Craig M. Brackett

Enhancement of anti-tumor immunity by photodynamic therapy

Photodynamic therapy (PDT) is an FDA-approved modality that rapidly eliminates local tumors, resulting in cure of early disease and palliation of advanced disease. PDT was originally considered to be a local treatment; however, both pre-clinical and clinical studies have shown that local PDT treatment of tumors can enhance systemic anti-tumor immunity. The current state of investigations into the ability of PDT to enhance anti-tumor immunity, the mechanisms behind this enhancement and the future of PDT as an immunotherapy are addressed in this review.

Central role of liver in anticancer and radioprotective activities of Toll-like receptor 5 agonist

Significance Toll-like receptor 5 (TLR5) is an innate immunity receptor that specifically recognizes and triggers immune response to bacterial flagellins. In addition to resistance to Salmonella infection, TLR5 agonists protect mammals from radiation and have anticancer effects, including suppression of tumor metastases. Using mouse models, we defined the liver as a major target for TLR5 agonists. Administration of pharmacologically optimized flagellin derivative CBLB502 leads to rapid activation of prosurvival nuclear factor kappa B (NF-κB) and STAT3 pathways in the liver and rescues mice from lethal doses of hepatotoxic Fas-agonistic antibodies. Thus, TLR5 agonists can be considered for treatment and prevention of liver metastasis and hepatoprotective applications. 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.

Photodynamic therapy enhancement of anti-tumor immunity

Photodynamic therapy (PDT) is an FDA-approved modality for the treatment of early-stage disease and palliation of late-stage disease. Pre-clinical studies using mouse models and clinical studies in patients have demonstrated that PDT is capable of influencing the immune system. The effect of PDT on the generation of anti-tumor immunity is regimen-dependent and is tightly linked to the degree and nature of inflammation induced by PDT. However, the precise mechanism underlying PDT-regulated adaptive anti-tumor immunity remains unclear. This review will focus on the current knowledge of immune regulation by PDT.

IL-17 Promotes Neutrophil Entry into Tumor-Draining Lymph Nodes following Induction of Sterile Inflammation

Blood-borne neutrophils are excluded from entering lymph nodes across vascular portals termed high endothelial venules (HEVs) because of lack of expression of the CCR7 homeostatic chemokine receptor. Induction of sterile inflammation increases neutrophil entry into tumor-draining lymph nodes (TDLNs), which is critical for induction of antitumor adaptive immunity following treatments such as photodynamic therapy (PDT). However, the mechanisms controlling neutrophil entry into TDLNs remain unclear. Prior evidence that IL-17 promotes neutrophil emigration to sites of infection via induction of CXCL2 and CXCL1 inflammatory chemokines raised the question of whether IL-17 contributes to chemokine-dependent trafficking in TDLNs. In this article, we demonstrate rapid accumulation of IL-17–producing Th17 cells in the TDLNs following induction of sterile inflammation by PDT. We further report that nonhematopoietic expression of IL-17RA regulates neutrophil accumulation in TDLNs following induction of sterile inflammation by PDT. We show that HEVs are the major route of entry of blood-borne neutrophils into TDLNs through interactions of l-selectin with HEV-expressed peripheral lymph node addressin and by preferential interactions between CXCR2 and CXCL2 but not CXCL1. CXCL2 induction in TDLNs was mapped in a linear pathway downstream of IL-17RA–dependent induction of IL-1β. These results define a novel IL-17–dependent mechanism promoting neutrophil delivery across HEVs in TDLNs during acute inflammatory responses.

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

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. 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. Toll-like 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.

IL-6 Potentiates Tumor Resistance to Photodynamic Therapy (PDT)

Photodynamic therapy (PDT) is an anticancer modality approved for the treatment of early disease and palliation of late stage disease. PDT of tumors results in the generation of an acute inflammatory response. The extent and duration of the inflammatory response is dependent upon the PDT regimen employed and is characterized by rapid induction of proinflammatory cytokines, such as IL‐6, and activation and mobilization of innate immune cells. The importance of innate immune cells in long‐term PDT control of tumor growth has been well defined. In contrast the role of IL‐6 in long‐term tumor control by PDT is unclear. Previous studies have shown that IL‐6 can diminish or have no effect on PDT antitumor efficacy.

