Bill Hanson

Listeria monocytogenes triggers AIM2-mediated pyroptosis upon infrequent bacteriolysis in the macrophage cytosol.

A host defense strategy against pathogens is the induction of cell death, thereby eliminating the pathogens intracellular niche. Pyroptosis, one such form of cell death, is dependent on inflammasome activation. In a genetic screen to identify Listeria monocytogenes mutants that induced altered levels of host cell death, we identified a mutation in lmo2473 that caused hyperstimulation of IL-1beta secretion and pyroptosis following bacteriolysis in the macrophage cytosol. In addition, strains engineered to lyse in the cytosol by expression of both bacteriophage holin and lysin or induced to lyse by treatment with ampicillin stimulated pyroptosis. Pyroptosis was independent of the Nlrp3 and Nlrc4 inflammasome receptors but dependent on the inflammasome adaptor ASC and the cytosolic DNA sensor AIM2. Importantly, wild-type L. monocytogenes were also found to lyse, albeit at low levels, and trigger AIM2-dependent pyroptosis. These data suggested that pyroptosis is triggered by bacterial DNA released during cytosolic lysis.

Listeria monocytogenes engineered to activate the Nlrc4 inflammasome are severely attenuated and are poor inducers of protective immunity

Inflammasomes are intracellular multiprotein signaling complexes that activate Caspase-1, leading to the cleavage and secretion of IL-1β and IL-18, and ultimately host cell death. Inflammasome activation is a common cellular response to infection; however, the consequences of inflammasome activation during acute infection and in the development of long-term protective immunity is not well understood. To investigate the role of the inflammasome in vivo, we engineered a strain of Listeria monocytogenes that ectopically expresses Legionella pneumophila flagellin, a potent activator of the Nlrc4 inflammasome. Compared with wild-type L. monocytogenes, strains that ectopically secreted flagellin induced robust host cell death and IL-1β secretion. These strains were highly attenuated both in bone marrow-derived macrophages and in vivo compared with wild-type L. monocytogenes. Attenuation in vivo was dependent on Nlrc4, but independent of IL-1β/IL-18 or neutrophil activity. L. monocytogenes strains that activated the inflammasome generated significantly less protective immunity, a phenotype that correlated with decreased induction of antigen-specific T cells. Our data suggest that avoidance of inflammasome activation is a critical virulence strategy for intracellular pathogens, and that activation of the inflammasome leads to decreased long-term protective immunity and diminished T-cell responses.

Constitutive Activation of the PrfA regulon enhances the potency of vaccines based on live-attenuated and killed but metabolically active Listeria monocytogenes strains.

ABSTRACT Recombinant vaccines derived from the facultative intracellular bacterium Listeria monocytogenes are presently undergoing early-stage clinical evaluation in oncology treatment settings. This effort has been stimulated in part due to preclinical results that illustrate potent activation of innate and adaptive immune effectors by L. monocytogenes vaccines, combined with efficacy in rigorous animal models of malignant and infectious disease. Here, we evaluated the immunologic potency of a panel of isogenic vaccine strains that varied only in prfA. PrfA is an intracellularly activated transcription factor that induces expression of virulence genes and encoded heterologous antigens (Ags) in appropriately engineered vaccine strains. Mutant strains with PrfA locked into a constitutively active state are known as PrfA* mutants. We assessed the impacts of three PrfA* mutants, G145S, G155S, and Y63C, on the immunologic potencies of live-attenuated and photochemically inactivated nucleotide excision repair mutant (killed but metabolically active [KBMA]) vaccines. While PrfA* substantially increased Ag expression in strains grown in broth culture, Ag expression levels were equivalent in infected macrophage and dendritic cell lines, conditions that more closely parallel those in the immunized host. However, only the prfA(G155S) allele conferred significantly enhanced vaccine potency to KBMA vaccines. In the KBMA vaccine background, we show that PrfA*(G155S) enhanced functional cellular immunity following an intravenous or intramuscular prime-boost immunization regimen. These results form the basis of a rationale for including the prfA(G155S) allele in future live-attenuated or KBMA L. monocytogenes vaccines advanced to the clinical setting.

Priming and activation of human ovarian and breast cancer-specific CD8+ T cells by polyvalent Listeria monocytogenes-based vaccines.

Immunotherapeutic vaccine is potentially an effective strategy to combat cancer. Essential components of an effective vaccine must include antigens that are processed by the major histocompatibility complex class I pathway, presented by the tumor major histocompatibility complex molecules, and an effective antigen delivery platform that is capable of breaking self-tolerance. In this study, we characterized a set of ovarian cancer-specific T-cell epitopes delivered by live-attenuated recombinant Listeria monocytogenes (Lm ΔactAΔinlB) as a vaccine vector. We present data that peptide-specific T cells recognize the human monocytic cell line THP-1 infected with recombinant Lm ΔactAΔinlB encoding the epitopes. Furthermore, we demonstrate that recombinant L. monocytogenes (Lm)-infected antigen-presenting cells can prime and expand epitope-specific CD8+ T cells in vitro and such CD8+ T cells recognize not only peptide-loaded targets but also ovarian and breast tumor cells presenting endogenous epitopes. Finally, peptide-specific T cells generated using peripheral blood mononuclear cell from ovarian cancer patients recognize target cells infected with recombinant Lm ΔactAΔinlB encoding the epitopes. Our results demonstrate that live-attenuated recombinant Lm can be used effectively as a vehicle to deliver cancer peptide antigens singly or as a multiepitope construct. Thus, the use of recombinant live-attenuated Lm strains encoding endogenously processed and presented tumor epitopes/antigens represents an attractive strategy for active cancer immunotherapy in a clinical setting.

