TAK-676: A Novel Stimulator of Interferon Genes (STING) Agonist Promoting Durable IFN-dependent Antitumor Immunity in Preclinical Studies

Oncology therapies targeting the immune system have improved patient outcomes across a wide range of tumor types, but resistance due to an inadequate T-cell response in a suppressive tumor microenvironment (TME) remains a significant problem. New therapies that activate an innate immune response and relieve this suppression may be beneficial to overcome this hurdle. TAK-676 is a synthetic novel stimulator of interferon genes (STING) agonist designed for intravenous administration. Here we demonstrate that TAK-676 dose-dependently triggers activation of the STING signaling pathway and activation of type I interferons. Furthermore, we show that TAK-676 is a highly potent modulator of both the innate and adaptive immune system and that it promotes the activation of dendritic cells, natural killer cells, and T cells in preclinical models. In syngeneic murine tumor models in vivo, TAK-676 induces dose-dependent cytokine responses and increases the activation and proliferation of immune cells within the TME and tumor-associated lymphoid tissue. We also demonstrate that TAK-676 dosing results in significant STING-dependent antitumor activity, including complete regressions and durable memory T-cell immunity. We show that TAK-676 is well tolerated, exhibits dose-proportional pharmacokinetics in plasma, and exhibits higher exposure in tumor. The intravenous administration of TAK-676 provides potential treatment benefit in a broad range of tumor types. Further study of TAK-676 in first-in-human phase I trials is ongoing. Significance: TAK-676 is a novel systemic STING agonist demonstrating robust activation of innate and adaptive immune activity resulting in durable antitumor responses within multiple syngeneic tumor models. Clinical investigation of TAK-676 is ongoing.


Introduction
Immuno-oncology checkpoint inhibitor therapies are revolutionizing cancer treatment and have improved patient outcomes across a broad range of tumor types; however, only a small fraction of patients respond to therapy, I IFN production has been associated with the host detection of danger signals that are released from stressed, injured, or necrotic cells through a diverse family of innate pattern recognition sensors (1). One such sensor is the stimulator of interferon genes (STING). STING is an endoplasmic reticulum signaling protein, that is broadly expressed in both immune and nonimmune cell types. STING binds 2 ,3 -cGAMP produced by cyclic GMP-adenosine monophosphate (AMP) synthase in response to cytosolic DNA (4) and rapidly induces type I IFNs and proinflammatory cytokines (5)(6)(7). It has been shown that productive type I IFN-mediated antitumor immunity requires an intact STING pathway within the DC population (8). Collective evidence highlights that agonism of the host STING pathway plays a critical role in eliciting robust and durable antitumor immunity (9,10).
Recognizing the therapeutic potential of targeting the STING pathway for its positive immune-modulatory properties has prompted many groups to develop STING-activating drugs utilizing intratumoral injection as an administration route (9). Poor pharmacokinetic and physicochemical properties of 2 ,3 -cGAMP and its derivatives are hurdles to the development of systemically administered STING agonists (9) . Therefore, overcoming these obstacles would facilitate the clinical development of STING agonists as novel anticancer therapeutics and potentially increase the number of patients that can benefit. TAK-676 is a novel synthetic STING agonist that is administered as an intravenous infusion and is a highly potent modulator of the innate immune system (11,12). TAK-676 is currently under investigation in cancer patients with locally advanced or metastatic solid tumors (ref. 13; NCT04420884, NCT04879849, NCT04541108). These trials are investigating the antitumor effects of TAK-676 as a monotherapy or with combination therapies such as checkpoint inhibitors, radiation, and chemotherapy.
Here we present the first report of the structure of TAK-676, and the activity of TAK-676 in preclinical studies. We demonstrate that TAK-676 dosedependently triggers activation of the STING signaling pathway and activation of type I IFNs, as well as robust activation of innate and adaptive immune activity both in vitro and in vivo. In syngeneic murine tumor models in vivo, TAK-676 induced dose-dependent cytokine responses and increased the activation and proliferation of immune cells within the TME and tumorassociated lymphoid tissue. Ultimately this immune activation resulted in durable antitumor responses within multiple syngeneic tumor models.

