Novel TLR9 agonists

Abstract

The present application provides novel oligonucleotides and their therapeutic uses. The oligonucleotides of the present invention can be used for activating or modulating immunity in a subject.

Description

[Technical Field]

[0001] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0002] The present invention relates to novel oligonucleotides or oligonucleotide derivatives capable of binding to nucleic acid receptors, including TLR9. The present invention further relates to pharmaceutical compositions comprising said oligonucleotides. The present invention further relates to methods for preventing or treating target diseases, including cancer or infectious diseases, using said oligonucleotides. [Background technology]

[0003] TLRs (Toll-like receptors) are a family of receptors that recognize pathogen-associated molecular patterns (PAMPs) and are localized on the surface of cell membranes or endosomal membranes. TLR activation leads to the secretion of type I interferons and proinflammatory cytokines. Ten TLRs, designated TLR1 through TLR10, are known in humans. TLR3, TLR7, TLR8, and TLR9 reside in endosomes and primarily detect nucleic acids. TLR3 primarily recognizes double-stranded RNA and a synthetic RNA called poly(I:C), which resembles viral RNA, whereas TLR7 and TLR8 primarily recognize single-stranded RNA. On the other hand, TLR9 primarily recognizes single-stranded DNA. In humans, TLR9 is expressed in plasmacytoid dendritic cells (pDCs), B cells, eosinophils, basophils, macrophages, and NK cells (Non-Patent Document 1; Non-Patent Document 2; Non-Patent Document 3).

[0004] TLR9 agonists activate pDCs and B cells, promoting their proliferation, production of inflammatory cytokines and antibodies, antigen presentation, and expression of costimulatory molecules and MHC molecules. Upon receiving stimulation from a TLR9 agonist, pDCs and B cells also increase the expression of chemokine receptors, resistance to apoptosis, and production of cytokines, including Th1-promoting chemokines such as macrophage inflammatory protein 1 (MIP1) and 10-kDa interferon-inducible protein (IP10). Memory B cells, in particular, differentiate into antibody-secreting plasma cells solely dependent on TLR9 activation. One example of a TLR9 agonist is CpG DNA (CpG). CpG DNA contains unmethylated cytosines and guanines, and was initially found in bacterial DNA as an important motif constituting pathogen-associated molecular patterns (PAMPs) (Non-Patent Document 4).

[0005] CpG-oligodeoxynucleotides (ODNs) have been reported to be classified into four classes based on their structure and function. Class A ODNs, also known as class D ODNs, have a phosphorothioated backbone at several nucleotides at the 3' end and several nucleotides at the 5' end. Class A ODNs contain a palindromic sequence containing CpG, which can form a stem-loop structure. The termini of class A ODNs contain a poly-G sequence, which can form a parallel quadruplex structure called a G-tetrad (Non-Patent Document 5). Class A ODNs strongly promote interferon-α (IFN-α) production from pDCs but only weakly activate B cells. Class B ODNs, also known as class K ODNs, have one or more CpG sequences and a backbone in which the majority of nucleotides are phosphorothioated. Class B ODNs strongly induce B cell proliferation, promote the secretion of IgM antibodies from B cells, and induce the differentiation and maturation of pDCs. Class C ODNs have a phosphorothioated backbone. Class C ODNs have a palindromic sequence with CpG at the 3' end, which can anneal intramolecularly to form a hairpin structure. Class C ODNs promote IFN-α production from pDCs and IL-6 production from B cells. Thus, class C ODNs have functions similar to those of both class A and class B ODNs. Class P ODNs have two palindromic sequences. Class P ODNs can anneal intermolecularly to form concatemers at the palindromic sequence at the 5' end or form intramolecular hairpin structures at the GC-rich 3' end. Class P ODNs strongly induce type I IFN production (Non-Patent Document 6).

