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. Proteins 2014; 82:975–989.

Non-Canonical Interleukin 23 Receptor Complex Assembly

“…To the best of our knowledge, it is not known whether IL-12p35 needs p40 for interaction with IL-12R␤1 and IL-12R␤2, but binding of the other cytokine members of the IL-6/IL-12 type cytokine family to their ␤ receptors depends on the preceding complex formation with the ␣ receptor subunit (28 -30). Although, a recent report showed that bacterially expressed and refolded p19 is able to bind to IL-23R in the absence of p40 (31), this study lacks the general demonstration of the biological activity of bacterially produced p19 in complex with p40 and IL-23R/IL-12R␤1. As observed previously by Oppmann et al (8), secretion of p19 was promoted by p40 coexpression (Fig.…”

Targeting Human Thrombus by Liposomes Modified with Anti-Fibrin Protein Binders

“…In this work, we generated insoluble fibrin-targeted protein binders useful for development of thrombus imaging agents. For this non-immunoglobulin approach, combinatorial library derived from 5.5 kDa albumin-binding domain of streptococcal protein G was chosen as this library of theoretical complexity up to 10 14 protein variants has already provided binders of several important protein targets [31,34,41], some of them with nanomolar or even sub-nanomolar binding affinity [30,32,33]. To stabilize small protein binders, 305 amino acid helical TolA protein is being used to form 38 kDa fusion binding variants.…”

“…Combinatorial DNA library was generated as described previously [33,34] with some modifications. The assembled library was in vitro transcribed/translated in a single step reaction using E. coli extract (EasyXpress E. coli kit, biotechrabbit, Hennigsdorf, Germany) and used for the pre-selection in 96well Maxisorp plates (NUNC, Roskilde, Denmark).…”

Development of Recombinant Lactococcus lactis Displaying Albumin-Binding Domain Variants against Shiga Toxin 1 B Subunit

“…TSA was performed with the real-time PCR Detection System CFX touch (BIO-Rad Laboratories, Hercules, USA) according to [12]. The data were analyzed with CFX Manager Software and the melting temperatures (Tm) determined using the first derivative spectra.…”