Novel th1-inducing adjuvant by combining different nucleic acid adjuvant and uses thereof

Abstract

To provide technologies for the induction of de novo Th1 responses, the induction of cytotoxic T cells, and anti-cancer and anti-allergic action.SOLUTION: Provided is a combination of a CpG oligonucleotide and a STING agonist. Alternatively, provided is a composition for use as a type I adjuvant comprising a STING agonist, the STING agonist being administered with a CpG oligonucleotide. Alternatively, provided is an anticancer agent comprising a CpG oligonucleotide, the CpG oligonucleotide being administered with a STING agonist. Also provided is a composition for reducing or eliminating the IgE-induced effect of a STING agonist comprising a CpG oligonucleotide.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the field of immunology, more particularly to a novel Th1-inducing adjuvant and its uses. [Background technology]

[0002] Adjuvants are immune enhancing agents added to vaccines to increase their effectiveness, and with recent advances in immunology, the mechanism of action of adjuvants is gradually becoming clear. Recently, due to the diverse immune control properties of adjuvants, they are expected to be used not only in the prevention and treatment of infectious diseases, but also in the prevention and treatment of allergies, cancer, and autoimmune diseases.

[0003] So far, most vaccine adjuvants have been developed to induce antibody production (humoral immunity). Therefore, most current adjuvants, including alum adjuvants, are humoral immunity-inducing adjuvants called Th2 adjuvants (type II adjuvants). However, in the prevention and treatment of cancer and allergies, it is more important to induce cellular immunity than humoral immunity, and these adjuvants are called Th1 adjuvants (type I adjuvants) (Figure 9).

[0004] To date, many candidate substances for Th1 adjuvants have been reported, but CpG oligonucleotides (CpG ODN) are considered to be the most effective. CpG ODN has been shown to be effective as a vaccine adjuvant for cancer and infectious diseases, and efforts are currently being made to develop and improve even more effective CpG ODN (Non-Patent Documents 1 and 2). If an effective improved CpG ODN can be developed, it may be applied to the treatment of cancer and allergies more than ever before. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Tougan, T. TLR9 adjuvants enhance immunogenicity and protective efficacy of the SE36 / AHG malaria vaccine in nonhuman primate models. Hum Vaccin Immunother. 2013, 9(2):283-90. [Non-Patent Document 2] Kobiyama, K. et. al. Nonagonistic dectin-1 ligand transforms CpG into a multitask nanoparticulate TLR9 agonist. Proc Natl Acad Sci US A. 2014, 111(8):3086-91. Summary of the Invention [Means for solving the problem]

[0006] The present inventors have analyzed the induction of Th1 responses, induction of cytotoxic T cells, and anti-cancer and anti-allergic effects by the combination of CpG ODN and STING agonist, and found that the specific combination of the present invention can be used to induce Th1 immune responses (Fig. 1A), to prevent and treat cancer (Fig. 5), and to prevent and treat allergies (Fig. 8). It was also found that it enables the switch between type I immune responses (cellular immunity) and type II immune responses (humoral immunity) (Fig. 11).

[0007] The present inventors attempted to overcome the problems that K3 CpG and cGAMP have individually by combining K3 CpG and 3'3'-cGAMP. The present inventors investigated the immunological characteristics, efficacy as a vaccine adjuvant, and potential of this combination as an antitumor immunotherapy, as well as the mechanism of action in vitro and in vivo. The effect of combining K3 CpG and cGAMP in vitro was analyzed using human and mouse PBMCs. Furthermore, the effect of this combination was analyzed in vivo through an immunization model and by measuring the induction of antigen-specific T cell and B cell responses after combined immunization. Finally, the present inventors evaluated the ability of the combined K3 CpG and cGAMP to suppress tumor growth in a mouse tumor model. Our results show that the combination of K3 CpG and cGAMP creates a potent type 1 adjuvant and a promising immunotherapeutic agent for cancer.

[0008] The inventors found that the combination of CpG ODN and a STING agonist synergistically induced a Th1-type immune response (Figure 10), suppressed IgE production induced as a side effect by the STING agonist, and required MyD88 signaling and STING-type I interferon signaling for Th1 responses. They also found that simultaneous administration of CpG ODN and a STING agonist effectively suppressed cancer cell proliferation in vivo in cancer-bearing model mice (Figure 5), and stimulated human peripheral blood mononuclear cells (PBMCs) and potently induced IFN-γ, an indicator of a Th1 response (Figure 1A).

[0009] Various STING agonists were also evaluated, and it was found that the endogenous STING agonist 2'3'-cGAMP, the bacterial-derived 3'3'-cGAMP and c-di-GMP, as well as the synthetic STING agonist DMXAA, all induced Th1-type immune responses when administered together with CpG to mice.

[0010] These results clarified that the combination of TLR9 signaling and STING-type I interferon signaling by CpG ODN is an important signal that induces a strong Th1-type immune response, and suggested that a similar effect is observed in humans. By using these as adjuvants, it is thought that vaccine therapy for cancer and allergies, which was previously difficult, will become possible.

[0011] Therefore, the present invention provides a method for inducing a Th1 adjuvant that is more effective than conventional methods by combining CpG ODN with another nucleic acid adjuvant (STING agonist), and further provides a novel method for treating cancer or allergies. The present invention relates to a marker, detection agent, inhibitor, etc. for identifying the Th1 response induced by the combination of an adjuvant, such as a signal transduction system factor or its expression product or a fragment or derivative thereof related to the induction of Th1 responses by the combination of CpG ODN and a STING agonist, and an anti-cancer or anti-allergic effect, and a composition for preventing or treating cancer or allergies.

[0012] Thus, agonists (STING agonists or STING ligands) for TLR9 and stimulator of IFN genes (STING) can function as vaccine adjuvants. However, currently available CpG ODN (K / B type) weakly induce IFN, and STING agonists induce type 2 immune responses, limiting their potential therapeutic applications. In this invention, we found a strong synergy between TLR9-agonists and STING-agonists. Combining them, we succeeded in creating an effective type 1 adjuvant and anti-cancer drug. Our in vitro studies suggest that the synergy between CpG ODN (e.g., K3) and STING agonists (e.g., cGAMP), which results in the production of IFNγ (type II IFN) in NK cells, is due to the simultaneous action of IL-12 and type I IFN, and is separately controlled by IRF3 / 7, STING, and MyD88. Our in vivo immunization model induced CD8 + As exemplified by the promotion of T cell responses, we have revealed that the combination of cGAMP and CpG ODN functions as a strong type 1 adjuvant that can strongly induce Th1-type responses. In our mouse tumor model, intratumoral injection of CpG ODN and cGAMP together significantly reduced tumor size compared to treatment alone, and CpG ODN and cGAMP functioned as antigen-free anticancer agents. Thus, the combination of CpG ODN and STING agonist provides therapeutic application as a strong type II IFN inducer. Thus, the present invention provides the following. <Inventions series focusing on "combinations"> (1) A combination of a CpG oligonucleotide and a STING agonist. (2) The combination according to item 1, which is for use as a type I adjuvant. (3) The combination according to item 1 or 2, which is for suppressing the IgE-inducing effect of the STING agonist. (4) The combination according to any one of items 1 to 3, wherein the CpG oligonucleotide is a K / B type oligonucleotide. (5) The combination according to any one of items 1 to 4, wherein the CpG oligonucleotide is a CpG oligonucleotide selected from the group consisting of K3 CpG (SEQ ID NO: 1 = 5'-atcgactatcgagagttctc-3'), CpG1826 (SEQ ID NO: 2 = 5'-tccatgacgttcctgacgtt-3'), and D35 CpG (SEQ ID NO: 3 = 5'-ggtgcatcgatgcagggggg-3'). (6) The combination according to any one of items 1 to 5, wherein the STING agonist is selected from cGAMP, 3'3'-cGAMP, c-di-GAMP, c-di-AMP, 2'3'-cGAMP, and DMXAA. (7) The combination according to any one of items 1 to 6, which is for use as an anticancer agent. (8) The combination according to item 7, wherein the anticancer drug targets a cancer selected from lymphoma and melanoma. (9) The combination according to any one of items 1 to 8, which is for reducing or eliminating a type II immune response and for expressing or enhancing a type I immune response. (10) The combination according to any one of items 1 to 9, which is for inducing interferon-γ (IFN-γ). (11) The combination according to any one of items 1 to 10, which is for use as a vaccine adjuvant. (A1) A method for treating or preventing a disease in a subject, the method comprising administering to the subject an effective amount of a CpG oligonucleotide in combination with a STING agonist. (A2) The method according to item A1, wherein the combination is used as a type I adjuvant. (A3) The method according to item A1 or A2, wherein the CpG oligonucleotide is provided in an amount effective to inhibit the IgE-inducing effect of the STING agonist. (A4) The method according to any one of items A1 to A3, wherein the CpG oligonucleotide is a K / B type oligonucleotide. (A5) The method according to any one of items A1 to A4, wherein the CpG oligonucleotide is a CpG oligonucleotide selected from the group consisting of K3 CpG (SEQ ID NO: 1 = 5'-atcgactatcgagagttctc-3'), CpG1826 (SEQ ID NO: 2 = 5'-tccatgacgttcctgacgtt-3'), and D35 CpG (SEQ ID NO: 3 = 5'-ggtgcatcgatgcagggggg-3'). (A6) The method according to any one of items A1 to A5, wherein the STING agonist is a STING agonist selected from cGAMP, 3'3'-cGAMP, c-di-GAMP, c-di-AMP, 2'3'-cGAMP, and DMXAA. (A7) The method according to any one of items A1 to A6, wherein the treatment or prevention is directed to cancer. (A8) The method according to item A7, wherein the cancer is selected from lymphoma and melanoma. (A9) The method according to any one of items A1 to A8, wherein the treatment or prevention reduces or eliminates a type II immune response and expresses or enhances a type I immune response. (A10) The method according to any one of items A1 to A9, wherein the treatment or prevention induces interferon γ (IFN-γ). (A11) The method according to any one of items A1 to A10, wherein the combination is used as a vaccine adjuvant. <Series of inventions emphasizing the use of STING agonist itself as a type I adjuvant formulation> (12) A composition for use as a type I adjuvant comprising a STING agonist, wherein the STING agonist is administered together with a CpG oligonucleotide. (13) The composition according to item 12, having any one or more of the features of items 2 to 11. (A12) A method for exerting the type I adjuvant effect of a STING agonist, the method comprising the step of administering the STING agonist together with a CpG oligonucleotide. (A13) The method according to item A12, having any one or more of the features of items 2 to 11 or A2 to A11. <Series of inventions emphasizing the enhancing effect of STING agonist on the type I adjuvant formulation of CpG> (14) An enhancer for enhancing the action of a type I adjuvant of a CpG oligonucleotide, comprising a STING agonist. (15) The enhancer according to item 14, having any one or more of the features of items 2 to 11. (A14) A method for enhancing the action of a type I adjuvant of a CpG oligonucleotide, the method comprising the step of administering the CpG oligonucleotide together with a STING agonist. (A15) The method according to item A14, having any one or more of the features of items 2 to 11 or A2 to A11. <Series of inventions emphasizing the anti-cancer agent aspect of STING agonist> (16) An anti-cancer agent comprising a STING agonist, wherein the STING agonist is administered together with a CpG oligonucleotide. (17) The anti-cancer agent according to item 16, having any one or more of the features of items 2 to 11. (A16) A method for treating or preventing cancer, the method comprising: The step of administering an anti-cancer agent comprising a STING agonist together with a CpG oligonucleotide. (A17) The method according to item A16, having any one or more of the features of items 2 to 11 or A2 to A11. <Series of inventions emphasizing the anti-cancer agent aspect of CpG oligonucleotide> An anti-cancer agent containing a CpG oligonucleotide, wherein the CpG oligonucleotide is administered together with a STING agonist. The anti-cancer agent according to item 18, having any one or more of the features of items 2 to 11. (A18) A method for treating or preventing cancer, the method comprising the step of administering an anti-cancer agent containing a CpG oligonucleotide together with a STING agonist. (A19) The method according to item A18, having any one or more of the features of items 2 to 11 or A2 to A11. <Claim series emphasizing the inhibitory effect of CpG on the allergy (IgE) - inducing action of STING agonist> (20) A composition for reducing or eliminating the IgE - inducing action of a STING agonist, containing a CpG oligonucleotide. (21) The composition according to item 20, wherein the allergy - inducing action of the STING agonist is reduced or eliminated. (22) The composition according to item 20 or 21, having any one or more of the features of items 2 to 11. (A20) A method for reducing or eliminating the IgE - inducing action of a STING agonist, the method comprising the step of administering a CpG oligonucleotide when using the STING agonist. (A21) The method according to item A20, wherein the allergy - inducing action of the STING agonist is reduced or eliminated. (A22) The method according to item A20 or A21, having any one or more of the features of items 2 to 11 or A2 to A11.

[0013] In the present invention, it is intended that, in addition to the explicitly stated combinations, any one or more of the above - described features may be further provided in combination.

