Weiqun Liu

STING agonist formulated cancer vaccines can cure established tumors resistant to PD-1 blockade

Cyclic dinucleotide formulated cancer vaccine combined with PD-1 blockade can induce regression of tumors that do not express PD-L1 constitutively. A therapy that STINGs tumors Stimulator of interferon genes, or STING, is a receptor that is found on a variety of cell types and activates an immune response in response to cyclic dinucleotides. Fu et al. found that combining cyclic dinucleotides with a cellular cancer vaccine called STINGVAX was effective against multiple types of tumors in mouse models. The authors then modified the cyclic dinucleotides to strengthen their binding to human STING, increasing their antitumor activity. The authors also showed that treatment with STINGVAX caused cancer cells to up-regulate PD-L1, a protein that suppresses the immune response. Inhibiting the PD-L1 pathway in mice treated with STINGVAX was very effective at killing even poorly immunogenic tumors. Stimulator of interferon genes (STING) is a cytosolic receptor that senses both exogenous and endogenous cytosolic cyclic dinucleotides (CDNs), activating TBK1/IRF3 (interferon regulatory factor 3), NF-κB (nuclear factor κB), and STAT6 (signal transducer and activator of transcription 6) signaling pathways to induce robust type I interferon and proinflammatory cytokine responses. CDN ligands were formulated with granulocyte-macrophage colony-stimulating factor (GM-CSF)–producing cellular cancer vaccines—termed STINGVAX—that demonstrated potent in vivo antitumor efficacy in multiple therapeutic models of established cancer. We found that rationally designed synthetic CDN derivative molecules, including one with an Rp,Rp dithio diastereomer and noncanonical c[A(2′,5′)pA(3′,5′)p] phosphate bridge structure, enhanced antitumor efficacy of STINGVAX in multiple aggressive therapeutic models of established cancer in mice. Antitumor activity was STING-dependent and correlated with increased activation of dendritic cells and tumor antigen–specific CD8+ T cells. Tumors from STINGVAX-treated mice demonstrated marked PD-L1 (programmed death ligand 1) up-regulation, which was associated with tumor-infiltrating CD8+IFNγ+ T cells. When combined with PD-1 (programmed death 1) blockade, STINGVAX induced regression of palpable, poorly immunogenic tumors that did not respond to PD-1 blockade alone.

Cytosolic Entry Controls CD8+-T-Cell Potency during Bacterial Infection

ABSTRACT Interaction with host immunoreceptors during microbial infection directly impacts the magnitude of the ensuing innate immune response. How these signals affect the quality of the adaptive T-cell response remains poorly understood. Utilizing an engineered strain of the intracellular pathogen Listeria monocytogenes that infects cells but fails to escape from the phagosome, we demonstrate the induction of long-lived memory T cells that are capable of secondary expansion and effector function but are incapable of providing protective immunity. We demonstrate that microbial invasion of the cytosol is required for dendritic cell activation and integration of CD40 signaling, ultimately determining the ability of the elicited CD8+-T-cell pool to protect against lethal wild-type L. monocytogenes challenge. These results reveal a crucial role for phagosomal escape, not for delivery of antigen to the class I major histocompatibility complex pathway but for establishing the appropriate cellular context during CD8+-T-cell priming.

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.

Impact of Preexisting Vector-Specific Immunity on Vaccine Potency: Characterization of Listeria monocytogenes-Specific Humoral and Cellular Immunity in Humans and Modeling Studies Using Recombinant Vaccines in Mice

ABSTRACT Recombinant live-attenuated Listeria monocytogenes is currently being developed as a vaccine platform for treatment or prevention of malignant and infectious diseases. The effectiveness of complex biologic vaccines, such as recombinant viral and bacterial vectors, can be limited by either preexisting or vaccine-induced vector-specific immunity. We characterized the level of L. monocytogenes-specific cellular and humoral immunity present in more than 70 healthy adult subjects as a first step to understanding its possible impact on the efficacy of L. monocytogenes-based vaccines being evaluated in early-phase clinical trials. Significant L. monocytogenes-specific humoral immunity was not measured in humans, consistent with a lack of antibodies in mice immunized with wild-type L. monocytogenes. Cellular immune responses specific for listeriolysin O, a secreted bacterial protein required for potency of L. monocytogenes-derived vaccines, were detected in approximately 60% of human donors tested. In mice, while wild-type L. monocytogenes did not induce significant humoral immunity, attenuated L. monocytogenes vaccine strains induced high-titer L. monocytogenes-specific antibodies when given at high doses used for immunization. Passive transfer of L. monocytogenes-specific antiserum to naïve mice had no impact on priming antigen-specific immunity in mice immunized with a recombinant L. monocytogenes vaccine. In mice with preexisting L. monocytogenes-specific immunity, priming of naïve T cells was not prevented, and antigen-specific responses could be boosted by additional vaccinations. For the first time, our findings establish the level of L. monocytogenes-specific cellular immunity in healthy adults, and, together with modeling studies performed with mice, they support the scientific rationale for repeated L. monocytogenes vaccine immunization regimens to elicit a desired therapeutic effect.

