COMBINED USE OF A CHEMOTHERAPEUTIC AGENT AND A CYCLIC DINUCLEOTIDE FOR CANCER TREATMENT

Abstract

A kit of parts includes a) gemcitabine or a pharmaceutically acceptable salt thereof and b) a cyclic dinucleotide or pharmaceutically acceptable salt thereof, wherein the cyclic dinucleotide or pharmaceutically acceptable salt thereof is an agonist of the receptor known as “stimulator of interferon genes” (STING), for use in the treatment of solid pancreatic cancer.

Claims

1-12. (canceled)

13. A method for treating a solid pancreatic tumor in a patient comprising administering to said patient a therapeutically effective amount of gemcitabine or a pharmaceutically acceptable salt thereof; and a therapeutically effective amount of a cyclic dinucleotide or pharmaceutically acceptable salt thereof, said cyclic dinucleotide or pharmaceutically acceptable salt thereof being an agonist of the receptor known as “stimulator of interferon genes” (STING), wherein said gemcitabine or a pharmaceutically acceptable salt thereof and said cyclic dinucleotide or a pharmaceutically acceptable salt thereof are administered to said patient in a separate form, either simultaneously or sequentially.

14. The method according to claim 13, wherein the nitrogenous base of each nucleoside of the cyclic dinucleotide is a purine that is substituted only in position 6.

15. The method according to claim 13, wherein one nucleoside of said cyclic dinucleotide is adenosine and the other nucleoside is inosine.

16. The method according to claim 13, wherein the linkage between the two nucleosides of said cyclic dinucleotide is a (3′,5′)(3′,5′), a (3′,5′)(2′,5′), a (2′,5′)(3′,5′) or a (2′,5′),(2′,5′) phosphodiester and/or phosphorothioate diester linkage.

17. The method according to claim 13, wherein said cyclic dinucleotide is represented by the following formula: ##STR00013##

18. The method according to claim 13, wherein said cyclic dinucleotide is represented by the following formula: ##STR00014##

19. The method according to claim 13, wherein said cyclic dinucleotide is represented by the following formula: ##STR00015##

20. The method according to claim 13, wherein said cyclic dinucleotide is represented by the following formula: ##STR00016##

21. The method according to claim 13, wherein said cyclic dinucleotide is represented by the following formula: ##STR00017##

22. The method according to claim 13, wherein gemcitabine is administered by intravenous perfusion.

23. The method according to claim 13, wherein the cyclic dinucleotide is administered by intravenous perfusion or by intratumoral injection.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0083] FIG. 1. STING signaling in the cell. Activation of STING by cyclic dinucleotides (CDN) leads to activation of the IRF3 and NF-κB pathways and consequently, to induction of Type I interferons and of pro-inflammatory cytokines, respectively.

[0084] FIG. 2. In vitro Type I interferon induction activity in THP1-Dual™ cells. Values measured after 24 h incubation of the cyclic dinucleotides with the cells.

[0085] FIG. 3. In vitro Type I interferon induction activity in wild-type vs. STING knockout B16 cells. Relative ISG54 activity (as an indirect measurement of Type I interferon induction) of cyclic dinucleotides incubated with cultures of wild-type (right-side of graph) or STING-knockout (left-side of graph) B16 cells for 24 h. WT: wild-type; SKO: STING knockout (homozygous).

[0086] FIG. 4. In vitro Type I interferon induction activity in wild-type vs. STING-knockout RAW cells. Relative ISG54 activity (as an indirect measurement of Type I interferon induction) of cyclic dinucleotides incubated in cultures of wild-type (right-side of graph) or STING-knockout (left-side of graph) RAW cell for 24 h. WT: wild-type; SKO: STING knockout (homozygous).

[0087] FIG. 5. Type I interferon induction activity of cyclic dinucleotides in mice. Measurement of Type I interferon induction in sera from mice at 4 h post-treatment.

[0088] FIG. 6. IL-6 induction activity of cyclic dinucleotides in mice. Measurement of IL-6 induction in sera from mice at 4 h post-treatment.

[0089] FIG. 7. Tumor-growth inhibition in a murine model of Panc02 tumors. The mice were treated with saline (control), gemcitabine monotherapy, c-AIMP monotherapy, or gemcitabine combined with c-AIMP. *The Data for Day 28 are shown only for Group 1, as all the mice in this group had died by that day. GemC: gemcitabine; i.t.: intratumoral; i.v.: intravenous.

[0090] FIG. 8. Mean tumor volume in a hamster model of orthotopic PC-1.0 tumors (on Day 22). The hamsters were treated with saline, gemcitabine monotherapy, or gemcitabine combined with c-AIMP. Tumor volume was measured at the end of the experiment. GemC: gemcitabine; i.t.: intratumoral; i.v.: intravenous.

[0091] FIG. 9. Survival rate in a hamster model of orthotopic PC-1.0 tumors. The hamsters were treated with saline, gemcitabine monotherapy, or a combination of c-AIMP and gemcitabine. GemC: gemcitabine; i.t.: intratumoral; i.v.: intravenous.

