Justin J. Leong

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.

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.

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 B020: STING activation in the tumor microenvironment using a synthetic human STING-activating cyclic dinucleotide induces potent antitumor immunity

Stimulator of Interferon Genes (STING) is a critical component of the cytosolic DNA sensing pathway of the innate immune system. STING is activated by cyclic dinucleotides (CDNs), a product of the intracellular enzyme, cyclic GMP-AMP synthase (cGAS), in response to presence of cytosolic DNA, including tumor-derived DNA. Production of type I interferon within the tumor microenvironment (TME), mediated by the STING pathway, leads to the priming and activation of systemic tumor antigen-specific CD8 + T-cell immunity and tumor regression. Therapeutic activation of STING through intratumoral (IT) administration of CDNs results in anti-tumor efficacy and long-lived survival in several mouse syngeneic tumor models. Rational design of synthetic CDN derivatives has shown that certain modifications alter STING binding, increase cellular potency, enhance maturation of human dendritic cells to promote in vitro T cell expansion, and are able to broadly activate all human STING haplotypes. Mechanistic studies in mouse tumor models demonstrate that CDNs mediate anti-tumor immunity by inducing an acute innate immune response, leading to collapse of the injected tumor, and promoting a tumor-specific CD8 + T cell response that protects against tumor re-challenge. Anti-tumor efficacy is enhanced by combination with immune checkpoint inhibitors, informing future clinical development. The ability to elicit innate and adaptive anti-tumor immunity via activation of STING in the TME demonstrates that CDNs have high translational potential for the treatment of patients with advanced/metastatic solid tumors. The design of an ongoing Phase 1 first-in-human clinical study to evaluate the safety, tolerability and possible antitumor activity of ADU-S100 in subjects with cutaneously accessible tumors and lymphomas will also be presented. Citation Format: Sarah M. McWhirter, Laura Hix Glickman, Tony Desbien, Kelsey Sivick Gauthier, David Kanne, Shailaja Kasibhatla, Jie Li, AnneMarie Culazzo Pferdekamper, George Katibah, Ed Lemmens, Leticia Corrales, Meredith Leong, Chudi Ndubaku, Justin Leong, Leonard Sung, Lianxing Zheng, Charles Cho, Yan Feng, Jeffery M. McKenna, John A. Tallarico, Steven L. Bender, Thomas W. Dubensky, Jr.. STING activation in the tumor microenvironment using a synthetic human STING-activating cyclic dinucleotide induces potent antitumor immunity [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 B020.

Abstract SY39-02: Direct activation of STING in the tumor microenvironment leads to potent and systemic tumor regression and immunity

