serum. These results demonstrate the considerable potential of TROS and justify the evaluation of TROS in relevant disease animal models of both acute and chronic inflammation and eventually in patients.
authors.affiliation: From the Inflammation Research Center, VIB, 9052 Ghent, the Department of Biomedical Molecular Biology, Ghent University, 9000 Ghent.
authors.forename: Sophie
authors.initials: S
authors.lastname: Steeland
authors.affiliation: From the Inflammation Research Center, VIB, 9052 Ghent, the Department of Biomedical Molecular Biology, Ghent University, 9000 Ghent.
authors.forename: Leen
authors.initials: L
authors.lastname: Puimège
authors.affiliation: From the Inflammation Research Center, VIB, 9052 Ghent, the Department of Biomedical Molecular Biology, Ghent University, 9000 Ghent.
authors.forename: Roosmarijn E
authors.initials: RE
authors.lastname: Vandenbroucke
authors.affiliation: From the Inflammation Research Center, VIB, 9052 Ghent, the Department of Biomedical Molecular Biology, Ghent University, 9000 Ghent.
authors.forename: Filip
authors.initials: F
authors.lastname: Van Hauwermeiren
authors.affiliation: the Protein Service Facility, Inflammation Research Center, VIB, Ghent University, 9052 Ghent.
authors.forename: Jurgen
authors.initials: J
authors.lastname: Haustraete
authors.affiliation: the In Vivo Cellular and Molecular Imaging Laboratory and Cellular and Molecular Immunology Laboratory, Vrije Universiteit Brussel, 1000 Brussels, the Center for Vaccinology.
authors.forename: Nick
authors.initials: N
authors.lastname: Devoogdt
authors.affiliation: From the Inflammation Research Center, VIB, 9052 Ghent, the Department of Biomedical Molecular Biology, Ghent University, 9000 Ghent.
authors.forename: Paco
authors.initials: P
authors.lastname: Hulpiau
authors.affiliation: Departments of Clinical Chemistry and.
authors.forename: Geert
authors.initials: G
authors.lastname: Leroux-Roels
authors.affiliation: Gastroenterology, Ghent University Hospital, 9000 Ghent, Belgium.
authors.forename: Debby
authors.initials: D
authors.lastname: Laukens
authors.affiliation: Departments of Clinical Chemistry and.
authors.forename: Philip
authors.initials: P
authors.lastname: Meuleman
authors.affiliation: Gastroenterology, Ghent University Hospital, 9000 Ghent, Belgium.
authors.forename: Martine
authors.initials: M
authors.lastname: De Vos
authors.affiliation: From the Inflammation Research Center, VIB, 9052 Ghent, the Department of Biomedical Molecular Biology, Ghent University, 9000 Ghent, Claude.Libert@irc.VIB-UGent.be.
authors.forename: Claude
authors.initials: C
authors.lastname: Libert
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003106:Colon / Q000187:drug effects* / Q000276:immunology / Q000473:pathology; D003424:Crohn Disease / Q000276:immunology / Q000473:pathology / Q000517:prevention & control*; D016207:Cytokines / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D005260:Female; D006801:Humans; D007249:Inflammation / Q000276:immunology / Q000473:pathology / Q000517:prevention & control*; D008099:Liver / Q000187:drug effects* / Q000276:immunology / Q000473:pathology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D016513:Mice, SCID; D008969:Molecular Sequence Data; D011487:Protein Conformation; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000037:antagonists & inhibitors* / Q000276:immunology* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology*; D020349:Surface Plasmon Resonance; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D016207:Cytokines; D047888:Receptors, Tumor Necrosis Factor, Type I; D061905:Single-Domain Antibodies; D014409:Tumor Necrosis Factor-alpha
keywords: Autoimmune Disease; Immunotherapy; Inflammation; Inflammatory Bowel Disease; Inhibitor; Nanobody; TNF Receptor; TNFR1; Tumor Necrosis Factor (TNF)