The active metabolite of the TLR7 agonist prodrug ANA773 activates anti-tumor activity by natural killer cells

3556 The innate immune response provides the first line of defense against tumor cells by inducing a cascade of immunomodulatory events that includes the secretion of cytokines and the stimulation of natural killer (NK) cells. NK cells mediate anti-tumor activity by two different mechanisms. First, NK cells direct the lysis of tumor cells through the secretion of cytotoxic granules and promote apoptosis through the production of IFN-γ. The latter also increases the anti-tumor response of other immune cells. Second, NK cells also recognize and kill transformed cells through antibody-dependent cell-mediated cytotoxicity (ADCC). NK cells are activated by diverse signals, including type I interferons (IFN). Large amounts of type I IFN are produced by plasmacytoid dendritic cells (pDCs) upon engagement of the pattern recognition receptor toll-like receptor 7 (TLR7). The natural ligand for TLR7 is single stranded RNA, although a variety of small molecule agonists have also been discovered. This study investigated the effects of such a low molecular weight TLR7 agonist on human NK cell activity. ANA773 is an oral prodrug of a selective TLR7 agonist. This biologically active metabolite of ANA773 induced the secretion of IFN-α and various other cytokines from human peripheral blood mononuclear cells (PBMC) cultured in vitro. Cytokine levels were also elevated in the plasma of treated primates. Human PBMC stimulated with the active metabolite of ANA773 augmented NK cell cytotoxicity and cytokine secretion against both K562 erythroleukemic cells and transformed B cell lines. These effects were shown to be dependent on the presence of type I IFN, confirming the importance of IFN-α in mediating NK responses to TLR7 agonists. In addition to enhancing NK activation through the direct recognition of tumor cells, this small molecule TLR7 agonist also enhanced ADCC; treatment of human PBMC with the active metabolite augmented cytolysis of rituximab (anti-CD20) bound, CD20-expressing B cell tumor lines. Thus, the active metabolite of ANA773 promotes NK cell mediated anti-tumor response by promoting cytokine secretion, cytolysis of tumor cells, and antibody-dependent tumor cell lysis. These data indicate that TLR7 is an attractive therapeutic target for the treatment of tumor malignancies.