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Supplemental Figure 2: STING agonists stimulate cytokines in tumor, plasma and peripheral tissues following IT dosing.

Supplementary Methods and Figures

Supplemental Figure 3: MC38 tumor growth in C57BL/6, Nude or NSG mice and comparison of systemic cytokines

Supplemental Figure 1: Day 10 tumor volume of injected (InjT) and noninjected (Non-injT) tumors following intratumoral (IT) injection with MSA-1

Supplemental Table 1: Log2Ratio and p values for all 310 genes shown in Figure 6

Stereochemically and structurally complex cyclic dinucleotide-based stimulator of interferon genes (STING) agonists were designed and synthesized to access a previously unexplored chemical space. The assessment of biochemical affinity and cellular potency, along with computational, structural, and biophysical characterization, was applied to influence the design and optimization of novel STING agonists, resulting in the discovery of MK-1454 as a molecule with appropriate properties for clinical development. When administered intratumorally to immune-competent mice-bearing syngeneic tumors, MK-1454 exhibited robust tumor cytokine upregulation and effective antitumor activity. Tumor shrinkage in mouse models that are intrinsically resistant to single-agent therapy was further enhanced when treating the animals with MK-1454 in combination with a fully murinized antimouse PD-1 antibody, mDX400. These data support the development of STING agonists in combination with pembrolizumab (humanized anti-PD-1 antibody) for patients with tumors that are partially responsive or nonresponsive to single-agent anti-PD-1 therapy.

The innate immune agonist STING (STimulator of INterferon Genes) binds its natural ligand 2′3′-cGAMP (cyclic guanosine-adenosine monophosphate) and initiates type I IFN production. This promotes systemic antigen-specific CD8+ T-cell priming that eventually provides potent antitumor activity. To exploit this mechanism, we synthesized a novel STING agonist, MSA-1, that activates both mouse and human STING with higher in vitro potency than cGAMP. Following intratumoral administration of MSA-1 to a panel of syngeneic mouse tumors on immune-competent mice, cytokine upregulation and its exposure were detected in plasma, other tissues, injected tumors, and noninjected tumors. This was accompanied by effective antitumor activity. Mechanistic studies in immune-deficient mice suggested that antitumor activity of intratumorally dosed STING agonists is in part due to necrosis and/or innate immune responses such as TNF-α activity, but development of a robust adaptive antitumor immunity is necessary for complete tumor elimination. Combination with PD-1 blockade in anti–PD-1–resistant murine models showed that MSA-1 may synergize with checkpoint inhibitors but can also provide superior tumor control as a single agent. We show for the first time that potent cyclic dinucleotides can promote a rapid and stronger induction of the same genes eventually regulated by PD-1 blockade. This may have contributed to the relatively early tumor control observed with MSA-1. Taken together, these data strongly support the development of STING agonists as therapy for patients with aggressive tumors that are partially responsive or nonresponsive to single-agent anti–PD-1 treatment by enhancing the anti–PD-1 immune profile.

Pharmacological activation of the STING (stimulator of interferon genes)-controlled innate immune pathway is a promising therapeutic strategy for cancer. Here we report the identification of MSA-2, an orally available non-nucleotide human STING agonist. In syngeneic mouse tumor models, subcutaneous and oral MSA-2 regimens were well tolerated and stimulated interferon-β secretion in tumors, induced tumor regression with durable antitumor immunity, and synergized with anti-PD-1 therapy. Experimental and theoretical analyses showed that MSA-2 exists as interconverting monomers and dimers in solution, but only dimers bind and activate STING. This model was validated by using synthetic covalent MSA-2 dimers, which were potent agonists. Cellular potency of MSA-2 increased upon extracellular acidification, which mimics the tumor microenvironment. These properties appear to underpin the favorable activity and tolerability profiles of effective systemic administration of MSA-2.

Activated STING (STimulator of INterferon Genes) bound to its natural ligand 2,3-cGAMP (cyclic guanosine-adenosine monophosphate), initiates type I interferon (IFN) and pro-inflammatory cytokine production. IFN upregulation is essential to promote antigen-specific CD8+ T-cell priming and leads to potent anti-tumor activity. To exploit this mechanism we synthesized a new STING agonist, MSA-1, that potently activates both mouse and human STING. Intratumoral (IT) administration of MSA-1 to MC38 syngeneic tumor-bearing mice increased tumor and plasma cytokine levels and was effective at driving complete responses (CRs) in 100% of the animals. Most surviving animals developed tumor-specific adaptive immune memory as demonstrated by robust protection against re-challenge with the same tumor type. Mechanistic studies in immune-deficient mice suggested that the initial antitumor activity is in part due to cytokine-driven cytotoxicity and/or other innate immune mechanisms, which may have contributed to some animals not developing an adaptive immune memory. Importantly, MSA-1 caused long-term tumor regressions or CRs in CT26 and B16-F10 tumor models, both of which are intrinsically resistant to single-agent therapy with a fully murinized anti-mouse PD-1 antibody (muDX400). The antitumor immune response in these models was further enhanced when treating the animals with MSA-1 in combination with muDX400. This combination restored T-cell responses in both blood and tumors of the treated mice and provided long-lived immunologic memory in a majority of the animals. Taken together, these data strongly support the development of STING agonists in combination with Keytruda (humanized anti-PD-1 antibody) for patients with tumors that are partially responsive or nonresponsive to single agent anti-PD-1 therapy. Citation Format: Samanthi A. Perera, Johnny E. Kopinja, Yanhong Ma, Jason Laskey, Kalyan Chakravarthy, Long Cui, Yiping Chen, Jeremy Presland, Sharad Sharma, Shuxia Zhao, Jennifer Piesvaux, Ellen C. Minnihan, Heidi Ferguson, Hyun Chong Woo, Ian Knemeyer, Ilona Kariv, Archie Tse, Saso Cemerski, Jared Cumming, B. Wesley Trotter, Bo-Sheng Pan, George H. Addona, Brian J. Long. Combination with a novel STING agonist significantly improves efficacy of anti-PD1 therapy in mouse syngeneic tumor models [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A08.