KBMA Listeria monocytogenes is an effective vector for DC-mediated induction of antitumor immunity

Vaccine strategies that utilize human DCs to enhance antitumor immunity have yet to realize their full potential. Approaches that optimally target a spectrum of antigens to DCs are urgently needed. Here we report the development of a platform for loading DCs with antigen. It is based on killed but metabolically active (KBMA) recombinant Listeria monocytogenes and facilitates both antigen delivery and maturation of human DCs. Highly attenuated KBMA L. monocytogenes were engineered to express an epitope of the melanoma-associated antigen MelanA/Mart-1 that is recognized by human CD8+ T cells when presented by the MHC class I molecule HLA-A*0201. The engineered KBMA L. monocytogenes induced human DC upregulation of costimulatory molecules and secretion of pro-Th1 cytokines and type I interferons, leading to effective priming of Mart-1-specific human CD8+ T cells and lysis of patient-derived melanoma cells. KBMA L. monocytogenes expressing full-length NY-ESO-1 protein, another melanoma-associated antigen, delivered the antigen for presentation by MHC class I and class II molecules independent of the MHC haplotype of the DC donor. A mouse therapeutic tumor model was used to show that KBMA L. monocytogenes efficiently targeted APCs in vivo to induce protective antitumor responses. Together, our data demonstrate that KBMA L. monocytogenes may be a powerful platform that can both deliver recombinant antigen to DCs for presentation and provide a potent DC-maturation stimulus, making it a potential cancer vaccine candidate.

Killed but Metabolically Active Bacillus anthracis Vaccines Induce Broad and Protective Immunity against Anthrax

ABSTRACT Bacillus anthracis is the causative agent of anthrax. We have developed a novel whole-bacterial-cell anthrax vaccine utilizing B. anthracis that is killed but metabolically active (KBMA). Vaccine strains that are asporogenic and nucleotide excision repair deficient were engineered by deleting the spoIIE and uvrAB genes, rendering B. anthracis extremely sensitive to photochemical inactivation with S-59 psoralen and UV light. We also introduced point mutations into the lef and cya genes, which allowed inactive but immunogenic toxins to be produced. Photochemically inactivated vaccine strains maintained a high degree of metabolic activity and secreted protective antigen (PA), lethal factor, and edema factor. KBMA B. anthracis vaccines were avirulent in mice and induced less injection site inflammation than recombinant PA adsorbed to aluminum hydroxide gel. KBMA B. anthracis-vaccinated animals produced antibodies against numerous anthrax antigens, including high levels of anti-PA and toxin-neutralizing antibodies. Vaccination with KBMA B. anthracis fully protected mice against challenge with lethal doses of toxinogenic unencapsulated Sterne 7702 spores and rabbits against challenge with lethal pneumonic doses of fully virulent Ames strain spores. Guinea pigs vaccinated with KBMA B. anthracis were partially protected against lethal Ames spore challenge, which was comparable to vaccination with the licensed vaccine anthrax vaccine adsorbed. These data demonstrate that KBMA anthrax vaccines are well tolerated and elicit potent protective immune responses. The use of KBMA vaccines may be broadly applicable to bacterial pathogens, especially those for which the correlates of protective immunity are unknown.

Activation of immature hepatic NK cells as immunotherapy for liver metastatic disease.

NK cells can identify and eliminate emerging tumors due to altered expression of activating and inhibitory ligands on aberrant cells, a process that is greatly enhanced following NK cell activation. As a principal site of both tumor metastases and immature NK cells, the liver represents a unique anatomic location in which activation of the innate immune system could provide substantial therapeutic benefit. We describe here the NK cell-dependent destruction of a primary hepatic tumor following infection with an attenuated intracellular bacterium derived from Listeria monocytogenes. NK cell-mediated immunity correlated with the ordered migration and maturation of NK cells within the liver. Cytolytic activity was partially dependent on NKG2D-mediated tumor cell recognition, but surprisingly was still effective in the absence of type I IFN. Significantly, NK cell-mediated destruction of a primary hepatic tumor in infected mice led to long-lived CD4- and CD8 T cell-dependent tumor-specific adaptive immunity. These findings establish that activation and differentiation of immature NK cells using complex microbial stimuli can elicit potent anti-tumor activity within the liver, promote cross-presentation of tumor-derived Ags leading to long-lived systemic anti-tumor immunity, and suggests a paradigm for clinical intervention of liver metastatic carcinoma.

