SR-717

Antitumor activity of a systemic STING-activating non-nucleotide cGAMP mimetic
Emily N. Chin1, Chenguang Yu1,2*, Vincent F. Vartabedian3*, Ying Jia1,3*, Manoj Kumar2, Ana M. Gamo2, William Vernier2, Sabrina H. Ali1, Mildred Kissai1, Daniel C. Lazar3, Nhan Nguyen3, Laura E. Pereira1, Brent Benish2, Ashley K. Woods2, Sean B. Joseph2, Alan Chu2, Kristen A. Johnson2,
Philipp N. Sander1, Francisco Martínez-Peña1, Eric N. Hampton2, Travis S. Young2, Dennis W. Wolan4, Arnab K. Chatterjee2, Peter G. Schultz1,2, H. Michael Petrassi2†, John R. Teijaro3†, Luke L. Lairson1†

Stimulator of interferon genes (STING) links innate immunity to biological processes ranging from antitumor immunity to microbiome homeostasis. Mechanistic understanding of the anticancer potential for STING receptor activation is currently limited by metabolic instability of the natural cyclic dinucleotide (CDN) ligands. From a pathway-targeted cell-based screen, we identified a non-nucleotide, small-molecule STING agonist, termed SR-717, that demonstrates broad interspecies and interallelic specificity. A 1.8-angstrom cocrystal structure revealed
that SR-717 functions as a direct cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) mimetic that induces the same “closed” conformation of STING. SR-717 displayed antitumor activity; promoted the activation of CD8+ T, natural killer, and dendritic cells in relevant tissues; and facilitated antigen cross-priming. SR-717 also induced the expression
of clinically relevant targets, including programmed cell death 1 ligand 1 (PD-L1), in a STING- dependent manner.
T
(ISG-THP1 cGAS KO) cell lines (fig. S1) to determine pathway specificity and potential target identity, respectively. Protein thermal shift assays, involving recombinant human and mouse STING protein (hSTING and mSTING), were used to profile compounds for direct on-target binding activity, cross- species activity, and human allele specificity. This resulted in the identification of SR-001 (Fig. 1A), which was found to robustly induce reporter signal in ISG-THP1 cells [fig. S2A; mean effective concentration (EC50) ~1.1 mM] and ISG-THP1 cGAS KO cells (fig. S2B) but was completely inactive in ISG-THP1 STING KO cells (fig. S2, A to C), suggesting that the com- pound acts downstream of cGAS with a de- pendence on STING expression. Commercial SR-001 was also found to increase the ther- mal stability of the soluble C-terminal CDN- binding domain of recombinant human STING protein (hSTINGREF) (fig. S2E), which is re- sponsible for recruiting downstream signaling proteins. Chemical resynthesis of SR-001 af- forded a compound that was consistently active in cell-based assays (fig. S2D) but was now devoid of activity in the STING thermal

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he cyclic guanosine monophosphate (GMP)–adenosine monophosphate (AMP) synthase (cGAS)–stimulator of inter- feron genes (STING) (cGAS-STING) sig- naling pathway plays a critical role in the innate response to infection (1, 2). It also serves as a direct link between inflammation and diverse physiological processes, includ- ing micronuclei surveillance in the context of DNA damage (3, 4), age-associated in- flammation (5), mitochondrial DNA–related inflammatory phenotypes (6), and microbiome- dependent intestinal homeostasis (7). STING is an endoplasmic reticulum signaling protein, partially localized to mitochondria-associated membranes, that is broadly expressed in both immune and nonimmune cell types. STING binds cyclic dinucleotides (CDNs)—including 2′,3′–cyclic GMP-AMP (2′,3′-cGAMP) produced by cGAS in response to cytosolic DNA (8)— and the scaffolding function rapidly induces type I interferon (IFN) and proinflamma- tory cytokines in a TBK1–IRF3-dependent fashion (9, 10). STING has been demonstrated to play essential roles in antitumor immu- nity. For example, efficient tumor-initiated T cell activation requires STING pathway–

1Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. 2California Institute for Biomedical Research, 11119 North Torrey Pines Road, La Jolla, CA 92037, USA. 3Department of Immunology and Microbiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. 4Department of Molecular Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
*These authors contributed equally to this work.
†Corresponding author. Email: [email protected] (H.M.P.); [email protected] (J.R.T.); [email protected] (L.L.L.)
dependent IFN-b expression, as well as ex- pression of STING in dendritic cells (DCs) (11, 12).
Initial STING agonist small molecules were synthesized as derivatives of the CDN natural ligand; however, because of poor stability prop- erties, CDN-based agonist administration is limited to intratumoral delivery. Although intratumoral delivery of CDN agonists has consistently shown regression of established tumors in syngeneic models (13, 14), intra- tumor CDN administration in humans has been met with mixed success. Activation of the STING pathway has also been demon- strated to contribute notably to the antitumor effect of radiation and chemotherapeutics (4, 15, 16). As such, a systemic STING-activating agent has considerable potential utility, not only as a therapeutic for cancer and infec- tious disease but also as a pharmacological probe to enable mechanistic discovery in the context of STING-dependent antitumor im- munity and diverse STING-dependent bio- logical processes.
We used a cGAS-STING pathway–targeted cell-based phenotypic screening approach to identify functional non-nucleotide small- molecule STING agonists. Human monocytic THP-1 cells, harboring an interferon regulatory factor (IRF)–inducible luciferase reporter con- struct (ISG-THP1), were used to screen a col- lection of ~100,000 commercially available structurally diverse druglike small molecules in 1536-well plate format [5 mM, 0.1% dimethyl sulfoxide (DMSO)]. Confirmed primary hits (plate-based z score >3) were evaluated in secondary assays involving STING knockout (ISG-THP1 STING-KO) or cGAS knockout
shift binding assay (fig. S2E). Analytical char- acterization of the commercial material re- vealed the presence of a minor but notable amount of the de-esterified derivative SR- 012 (Fig. 1A). This suggested that SR-001 was acting as a prodrug, with ester substi- tution being a requisite for cell permeabil- ity and the active STING-binding species being the carboxylic acid.
Consistent with this hypothesis, synthetic SR-012 was found to bind both recombinant hSTING and mSTING protein (fig. S2E) but was inactive in cell-based assays (fig. S2D). SR-001 was also observed to be rapidly con- verted to SR-012 in cells (fig. S2F). To address the cell permeability of SR-012 and intrac- table rodent exposure properties of the SR-001 prodrug, we completed a medicinal chem- istry effort focused on improved prodrug stability or bioisosteric replacement of the carboxylic acid or improved cell permeability for the carboxylic acid. This resulted in the identification of a carboxylic acid–containing analog, containing difluoro-substitution of the aniline ring system (SR-717; Fig. 1B), which was found to possess equivalent cell- based activity when compared with SR-001 prodrug (Fig. 1C; ISG-THP1, EC50 = 2.1 mM; ISG-THP1 cGAS KO, EC50 = 2.2 mM; ISG-
THP1 STING KO, no activity up to the limit of solubility). Critically, SR-717 increased the thermal stability of the common human al- leles of hSTING (Fig. 1D and fig. S3A), as well as that of recombinant soluble mSTING pro- tein (Fig. 1D), suggesting that the binding mode of SR-717 is not affected by interallelic or interspecies differences in amino acid sequence. This latter issue was ultimately

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Fig. 1. Discovery and profile of small-molecule STING agonist SR-717.
(A) Structure of prodrug screening hit SR-001 and elucidated STING agonist SR-012. Me, methyl. (B) Structure of optimized STING agonist SR-717.
(C) Cell-based activity of SR-717 in ISG-THP1 (WT), ISG-THP1 cGAS KO (cGAS KO), and ISG-THP1 STING KO (STING KO) cell lines was quantified by normalizing SR-717–induced reporter activity to DMSO-treated reporter activity for each
cell and reported as relative light units (RLU). (D) Impact of SR-717 (100 mM) or cGAMP (100 mM) on the melting temperature (DTm) of recombinant common human alleles of STING protein (hSTING), as well as recombinant mSTING. REF, R232H; AQ, G230A, R293Q; Q, R293Q; d(Fluor)/dT, first derivative of
fluorescence melt curve. (E) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of time-dependent target gene expression in THP1 cells treated with SR-717 (EC80 = 3.6 mM) normalized to DMSO-treated
control. (F) qRT-PCR analysis of time-dependent target gene expression in freshly isolated human PBMCs treated with SR-717 (10 mM) normalized to DMSO-treated control. (G) Western blot analysis of the kinetics of activation of the cGAS-STING and FN-a/b receptor (IFNAR) signaling pathways in THP1 cells stimulated with SR-717 (EC80 = 3.6 mM). g-Tubulin was used as a loading control. Data are representative of three independent experiments [(C) to (G)], and values are
the mean of three replicates ± SD [(C) to (F)].

