Structural Evaluations of a Selective Human STING^A230 Agonist and Its Use in Macrophage Immunotherapies

Previously we identified a non-nucleotide agonist BDW568 that selectively activates the human STING^A230 allele. Here, we further characterized the mechanism of BDW568 and highlighted its potential use for selectively controlling the activation of engineered macrophages that constitutively express STING^A230 as a genetic adjuvant. We obtained the crystal structure of the C-terminal domain of STING^A230 complexed with BDW-OH (active metabolite) at 1.95 Å resolution. Structure-activity relationship studies revealed that all three heterocycles in BDW568 and the S-acetate side chain are critical for retaining activity. We demonstrated that BDW568 could robustly activate type I interferon signaling in purified human primary macrophages that were transduced with lentivirus expressing STING^A230. In contrast, BDW568 could not stimulate innate immune responses in human primary peripheral blood mononuclear cells in healthy donors in the absence of a STING^A230 allele. This high STING variant specificity suggested a promising application of STING^A230 agonists in macrophage-based therapeutic approaches.