[0006] The mechanism by which CpG-ODN activates TLR9-expressing cells has been reported as follows: First, internalized CpG-ODN binds to TLR9 in endosomes. Once CpG-ODN binds to TLR9, the adaptor protein MyD88, which is bound to the cytoplasmic side of TLR9, is activated by phosphorylation. Activated MyD88 induces the transcription of cytokines, including type I interferons (IFNs), through activation of transcription factors such as IRF3, 5, and 7. Activated MyD88 also signals through the nuclear factor-kappa B (NF-kB) pathway. Downstream signals from MyD88 ubiquitinate IkB (inhibitor of kappa B or kappa-beta inhibitor), inducing its degradation and thereby activating NF-kB. Activated NF-kB binds to the NF-kB promoter and activates the expression of target genes. As a result, the production of inflammatory cytokines such as interleukin-1beta (IL-1β), TNF-alpha (TNF-α) and interleukin-6 (IL-6) is induced.

[0007] Since CpG-ODN has the above-mentioned biological TLR9 activating activity, administration of CpG-ODN is suitable for treating and / or preventing several diseases or disorders such as those listed below.

[0008] <Anti-cancer (tumor) activity> CpG-ODN can affect the tumor microenvironment (TME) and convert cold tumors into hot tumors (Non-Patent Document 7). The TME is an environment constructed by tumor cells and non-tumor cells such as immune cells, fibroblasts, and vascular cells surrounding the tumor. The state of the TME significantly affects the progression of tumors. Cold tumors are tumors that contain immunosuppressive cells such as tumor-associated macrophages (TAM), myeloid-derived suppressor cells (MDSC), and regulatory T cells (Treg) that constitute an immunosuppressive TME, and only slightly activated tumor infiltrating lymphocytes (TIL). Cold tumors often show resistance to cancer treatments including immunotherapy. Hot tumors are tumors that contain anti-tumor immune cells including TIL and M1 macrophages that constitute an immunologically activated TME. Hot tumors generally respond to cancer treatments. For example, intratumoral administration of CpG-ODN has been reported to result in an increase in T cells, pDC, and NK cells, a decrease in Treg, and suppression of MDSC (Non-Patent Document 8). Furthermore, TLR9 agonists have been reported to re-educate tumor-promoting M2-like macrophages to induce anti-tumor M1-like macrophage polarization (Non-Patent Document 9). CpG-ODN has been reported to enhance the anti-tumor activity of macrophages to engulf cancer cells that express "don't eat me signals" such as CD47 (Non-Patent Document 2).

[0009] CpG-ODN can also suppress the immunosuppressive properties of monocytic (CD11b+, Ly6G-, Ly6C high , ,

[0010] , , , , ,

[0009] ) MDSC, such as the inhibitory activity of T cell function (Non-Patent Document 10).

[0010] <Prevention or treatment of Th2- or Th17-mediated diseases> CpG-ODN is suitable for the effective prevention or treatment of Th2 or Th17 mediated diseases. Conventional CpG ODN has been shown to regulate the balance of immune response by activating Th1 cells and Tregs in terms of their immunoregulatory effects, and as a result, suppress Th2 cells and Th17 cells.Th1 cells and Th2 cells mutually inhibit the differentiation into Th2 cells or Th1 cells, respectively, so it is thought that the differentiation induction into Th1 cells by CpG-ODN leads to the reduction of Th2 cells.

[0011] Recently, many factors have been reported that regulate the balance between Th17 cells and Tregs, including downstream signals of the T cell receptor, costimulatory molecules, cytokines, metabolic pathways, and the intestinal microbiota (Non-Patent Document 11). It has been reported that LPS, a TLR4 agonist, induces the differentiation of Th17 cells, and peptidoglycan, a TLR2 agonist, induces the differentiation of Th17 cells and Th1 cells to some extent. On the other hand, CpG-ODN has been reported to preferentially induce the differentiation of Th1 cells through IFN-α production (Non-Patent Document 12).

[0012] The feasibility of therapy to maintain immune balance has also been demonstrated in vivo. For example, when CpG-ODN was administered to ulcerative colitis model mice and patients, the number of Th17 cells and the production of IL-17 and IL-6 were reduced, but the number of Treg cells and the production of IL-10 were increased, resulting in symptomatic improvement (Non-Patent Document 13). It was also reported that the addition of CpG alleviated the symptoms of EAE in mice treated with complete Freund's adjuvant, revealing the effect of CpG in attenuating the onset of Th17-mediated disease (Non-Patent Document 14).