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responsible for the clinical failure of 5,6- dimethylxanthone-4-acetic acid (DMXAA), a vascular disrupting agent that can function as a murine STING agonist but fails to bind to the human form of the protein (17, 18). Characterization of SR-717 in a homogeneous time-resolved fluorescence-based cGAMP competition assay, involving wild-type (WT) human STING protein, revealed that SR-717 binds to STING with an apparent affinity [mean inhibitory concentration (IC50)= 7.8 mM; fig. S3B] that is comparable to its observed cell-based potency. Compound washout ex- periments revealed that a robust induction of reporter signal is achieved within 1 hour of exposure (fig. S3C). Further, SR-717 was found to possess mouse pharmacokinetic properties that would enable its in vivo characterization in the context of systemic administration (fig. S4A). Cell-free profiling of SR-717 at a concen-

tration of 5 mM against a panel of 468 kinases revealed the compound to be exceptionally selective, with only a single kinase (serine/ threonine kinase PIM3) being identified as an interacting partner (fig. S5; percent of control < 35). Kinetic characterization of the cell-based activity of SR-717, at the calculated EC80 concentration (3.6 mM), revealed that interferon-stimulated response element (ISRE)– luciferase reporter activity translated to ac- tivation of target gene expression. Specifically, SR-717 stimulates IFNB1 expression within 1 hour of incubation and achieves maximal activation by 2 hours of treatment, whereas activation of CXCL10, a transcript activated by both IRF3 and IFN-b signaling, peaks at
6 hours (Fig. 1E). Importantly, these ki- netics of activation are maintained in primary human peripheral blood mononuclear cells (PBMCs) (Fig. 1F). On-target pathway acti-

vation by SR-717 was further validated by analyzing the phosphorylation of TBK1, IRF3, and p65 signal transduction proteins (Fig. 1G), which are downstream of STING, as well as the secondary activation of STAT1 and STAT3 (Fig. 1G), which are downstream of IFN-b and IL-6 signaling, respectively. Further, inhibition of the downstream effector TBK1, using small molecule inhibitor BX795, was observed to block the induction of reporter signal (fig. S6, A and B) and downstream IRF-3 and secondary STAT1 phosphorylation events (fig. S6C) by SR-717.
The soluble cytosolic C-terminal region of hSTING exists in solution as a dimer and binds to naturally occurring and synthetic CDNs with varying degrees of affinity. X-ray crystallographic analysis of this CDN-binding domain has revealed that STING exists as a closely associated dimer that takes the shape

of a “pair of wings,” with an approximate antiparallel four-helix bundle making up an extensive buried dimer interface (19, 20). The solved cocrystal structures of cGAMP bound to hSTING revealed that cGAMP binds in a deep cleft at the dimer interface and causes a substantial conformational change, which leads to the formation of an antiparallel four- strand b-sheet overhead cap element that serves to completely envelop the CDN [fig. S7A, (18, 21)]. In the cGAMP-bound “closed” confor- mation, the distance between the tips of the symmetry-related a2 helices is substantially re- duced when compared with the unbound pro- tein structure [fig. S7B; Protein Data Bank (PDB) ID 4F9E (19)]. By contrast, binding of
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di-GMP CDN induces an increase in the angle and distance between the tips of the a2 heli- ces, leading to the formation of an “open” con- formation that lacks the b-sheet overhead cap element [fig. S7C; PDB ID 4F9G (19)]. Mech- anistic and structural studies involving cGAMP and DMXAA (18, 21) suggested that ligands that induce the closed conformation lead to STING activation. Interestingly, diABZI-2 was recently described as eliciting the phosphoryl-
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ation of IRF3 and downstream target expres-
sion leading to antitumor immunity by binding to STING and inducing an open conformation akin to that induced by di-GMP [fig. S7D; PDB ID 6DXL (22)]. It is intriguing that CDNs pro- duced directly by bacteria (i.e., di-GMP) can activate STING by stabilizing an open con- formation that is so substantially different to that of the closed conformation, which is in- duced by the endogenously produced second- ary message of cytosolic double-stranded DNA (i.e., cGAMP). The biological consequences of differing CDN-induced conformation-dependent
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The 1.8-Å cocrystal structure of SR-717 bound to hSTINGR232 (residues 155 to 341) is shown in Fig. 2A (supporting crystallography statis- tics are shown in table S1), with simulated an-