[0014] Still further embodiments and advantages of the present invention will be recognized by those skilled in the art by reading and understanding the following detailed description as needed. [[Effect of the Invention]]

[0015] The present invention provides information on novel adjuvants that induce Th1-type immune responses, which has been difficult to achieve until now, and the signals that induce them. Many of the current adjuvants are Th2 adjuvants that induce antibody production, but these adjuvants are not suitable for vaccine therapy for cancer or allergies. Furthermore, STING agonists induce IgE as a side reaction, and therefore may cause allergic inflammation. However, in the present invention, a strong Th1 adjuvant effect can be induced while suppressing IgE induction simply by combining two types of adjuvants. In addition, since STING agonists such as DMXAA themselves function as Th2 adjuvants that activate humoral immunity, similar to alum adjuvants, by using STING agonists as a platform, they can be used as a conventional humoral immunity-inducing Th2 adjuvant when used as is, and as a strong cellular immunity-inducing Th1 adjuvant when combined with CpG ODN (Figure 11). There are no examples of such versatile adjuvants that can be used against diseases of different qualities, and as a next-generation adjuvant, it has a ripple effect both technically and economically. [Brief description of the drawings]

[0016] [Figure 1A]Figure 1A shows the concentration of IFNγ produced in the supernatant of human PBMC cultures from each donor. From the left, no adjuvant, K3 CpG, K3 CpG+cGAMP, and cGAMP are shown. The vertical axis shows the concentration of IFNγ (ng / ml) in the culture supernatant. K3 CpG and cGAMP (TLR9-agonist and STING-agonist, respectively) synergistically induce innate IFNγ production by NK cells. Human PBMCs from two healthy donors were incubated with K3 CpG (10 μg / ml), cGAMP (10 μM), or K3 CpG+cGAMP for 24 hours, and the concentration of IFNγ in the supernatant was measured by ELISA. Data are representative of at least two independent experiments and are shown as the mean + SD of duplicates. *p<0.05; **p<0.01 (one-way ANOVA with Bonferroni's multiple comparison test). [Figure 1B] Figure 1B shows the results analyzed by flow cytometry. From the top, the panels show no adjuvant, K3 CpG, K3 CpG+cGAMP, and cGAMP. The left panel shows forward scatter (FSC) intensity of CD16+CD56+ natural killer cells (NK cells) on the vertical axis and fluorescence intensity of IFNγ-APC on the horizontal axis. The right panel shows fluorescence intensity of CD8-PE on the vertical axis and fluorescence intensity of IFNγ-APC on the horizontal axis. K3 CpG and cGAMP (TLR9-agonist and STING-agonist, respectively) synergistically induce the production of innate IFNγ by NK cells. Human PBMCs from three healthy donors were stimulated with K3 CpG, cGAMP, or K3 CpG+cGAMP for 16 hours, and the last 4 hours were stimulated in the presence of brefeldin A. After stimulation, cells were analyzed by flow cytometry to detect IFNγ-producing cells. The graph shows the percentage of IFNγ-producing CD3+CD8+ T cells, CD3+CD8- T cells (including CD4+ T cells) and CD3-CD56+CD16+ NK cells. [Figure 1C]Figure 1C shows the concentration of IFNγ produced in the supernatant of human PBMC cultures from each donor when treated with neutralizing antibodies. From the left, no adjuvant, K3 CpG, K3 CpG+cGAMP, and cGAMP are shown. From the left, each group shows isotype control, α-type I IFN, α-IL-12 / 23p40, and α-type I IFN+α-IL-12 / 23p40. K3 CpG and cGAMP (TLR9-agonist and STING-agonist, respectively) synergistically induce the production of innate IFNγ by NK cells. Human PBMC from two healthy donors were treated with 5 μg / ml of isotype control, type I IFN neutralizing antibody, IL-12 / 23p40 neutralizing antibody, or type I IFN+IL-12 / 23p40 neutralizing antibody for 30 s prior to 24 h stimulation with K3 CpG, cGAMP, or K3 CpG+cGAMP. IFNγ production was measured by ELISA. Data are representative of at least two independent experiments and shown as mean + SD of duplicates. *p<0.05; **p<0.01 (one-way ANOVA with Bonferroni's multiple comparison test). [Figure 2A] FIG. 2A shows the concentration of IFNγ produced in the supernatant of mouse PBMC culture. From the left group, no adjuvant, K3 CpG, K3 CpG+cGAMP and cGAMP are shown. For each group, the left side is from wild-type mice, and the right side is from IRF3 / 7 DKO mice. The vertical axis shows the concentration of IFNγ (ng / ml) in the culture supernatant. The combination of K3 CpG and cGAMP causes synergistic induction of innate IFNγ in mPBMCs in an IRF3 / 7-dependent manner, and the production of IFNα and IL-12 by dendritic cells. Mouse PBMCs from wild-type and IRF3 / 7 DKO mice were stimulated with K3 CpG, cGAMP, or K3 CpG+cGAMP for 24 hours, and the production of IFNγ was measured by ELISA. Data are representative of at least two independent experiments and are shown as the mean + SEM of duplicates. ***p<0.001; (Student's t-test). [Figure 2B]FIG. 2B shows the concentration of IL-12p40 produced by DM-DC. From the left, no adjuvant, K3 CpG, K3 CpG+cGAMP, and cGAMP are shown. The vertical axis shows the concentration of IL-12p40 (ng / ml). The combination of K3 CpG and cGAMP induces synergistic induction of innate IFNγ in mPBMCs in an IRF3 / 7-dependent manner, and IFNα and IL-12 production by dendritic cells. GM-DCs were stimulated with K3 CpG, cGAMP, or K3 CpG+cGAMP for 24 hours, and the production of IL-12p40 was measured by ELISA. Data are representative of at least two independent experiments and are shown as the mean + SD of duplicates. ***p<0.001; (one-way ANOVA with Bonferroni's multiple comparison test). [Figure 2C] Figure 2C shows the concentration of IL-12p40 produced by FL-DCs. From the left, no adjuvant, K3 CpG, K3 CpG+cGAMP, and cGAMP are shown. The vertical axis shows the concentration of IL-12p40 (ng / ml). The combination of K3 CpG and cGAMP induces synergistic induction of innate IFNγ in mPBMCs in an IRF3 / 7-dependent manner, and IFNα and IL-12 production by dendritic cells. FL-DCs were stimulated with K3 CpG, cGAMP, or K3 CpG+cGAMP for 24 hours, and the production of IL-12p40 was measured by ELISA. Data are representative of at least two independent experiments and are shown as the mean + SD of duplicates. ***p<0.001; (one-way ANOVA with Bonferroni's multiple comparison test). [Figure 2D]Figure 2D shows the concentration of IL-12p40 produced by FL-DCs. From the left, no adjuvant, K3 CpG, K3 CpG+cGAMP, and cGAMP are shown. The vertical axis shows the concentration of IFNα (ng / ml). The combination of K3 CpG and cGAMP induces synergistic induction of innate IFNγ in mPBMCs in an IRF3 / 7-dependent manner, and IFNα and IL-12 production by dendritic cells. FL-DCs were stimulated with K3 CpG, cGAMP, or K3 CpG+cGAMP for 24 hours, and IFNα production was measured by ELISA. Data are representative of at least two independent experiments and are shown as the mean + SD of duplicates. ***p<0.001; (one-way ANOVA with Bonferroni's multiple comparison test). [Figure 3A] FIG. 3A shows the concentration of IFNγ produced in the supernatant of mouse PBMC culture. From the left, no adjuvant, K3 CpG, K3 CpG+3'3'-cGAMP, 3'3'-cGAMP, K3 CpG+c-di-GMP, c-di-GMP, K3 CpG+2'3'-cGAMP, and 2'3'-cGAMP are shown. The vertical axis shows the concentration of IFNγ (ng / ml) in the culture supernatant. The combination of TLR9-agonist and STING-agonist is a potent type 1 adjuvant that also suppresses type 2 immune responses in vivo. Mouse PBMC were stimulated with K3 CpG (10 μg / ml), STING-agonist (10 μM), or K3 CpG+STING-agonist for 24 hours, and IFNγ production was measured by ELISA. Data are representative of at least two independent experiments and are shown as the mean + SEM of duplicates. [Figure 3B]Figure 3B shows the anti-OVA antibody titers in serum from mice immunized with OVA. The left panel shows the anti-OVA antibody titers of IgG2c, and the right panel shows the anti-OVA antibody titers of IgG1. From the left, saline, K3 CpG, K3 CpG+3'3'-cGAMP, 3'3'-cGAMP, K3 CpG+c-di-GMP, c-di-GMP, K3 CpG+2'3'-cGAMP, 2'3'-cGAMP, and DMXAA are shown. The combination of TLR9-agonist and STING-agonist is a potent type 1 adjuvant that also suppresses type 2 immune responses in vivo. Mice were immunized intramuscularly on days 0 and 10 with K3 CpG (10 μg), 3'3' / 2'3'-cGAMP (1 μg), c-di-GMP (1 μg), DMXAA (50 μg) or OVA (10 μg) with or without K3+3'3' / 2'3'-cGAMP / c-di-GMP / DMXAA. On day 17, OVA-specific serum IgG1 and IgG2c were measured by ELISA. Data are representative of at least two independent experiments and are shown as the mean + SD of duplicates. *p<0.05; **p<0.01 (Mann-Whitney U test). [Figure 3C]Figure 3B shows the concentrations of IFNγ and IL-13 produced by OVA-stimulated splenocytes. The vertical axis of the left panel shows the concentration of IFNγ (ng / ml), and the vertical axis of the right panel shows the concentration of IL-13 (ng / ml). From the left, saline, K3 CpG, K3 CpG+3'3'-cGAMP, 3'3'-cGAMP, K3 CpG+c-di-GMP, c-di-GMP, K3 CpG+2'3'-cGAMP, 2'3'-cGAMP, and DMXAA are shown. The combination of TLR9-agonist and STING-agonist is a potent type 1 adjuvant that also suppresses type 2 immune responses in vivo. (BC) Mice were immunized intramuscularly on days 0 and 10 with K3 CpG (10 μg), 3'3' / 2'3'-cGAMP (1 μg), c-di-GMP (1 μg), DMXAA (50 μg) or OVA (10 μg) with or without K3+3'3' / 2'3'-cGAMP / c-di-GMP / DMXAA. Spleen cells were stimulated with OVA (10 μg / ml) protein for 48 h. IFNγ and IL-13 production was measured by ELISA. Data are representative of at least two independent experiments and are shown as the mean + SD of duplicates. *p<0.05; **p<0.01 (Mann-Whitney U test). [Figure 4A]Figure 4A shows the serum anti-OVA antibody titers in each mouse immunized with OVA. The left panel shows the anti-OVA antibody titers of IgG2c, and the right panel shows the anti-OVA antibody titers of IgG1. From the left, K3 CpG, K3 CpG+cGAMP, and cGAMP are shown. From the left, wild-type, Tmem173gt, MyD88 KO, IRF3 / 7 DKO, and IFNAR KO mice are shown. The synergistic effect of the combination of K3 CpG and cGAMP in inducing antigen-specific IFNγ is dependent on IRF3 / 7, STING, MyD88, IL-12, and type I IFN signaling. Wild-type, Tmem173gt, IRF3 / 7 DKO, MyD88 KO and IFNAR KO C57BL / 6J mice were immunized with OVA and K3 CpG, cGAMP, or K3 CpG+cGAMP via the intramuscular route on days 0 and 10. OVA-specific serum IgG2c and IgG1 were measured by ELISA on day 17. Data are representative of at least two independent experiments and shown as the mean + SD of duplicates. *p<0.05; **p<0.01; ***p<0.001 (one-way ANOVA with Bonferroni's multiple comparison test). [Figure 4B] Figure 4B shows the concentrations of IFNγ and IL-13 produced by OVA-stimulated splenocytes. The vertical axis of the left panel shows the concentration of IFNγ (ng / ml), and the vertical axis of the right panel shows the concentration of IL-13 (ng / ml). From the left group, K3 CpG, K3 CpG+cGAMP, and cGAMP are shown. From the left, wild-type, Tmem173gt, MyD88 KO, IRF3 / 7 DKO, and IFNAR KO mice are shown. The synergistic effect of the combination of K3 CpG and cGAMP in inducing antigen-specific IFNγ is dependent on IRF3 / 7, STING, MyD88, IL-12, and type I IFN signaling. Spleen cells were stimulated with OVA for 48 hours. IFNγ production was measured by ELISA. Data are representative of at least two independent experiments and are shown as the mean + SD of duplicates. *p<0.05; **p<0.01; ***p<0.001 (one-way ANOVA with Bonferroni's multiple comparison test). [Figure 4C]Figure 4C shows serum anti-OVA antibody titers in OVA-immunized C57BL / 6J mice. The left panel shows IgG2c anti-OVA antibody titers, and the right panel shows IgG1 anti-OVA antibody titers. From the left group, K3 CpG, K3 CpG+cGAMP, and cGAMP are shown. The left side of each group shows IL-12p40+ / -, and the right side shows IL-12p40 KO. The synergistic effect of the combination of K3 CpG and cGAMP in inducing antigen-specific IFNγ is dependent on IRF3 / 7, STING, MyD88, IL-12, and type I IFN signaling. (C) IL-12p40+ / - and - / - C57BL / 6J mice were immunized with OVA and K3 CpG, cGAMP, or K3 CpG+cGAMP via the intramuscular route on days 0 and 10. OVA-specific serum IgG2c and IgG1 were measured by ELISA on day 17. Data are representative of at least two independent experiments and are shown as the mean + SD of duplicates. [Figure 4D] Figure 4D shows the concentrations of IFNγ and IL-13 produced by OVA-stimulated splenocytes. The vertical axis of the left panel indicates the concentration of IFNγ (ng / ml), and the vertical axis of the right panel indicates the concentration of IL-13 (ng / ml). From the left group, K3 CpG, K3 CpG+cGAMP, and cGAMP are shown. In each group, the left side indicates IL-12p40+ / -, and the right side indicates IL-12p40 KO. The synergistic effect of the combination of K3 CpG and cGAMP in inducing antigen-specific IFNγ is dependent on IRF3 / 7, STING, MyD88, IL-12, and type I IFN signaling. (D) Splenocytes were stimulated with OVA protein for 48 hours. IFNγ production was measured by ELISA. Data are representative of at least two independent experiments and are shown as the mean + SD of duplicates. *p<0.05 (Mann-Whitney U test). [Diagram 5]FIG. 5 shows a graph measuring tumor growth in an EG-7 mouse tumor model. The vertical axis shows tumor size (cm3), and the horizontal axis shows the number of days after inoculation of EG-7 lymphoma cells. The combination of K3 CpG and cGAMP efficiently suppresses tumors in an EG-7 mouse tumor model. Mice were subcutaneously injected with 1×106 EG-7 lymphoma cells (in 100 μl PBS) on day 0. On days 7 and 10, mice received intratumoral injections of PBS (n=8), K3 CpG (n=8), cGAMP (n=8), or K3 CpG+cGAMP (n=9), and mice were monitored for tumor growth for 22 days. Data are representative of two independent experiments. *p<0.05; **p<0.01 (Mann-Whitney U test). [Figure 6] Figure 6 shows the concentration of IFNγ produced by splenocytes stimulated with OVA254 or OVA323. From the left group, saline, K3 CpG, K3 CpG+cGAMP, and cGAMP are shown. The vertical axis shows the concentration of IFNγ (ng / ml). The combination of K3 CpG and cGAMP induces potent CD8+ T cell activity in vivo. Mice were immunized on days 0 and 10 via the intramuscular route with either OVA alone (n=2), or OVA and K3 CpG (n=4), cGAMP (n=4), or K3+cGAMP (n=4). On day 17, splenocytes were isolated and stimulated for 48 h with OVA or OVA peptides specific for MHC class I (OVA257) or MHC class II (OVA323). IFNγ production was measured by ELISA. Data are representative of at least two independent experiments. *p<0.05; **p<0.01 (Student's t-test). [Figure 7] Figure 7 shows the gating strategy for intracellular IFNγ staining experiments. First, CD3+ / - cells were gated from the lymphocyte gate. Then, IFNγ+CD8+ / - cells were gated from the CD3+ population and shown in the results. NK cells were gated from the CD3-CD56+CD16+ population and IFNγ+ NK cells are shown in the results. [Figure 8]Figure 8 shows the serum IgE concentration in mice immunized with OVA. From the left, saline, K3 CpG, cGAMP, and K3 CpG+cGAMP are shown. The vertical axis shows the IgE concentration. STING agonists induce IgE, but when combined with CpG, this is suppressed. [Figure 9] Figure 9 shows an overview of cellular and humoral immunity. Up until now, many vaccine adjuvants have been developed primarily to induce antibody production (humoral immunity). Therefore, many current adjuvants, including alum adjuvants, are humoral immunity-inducing adjuvants called Th2 adjuvants (type II adjuvants). However, in the prevention and treatment of cancer and allergies, the induction of cellular immunity is more important than humoral immunity, and these are called Th1 adjuvants (type I adjuvants). [Figure 10] FIG. 10 shows the production of IgG2c and IFNγ, which are indicators of type I adjuvants. The left panel shows anti-OVA IgG2c titers in serum from mice immunized with OVA. From the left, saline, K3 CpG, cGAMP, and K3 CpG+cGAMP are shown. The right panel shows the production of IFNγ when stimulated with OVA or not stimulated with OVA. From the left group, saline, K3 CpG, cGAMP, and K3 CpG+cGAMP are shown. The left side of each group shows the IFNγ produced when not stimulated with OVA, and the right side shows the IFNγ produced when stimulated with OVA. The vertical axis shows the concentration of IFNγ (ng / ml). The combination of CpG ODN and STING agonist synergistically induces Th1-type immune responses. [Figure 11] Figure 11 shows an overview of the new next-generation adjuvants that use STING agonists as a platform. STING agonists such as DMXAA themselves function as Th2 adjuvants that activate humoral immunity, similar to alum adjuvants. Therefore, by using STING agonists as a platform, they can be used as conventional humoral immunity-inducing Th2 adjuvants when used as is, and as powerful cellular immunity-inducing Th1 adjuvants when combined with CpG ODN. [Figure 12]12 is a diagram showing the synergistic effect of the combination of CpG1826 and 3'3'-cGAMP or the combination of D35 CpG and 3'3'-cGAMPc-di-GMP on interferon gamma production in splenocytes. The upper row shows the combination of CpG1826 and 3'3'-cGAMP, and the lower row shows the combination of D35 CpG and 3'3'-cGAMPc-di-GMP. The y-axis shows the amount of interferon gamma (IFNγ) produced (pg / ml), and the x-axis shows, from the left, no stimulation, the combination of CpG1826 and 3'3'-cGAMP or the combination of D35 CpG and 3'3'-cGAMPc-di-GMP, and 3'3'-cGAMP alone. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present invention will be described below while showing the best mode. Throughout this specification, the expression of the singular form should be understood to include the concept of the plural form, unless otherwise specified. Therefore, the singular article (for example, in the case of English, "a", "an", "the", etc.) should be understood to include the concept of the plural form, unless otherwise specified. In addition, it should be understood that the terms used in this specification are used in the sense commonly used in the field, unless otherwise specified. Therefore, unless otherwise defined, all technical terms and scientific and technical terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In case of conflict, this specification (including definitions) will take precedence.