[0092] FIG. 10. Tumor growth inhibition in the right-flank tumor in a hamster model of bilateral PC-1.0 tumors. The hamsters were treated in the right-flank tumor with saline, gemcitabine monotherapy, c-AIMP monotherapy, or gemcitabine combined with c-AIMP. GemC: gemcitabine.

[0093] FIG. 11. Tumor growth in mice implanted with orthotopic DT6606 pancreatic tumors. Pancreatic tumor (DT6606) growth at Day 36 post-implantation in mice treated with either gemcitabine (GemC) or an intercalated combination of CL592 and gemcitabine (CL592+GemC).

[0094] FIG. 12. Mean tumor volume in mice implanted with orthotopic Panc02 pancreatic tumors. Average tumor volume at Day 30 post-implantation was calculated for each group. Gem: gemcitabine.

EXAMPLES

Biological Assays

[0095] Before investigating the combination of gemcitabine with any of the cyclic dinucleotides encompassed by the present invention, the immunomodulatory activity of these cyclic dinucleotides was ascertained when used alone. These compounds induced the production of multiple cytokines in live human or animal cells. Specifically, these cyclic dinucleotides induce the production of Type I interferons and/or pro-inflammatory cytokines. The in vitro cytokine-induction activity of a representative set of these cyclic dinucleotides is reported here to require the presence of the eukaryotic cellular receptor known as “stimulator of interferon genes” (STING).

In Vitro Cytokine Induction

[0096] The cytokine-induction activities of the cyclic dinucleotides disclosed in Table 1 have been demonstrated by using different reporter cell lines. The cell lines and experiments are explained below.

Cell Lines

[0097] All the cell lines were obtained from InvivoGen. They are described here and provided with their corresponding InvivoGen catalog code.

[0098] THP1-Dual™ (Catalog Code: Thpd-Nfis):

[0099] These cells were derived from the human monocytic cell line THP-1 by stable integration of two inducible reporter constructs. They enable simultaneous study of the two main signaling pathways for STING: the NF-κB pathway, by monitoring the activity of secreted embryonic alkaline phosphatase (SEAP); and the IRF pathway, by assessing the activity of a secreted luciferase (Lucia).

[0100] Both reporter proteins are readily measurable in the cell culture supernatant when using QUANTI-Blue™ (InvivoGen catalog code: rep-qb1), a SEAP detection reagent that turns purple/blue in the presence of SEAP (quantified by measuring the optical density from 620 nm to 655 nm), and QUANTI-Luc™ (InvivoGen; catalog code: rep-q1c1), a luminometric enzyme assay that measures luciferase expression to report on ISG54 expression (as an indicator of IFN-α/β production).

[0101] Lucia ISG Cell Lines:

[0102] Each of the following three cell lines expresses a secreted luciferase (Lucia) reporter gene under control of an IRF-inducible promoter. This composite promoter comprises five IFN-stimulated response elements (ISREs) fused to a minimal promoter of the human ISG54 gene, which is unresponsive to activators of the NF-kB or AP-1 pathways. Hence, these cells enable monitoring of the IRF pathway based on luciferase (Lucia) activity.

[0103] In the present invention, monitoring of the IRF pathway is used to measure the STING agonist activity of the subject cyclic dinucleotides. [0104] 1. RAW-Lucia™ ISG (catalog code: rawl-isg): These cells were generated from the murine RAW 264.7 macrophage cell line. [0105] 2. RAW-Lucia™ ISG-KO-STING (catalog code: rawl-kostg): These cells were generated from the RAW-Lucia™ ISG54 cell line (see above), through stable homozygous knockout of the STING gene.

[0106] Blue™ Cell Lines:

[0107] Each of the following three cell lines expresses a SEAP reporter gene under a promoter: either I-ISG54, which comprises the IFN-inducible ISG54 promoter enhanced by a multimeric ISRE; or the IFN-β minimal promoter fused to five NF-κB (and five AP-1) binding sites. Stimulation of these cells with interferons, or inducers of type I interferons or of the NF-κB pathway, triggers activation of the I-ISG54 promoter (and consequently, production of SEAP) or of the IFN-β minimal promoter (and consequently, production of TNF-α). The levels of SEAP in the supernatant can be easily determined using QUANTI-Blue™ (InvivoGen catalog code: rep-qb1), a reagent that turns purple/blue in the presence of SEAP, by measuring the optical density from 620 nm to 655 nm. [0108] 1. B16-Blue™ ISG (catalog code: bb-ilhabg): These cells are derived from the murine B16 F1 melanoma cell line. Production of Type I interferons in these cells is measured using QUANTI-Blue™. [0109] 2. B16-Blue™ ISG-KO-STING (catalog code: bb-kostg): These cells were generated from the B16-Blue™ ISG cell line (see above), through stable homozygous knockout of the STING gene. Production of Type I interferons in these cells is measured using QUANTI-Blue™.