Phase I study of safety and immunogenicity of ADU-623, a live-attenuated listeria monocytogenes vaccine (ΔactA/ΔinlB) expressing EGFRVIII and NY-ESO-1, in patients with who grade III/IV astrocytomas

Meeting abstracts The neo-antigen EGFRvIII is expressed in multiple tumor types, including high-grade astrocytomas. It is associated with a poor prognosis and resistance to conventional therapies such as chemotherapy and radiation that are part of the standard treatment. We propose that

Abstract B50: Synergistic antitumor efficacy in mice with immunotherapy regimens combining recombinant live-attenuated Listeria with immune checkpoint inhibitors

Development of effective and durable antitumor immunity requires the activation, expansion and maintenance of function of tumor antigen-specific effector T cells. The mechanism of action of recently FDA approved monoclonal antibody (mAb) therapies which target immune checkpoints such as PD-1 and CTLA-4 and uncouple inhibitory pathways from the activation of antigen-specific T cells underlines the critical requirement for antigen-specific priming to elicit potent and long-lasting anti-tumor immunity. However, immune checkpoint blockade results in significant clinical benefit largely in malignancies with high mutational burden, where the tumor itself can initiate T cell priming, but is less effective when used as a single agent among cancers with lower mutational burden, associated typically with a low level of lymphocyte infiltration into the tumor microenvironment (TME). We are evaluating clinical immunotherapy regimens combining immune checkpoint blockade with recombinant live-attenuated double-deleted strains of the intracellular bacterium Listeria monocytogenes (LADD), based on a hypothesis that effective immunotherapy will result from agents that in combination re-polarize the TME to facilitate immune effector cell function, prime functional tumor-specific T cells in the appropriate context, and block inhibitory signaling pathways. Here we show that tumor antigen-expressing LADD therapy enhanced CD8+ T cell effector function, resulting in significant tumor eradication in several preclinical mouse models. LADD treatment in addition induced favorable changes in the TME, as shown by enhanced CD8+ T effector function, recruitment of antigen presenting cells and reduction of regulatory T cells. Treatment regimens combining LADD-based immunotherapy with PD-1 immune checkpoint blockade significantly enhanced antitumor efficacy in CT26, 4T1 and MC38 tumor models. Together these data support the rationale for integrating LADD-based immunotherapy into clinical regimens with immune checkpoint blockade on the basis that TME modification and priming of tumor Ag-specific T cells significantly enhances the activity of mAb therapies blocking T cell inhibitory pathways. Citation Format: Weiwen Deng, Weiqun Liu, Thomas Hudson, Chris S. Rae, Ed Lemmens, Anthony Desbien, Bill Hanson, Pete Lauer, Thomas W. Dubensky, Jr., Meredith Leong. Synergistic antitumor efficacy in mice with immunotherapy regimens combining recombinant live-attenuated Listeria with immune checkpoint inhibitors. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B50.

Abstract 2837: Combined targeting of antigens expressed in prostate cancer and prostate stem cells using Listeria-based cancer vaccines for the treatment of prostate cancer.

Immune-based therapies have demonstrated benefit in multiple clinical trials, and as we gain better knowledge of the mechanisms by which tumors avoid immune detection, new strategies can be designed to more effectively target tumor-specific antigens. Aduro BioTech, Inc. has engineered attenuated Listeria monocytogenes (Lm), to be safe for human use and to also induce potent and broad innate and adaptive immune responses including CD4 and CD8 T cells specific for tumor-associated antigens. Aduro9s lead therapeutic, CRS-207, is now being evaluated in a randomized, controlled Phase 2 study in patients with metastatic pancreas cancer. Prostate cancer is the only type of cancer for which a targeted therapeutic cancer vaccine has been approved by the U.S. Food and Drug Administration (FDA). Provenge®, an autologous cellular vaccine targeting prostatic acid phosphatase (PAP) developed by the Dendreon Corporation, was approved in 2010 for patients with metastatic, castrate-resistant prostate cancer (CRPC), validating the benefits of immunotherapy and providing a new treatment for CRPC with significantly less toxicity than chemotherapy. We have developed a candidate therapeutic vaccine strain for prostate cancer, named Lm-PCaVx, in which Aduro9s proprietary Lm platform has been engineered to express three antigens, PAP, NY-ESO-1 and NKX3.1. The goal is to concurrently target antigens that are expressed by tumor cells (PAP and NY-ESO-1) as well as an antigen that can be expressed by cancer stem cells (NKX3.1). PAP has been clinically validated as the target of the Provenge® vaccine; NY-ESO-1 has been shown to a tumor-associated immunological target by other vaccine approaches; and NKX3.1 is over-expressed in prostate tissue that regenerates after castration therapy. In this study, we report the construction of an Lm vaccine strain encoding these three antigens and demonstrate expression of the encoded antigens in Lm-infected antigen-presenting cells. Immunization with Lm-PCaVx induced PAP-, NY-ESO-1- and NKX3.1-specific T-cell responses in mice as well as anti-tumor-type responses in HLA-A2/HLA-DR1 transgenic mice. Together these findings support the further development of a multivalent Lm-based vaccine targeting cell-mediated immune responses against multiple tumor-associated antigens as a novel, cost-effective and rational approach for the treatment of patients with prostate cancer. Citation Format: Douglas McNeel, Marcella Fasso, Laura Johnson, Ed Lemmens, Bill Hanson, Pete Lauer, Steven Bodovitz, Charles Drake, Dirk G. Brockstedt. Combined targeting of antigens expressed in prostate cancer and prostate stem cells using Listeria-based cancer vaccines for the treatment of prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2837. doi:10.1158/1538-7445.AM2013-2837