Cell experiment:

Spleen B cells from C57BL/6 mice are labeled with Cell Trace Violet and incubated for 72 hours with either anti-IgM (10 mg/mL) or anti-CD40 (5 mg/mL), supplemented with IL-4 (5 ng/mL) or LPS (2.5 mg/mL) in the presence of different concentrations of AX-024. Proliferation is calculated according to the total number of cell divisions[1].

Animal experiment:

Eight-week-old CD-1 mice are injected intraperitoneally with different amounts of the AX-024 dissolved in 0.5 mL of saline. All animals are observed clinically for the appearance of macroscopically visible adverse reactions twice daily over 14 days, as well as immediately after AX-024 administration. A necropsy is carried out on each animal on day 14, and the abdominal, thoracic, and cranial cavities are examined in situ, together with their associated organs[1].

AX-024 is an cytokine release inhibitor which can strongly inhibit the production of interleukin-6 (IL-6), tumor necrosis factor-α (TNFα), interferon-γ (IFN-γ), IL-10 and IL-17A. IL-6, TNFα, IFN-γ, IL-10 and IL-17A[1]

Compound AX-024 is >10,000-fold more potent than the AX-000 hit in terms of inhibition of TCR-triggered T cell proliferation. The IC50 of AX-024 in this assay is 1 nM, although it shows inhibitory effects at a concentration of 1 pM or less. AX-024 is also a much more potent inhibitor of cytokine release by human peripheral blood mononuclear cells stimulated with anti-CD3 than AX-000, strongly hindering interleukin-6 (IL-6), tumor necrosis factor-α (TNFα), interferon-γ (IFN-γ), IL-10, and IL-17A production at a concentration of 10 nM. In CD8+ T cells of OT1 TCR transgenic (OT1Tg) mice bearing wild-type (WT) AX-024 strongly inhibits T cell proliferation at a concentration of 0.1 nM when OT1Tg T cells are WT for the PRS mutation. Coimmunoprecipitation experiments in these cells show that Nck recruitment to the TCR is induced upon stimulation in the absence of drug but is inhibited in the presence of AX-024 in a dose-dependent manner at concentrations starting from 1 nM[1].

The AX-024-treated group presents less scales and reduces skin thickening compare to the vehicle group. Treatment with AX-024 significantly reduces thickening of both skin layers, but more effectively of the dermis, which rather resembles that of mice treated with a control cream lacking imiquimod (IMQ). Treatment with AX-024 significantly diminishes the number of airway inflammatory cells in both assays. Mice receiving AX-024 rapidly recovers from neurological impairment and weight loss, becoming symptom-free by day 30, unlike mice that receives the vehicle, in which ataxia and loss of the righting reflex persist[1].

[1]. Borroto A, et al. First-in-class inhibitor of the T cell receptor for the treatment of autoimmune diseases. Sci Transl Med. 2016 Dec 21;8(370):370ra184.