[0013] Based on the finding that CpG ODN induced the production of indoleamine 2,3-dioxygenase (IDO) from dendritic cells and the activation and proliferation of Tregs, it is also suggested that CpG-ODN may be effective in treating asthma and atopic diseases, which belong to Th17-mediated diseases (Non-patent Document 15).

[0014] <Combination therapy> Furthermore, because CpG-ODNs exhibit the immune-modulating activity described above, they have also been reported to be suitable for combination therapy with (i) vaccines, (ii) antibody drugs, (iii) conventional chemotherapy, (iv) molecular-targeted drugs, (v) surgical procedures, (vi) cytokines, (vii) adoptive immune cell therapy (also known as adoptive cell transfer (ACT)), and (viii) therapies containing agonists for nucleic acid receptors.

[0015] (i) Use in combination with a vaccine CpG-ODN can improve the immunogenicity of vaccine.For example, CpG-ODN can be administered together with anti-cancer vaccines such as melanoma antigen vaccines (Non-Patent Document 6; Non-Patent Document 8) and vaccines against viruses such as cytomegalovirus, malaria, anthrax and influenza virus (Non-Patent Document 6).HepliSab-B (registered trademark), a hepatitis B vaccine containing CpG-ODN and hepatitis B antigen, has been approved by FDA.

[0016] (ii) Combination with antibody drugs (immune checkpoint inhibitors (CPIs) and cytotoxic antibodies, etc.) CPIs are drugs that alter the immunosuppressive state of the tumor microenvironment or the environment surrounding infected cells by binding to checkpoint molecules or their ligands. For example, the combination of CpG-ODN and Keytruda was investigated in a clinical trial for advanced melanoma (Non-Patent Document 16). A clinical trial of the combination with Yervoy was also conducted for the treatment of advanced solid tumors (Non-Patent Document 17). Interestingly, the combination of CpG-ODN and CPIs has been suggested to be effective in patients resistant to standard treatments. For example, it has been reported that the combination of CpG-ODN and CPIs exhibits antitumor activity even in patients who are resistant to CPIs such as PD-1 antibodies (Non-Patent Document 18). Cytotoxic antibodies are drugs that have the ability to induce antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cellular cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP). Synergistic effects of the combined use of CpG-ODN and cytotoxic antibodies have been reported (Non-Patent Document 19). For example, the combination of CpG-ODN and Rituxan was investigated in a clinical trial for non-Hodgkin's lymphoma (Non-Patent Document 20). It has also been shown that the combination of CpG oligonucleotides, poly(I:C), and cytotoxic antibodies had antitumor activity even in patients who were resistant to cytotoxic antibodies such as Herceptin (Non-Patent Document 21).

[0017] (iii) Combination with conventional chemotherapy Conventional chemotherapy drugs consist of chemicals that inhibit the proliferation of fast-growing tumor cells and kill such cells. For example, the combination of platinum-based drugs, taxane-based drugs, and CpG-ODN has been investigated in clinical trials (Non-Patent Document 22).

[0018] (iv) Combination with molecular targeted drugs Conventional molecular targeted drugs are generally formed from small molecules that bind to specific target molecules and regulate their function. Examples of target molecules include those that cause carcinogenesis, those that are involved in the induction of mutations, or those that are involved in the homeostasis of tumor cells. For example, the synergistic effect between CpG and bortezomib in multiple myeloma has been reported in preclinical trials, and it has been concluded that this forms the basis for feasibility testing in the clinical stage (Non-Patent Document 23).

[0019] (v) in combination with surgical procedures (including radiation therapy, cryoablation, and radiofrequency ablation) It has been reported that administration of CpG-ODN after surgical resection improved survival rates (Non-Patent Document 24). Radiotherapy is a treatment that uses radiation to damage the DNA of proliferating tumor cells and kill such cells. Cryoablation is a treatment that freezes and kills tumor cells. Radiofrequency ablation is a treatment that coagulates tumor cells with heat generated by radiofrequency waves and kills them. CpG-ODN is thought to be useful for eliciting immunity against tumor antigens released from dead cells killed by these treatments (Non-Patent Document 25).