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neal omit maps of unbiased electron density for bound ligands shown in fig. S8A. Binding of two molecules of SR-717 at the base of the STING dimer intersubunit cleft closely mimics the binding mode of cGAMP. It induces the same closed conformation (Fig. 2B and fig. S7A), involving a near identical characteristic reduc- tion in distance between the tips of the a2 helices (Fig. 2C and fig. S7A), as well as the formation of the four-strand b-sheet overhead cap element (Fig. 2, A, B, and D; and fig. S7A). As occurs with both purine bases of bound cGAMP (18, 21), the pyridazine ring of each molecule of SR-717 is “bracketed” by stacking interactions with the side chains of Tyr167 (Y167) and Arg238 (R238) from opposing monomers (Fig. 2, D and F, and fig. S9A). The structure of SR-717 facilitates a binding mode in which Thr263 (T263) side chain hydroxyls from both
Fig. 2. Crystal structure of SR-717 bound to hSTINGR232 and comparison to existing hSTING
structures and intermolecular contacts. (A) The 1.8-Å structure of SR-717 bound to hSTINGR232 (residues 155 to 341). Ribbon representation of the symmetrical hSTINGR232 dimer and stick representations of
SR-717 are shown. (B) Superposition of the structure of SR-717 bound to hSTINGR232 with the structure
of cGAMP bound to hSTING [PDB ID 4KSY (21)], shown as yellow or blue ribbon representations, respectively, highlighting near identical closed conformations and associated formation of characteristic b-sheet overhead cap elements. Stick representations of bound SR-717 or cGAMP are shown. (C) Superposition
of the structure of SR-717 bound to hSTINGR232 with the unbound (APO) structure of hSTING [PDB ID 4F9E (19)], shown as yellow or purple ribbon representations, respectively. (D) Details of the intermolecular contacts and hydrogen-bond (dashed red line) network associated with the binding of two molecules of
SR-717 to the cGAMP binding site of hSTINGR232. Residues from individual monomers of the hSTINGR232 dimer are shown in stick format in yellow and purple. The a and b superscripts indicate protein monomer or individual ligand identity. (E) Superposition of the structure of SR-717 bound to hSTINGR232 with the structure of di-GMP bound to hSTING [PDB ID 4F9G (19)], shown as yellow or orange ribbon representations, respectively. Stick representations of SR-717 and di-GMP are shown. (F) Detailed view of the overlay and conserved intermolecular contacts of bound SR-717 and cGAMP ligands from the overlaid hSTING structures shown in (B). Side chains from the SR-717–bound hSTINGR232 structure are shown in yellow or purple,
and those from the cGAMP structure are shown in blue. Stick representations of two molecules of SR-717 are shown in gray, and cGAMP is shown in light blue.

monomers are positioned to form hydrogen- bond interactions with both the carboxylic acid of one bound molecule of SR-717 and the amide carbonyl of the other bound SR-717
molecule (Fig. 2D and fig. S9B). These side chains, at the base of the cGAMP binding site, form hydrogen-bonding interactions with the purine bases of bound cGAMP (PDB ID 4KSY).
Consistent with the observed requisite of a carboxylic acid for binding (fig. S2E), the car- boxylic acid of each molecule of bound SR-717 is directly positioned in the location of a

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Fig. 3. STING-dependent pharmacodynamic and antitumor activities of systemic SR-717. (A) Dose escalation of SR-717 by intraperitoneal injection and corresponding plasma IFN-b levels in C57BL/6 mice (n = 4) 4 hours after dosing, after 4 days of daily dosing (n = 4). (B) Plasma concentrations of cGAS-STING signaling target cytokines after dosing with SR-717 (15 mg/kg intraperitoneally) in WT (n = 4) or Stinggt/gt mice (n = 4). (C) Schematic of therapeutic treatment strategy of B16.F10 tumor-bearing mice used to evaluate SR-717.
I.P., intraperitoneal. (D) Impact of SR-717 [30 mg/kg intraperitoneally, using dosing regimen described in (C)] on B16.F10 tumor growth in WT (n = 8) or Stinggt/gt mice (n = 8). (E) Kaplan-Myer survival curve of WT (n = 8) or Stinggt/gt B16.F10 tumor-bearing mice (n = 8) after treatment with SR-717 as described in (D). Mice were euthanized when tumor area exceeded 2000 mm3.
(F) Impact of SR-717 or DMXAA positive control (both dosed at 15 mg/kg intraperitoneally, once per day) on metastasized B16.F10 lung nodule formation in C57BL/6 mice. Pulmonary nodules were quantified 7 days after intravenous
tail vein administration of B16.F10 cells (n = 5 mice for vehicle, n = 5 for SR-717, and n = 3 for DMXAA). Each data point represents the number of nodules per set of lungs in each mouse. **P ≤ 0.01; n.d., no difference. (G) Representative images of isolated lungs from studies described in (F). (H) Impact of SR-717
[n = 8, 30 mg/kg intraperitoneally, using dosing regimen described in (C) starting on day 10]; anti-PD-1 antibody (n = 8, 200 mg on days 10, 14, and 17); or combination SR-717 plus anti–PD-1 treatment (n = 8) on B16F.10 tumor growth in WT C57BL/6 mice. (I) Kaplan-Myer survival curve of WT B16.F10 tumor-bearing mice (n = 8) after treatments described in (H). (J) Impact of SR-717 [30 mg/kg intraperitoneally, using dosing regimen described in (C) starting on day 11] (n = 8); anti-PDL1 antibody (200 mg on days 11, 14, and 17)
(n = 8); or combination thereof (n = 8) on B16F.10 tumor growth in WT C57BL/6 mice. (K) Kaplan-Myer survival curve of WT B16.F10 tumor-bearing mice (n = 8) after treatments described in (J). Data are representative of three independent experiments, and values are the mean ± SEM [(A), (B), and (D) to (K)].

