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

• Published in: Proteins, structure, function, and bioinformatics structure, function, and bioinformatics, 2014-06, Vol.82 (6), p.975-989
• Main Authors: Kuchař, Milan, Vaňková, Lucie, Petroková, Hana, Černý, Jiří, Osička, Radim, Pelák, Ondřej, Šípová, Hana, Schneider, Bohdan, Homola, Jiří, Šebo, Peter, Kalina, Tomáš, Malý, Petr
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Anti-Inflammatory Agents - chemistry; Anti-Inflammatory Agents - pharmacology; Bacterial Proteins - chemistry; Binding, Competitive; combinatorial library; cytokine; Drug Evaluation, Preclinical; engineered binding protein; Humans; Immunologic Factors - chemistry; Immunologic Factors - pharmacology; Interleukin-23 - chemistry; Interleukin-23 - physiology; Jurkat Cells; K562 Cells; Molecular Sequence Data; Peptide Fragments - chemistry; Peptide Fragments - pharmacology; Protein Binding; protein scaffold; psoriasis; Receptors, Interleukin - antagonists & inhibitors; Receptors, Interleukin - physiology; ribosome display; Sequence Homology, Amino Acid; Streptococcus; Th17 Cells - drug effects; Th17 Cells - metabolism
• ISSN: 0887-3585; 1097-0134
• DOI: 10.1002/prot.24472

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. Proteins 2014; 82:975–989. © 2013 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.