p19-targeted ABD-derived protein variants inhibit IL-23 binding and exert suppressive control over IL-23-stimulated expansion of primary human IL-17+ T-cells

Abstract

Interleukin-23 (IL-23), a heterodimeric cytokine of covalently bound p19 and p40 proteins, has recently been closely associated with development of several chronic autoimmune diseases such as psoriasis, psoriatic arthritis or inflammatory bowel disease. Released by activated dendritic cells, IL-23 interacts with IL-23 receptor (IL-23R) on Th17 cells, thus promoting intracellular signaling, a pivotal step in Th17-driven pro-inflammatory axis. Here, we aimed to block the binding of IL-23 cytokine to its cell-surface receptor by novel inhibitory protein binders targeted to the p19 subunit of human IL-23. To this goal, we used a combinatorial library derived from a scaffold of albumin-binding domain (ABD) of streptococcal protein G, and ribosome display selection, to yield a collection of ABD-derived p19-targeted variants, called ILP binders. From 214 clones analyzed by ELISA, Western blot and DNA sequencing, 53 provided 35 different sequence variants that were further characterized. Using in silico docking in combination with cell-surface competition binding assay, we identified a group of inhibitory candidates that substantially diminished binding of recombinant p19 to the IL-23R on human monocytic THP-1 cells. Of these best p19-blockers, ILP030, ILP317 and ILP323 inhibited IL-23-driven expansion of IL-17-producing primary human CD4T-cells. Thus, these novel binders represent unique IL-23-targeted probes useful for IL-23/IL-23R epitope mapping studies and could be used for designing novel p19/IL-23-targeted anti-inflammatory biologics.

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).…”

“…After 1 h incubation at 4 • C, selection well was in the washed 5 times (10 times in second and third round) with wash buffer (50 mM Tris, 150 mM NaCl, 50 mM Mg-acetate, pH 7.5) supplemented with 0.05% Tween 20 (0.05% and 0.25% for second and third round, respectively). Collection of cDNA, obtained by reverse transcription after the third round of selection campaign, was cloned as NcoI and BamHI fragments in a pET-28b-TolA-Avi vector containing DNA sequences for spacer TolA and C-terminal Avitag [34]. The final TolA-Avi fusion proteins were expressed in E. coli BL21 (λDE3) GOLD strain.…”

New opportunities in endocrine ophthalmopathy diagnostics (review)

“…Результаты исследований показали, что ИЛ-23 играет важную роль в поддержа-нии функциональной активности T-хелперов-17, так как рецепторы к нему экспрессируются только на активированных клетках [55,56].…”

ABD-Derived Protein Blockers of Human IL-17 Receptor A as Non-IgG Alternatives for Modulation of IL-17-Dependent Pro-Inflammatory Axis

“…As the critical residues for a high-affinity binding to human serum albumin (HSA) are located on the second and third helices, we selected this surface for the randomization of 11 residues, thus disrupting the original HSA binding sites and providing a library of a theoretical complexity 2 × 10 14 protein variants. The validity of the library concept has already been demonstrated by the generation of ABD-derived binders targeting human interferon-γ with a sub-nanomolar affinity [ 32 ], by immunosuppressive protein blockers of human IL-23 receptor [ 35 ], by development of ABD-derived binders of human prostate specific protein 94 (PSP94, MSMB) [ 36 ] and by Shiga toxin 1B subunit-specific binders [ 37 ], and recently by immunomodulatory binders of human IL-23 cytokine [ 38 ].…”