Recent reports have provided the mechanistic insight of how innate immune activation promotes priming of anti-tumor immunity and inform the development of clinical approaches to facilitate this process. Spontaneous T cell infiltration of melanoma lesions in humans is correlated with a type I interferon (IFN) transcriptional profile in the tumor microenvironment (TME) and infiltration of lymphocytes, indicative of ongoing innate immune recognition within the tumor. Substantial evidence indicates that tumor infiltrating lymphocytes (TILs), including activated CD8+ T cells, is predictive of a positive clinical outcome in response to several immunotherapy strategies. Similarly, in mice bearing melanoma, there is a correlation between expression of IFN-β by tumor-resident dendritic cells (DCs), and spontaneous priming of tumor-specific immunity. Induction of IFN-β expression and co-regulated IFN-responsive genes and pro-inflammatory chemokines is dependent upon activation of the STING (Stimulator of Interferon Genes) pathway, mediated through sensing of tumor dsDNA in TME-resident CD8α+ DCs by cyclic GMP-AMP (cGAMP) synthase (cGAS), which in turn synthesizes cGAMP. The cyclic dinucleotide (CDN) cGAMP produced by cGAS is the natural STING agonist ligand. Thus, the cGAS-STING signaling axis has emerged as a central node for sensing damage in the host. We hypothesized that direct activation of the STING pathway in the TME by intratumoral (IT) injection of specific CDNs would be an effective therapeutic strategy to promote broad tumor-initiated T cell priming against an individual9s tumor antigen repertoire. There are five variant human STING alleles that exist at varying frequencies. While the natural STING ligand cGAMP activates signaling in all variants, other natural CDNs, including those produced by bacteria, have structural differences and are unable to activate particular STING variants, such as the REF (R232H) allele, informing the development of compounds that activate all human STING alleles. We sought to develop synthetic CDN compounds with increased activity in human cells as well as the ability to engage all known polymorphic human STING molecules. Using human 293T cell lines engineered to express the various STING proteins, we screened a large panel of CDN derivatives that varied in purine nucleotide base, structure of the phosphate bridge linkage, and substitution of the non-bridging oxygen atoms at the phosphate bridge with sulfur atoms. ADU-S100 is composed of two adenosine monophosphate (AMP) analogues cyclized via a 2’-5’ (non-canonical) and a 3’-5’ (canonical) phosphodiester bond, and was selected for clinical translation based on properties of enhanced cellular uptake, human STING activation, stability and anti-tumor efficacy, 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 haplotypes, including donors with a homozygous haplotype for the refractory human REF allele, indicates that ADU-S100 activates STING across a diverse human population. Direct engagement of STING through 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 provided long-lived immunologic memory. A bell-shaped ADU-S100 dose response curve (which varied based on tumor model) delineated regression of injected tumor, induction of tumor-specific CD8+ T cell immunity, and regression of distal non-injected tumors (abscopal effect), and/or protection against autologous tumor challenge. At low dose levels, regression of the treated tumor was suboptimal. At optimal doses, regression of the treated and distal untreated tumors, or protection against tumor re-challenge was observed, and correlated with induction of a robust tumor Ag-specific CD8+ T cell response. At higher dose levels there was a loss of protection against tumor re-challenge which correlated with increases in acute systemic cytokines and reduction in CD8+ T cell responses even though growth of the treated tumor was inhibited. These results suggest that the mechanism of ADU-S100-induced tumor regression is due to both an acute pro-inflammatory cytokine response and also tumor-specific CD8+ T cell immunity. The local anti-tumor effect without systemic immunity is consistent with well-established data in which excessive innate immune stimulation and induction of pro-inflammatory cytokines such as TNF-α are known to inhibit both priming of CD8+ T cell immunity and establishment of a stable and self-renewing memory CD8+ T cell population. In addition, anti-tumor efficacy was enhanced by combination with immune checkpoint inhibitors, for example α-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. A Phase 1 clinical study to evaluate the safety and tolerability and possible anti-tumor effects in subjects with cutaneously accessible non UV-induced and UV-induced malignancies or lymphomas given repeated IT doses of ADU-S100 is planned. Citation Format: Laura Hix Glickman, Leticia Corrales, David B. Kanne, Shailaja Kasibhatla, Jie Li, Anne Marie Culazzo Pferdekamper, Kelsey Sivick Gauthier, George E. Katibah, Justin J. Leong, Leonard Sung, Ken Metchette, Weiwen Deng, Anthony L. Desbien, Chudi Ndubaku, Lianxing Zheng, Charles Cho, Yan Feng, Jeffery M. McKenna, John A. Tallarico, Steven L. Bender, Sarah M. McWhirter, Thomas F. Gajewski, Thomas W. Dubensky. Direct activation of STING in the tumor microenvironment leads to potent and systemic tumor regression and 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 SY39-02.

Abstract IA10: Effective immunotherapy regimens incorporating highly active human STING-activating cyclic dinucleotide derivatives