Quantification of IL-6 in Experiments

[0110] Interleukin-6 was quantified using an enzyme-linked immunoassay (ELISA) according to the manufacturer's instructions (R&D Systems).

In Cell Cultures

[0111] In various experiments in which different cell cultures were separately incubated with a cyclic dinucleotide, the cyclic dinucleotide induced production of Type I interferons and/or pro-inflammatory cytokines in those cells, as indirectly determined by an ISG54 (interferon-stimulated gene) reporter assay (Fensterl, White, Yamashita, & Sen, 2008). These experiments were performed as described below.

Example 1: Measuring Cytokine Induction in Treated Cell Cultures

[0112] Cytokine reporter cell lines used: THP1-Dual™ [0113] Cyclic dinucleotides tested: CL602, CL604, CL606, CL609, CL611, CL614, CL647, CL655, CL656 and CL659 [0114] Reference compound: c-AIMP [0115] Cytokines evaluated: IFN-α/β

[0116] To each well of a flat-bottom 96-well plate were added 20 μL of a solution a cyclic dinucleotide (100 μg/mL in sterile water), followed by 180 μL of a suspension of a single cell line (THP1-Dual™: ca. 100,000 cells per well). The plate was incubated for 18 h to 24 h at 37° C. in 5% CO.sub.2. The level of IFN-α/β in each well was indirectly quantified using QUANTI-Luc™ (as an indicator of IFN-β production), which was prepared and used according to the manufacturer's instructions (InvivoGen).

[0117] The results from this experiment are shown in FIG. 2, which illustrates that each one of the tested cyclic dinucleotides induces production of Type I interferons in THP1 cells.

Cytokine Induction Activity is STING-Dependent

[0118] The cyclic dinucleotides disclosed in the present invention do not induce cytokine production in vitro in the supernatant of cells that lack the receptor STING.

[0119] In an experiment in which wild-type (WT) reporter cells and homozygous STING knockout (SKO) reporter cells were each separately incubated with the cyclic dinucleotide for 18 h to 24 h, the cyclic dinucleotide induced production of Type I interferons in the WT cells but not in the STING KO cells. This finding demonstrated that STING is required for the cytokine-induction activity of the cyclic dinucleotide in vitro in cells. These experiments were performed as described below:

Example 2: Measuring Cytokine Induction in CDN-Treated Wild-Type or STING Knockout Cells

[0120] Cyclic dinucleotides tested: CL604, CL609, CL614, CL647, CL655 and CL656 [0121] Reference compounds: c-AIMP [0122] Cytokines evaluated: IFN-α/β [0123] Cell lines used: RAW-Lucia™ ISG, RAW-Lucia™ ISG-KO-STING, B16-Blue™ ISG, and B16-Blue™ ISG-KO-STING (depending on experiment)

[0124] To each well of a flat-bottom 96-well plate were added 20 μL of a solution a cyclic dinucleotide (100 μg/mL in sterile water), followed by 180 μL of a suspension of a single cell line (RAW-Lucia™ ISG: ca. 100,000 cells per well; B16-Blue™ ISG: ca. 50,000 cells per well). The plate was incubated for 18 h to 24 h at 37° C. in 5% CO.sub.2. For the RAW cell lines, the level of IFN-α/β in each well was indirectly quantified using QUANTI-Luc™ (as an indicator of IFN-β production), which was prepared and used according to the manufacturer's instructions. For the B16 cell lines, the level of IFN-α/β in each well was indirectly quantified using QUANTI-Blue™, as described above.

[0125] The results from this experiment are shown in FIGS. 3 and 4, which reveal three important findings. Firstly, each one of the tested cyclic dinucleotides induces production of Type I interferons in WT B16 (FIG. 3) and WT RAW (FIG. 4) cells. Secondly, none of the compounds exhibits this activity in STING knockout B16 (FIG. 3) or STING knockout RAW (FIG. 4) cells, thereby indicating that this activity requires the presence of STING. Lastly, the majority of the fluorinated cyclic dinucleotides are more active than is the reference compound (c-AIMP), as observed in the WT B16 (FIG. 3) and WT RAW (FIG. 4) cells.

In Vivo Cytokine Induction

[0126] The cyclic dinucleotides disclosed in the present invention induce cytokines in vivo in mice.

Example 3: Measuring Cytokine Induction in CDN-Treated Mice

[0127] Species evaluated: mouse [0128] Cyclic dinucleotides tested: CL604, CL606, CL609, CL611 and CL614 [0129] Reference compound: c-AIMP and saline [0130] Cytokines evaluated: IFN-α/β (using RAW ISG54 reporter cells) and IL-6 (by ELISA)

[0131] Twenty-one mice (Swiss; female; mean age: 8 weeks) were divided into seven groups of three: one group served as control (saline) and the other six groups were each treated with a cyclic dinucleotide (either c-AIMP, CL604, CL606, CL609, CL611 or CL614). On Day −7, blood samples for basal cytokine levels were collected from all mice and stored at −20° C. until analysis. On Day 1, the mice were treated with either 200 μL of physiologic serum (containing 0.9% NaCl) or 200 μL of a solution of a cyclic dinucleotide (dose: 10 mg/kg) in physiologic serum (containing 0.9% NaCl), by intravenous (i.v.) injection. Blood samples were collected from the mice at 4 h post-injection, and then stored at −20° C. until analysis. Cytokine induction was measured in the sera from the blood samples.

