Toll-like receptor-5 agonist, entolimod, suppresses metastasis and induces immunity by stimulating an NK-dendritic-CD8+T-cell axis

Craig M. Brackett, Bojidar Kojouharov, Jean Veith, Kellee F. Greene, Lyudmila G. Burdelya, Sandra O. Gollnick, Scott I. Abrams, Andrei V. Gudkov
2016 Proceedings of the National Academy of Sciences of the United States of America  
Activation of an anticancer innate immune response is highly desirable because of its inherent ability to generate an adaptive antitumor T-cell response. However, insufficient safety of innate immune modulators limits clinical use to topical applications. Tolllike receptor 5 (TLR5) agonists are favorably positioned as potential systemic immunotherapeutic agents because of unusual tissue specificity of expression, uniquely safe profile of induced cytokines, and antitumor efficacy demonstrated in
more » ... a number of animal models. Here, we decipher the molecular and cellular events underlying the metastasis suppressive activity of entolimod, a clinical stage TLR5 agonist that activates NF-κB-, AP-1-, and STAT3-driven immunomodulatory signaling pathways specifically within the liver. Used as a single agent in murine colon and mammary metastatic cancer models, entolimod rapidly induces CXCL9 and -10 that support homing of blood-borne CXCR3-expressing NK cells to the liver predominantly through an IFN-γ signaling independent mechanism. NK cell-dependent activation of dendritic cells is followed by stimulation of a CD8 + T-cell response, which exert both antimetastatic effect of entolimod and establishment of tumor-specific and durable immune memory. These results define systemically administered TLR5 agonists as organ-specific immunoadjuvants, enabling efficient antitumor vaccination that does not depend on identification of tumor-specific antigens. cancer immunotherapy | liver | colorectal cancer | breast cancer | innate immunity R ecent advancements in the field of anticancer immunotherapy have been primarily focused on development of T-cellbased approaches because of recognition of the inherent ability of adaptive immunity to efficiently eradicate neoplastic disease (1, 2). Innate immune responses play important roles in T-cell activation, but their potential relevance for prevention and treatment of cancer remains underappreciated (3-5). Toll-like receptors (TLRs) are gaining attention as potential therapeutic targets capable of stimulating antitumor immunity by initiating innate responses (6) and subsequent adaptive T-cell-based immunity (7) . Although proof-ofprinciple for this concept has been demonstrated with agonists of several TLRs (TLR3, -7, and -9) (8), only one, the TLR7 agonist Imiquimod, has been approved for clinical use [however, this is limited to topical treatment of basal cell carcinoma (9)]. The major clinical limitations of many TLR agonists are the risk of dose-limiting toxicities associated with their systemic delivery (10-12) and metastasis stimulation (13-15). Furthermore, some previously investigated TLR agonists are restricted to injection directly into tumor tissue (3, 16-18), an approach that will likely have limited therapeutic value in cancer patients with metastatic disease. TLR5 is unique among TLRs as a potential target for systemic anticancer immunotherapy. Studies have shown that the only known natural TLR5 agonist, flagellin, flagellin-expressing Salmonella bacteria, and a pharmacologically optimized flagellin derivative named entolimod (CBLB502) have antitumor effects in several tumor models (19-23), including mouse models of liver metastases (24-26). Moreover, systemic administration of TLR5 agonists is uniquely safe because of the restricted pattern of expression of TLR5 (primarily in the gut, liver, and bladder) and the nature of the cytokines induced following TLR5 stimulation. In particular, TLR5 agonists are significantly less toxic than agonists of some other TLRs as a result of the lack of induction of self-amplifying "cytokine storm"-inducing cytokines, such as TNF-α, IL-1β, and IL-2, which can cause septic shock (27) (28) (29) . Instead, TLR5 agonists induce rapid and short-lived production of high levels of G-CSF, IL-6, IL-8, and IL-10 in all tested species, including rodents, nonhuman primates, and humans (29-31). The liver shows the strongest TLR5 activation response following systemic entolimod administration characterized by dramatic activation of NF-κB-, STAT3-, and AP-1-driven transcription leading to cytokine production (as described above) and mobilization of different classes of immune cells into the liver (26). In particular, entolimod-driven recruitment of natural killer (NK) cells to the liver was shown to be critical for the antitumor efficacy of the drug in murine tumor models (26, 32) and for its antiviral activity in a mouse model of cytomegalovirus infection (33). The liver is a common site of colorectal cancer (CRC) tumor metastasis (34) and the location of large numbers of NK cells (35), which have been reported to have antitumor activity in the liver (36, 37). NK cells are Significance Innate immune modulators can generate a potent antitumor T-cell response and are thus a desirable approach to immunotherapy. However, their use is limited by the risk of induction of acute inflammation. In this regard, bacterial flagellin is unique among other innate immune modulators because of a significantly safer cytokine profile induced upon activation by its target, Toll-like receptor 5 (TLR5). We show here that systemic administration of entolimod, a pharmacologically optimized flagellin derivative, induces a cascade of cell-cell signaling resulting in mobilization to the liver of various components of innate and adaptive immunity, followed by suppression of liver metastases and development of long-term antitumor immune memory. Thus, TLR5 agonists can be considered as an organ-specific immunotherapy for the treatment and prevention of metastases.
doi:10.1073/pnas.1521359113 pmid:26831100 pmcid:PMC4763744 fatcat:dbhi6qxyvrga7c6x5dzi6gd6cu