[0018] The following provides definitions of terms particularly used in this specification and / or explains basic technical content as appropriate.

[0019] As used herein, "CpG oligonucleotide" or "CpG oligodeoxynucleotide", "CpG ODN" or simply "CpG" are used interchangeably and refer to a polynucleotide, preferably an oligonucleotide, that contains at least one unmethylated CG dinucleotide sequence. An oligonucleotide that contains at least one CpG motif may contain multiple CpG motifs. As used herein, the phrase "CpG motif" refers to an unmethylated dinucleotide portion of an oligonucleotide that contains a cytosine nucleotide followed by a guanosine nucleotide. 5-methylcytosine may also be used in place of cytosine.

[0020] CpG oligonucleotides (CpG ODN) are short (approximately 20 base pairs), single-stranded synthetic DNA fragments containing immunostimulatory CpG motifs. They are potent agonists of Toll-like receptor 9 (TLR9), activate dendritic cells (DCs) and B cells to produce type I interferons (IFNs) and inflammatory cytokines (Hemmi, H., et al. Nature 408, 740-745 (2000); Krieg, AM Nature reviews. Drug discovery 5, 471-484 (2006).), and act as adjuvants for Th1-type humoral and cellular immune responses, including cytotoxic T lymphocyte (CTL) responses (Brazolot Millan, CL, Weeratna, R., Krieg, AM, Siegrist, CA & Davis, HL Proceedings of the National Academy of Sciences of the United States of America 95, 15553-15558 (1998).;Chu, RS, Targoni, OS, Krieg, AM, Lehmann, PV & Harding, CV The Journal of experimental medicine 186, 1623-1631 (1997)). Thus, CpG ODNs have been considered as potential immunotherapeutic agents for infectious diseases, cancer, asthma and hay fever (Krieg, AM Nature reviews. Drug discovery 5, 471-484 (2006);Klinman, DM Nature reviews. Immunology 4, 249-258 (2004)).

[0021] CpG oligodeoxynucleotides (CpG ODNs) are synthetic single-stranded DNAs containing unmethylated CpG motifs with immunostimulatory characteristics due to similarity to bacterial genomes, and are recognized by TLR9 in certain types of innate immune cells [Hartmann et al., J. Immunol.(2000)164:944-953;Wagner et al., Trends Immunol.(2004)25:1-6]. Upon ligand binding, TLR9 transmits signals through the adaptor molecule myD88, leading to the production of IRF7-dependent type I IFN and NF-κB-dependent cytokines [Krieg et al., Nat. Rev. Drug Discov.(2006)5:471-84]. Moreover, it has been reported that in vivo, CpG ODN induces Th1-type response due to the type of cytokine induced by CpG ODN in APC [Krieg et al., Nat. Rev. Drug Discov.(2006)5:471-84]. Among different types of CpG ODN, D-type CpG ODN strongly induces both type I and type II IFN, but cannot induce B cell activation [Krieg et al., Nat. Rev. Drug Discov.(2006)5:471-84;Klinman et al., Nat. Rev. Immunol.(2004)4:1-10]. K-type CpG ODN (K3 CpG) strongly induces B cell activation, resulting in the production of IL-6 and antibodies, while they only weakly induce type I and type II IFN. However, because D-type CpG ODNs form aggregates, only K-type CpGs are available for clinical use [Krieg et al., Nat. Rev. Drug Discov.(2006)5:471-84;Klinman et al., Nat. Rev. Immunol.(2004)4:1-10].

[0022] Pathogen-derived factors such as LPS or unmethylated CpG DNA (CpG) (CpG ODN) stimulate innate immune cells to produce cytokines such as IL-12 and type I or type II IFN, which help generate Th1-type responses and cellular immunity [Kawai et al., Immunity.(2011)34:637-650;Trinchieri et al., Immunol.(2007)7:179-190]. IL-12 stimulates naive CD4 + It acts on T cells to drive Th1 development and IFNγ production [Seder et al., Proc. Natl. Acad. Sci. USA(1993)90:10188-92;Hsieh et al., Science.(1993)260:547-579]. And IFNγ-producing Th1 cells are the main players in the induction of type 1 immunity, which is distinguished by high phagocytic activity [Spellberg et al., Clin. Infect. Dis.(2001)90509:76-102;Mantovani et al., Curr. Opin. Immunol.(2010)22:231-237]. Moreover, Th1 cells play a key role in the development of antitumor immunity, serving for proper activation and effector function of CTLs, including the production of IFNγ [Hung et al., J. Exp. Med.(1998)188:2357-68; Vesely et al., Annu. Rev. Immunol.(2011)29:235-271]. Therefore, agents capable of inducing strong Th1-type responses, CTLs, and NK cells [Vitale et al., Eur. J. Immunol.(2014)44:1582-1592] are immediately required, as they may play a crucial role in the development of efficient vaccine adjuvants or immunotherapeutic agents against intracellular pathogens or cancer.

[0023] They are classified into D / A, K / B, C, and P types based on differences in skeletal modifications and surrounding sequences (Vollmer, J. & Krieg, AM Advanced drug delivery reviews 61, 195-204 (2009).). It has been suggested that the D / A type mainly induces the production of type I interferon from plasmacytoid dendritic cells (called "plasmacytoid DC" or "pDC"), while the K / B type induces the proliferation of B cells and the production of IgM, IL-6, etc. D / A type CpG-DNA strongly induces IFN-α production, but has low pDC maturation induction activity and does not show direct immunostimulatory activity on B cells. The K / B type exhibits immunostimulatory activity on B cells, strongly promotes pDC maturation, and has high IL-12 induction ability, but has low IFN-α induction ability. The C-type sequence, which has a repeat sequence of TCG and is entirely thiolated, induces polyclonal B cell activation and IFN-α production by pDC.

[0024] D / A type CpG ODN (also called A type, D type, etc., and also displayed as CpG-A ODN) is an oligonucleotide characterized by a poly-G motif with phosphorothioate (PS) bonds at the 5' and 3' ends and a palindromic CpG-containing sequence of phosphodiester (PO) at the center. The presence of phosphorothioate (PS) at the 5' and 3' ends is said to facilitate cellular uptake. CpG D / A type produces large amounts of interferon alpha (IFN-α) in pDCs (a feature different from CpG K / B type). This results in strong activation and interferon gamma production in NK cells and γδT cells. However, B cells are not activated and do not undergo pDC maturation (Krug, A., et al. European journal of immunology 31, 2154-2163 (2001).; and Verthelyi, D., Ishii, KJ, Gursel, M., Takeshita, F. & Klinman, DM Journal of immunology 166, 2372-2377 (2001).).

[0025] The other three types of ODN consist of a PS backbone.

[0026] K / B type CpG ODNs, also called CpG-B type or CpG-K type, are those that lack poly-G motifs, have one or more CpG motifs, and all have a phosphorothioate (PS) backbone. They typically contain multiple CpG motifs that are non-palindromic. K / B type CpG has weak (almost no) IFN-α induction activity, but is a very strong Th1 adjuvant and a strong B cell response stimulator, activating and maturing pDCs to produce IL-6 (Verthelyi, D., Ishii, KJ, Gursel, M., Takeshita, F. & Klinman, DM Journal of immunology 166, 2372-2377 (2001); and Hartmann, G. & Krieg, AM Journal of immunology 164, 944-953 (2000).). K / B-type CpG ODNs have the function of promoting survival, activation, and maturation of both monocyte-derived dendritic cells and pDCs.

[0027] Recently, C-type and P-type CpG ODNs have been developed, which contain one and two palindromic CpG sequences, respectively. Both can activate B cells like K-type and pDCs like D-type, but compared with P-type CpG ODN, C-type CpG ODN induces IFN-α production less strongly (Hartmann, G., et al. European journal of immunology 33, 1633-1641 (2003); Marshall, JD, et al. Journal of leukocyte biology 73, 781-792 (2003).; and Samulowitz, U., et al. Oligonucleotides 20, 93-101 (2010)).

[0028] D / K-type and P-type CpG ODNs have been shown to form higher-order structures, such as Hoogsteen base pairs that form parallel quadruple structures called G-tetrads, and Watson-Crick base pairs between cis- and trans-palindromic regions, which are required for potent IFN-α production by pDCs (Samulowitz, U., et al. Oligonucleotides 20, 93-101 (2010).; Kerkmann, M., et al. The Journal of biological chemistry 280, 8086-8093 (2005).; and Klein, DC, Latz, E., Espevik, T. & Stokke, BT Ultramicroscopy 110, 689-693 (2010)). Due to their higher-order structures, only K-type and C-type CpG ODNs are considered to be generally available for use as immunotherapeutic agents and vaccine adjuvants in humans (Puig, M., et al. Nucleic acids research 34, 6488-6495 (2006); Bode, C., Zhao, G., Steinhagen, F., Kinjo, T. & Klinman, DM Expert review of vaccines 10, 499-511 (2011); and McHutchison, JG, et al. Hepatology 46, 1341-1349 (2007)).

[0029] In contrast to A-type CpG ODN, C-type CpG has a completely phosphorothioate (PS) backbone without poly-G motifs, but contains an A-type palindromic sequence of CpG in combination with stimulatory CpG motifs. In vivo studies have reported that C-type CpG ODN is a highly potent Th1 adjuvant.

[0030] In a preferred embodiment of the present invention, the K-type CpG ODN is 10 nucleotides or more in length and has the formula:

[0031] [ka]

[0032] wherein the central CpG motif (written as CpG) is unmethylated, W is A or T, and N1, N2, N3, N4, N5 and N6 can be any nucleotide.

[0033] In one embodiment, the K-type CpG ODN of the present invention is 10 nucleotides or more in length and includes the nucleotide sequence of the above formula. However, in the above formula, the CpG motif (TCpGW) of the central four bases only needs to be included in the 10 nucleotides, and does not necessarily need to be located between N3 and N4 in the above formula. In addition, in the above formula, N1, N2, N3, N4, N5 and N6 may be any nucleotide, and at least one (preferably one) combination of N1 and N2, N2 and N3, N3 and N4, N4 and N5, and N5 and N6 may be a two-base CpG motif. When the four-base CpG motif is not located between N3 and N4, in the above formula, any two consecutive bases in the central four bases (the fourth to seventh bases) are a CpG motif, and the other two bases may be any nucleotide. In addition, some or all of the phosphodiester bonds of the oligodeoxynucleotide may be replaced with phosphorothioate bonds. Preferably, all of the phosphodiester bonds of the oligodeoxynucleotide are replaced by phosphorothioate bonds.