Innate immune sensing in the tumor microenvironment is a critical step in promoting tumor infiltrating lymphocytes (TILs) and spontaneous anti-tumor T cell priming. Transcriptional profiling analysis of melanoma patients has revealed that tumors with a T cell-inflamed immunophenotype are characterized by a type I IFN (IFN) transcriptional signature. Studies in mice support the notion that IFN produced by tumor-resident dendritic cells (DCs) plays a critical role in spontaneous T cell priming against tumor antigens, which is dependent upon the host Stimulator of Interferon Genes (STING) pathway. STING mediates host innate defense by responding to cytosolic nucleic acids, either through direct binding of cyclic dinucleotides (CDNs) produced by bacteria, or through binding of a structurally distinct CDN produced by host cyclic GMP-AMP synthetase in response to cytosolic double-stranded DNA. While CDN adjuvants have been explored previously in mice, we sought to develop compounds that activate human STING. We therefore synthesized a panel of cyclic dinucleotides (CDNs) that varied by purine nucleotide base, internucleotide phosphate bridge linkage, or by substitution of non-bridging oxygen atoms in the phosphate bridge with sulfur. We screened and selected from among these compounds based on their capacity to activate all known human STING alleles expressed in stably transformed reporter cell lines, stimulate the activation of human PBMCs, and impact significant antitumor efficacy in several mouse tumor models, without significant local or systemic toxicity. Strikingly, direct IT injection of particular CDN derivative molecules into two-week established flank B16 melanoma, CT26 colon, or 4T1 breast tumors profoundly inhibited tumor growth that was durable and correlated with induction of lasting systemic antigen-specific CD8+ T cell immunity that conferred complete protection against tumor re-challenge, or significantly inhibited the growth of distal untreated tumors. Induction of cytokines, tumor antigen-specific immunity, and antitumor efficacy was entirely STING-dependent. We selected dithio-[Rp,Rp]-c[A(29,59)pA(39,59)p], a synthetic CDN molecule that has significantly higher activity than natural STING ligands, as the lead molecule for continued development. We next tested whether direct activation of STING within the tumor microenvironment would enhance the absocopal effect resulting from irradiating the CDN-treated tumor. Treatment of one tumor in mice bearing established bilateral Panc02 flank tumors, with a suboptimal dose of 10 Gy of radiotherapy (RT) in combination with IT CDN injection resulted in rapid local and systemic induction of inflammatory mediators, and vascular damage that spread through the injected tumor without causing detectable damage to normal tissues. Compared to RT alone, CDN injection resulted in significantly enhanced adaptive-immune mediated control of the contralateral tumor. The synthetic CDN molecule described here was significantly more potent than IT TLR ligands, indicating its high translational potential as an approach to elicit effective unbiased T cell priming against an individual9s unique tumor antigen repertoire. Citation Format: Laura Hix Glickman, Leticia Corrales, Sarah M. McWhirter, David B. Kanne, Kelsey E. Sivick, Jason R. Baird, Edward Lemmens, Justin J. Leong, Ken Metchette, Mark Crittenden, Michael Gough, Thomas F. Gajewski, Thomas W. Dubensky, Jr.. Effective immunotherapy regimens incorporating highly active human STING-activating cyclic dinucleotide derivatives. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr IA10.

Abstract 4272: Potent in situ cancer immunotherapy with synthetic human STING-activating cyclic dinucleotides

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Spontaneous tumor-initiated T cell priming is dependent on IFN-β production by tumor-resident dendritic cells. Based on recent observations indicating that IFN-β expression was dependent upon activation of the host STING (Stimulator of Interferon Genes) pathway, we hypothesized that direct engagement of STING through intratumoral administration of specific agonists would result in effective anti-tumor therapy. To this end, we generated novel synthetic cyclic dinucleotide (CDN) derivatives with superior STING-activating and anti-tumor properties. The most potent molecule contains a 2′-5′ and 3′-5′ phosphate bridge that increases its affinity for STING at least 10-fold. Additionally, sulfur atoms at the non-bridging oxygens of the internucleotide phosphate bridge enable both superior STING binding affinity, as well as resistance to host cell phosphodiesterases. Importantly, the lead molecule activates PBMCs from a panel of human donors representing all known STING alleles, including homozygotes for the most refractory allele. Intratumoral injection of the lead CDN molecule induced profound regression of established B16 melanoma tumors, generated substantial systemic immune responses capable of rejecting distant metastases and provided long-lived immunologic memory. Cures in entire cohorts of mice bearing well-established 4T1 mammary and CT26 colon carcinoma models correlated with induction of cytokine-mediated innate and adaptive antigen-specific T cell immunity, both systemically and in the tumor microenvironment. Immune activation and anti-tumor efficacy was lost in mice encoding non-functional STING. In vivo activation of STING with the lead CDN administered by multiple routes demonstrated a high safety and tolerability margin in preclinical toxicology studies. In addition, pharmacokinetic analysis indicates that the lead CDN compound was rapidly cleared from systemic circulation, consistent with pulsatile STING activation and transient systemic cytokine and hematological changes. These results demonstrate that CDNs have high translation potential for treatment strategies to induce activation of the tumor microenvironment, leading to effective tumor-initiated T cell priming and anti-tumor immunity. Citation Format: Laura Hix Glickman, David B. Kanne, Kelsey E. Gauthier, George E. Katibah, Justin J. Leong, Ken Metchette, Thomas W. Dubensky, Sarah M. McWhirter. Potent in situ cancer immunotherapy with synthetic human STING-activating cyclic dinucleotides. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4272. doi:10.1158/1538-7445.AM2015-4272