Abstract
Interleukin 17 (IL-17) and its cognate receptor A (IL-17RA) play a crucial role in Th17 cells-mediated pro-inflammatory pathway and pathogenesis of several autoimmune disorders including psoriasis. IL-17 is mainly produced by activated Th-17 helper cells upon stimulation by IL-23 and, via binding to its receptors, mediates IL-17-driven cell signaling in keratinocytes. Hyper-proliferation of keratinocytes belongs to major clinical manifestations in psoriasis. To modulate IL-17-mediated inflammatory cascade, we generated a unique collection of IL-17RA-targeting protein binders that prevent from binding of human IL-17A cytokine to its cell-surface receptor. To this goal, we used a highly complex combinatorial library derived from scaffold of albumin-binding domain (ABD) of streptococcal protein G, and ribosome display selection, to yield a collection of ABD-derived high-affinity ligands of human IL-17RA, called ARS binders. From 67 analyzed ABD variants, 7 different sequence families were identified. Representatives of these groups competed with human IL-17A for binding to recombinant IL-17RA receptor as well as to IL-17RA-Immunoglobulin G chimera, as tested in enzyme-linked immunosorbent assay (ELISA). Five ARS variants bound to IL-17RA-expressing THP-1 cells and blocked binding of human IL-17 cytokine to the cell surface, as tested by flow cytometry. Three variants exhibited high-affinity binding with a nanomolar Kd value to human keratinocyte HaCaT cells, as measured using Ligand Tracer Green Line. Upon IL-17-stimulated activation, ARS variants inhibited secretion of Gro-α (CXCL1) by normal human skin fibroblasts in vitro. Thus, we identified a novel class of inhibitory ligands that might serve as immunosuppressive IL-17RA-targeted non-IgG protein antagonists.
Highlights
Interleukin 23/Interleukin 17 (IL-23/IL-17) pro-inflammatory axis plays a pivotal role in the pathogenesis of several chronic autoimmune diseases [1,2,3]
IL-17A and IL-17F have been described to closely associate with development of autoimmune diseases via interactions with a receptor complex formed by IL-17 receptor A and C [9]
In our previous work [35] we demonstrated that the extracellular moiety of the human IL-23 receptor gene can be successfully produced as a refolded bacterial protein, which can serve as a target for the selection of protein binders from a highly complex combinatorial library
Summary
Interleukin 23/Interleukin 17 (IL-23/IL-17) pro-inflammatory axis plays a pivotal role in the pathogenesis of several chronic autoimmune diseases [1,2,3]. Heterodimer of a unique p19 subunit and a common p40 protein, both known as α- and β-subunits of IL-23 cytokine [4], is secreted by activated dendritic cells and macrophages, and stimulates the differentiation of naive Th cells into Th17 cell population. This action is mediated via interactions with its cognate receptor complex, formed by IL-23R and IL-12Rβ1 receptor units. IL-17A and IL-17F have been described to closely associate with development of autoimmune diseases via interactions with a receptor complex formed by IL-17 receptor A and C [9]. IL-17A and F are mostly secreted by activated Th17 cells and γδT cells, but can be produced by NKT cells or iLCs [11,12]
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