Materials and Methods
A summary methodology for all components of the analyses reported is provided below; full methodologic details for all analyses are provided in the Supplementary Appendix. For details on cell lines and use, see "Cell lines and mouse models" section at the end of this Materials and Methods section.

Chemical Synthesis and Characterization of TAK-676
Detailed methods for the synthesis and purification of TAK-676 can be found within the Supplementary Appendix.

STING Binding and Pathway Activation
To measure the binding of TAK-676 to mouse, rat, monkey, and human STING orthologs, in vitro time-resolved fluorescence resonance energy transfer (TR-FRET) assays were used. To assess pathway activation, the stimulatory effect of TAK-676 on the STING signaling pathway in vitro in the THP1-Dual human acute myeloid leukemia (AML) and CT26.wild type (WT) and STINGdeficient CT26.KO mouse colon carcinoma cell lines was evaluated. Cells were treated with increasing concentrations of TAK-676 for 3 hours, subsequently lysed, and the proteins analyzed by SDS-PAGE and immuno-blotting. For experiments in STING-deficient CT26.KO cell lines, 5,6-dimethylxanthenone-4-acetic acid (DMXAA; 50 μg/mL) was used as a control for a treatment time of 1 hour prior to cell lysis and blotting. Three STING-induced phosphorylation events, part of a signaling cascade critical for the induction of type I IFNs, were monitored by Western blotting: TANK-binding kinase 1 (TBK1) on S172 in the kinase activation loop, STING on S365/366 (mouse/human numbering), and interferon regulatory factor (IRF) 3 on S396.
STING agonists trigger the production of type I IFNs and the induction of interferon-stimulated genes (ISG) through IRFs. The potency of the STING agonist was evaluated in reporter assays using the ISRE_NanoLuc human embryonic kidney 293 (HEK293T) cells, the more physiologically relevant human monocyte derived THP1-Dual cells, and the mouse macrophage RAW-Lucia ISG cells. These cell lines, that are of mouse or human origin, allow measurement of STING agonist activity using the NanoLuc luciferase (HEK293T) or IRF-inducible Lucia luciferase as readouts (THP1-Dual and RAW-Lucia). In HEK293T cells, which lack endogenous STING, human and mouse STING isoforms were transiently transfected, and a reporter assay was developed by generating an ISRE_NanoLuc HEK293T stable cell line using the pNL (NLucP/ISRE/Hygro) vector to monitor the resulting increase in type I IFNs.
The concentration at inflection point of the curve producing a half-maximal response (EC 50 ) was calculated in cultured ISRE_NanoLuc HEK293T cells that were transiently transfected with human WT STING (R232), mouse STING (WT), and the four other isoforms of STING-R232H, R293Q, G230A-R293Q (AQ), and R71H-G230A-R293Q (HAQ)-that exist in human populations (14). The EC 50 was also calculated in RAW-Lucia ISG cells and THP1-Dual cells.
Digitonin was used to permeabilize the HEK293T and RAW-Lucia ISG cells, but not the THP1-Dual cell line as digitonin was toxic to these cells, thus preventing accurate measurement of type I IFNs.

Generation of STING Knockout and WT Cell Line Clones in CT26.WT and B16F10 Cells
The "hit-and-run" clustered regularly interspaced short palindromic repeats (CRISPR) method was employed for creation of STING knockout (KO) cell clones and their WT counterparts in CT26.WT and B16F10 cells by following the Lipofectamine CRISPRMAX Transfection Reagent protocol (Thermo Fisher Scientific). Confluent clones were harvested and evaluated by Western blotting for mSTING protein and PCR analysis of mSTING genomic DNA to reconfirm efficient knockdown. Several confirmed clonal mSTING KOs, as well as matched WT controls shown to have intact mSTING, were expanded, banked, tested for murine pathogens, and cryopreserved for use in subsequent in vitro and in vivo studies.