The Toll-like receptor 5 agonist entolimod suppresses hepatic metastases in a murine model of ocular melanoma via an NK cell-dependent mechanism

Uveal melanoma (UM) is the most common primary cancer of the eye in adults and progresses to metastatic disease predominantly of the liver in ∼50% of patients. In these cases, life expectancy averages just 9 months due to the lack of effective treatment options. The Toll-like receptor 5 (TLR5) agonist entolimod (former name CBLB502) rapidly activates TLR5-NF-κB signaling in hepatocytes and suppresses growth of both TLR5-expressing and non-expressing tumors in the liver through mobilization and activation of innate and adaptive immune mechanisms. The goal of this study was to explore the potential of entolimod as an immunotherapeutic agent against hepatic metastasis of UM using the TLR5-positive B16LS9 mouse model of ocular melanoma. Mice were given seven subcutaneous injections of vehicle or entolimod given 72 h apart started one day before, on the same day or three days after intraocular injection of B16LS9 cells. All tested regimens of entolimod treatment resulted in significantly reduced B16LS9 metastasis to the liver. Entolimod induced mobilization of natural killer (NK) cells to the liver and stimulated their maturation, differentiation and activation. Antibody-mediated depletion of NK cells from mice abrogated entolimods antimetastatic activity in the liver and eliminated the entolimod-elicited in vitro cytotoxic activity of hepatic lymphocytes against B16LS9 cells. These results provide pre-clinical evidence of entolimods efficacy against hepatometastasis of UM and support its further development as an anticancer immunotherapeutic drug.

Abstract B090: A Toll-like receptor 5 agonist entolimod as a potential anticancer immunotherapeutic agent

Liver metastases are a major common cause of mortality of patients with different types of cancer due to lack of effective treatment options. Anticancer immunotherapy provides an attractive approach for developing a more effective systemic therapy for patients with metastatic liver cancer. We generated a Toll-like receptor 5 (TLR5) agonistic agent entolimod™ (CBLB502), a pharmacologically optimized derivative of bacterial Salmonella flagellin that is now at advanced stage of clinical development as a medical countermeasure to reduce adverse side effects of radiation therapy. We also found that entolimod, in addition to its radioprotective properties, has anticancer effects against a variety of TLR5-positive tumors and TLR5-negative tumors residing in a TLR5-expressing environment such as the liver in mice. We hypothesized that entolimod can suppress tumor growth in a TLR5-responsive microenvironment such as the liver independently of the TLR5 status of tumor cells. We tested the therapeutic potential of entolimod against TLR5 responsive and non-responsive tumors using mouse triple negative 4T1 breast and CT26 colorectal adenocarcinoma models mimicking clinically occurring development of post-surgery liver metastases in cancer patients. Our results demonstrate that entolimod treatment was efficient in suppressing metastatic growth when injected systemically in both models. Investigating the mechanism of entolimod9s antitumor activity we found that several major transcriptional factors, such as NF-κB, STAT-3 and AP-1, are rapidly activated in hepatocytes leading to a short-term neutrophilic infiltration and long-term NK cell accumulation and activation that coordinate CD8+ and CD4+ T cell immunity against liver metastases. This resulted in significant improvement of long-term tumor-free survival and durable, tumor-specific T cell memory. Importantly, unlike other TLR agonists entolimod is safe and does not show signs of septic shock even with systemic administration. Moreover, the results of two phase 1 clinical trials with 150 healthy volunteers and 26 patients with advanced cancers demonstrated good tolerability and safe profile of the response, which supports entolimod9s potential as an immunotherapeutic agent. This also suggests that entolimod can be potentially combined with radiation, chemotherapeutic, targeted, and other immunotherapeutic agents to improve the efficacy of anticancer therapy. Citation Format: Craig Brackett, Bojidar Kojouharov, Sandra Gollnick, Andrei Gudkov, Lyudmila Burdelya. A Toll-like receptor 5 agonist entolimod as a potential anticancer immunotherapeutic agent. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B090.