Human IL-23 receptor antagonists derived from an albumin-binding domain scaffold inhibit IL-23-dependent ex vivo expansion of
IL-17-producing T-cells

Lucie Vaňková¹, Milan Kuchař¹, Hana Petroková¹, Jiří Černý¹, Radim Osička², Ondřej Pelák³, Bohdan Schneider¹, Tomáš Kalina³, Peter Šebo¹, Petr Malý¹

¹Institute of Biotechnology AS CR, v. v. i. and ² Institute of Microbiology AS CR, v. v. i., Vídeňská 1083, 142 20 Prague, Czech Republic

³Department of Pediatric Hematology and Oncology, 2nd Faculty of Medicine, Charles University and Motol University Hospital, Prague, Czech Republic

lucie.vankova@img.cas.cz, petr.maly@img.cas.cz

Interleukin-23 (IL-23), a heterodimeric cytokine of covalently bound p19 and p40 subunits, has been recently described to play a pivotal role in the development of chronic autoimmune diseases.
As an alternative to antibody-based drugs, we engineer novel protein binders suppressing IL-23-mediated signaling. Library of three-helix bundle variants of albumin-binding domain (ABD) from streptococcal protein G (Ahmad et al, 2012) was used to identify binders of extracellular human
IL-23R via ribosome display selection. To screen the library of ABD variants for high affinity binders of IL-23 receptor, we produced bacterial extracellular N-terminal poly-his-modified IL-23 receptor.
The collection of variants (REX series) was tested for binding affinity in ELISA and selected clones were modified by a C-terminal AviTag for in vivo biotinylation. Finally we identified a group of clones that inhibited binding of p19-subunit of IL-23 to refolded IL-23R or glycosylated IL-23R Fc chimera, secreted by murine NSO-derived cells. We further demonstrate that REX variants bind to
K-562, Jurkat and THP-1 human cell lines and this binding correlates with IL-23R cell-surface expression. As binding of the REX clones to THP-1 cells can be substantially diminished by a high dose of p19 protein and the three best-performing REX binders inhibited the IL-23-driven expansion of IL-17-producing primary human CD4⁺ T-cells, we conclude that unique IL-23R antagonists derived from the ABD scaffold were generated that might be useful in designing novel anti-inflammatory biologicals.

Funding by the grant GACR P303/10/1849 and the institutional research concept AV0Z50520701.

Reference: Novel high-affinity binders of human interferon gamma derived from albumin-binding domain of protein G. Jawid N. Ahmad, Jingjing Li, Lada Biedermannová, Milan Kuchař, Hana Šípová, Alena Semerádtová, Jiří Černý, Hana Petroková, Pavel Mikulecký, Jiří Polínek, Ondřej Staněk, Jiří Vondrášek, Jiří Homola, Jan Malý, Radim Osička, Peter Šebo, Petr Malý. Proteins: Structure, Function, and Bioinformatics. Volume 80, Issue 3, pages 774–789, March 2012. DOI: 10.1002/prot.23234