Lactococcus lactis, a probiotic bacterium of food origin, has recently been demonstrated as a suitable strain for the production and in vivo delivery of therapeutically important proteins into the gut. We aimed to engineer recombinant L. lactis cells producing/secreting REX binding proteins that have been described as IL-23 receptor (IL-23R) blockers and IL-23R antagonists suppressing the secretion of cytokine IL-17A, a pivotal step in the T-helper Th17-mediated pro-inflammatory cascade, as well as in the development of autoimmune diseases, including inflammatory bowel disease (IBD). To reach this goal, we introduced cDNA sequences coding for REX009, REX115, and REX125 proteins into plasmid vectors carrying a Usp45 secretion signal, a FLAG tag sequence consensus, and a LysM-containing cA surface anchor (AcmA), thus allowing cell-surface peptidoglycan anchoring. These plasmids, or their non-FLAG/non-AcmA versions, were introduced into L. lactis host cells, thus generating unique recombinant L. lactis-REX strains. We demonstrate that all three REX proteins are expressed in L. lactis cells and are efficiently displayed on the bacterial surface, as tested by flow cytometry using an anti-FLAG antibody conjugate. Upon 10-fold concentration of the conditioned media, a REX125 secretory variant can be detected by Western blotting. To confirm that the FLAG/non-FLAG REX proteins displayed by L. lactis retain their binding specificity, cell-surface interactions of REX proteins with an IL-23R-IgG chimera were demonstrated by flow cytometry. In addition, statistically significant binding of secreted REX009 and REX115 proteins to bacterially produced, soluble human IL-23R was confirmed by ELISA. We conclude that REX-secreting L. lactis strains were engineered that might serve as IL-23/IL-23R blockers in an experimentally induced mouse model of colitis.

• Klíčová slova
• IL-23 cytokine, IL-23R, albumin-binding domain, binding protein, lactococcus, surface display,
• Publikační typ
• časopisecké články MeSH

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.

• Klíčová slova
• IL-17 receptor, albumin-binding domain, binding protein, combinatorial library, cytokine,
• MeSH
• bakteriální proteiny chemie   metabolismus   MeSH
• buněčné linie MeSH
• cytokiny metabolismus   MeSH
• interakční proteinové domény a motivy MeSH
• interleukin-17 metabolismus   MeSH
• konformace proteinů MeSH
• lidé MeSH
• mediátory zánětu metabolismus   MeSH
• molekulární modely MeSH
• receptory interleukinu-17 antagonisté a inhibitory   chemie   metabolismus   MeSH
• rekombinantní proteiny metabolismus   MeSH
• stabilita proteinů MeSH
• transportní proteiny metabolismus   MeSH
• vazba proteinů MeSH
• zánět imunologie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• cytokiny MeSH
• IgG Fc-binding protein, Streptococcus MeSH Prohlížeč
• IL17A protein, human MeSH Prohlížeč
• interleukin-17 MeSH
• mediátory zánětu MeSH
• receptory interleukinu-17 MeSH
• rekombinantní proteiny MeSH
• transportní proteiny MeSH