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backbone phosphate of bound cGAMP (Fig. 2F). This facilitates formation of direct hydrogen-bond contacts with the guanidi- nium side chains of R238 from both STING monomers (Fig. 2, D and F). These residues are located on opposing ends of the b strands from each monomer that make the intersu- bunit interactions within the b-sheet cap element (Fig. 2D). By inducing compaction at the base of the binding site (i.e., through interactions with both T263 side chains), mimicking intersubunit stacking interactions and orientating both R238 side chains to induce formation of the b-sheet cap element, SR-717 is able to serve as a direct cGAMP mimetic that activates STING by inducing it to adopt the closed conformation. SR-717 also forms hydrogen-bond interactions with the side chain of R232 from only one monomer (fig. S9B), exactly as is observed for bound cGAMP (PDB ID 4KSY). Additional stabiliz- ing interactions are derived from a water- mediated hydrogen-bond network involving Val239 (V239), Ser241 (S241), Asn242 (N242),
and Y240 (fig. S9B). The near identical con- formational change induced by SR-717, as well as its ability to directly mimic the binding mode of cGAMP, is highlighted by an overlay of the SR-717–bound hSTING co- crystal structure with the cGAMP-bound com- plex (Fig. 2, B and F). By contrast, the SR-717 complex clearly does not correspond to the open conformation induced by di-GMP (Fig. 2E; PDB ID 4F9G). Consistent with the ob- served impact of SR-717 on the thermal sta- bility of mSTING (Fig. 1D), the 2.5-Å cocrystal structure of SR-717 bound to mSTING (resi- dues 154 to 340) (figs. S8B and S10A) is nearly identical to the induced closed-conformation hSTING complex (figs. S10B and S11). SR-717 is a stable cGAMP mimetic that activates STING by inducing the same closed confor- mation, which thereby provides an avenue to explore this class of systemic STING agonist in diverse contexts, including antitumor immunity.
We used IFN-b protein concentrations in circulating plasma as a pharmacodynamic marker of STING target engagement by SR- 717, which displayed favorable mouse phar- macokinetic properties (fig. S4A), as well as robust antitumor activity after intratumoral delivery in syngeneic B16.F10 melanoma or MC38 colorectal adenocarcinoma mouse mod- els (fig. S12, A and B, respectively). Dose- dependent induction of IFN-b was observed after intraperitoneal administration of SR-717 in WT C57BL/6 mice (Fig. 3A). By contrast, SR-717 did not affect circulating IFN-b or associated proinflammatory cytokine expres- sion in Stinggt/gt mice (Fig. 3B and fig. S16A), thereby demonstrating its selective on-target in vivo activity. Based on the known ability of cGAS-STING pathway activation in DCs to
stimulate CD8 T cell priming (11, 12, 23), we elected to use the poorly immunogenic and highly aggressive syngeneic B16.F10 murine melanoma model (24) to evaluate and char- acterize the antitumor activities of system- ically delivered closed conformation–inducing STING agonists. A therapeutic mode of treat- ment was modeled by initiating treatment with SR-717 on day 11 when B16.F10 mela- noma tumors are well established (Fig. 3C). Notably, efficacious doses of diABZI-2 (22), an open conformation–inducing STING ago- nist that induces equivalent maximal levels of cell-based activity when compared with SR-717 (fig. S13, A to C), were found to in- duce ~20 ng/ml of IFN-b (fig. S13D). By con- trast, systemic dosing regimens of the closed conformation–inducing STING agonist SR-717 that resulted in the induction of >0.2 ng/ml of circulating IFN-b (Fig. 3A and fig. S13D) were found to be well tolerated and effica- cious. Specifically, a 30 mg/kg intraperitoneal once-per-day regimen of SR-717 for 1 week (Fig. 3C) was found to maximally inhibit tumor growth (Fig. 3D), as well as lengthen survival time in tumor-bearing mice (Fig. 3E). The antitumor efficacy of SR-717 displayed an obligatory dependence on STING expres- sion, based on the observed lack of activity in Stinggt/gt host mice (Fig. 3, D and E).
To establish if efficacy was restricted to subcutaneous tumors, as well as to determine utility in the context of metastasis, we treated C57BL/6 mice that had been injected intra- venously with B16.F10 cells, which are reported to home to and colonize lung tissue (25). SR-717 was observed to significantly inhibit the formation of pulmonary nodules in this model of metastasis (Fig. 3, F and G), consistent with the ability of a systemic STING agonist to control metastasis and B16.F10 tumori- genesis in a manner that is independent of tissue type. We investigated the efficacy of this compound series in the context of oral delivery, using the active STING agonist ana- log SR-301 (fig. S14, A and B; EC50 = 0.6 mM),
which has appreciable bioavailability (%F = 32.2) and suitable rodent exposure proper- ties (fig. S4B).
Encouragingly, 15 mg/kg oral once-per-day dosing with SR-301 for 18 days was found to maximally reduce tumor burden in the B16. F10 model (fig. S14, C to E). Based on the more favorable physicochemical properties, as well as enhanced uniformity with respect to dos- age to pharmacodynamic relationships, intra- peritoneal administration of SR-717 was used to further characterize antitumor immunity. Using the B16.F10 model, we compared the antitumor activity of SR-717 to that observed for anti–programmed cell death 1 (anti–PD-1) or anti–programmed cell death 1 ligand 1 (anti–PD-L1) antibody therapy. Under the constraints and limitations of this model,
maximal achievable efficacy was observed with a single-agent therapy consisting of SR-717 STING agonist. Specifically, SR-717 dis- played a better level of efficacy to that achieved by anti–PD-1 or anti–PD-L1 antibody therapy in this model, with respect to tumor burden (Fig. 3, H and J, respectively) or overall survival (Fig. 3, I and K, respectively). The observed activity profiles of PD-1–based check- point blockade in this poorly immunogenic model is consistent with previous reports (24, 26).
We investigated the impact of effective sys- temic exposure levels of SR-717 on immune cell activation and immunological mechanisms, in the context of tumor-bearing mice. Consist- ent with previous reports associated with in- tratumoral injection of 2′3′-cGAMP (13, 27), systemic delivery of SR-717 increased the fre- quency of activated CD69+CD8 T cells among isolated tumor infiltrating lymphocytes (TILs) and within isolated spleens and inguinal lymph nodes (Fig. 4A). A similar increase was observed in the frequency of activated CD69+ natural killer (NK) cells within isolated spleens and inguinal lymph nodes (Fig. 4B), con- sistent with previous findings describing the ability of the STING pathway to activate this cell type (28). Within the CD45.2+ popula- tion, SR-717 treatment resulted in a signifi- cant increase in the frequency of CD8 T cells among TILs and a decrease in the frequen- cies of NK cells within the draining lymph node (dLN) and spleen (fig. S16B). Because of the potential for STING agonism to in- duce pathological consequences, we next assessed the infiltration of CD8 T cells into peripheral tissues. We observed no signif- icant differences in lymphocyte or CD8 T cell infiltration into the lung after SR-717 treatment (30 mg/kg intraperitoneally for 7 days) (fig. S15, A and D), although a small but statistically significant increase in fre- quencies of CD44+PD-1+ T cells was observed (fig. S15A). In the liver, a small but statistically significant increase in CD8 T cell infiltration was observed (fig. S15, B and D). Measure- ment of liver enzymes revealed a transient up- regulation of alanine aminotransferase (ALT) levels on day 4 after treatment, which returned to vehicle levels by day 7 after treatment, and no impact on aspartate transaminase (AST) lev- els was observed (fig. S15C). Further assess- ment of T cell function revealed that SR-717 treatment significantly increased the fre- quency of granzyme B and CD107a+ CD8 T cells in both spleen and tumor (Fig. 4, C and E). Although we did not observe increases in granzyme B+ NK cells in spleen or tumor (Fig. 4D), we did detect increases in CD107a ex- pression in NK cells in both tissues (Fig. 4F), suggesting that at the time of analysis, NK cells had already degranulated in SR-717– treated animals. Analysis of cytokine-producing