[0132] The results from this experiment are shown in FIGS. 5 and 6, which reveal two important findings: firstly, at the indicated dose, within 4 h post-treatment, all of the tested cyclic dinucleotides except CL611 strongly induced Type I interferons (FIG. 5) in mice; and secondly, all of the cyclic dinucleotides except CL611 induced IL-6 (FIG. 6).

In Vivo Efficacy of c-AIMP Combined with Gemcitabine

[0133] In experiments in which animal models of pancreatic cancer were treated with either gemcitabine monotherapy, c-AIMP monotherapy or chemoimmunotherapy (gemcitabine combined with c-AIMP), those animals that had received the combination therapy exhibited the greatest shrinkage in tumor volume, the lowest incidence of metastasis and/or the lowest mortality by the end of the experiment. Interestingly, in hamsters with bilateral subcutaneous pancreatic tumors, treatment of the right-flank tumor with chemoimmunotherapy (gemcitabine combined with c-AIMP) led to shrinkage of it as well as of the left-flank (distal) tumor.

[0134] The aforementioned experiments were performed as described below:

Example 4: In Vivo Efficacy of Gemcitabine Combined with c-AIMP in a Murine Model of Pancreatic Cancer

[0135] Species evaluated: mouse [0136] Tumor model: Panc02 (murine pancreatic tumor cell line) [0137] Treatments tested: gemcitabine monotherapy, c-AIMP monotherapy, and gemcitabine combined with c-AIMP [0138] Clinical parameters evaluated: tumor volume, incidence of metastasis and mortality [0139] Administration routes evaluated: intravenous (i.v.) or intratumoral (i.t.) injection (depending on experiment) [0140] On Day 1, 30 mice (C57BL/6; male) received an orthotopic injection of Panc02 tumor cells (1×10.sup.6) in their pancreas. The mice were then divided into six groups of five animals. Each group received a different treatment, as outlined below: [0141] Group 1: saline (by i.v. injection) on Days 7, 10, 14, 17, 21 and 24; [0142] Group 2: gemcitabine monotherapy (100 mg/kg; i.p.); on Days 7, 10, 14, 17, 21 and 24; [0143] Group 3: c-AIMP monotherapy (25 mg/kg; i.t.) on Days 7 and 21; [0144] Group 4: c-AIMP monotherapy (25 mg/kg; i.v.) on Days 7, 14 and 21; [0145] Group 5: c-AIMP (25 mg/kg; i.t.) followed (5 h later) by gemcitabine (100 mg/kg; i.p.) on Day 7; and gemcitabine (100 mg/kg; i.p.) on Days 10, 14, 17, 21 and 24; [0146] Group 6: c-AIMP (25 mg/kg; i.v.) followed (5 h later) by gemcitabine (100 mg/kg; i.p.) on Day 7; and gemcitabine (100 mg/kg; i.p.) on Days 10, 14, 17, 21 and 24;

[0147] At days 7, 21/24, 28 and 34, the mice were assessed for tumor volume, incidence of metastasis and mortality.

TABLE-US-00002 TABLE 2 Incidence of metastasis in a murine model of Panc02 tumors. The mice were treated with saline (control), gemcitabine monotherapy, c-AIMP monotherapy, or gemcitabine combined with c-AIMP. All data from Day 34, except those for Group 1 (Day 28). GemC: gemcitabine; i.t.: intratumoral; i.v.: intravenous. INCIDENCE OF TREATMENT GROUP METASTASIS Group 1: Saline 100% Group 2: GemC 50% Group 3: cAIMP (i.t.) 0% Group 4: cAIMP (i.v.) 0% Group 5: cAIMP (i.t.) + GemC 0% Group 6: cAIMP (i.v.) + GemC 40%

TABLE-US-00003 TABLE 3 Mortality in a murine model of Panc02 tumors. The mice were treated with saline (control), gemcitabine monotherapy, c-AIMP monotherapy, or gemcitabine combined with c- AIMP. All data from Day 34, except those for Group 1 (Day 28). PRE-SACRIFICE TREATMENT GROUP MORTALITY Group 1: Saline 100% Group 2: GemC 20% Group 3: cAIMP (i.t.) 0% Group 4: cAIMP (i.v.) 0% Group 5: cAIMP (i.t.) + GemC 0% Group 6: cAIMP (i.v.) + GemC 0%

[0148] The results from this experiment are shown in FIG. 7 and in Tables 2 and 3. FIG. 7 reveals that among all of the treatments tested, the most effective ones at reducing tumor growth were c-AIMP monotherapy and the two combination treatments (gemcitabine plus c-AIMP [i.v. or i.t.]). Table 2 indicates that among the six treatment groups, the lowest incidences of metastasis were found in all four groups that had received c-AIMP (either alone or in combination with gemcitabine). Likewise, Table 3 shows that in these same four groups, the pre-sacrifice mortality rate by Day 34 was 0%, compared to 20% for the gemcitabine monotherapy group and 100% (by Day 28) for the saline group.

