A Novel Method for Screening Adenosine Receptor Specific Agonists for Use in Autoimmune Diseases

Graft versus host disease (GvHD) is a common complication of allogeneic transplantation. In GvHD, donor T cells recognize host tissues as foreign, causing an increase in the number of donor-derived effector T cells in target tissues and increased production of pro-inflammatory cytokines. First line treatment to date remains high dose corticosteroids. Unfortunately, some patients with GvHD remain steroid refractory and even when effective, high dose corticosteroids can result in significant side effects including opportunistic infections. A more ideal method involves the induction of immune tolerance via FoxP3+ regulatory T-cells (Tregs) in host tissues, preventing the donor cells from reacting to host tissues while preserving the overall immune reaction to infection. Recently, we have published data involving the use of adenosine 2A receptor (A2AR) agonists to treat a mouse model of GvHD and have successfully induced FoxP3 expression while significantly reducing GvHD mediated tissue damage in treated mice. In mouse models, the small molecule A2AR agonists most potent at preventing GvHD and inducing production of Tregsare those that are highly specific for the murine A2AR, but lack agonist activity to other adenosine receptors. In order to develop A2AR agonists for treatment of GvHD in humans, it is important to have assays to screen small molecule agonists to demonstrate high specificity for the human A2AR. As the A2AR is a known mediator of intracellular calcium release, our lab has created a new calcium release detection method for determining specificity of synthetic A2AR agonist compounds for the A2AR in human cells. A2AR expression was effectively removed from K562 cells using CRISPR technology, resulting in an A2AR null line. Using lentivector, an A2AR overexpressor line was created from K562 cells. With a microplate reader we were then able to read changes in intracellular free calcium levels upon compound addition to our knockout and overexpressor lines. Those compounds found to be specific to the A2AR are then screened for efficacy using human naïve T-cells, evaluating them on their ability to induce differentiation of FoxP3+ Tregs. We evaluated a number of compounds and two, LNC-3025 and LNC-3047, were found to be A2AR specific, with no calcium release from knockout cells and significantly higher calcium release over baseline in overexpressing cells. These two compounds also significantly increased differentiation of Tregs from human T-cells compared to untreated controls (LNC-3025, p<0.05, LNC-3047, p<0.005). Our lab is now evaluating these two compounds for their ability to reduce pro-inflammatory cytokines as well as testing them in humanized mouse models of GvHD to determine their efficacy in vivo. Through these studies, we will be able to isolate the most specific and effective agonist compounds and will be one step closer to developing an A2AR agonist therapy for GvHD patients.