David B. Kanne

Direct Activation of STING in the Tumor Microenvironment Leads to Potent and Systemic Tumor Regression and Immunity

Spontaneous tumor-initiated T cell priming is dependent on IFN-β production by tumor-resident dendritic cells. On the basis of recent observations indicating that IFN-β expression was dependent upon activation of the host STING pathway, we hypothesized that direct engagement of STING through intratumoral (IT) administration of specific agonists would result in effective anti-tumor therapy. After proof-of-principle studies using the mouse STING agonist DMXAA showed a potent therapeutic effect, we generated synthetic cyclic dinucleotide (CDN) derivatives that activated all human STING alleles as well as murine STING. IT injection of STING agonists induced profound regression of established tumors in mice and generated substantial systemic immune responses capable of rejecting distant metastases and providing long-lived immunologic memory. Synthetic CDNs have high translational potential as a cancer therapeutic.

The reaction of the psoralens with deoxyribonucleic acid

Psoralen photochemistry is specific for nucleic acids and is better understood at the molecular level than are all other methods of chemical modification of nucleic acids. These compounds are used both for in vivo structure analysis and for photochemotherapy since they easily penetrate both cells and virus particles. Apparently, natural selection has selected for membrane and virus penetrability during the evolution of these natural products. Most cells are unaffected by relatively high concentrations of psoralens in the absence of ultraviolet light, and the metabolites of the psoralens have thus far not created a problem. Finally, psoralens form both monoadduct and cross-links in nucleic acid helices, the yield of each being easily controlled by the conditions used during the photochemistry.

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.

Structure of a psoralen-thymine monoadduct formed in photoreaction with DNA☆

Abstract A photoreaction product between calf thymus DNA and 8-methoxypsoralen, a naturally occurring psoralen, was hydrolyzed with acid to isolate a major species of the reaction, 8-methoxypsoralen-thymine monoadduct. The crystal structure of the monoadduct has been determined. The structure confirms the results of other spectroscopic studies and allows one to conclude or suggest that: (1) the photoreaction occurs preferentially at both T-A and A-T sequences in natural DNA; (2) the photocrosslink introduces a substantial kink in the DNA structure; (3) the configuration of the photoproduct is “cis-syn” at both ends of the psoralen; (4) the thymine and psoralen moieties remain planar even after the photoreaction; and (5) the angle between psoralen and thymine in the photoproducts is not fixed but has a limited range of flexibility.

Radiotherapy Combined with Novel STING-Targeting Oligonucleotides Results in Regression of Established Tumors

Cytotoxic therapies prime adaptive immune responses to cancer by stimulating the release of tumor-associated antigens. However, the tumor microenvironment into which these antigens are released is typically immunosuppressed, blunting the ability to initiate immune responses. Recently, activation of the DNA sensor molecule STING by cyclic dinucleotides was shown to stimulate infection-related inflammatory pathways in tumors. In this study, we report that the inflammatory pathways activated by STING ligands generate a powerful adjuvant activity for enhancing adaptive immune responses to tumor antigens released by radiotherapy. In a murine model of pancreatic cancer, we showed that combining CT-guided radiotherapy with a novel ligand of murine and human STING could synergize to control local and distant tumors. Mechanistic investigations revealed T-cell-independent and TNFα-dependent hemorrhagic necrosis at early times, followed by later CD8 T-cell-dependent control of residual disease. Clinically, STING was found to be expressed extensively in human pancreatic tumor and stromal cells. Our findings suggest that this novel STING ligand could offer a potent adjuvant for leveraging radiotherapeutic management of pancreatic cancer.