The innate immune agonist STING (STimulator of INterferon Genes) binds its natural ligand 2'3'-cGAMP (cyclic guanosine-adenosine monophosphate) and initiates type I interferon production. This promotes systemic antigen-specific CD8+ T-cell priming that eventually provides potent anti-tumor activity. To exploit this mechanism we synthesized a novel STING agonist, MSA-1, that activates both mouse and human STING with higher in vitro potency than cGAMP. MSA-1 was administered to immune-competent mice bearing MC38 syngeneic tumors to monitor pharmacodynamics, pharmacokinetics and in vivo efficacy. Intratumoral (IT) dosed MSA-1 demonstrated robust tumor and plasma cytokine upregulation and effective anti-tumor activity. The highest tolerated doses provided complete responses (CRs) in 100% of MC38 tumors. Tumor models such as CT26 and B16-F10 that are intrinsically resistant to single-agent therapy with a fully murinized mouse anti-PD-1 antibody (mDX400) also demonstrated long-term tumor regressions or CRs. Mechanistic studies in immune-deficient mice suggested that anti-tumor activity of IT dosed STING agonists are in part due to cytotoxicity and/or innate immune responses rather than development of robust adaptive anti-tumor immunity. To enhance the adaptive immune response, we combined MSA-1 with mDX400 in mouse syngeneic tumor models previously characterized to be unresponsive to anti-PD-1 blockade. This combination restored T-cell responses in both blood and tumors of the treated mice and provided long-lived immunologic memory in a majority of the animals. Taken together, these data strongly support the development of STING agonists in combination with Keytruda for patients with tumors that are partially responsive or non-responsive to single agent anti-PD-1 therapy. Citation Format: Samanthi A. Perera, Johnny E. Kopinja, Yanhong Ma, Jason Laskey, Kalyan Chakravarthy, Yiping Chen, Long Cui, Jeremy Presland, Shuxia Zhao, Ellen Minnihan, Heidi Ferguson, Jennifer Piesvaux, Bo-Sheng Pan, Hyun Chong Woo, Ian Knemeyer, Saso Cemerski, Jared Cumming, Wesley Trotter, Archie Tse, George H. Addona, Brian J. Long. Combining STING agonists with an anti-PD-1 antagonist results in marked antitumor activity in immune-excluded tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4721.

Polycythemia vera (PV) is a myeloproliferative disorder characterized by increased red cell mass and splenomegaly in the absence of secondary causes [Tefferi A., Spivak J.L., Polycythemia vera: scientific advances and current practice. Semin Hematol 2005;42(4):206-20.]. Recently, several laboratories have discovered that the vast majority of patients with PV carry a single, activating mutation (V617F) in the pseudokinase domain of Janus kinase 2 (Jak2) [Zhao R, Xing S, Li Z, Fu X, Li Q, Krantz SB, et al., Identification of an acquired JAK2 mutation in polycythemia vera. J Biol Chem 2005;280(24):22788-92; James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Lacout C, et al., A unique clonal JAK2 mutation leading to constitutive signalling causes polycythemia vera. Nature 2005;434(7037):1144-8; Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR, et al., A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005;352(17):1779-90; Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ, et al., Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 2005;7(4):387-97.]. This discovery has spurred interest in developing therapies for PV via inhibition of Jak2. We induced polycythemia in mice by administering high dose recombinant erythropoietin (Epo) and determined that administration recapitulates almost all of the major and minor diagnostic features of human PV. We then tested a selective, small molecule inhibitor of Jak2 (Jak2i) and showed that this treatment prevents polycythemia. This prevention of polycythemia was accompanied by lower hematocrits, reduced spleen sizes and reductions in Stat5 phosphorylation (pStat5). Surprisingly, Epo rapidly (

A high percentage of patients with the myeloproliferative disorder polycythemia vera (PV) harbor a Val617→Phe activating mutation in the Janus kinase 2 (JAK2) gene, and both cell culture and mouse models have established a functional role for this mutation in the development of this disease. We describe the properties of MRLB-11055, a highly potent inhibitor of both the WT and V617F forms of JAK2, that has therapeutic efficacy in erythropoietin (EPO)-driven and JAK2V617F-driven mouse models of PV. In cultured cells, MRLB-11055 blocked proliferation and induced apoptosis in a manner consistent with JAK2 pathway inhibition. MRLB-11055 effectively prevented EPO-induced STAT5 activation in the peripheral blood of acutely dosed mice, and could prevent EPO-induced splenomegaly and erythrocytosis in chronically dosed mice. In a bone marrow reconstituted JAK2V617F-luciferase murine PV model, MRLB-11055 rapidly reduced the burden of JAK2V617F-expressing cells from both the spleen and the bone marrow. Using real-time in vivo imaging, we examined the kinetics of disease regression and resurgence, enabling the development of an intermittent dosing schedule that achieved significant reductions in both erythroid and myeloid populations with minimal impact on lymphoid cells. Our studies provide a rationale for the use of non-continuous treatment to provide optimal therapy for PV patients.

Nat. Genet 33, 401–406 (2003); published online 18 February 2003; corrected after print 14 May 2007 The authors wish to remove panels b, d and e of Figure 2 because they were falsified or fabricated. Figure 2 shows the functional silencing of genes in primary cells by lentiviral-mediated RNAi. The authors showed that primary activated T cells can be successfully transduced by lentivirus and that RNAi-targeted surface markers are downregulated in transduced cells (Fig.