title: Human interleukin-23 receptor antagonists derived from an albumin-binding domain scaffold inhibit IL-23-dependent ex vivo expansion of IL-17-producing T-cells.
abstract: Engineered combinatorial libraries derived from small protein scaffolds represent a powerful tool for generating novel binders with high affinity, required specificity and designed inhibitory function. This work was aimed to generate a collection of recombinant binders of human interleukin-23 receptor (IL-23R), which is a key element of proinflammatory IL-23-mediated signaling. A library of variants derived from the three-helix bundle scaffold of the albumin-binding domain (ABD) of streptococcal protein G and ribosome display were used to select for high-affinity binders of recombinant extracellular IL-23R. A collection of 34 IL-23R-binding proteins (called REX binders), corresponding to 18 different sequence variants, was used to identify a group of ligands that inhibited binding of the recombinant p19 subunit of IL-23, or the biologically active human IL-23 cytokine, to the recombinant IL-23R or soluble IL-23R-IgG chimera. The strongest competitors for IL-23R binding in ELISA were confirmed to recognize human IL-23R-IgG in surface plasmon resonance experiments, estimating the binding affinity in the sub- to nanomolar range. We further demonstrated that several REX variants bind to human leukemic cell lines K-562, THP-1 and Jurkat, and this binding correlated with IL-23R cell-surface expression. The REX125, REX009 and REX128 variants competed with the p19 protein for binding to THP-1 cells. Moreover, the presence of REX125, REX009 and REX115 variants significantly inhibited the IL-23-driven expansion of IL-17-producing primary human CD4(+) T-cells. Thus, we conclude that unique IL-23R antagonists derived from the ABD scaffold were generated that might be useful in designing novel anti-inflammatory biologicals.
authors.affiliation: Laboratory of Ligand Engineering, Institute of Biotechnology AS CR, v. v. i., Vídeňská 1083, 142 20 Prague, Czech Republic.
authors.forename: Milan
authors.initials: M
authors.lastname: Kuchař
authors.forename: Lucie
authors.initials: L
authors.lastname: Vaňková
authors.forename: Hana
authors.initials: H
authors.lastname: Petroková
authors.forename: Jiří
authors.initials: J
authors.lastname: Cerný
authors.forename: Radim
authors.initials: R
authors.lastname: Osička
authors.forename: Ondřej
authors.initials: O
authors.lastname: Pelák
authors.forename: Hana
authors.initials: H
authors.lastname: Sípová
authors.forename: Bohdan
authors.initials: B
authors.lastname: Schneider
authors.forename: Jiří
authors.initials: J
authors.lastname: Homola
authors.forename: Peter
authors.initials: P
authors.lastname: Sebo
authors.forename: Tomáš
authors.initials: T
authors.lastname: Kalina
authors.forename: Petr
authors.initials: P
authors.lastname: Malý
mesh_terms: D000595:Amino Acid Sequence; D000893:Anti-Inflammatory Agents / Q000737:chemistry / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000737:chemistry; D001667:Binding, Competitive; D004353:Drug Evaluation, Preclinical; D006801:Humans; D007155:Immunologic Factors / Q000737:chemistry / Q000494:pharmacology; D053759:Interleukin-23 / Q000737:chemistry / Q000502:physiology; D019169:Jurkat Cells; D020014:K562 Cells; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology*; D011485:Protein Binding; D018123:Receptors, Interleukin / Q000037:antagonists & inhibitors* / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D058504:Th17 Cells / Q000187:drug effects* / Q000378:metabolism
chemical_list: D000893:Anti-Inflammatory Agents; D001426:Bacterial Proteins; C515500:IL23R protein, human; C034284:IgG Fc-binding protein, Streptococcus; D007155:Immunologic Factors; D053759:Interleukin-23; D010446:Peptide Fragments; D018123:Receptors, Interleukin
keywords: combinatorial library; cytokine; engineered binding protein; protein scaffold; psoriasis; ribosome display