Example 5: In Vivo Efficacy of c-AIMP Combined with Gemcitabine in a Hamster Model of Pancreatic Cancer (Orthotopic Tumor)

[0149] Species evaluated: hamster [0150] Tumor model: PC-1.0 (hamster pancreatic tumor cell line (Egami, Tomioka, Tempero, Kay, & Pour, 1991)) [0151] Treatments tested: gemcitabine monotherapy, and gemcitabine combined with c-AIMP [0152] Clinical parameters evaluated: tumor volume, incidence of metastasis and mortality [0153] Administration routes evaluated: intravenous (i.v.) vs. intratumoral (i.t.) injection (depending on experiment) for CL592 [0154] On Day 1, 22 hamsters (Golden Syrian) received an orthotopic injection of PC-1.0 tumor cells (1×10.sup.6) in the tail of their pancreas. The hamsters were then divided into four groups of five or six animals. Each group received a different treatment, as outlined below: [0155] Group 1 (n=5) received saline (by i.v. injection) on Days 8, 15 and 22; [0156] Group 2 (n=6): c-AIMP (25 mg/Kg; i.v.) followed by gemcitabine (50 mg/Kg; i.p.) on Day 8; and gemcitabine (50 mg/Kg; i.p.) on Days 15 and 22; [0157] Group 3 (n=6): c-AIMP (25 mg/Kg; i.t.) followed by gemcitabine (50 mg/Kg; i.p.) on Day 8; and gemcitabine (50 mg/Kg; i.p.) on Days 15 and 22; [0158] Group 4 (n=5): gemcitabine monotherapy (50 mg/Kg; i.p.) at Days 8, 15 and 22.

[0159] At days 8, 21/24, 28 and 34, the mice were assessed for tumor volume, incidence of metastasis and mortality.

TABLE-US-00004 TABLE 4 Incidence of metastases in a hamster model of orthotopic PC-1.0 tumors. The hamsters were treated with saline, gemcitabine monotherapy, or a combination of c-AIMP and gemcitabine. GemC: gemcitabine; i.t.: intratumoral; i.v.: intravenous. INCIDENCE TREATMENT GROUP OF METASTASES Group 1: Saline 100% Group 2: cAIMP (i.v.) + GemC 0% Group 3: cAIMP (i.t.) + GemC 0% Group 4: GemC 100%

TABLE-US-00005 TABLE 5 Number of metastases in a hamster model of orthotopic PC-1.0 tumors. The hamsters were treated with saline, gemcitabine monotherapy, or a combination of c-AIMP and gemcitabine. GemC: gemcitabine; i.t.: intratumoral; i.v.: intravenous. TREAT- MENT NUMBER OF METASTASES PER HAMSTER GROUP Hamster 1 Hamster 2 Hamster 3 Hamster 4 Hamster 5 Group 1: 28 10 17 3 20 Saline Group 2: 0 0 0 0 0 cAIMP (i.v.) + GemC Group 3: 0 0 0 0 0 cAIMP (i.t.) + GemC Group 4: 13 15 3 10 5 GemC

[0160] The results from this experiment are shown in FIGS. 8 and 9, and in Tables 4 and 5. FIG. 8 reveals that among the four treatments tested, both combination therapies were better at reducing tumor growth than was gemcitabine monotherapy, and that the better of the combination therapies was gemcitabine plus c-AIMP (i.t.). Similarly, FIG. 9 illustrates that gemcitabine plus c-AIMP (i.t.) provided the highest survival rate. Table 4 shows that none (0% incidence) of the hamsters in the two combination-treatment groups exhibited any metastases, whereas all (100% incidence) of the hamsters in both the gemcitabine monotherapy group and the saline group exhibited metastases. Table 5 lists the number of metastases per hamster in each group, showing a value of zero for every hamster in the two combination-treatment groups.

Example 6: In Vivo Efficacy of c-AIMP Combined with Gemcitabine in a Hamster Model of Subcutaneous Pancreatic Tumors (Bilateral)