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Fig. 4. Impact of systemic SR-717 administration on antitumor immunity in mice. (A) Impact of systemic SR-717 delivery (administered as described
in Fig. 3C) on surface CD69 expression, assessed by flow cytometry, on CD8 T cells isolated from tumors (TILs), spleens, or dLN of B16.F10 tumor- bearing mice (n = 4). (B) Impact of systemic SR-717 delivery (administered as described in Fig. 3C) on surface CD69 expression, assessed by flow cytometry, on NK cells isolated from tumors (TILs), spleens, or dLN of B16. F10 tumor-bearing mice (n = 4). (C) Impact of systemic SR-717 delivery (administered as described in Fig. 3C) on granzyme B expression, assessed by flow cytometry, on restimulated intratumoral and splenic CD8 T cells
(n = 5). (D) Impact of systemic SR-717 delivery (administered as described in Fig. 3C) on granzyme B expression, assessed by flow cytometry, on restimulated intratumoral and splenic NK cells (n = 5). (E) Impact of systemic SR-717 (administered as described in Fig. 3C) on surface CD107a

expression on restimulated intratumoral and splenic CD8 T cells (n = 5).
(F) Impact of systemic SR-717 (administered as described in Fig. 3C) on surface CD107a expression on restimulated intratumoral and splenic NK cells (n = 5). (G) Impact of systemic SR-717 delivery (administered as described in Fig. 3C) on surface expression of CD80 and CD86, determined by flow cytometry, and on CD8a+ DCs from dLN of B16.F10 tumor-bearing mice
(n = 4). (H) Western blot analysis of the impact of SR-717 delivery (3.8 mM) on PD-L1 expression in WT THP1 or ISG-THP1 STING KO (STING KO) cells. Vinculin was used as a loading control. (I) Impact of systemic SR-717 delivery (administered as described in Fig. 3C) on PD-L1 surface expression on CD8a− DCs from the dLN of B16.F10 tumor-bearing mice (n = 4). Data are representative of three independent experiments, and values indicate mean ± SEM with the exception of values in (I), which indicate mean ± SD. *P ≤ 0.05;
**P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; n.d., no difference.