Rationale, progress and development of vaccines utilizing STING-activating cyclic dinucleotide adjuvants

A principal barrier to the development of effective vaccines is the availability of adjuvants and formulations that can elicit both effector and long-lived memory CD4 and CD8 T cells. Cellular immunity is the presumptive immune correlate of protection against intracellular pathogens: a group composed of bacteria, viruses and protozoans that is responsible for a staggering level of morbidity and mortality on a global scale. T-cell immunity is also correlated with clinical benefit in cancer, and the development of therapeutic strategies to harness the immune system to treat diverse malignancies is currently undergoing a renaissance. Cyclic dinucleotides (CDNs) are ubiquitous small molecule second messengers synthesized by bacteria that regulate diverse processes and are a relatively new class of adjuvants that have been shown to increase vaccine potency. CDNs activate innate immunity by directly binding the endoplasmic reticulum-resident receptor STING (stimulator of interferon genes), activating a signaling pathway that induces the expression of interferon-β (IFN-β) and also nuclear factor-κB (NF-κB) dependent inflammatory cytokines. The STING signaling pathway has emerged as a central Toll-like receptor (TLR) independent mediator of host innate defense in response to sensing cytosolic nucleic acids, either through direct binding of CDNs secreted by bacteria, or, as shown recently, through binding of a structurally distinct CDN produced by a host cell receptor in response to binding cytosolic double-stranded (ds)DNA. Although this relatively new class of adjuvants has to date only been evaluated in mice, newly available CDN-STING cocrystal structures will likely intensify efforts in this field towards further development and evaluation in human trials both in preventive vaccine and immunotherapy settings.

A STING Agonist Given with OX40 Receptor and PD-L1 Modulators Primes Immunity and Reduces Tumor Growth in Tolerized Mice

The efficacy and immune dynamics of STING modulation in the toleragenic tumor microenvironment were examined. Combining a STING agonist, PD-L1 blockade, and OX40R stimulation created an inflamed tumor microenvironment that recruited T cells and activated tumor-specific immunity. Stimulator of interferon genes (STING) signaling induces IFNβ production by intratumoral dendritic cells (DC), driving T-cell priming and recruitment into the tumor microenvironment (TME). We examined to what extent preexisting antigen-specific tolerance influenced the efficacy of in situ delivery of a potent STING-activating cyclic dinucleotide (CDN), ADU S-100, against established HER-2+ breast tumors. ADU S-100 induced HER-2–specific CD8+ T-cell priming and durable tumor clearance in 100% of nontolerant parental FVB/N mice. In contrast, ADU S-100 did not sufficiently prime HER-2–specific CD8+ T cells in tolerant neu/N mice, resulting in only delayed tumor growth and tumor clearance in 10% of the mice. No differences in IFNβ production, DC priming, or HER-2–specific CD8+ T-cell trafficking were detected between FVB/N and neu/N mice. However, activation and expansion of HER-2–specific CD8+ T cells were defective in neu/N mice. Immune cell infiltrates of untreated tumor-bearing neu/N mice expressed high numbers of PD1 and OX40 receptors on their CD8+ T cells, and PD-L1 was highly expressed on both myeloid and tumor cells. Modulating PD-L1 and OX40 receptor signaling combined with intratumoral ADU S-100 administration enhanced HER-2–specific CD8+ T-cell activity, clearing tumors in 40% of neu/N mice. Thus, intratumoral STING agonists could potently prime tumor antigen–specific CD8+ T-cell responses, and adding PD-L1 blockade and OX40 receptor activation can overcome antigen-enforced immune tolerance to induce tumor regression. Cancer Immunol Res; 5(6); 468–79. ©2017 AACR.

Modified STING-activating cyclic dinucleotide derivatives significantly enhance the anti-tumor activity of therapeutic vaccines

