960 TT-816, a novel small molecule immune checkpoint inhibitor targeting cannabinoid CB2receptor, stimulates innate and adaptive immunity for cancer therapy

Background The endocannabinoid system is widely expressed in the human body, including the innate and adaptive immune system, where endocannabinoids, D 9-tetrahydrocannabinol and synthetic ligands regulate immune response. The effects of endocannabinoids on immune regulation are primarily medi-ated by G-protein coupled cannabinoid CB 2 receptors (CB 2 R) via several mechanisms, including development, migration, proliferation and effector functions. The upregulated expression of CB 2 R and elevated levels of endocannabinoids have been observed in a variety of tumor microenvironments and are associated with the aggressiveness of cancer. Methods Membranes prepared from CHO-K1 cells stably expressing human CB 2 R were used for receptor binding assays in the presence of TT-816 and [3H]CP-55,940, and for GTP g S binding assay in the presence of TT-816, 10 m M GDP and 0.3 nM [35S]GTP g S. cAMP assay was performed by incu-bating the CHO-K-1 cells for 30 min with TT-816, 25 m M forskolin and 12 nM CP-55,940, or with TT-816 and 5 m M forskolin. NK cell function was determined by co-culturing TT-816 pretreated NK cells with K562 cancer cells for 24 hours. The mixed lymphocyte reaction assay was conducted by co-culturing human CD4+ T cells with monocyte-derived dendritic cells. Cell viability was measured by FACS and IFN-g by MSD. Results TT-816 is a competitive and selective CB 2 R antagonist. human CB an IC50 26.2 nM, showing greater than 380-fold selectivity cannabinoid CB 1 receptors.