Downloaded from http://science.sciencemag.org/ on August 21, 2020
NK cells revealed significantly reduced IFN-g+ NK cells in TILs (fig. S16C), with a modest but significant increase of IFN-g+ NK cells in spleen (fig. S16C). Moreover, SR-717 treatment had no significant effect on the frequency of polyfunctional CD8 T cell responses (fig. S16D). We also observed increases in the fre- quencies of CD8 T cells undergoing apopto- sis within the dLN (fig. S16E), the magnitude of which being consistent with those previ- ously reported for efficacious doses of cGAMP after intratumoral injection (14). The anti- tumor activity derived from STING activation is dependent on CD11c+CD8a DCs (12) and involves tumor antigen cross-presentation lead- ing to the activation of CD8 T cells within the draining lymph node, as well as the activation of type I interferon signaling (11, 23). Sys- temic SR-717 administration induces the ac- tivation of CD11c+CD8a DCs, as determined by CD80 and CD86 staining intensity (Fig. 4G), and enhances cross-priming of CD8 T cells, as determined by monitoring the in vivo proliferation of transferred Thy1.1+ OT-I CD8 T cells isolated from mice pretreated with SR-717 and subsequently injected with oval- bumin protein (fig. S16, F and G). At this stage of treatment, OT-I CD8 T cell activation state was determined to be significantly en- hanced by treatment with SR-717, as deter- mined based on the evaluation of CD44+PD-1+ and granzyme B+ CD8 T cell populations, as well as polyfunctional CD8 T cell responses (fig. S16, H to J).
Finally, we examined the impact of SR-717 on the expression of critical targets associated with antitumor immunity. STING pathway activation can induce mechanisms known to regulate immune checkpoint protein expres- sion. STING activation, and the subsequent induction of type I interferon, can induce STAT3 phosphorylation (29), a key regulator of interferon-dependent PD-L1 expression (30, 31). SR-717 was found to induce the ex- pression of PD-L1 in THP1 cells (Fig. 4H and fig. S17A) and in primary human PBMCs (fig. S17B) in a STING-dependent manner (Fig. 4H). Consistent with previous findings describing the impact of cGAMP on STING protein lev- els (32), and indicative of negative feedback mechanisms associated with pathway activa- tion, total STING protein levels were observed to decrease after treatment with SR-717 (Fig. 4H). In vivo, we observed that intraperitoneal injection of SR-717 resulted in increased cell- surface levels of PD-L1 on CD11c+CD8− DCs but not on CD8+ DCs isolated from the inguinal lymph nodes of B16.F10 tumor bearing mice (Fig. 4I), even though SR-717 clearly activates CD8+ DCs, which suggests cell type–selective differences in downstream STING-dependent signaling. Indoleamine 2,3-dioxygenase 1 (IDO1) in vivo expression has been demonstrated to be induced in a STING-dependent manner
(33). SR-717 STING agonist was found to in- duce IDO1 expression in primary human PBMCs (fig. S17C). Taken together, our results demonstrate that although STING activation with SR-717 induces the expected stimulatory events, a corresponding induction of molecules known to suppress immune responses was also elicited, albeit in a cell type–selective manner. These observations have important implications for the selection of agents and the temporal design of combination-based clinical trials in- volving a systemically delivered STING agonist. To address the limitations of intratumoral delivery, we have identified the SR-717 chemi- cal series of functional cGAMP mimetic STING agonists, which, after systemic administra- tion, were demonstrated to promote anti- tumor immunity and activate CD8+ T cells within tumors and the dLN, as well as ac- tivate NK cells within the dLN. The systemic administration of SR-717 reduced tumor bur- den in the B16.F10 melanoma model with a level of efficacy that was observed to be superior than what is observed for anti– PD-1 or anti–PD-L1 therapy in this particular poorly immunogenic model. Importantly, sys- temic administration of SR-717 produced substantial efficacy despite inducing mod- est levels of IFN-b, suggesting that the threshold for efficacy in tumor models may be far lower than previously reported and can be achieved without considerable tox- icity. It is also of potential critical impor- tance that STING activation by SR-717 was found to induce the expression of PD-L1 in a STING-dependent fashion. These results have important implications for the choice of agent to be combined with a STING agonist, as well as the relative timing of a dosing reg- imen, in the context of cancer treatment. Pre- sumably, it would be unproductive to treat with an agent that increases the relative abundance of the target of the second agent. The ability of SR-717 to induce the cGAMP- induced closed STING conformation, in con- trast to open conformation–inducing ligands, enables exploration of the relative impor- tance of different potential scaffolding func- tions in vivo and in the context of systemic distribution in settings of antitumor immu- nity and beyond. Differential pathway acti- vation associated with the recognition of bacterial-derived CDNs [e.g., di-GMP derived from commensal bacteria (34)] as compared with endogenously produced cGAMP, derived from cytosolic DNA as a result of diverse pathological events (e.g., genomic instabil- ity), is readily conceivable and most likely probable. Each class of agonist may provide differential therapeutic benefits depending
on the setting.