[0034] The K-type CpG ODN more preferably used in the present invention contains a non-palindrome containing one or more CpG motifs. The K-type CpG ODN further preferably used consists of a non-palindrome containing one or more CpG motifs.

[0035] The K-type CpG ODN contained in the oligodeoxynucleotide of the present invention is preferably humanized. "Humanized" means having agonistic activity against human TLR9. Therefore, the oligodeoxynucleotide of the present invention containing the humanized K-type CpG ODN has an immunostimulatory activity specific to K-type CpG ODN in humans (e.g., activity of activating human B cells to produce IL-6).

[0036] Humanized K-type CpG ODN is generally characterized by a four-base CpG motif consisting of TCGA or TCGT. In many cases, two or three of these four-base CpG motifs are contained in one humanized K-type CpG ODN. Therefore, in a preferred embodiment, the K-type CpG ODN contained in the oligodeoxynucleotide of the present invention contains at least one, more preferably two or more, and even more preferably two or three, four-base CpG motifs consisting of TCGA or TCGT. When the K-type CpG ODN has two or three four-base CpG motifs, these four-base CpG motifs may be the same or different. However, there is no particular limitation as long as it has agonistic activity against human TLR9.

[0037] The K-type CpG ODN contained in the oligodeoxynucleotide of the present invention more preferably contains a nucleotide sequence represented by the sequence (atcgactctc gagcgttctc (SEQ ID NO: 1)).

[0038] The length of the K-type CpG ODN is not particularly limited as long as the oligodeoxynucleotide of the present invention has an immunostimulatory activity (e.g., activity of activating B cells (preferably human B cells) to produce IL-6) or an anticancer activity, but is preferably 100 nucleotides or less (e.g., 10-75 nucleotides). The length of the K-type CpG ODN is more preferably 50 nucleotides or less (e.g., 10-40 nucleotides). The length of the K-type CpG ODN is further preferably 30 nucleotides or less (e.g., 10-25 nucleotides). The length of the K-type CpG ODN is most preferably 12-25 nucleotides.

[0039] As used herein, the terms "ligand" and "agonist" are used interchangeably and refer to a substance that exerts or enhances the biological action of a target entity (e.g., a receptor) on that receptor. In addition to natural agonists (also called ligands), synthetic and modified agonists can be included.

[0040] "STING" (Stimulator of IFN genes (adapter molecule) ( st emulator of in terferon gSTING plays an important role in the host defense mechanism against infection by various RNA and DNA viruses. It has also been reported that STING plays an important role in inducing innate immune responses to DNA components derived from viruses and bacteria, but the molecular mechanism behind this has not been elucidated. In this paper, we elucidate that STING can form complexes not only with genomic DNA derived from viruses, but also with synthetic double-stranded DNA of 45-90 base pairs called ISD, and with self-DNA components derived from apoptotic cells. Analysis of the DNA interaction region in vitro demonstrated that the C-terminal region of STING is important. It was shown that recognition of various DNA components by STING induces dynamic localization changes of STING to the peripheral region of the nuclear envelope and induces interferon production via activation of TBK1. Furthermore, it was suggested that STING may be involved in the control of chronic inflammatory responses through recognition of not only non-self DNA components derived from microorganisms, but also self-DNA components.

[0041] As used herein, "STING ligand" and "STING agonist" are used interchangeably and refer to a stimulator of (adapter molecule) IFN genes (STING= st emulator of in terferon gSTING agonists are believed to be membrane proteins localized in the endoplasmic reticulum. In addition to cGAMP, the bacterial-origin cyclic dinucleotides c-di-AMP and c-di-GMP are ligands for the adaptor molecule stimulator of IFN genes (STING), which signal through the TBK1-IRF3 axis to induce type I IFN production and NF-κB-mediated cytokine production [Burdette et al., Nature.(2011)478:515-8;Mcwhirter et al., J. Exp. Med.(2009)206:1899-1911]. Recent studies have shown that these cyclic dinucleotides function as potent vaccine adjuvants due to their ability to enhance antigen-specific T cell and humoral immune responses. Nevertheless, our group previously showed that the STING agonist DMXAA unexpectedly induces type 2 immune responses through STING-IRF3-mediated production of type I IFN [Tang et al., PLoS One.(2013)8:1-6]. The clinical utility of STING agonists containing cyclic dinucleotides has been controversial, since type 2 immune responses may fail to induce type 1 immune responses. For example, the most common adjuvant aluminum salt (alum) lacks the ability to induce cell-mediated immunity that is thought to protect against diseases caused by intracellular pathogens or cancer [Hogenesch et al., Front. Immunol.(2013)3:1-13]. To overcome this limitation, alum has been combined with many different types of adjuvants, including monophosphoryl lipid A [Macleod et al., Proc. Natl. Acad. Sci. USA(2011)108:7914-7919] and CpG ODN [Weeratna et al., Vaccine.(2000)18:1755-1762].For STING-related technologies, host DNA, like microbial DNA, can also be a danger signal, especially if it is inappropriately present in the cytosol, thereby resulting in the production of interferons and inflammatory cytokines [Desmet et al., Nat. Rev. Immunol.(2012)12:479-491;Barber et al., Immunol. Rev.(2011)243:99-108]. One recently identified cytosolic DNA sensor is cyclic GMP-AMP synthase (cGAS), which catalyzes the production of the non-canonical cyclic dinucleotide cGAMP (2'3'-cGAMP), which contains non-canonical 2',5' and 3',5' linkages between its purine nucleosides [Sun et al., Science.(2013)339:786-91]. Canonical cGAMP(3'3') is synthesized in bacteria and has a more variable linkage than mammalian 2'3'-cGAMP, in which the GMP and AMP nucleosides are linked by a bis-(3',5') bond [Wu et al., Science.(2013)339:826-30;Zhang et al., Mol. Cell.(2013)51:226-35].

[0042] Thus, examples of STING agonists that can be used in the present invention include cyclic dinucleotides (CDNs) such as 2'3'-cGAMP, c-di-AMP, 3'3'-cGAMP, 3'2'-cGAMP, xanthenone derivatives such as DMXAA, etc. STING agonists are also described in WO2010 / 017248, the contents of which are incorporated by reference in their entirety.

[0043] As used herein, the term "adjuvant" refers to an immune enhancing agent added to a vaccine to enhance its effectiveness, and is not a specific antigen but rather a drug that boosts the immune response to an administered antigen. With the recent development of immunology, the mechanism of action of adjuvants is gradually becoming clear. Recently, due to the diverse immune control properties of adjuvants, they are expected to be used not only for the prevention and treatment of infectious diseases, but also for allergies, cancer, and autoimmune diseases.

[0044] So far, most vaccine adjuvants have been developed to induce antibody production (humoral immunity). Therefore, most current adjuvants, including alum adjuvants, are humoral immunity-inducing adjuvants called Th2 adjuvants. However, in the prevention and treatment of cancer and allergies, it is more important to induce cellular immunity than humoral immunity, and these adjuvants are called Th1 adjuvants or type I adjuvants.

[0045] To date, many candidate substances for Th1 adjuvants have been reported, but CpG oligonucleotides (CpG ODN) are considered to be the most effective. CpG ODN has been shown to be effective as a vaccine adjuvant for cancer and infectious diseases (Non-Patent Document 1), and efforts are currently being made to develop and improve even more effective CpG ODN (Non-Patent Document 2). If an effective improved CpG ODN can be developed, it may be applied to the treatment of cancer and allergies more than ever before.

[0046] As used herein, the term "type I adjuvant" is also called a Th1 adjuvant and refers to an adjuvant that induces a type I immune response. Typically, it is characterized by an antitumor effect mediated by natural killer cells or cytotoxic T cells.

[0047] As used herein, the term "type II adjuvant" refers to an adjuvant that induces a type II immune response, also known as a Th2 adjuvant, and is typically characterized by an infection prevention effect due to antibody induction.

[0048] In this specification, "type I immune response" is also called cellular immunity, and is an immune system in which phagocytes, cytotoxic T lymphocytes (CTLs), and natural killer cells are responsible for eliminating foreign substances from the body. T cells are involved. Helper T cells include Th1 cells and Th2 cells, which are thought to suppress each other's functions by releasing cytokines. These cells originally differentiate from a single cell, which is called a naive T cell (Th0). For specific differentiation mechanisms, see the T cell section. Cells that differentiate from Th0 cells to Th1 cells produce so-called Th1 cytokines such as IL-2, and activate CTLs that destroy virus-infected cells. Cellular immunity is also involved not only in the elimination of virus-infected cells but also in tumor immunity and transplant immunity.

[0049] In this specification, the "type II immune response" is also called humoral immunity, and is an immune system centered on antibodies and complement. It is so named because antibodies are dissolved and exist in serum. Macrophages and dendritic cells function as antigen-presenting cells by taking up antigens into the cells, decomposing them, and presenting the fragments on the cell surface. Antigen-presenting cells present antigen fragments via MHC class II molecules and transmit signals to T cell antigen receptors (TCR; T cell receptors) on the surface of naive T cells (Th0). The ratio of Th0 cells that differentiate into type 1 helper T cells (Th1) or type 2 helper T cells (Th2) varies depending on the disease, but in the presence of Th2 cytokines such as interleukin (IL; Interleukin)-4 and IL-5, Th0 differentiates into Th2 and induces humoral immunity.

[0050] As used herein, "immunoglobulin E" or "IgE" refers to a glycoprotein found only in mammals, and is a type of immunoglobulin. An IgE molecule is composed of two heavy chains (ε chains) and two light chains (κ chains and λ chains), and has two antigen-binding sites. The IgE concentration in serum of healthy individuals is in the ng / ml range, which is very low compared to other types of immunoglobulins, but the concentration increases in the serum of patients with allergic diseases, and it induces the rapid release (degranulation reaction) of physiologically active substances stored in intracellular granules of mast cells and basophils. For these reasons, IgE is counted as one of the molecules that play a central role in allergic reactions, along with histamine.

[0051] In this specification, "allergy" refers to an excessive immune response to a specific antigen. Environmental antigens that cause allergies are specifically called allergens. "Allergic disease" refers to a disease in which an immune response occurs to an external antigen. However, the antigen is often harmless in the amounts exposed in normal life (for example, early spring pollen itself is not toxic), and it can be said that an immune response is occurring that brings about unnecessarily unpleasant results. It is also called allergic disease. Representative diseases include atopic dermatitis, allergic rhinitis (hay fever), allergic conjunctivitis, allergic gastroenteritis, bronchial asthma, childhood asthma, food allergies, drug allergies, and urticaria. Recently, diseases have been reported in which type 1 allergy symptoms such as asthma and facial flushing can be seen in response to the smell of citrus fruits or perfumes such as gum. The attitude is attracting attention.

[0052] On the other hand, an "autoimmune disease" is a disease in which an immune response occurs against substances that compose one's own body as antigens. It can cause damage or inflammation in specific organs or parts of the body, or can cause systemic symptoms. Typical examples of this disease include collagen diseases such as rheumatoid arthritis and alopecia areata.

[0053] As used herein, "interferon (IFN)" refers to a protein (cytokine) secreted by cells in response to the invasion of foreign substances such as pathogens (particularly viruses) and tumor cells into the animal body, and among these, IFN-γ is a Th1 type cytokine.

[0054] As used herein, the term "subject" refers to a subject that is the target of diagnosis, detection, or treatment, etc., of the present invention (for example, an organism such as a human, or cells, blood, serum, etc. extracted from an organism).

[0055] In this specification, the term "drug," "agent," or "factor" (all of which are equivalent to the English term "agent") In a broad sense, the terms "energy" and "energy source" are used interchangeably and may refer to any substance or other element (e.g., energy such as light, radioactivity, heat, electricity, etc.) as long as it can achieve the intended purpose. Such substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (including, for example, DNA such as cDNA and genomic DNA, and RNA such as mRNA), polysaccharides, oligosaccharides, and the like. Examples of the factor specific to a polynucleotide include, but are not limited to, a polynucleotide having a certain sequence homology (e.g., 70% or more sequence identity) with respect to the sequence of the polynucleotide, a polypeptide such as a transcription factor that binds to a promoter region, and the like. Examples of the factor specific to a polypeptide include, but are not limited to, an antibody specifically directed against the polypeptide or a derivative or analog thereof (e.g., a single-chain antibody), a specific ligand or receptor when the polypeptide is a receptor or ligand, and a substrate when the polypeptide is an enzyme.

[0056] As used herein, the term "treatment" refers to preventing, preferably maintaining, more preferably alleviating, and even more preferably eradicating a disease or disorder (e.g., cancer, allergy) that has developed into a certain condition, and is intended to have an effect of improving or preventing symptoms of the patient's disease or one or more symptoms associated with the disease. Making a diagnosis in advance and then providing appropriate treatment is called "companion therapy," and the diagnostic drugs used for this purpose are sometimes called "companion diagnostics."

[0057] In the present specification, the term "therapeutic agent (agent)" refers, in a broad sense, to any drug capable of treating a target condition (e.g., a disease such as cancer or allergy). In one embodiment of the present invention, the "therapeutic agent" may be a pharmaceutical composition containing an active ingredient and one or more pharmacologically acceptable carriers. The pharmaceutical composition may be produced, for example, by mixing the active ingredient with the above-mentioned carrier and using any method known in the technical field of pharmaceutical formulations. The therapeutic agent may be in any form as long as it is used for treatment, and may be an active ingredient alone or a mixture of the active ingredient and any ingredient. The shape of the carrier is not particularly limited, and may be, for example, a solid or liquid (e.g., a buffer solution). The therapeutic agent for cancer, allergy, etc. includes drugs (prophylactic drugs) used for the prevention of cancer, allergy, etc., or suppressants of cancer, allergy, etc.

[0058] As used herein, the term "prevention" refers to preventing a certain disease or disorder (e.g., allergy) from occurring before such a state occurs. The agent of the present invention can be used to perform diagnosis, and if necessary, the agent of the present invention can be used to prevent, for example, allergies, or measures for prevention can be taken.

[0059] As used herein, the term "prophylactic drug (agent)" refers broadly to any drug that can prevent a target condition (eg, a disease such as allergy).

