title: p19-targeted ABD-derived protein variants inhibit IL-23 binding and exert suppressive control over IL-23-stimulated expansion of primary human IL-17+ T-cells.
abstract: Interleukin-23 (IL-23), a heterodimeric cytokine of covalently bound p19 and p40 proteins, has recently been closely associated with development of several chronic autoimmune diseases such as psoriasis, psoriatic arthritis or inflammatory bowel disease. Released by activated dendritic cells, IL-23 interacts with IL-23 receptor (IL-23R) on Th17 cells, thus promoting intracellular signaling, a pivotal step in Th17-driven pro-inflammatory axis. Here, we aimed to block the binding of IL-23 cytokine to its cell-surface receptor by novel inhibitory protein binders targeted to the p19 subunit of human IL-23. To this goal, we used a combinatorial library derived from a scaffold of albumin-binding domain (ABD) of streptococcal protein G, and ribosome display selection, to yield a collection of ABD-derived p19-targeted variants, called ILP binders. From 214 clones analyzed by ELISA, Western blot and DNA sequencing, 53 provided 35 different sequence variants that were further characterized. Using in silico docking in combination with cell-surface competition binding assay, we identified a group of inhibitory candidates that substantially diminished binding of recombinant p19 to the IL-23R on human monocytic THP-1 cells. Of these best p19-blockers, ILP030, ILP317 and ILP323 inhibited IL-23-driven expansion of IL-17-producing primary human CD4+T-cells. Thus, these novel binders represent unique IL-23-targeted probes useful for IL-23/IL-23R epitope mapping studies and could be used for designing novel p19/IL-23-targeted anti-inflammatory biologics.
authors.affiliation: a Laboratory of Ligand Engineering , Institute of Biotechnology, Czech Academy of Sciences, v. v. i, BIOCEV Research Center , Vestec , Czech Republic.
authors.forename: Lucie
authors.initials: L
authors.lastname: Křížová
authors.affiliation: a Laboratory of Ligand Engineering , Institute of Biotechnology, Czech Academy of Sciences, v. v. i, BIOCEV Research Center , Vestec , Czech Republic.
authors.forename: Milan
authors.initials: M
authors.lastname: Kuchař
authors.affiliation: a Laboratory of Ligand Engineering , Institute of Biotechnology, Czech Academy of Sciences, v. v. i, BIOCEV Research Center , Vestec , Czech Republic.
authors.forename: Hana
authors.initials: H
authors.lastname: Petroková
authors.affiliation: b Laboratory of Molecular Biology of the Bacterial Pathogens , Institute of Microbiology, Czech Academy of Sciences , Prague , Czech Republic.
authors.forename: Radim
authors.initials: R
authors.lastname: Osička
authors.affiliation: a Laboratory of Ligand Engineering , Institute of Biotechnology, Czech Academy of Sciences, v. v. i, BIOCEV Research Center , Vestec , Czech Republic.
authors.forename: Marie
authors.initials: M
authors.lastname: Hlavničková
authors.affiliation: c CLIP, Department of Pediatric Hematology and Oncology, 2nd Faculty of Medicine, Charles University and University Hospital Motol , Prague , Czech Republic , and.
authors.forename: Ondřej
authors.initials: O
authors.lastname: Pelák
authors.affiliation: d Laboratory of Biomolecular Recognition , Institute of Biotechnology, Czech Academy of Sciences, v. v. i, BIOCEV Research Center , Vestec , Czech Republic.
authors.forename: Jiří
authors.initials: J
authors.lastname: Černý
authors.affiliation: c CLIP, Department of Pediatric Hematology and Oncology, 2nd Faculty of Medicine, Charles University and University Hospital Motol , Prague , Czech Republic , and.
authors.forename: Tomáš
authors.initials: T
authors.lastname: Kalina
authors.affiliation: a Laboratory of Ligand Engineering , Institute of Biotechnology, Czech Academy of Sciences, v. v. i, BIOCEV Research Center , Vestec , Czech Republic.
authors.forename: Petr
authors.initials: P
authors.lastname: Malý
mesh_terms: D002460:Cell Line; D003713:Dendritic Cells / Q000276:immunology / Q000378:metabolism; D006801:Humans; D053759:Interleukin-23 / Q000737:chemistry / Q000378:metabolism*; D053760:Interleukin-23 Subunit p19 / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000276:immunology*; D008264:Macrophages / Q000276:immunology / Q000378:metabolism; D008958:Models, Molecular; D010586:Phagocytes / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D018123:Receptors, Interleukin / Q000378:metabolism; D011994:Recombinant Proteins; D015398:Signal Transduction; D016176:T-Lymphocyte Subsets / Q000276:immunology / Q000378:metabolism; D058504:Th17 Cells / Q000276:immunology / Q000378:metabolism*
chemical_list: C515500:IL23R protein, human; D053759:Interleukin-23; D053760:Interleukin-23 Subunit p19; D018123:Receptors, Interleukin; D011994:Recombinant Proteins
keywords: IL-23; Psoriasis; Th17 cell; autoimmune disease; protein binder