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ACKNOWLEDGMENTS
We thank A. Theofilopoulos for invaluable discussion and insight. Author contributions: L.L.L. and E.N.C. conceived of, initiated, and coordinated the project. L.L.L., E.N.C., J.R.T., H.M.P., P.G.S., and A.K.C. contributed to conceptualization.
E.N.C., C.Y., V.F.V., Y.J., M.K., A.M.G.A., W.V., S.A., D.L., N.N.,
L.P., B.B., P.S., F.M.-P., and E.H. conducted research. L.L.L.,
J.R.T., H.M.P., P.G.S., A.C., T.Y., K.J., S.J., and A.K.W. were
involved with project administration. L.L.L., E.N.C., J.R.T., V.F.V., H.M.P., P.G.S., C.Y., D.W., and M.Ki. were involved with the interpretation of data. L.L.L. and E.N.C. wrote the original draft. L.L.L., E.N.C., J.R.T., V.F.V., and H.M.P. were involved with review and editing. Competing interests: L.L.L., E.N.C., A.K.C., M.Ku., A.M.G.A., H.M.P., P.G.S., C.Y., and W.V. are inventors on patent application PCT/US2019/018899 submitted by The Scripps Research Institute that covers small-molecule agonists of STING. Data and materials availability: All data are available in the manuscript or in the supplementary materials. Noncommercial reagents described in this manuscript are available from L.L.L., H.M.P., or J.R.T. under a material transfer agreement with The Scripps Research Institute. Coordinates for the following crystal structure complexes have been deposited in the RCSB Protein Data Bank: hSTING:SR-717
(PDB ID 6XNP) and mSTING:SR-717 (PDB ID 6XNN).

SUPPLEMENTARY MATERIALS
science.sciencemag.org/content/369/6506/993/suppl/DC1 Materials and Methods
Figs. S1 to S19 Table S1 References
View/request a protocol for this paper from Bio-protocol.

24 February 2020; accepted 9 July 2020 10.1126/science.abb4255

Antitumor activity of a systemic STING-activating non-nucleotide cGAMP mimetic
Emily N. Chin, Chenguang Yu, Vincent F. Vartabedian, Ying Jia, Manoj Kumar, Ana M. Gamo, William Vernier, Sabrina H. Ali, Mildred Kissai, Daniel C. Lazar, Nhan Nguyen, Laura E. Pereira, Brent Benish, Ashley K. Woods, Sean B. Joseph, Alan Chu, Kristen A. Johnson, Philipp N. Sander, Francisco Martínez-Peña, Eric N. Hampton, Travis S. Young, Dennis W. Wolan, Arnab
K. Chatterjee, Peter G. Schultz, H. Michael Petrassi, John R. Teijaro and Luke L. Lairson

Science 369 (6506), 993-999.
DOI: 10.1126/science.abb4255

Targeting STING for cancer therapy
Activation of the STING (stimulator of interferon genes) protein by cyclic dinucleotide metabolites plays a critical role in antitumor immunity. The development of synthetic STING agonists is therefore being pursued as a strategy for cancer therapy, but the inherent instability of dinucleotides has limited current efforts. Pan et al. and Chin et al. identified stable STING agonists that act in a ”closed” conformation similar to the natural STING ligand, cyclic guanosine monophosphateadenosine monophosphate (see the Perspective by Gajewski and Higgs). The small molecules can be given orallyan advantage over previously developed STING agonists, which required intratumoral administration. After oral or systemic administration in mice, the agonists activated STING and diverse immune cell types to promote antitumor immunity. These studies represent progress toward clinically viable STING agonists for cancer immunotherapy.
Science, this issue p. eaba6098, p. 993; see also p. 921

ARTICLE TOOLS http://science.sciencemag.org/content/369/6506/993

SUPPLEMENTARY http://science.sciencemag.org/content/suppl/2020/08/19/369.6506.993.DC1
MATERIALS

Cathepsin K

Cathepsin K is a cysteine protease expressed predominantly in osteoclasts

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