[0060] As used herein, a "kit" refers to a set of components to be provided, usually divided into two or more compartments. This refers to a unit in which a part (e.g., a test agent, a diagnostic agent, a therapeutic agent, an antibody, a label, instructions, etc.) is provided. This kit form is preferred when the purpose is to provide a composition that should not be provided in a mixed state for reasons of stability, etc., but is preferably mixed and used immediately before use. Such a kit advantageously includes instructions or instructions describing how to use the provided part (e.g., a test agent, a diagnostic agent, a therapeutic agent, or how the reagent should be handled. When the kit is used as a reagent kit in this specification, the kit usually includes instructions describing how to use the test agent, diagnostic agent, therapeutic agent, antibody, etc.

[0061] In the present specification, the term "instructions" refers to a description of a method for using the present invention to a physician or other user. The instructions include a description of the detection method of the present invention, a description of how to use a diagnostic agent, or a description of how to administer a medicine or the like. The instructions may also include a description of the administration site, such as oral or esophageal administration (e.g., by injection). The instructions are prepared in accordance with a format prescribed by a regulatory agency of the country in which the present invention is implemented (e.g., the Ministry of Health, Labor and Welfare in Japan, the Food and Drug Administration (FDA) in the United States, etc.), and it is clearly stated that the instructions have been approved by the regulatory agency. The instructions are so-called package inserts, and are usually provided in paper form, but are not limited thereto, and may also be provided in the form of, for example, electronic media (e.g., a homepage provided on the Internet, e-mail).

[0062] (Preferred embodiment) Preferred embodiments of the present invention will be described below. It should be understood that the embodiments provided below are for better understanding of the present invention, and the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention with reference to the description in this specification. Also, it is understood that the following embodiments of the present invention can be used alone or in combination with each other.

[0063] <Combination of CpG and STING agonist> In one aspect, the present invention provides a combination of a CpG oligonucleotide and a STING agonist (also referred to as a STING ligand). Using the combination of the present invention, various treatments or preventions, for example, treatments or preventions for cancer, allergies, etc., or treatments or preventions using immune activation can be performed.

[0064] Therefore, the present invention provides a method for treating or preventing a disease, which includes the step of administering to a subject in need thereof an effective amount of a combination of a CpG oligonucleotide and a STING agonist.

[0065] In one embodiment, the combination of the present invention is for use as a type I adjuvant. This phenomenon is unexpected from both aspects. That is, from the perspective of the STING agonist, usually this ligand is considered to only produce an infection prevention effect by humoral immunity, that is, antibody induction. However, by adding CpG, cellular immunity is induced. Such a "switch" effect can be said to be truly unexpected. Also, the cellular immunity induction effect of CpG was significantly enhanced by the STING agonist. Considering the cellular immunity induction effect of CpG, it can be said that there was an unexpectedly significant enhancement in terms of quantity even when considering that the STING agonist has almost no cellular immunity induction effect.

[0066] In another embodiment, the combination of the present invention is for suppressing the IgE-inducing effect of the STING agonist. Without wishing to be bound by theory, it was unexpectedly discovered in the present invention that STING agonists have IgE-inducing effect and may have allergic side effects. Therefore, it is unexpected that such a problem needs to be solved, and the solution is achieved by CpG.

[0067] In one embodiment, the STING agonist may include 3'3'-cGAMP, 2'3'-cGAMP, 2'2'-cGAMP, DMXAA, c-di-AMP and c-di-GMP. DMXAA is a mouse-specific STING agonist and is considered to be less effective in humans. 3'3'-cGAMP is bacterial cGAMP, 2'3'-cGAMP is mammalian cGAMP, and 2'2'-cGAMP is a non-natural cGAMP.

[0068] In one embodiment, the CpG oligonucleotide of the present invention is a K / B type oligonucleotide. Without wishing to be bound by theory, it has been shown that K / B type oligonucleotides, particularly CpG oligonucleotides, are effective as vaccine adjuvants for cancer and infectious diseases, and this can be further enhanced by the present invention.

[0069] In one embodiment, the CpG oligonucleotide that can be used in the present invention may be any common CpG, including, but not limited to, K3 CpG (SEQ ID NO: 1 (5'-atcgactatcgagagttctc-3')), CpG1826 (SEQ ID NO: 2 (5'-tccatgacgttcctgacgtt-3')), and D35 CpG (SEQ ID NO: 3 (5'-ggtgcatcgatgcagggggg-3')).

[0070] In one embodiment, STING agonists that may be used in the present invention include cGAMP, 3'3'-cGAMP, c-di-AMP, c-di-GAMP, 2'3'-cGAMP, DMXAA, and the like.

[0071] In one particular embodiment, the combination of the present invention is used as an anticancer agent. In this case, the method for treatment or prevention of the present invention is provided as a method for treating or preventing cancer.

[0072] In one embodiment, the anti-cancer agent may target cancers such as lymphoma, melanoma, etc.

[0073] In another embodiment, the combination of the present invention is for reducing or eliminating a type II immune response and / or for developing or enhancing a type I immune response.

[0074] In another embodiment, the combination of the present invention is for inducing interferon gamma (IFN-γ). Alternatively, the combination of the present invention is for use as a vaccine adjuvant. In this case, the method for treatment or prevention of the present invention is provided as a method for inactivating immunity.

[0075] These two factors (CpG oligonucleotide and STING agonist) may be administered simultaneously or at different times, in the same formulation or in combination with different formulations, sequentially or separately.

[0076] Assays for T cell activation can be performed, for example, as follows. In some embodiments, various assays can be utilized to determine whether an immune response has been stimulated in a T cell or group of T cells (i.e., whether a T cell or group of T cells is "activated"). In some embodiments, stimulation of an immune response in T cells can be determined by measuring antigen-induced production of cytokines by T cells. In some embodiments, stimulation of an immune response in T cells can be determined by measuring antigen-induced production of IFNγ, IL-4, IL-2, IL-10, IL-17, and / or TNFα by T cells. In some embodiments, antigen-induced production of cytokines by T cells can be measured by flow cytometry after intracellular cytokine staining. In some embodiments, antigen-induced production of cytokines by T cells can be measured by flow cytometry after surface capture staining. In some embodiments, antigen-induced production of cytokines by T cells can be determined by measuring cytokine concentration in the supernatant of activated T cell cultures. In some embodiments, this can be measured by ELISA.

[0077] In some embodiments, antigen-generated production of cytokines by T cells can be measured by ELISPOT assay. In general, ELISPOT assays use a technique very similar to that of sandwich enzyme-linked immunosorbent assay (ELISA). Antibodies (e.g., monoclonal, polyclonal, etc.) are aseptically coated onto PVDF (polyvinylidene fluoride) backed microplates. Antibodies are selected for specificity to the cytokine of interest. The plates are blocked (e.g., with serum proteins that are not reactive with any of the antibodies in the assay). Cells of interest are plated at various densities with antigen or mitogen, then incubated in a humidified 37°C CO2-incubated 5% ... 2The wells are then placed in an incubator for a specified time. Cytokines secreted by the activated cells are captured locally by the coated antibodies on the large surface area PVDF membrane. After washing the wells to remove cells, debris and medium components, a second antibody specific for the cytokine (e.g., a biotinylated polyclonal antibody) is added to the wells. This antibody is reactive with a different epitope of the target cytokine and is therefore used to detect the captured cytokine. After washing to remove any unbound biotinylated antibody, the detected cytokine is visualized using avidin-HRP and a promoting substrate (e.g., AEC, BCIP / NBT). The colored end product (spots, usually dark blue) typically represents individual cytokine-producing cells. Spots can be counted manually (e.g., using a dissecting microscope) or using an automated reader that captures images of the microwells and analyzes the number and size of the spots. In some embodiments, each spot corresponds to a single cytokine-producing cell.

[0078] In some embodiments, an immune response in T cells is said to be stimulated if about 1% to about 100% of antigen-specific T cells are producing cytokines. In some embodiments, an immune response in T cells is said to be stimulated if at least about 1%, at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, at least about 95%, at least about 99% or about 100% of antigen-specific T cells are producing cytokines.

[0079] In some embodiments, an immune response in T cells is said to be stimulated when an immunized subject contains at least about 10 times, at least about 50 times, at least about 100 times, at least about 500 times, at least about 1000 times, at least about 5000 times, at least about 10,000 times, at least about 50,000 times, at least about 100,000 times, or at least about 100,000 times more cytokine-producing cells than an untreated control.

[0080] In some embodiments, stimulation of an immune response in T cells can be determined by measuring antigen-induced proliferation of T cells. In some embodiments, antigen-induced proliferation is determined by measuring H in dividing T cells. 3 -thymidine incorporation (sometimes referred to as "lymphocyte transformation test" or "LTT"). In some embodiments, H 3 Antigen-induced proliferation is said to have occurred if thymidine incorporation (given as counts from a gamma counter) is at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, at least about 1000-fold, at least about 5000-fold, at least about 10,000-fold or at least about 10,000-fold greater than untreated controls.

[0081] In some embodiments, antigen-induced proliferation may be measured by flow cytometry. In some embodiments, antigen-induced proliferation may be measured by carboxyfluorescein succinimidyl ester (CFSE) dilution assay. CFSE is a non-toxic, fluorescent, membrane-permeable dye that binds to amino groups of cytoplasmic proteins (e.g., T cell proteins) through its succinimidyl reactive group. When cells divide, CFSE-labeled proteins are equally distributed to daughter cells, so that cell fluorescence is halved with each division. As a result, antigen-specific T cells lose their fluorescence (CFSE) after culture in the presence of the respective antigen. 低 ), and other cells in culture (CFSE 高In some embodiments, CFSE dilution (all CFSE + CFSE on cells 低 Antigen-induced proliferation is said to have occurred when the percentage of cells that are positive for antigen (given as a percentage of cells) is at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, at least about 95% or at least about 100%.

[0082] In some embodiments, an immune response in T cells is said to be stimulated when cellular markers of T cell activation are expressed at different levels (e.g., higher or lower levels) than in unstimulated cells. In some embodiments, CD11aCD27, CD25, CD40L, CD44, CD45RO and / or CD69 are more highly expressed in activated T cells than in unstimulated T cells. In some embodiments, L-selectin (CD62L), CD45RA and / or CCR7 are less highly expressed in activated T cells than in unstimulated T cells.

[0083] In some embodiments, the immune response in T cells is effector CD8 T cells against antigen-pulsed target cells. + This is measured by assaying T cell cytotoxicity. For example, 51 chromium( 51 A CD8+ (Cr) release assay can be performed. In this assay, effector CD8 + T cells bind to infected cells presenting viral peptides on class I MHC and signal the infected cells to undergo apoptosis. Effector CD8 + Before the T cells were added, the cells 51 If labeled with Cr, it is released into the supernatant 51 The amount of Cr is proportional to the number of targets killed.

[0084] One of ordinary skill in the art will understand that the above assays are merely exemplary methods that can be utilized to determine whether T cell activation has occurred. Any assay known to one of ordinary skill in the art that can be used to determine whether T cell activation has occurred is encompassed within the scope of the present invention. Assays utilized in the present invention as well as additional assays that can be used to determine whether T cell activation has occurred are described in Current Protocols in Immunology (John Wiley & Sons, Hoboken, NY, 2007; incorporated herein by reference).

[0085] <Use of the STING agonist itself as a type I adjuvant formulation> In another aspect, the present invention provides a composition for use as a type I adjuvant comprising a STING agonist, wherein the STING agonist is administered together with a CpG oligonucleotide. It is understood that the STING agonist and CpG oligonucleotide used herein can be employed in one or a combination of any forms described elsewhere in this specification, including <Combination of CpG and STING agonist> and the examples.

[0086] Accordingly, the present invention provides a method for the treatment or prevention of a disease in a subject, the method comprising administering to a subject in need thereof a composition for use as a type I adjuvant comprising an effective amount of a STING agonist, wherein the STING agonist is administered together with a CpG oligonucleotide. In this case, the CpG oligonucleotide can be administered simultaneously with, before, or after the administration of the STING agonist. Accordingly, the present invention provides a method for exerting the type I adjuvant effect of a STING agonist, the method comprising the step of administering the STING agonist together with a CpG oligonucleotide.

[0087] In one embodiment, diseases that can be treated or prevented by the present invention utilizing the adjuvant enhancing effect include cancer, allergy, viral or bacterial infections, etc. More specifically, diseases that can be treated or prevented by the present invention utilizing the adjuvant enhancing effect are cancer, and such cancers include lymphoma, melanoma, etc.

[0088] These two factors (CpG oligonucleotide and STING agonist) may be administered simultaneously, at different times, in the same formulation, in combination with different formulations, or sequentially or separately.

[0089] <Enhancing effect of type I adjuvant formulation of CpG by STING agonist> In another aspect, the present invention provides an enhancer of the action of a type I adjuvant of CpG oligonucleotide, which contains a STING agonist. It is understood that the STING agonist and CpG oligonucleotide used herein can adopt one or a combination of any forms described elsewhere in this specification, including <Combination of CpG and STING agonist> and examples.

[0090] Therefore, the present invention provides a method for enhancing the action of a type I adjuvant of CpG oligonucleotide, which includes the step of administering to a subject a composition containing an effective amount of a STING agonist. Therefore, the present invention also provides a method for enhancing the action of a type I adjuvant of CpG oligonucleotide, which includes the step of administering the CpG oligonucleotide together with a STING agonist.

[0091] These two factors (CpG oligonucleotide and STING agonist) may be administered simultaneously, at different times, in the same formulation, in combination with different formulations, or sequentially or separately.

[0092] <Use of a STING agonist as an anti-cancer agent> In another aspect, the present invention provides an anti-cancer agent comprising a STING agonist, wherein the STING agonist is administered together with a CpG oligonucleotide. It is understood that the STING agonist and CpG oligonucleotide used herein can be employed in one or a combination of any forms described elsewhere in this specification, including <Combination of CpG and STING agonist> and the examples.

[0093] Accordingly, the present invention provides a method for treating or preventing cancer in a subject, which includes the step of administering to the subject an effective amount of an anti-cancer agent comprising a STING agonist, wherein the STING agonist is administered together with a CpG oligonucleotide. Accordingly, the present invention provides a method for treating or preventing cancer, the method comprising the step of administering an anti-cancer agent comprising a STING agonist together with a CpG oligonucleotide.

[0094] In one embodiment, cancers that can be treated or prevented by the present invention using a STING agonist can be targeted cancers such as lymphoma, melanoma, etc.