[0161] Species evaluated: hamster [0162] Tumor model: PC-1.0 (see above) [0163] Treatments tested: gemcitabine monotherapy, c-AIMP monotherapy, and gemcitabine combined with c-AIMP [0164] Clinical parameters evaluated: tumor volume at right (treated) flank, incidence of metastasis and mortality [0165] Administration routes evaluated: intravenous (i.v.) or intratumoral (i.t.) injection (depending on experiment) [0166] On Day 1, 25 hamsters (Golden Syrian) received a subcutaneous injection of PC-1.0 cells (1×10.sup.6) in the right flank. On Day 6, the hamsters received a subcutaneous injection of PC-1.0 cells (1×10.sup.5) in their left flank. On Day 7, 25 of the hamsters were randomly assigned (based on right-flank tumor size) to groups of five animals each. Each group received a different treatment, as outlined below: [0167] Group 1: saline (i.t.) on Day 8; [0168] Group 2: saline (i.t.) followed (3 h later) by gemcitabine (50 mg/kg; i.p.) in saline on Day 8; gemcitabine (50 mg/kg; i.p.) in saline on Days 15 and 22; [0169] Group 3: c-AIMP (25 mg/kg; intratumoral injection in right-flank tumor) on Day 8 and, if a tumor was present, on Day 22; [0170] Group 4: c-AIMP (25 mg/kg; intratumoral injection in right-flank tumor) followed (3 h later) by gemcitabine (50 mg/kg; i.p.) on Day 8; gemcitabine (50 mg/kg; i.p.) on Day 15; if a tumor was present, c-AIMP (25 mg/kg; intratumoral injection in right-flank tumor) on Day 22 and in all cases, gemcitabine (50 mg/kg; i.p.) on Days 15 and 22.

[0171] The results from this experiment are shown in FIG. 10, which reveals that over the course of the experiment, the most effective treatment at reducing tumor growth was the combination of gemcitabine and c-AIMP. In fact, the hamsters treated with this combination treatment exhibited the smallest tumor volume at all time points measured except for one (Day 11 post-injection).

Example 7: Comparison of Gemcitabine with an Intercalated Combination of CL592 and Gemcitabine in an Orthotopic Murine Model of Pancreatic Cancer

[0172] Tumor line evaluated: DT6606 (Partecke, 2011) [0173] Treatment tested: intercalated combination of CL592 and gemcitabine [0174] Reference compound: gemcitabine [0175] Parameter evaluated: tumor growth

[0176] On Day 1, 20 mice (C57/BL6; female; 10 weeks old; 18 g to 22 g) received an intrapancreatic injection of DT6606 cells (5×10.sup.5 cells in 30 μL serum-free medium). One mouse was sacrificed before treatment due to a renal deformation. The remaining mice were divided into four groups (n=5, except for Group 2: n=4), as shown in the table below:

TABLE-US-00006 Group Treatment 1 saline (control) 2 gemcitabine 3 CL 592 4 CL592 + gemcitabine

[0177] On Day 13, tumor growth was confirmed in all the mice and the volume of each tumor was measured. The groups were then treated according to the treatment regimen below.

Treatment Regimen

[0178] Day 13: Groups 1 and 2 received an intratumoral injection of saline solution (50 μL), and Groups 3 and 4, an intratumoral injection of CL592 (50 μL; 2.5 mg/mL in saline buffer;).

[0179] Day 16: Groups 2 and 4 received an intraperitoneal tail injection of gemcitabine (100 μL solution/100 g body mass; 10 mg/mL in saline buffer; dose: 100 mg/kg).

[0180] Day 20: Groups 3 and 4 received an intravenous tail injection of CL592 (200 μL; 0.5 mg/mL in saline buffer; dose: 5 mg/kg).

[0181] Day 23: Groups 2 and 4 were treated as on Day 16.

[0182] Day 27: Groups 3 and 4 were treated as on Day 20.

[0183] Day 30: Groups 2 and 4 were treated as on Days 16 and 23.

[0184] Day 36: Each mouse was checked for tumor presence. The volume of each observed tumor was measured and the mice were then sacrificed.

[0185] Tumor growth (expressed as a percentage) was calculated as follows:

(([tumor volume at day 36]−[pre-treatment tumor volume])/[pre-treatment tumor volume])×100%

[0186] The principal result from this experiment is shown in FIG. 11, which reveals that the intercalated combination of CL592 and gemcitabine was markedly more effective at stopping tumor growth than was gemcitabine monotherapy. Specifically, by the end of the experiment (Day 36), the tumors in the combination group had shrunk drastically (mean growth: −94%), whereas those in the gemcitabine group had actually grown slightly (mean growth: 22%).

Example 8: Evaluation of Different Intercalated Combinations of a CDN and Gemcitabine in an Orthotopic Murine Model of Pancreatic Cancer

[0187] Species evaluated: mouse [0188] Tumor model: Panc02 [0189] Treatment tested: intercalated combinations of a CDN (either CL592, CL614 or CL656) and gemcitabine [0190] Reference compounds: gemcitabine, CL592, CL614 and CL656 [0191] Parameters evaluated: tumor growth, and incidence of metastases

[0192] On Day 1, 55 mice (C57/BL6; male; 10 weeks old; 23 g to 25 g) each received an intrapancreatic injection of Panc02 cells (1×10.sup.6 cells in 50 μL serum-free medium). The mice were divided into eight groups, as shown in the table below:

TABLE-US-00007 Number Group Treatment of mice 1 saline (control) 8 2 gemcitabine 8 3 CL592 5 4 CL614 5 5 CL656 5 6 CL592 + gemcitabine 8 7 CL614 + gemcitabine 8 8 CL656 + gemcitabine 8

[0193] The groups were treated according to the treatment regimen below.

