Directed Evolution of a Soluble Human IL-17A Receptor for the Inhibition of Psoriasis Plaque Formation in a Mouse Model

Abstract

Interleukin-17 (IL-17) is a T-cell-derived cytokine that promotes inflammatory pathology in autoimmune diseases. Blocking IL-17A interactions with its endogenous IL-17 receptor (IL-17RA) can constitute an important target for therapeutic intervention. Here, we utilized a directed evolution approach to generate soluble IL-17RA mutants that exhibit increased IL-17A binding affinity and thermostability, relative to the wild-type. Human fibroblast cell-based assay and in vivo analysis in mice indicated that two improved IL-17RA mutants efficiently inhibit the secretion of IL-17A-induced proinflammatory cytokines. Analysis of one of these mutants in a psoriasis mouse model showed its efficacy in promoting the recovery of psoriasis plaques. This mutant can be used as a promising drug candidate for the treatment of psoriasis and may be a therapeutic agent for various other autoimmune diseases.

Directed evolution of a soluble human DR3 receptor for the inhibition of TL1A induced cytokine secretion

“…coli Clooni strain (Lucigen) was used for cloning and plasmid extraction. For the display of DR3 on the yeast cell surface, the DR3 variants were cloned into the pCTCON plasmid using the Nhe I and Bam HI sites, as previously described [17,22]. For mammalian cell expression, the pFUSE (Invivogen) vector was used to yield DR3-ECD fused with human IgG1 Fc.…”

“…The DR3 variants were displayed on the yeast cell surface of EBY100 strain cells and analyzed the cells by flow cytometry, essentially as described [17,22]. For this, yeast cells were transformed with the pCTCON plasmid encoding for DR3 variants.…”

“…For this, yeast cells were transformed with the pCTCON plasmid encoding for DR3 variants. Following transformation, the cells were grown in SDCAA media (20 g sucrose, 6.7 g yeast nitrogen base, 5 g casamino acids, 5.4 g Na 2 HPO 4 and 8.6 g NaH 2 PO 4 ) at 30°C to logarithmic phase and induced as previously described [17]. The cells were subsequently collected, washed with PBSF (PBS + 1 g/L BSA) and labeled with 0.2 μM TL1A (R&D Systems) that was biotinylated using a biotin labeling kit (Pierce) according to manufacturer’s procedure.…”

“…Next, cells were washed and labeled with FITC-conjugated α-mouse IgG (Sigma, 1 μl/50 μl PBSF) and allophycocyanin-conjugated streptavidin (Jackson Immunoresearch, 1 μl/50 μl PBSF). The labeled cells were washed, resuspended with PBSF and analyzed by flow cytometry (FACS Accury, BD) as previously described [17]. …”

“…Indeed, we have previously utilized directed evolution to engineer soluble IL-17A receptor for efficient blocking of IL-17A-mediated inflammatory responses. Our engineered IL-17 receptor exhibits high biological activity and effectively inhibits psoriasis plaque formation in a humanized mice model [17]. …”