[0095] These two factors (CpG oligonucleotide and STING agonist) may be administered simultaneously, at different times, in the same formulation, in combination with different formulations, or sequentially or separately.

[0096] <Use of a CpG oligonucleotide as an anti-cancer agent> In another aspect, the present invention provides an anti-cancer agent comprising a CpG oligonucleotide, wherein the CpG oligonucleotide is administered together with a STING agonist. It is understood that the STING agonist and the CpG oligonucleotide used herein can be employed in one or a combination of any forms described elsewhere in this specification, including <the combination of CpG and STING agonist> and the examples.

[0097] Accordingly, the present invention provides a method for treating or preventing cancer, which comprises the step of administering to a subject in need thereof an effective amount of an anti-cancer agent comprising a CpG oligonucleotide, wherein the CpG oligonucleotide is administered together with a STING agonist. Accordingly, the present invention provides a method for treating or preventing cancer, which method comprises the step of administering an anti-cancer agent comprising a CpG oligonucleotide together with a STING agonist.

[0098] In one embodiment, cancers that can be treated or prevented by the present invention using CpG oligonucleotides include, but are not limited to, cancers such as lymphoma and melanoma.

[0099] These two factors (CpG oligonucleotide and STING agonist) may be administered simultaneously, at different times, in the same formulation, in a combination of different formulations, sequentially, or separately.

[0100] <Inhibition of the allergy (IgE)-inducing effect of STING agonist by CpG> In another aspect, the present invention provides a composition for reducing or eliminating the IgE-inducing effect of a STING agonist, which composition contains a CpG oligonucleotide. It is understood that the STING agonist and the CpG oligonucleotide used herein can be employed alone or in combination of one or more of any forms described elsewhere in this specification, including <the combination of CpG and STING agonist> and the examples.

[0101] Accordingly, the present invention provides a method for reducing or eliminating the IgE-inducing effect of a STING agonist, which method includes the step of administering to a subject a composition containing an effective amount of a CpG oligonucleotide. Accordingly, the present invention provides a method for reducing or eliminating the IgE-inducing effect of a STING agonist, the method including the step of administering a CpG oligonucleotide when using the STING agonist.

[0102] In one embodiment, the allergies that can be treated or prevented by the present invention include, but are not limited to, allergies as side effects of STING agonists, and other atopic dermatitis, allergic rhinitis (hay fever), allergic conjunctivitis, allergic gastroenteritis, bronchial asthma, childhood asthma, food allergy, drug allergy, urticaria, etc.

[0103] These two factors (CpG oligonucleotide and STING agonist) may be administered simultaneously, at different times, in the same formulation, in combination with different formulations, in sequence or separately.

[0104] In one embodiment, the allergy-inducing effect of the STING agonist is reduced or eliminated.

[0105] (Pharmaceutical, dosage form, etc.) The present invention is provided as a medicament (therapeutic or prophylactic agent) in the above various forms.

[0106] The administration route of the therapeutic agent, prophylactic agent, etc. is preferably one that is effective for treatment, and may be, for example, intravenous, subcutaneous, intramuscular, intraperitoneal, or oral administration. The administration form may be, for example, an injection, capsule, tablet, granule, etc. When administering the component of the present invention, it is effective to use it as an injection. The aqueous solution for injection may be stored, for example, in a vial or a stainless steel container. The aqueous solution for injection may also be mixed with, for example, physiological saline, sugar (for example, trehalose), NaCl, or NaOH. The therapeutic agent may also be mixed with, for example, a buffer (for example, a phosphate buffer), a stabilizer, etc.

[0107] Generally, the compositions, medicaments, therapeutic agents, prophylactic agents, etc. of the present invention comprise a therapeutically effective amount of a therapeutic agent or active ingredient, and a pharma- ceutically acceptable carrier or excipient. As used herein, "pharmaceutically acceptable" means approved by a government regulatory agency or listed in a pharmacopoeia or other generally recognized pharmacopoeias for use in animals, and more particularly in humans. As used herein, "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, including, but not limited to, peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the medicament is administered orally, water is the preferred carrier. When the pharmaceutical composition is administered intravenously, saline and aqueous dextrose are the preferred carriers. Preferably, saline solutions, as well as aqueous dextrose and glycerol solutions, are used as liquid carriers for injectable solutions. Suitable excipients include light anhydrous silicic acid, crystalline cellulose, mannitol, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, glycol, water, ethanol, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl acetal diethyl amino acetate, polyvinyl pyrrolidone, gelatin, medium chain triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, inorganic salts, etc. The composition can also contain a small amount of a wetting or emulsifying agent, or a pH buffer, if desired. These compositions can take the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained release formulation, etc. The composition can also be formulated as a suppository, using traditional binders and carriers, such as triglycerides.Oral formulations may contain standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable carriers are described in EW Martin, Remington's Pharmaceutical Sciences (Mark Publishing Company, Easton, USA). Such compositions contain a therapeutically effective amount of the therapeutic agent, preferably in purified form, together with an appropriate amount of carrier to provide a form that is properly administered to the patient. The formulation must be suitable for the mode of administration. In addition to these, for example, surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffers, suspending agents, isotonicity agents, binders, disintegrants, lubricants, flow enhancers, flavoring agents, etc. may be included.

[0108] In one embodiment of the present invention, the term "salt" includes, for example, an anionic salt formed with any acidic (e.g., carboxyl) group, or a cationic salt formed with any basic (e.g., amino) group. Salts include inorganic salts or organic salts, such as those described in Berge et al., J. Pharm.Sci., 1977, 66, 1-19. Examples of salts include metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, and salts with organic acids. In one embodiment of the present invention, a "solvate" is a compound formed by a solute and a solvent. For solvates, see, for example, J. Honig et al., The Van Nostrand Chemist's Dictionary P650 (1953). If the solvent is water, the solvate formed is a hydrate. It is preferable that the solvent does not interfere with the biological activity of the solute. Examples of such preferable solvents include, but are not limited to, water or various buffers. In one embodiment of the present invention, the "chemical modification" includes, for example, modification with PEG or a derivative thereof, fluorescein modification, biotin modification, and the like.

[0109] When administering the present invention as a pharmaceutical, various delivery systems are known and such systems can be used to administer the therapeutic agent of the present invention to the appropriate site (e.g., esophagus), such as encapsulation in liposomes, microparticles, and microcapsules; using recombinant cells capable of expressing the therapeutic agent (e.g., polypeptides); using receptor-mediated endocytosis; constructing the therapeutic nucleic acid as part of a retroviral or other vector. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The pharmaceutical agent can be administered by any suitable route, such as by infusion, by bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral, rectal, and intestinal mucosa, etc.), using an inhaler or nebulizer, if necessary with an aerosolizing agent, and can be administered together with other biologically active agents. Administration can be systemic or local. When the present invention is used for cancer, it may further be administered by any suitable route, such as by direct injection into the cancer (lesion).

[0110] In a preferred embodiment, the composition can be formulated as a pharmaceutical composition adapted for administration to humans according to known methods. Such compositions can be administered by injection. Typically, compositions for administration by injection are solutions in sterile isotonic aqueous buffer. If necessary, the composition can also include a solubilizing agent and a local anesthetic, such as lidocaine, to ease pain at the injection site. Generally, the ingredients are supplied separately or mixed together in unit dosage form, for example, as a lyophilized powder or water-free concentrate in a sealed container, such as an ampoule or sachet indicating the quantity of active agent. If the composition is to be administered by injection, it can be dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline. If the composition is to be administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0111] The compositions, medicines, therapeutic agents, and preventive agents of the present invention can be formulated in neutral or salt form or as other prodrugs (e.g., esters, etc.). Pharmaceutically acceptable salts include those formed with free carboxyl groups derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., those formed with free amine groups such as those derived from isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc., and those derived from sodium, potassium, ammonium, calcium, and ferric hydroxide, etc.

[0112] The amount of the therapeutic agent of the present invention effective for treating a particular disorder or condition may vary depending on the nature of the disorder or condition, but can be determined by one of skill in the art using standard clinical techniques based on the description herein. In addition, in vitro assays may be used in some cases to help identify optimal dosage ranges. The exact dose to be used in the formulation may also vary depending on the route of administration and the severity of the disease or disorder, and should be determined according to the judgment of the attending physician and the circumstances of each patient. However, the dosage is not particularly limited, and may be, for example, 0.001, 1, 5, 10, 15, 100, or 1000 mg / kg body weight per dose, or within any two of these values. The administration interval is not particularly limited, and may be, for example, 1 or 2 doses per 1, 7, 14, 21, or 28 days, or 1 or 2 doses per two of these values. The dosage, administration interval, and administration method may be appropriately selected depending on the age, weight, symptoms, target organ, etc. of the patient. The therapeutic agent preferably contains an active ingredient in a therapeutically effective amount, or in an amount effective to exert a desired effect. The therapeutic effect may be judged to have been achieved if the malignant tumor marker is significantly reduced after administration. The effective dose can be estimated from a dose-response curve obtained from an in vitro or animal model test system.

[0113] In one embodiment of the invention, a "patient" or "subject" includes a human or a non-human mammal (e.g., one or more of a mouse, guinea pig, hamster, rat, mouse, rabbit, pig, sheep, goat, cow, horse, cat, dog, marmoset, monkey, or chimpanzee, etc.).

[0114] The pharmaceutical composition or therapeutic or prophylactic agent of the present invention can be provided as a kit.

[0115] In certain embodiments, the invention provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the compositions or medicaments of the invention, optionally associated with such containers may be information indicating approval by a governmental agency for the manufacture, use or sale for human administration, in a manner prescribed by the governmental agency regulating the manufacture, use or sale of pharmaceutical or biological products.

[0116] In certain embodiments, pharmaceutical compositions containing the components of the present invention can be administered via liposomes, microparticles, or microcapsules. In various aspects of the present invention, it may be useful to use such compositions to achieve sustained release of the components of the present invention.

[0117] Procedures for formulating the therapeutic agent, prophylactic agent, and other agents of the present invention into pharmaceuticals and the like are known in the art and are described, for example, in the Japanese Pharmacopoeia, the United States Pharmacopoeia, the Pharmacopoeias of other countries, etc. Thus, given the description herein, a person skilled in the art can determine the embodiments, such as the amount to be used, without undue experimentation.

[0118] (General technology) The molecular biological, biochemical and microbiological techniques used herein are well known and commonly used in the art, and are described, for example, in Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, FM (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, FM (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, MA (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, FM (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, FM (1995).Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, MA et al.(1995).PCR Strategies, Academic Press; Ausubel, FM(1999).Short Protocols in These methods are described in, for example, Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, JJ et al. (1999); PCR Applications: Protocols for Functional Genomics, Academic Press, and Special Edition of Experimental Medicine, "Gene Introduction & Expression Analysis Experimental Methods," Yodosha, 1997, and the relevant portions (which may be in their entirety) of these are incorporated by reference in this specification.

[0119] DNA synthesis techniques and nucleic acid chemistry for producing artificially synthesized genes are described, for example, in Gait, MJ (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, MJ (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, RL et al. (1992). The Biochemistry of the Nucleic Acids, Chapman & Hall; Shabarova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, GM et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, GT (I996). Bioconjugate Techniques, Academic Press, and the like, the relevant portions of which are incorporated herein by reference.

[0120] For example, the oligonucleotides of the invention can be synthesized by standard methods known in the art, such as by use of an automated DNA synthesizer (such as those commercially available from Biosearch, Applied Biosystems, etc.), phosphorothioate oligonucleotides can be synthesized by the method of Stein et al. (Stein et al., 1988, Nucl. Acids Res. 16:3209), and methylphosphonate oligonucleotides can be prepared using controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. USA 85:7448-7451), or the like.

[0121] In this specification, "or" is used when "at least one or more" of the items listed in the sentence can be employed. The same applies to "alternative." In this specification, when it is specified that "within a range of two values," the range includes the two values ​​themselves.

[0122] All references cited herein, including scientific literature, patents, patent applications, and the like, are hereby incorporated by reference in their entirety to the same extent as if each was specifically set forth.

[0123] The present invention has been described above by showing preferred embodiments for ease of understanding. The present invention will be described below based on examples, but the above description and the following examples are provided for illustrative purposes only and are not provided for the purpose of limiting the present invention. Therefore, the scope of the present invention is not limited to the embodiments or examples specifically described in this specification, but is limited only by the scope of the claims. EXAMPLES

[0124] Examples are described below. When necessary, the animals used in the following examples were handled in accordance with the standards set forth by the National Institute of Biomedical Innovation and / or Osaka University, and in accordance with the Declaration of Helsinki. The reagents used were specifically the products described in the examples, but equivalent products from other manufacturers (Sigma-Aldrich, Wako Pure Chemical Industries, Nakarai, R&D Systems, USCN Life Science INC, etc.) can also be used.

[0125] (General method) (material and method) (mouse) Female C57BL / 6J mice aged 7–10 weeks were purchased from CLEA JAPAN INC. (Osaka, Japan). MyD88 KO mice were purchased from Oriental BioService Inc. (Kyoto, Japan). IL-12p40 KO and STING mutant mice (Tmem173gt), which have loss-of-function mutations in the ligand-binding site of STING [Sauer et al., Infect. Immun.(2011)79:688-94], were purchased from Jackson Laboratories (Bar Harbor, ME, USA). IRF3 / 7 DKO mice were generated from IRF3 KO [Tang et al., PLoS One.(2013)8:1-6] and IRF7 KO mice, the latter of which was provided by RIKEN BRC (Ibaraki, Japan) through the National Bio-Resource Project of MEXT. IFNAR2 KO mice were obtained from B&K Universal. All animal experiments were performed in accordance with the Animal Care and Use Committe of RIMD and the guidelines of the Osaka University Immunology Frontier Research Center (IFReC), and animal use was approved by Osaka University.

[0126] (reagent) 2'3'-cGAMP, c-di-GMP and 3'3'-cGAMP were purchased from Invivogen (San Diego, CA, USA). DMXAA was purchased from Sigma-Aldrich (St. Louis, MO, USA) and diluted with 5% NaHCO 3 The c-di-GMP was dissolved in 100 mM NaCl. Yamasa (Chiba, Japan) kindly provided c-di-GMP. OVA was purchased from Kanto Chemical (Osaka, Japan) and endotoxin levels were determined as less than 1 EU / mg by Toxicolor® (Seikagaku Corporation, Tokyo, Japan). K3 CpG ODN (SEQ ID NO: 1) was synthesized by Gene Design (Ibaraki, Osaka, Japan) as previously described.