In Vitro Immune Cell Activation Assays
As STING proteins are highly expressed in DCs, the activation of DCs derived from human peripheral blood mononuclear cells from 5 healthy donors and mouse bone marrow (BMDC) was evaluated using flow cytometry to detect Mean fluorescence intensity (MFI) of CD86 + DCs from each sample was evaluated by flow cytometry and MoDC viability (defined as LIVE/DEAD Fixable Near-IR Dead Cell Staining Dye negative population from the parent gate) was recorded. Natural killer (NK) and T-cell activation was assessed in whole blood from 5 healthy human donors using flow cytometry to detect expression of the activation marker CD69 on CD4 + and CD8 + T cells and CD56 + /CD16 + NK cells. CD69 expression (as evaluated by MFI) was measured for each cell population, and mean plasma-based EC 50 , Hill slope, and concentration producing 10% of maximal response (EC 10 ) were calculated.

Assessment of Pharmacokinetics
Female BALB/c mice were inoculated subcutaneously with mouse A20 (B-cell lymphoma) tumor cells in the right flank and TAK-676 dosing was initiated when estimated tumor volumes reached 300-800 mm 3 . Mice (n = 3/group) were dosed via intravenous injection with single doses of TAK-676 at 0.025, 0.125, 0.25, 0.5, and 2 mg/kg formulated in PBS as a single agent. Exposure to TAK-676 was determined by measuring plasma and tumor concentrations of TAK-676 at various timepoints using a LC-MS/MS method.

In Vivo Assessment of TAK-676 Efficacy in Mouse Models
TAK-676 activity was evaluated in syngeneic CT26.WT and A20 mouse models. In addition, a B16F10_WT (melanoma) tumor model and STINGdeficient B16F10_STING KO tumor model were used to demonstrate the STING dependence of antitumor activity using both WT and STING-deficient C57BL/6J-Tmem173gt/J (Goldenticket; Jackson Laboratory) models. Mice were inoculated subcutaneously into the right flank with CT26.WT tumor cells, A20 tumor cells, or B16F10 tumor cells and were treated with either vehicle (PBS) or TAK-676. Mice in the vehicle and TAK-676 (1 or 2 mg/kg) groups received intravenous doses once every 3 days for three doses (Q3D×3, days 0, 3, and 6). Effects on tumor growth were evaluated by measuring growth rate inhibition (GRI) and tumor regression. Tolerability was assessed by measuring percent body weight loss (BWL; body weights were measured twice weekly), mortality, or any clinical signs of adverse treatment-related side effects (see Supplementary Appendix).

In Vivo Immune Cell Activation and Proliferation
Female C57BL/6 mice bearing B16F10 tumors were treated with TAK-676 intravenously at 0.3, 1.0, 2.0 mg/kg or vehicle on day 0 (n = 4/group). In addition, female BALB/c mice bearing CT26 tumors were treated with vehicle or 0.25 mg/kg TAK-676 on day 0 (n = 4/group). Pooled tumor-draining lymph nodes (axillary, brachial, and inguinal) and tumors were collected on day 3, and immune cell populations were analyzed by flow cytometry. Samples were initially gated off total live cells (within the live/dead negative gate) and subsequently for CD45 + cells. In the tumor, total T cells, defined as CD3 + , were then examined further to specifically identify tumor-infiltrating CD8 + T cells. Because of the strong correlation between the presence of cytotoxic CD8 + T cells within the tumor and clinical prognosis (15,16), CD8 + T cells in the tumor samples were further characterized for markers of activation and proliferation.
CD8 + T-cell populations were evaluated for either CD69 (A20 tumors) or Ki67 and CD25 expression (CT26 tumors). In addition, to assess the functionality of the CD8 + T-cell population within the A20 tumors, expression of the proinflammatory cytokine IFNγ was evaluated ex vivo following an overnight stimulation with plate bound α-CD3 and soluble α-CD28 antibodies. In the lymph node, CD8 + T cells were also assessed for IFNγ expression, as well as Ki-67 and CD69 expression, following overnight stimulation with α-CD3/αCD28. Lymph nodes were also evaluated for the CD11c + MHC1 + DC population as a fraction of all viable CD45 + cells, as well as for the expression of DC activation markers CD80 and CD86. Lymph nodes from mice inoculated with CT26 tumors were also evaluated for the expression of CD69 on NK cells.