[0020] (vi) Combination use with cytokines Examples of cytokines are IFN-α and IL-18, which activate NK cells or dendritic cells. It has been reported that the combination of IL-18 and CpG-ODN induces apoptosis of malignant B cells, and these B cells secrete granzymes, which further kill nearby malignant B cells (Non-Patent Document 25).

[0021] (vii) Combination with adoptive immune cell therapy Adoptive cell therapy is a type of therapy that administers immune cells that have been improved ex vivo. Examples of such therapies include CAR-T therapy, which uses genetically modified T cells transduced with TCRs against specific cancer antigens and immune cells such as patient- or donor-derived TILs or dendritic cells stimulated ex vivo to have antitumor effects (Non-Patent Document 26).

[0022] Combination use with "(viii) an agonist for nucleic acid receptors" Combination with TLR7 / 8 agonists has been reported to show a synergistic effect in clinical trials (Non-Patent Document 8). In addition, synergistic effects between TLR9 agonists such as CpG and STING (Stimulator of interferon genes) agonists have also been reported to enhance Th1-biased immune responses such as production of antigen-specific IgG and IFN-γ and cytotoxic CD8(+) T cell responses in PBMCs (Non-Patent Document 27).

[0023] <Mechanisms Supporting Combination Therapies Involving Administration of CpG> Some of the above treatments or drugs have been shown to induce immunogenic cell death (ICD), and combination with CpG-ODN has been investigated. ICD is a type of cell death in which corpses of cells damaged by ICD or dying cells induce a strong immune response by secreting DAMPs, and molecules constituting DAMPs are reported to be, for example, calreticulin, HMGB (high-mobility group box), heat shock protein, or ATP. An increase in the amount of such DAMPs was observed compared with normal cell death (Non-Patent Document 28). Some chemotherapeutic drugs and some molecular target drugs are known as inducers of ICD, and such ICD inducers include doxorubicin, mitoxantrone, oxaliplatin, and bortezomib. Radiation and photodynamic therapy (PDT) are also known to be inducers of ICD (Non-Patent Document 29).

[0024] Some treatments or drugs that suppress or kill Tregs have been investigated regarding combination with the above CpG-ODN. For example, CpG-ODN synergistically enhances the immune response after depletion of Tregs by anti-CD25 antibody (Non-Patent Document 3).

[0025] In conclusion, CpG-ODN can be administered alone or in combination with at least one other active ingredient for the following pharmaceutical purposes: (i) immunostimulatory effects for preventing and treating neoplasms and infectious diseases, including cancer (e.g., metastatic solid tumors, melanoma, cutaneous T-cell lymphoma, chronic lymphocytic leukemia, etc.); (ii) immunomodulatory effects for preventing and treating immune-mediated diseases, such as Th2- or Th17-related diseases, including some types of autoimmune and allergic diseases; and (iii) modulation of the responsiveness of TLR7 / 9-expressing tumor cells to anticancer drugs and immune cells. [Prior art documents] [Patent documents]

[0026] [Patent Document 1] International Publication No. 2014082254 [Patent Document 2] Patent Publication No. 5011520 [Patent Document 3] International Publication No. 2004016805 [Non-patent literature]