The STING signaling pathway has emerged as a central TLR-independent mediator of host innate defense in response to sensing cytosolic nucleic acids, either through direct binding of CDNs secreted by intracellular bacteria, or through binding of a structurally distinct CDN produced by a host cell receptor, cyclic GMP-AMP (cGAMP) synthase (cGAS), in response to binding cytosolic double-stranded DNA. Binding of CDNs to STING initiates a signaling cascade through the TBK-1/IRF-3 axis to induce type I interferon (IFN) and other co-regulated genes. Discovered recently, CDNs synthesized by cGAS and bacteria are structurally distinct. The cGAS product, termed non-canonical CDN, has a phosphate bridge with both 2’-5’ and 3’-5’ linkages that reportedly increases its affinity for STING by 200-fold. Here we show in a panel of human donors that the non-canonical linkage confers significantly increased activity of CDNs to activate PBMCs, as measured by expression of IFN and NF-κB dependent cytokines, as compared to CDNs with canonical 3’-5’ phosphate linkages. By conducting Immunogenicity-Structure Activity Relationship studies in vitro and in various animal models of infection and cancer, we selected synthetic derivatives of native CDNs produced by bacteria or eukaryotic cells with increased potency. CDN derivatives with sulfur atoms at non-bridging oxygens of the internucleotide phosphate bridge were resistant to digestion with phosphodiesterase. Surprisingly, R,R di-thio CDN diastereomers induced higher levels of IFN in vitro, and induced a higher magnitude of peak and memory antigen-specific CD4 and CD8 T cell responses correlated with protective immunity, as compared to either the R,S di-thio CDN diastereomer, or di-oxo CDN. CDNs based on adenosine nucleotides were comparatively independent of host cell permeabilization to activate STING signaling. We applied this finding to enhance the activity of immunotherapy regimens utilizing irradiated GM-CSF expressing tumor cell vaccines. Co-formulation with R,R dithio-c-di-AMP significantly inhibited tumor growth in several models, correlated with increased mobilization and activation of dendritic cells, increased tumor lymphocyte infiltration, and T cell immunity. Immunologic potency of CDNs was essentially lost in mice encoding a non-functional STING allele. Collectively, CDNs have high translation potential for diverse applications in clinical oncology.

Abstract 1445: STING activation in the tumor microenvironment with a synthetic human STING-activating cyclic dinucleotide leads to potent anti-tumor immunity

Stimulator of Interferon Genes (STING) is a critical signaling sensor of the innate immune system. STING binds cyclic dinucleotides (CDN) produced by an intracellular enzyme in response to presence of intracellular DNA, including tumor-derived DNA. STING-mediated production of host type I interferon within the tumor microenvironment (TME) leads to the priming and activation of systemic tumor antigen-specific CD8+ T-cell immunity and tumor regression. A novel synthetic CDN derivative (ADU-S100), with superior STING-activating and anti-tumor properties, was developed for clinical translation. ADU-S100 has enhanced cellular uptake properties and stability, as compared to bacterial- and mammalian-derived CDNs. Induced cytokine expression from a panel of donor human peripheral blood mononuclear cells (PBMCs) expressing a variety of STING alleles, including a homozygous haplotype for the most refractory human allele (R232H), indicate that ADU-S100 activates STING across a diverse human population. Direct engagement of STING through intratumoral (IT) administration of ADU-S100 results in effective anti-tumor therapy and long-term survival in various mouse syngeneic tumor models. IT injection of ADU-S100 also generates substantial systemic immune responses capable of rejecting distant metastases and provides long-lived immunologic memory. Mechanistic studies demonstrate that STING-mediated anti-tumor immunity is due in part to an acute pro-inflammatory cytokine response as well as a tumor-specific CD8+ T cell response. Anti-tumor efficacy is enhanced by combination with immune checkpoint inhibitors, for example anti-PD1, informing future clinical development. By virtue of the ability to elicit innate and T cell-mediated anti-tumor immunity in the TME, these results demonstrate that CDNs have high translational potential for the treatment of patients with advanced/metastatic solid tumors. Citation Format: Laura Hix Glickman, David B. Kanne, Shailaja Kasibhatla, Jie Li, AnneMarie Culazzo Pferdekamper, Kelsey Sivick Gauthier, Weiwen Deng, Anthony L. Desbien, George E. Katibah, Justin J. Leong, Leonard Sung, Ken Metchette, Chudi Ndubaku, Lianxing Zheng, Charles Cho, Yan Feng, Jeffrey M. McKenna, John A. Tallarico, Steven L. Bender, Thomas W. Dubensky, Sarah M. McWhirter. STING activation in the tumor microenvironment with a synthetic human STING-activating cyclic dinucleotide leads to potent anti-tumor immunity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1445.

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