Background The endocannabinoid system is widely expressed in the human body, including the innate and adaptive immune system, where endocannabinoids, Δ9-tetrahydrocannabinol and synthetic ligands regulate immune response. The effects of endocannabinoids on immune regulation are primarily mediated by G-protein coupled cannabinoid CB₂ receptors (CB₂R) via several mechanisms, including development, migration, proliferation and effector functions. The upregulated expression of CB₂R 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 CB₂R were used for receptor binding assays in the presence of TT-816 and [3H]CP-55,940, and for GTPγS binding assay in the presence of TT-816, 10 μM GDP and 0.3 nM [35S]GTPγS. cAMP assay was performed by incubating the CHO-K-1 cells for 30 min with TT-816, 25 μM forskolin and 12 nM CP-55,940, or with TT-816 and 5 μM 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-γ by MSD.

Results TT-816 is a competitive and selective CB₂R antagonist. It bound to human CB₂R with an IC50 26.2 nM, showing greater than 380-fold selectivity over cannabinoid CB₁ receptors. The ability of TT-816 to inhibit the constitutive activity of CB₂R was characterized in both GTPγS and cAMP assays. TT-816 concentration-dependently inhibited the basal GTPγS binding response, antagonized CB₂R agonist-mediated cAMP production and enhanced the forskolin response on basal cAMP level. Consistent with its inhibition of CB₂R function, TT-816 inhibited the growth of human breast, colorectal and lung cancer cells. In addition, TT-816 concentration-dependently enhanced the functions of NK cells, dendritic cells and T cells. It increased NK cell killing of the human cancer cells and IFN-γ production, significantly stimulated the expression of CD86, HLA-DR, IL-12 and TNF-a in monocyte-derived dendritic cells, and enhanced CD4+ T cell proliferation and IFN-γ production in a mixed lymphocyte reaction assay.

Conclusions TT-816 is a novel, oral small molecule immune checkpoint inhibitor that selectively blocks CB₂R on cancer cells and immune cells. Preclinical data have demonstrated that it stimulates antitumor innate and adaptive immune response and inhibits cancer cell proliferation. TT-816 is currently undergoing phase 1 clinical trials for the treatment of a broad range of solid tumors.