Inhibitory protein variants derived from an albumin-binding domain scaffold targeting human IL-23 cytokine

L. Vaňková1, M. Kuchař1, R. Osička2, H. Petroková1, J. Černý1, P. Šebo1,2 and P. Malý1

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



This work was aimed to generate a collection of recombinant binders of human interleukin-23 (IL-23), which is a key element of pro-inflammatory IL-23-mediated signaling. IL-23, a heterodimeric cytokine of covalently bound p19 and p40 subunits, plays a pivotal role in the development of chronic autoimmune diseases, such as psoriasis, inflammatory bowel disease and multiple sclerosis. Binding of the IL-23 to its receptor (IL-23R) leads to triggering of the Jak/Stat signaling cascade that results in the secretion of inflammatory modulators such as IL-17A, IL-17F, IL-22 that thereby play a major role in downstream pro-inflammatory processes.

A high-complex combinatorial library derived from a 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 human p19 protein, the alpha-subunit of the IL-23 cytokine. A collection of p19-binding proteins (called ILP binders) was used to identify a group of binding proteins that inhibited binding of p19 (IL-23) to its cognate receptor.

The binding of ILP binders to the p19/IL-23 is demonstrated using ELISA and the prediction of blocking function for several selected ILP variants is documented using docking of p19 to IL-23R homology model based on known IL-6/IL-6 receptor crystal structure. Inhibitory function of these variants is further confirmed using cell-surface competition binding assay. Our data document that the three-helix bundle scaffold of ABD is useful for generation of novel epitope-mapping tools important for development of novel IL-23-based next-generation therapeutics.