Cytokines: an open gate to autoimmune disease therapies

Life Science stories
Mathis L.
By Mathis L.    time to read 3 min

Cytokines are the main mediators of inflammation and one of the most successful approaches to inflammatory disorder or autoimmune disease research. Cytokines have been used to screen for compounds inhibiting their secretion or blocking their receptors and signal transduction.

RORγt antagonist improves psoriasis mouse models

In 2016, Takaishi’s team aimed to develop an antagonist to the master transcription factor RORγt (Retinoic acid-related Orphan Receptor gamma T), which is responsible for the expression of helper T lymphocytes Th17, whose misregulated proliferation is thought to contribute to the pathogenesis and persistence of psoriasis (Ref 1).

The researchers identified a promising drug candidate designated in A213 through the screening of a chemical library with the HTRF IL-17 assay, which is the landmark of Th17 phenotype. Comparison of A213 inhibitory potency to that of digoxin, another potent RORγt antagonist, were very encouraging as they showed the former achieves results of similar magnitude with doses over 10-fold smaller.

Challenging their findings furthers, the team investigated the effects of A213 in mouse models of psoriasis upon oral administration and were able to observe a decrease in psoriasis-like symptoms such as skin lesions and inflammation. HTRF-monitored IL-17 expression was also reduced in the inflamed areas of the skin. Such in-vivo results were quite promising and hint at a strong potential application of A213 for psoriasis.

RORgt inhibition reduces IL-17 expression
Effects of A213 on TPA-induced psoriasis-like symptoms in mice ears. Methylcellulose (MC) for control.

Novel antibodies variants screened against a cytokine receptor

Though most cytokine-related assays in literature focus on identifying inhibitors of cytokine secretion, other drug discovery approaches are possible, such as reducing a cytokine activity via blocking its receptor or transduction signal pathway.

IL-21 is a cytokine mainly secreted by CD4+ T-cells and NKT cells. It has been increasingly thought to be part of the pathogenesis of autoimmune diseases characterized by chronic inflammation and neutralizing its bioactivity has already shown to decrease the diseases’ symptoms in murine models. In 2010, Vugmeyster et al sought to develop optimized antibodies suited for the blockade of that cytokine receptor (IL-21R) and produced numerous antibody variants (Single-chain variant fragments or ScFv) from the 18A5 anti-IL-21R antibody (Ref 2).

These ScFv were tested against the original 18A5 antibody in a TR-FRET competitive assay to identify the ones exhibiting a higher affinity for the receptor (assay built using the Europium Cryptate labeling kit and Streptavidin-XL665 kit). Thanks to this assay, the authors were able to identify a set of potential antibody variants, two of which stood out while differing by only four amino acids. Further investigations and tests of these two in mouse lupus models led the team to conclude that very minor variations in such antibodies could results in significant in vitro and in vivo effects in terms of pharmacological activity and pharmacodynamic profiles.

Protein-protein interaction assay for antibody characterization
Biotin-tagged IL-21R are incubated in presence of XL665 coupled streptavidin and cryptate coupled anti-IL-21R antibodies. The streptavidin binds to to the biotin tag while the antibodies bind to IL-21R, which brings the cryptate and XL665 in proximity and triggers FRET. Single-chain fragments are introduced to the mix and those exhibiting a potential IL-21R binding profile compete with the anti-IL-21R antibodies, which decreases the FRET signal.

Conclusion

Cytokines offer multiple screening targets for drug discovery in autoimmune diseases and the development of molecules susceptible of regulating the activity and signaling potential of these key mediators is now central in inflammatory related research. As the prevalence of autoimmune disorders grows, the publications mentioned in this post bring evidence that homogeneous fluorescence assays have now become relevant tools to enhance our drug discovery arsenal and advance therapeutic research.

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