Abstract 4573: STINGVAX - A novel tumor vaccine with cyclic dinucleotides - can induce potent anti-tumor responses in vivo.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Cyclic dinucleotides (CDN) are ubiquitous bacterial intracellular messengers, and they have recently been shown to also function as a pathogen associated molecular pattern (PAMP) molecules that are sensed by eukaryotic host Stimulator of Interferon Genes (STING) that can activate the TBK-1/IRF-3 signaling pathway to induce type I interferon and other co-regulated genes. CDN was found to induce STING dependent augmentation of T-cell priming in multiple vaccination models, potentially by activating dendritic cells. We formulated CDN with a GM-CSF secreting tumor cell vaccine (STINGVAX) to mobilize and as well as activate dendritic cells both in vitro and in vivo. STINGVAX was tested in a stringent B16 melanoma treatment model, and we demonstrated significant reduction of tumor growth rate in vivo. STINGVAXs anti-tumor response was correlated with increased tumor infiltrating CD8 T-cells as well as increased number of p15E tumor-specific cytotoxic T-cells. STINGVAXs in vivo anti-tumor response was T-cell dependent as well as STING dependent. When we combined STINGVAX with multiple TLR 4/7/8 agonists signaling through the MyD88/TRIF pathway that is distinct from CDN/STING/TBK-1 signaling treatment of palpable B16 tumor resulted in regression of 30-60% of these non-immunogenic tumors. When the STINGVAX treated tumor microenvironment was examined, both IFNγ+CD8+ and PD-L1 was upregulated, potentially demonstrating an adaptive immune resistance mechanism which would render STINGVAX an excellent candidate to be combined with anti-PD-1 blockade. Cumulatively, STINGVAX is a novel tumor vaccine with a high potential for translation in clinical oncology. Citation Format: Thomas W. Dubensky, Meredith L. Leong, David B. Kanne, Edward E. Lemmens, Ken Metchette, Weiqun Liu, Marcella Fasso, Juan Fu, Joshua J. Woodward, Drew Pardoll, Daniel A. Portnoy, Young J. Kim. STINGVAX - A novel tumor vaccine with cyclic dinucleotides - can induce potent anti-tumor responses in vivo . [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 4573. doi:10.1158/1538-7445.AM2013-4573

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 A013: Favorable changes in tumor microenvironment following intravenous dosing with live attenuated Listeria monocytogenes-based immunotherapy

Modification of the tumor microenvironment (TME) to promote immune-mediated tumor cell destruction is considered to be an essential step for effective immunotherapy. We are evaluating recombinant live-attenuated, double deleted Listeria monocytogenes (LADD) as an immunotherapy platform for the treatment of cancer in several clinical trials in diverse indications. One LADD strain, known as CRS-207, has been engineered to express the tumor-associated antigen mesothelin and is being tested in pancreatic, ovarian and mesothelioma malignancies. Using multi-dimensional immunohistochemistry of paired biopsies from three patients with mesothelioma, we demonstrate the recruitment and expansion of effector tumor-infiltrating lymphocytes, including CD8+ T cells, mature DCs, CD163− macrophages and NK cells, following two prime infusions of CRS-207. In several different syngeneic mouse tumor models, we demonstrate that treatment with LADD engineered to express endogenous tumor antigens also induced significant changes in the TME that were consistent with changes observed in cancer patients, including enhanced CD8+ T cell effector function, recruitment of critical antigen presenting cells and reduction of regulatory T cells, and these changes correlated with significant therapeutic benefit in the mouse. LADD-induced changes to the TME were required for synergistic therapeutic antitumor efficacy combined with immune checkpoint blockade, including targeting MC38 tumor-specific neoantigens. Together, these findings demonstrate that intravenous administration of recombinant LADD therapy induces favorable changes in the tumor microenvironment in mice and humans with promise for effective outcomes in human clinical trials. Citation Format: Weiwen Deng, Takahiro Tsujikawa, Nitya Nair, Thomas Hudson, Weiqun Liu, Chris S. Rae, Edward E. Lemmens, Anthony W. Desbien, William Hanson, Peter Lauer, Lisa M. Coussens, Dirk G. Brockstedt, Thomas W. Dubensky, Jr., Meredith L. Leong. Favorable changes in tumor microenvironment following intravenous dosing with live attenuated Listeria monocytogenes-based immunotherapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A013.