Cell Lines and Mouse Models
All animal experiments were performed in compliance with protocols as approved by an Institutional Animal Care and Use Committee. All cell lines used in the experiments were obtained from the indicated sources summarized below and included in Supplementary Table S1, cultured in appropriate media and expanded, and then banked into the Takeda Oncology Cell bank as highdensity frozen vials stored in liquid nitrogen (LN 2 ) between passage three and six. For cell line reporter assays, cells were thawed from high-density frozen vials and then used immediately for experiments following a short recovery period. Cells for in vitro Western blot assays and for in vivo implantation were thawed from frozen vials and cultured a short time before use in the experiment. During culture time, cells were fed and split as needed to avoid over confluence.
Cells were used for experiments as needed during the period of culture but never exceeding passage 10. Cells were Mycoplasma and murine pathogen tested via the IMPACT I assay by Idexx Bioresearch (the most recent testing dates are indicated in Supplementary Table S1 and

Statistical Analysis
For in vivo studies, the differences in the tumor growth trends over time between pairs of treatment groups were assessed by fitting each animal's data to a simple exponential growth model and comparing the mean growth rates of the two groups. The difference in the growth rates was summarized by the GRI,

AACRJournals.org
Cancer Res Commun; 2(6) June 2022 which is the reduction in growth rate experienced by the treatment group relative to that of the reference group, expressed as a fraction of the vehicle growth rate. A positive GRI indicates that the tumors in the treatment group grew at a reduced rate relative to the reference group. A statistically significant P value (<0.05) suggests that the trends over time for the two treatment groups were different. For all other relevant studies, the mean, SD, and P value (where significant; relative to control) were calculated using GraphPad Prism 7 software (GraphPad Software).

Data Availability Statement
Data are available upon reasonable request made to the corresponding authors.

TAK-676, a Synthetic STING Agonist, is a Potent and Selective Binder of STING Proteins of Multiple Species
The chemical structure of TAK-676 is shown in  Table S2). To assess the ability of TAK-676 to activate the STING pathway, the phosphorylation of STING and STING pathway proteins was evaluated in human THP1-Dual and murine CT26.WT cell lines following treatment with TAK-676, as shown in Fig. 2A and B, respectively. These data demonstrate a dosedependent induction in the expression of pSTING (S366), pTBK1 (S172), and pIRF3 (S396) in both murine and human cell lines following treatment with TAK-676. To confirm that the induction of STING pathway activation following treatment with TAK-676, STING-deficient cells were generated from CT26.WT cell lines. As shown in Fig. 2C, in the absence of STING expression neither TAK-676 nor control STING agonist DMXAA induced the phosphorylation of pTBK1 (S172) or pIRF3 (S396). Collectively, these data demonstrate the ability of TAK-676 to dose-dependently induce STING-TBK1-IRF3 activation is critically dependent on STING expression.

TAK-676-Mediated STING Pathway Agonism Activates DCs, NK, and T Cells In Vitro
STING protein is highly expressed in DCs, and activation of DCs following STING pathway activation is key to promote innate and adaptive immune response (17). TAK-676-mediated DC activation studies were completed by monitoring the induction of the DC maturation marker CD86 by flow cytometry in both mouse BMDCs and human MoDCs.  Fig. S1).
To further characterize the ability of TAK-676 to mediate both innate and adaptive immune cell activation, TAK-676-mediated NK-and T-cell activation was measured by evaluating the expression levels of CD69 on human peripheral blood NK and T cells (n = 5 healthy human donors), as shown in Fig. 3D-F.
TAK-676 treatment resulted in a dose-dependent increase in CD69 expression in both NK and T cells. The plasma-based EC 50 and Hill slope for each donor was analyzed, and the mean EC 50 was estimated to be 0.271, 0.216, and 0.249 μmol/L for NK, CD8 + , and CD4 + T cells, respectively. The mean plasma-based EC 10 was calculated from the mean plasma-based EC 50 to be 0.114, 0.0986, and 0.106 μmol/L for NK cells, CD8 + , and CD4 + T cells, respectively. Collectively, these data suggest that TAK-676 is a potent inducer of both innate and adaptive immune cell activation.