[0027] [Non-Patent Document 1] Roda, JM, et al., CpG-containing oligodeoxynucleotides act through TLR9 to enhance the NK cell cytokine response to antibody-coated tumor cells. J Immunol, 2005. 175(3): p.1619-27. [Non-patent document 2] Liu, M., et al., Metabolic rewiring of macrophages by CpG potentiates clearance of cancer cells and overcomes tumor-expressed CD47-mediated 'don't-eat-me' signal. Nat Immunol, 2019. 20(3): p.265-275. [Non-patent document 3] Hemmi, H., et al., A Toll-like receptor recognizes bacterial DNA. Nature, 2000. 408(6813): p.740-5. [Non-patent document 4] Krieg, AM, Therapeutic potential of Toll-like receptor 9 activation. Nat Rev Drug Discov, 2006. 5(6): p.471-84. [Non-Patent Document 5] Puig M. et al., Use of thermolytic protective groups to prevent G-tetrad formation in CpG ODN type D: structural studies and immunomodulatory activity in primates., Nucleic Acids Res. 2006, 34(22): p.6488-95 [Non-patent document 6] Scheiermann, J. and Klinman, DM, Clinical evaluation of CpG oligonucleotides as adjuvants for vaccines targeting infectious diseases and cancer. Vaccine, 2014. 32(48): p.6377-89. [Non-Patent Document 7] (Authors not listed), Warming “Cold” Melanoma with TLR9 Agonists. Cancer Discov, 2018. 8(6): p.670. [Non-patent document 8] Shirota, H., et al., CpG Oligonucleotides as Cancer Vaccine Adjuvants. Vaccines (Basel), 2015. 3(2): p.390-407. [Non-Patent Document 9] Mantovani, A., et al., The interaction of anticancer therapies with tumor-associated macrophages. J Exp Med, 2015. 212(4): p.435-45. [Non-Patent Document 10] Shirota, Y., et al., Intratumoral injection of CpG oligonucleotides induces the differentiation and reduces the immunosuppressive activity of myeloid-derived suppressor cells. J Immunol, 2012. 188(4): p.1592-9. [Non-Patent Document 11] Lee, GR, The Balance of Th17 versus Treg Cells in Autoimmunity. Int J Mol Sci, 2018. 19(3): p.730. [Non-Patent Document 12] Shi, G., et al., Differential involvement of Th1 and Th17 in pathogenic autoimmune processes triggered by different TLR ligands. J Immunol, 2013. 191(1): p.415-23. [Non-Patent Document 13] Schmitt, H et al., OP004 The TLR9 agonist cobitolimod induces anti-inflammatory effects and balances the Th17 / T-reg cell response in ulcerative colitis. J Crohns Colitis, 2018, 12(Suppl1): p.S003 [Non-Patent Document 14] Tigno-Aranjuez, JT, et al., Encephalitogenicity of complete Freund's adjuvant relative to CpG is linked to induction of Th17 cells. J Immunol, 2009, 183(9): p.5654-61 [Non-Patent Document 15] Kline, JN, et al., Toll-like receptor 9 activation with CpG oligodeoxynucleotides for asthma therapy. Drug News Perspect, 2008. 21(8): p.434-9. [Non-Patent Document 16] Ribas, A., et al., SD-101 in Combination with Pembrolizumab in Advanced Melanoma: Results of a Phase Ib, Multicenter Study. Cancer Discov, 2018. 8(10): p.1250-1257. [Non-Patent Document 17] Reilley, M., et al., Phase 1 trial of TLR9 agonist lefitolimod in combination with CTLA-4 checkpoint inhibitor ipilimumab in advanced tumors. Journal of Clinical Oncology, 2019. 37(15_suppl): p.TPS2669-TPS2669. [Non-Patent Document 18] Wang, S., et al., Intratumoral injection of a CpG oligonucleotide reverts resistance to PD-1 blockade by expanding multifunctional CD8+ T cells. Proc Natl Acad Sci U S A, 2016. 113(46): p.E7240-E7249. [Non-Patent Document 19] Hiramatsu, K., et al., CpG oligodeoxynucleotides potentiate the antitumor activity of anti-BST2 antibody. Cancer Sci, 2015. 106(10): p.1474-8. [Non-Patent Document 20] Friedberg, J.W., et al., Combination immunotherapy with a CpG oligonucleotide (1018 ISS) and rituximab in patients with non-Hodgkin lymphoma: increased interferon-alpha / beta-inducible gene expression, without significant toxicity. Blood, 2005. 105(2): p.489-95. [Non-Patent Document 21] Charlebois, R., et al., PolyI:C and CpG Synergize with Anti-ErbB2 mAb for Treatment of Breast Tumors Resistant to Immune Checkpoint Inhibitors. Cancer Res, 2017. 77(2): p.312-319. [Non-Patent Document 22] Krieg, AM, Development of TLR9 agonists for cancer therapy. J Clin Invest, 2007. 117(5): p.1184-94. [Non-Patent Document 23] Ray, A., et al., A novel TLR-9 agonist C792 inhibits plasmacytoid dendritic cell-induced myeloma cell growth and enhance cytotoxicity of bortezomib. Leukemia, 2014. 28(8): p.1716-24. [Non-Patent Document 24] Weigel, BJ, et al., CpG oligodeoxynucleotides potentiate the antitumor effects of chemotherapy or tumor resection in an orthotopic murine model of rhabdomyosarcoma. Clin Cancer Res, 2003. 9(8): p.3105-14. [Non-Patent Document 25] Jahrsdorfer, B. et al., CpG oligodeoxynucleotides as immunotherapy in cancer. Update Cancer Ther, 2008. 3(1): p.27-32. [Non-Patent Document 26] Xu, L., et al., CpG oligodeoxynucleotides enhance the efficacy of adoptive cell transfer using tumor infiltrating lymphocytes by modifying the Th1 polarization and local infiltration of Th17 cells. Clin Dev Immunol, 2010: p.410893. [Non-Patent Document 27] Temizoz B., et al, TLR9 and STING agonists synergistically induce innate and adaptive type-II IFN. Eur J Immunol, 2015, 45(4): p.1159-69 [Non-patent document 28] Bedognetti, D., et al., Toward a comprehensive view of cancer immune responsiveness: a synopsis from the SITC workshop. J Immunother Cancer, 2019. 7(1): p.131. [Non-Patent Document 29] Galluzzi, L., et al., Immunogenic cell death in cancer and infectious disease. Nat Rev Immunol, 2017. 17(2): p.97-111. [Non-Patent Document 30] Jarry, U., et al., Treg depletion followed by intracerebral CpG-ODN injection induce brain tumor rejection. J Neuroimmunol, 2014. 267(1-2): p.35-42. [Non-Patent Document 31] Maeda, T., et al., A novel plasmacytoid dendritic cell line, CAL-1, established from a patient with blastic natural killer cell lymphoma. Int J Hematol, 2005. 81(2): p.148-54.