[0127] (Cytokine measurement) Levels of mouse IL-12p40, mouse IL-13, and human IFNγ were measured using ELISA kits from BioLegend (San Diego, CA, USA).

[0128] (statistical analysis) One-way analysis of variance including Mann-Whitney U test, Student's t test, or Bonferroni multiple comparison test was used for statistical analysis ( * p < 0.05; ** p < 0.01; *** p<0.001). Statistical analysis was performed using GraphPad Prism software (La Jolla, CA, USA).

[0129] Example 1: Immunization and Spleen Cell Culture After anesthesia, C57BL / 6J mice were immunized intramuscularly with OVA (10 μg), or OVA and K3 CpG (10 μg), DMXAA (50 μg), c-di-GMP (1 μg), 2'3'-cGAMP or 3'3'-cGAMP (1 μg), or K3 CpG+2'3'-cGAMP / 3'3'-cGAMP / c-di-GMP / DMXAA on days 0 and 10. On day 17, OVA-specific serum IgG1 and IgG2c were measured by ELISA as previously described [Kuroda et al., Immunity.(2011)34:514-526]. The secondary antibodies used in the IgG2c and IgG1 ELISAs were horseradish peroxidase-conjugated goat anti-mouse IgG2c and IgG1 (Bethyl Laboratories, Montgomery, TX). On day 17, spleen cells were harvested and single cell suspensions were prepared using a gentleMACS dissociator (miltenyi Biotech, Gladbach, Germany). 4 After lysis of red blood cells with Cl2 buffer, adipose tissue was cultured in RPMI (containing 1% penicillin / streptomycin and 10% fetal calf serum (FCS)) and stimulated with whole OVA (10 μg / ml) or OVA peptides specific for MHC class I or MHC class II (OVA257, OVA323, respectively) (10 μg / ml) for 48 h. IFNγ and IL-13 production was measured by ELISA.

[0130] (Results: The combination of a TLR9-agonist and a STING-agonist is a type 1 adjuvant and suppresses type 2 immune responses) Given that the agonistic STING agonists DMXAA, c-di-GMP and mammalian 2'3'-cGAMP have been reported to induce type 2 immune responses [Zhang et al., Mol. Cell.(2013)51:226-35; Burdette et al., Nature.(2011)478:515-8; Tang et al., PLoS One.(2013)8:1-6], we next investigated the ability of K3 CpG to cooperate with these other STING agonists. Mouse PBMCs stimulated with 3'3'-cGAMP, as well as 2'3'-cGAMP and c-di-GMP, cooperated with K3 CpG to induce innate IFNγ production (Figure 3A).

[0131] To evaluate the adjuvant properties of these combinations in vivo, we immunized mice twice on days 0 and 10 with OVA protein and a combination of K3 CpG, STING-agonist, or K3 CpG and STING-agonist. On day 17, antigen-specific antibody responses and splenocyte responses were tested. All mouse groups adjuvanted with STING agonists such as cGAMP, c-di-GMP, and DMXAA had type 2 immune responses characterized by high serum anti-OVA IgG1 titers (Figure 3B) and OVA-specific IL-13 production by splenocytes (Figure 3C), whereas the group adjuvanted with the TLR9 agonist K3 CpG did not have type 2 immune responses. In sharp contrast, the addition of K3 CpG converted all type 2 immune responses induced by STING agonists into type 1 immune responses characterized by strong induction of OVA-specific serum IgG2c and splenocyte IFNγ, while shutting down the production of OVA-specific IgG1 and IL-13 (Figures 3B and 3C). We also found that OVA-specific CD8 +We observed synergistic induction of IFNγ by T cells (Figure 6). These results suggest that the combination of TLR9-agonist and STING-agonist induces synergistic adaptive IFNγ in antigen-stimulated splenocytes of mice immunized with the combination and generates a potent type 1 adjuvant capable of suppressing type 2 immune responses induced by STING agonists.

[0132] Example 2: Isolation and stimulation of human PBMCs All hPBMC experiments were performed in accordance with approval from the Institutional Review Board of the National Institute of Biomedical Innovation. Human PBMCs were isolated from the blood of healthy blood donors using human lymphoid separation medium (IBL, Japan) and cultured at 1 × 10 6 Cells were cultured in RPMI. PBMCs were stimulated with K3 CpG (10 μg / ml), cGAMP (10 μM), or K3 CpG+cGAMP for 24 h, and IFNγ production was measured by ELISA.

[0133] For in vitro neutralization experiments, hPBMCs cultured as described above were subjected to IL-12 / 23p40 neutralizing antibody (clone: ​​C8.6, BioLegend, San Diego, CA, USA), type I IFN neutralizing antibody (clone: ​​MMHAR-2, PBL Interferon Source, Piscataway, NJ, USA), or IL-12 / 23p40 neutralizing antibody and type I IFN neutralizing antibody treatment (5 μg / ml) for 30 min 24 h prior to stimulation.

[0134] Results: When combined, K3 CpG and cGAMP potently induce IFNγ in human PBMCs (hPBMCs). K3 CpG is a humanized K-type (also known as B-type) CpG ODN that has been reported to induce type 1 immune responses, but only weakly induces IFN [Klinman et al., Nat. Rev. Immunol.(2004)4:1-10;Verthelyi et al., J. Immunol.(2001)166:2372-2377]. On the other hand, cGAMP can induce strong type 1 IFN and function as an adjuvant [Li et al., Science.(2013)341:1390-4], and other STING agonists have been reported to induce type 2 immune responses [Tang et al., PLoS One.(2013)8:1-6]. To overcome these known limitations of K3 CpG and cGAMP, we investigated the immunostimulatory properties of the combination of K3 CpG and standard 3'3'-cGAMP in hPBMCs in vitro. After screening many cytokines using multiple hPBMCs to find interactions with TLR9- and STING-mediated signaling pathways (data not shown), we found that the combination of the present invention exhibited a strong synergistic effect (approximately 10-90 times higher than stimulation with K3 CpG or cGAMP alone) in inducing IFNγ (Figure 1A).

[0135] Next, to identify the major IFNγ-producing cell type in hPBMCs, we performed intracellular staining of IFNγ in hPBMCs stimulated with K3 CpG, CGAMP, or a combination of these. Our results showed that CD3 - CD56 + CD16 + We show that NK cells are the major producers of synergistic IFNγ in response to combined stimulation among hPBMCs (FIG. 1B).

[0136] In addition to inducing type 1 immune responses, type I IFN and IL-12 can activate NK cells for the production of IFNγ [Hunter et al., Immunol. Lett. (1997) 59: 1-5; Nguyen et al., J. Immunol. (2002) 169: 4279-4287]. Therefore, we next investigated the role of IL-12 and type I IFN in combination-induced innate IFNγ production in hPBMCs. Treatment with IL-12 neutralizing antibody reduced the synergistic IFNγ induction by combination stimulation (Figure 1C). Treatment with type I IFN neutralizing antibody had no effect on combination-induced IFNγ production, but neutralizing both type I IFN and IL-12 simultaneously further reduced synergistic IFNγ production (Figure 1C). These results indicate that IL-12, in combination with type I IFN, aids in the synergistic production of IFNγ by hPBMCs. Collectively, the above results indicate that, when combined, K3 CpG and cGAMP can be potent NK activators that lead to the production of large amounts of IFNγ through a mechanism that is partially dependent on IL-12 and type I IFN.

[0137] Example 3: Culture of mouse PBMCs and dendritic cells Mouse PBMCs were isolated from C57BL / 6J mice using mouse lymphoid separation medium (IBL, Japan) and 0.5 × 10 6 The cells were cultured in RPMI. Cultures of GM-DCs were prepared by flushing bone marrow cells from the tibias and femurs of C57BL / 6J mice and culturing these cells for 7 days in the presence of 20 ng / ml GM-CSF (Pepro Tech, Rocky Hill, NJ, USA). GM-DCs were cultured in RPMI containing 1% penicillin / streptomycin and 20% FCS. Cultures of FL-DCs were prepared from bone marrow cells of C57BL / 6J mice cultured for 7 days in the presence of 100 ng / ml human Flt3L (Pepro Tech). FL-DCs were cultured in RPMI containing 1% penicillin / streptomycin and 10% FCS.

[0138] Mouse PBMCs from wild-type and IRF3 / 7 DKO mice were stimulated with K3 CpG, cGAMP, or K3 CpG + cGAMP for 24 h, and IFNγ production was measured by ELISA.GM-DCs and FL-DCs were stimulated with K3 CpG, cGAMP, or K3 CpG + cGAMP for 24 h, and IL-12p40 and IFNα production was measured by ELISA.

[0139] (Results: Cellular and intracellular mechanisms of synergistic IFNγ induction by K3 CpG and cGAMP in mice) To investigate the synergy between the TLR9 agonist of the present invention and the STING-agonist on the induction of early (innate) IFNγ in mice, we stimulated mouse PBMCs (mPBMCs) with K3 CpG, cGAMP, or a combination of these in vitro. We observed the production of large amounts of IFNγ in a synergistic manner similar to what we observed in hPBMCs. Because IRF3 and IRF7 are necessary downstream molecules for the induction of cGAMP-mediated type I IFN and CpG-mediated type I IFN, respectively [Wu et al., Science.(2013)339:826-30; Kawai et al., Nat. Immunol.(2004)5:1061-8], we investigated the role of IRF3 and IRF7 in synergistic IFNγ production using mPBMCs derived from either wild-type mice or mice lacking both IRF3 and IRF7 (double knockout, DKO).Synergistic IFNγ production was suppressed in IRF3 / 7DKO mPBMCs (Figure 2A).

[0140] Because IL-12 and type I IFN are responsible for synergistic production of IFNγ in hPBMCs (Figure 1C), we investigated the ability of the combined K3 CPG and cGAMP to activate dendritic cells capable of producing IL-12 and / or type I IFN. When we incubated GM-CSF-derived dendritic cells (GM-DCs) and Flt3L-derived dendritic cells (FL-DCs) with K3 CpG, cGAMP, or a combination of these, we found synergy similar to that observed in mPBMCs (Figures 2B-D). The combination of K3 CpG and cGAMP induced significantly higher production of IL-12p40 by both GM-DCs (Figure 2B) and FL-DCs (Figure 2C), and significantly higher production of IFNα by FL-DCs (Figure 2D), than the amount induced by stimulation alone. This indicates a potential role for IL-12 and type I IFN in the synergistic induction of IFNγ by the combination of the present invention. Together, these results show that the synergy between K3 CpG and cGAMP, which potently induces IFNγ in hPBMCs, is reproduced in mice. The mechanism for this synergy involves IRF3 / 7-mediated intracellular signaling, and the synergy induces the production of type I IFN by plasmacytoid DCs (pDCs) and IL-12 by both classical DCs (cDCs) and pDCs.

[0141] Example 5: Staining of intracellular cytokines and cell surface molecules Human PBMCs were stimulated with K3 CpG (10 μg / ml), cGAMP (10 μM), or K3 CpG + cGAMP for 16 h, with the final 4 h stimulated in the presence of brefeldin A. After stimulation, cells were harvested and stained for surface molecules using CD16-PerCP-Cy5.5 antibody (BD Biosciences: Franklin Lake, NJ), CD56-BV421 antibody (BioLegend), CD3-FITC antibody (BD Biosciences), and CD8-PE antibody (Miltenyi Biotech). Fixed and permeabilized cells were stained with IFNγ-APC (BioLegend) for detection of intracellular IFNγ and analyzed using a BD FACSCANTO II flow cytometer.

[0142] Example 6: Tumor Cells and Treatments E.G7-OVA thymoma cells were purchased from the American Type Culture Collection (VA, USA) and cultured in RPMI. 6 Cells were injected subcutaneously into the backs of mice on day 0. On days 7 and 10, mice received PBS (50 μl), K3 CpG (10 μg), cGAMP (10 μg), or K3CpG+cGAMP intratumorally and mice were monitored for tumor growth for 22 days.

[0143] Results: K3 CpG and cGAMP together can suppress tumor growth in a mouse syngeneic explant tumor model. Th1 cells and CD8 +Since T cell responses are important for generating antitumor immunity, we investigated the immunotherapeutic potential of the combination of K3 CpG and cGAMP in a mouse tumor model. We inoculated mice with OVA-expressing EG-7 lymphoma cells by subcutaneous injection. On days 7 and 10, mice received intratumoral injections of PBS, K3 CpG (10 μg), cGAMP (10 μg), or K3 CpG and cGAMP. The combination treatment significantly suppressed tumor growth compared to PBS, K3 CpG, or cGAMP treatment ( FIG. 5 ), suggesting that the combination of the present invention can function as an antigen-free immunotherapeutic agent against cancer.

[0144] Example 7: IFNγ production in KO mice Wild-type, Tmem173gt, IRF3 / 7 DKO, MyD88 KO and IFNAR KO C57BL / 6J mice were immunized with OVA and K3 CpG, cGAMP, or K3 CpG+cGAMP via the intramuscular route on days 0 and 10. OVA-specific serum IgG2c and IgG1 were measured by ELISA on day 17. Spleen cells were also stimulated with OVA for 48 h and IFNγ production was measured by ELISA.

[0145] IL-12p40+ / - and - / - C57BL / 6J mice were also immunized with OVA and K3 CpG, cGAMP, or K3 CpG+cGAMP via the intramuscular route on days 0 and 10. OVA-specific serum IgG2c and IgG1 were measured by ELISA on day 17. Spleen cells were also stimulated with OVA protein for 48 h, and IFNγ production was measured by ELISA.

[0146] (Results: Synergistic induction of IFNγ dependent on IRF3 / 7, STING, MyD88, IL-12 and type I IFN signaling) We showed that the synergistic production of innate IFNγ in mPBMCs is completely dependent on IRF3 and IRF7, which are required for the induction of type I IFN by cGAMP and K3 CpG, respectively. Because cGAMP is a ligand for STING and K3 CpG is a ligand for TLR9, which signals through the adaptor molecule MyD88, we used IRF3 / 7 DKO mice, IFNα / β receptor (IFNAR) KO mice, MyD88 KO mice and STING mutant mice to evaluate the involvement of IRF3 / 7, MyD88, STING and type I IFN in the synergistic production of antigen-specific IFNγ induced by the combination. The combination-induced antigen-specific IgG2c in serum and IFNγ production by spleen were significantly reduced in STING mutant mice, IRF3 / 7 DKO mice, MyD88 KO mice and IFNAR KO mice compared to wild-type mice (Figure 4A and Figure 4B).