TAK-676 Exhibits Dose-Proportional Pharmacokinetics in Plasma and Greater Exposure in Tumor Tissue
To support systemic dosing of TAK-676, mean plasma and tumor concentration-time curves of TAK-676 in female BALB/c mice bearing A20 syngeneic tumors were calculated and are presented in Supplementary  Supplementary  Table S3.

TAK-676 Dosing Results in Significant T Cell-Dependent In Vivo Antitumor Activity
Dose-dependent in vivo efficacy of TAK-676 was tested in multiple mouse syngeneic tumor models, including A20 (Fig. 4A) and CT26.WT (Fig. 4B) syngeneic tumor models. In both models, the dosing of TAK-676 at both 1.0 and 2.0 mg/kg was well tolerated (Supplementary Fig. S3A and S3B). In mice, body weight is a meaningful safety readout to determine the MTD (18,19), and all experiments were conducted at or below the MTD. BALB/c mice bearing A20 syngeneic tumors showed significant antitumor activity compared with AACRJournals.org Cancer Res Commun; 2(6) June 2022

Concentration-dependent activation in human MoDCs from 5 donors. NK-cell activation (D)
and T-cell activation (E and F) in human whole blood from 5 donors following TAK-676 treatment for 24 hours. Conc., concentration; DMSO, dimethyl sulfoxide.
vehicle treatment when TAK-676 was dosed at 1 mg/kg intravenously Q3D×3 (GRI 72%, P < 0.001), with 1 of 10 mice achieving complete response (CR). Similar to the A20 tumor model, CT26 tumor-bearing animals treated at 1 mg/kg also displayed tumor control over vehicle treated animals (Fig. 4B). Notably, when the dose of TAK-676 was increased from 1.0 to 2.0 mg/kg, there was an observable increase in the antitumor activity in both A20 (GRI = 91%, P < 0.001) and CT26 (GRI = 132%, P < 0.001) models ( Fig. 4A and B; left), with more animals achieving CRs (two for A20 and four CRs for CT26). To assess the durability of the antitumor response elicited by TAK-676, mice with CRs in both groups were continuously monitored for ≥48 days with no tumor regrowth.
The ability of TAK-676 to promote durable CD8-dependent antitumor efficacy was assessed in a subsequent study in CT26.WT tumor-bearing BALB/c mice. In this study, two mice with CRs following treatment with 1 mg/kg of TAK-676 were rechallenged on the opposite flank with CT26.WT tumors on day 34. Following rechallenge, no tumor regrowth was observed in the complete responder mice, as compared with naïve control animals. To determine whether CD8 + T cells were responsible for the observed durable antitumor immunity in these two animals, on day 59, CD8 + T cells were depleted by antibody treatment, and regrowth was observed in one animal. At day 88, CD8 + T cells were depleted a second time, at which point regrowth was observed in the second animal (Fig. 4C). These data demonstrate that TAK-676 drives durable anti-tumor efficacy that is resistant to rechallenge and is dependent upon CD8 + T cells.

TAK-676's In Vivo Antitumor Activity is Dependent on STING Expression in Immune Cells
To assess whether TAK-676-driven antitumor response was dependent on either host or tumor cell STING expression, Goldenticket mice were utilized and compared with WT mice following implantation of either WT or STING KO B16F10 tumor cells. When TAK-676 was dosed at 2 mg/kg intravenously Q3D×3 in WT C57BL/6 mice bearing B16F10 WT tumors, significant antitumor activity was observed (GRI 136%, P < 0.001; Fig. 5A). In contrast, in STING-deficient mice bearing B16F10 WT tumors, TAK-676 treatment failed to induce significant antitumor activity, with a GRI of −2% (P = 0.862; Fig.  5B). In addition, although TAK-676 was well tolerated in both strains, STINGdeficient animals treated with TAK-676 showed reduced BWL when compared with their WT counterparts (see Supplementary Results and Supplementary  Fig. S4 for details of tolerability). These results underscore the importance of the STING signaling pathway in the host for TAK-676-driven efficacy. To determine the role of STING expression in tumor cells, STING KO B16F10 cells were implanted in WT C57BL/6 animals. Treatment with TAK-676 2 mg/kg intravenously Q3D×3 in this model resulted in reduced, but significant, antitumor activity (GRI 53%, P = 0.001). These data demonstrate that STING expression in the host is the major determinant in promoting robust antitumor immunity (Fig. 5C). In further support of this, treatment of STING KO B16F10 tumor-bearing STING-deficient mice with TAK-676 also resulted in a complete lack of antitumor activity similar to what was observed with WT B16F10 tumor-bearing Goldenticket mice (GRI −16%, P = 0.349; Fig. 5D).
To evaluate the role of host versus tumor cell STING expression on STING pathway activation, tumors from these experiments were assessed for expression of STING, pIRF3 (S396), and pTBK1 (S172) at 3 hours after treatment with TAK-676 2 mg/kg intravenously As shown in Fig. 5E (left), TAK-676 treatment induced expression of pIRF3 (S396) and pTBK1 (S172) in both WT and STINGdeficient mice implanted with B16F10 WT tumors. In animals implanted with STING KO B16F10 tumors ( Fig. 5E; right), activation of the STING pathway can be observed, albeit at a much reduced level in WT C57BL/6 mice, but is completely abrogated in STING-deficient mice.