Treatment Regimen

[0194] Day 9: Groups 3 to 8 each received an intratumoral injection of the appropriate CDN (CL592, CL614 or CL656, respectively; 50 μL; 5 mg/kg in 0.9% saline)

[0195] Day 12: Group 2 and Groups 6 to 8 each received an intraperitoneal injection of gemcitabine (200 μL; 100 mg/kg in 0.9% saline)

[0196] Day 16: Groups 3 to 8 each received an intravenous injection of the appropriate CDN (CL592, CL614 or CL656, respectively; 50 μL; 5 mg/kg in 0.9% saline)

[0197] Day 19: Group 2 and Groups 6 to 8 were treated as on Day 12.

[0198] Day 23: Groups 3 to 8 were treated as on Day 16.

[0199] Day 26: Group 2 and Groups 6 to 8 were treated as on Days 12 and 19.

[0200] Day 30: Each mouse was checked for tumor presence and metastases. The volume of each observed tumor was measured, any observed metastases were counted and then, the mice were sacrificed.

[0201] The principal results from this experiment are shown in FIG. 12 and Table 6, which reveal that the intercalated combination of any one of the CDNs and gemcitabine was markedly more effective at stopping tumor growth (FIG. 12) and preventing metastasis (Table 6) than was any of the tested single reference compounds (gemcitabine, CL592, CL614 or CL656). Specifically, by the end of the experiment (Day 30), the mean tumor volume in each combination group (Groups 3: 1.3 mm.sup.3±2.2 mm.sup.3; Group 4: 12.6 mm.sup.3±21.7 mm.sup.3; and Group 5: 26.1 mm.sup.3±55.3 mm.sup.3) was hundreds of times smaller than that of the gemcitabine group (380.7 mm.sup.3±140.9 mm.sup.3), the CL592 group (231.0 mm.sup.3±90.0 mm.sup.3), the CL614 group (318.6 mm.sup.3±93.8 mm.sup.3), the CL656 group (340.2 mm.sup.3±210. mm.sup.3) or the saline group (854.4 mm.sup.3±784.1 mm.sup.3).

[0202] Interestingly, the results from this and another experiment on Panc02 in mice provide important insight on the dosage of CL592 to be used: at lower doses (5 mg/kg; Example 8), the combination of gemcitabine and CL592 provides a clear beneficial effect relative to either component alone, whereas at a far higher dose (25 mg/kg; Example 4), this effect is less pronounced. This observation could ultimately have crucial implications for development of a clinical treatment regimen based on our proposed combination of gemcitabine and a CDN STING agonist: for example, in trying to maximize the efficacy of the combination while minimizing the respective toxicity of each component.

TABLE-US-00008 TABLE 6 Incidence of metastases in mice implanted with orthotopic Panc02 pancreatic tumors. The number of animals with metastasis and the average number of metastases per animal at Day 30 post-implantation were calculated for each group. Note that one of the mice in Group 1 had died before Day 30. # Mice with Average # metastases Group Treatment metastases per mouse 1 saline (control) 100% (7/7)  17 2 gemcitabine 88% (7/8) 13 3 CL592 20% (1/5) 2 4 CL614 40% (2/5) 5 5 CL656 40% (2/5) 3 6 CL592 + gemcitabine  0% (0/8) 0 7 CL614 + gemcitabine  0% (0/8) 0 8 CL656 + gemcitabine  0% (0/8) 0