[0147] Our in vitro studies in mice and human PBMCs also showed that IL-12 contributes to the synergistic induction of innate IFNγ. Therefore, we next investigated the involvement of IL-12 by using IL-12p40+ / - and - / - mice. We found that IL-12p40 is required for the synergistic induction of antigen-specific IFNγ but not for the induction of IgG2c antibody responses (Figure 4C and Figure 4D). All our results suggest that the combination of K3 CpG and cGAMP is a potent type 1 adjuvant, synergistically inducing the production of antigen-specific IFNγ in an IRF3 / 7, STING, MyD88, IL-12 and type I IFN signaling-dependent manner.

[0148] Example 9: Measurement of IgE antibody production This example is an example regarding suppression of allergies.

[0149] After anesthesia, mice were immunized intramuscularly with OVA (10 μg), or OVA and K3 CpG (10 μg), cGAMP (10 μM), or K3 CpG+GAMP on days 0 and 10. On day 17, OVA-specific serum IgE was measured by ELISA.

[0150] (Results: STING agonists induce IgE, but in combination with CpG this is suppressed.) Many of the current adjuvants are Th2 adjuvants that induce antibody production, but these adjuvants are not suitable for vaccine therapy of cancer or allergies. Furthermore, STING agonists induce IgE as a side effect, which may cause allergic inflammation. However, in the present invention, by simply combining two types of adjuvants, it is possible to induce a strong Th1 adjuvant effect while suppressing IgE induction (Figure 8).

[0151] (Consideration) Efficient vaccines against intracellular pathogens or cancer require adjuvants that induce type 1 immune responses. Cyclic dinucleotides such as cGAMP and c-di-GMP have attracted attention as potential vaccine adjuvants because they directly bind to the transmembrane molecule STING and activate the TBK1-IRF3-dependent signaling pathway to induce type I IFNs [Dubensky et al., Ther. Adv. Vaccines.(2013)1:131-143]. However, there is little evidence that STING-agonists induce type 2 rather than type 1 protective immune responses [Tang et al., PLoS One. (2013) 8:1-6], suggesting that this limits their potential therapeutic application. In this study, we address this issue by combining a STING-agonist with the TLR9 ligand K3 CpG. This combination synergistically enhances the production of innate and adaptive IFNγ. It functions as a potent type 1 adjuvant, inhibiting antibodies as well as CD4 + Th1 cells and CD8 +It potently induces T cells and functions as an antitumor agent that can efficiently suppress tumor growth in mouse tumor models.

[0152] This study shows that the combination of K3 CpG and cGAMP synergistically induces the production of innate IFNγ in both human and mouse PBMCs (Figures 1 and 2), suggesting that this phenomenon is conserved between humans and mice. Our in vitro results also show that the mechanism of action involves IL-12 and type I IFN. Specifically, during the synergy between K3 CpG and cGAMP, type I IFN was not required because its loss of effect was compensated for by IL-12 (Figure 1C). Previous reports have shown that type I IFN and IL-12 upregulate CD4+ IFN production in mice following infection with Listeria monocytogenes. + It was suggested that the combination of the present invention may synergistically induce the production of IFNγ by T cells. They showed that the synergy was significantly reduced in the absence of both cytokines, but was partially reduced in the absence of either one of the cytokines, which is consistent with our results [Way et al., Immunol.(2007)178:4498-4505]. Furthermore, we found that similar to the synergy observed in PBMCs, the combination of the present invention can synergistically induce the production of IL-12p40 in GM-DCs and FL-DCs (Figures 2C and 2D), suggesting a potential role for conventional dendritic cells and plasmacytoid dendritic cells in the synergy induced by the combination. A similar synergistic effect of IL-12 was reported by Krummen et al. with the combination of TLR ligands, CpG and poly I:C in bone marrow-derived DCs, which required MyD88 and TRIF-dependent signaling pathways [Krummen et al., J. Leukoc. Biol. (2010) 88: 189-99]. Our results also suggest that combinations of molecules that activate MyD88-dependent (TLR9) and -independent (STING) signaling pathways generate potent immunostimulants, and such combinations may be useful for immunotherapeutic applications.

[0153] According to the findings of the present inventors, NK cells are the major IFNγ-producing cells in the culture of hPBMCs and subsequent stimulation with the combination (FIG. 1B). On the other hand, previous reports have shown that NK cells express low levels of TLR9, but the cells that respond to CpG stimulation are TLR9-expressing pDCs and B cells in hPBMCs. It has also been reported that IL-12 and type I IFN regulate the production and cytotoxicity of IFNγ in NK cells [Nguyen et al., J. Immunol.(2002)169:4279-4287;Chace et al., Clin. Immunol. Immunopathol. (1997) 84:185-193]. Considering these reports and our in vitro data, we propose that the mechanism of synergistic induction of innate IFNγ is that pDCs respond to K3 CpG, and other cells such as cDCs or macrophages respond to cGAMP, and can produce large amounts of type I IFN and IL-12, which then cooperate to induce the production of IFNγ in NK cells by signaling through the IL-12 receptor and the type I IFN receptor.

[0154] The first report on the adjuvant effect of 2'3'-cGAMP showed that intramuscular cGAMP immunization could induce antigen-specific B and T cell responses in a STING-dependent manner [Li et al., Science.(2013)341:1390-4]. Our in vivo immunization study with 3'3'-cGAMP is also consistent with previous reports; it induces strong antigen-specific B and T cell responses in a STING-dependent manner (Figures 4A and 4B). We also showed that 3'3'-cGAMP is a type 2 adjuvant that can induce not only IgG1 but also IgG2c antibody responses and Th2-type cytokine responses in spleen cells (Figures 3B and 3C). Although type 2 adjuvants do not normally induce the production of Th1-like Ig isotypes (IgG2c), cGAMP can induce Th1-like Ig isotypes, likely due to its ability to induce type I IFN, since type I IFN induces IgG2c antibody responses [Swanson et al., J. Exp. Med.(2010)207:1485-500]. Furthermore, we found that distinct mechanisms are involved in the induction of B and T cell responses by cGAMP, in which cGAMP-induced antibody responses, but not Th2 responses, depend on type I IFN signaling (Figure 4B). Furthermore, since cGAMP is known to signal exclusively through the STING-IRF3 axis to induce the production of type I IFN [Wu et al., Science.(2013)339:826-30], we expected to observe a decrease in antibody and T cell responses in IRF3 / 7DKO mice. However, cGAMP-induced antibody responses were slightly decreased in IRF3 / 7DKO mice, whereas cGAMP-induced T cell responses were partially dependent on IRF3 / 7 and, surprisingly, on MyD88, but such effects were completely dependent on STING (Figures 4A and 4B). Therefore, we are further investigating the possibility that, in addition to the STING-IRF3 pathway, cGAMP may activate an unknown signal pathway involving the adaptor molecule MyD88.

[0155] Although K3 CpG was reported as an adjuvant capable of inducing a type 1 immune response [Chu et al., J. Exp. Med.(1997)186:1623-1631], we found that K3 CpG alone is a weak type 1 adjuvant, as it was unable to induce antigen-specific antibody or T cell responses at levels comparable to those of cGAMP or combination immunized groups. Interestingly, the combination of the weak type 1 adjuvant K3 CpG with the type 2 adjuvant cGAMP enhanced synergistic antigen-specific IFNγ production as well as strong Th1-like antibodies and CD8 +This resulted in a strong type 1 adjuvant that induces T cell responses (Figures 3 and 6). Our findings are also consistent with previous studies showing that the combination of type 2 adjuvants CpG and IFA induces type 1 immune responses while suppressing type 2 immune responses [Chu et al., J. Exp. Med.(1997)186:1623-1631]. Importantly, in addition to inducing a strong type 1 immune response by the combination of the present invention, we also showed that the combination of the present invention can suppress the type 2 immune response induced by cGAMP. This is important for increased safety, since a dominant type 2 response has been reported to cause many chronic diseases, such as allergies [Spellberg et al., Clin. Infect. Dis.(2001)90509:76-102;Muller et al., J. Immunol.(1993)150:5576-5584;Seki et al., Nat. Med.(2003)9:1047-1054]. Our results are also consistent with the findings of Lin et al., in that IgG2c production is enhanced, whereas IgG1 production is suppressed by CpG [Lin et al., Eur. J. Immunol.(2004)34:1483-7]. Furthermore, the synergistic effect of the combination of the present invention on the induction of antigen-specific IFNγ is dependent on IRF3 and IRF7 (Figure 4A and Figure 4B), indicating that type I IFN may play an important role in this synergy. This idea is further supported by the complete loss of synergy that we observed in IFNAR KO mice (Figure 4A and Figure 4B). Furthermore, since MyD88 is a downstream signaling molecule of TLR9 and cGAMP is a ligand of STING, we found that the type 1 immune induction effect of the combination is dependent on both MyD88 and STING as expected (Figure 4A and Figure 4B). On the other hand, we showed that IL-12p40 is required for the synergistic induction of Th1 type cytokine responses, but not for the induction of IgG2c antibody responses (Figure 4C and Figure 4D).Since IL-12 is important for the development of Th1 cells and the production of IFNγ, it is reasonable to observe the dependency of IL-12 in Th1 type cytokine response. A possible explanation for the IL-12-independent induction of IgG2c by the combination of the present invention is that the production of type I IFN in KO mice can compensate for the absence of IL-12. Previous reports have shown that type I IFN can induce IgG2c antibody responses in a T cell-independent manner [Swanson et al., J. Exp. Med.(2010)207:1485-500], while IL-12 induces IgG2c antibody responses by inducing IFNγ production from T cells or NK cells [Gracie et al., Eur. J. Immunol.(1996)26:1217-1221].

[0156] Finally, we found that the combination of K3 CpG and cGAMP had a strong antitumor effect, since treatment with this combination alone was able to effectively suppress tumor growth in the EG-7 mouse tumor model (Figure 5). Our in vivo results showed that the combination strongly inhibited CD8 + The results showed that CpG ODN induces CD8 T cell responses (Figure 6). + It has already been reported that the combination induces the generation of cytotoxic T cells [Krieg et al., Nat. Rev. Drug Discov. (2006) 5:471-84], so the antitumor effect of the combination of the present invention is expected to be strong CD8 + Our hypothesis is that vaccination with OVA-conjugated CpG ODN also induces activation of CD8 T cells. +This is supported by previous reports showing that K3 CpG has a strong antitumor effect that depends on T cells [Cho et al., Nat. Biotechnol.(2000)18:509-514]. However, the possibility of enhanced NK cell activation by the combination treatment of the present invention cannot be excluded, and as in the case of hPBMC culture, we identified NK cells as playing a major role in the synergistic effect of IFNγ. The advantage of the combination treatment of the present invention over previously reported CpG-based antitumor agents such as OVA-conjugated CpG ODN [Cho et al., Nat. Biotechnol.(2000)18:509-514] or nanoparticle-conjugated CpG ODN [De et al., Proc. Natl. Acad. Sci. USA(2013)110:19902-7] is that it does not require chemical conjugation between K3 CpG and cGAMP. Moreover, unlike these systems, the approach of the present invention does not require injection or conjugation of tumor antigens. It functions as an antigen-free antitumor agent rather than a prophylactic vaccine.

[0157] In conclusion, our study suggests that the combination of TLR9-agonist and STING-agonist is a type 1 adjuvant favorable for vaccines requiring a strong cellular immune response, and a promising antitumor agent that can also stimulate human NK cells for synergistic IFNγ production. Thus, our results provide insight into the combined mechanism of action of TLR9 and STING signaling pathways, which potentially enhances the immunotherapeutic and adjuvant properties of the combination of the present invention.

[0158] (Example 10: Experiments on various types of STING ligands) In this example, the same experiments as in Examples 1 to 9 are carried out. Using cGAMP, c-di-GMP, 2'3'-cGAMP, and DMXAA, similar experiments are carried out under the conditions of Examples 7 to 9, except that instead of 3'3'-cGAMP, cGAMP, c-di-GMP, 2'3'-cGAMP, and DMXAA are used.

[0159] It can be understood that, as seen in Examples 1 to 6, results similar to those of 3'3'-cGAMP can be obtained with cGAMP, c-di-GMP, 2'3'-cGAMP, and DMXAA.

[0160] (Example 11: Experiments on various types of CpG (other than K3)) In this example, the experiments of Examples 1 to 9 were carried out using CpGs other than K3, such as CpG1826 and D35 CpG.

[0161] (material and method) CpG1826 (SEQ ID NO:2 = 5'-tccatgacgttcctgacgtt-3') and D35 CpG (SEQ ID NO:3 = 5'-ggtgcatcgatgcagggggg-3') were purchased from Invivogen and synthesized by Gene Design (Ibaraki, Osaka, Japan), respectively. After synthesis or purchase, the lyophilized products were dissolved in sterile water before use.

[0162] (method) 1 × 10 mouse (c57BL / 6) splenocytes 7 The cells were adjusted to a concentration of 1000 cells / ml, and the cells were stimulated in vitro with the above-mentioned stimulants such as CpG for 24 hours, after which IFN-γ in the culture supernatant was measured by ELISA. These methods were performed as described in (General Methods). (result) The results are shown in Figure 12. As shown, it was demonstrated that each CpG has a synergistic effect on interferon production in spleen cells when combined with the STING agonist 3'3'-cGAMP. Therefore, it can be said that the effect of the present invention is not limited to a specific CpG but is widely exerted by CpG in general.

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[0164] The present invention is expected to have a ripple effect both technically and economically as a next-generation adjuvant, and can find industrial applicability in the pharmaceutical industry, bioindustry, etc. [Sequence List Free Text]

[0165] SEQ ID NO: 1: Sequence of K3 CpG: 5'-atcgactatcgagagttctc-3' SEQ ID NO: 2: Sequence of CpG1826: 5'-tccatgacgttcctgacgtt-3' SEQ ID NO: 3: Sequence of D35 CpG: 5'-ggtgcatcgatgcagggggg-3'

Claims

[Claim 1] The invention described in the examples.

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