TAK-676 Induces Dose-Dependent Cytokine Responses In Vivo
Type I IFNs modulate the activity of innate immune effector cells and promote DC maturation and antigen presentation to T cells, propagating an adaptive immune response (20,21) downstream of STING pathway activation. As such, we sought to investigate the potential of TAK-676 to induce type I IFNs, as well as other cytokine expression in the plasma and the tumor, following treatment in syngeneic tumor-bearing mice. As shown in Fig. 6A-C (IFNα, IFNγ, and IP-10) and Supplementary Fig. S5 (TNFα, MCP-1, and IL6), TAK-676 induced dosedependent cytokine responses in female BALB/c mice bearing A20 syngeneic tumors. To determine whether the induction of these cytokines depended on either tumor or immune cell STING expression, cytokine responses were evaluated in the serum and tumors from both WT and STING-deficient mice bearing WT or STING-deficient B16F10 tumors following treatment with TAK-676. As shown in Fig. 6 (right), TAK-676-driven induction of IFNα, IFNγ, and IP-10 in both serum and tumor is dependent upon host STING expression and less so in the tumor cells. Collectively, the in vivo and in vitro data suggest that host STING expression is a critical determinant of robust immune activation leading to effective antitumor response.

TAK-676 Increases the Activation and Proliferation of Immune Cells within the Tumor Microenvironment and Local Tumor-Associated Lymphoid Tissue
To evaluate the ability of TAK-676 to induce T-cell infiltration, proliferation, and activation in vivo, immune modulation in the tumor and lymph nodes was assessed following treatment in tumor-bearing mice. The in vivo T-cell responses to TAK-676 in C57BL/6 mice bearing B16F10 tumors and BALB/c mice bearing CT26 tumors are shown in Fig. 7 (left and right, respectively). At day 3, the frequency of live CD45 + cells increased in tumors isolated from all TAK-676-treated groups ( Fig. 7A and B). Intratumoral CD8 + T cells within the viable CD45 + CD3 + cells were slightly increased at day 3 in B16F10 tumors, and significantly increased in CT26 tumors ( Fig. 7C and D). While in vitro activation assays demonstrated an increase in CD69 + CD8 + T cells following treatment with TAK-676, this did not translate to in vivo assessments at day 3 in B16F10 tumors (Fig. 7G). However, an increase in IFNγ + CD8 + T cells was observed in B16F10 tumors, indicating activation of the CD8 + T cells (Fig. 7E).
Corresponding increases in CD25 and Ki67 expression were also observed in the CD8 + T cells of CT26 tumors, further demonstrating CD8 + T-cell activation as well as increased proliferation in these tumors as early as day 3 following TAK-676 dosing ( Fig. 7F and H).
Immune modulation following treatment with TAK-676 was also observed within the tumor-draining lymph nodes of B16F10 and CT26 tumor-bearing mice (Fig. 8). Within the tumor-draining lymph nodes of the B16F10 tumorbearing mice, a measurable increase in total viable cells was initially observed at day 3 (Fig. 8A). Notably, also at day 3, the frequency of total DCs (identified as CD45 + CD11c + MHCII + ) within the lymph node was increased in both B16F10 and CT26 tumor-bearing mice ( Fig. 8B and H Similarly to what was observed within the tumors, an increase in CD69 + CD8 + T cells was not seen in the lymph nodes from B16F10 tumor-bearing animals (Fig. 8G). However, the frequency of IFNγ + CD8 + T cells was again increased within the tumor-draining lymph nodes of B16F10 tumor-bearing mice (Fig. 8E). In addition, TAK-676 induced the proliferation (as measured by Ki67 + staining) of CD8 + T cells within the lymph nodes of B16F10 tumorbearing mice at day 3 following treatment (Fig. 8F). Finally, the frequency of CD69 + NK cells was increased in the lymph nodes from CT26 tumor-bearing animals, corresponding to the NK-cell activation observed in vitro (Fig. 8K).
Collectively, these data demonstrate increased NK-cell activation and DC frequency and activation in the lymph nodes accompanied by increased frequency, proliferation, and activation of CD8 + T cells in both the lymph nodes and the tumor following treatment with TAK-676. These data are consistent with the in vitro immune cell activation shown in Fig. 3, and confirm the ability of TAK-676 to induce both innate and adaptive immune responses in vivo.