REFERENCES

[0203] 1. Abdul-Sater, A. A. et al. Cyclic-di-GMP and cyclic-di-AMP activate the NLRP3 inflammasome. EMBO Rep 14, 900-6 (2013). [0204] 2. Ablasser, A. et al. cGAS produces a 2′-5′-linked cyclic dinucleotide second messenger that activates STING. Nature 498, 380-4 (2013). [0205] 3. Bundgaard, H. Design of prodrugs (Elsevier; Sole distributors for the USA and Canada, Elsevier Science Pub. Co., Amsterdam; New York; New York, N.Y., USA, 1985). [0206] 4. Chandra, D. et al. STING ligand c-di-GMP improves cancer vaccination against metastatic breast cancer. Cancer Immunol Res 2, 901-10 (2014). [0207] 5. Danilchanka, O. & Mekalanos, J. J. Cyclic dinucleotides and the innate immune response. Cell 154, 962-70 (2013). [0208] 6. Diner, E. J. et al. The innate immune DNA sensor cGAS produces a noncanonical cyclic dinucleotide that activates human STING. Cell Rep 3, 1355-61 (2013). [0209] 7. Downey, C. M., Aghaei, M., Schwendener, R. A. & Jirik, F. R. DMXAA causes tumor site-specific vascular disruption in murine non-small cell lung cancer, and like the endogenous non-canonical cyclic dinucleotide STING agonist, 2′3′-cGAMP, induces M2 macrophage repolarization. PLoS One 9, e99988 (2014). [0210] 8. Dubensky, T. W., Jr., Kanne, D. B. & Leong, M. L. Rationale, progress and development of vaccines utilizing STING-activating cyclic dinucleotide adjuvants. Ther Adv Vaccines 1, 131-43 (2013). [0211] 9. Fensterl, V., White, C. L., Yamashita, M. & Sen, G. C. Novel characteristics of the function and induction of murine p56 family proteins. J Virol 82, 11045-53 (2008). [0212] 10. Foundation, H. (2014). [0213] 11. Fritz, J. et al. In vitro immunomodulatory properties of gemcitabine alone and in combination with interferon-alpha. Immunol Lett 168, 111-9 (2015). [0214] 12. Gomelsky, M. cAMP, c-di-GMP, c-di-AMP and now cGMP: bacteria use them all! Mol Microbiol 79, 562-5 (2011). [0215] 13. Hirooka, Y. et al. A combination therapy of gemcitabine with immunotherapy for patients with inoperable locally advanced pancreatic cancer. Pancreas 38, e69-74 (2009). [0216] 14. Ishikawa, H., Ma, Z. & Barber, G. N. STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity. Nature 461, 788-92 (2009). [0217] 15. Karaolis, D. K. et al. 3′,5′-Cyclic diguanylic acid (c-di-GMP) inhibits basal and growth factor-stimulated human colon cancer cell proliferation. Biochem Biophys Res Commun 329, 40-5 (2005). [0218] 16. Kimura, Y. et al. Clinical and immunologic evaluation of dendritic cell-based immunotherapy in combination with gemcitabine and/or S-1 in patients with advanced pancreatic carcinoma. Pancreas 41, 195-205 (2012). [0219] 17. Klarquist, J. et al. STING-Mediated DNA Sensing Promotes Antitumor and Autoimmune Responses to Dying Cells. J Immunol (2014). [0220] 18. Li, L. et al. Hydrolysis of 2′3′-cGAMP by ENPP1 and design of nonhydrolyzable analogs. Nat Chem Biol 10, 1043-8 (2014). [0221] 19. Malvezzi, M., Bertuccio, P., Levi, F., La Vecchia, C. & Negri, E. European cancer mortality predictions for the year 2013. Ann Oncol 24, 792-800 (2013). [0222] 20. Mitzel, D. N., Lowry, V., Shirali, A. C., Liu, Y. & Stout-Delgado, H. W. Age-enhanced endoplasmic reticulum stress contributes to increased Atg9A inhibition of STING-mediated IFN-beta production during Streptococcus pneumoniae infection. J Immunol 192, 4273-83 (2014). [0223] 21. Miyabe, H. et al. A new adjuvant delivery system ‘cyclic di-GMP/YSK05 liposome’ for cancer immunotherapy. J Control Release 184, 20-7 (2014). [0224] 22. Murugesan, S. R. et al. Combination of human tumor necrosis factor-alpha (hTNF-alpha) gene delivery with gemcitabine is effective in models of pancreatic cancer. Cancer Gene Ther 16, 841-7 (2009). [0225] 23. Nishida, S. et al. Wilms tumor gene (WT1) peptide-based cancer vaccine combined with gemcitabine for patients with advanced pancreatic cancer. J Immunother 37, 105-14 (2014). [0226] 24. Ohkuri, T. et al. STING contributes to anti-glioma immunity via triggering type-I IFN signals in the tumor microenvironment. Cancer Immunol Res (2014). [0227] 25. Partecke, L. I. et al. A syngeneic orthotopic murine model of pancreatic adenocarcinoma in the C57/BL6 mouse using the Panc02 and 6606PDA cell lines. Eur Surg Res 47, 98-107 (2011). [0228] 26. Romling, U. & Amikam, D. Cyclic di-GMP as a second messenger. Curr Opin Microbiol 9, 218-28 (2006). [0229] 27. Shindo, Y. et al. Adoptive immunotherapy with MUC1-mRNA transfected dendritic cells and cytotoxic lymphocytes plus gemcitabine for unresectable pancreatic cancer. J Transl Med 12, 175 (2014). [0230] 28. Simm, R., Morr, M., Kader, A., Nimtz, M. & Romling, U. GGDEF and EAL domains inversely regulate cyclic di-GMP levels and transition from sessility to motility. Mol Microbiol 53, 1123-34 (2004). [0231] 29. Sloan, K. B. Prodrugs: topical and ocular drug delivery (M. Dekker, New York, 1992). [0232] 30. Society, A. C. (2014). [0233] 31. Suzuki, E., Kapoor, V., Jassar, A. S., Kaiser, L. R. & Albelda, S. M. Gemcitabine selectively eliminates splenic Gr-1+/CD11b+ myeloid suppressor cells in tumor-bearing animals and enhances antitumor immune activity. Clin Cancer Res 11, 6713-21 (2005). [0234] 32. Tomimaru, Y. et al. Synergistic antitumor effect of interferon-ss with gemcitabine in interferon-alpha-non-responsive pancreatic cancer cells. Int J Oncol 38, 1237-43 (2011). [0235] 33. Zhang, X. et al. Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING. Mol Cell 51, 226-35 (2013).