Vertebrate Toll-like receptor 5 (TLR5) recognizes bacterial flagellin proteins and activates innate immune responses to motile bacteria. In addition, activation of TLR5 signaling can inhibit growth of TLR5-expressing tumors and protect normal tissues from radiation and ischemia-reperfusion injuries. To understand the mechanisms behind these phenomena at the organismal level, we assessed nuclear factor kappa B (NF-κB) activation (indicative of TLR5 signaling) in tissues and cells of mice treated with CBLB502, a pharmacologically optimized flagellin derivative. This identified the liver and gastrointestinal tract as primary CBLB502 target organs. In particular, liver hepatocytes were the main cell type directly and specifically responding to systemic administration of CBLB502 but not to that of the TLR4 agonist LPS. To assess CBLB502 impact on other pathways, we created multireporter mice with hepatocytes transduced in vivo with reporters for 46 inducible transcription factor families and found that along with NF-κB, CBLB502 strongly activated STAT3-, phenobarbital-responsive enhancer module (PREM), and activator protein 1 (AP-1-) -driven pathways. Livers of CBLB502-treated mice displayed induction of numerous immunomodulatory factors and massive recruitment of various types of immune cells. This led to inhibition of growth of liver metastases of multiple tumors regardless of their TLR5 status. The changed liver microenvironment was not, however, hepatotoxic, because CBLB502 induced resistance to Fas-mediated apoptosis in normal liver cells. Temporary occlusion of liver blood circulation prevented CBLB502 from protecting hematopoietic progenitors in lethally irradiated mice, indicating involvement of a factor secreted by responding liver cells. These results define the liver as the key mediator of TLR5-dependent effects in vivo and suggest clinical applications for TLR5 agonists as hepatoprotective and antimetastatic agents.breast cancer | colon cancer | neutrophils | natural killer cells | Salmonella T oll-like receptors (TLRs) recognize and are activated by specific patterns in molecules that are produced by a broad range of microbial pathogens but are not present in host molecules. Activation of TLRs by these pathogen-associated molecular patterns leads to induction of infection-fighting innate immune responses (1). Various TLR agonists have been considered for multiple clinical applications, including cancer immunotherapy (2-4), and one, the TLR7 agonist imiquimod, is approved for topical treatment of basal cell carcinoma (5).Although signaling pathways induced by different TLRs all result in mobilization of an innate immune response and involve activation of nuclear factor kappa B (NF-κB), the key regulator of immunity (6, 7), TLR5 is a particularly attractive candidate for therapeutic targeting for several reasons. First, bacterial flagellin, the natural ligand of TLR5, was found to have strong radioprotective effects in rodents and nonhuman primates (8). CBLB502 is a rationally designed derivative of Salmon...

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 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)(17)(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 pharmacolog...

Myelosuppression and gastrointestinal damage are common side effects of cancer treatment limiting efficacy of DNA-damaging chemotherapeutic drugs. The Toll-like receptor 5 (TLR5) agonist Entolimod has demonstrated efficacy in mitigating damage to hematopoietic and gastrointestinal tissues caused by radiation. Here, using 5-Fluorouracil (5-FU) treated mice as a model of chemotherapy-induced side effects, we demonstrated significant reduction in the severity of 5-FU-induced morbidity and increased survival accompanied by the improved integrity of intestinal tissue and stimulated the restoration of hematopoiesis. Entolimod-stimulated IL-6 production was essential for Entolimod's ability to rescue mice from death caused by doses of 5-FU associated with hematopoietic failure. In contrast, IL-6 induction was not necessary for protection and restoration of drug-damaged gastrointestinal tissue by Entolimod. In a syngeneic mouse CT26 colon adenocarcinoma model, Entolimod reduced the systemic toxicity of 5-FU, but did not reduce its antitumor efficacy indicating that the protective effect of Entolimod was selective for normal, non-tumor, tissues. These results suggest that Entolimod has clinical potential to broaden the therapeutic window of genotoxic anticancer drugs by reducing their associated hematopoietic and gastrointestinal toxicities.

Activation of an anticancer innate immune response is an attractive immunotherapeutic opportunity that is challenged by insufficient safety of systemically administered innate immune modulators. Unusual tissue specificity of expression of TLR5 determines a uniquely safe profile of cytokines induced by the sole TLR5 agonist flagellin. Entolimod, a pharmacologically optimized flagellin derivative, was initially developed to treat and prevent acute radiation syndrome with demonstrated efficacy in rodents and non-human primates and safety in human healthy volunteers. In addition, Entolimod demonstrated antitumor effects in mouse models of uveal melanoma, lymphoma, breast, and colorectal carcinoma. Entolimod’s mechanism of action involves activation of NF-κB-, AP-1-, and STAT-3-driven immunomodulatory pathways in hepatocytes that initiates a cascade of cell-cell signaling events that mobilize innate and adaptive immunity to the liver. This includes CXCR3-dependent blood-borne NK cell homing followed by DC activation and antitumor CD8+ T cell memory formation. These results define systemically administered TLR5 agonists as an organ-specific immunoadjuvant enabling efficient antitumor vaccination that does not depend on identification of tumor-specific antigens. Therefore, Entolimod has strong promise as a safe, effective and broadly applicable immunotherapeutic agent against liver metastases, which are currently a major cause of cancer-associated mortality. Recent completion of a phase I trial of Entolimod in patients with advanced metastatic solid tumors has opened the opportunity to test whether efficacy demonstrated in animal cancer models could be translated into immunotherapy of human tumors.

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