Discussion
This is the first report of the chemical structure and biological activity of the synthetic STING agonist TAK-676, designed for intravenous administration by improving the drug-like properties of natural STING agonists through chemical modifications. Here we demonstrate that TAK-676 is a potent and selective binder of STING proteins and activator of the STING signal- activation and priming of T cells observed here and with other STING agonists (22) to induce antitumor activity is of known importance in immunotherapy. The additional activation of NK cells seen here promotes an adaptive immune response by increasing cytokine and chemokine production, which enhances DC recruitment and maturation, and allows NK cells to directly kill tumors that T cells have not targeted (23,24 (28). On the contrary, the intravenous administration with TAK-676 is potentially applicable to a broad population of patients with malignant cancers, including solid tumors and hematologic tumors, which may not be amenable to intratumoral administration. This expansion of tumor indications will afford opportunities to better understand how STING agonism modulates cancer immunity in humans.
Following administration in syngeneic tumor-bearing mice, TAK-676 dosedependently induced cytokine production in both plasma and tumor, and increased the activation and proliferation of immune cells within the TME. Importantly, TAK-676 also exerted its effects within the tumor-draining lymph nodes, demonstrated by the increase in total viable cells, increased frequency of DCs, and increased activation and proliferation of CD8 + T cells over that of vehicle treated mice. DCs play a critical role in bridging an innate immune response into a long-term adaptive response with potential for durable memory (29,30), and the ability of TAK-676 to increase the frequency of DCs within the tumor-draining lymph nodes is an important part of that process. The breadth of the immune response activated following TAK-676 administration suggests a broad-based effect that may be beneficial in terms of antitumor activity. Moreover, this response could be enhanced in combination with agents that disrupt DNA replication and increase antigen presentation, such as those used in radiation, chemotherapy, and anti-PD-1 therapy (31,32).
In summary, TAK-676 is a selective and potent novel synthetic STING agonist that demonstrates immune activation effects in preclinical models bearing solid tumors as demonstrated by increased production of proinflammatory cytokines and activation of both tissue and circulating immune cells. Importantly, peripheral detection of TAK-676-mediated immune activation can be monitored in clinical samples, thereby enabling analysis of pharmacodynamic activity of TAK-676 in clinical studies (13). Significant STING-dependent antitumor activity was observed with no tumor regrowth and acceptable tolerability, suggesting durable tumor suppression. TAK-676 exhibited greater exposure in the tumor tissue than the plasma with intravenous administration, highlighting a potential benefit regarding the convenience of treating future patients versus intratumoral administration. Our findings identify TAK-676 as a promising new therapeutic candidate and support the clinical studies that are currently ongoing. TAK-676 is currently being investigated in phase I studies of patients with locally advanced or metastatic solid tumors alone or in combination with pembrolizumab, an anti-PD-1 antibody (ref. 13; NCT04879849, NCT04420884), and with various other anticancer agents (NCT04541108).