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[0028] The present invention provides novel TLR agonists and their therapeutic uses. [Means for solving the problem]

[0029] The present inventors have newly discovered the following: (1) the oligonucleotides of the present invention can be used to activate or modulate immunity in a subject; and (2) The oligonucleotides of the present invention are particularly useful for treating or preventing subjects suffering from diseases including tumors, microbial infections, primary immunodeficiency diseases, and Th2 / Th17-related diseases.

[0030] The present invention includes the following embodiments: (1) A single-stranded oligonucleotide containing the sequence motifs 5'-tcgcaacgttt-3' (SEQ ID NO: 1) and 5'-cgacg-3'.

[0031] (2) The oligonucleotide according to (1), comprising the nucleotide sequence motif 5'-tcgcaacgttt-n-cgacg-n-cg-nn-cg-3' (SEQ ID NO: 2) (n represents any base).

[0032] (3) The oligonucleotide according to (1) or (2), having a total of 20 to 25, preferably 21 to 24, more preferably 22 to 23 nucleotides.

[0033] (4) 5'-tcgcaacgtttgcgacgtcggtcga (SEQ ID NO: 54); 5'-tcgcaacgtttgcgacggcgctcga (SEQ ID NO: 55); 5'-tcgcaacgtttgcgacgtcgttcga (SEQ ID NO: 56); 5'-tcgcaacgtttgcgacggcgttcga (SEQ ID NO: 57); 5'-tcgcaacgtttgcgacgtcgttcg (SEQ ID NO: 58); 5'-tcgcaacgtttgcgacgtcgttcgg (SEQ ID NO: 59); 5'-tcgcaacgtttacgacgtcggtcga (SEQ ID NO: 60); 5'-tcgcaacgtttacgacggcgctcga (SEQ ID NO: 61); 5'-tcgcaacgtttacgacgtcgttcga (SEQ ID NO: 62); and 5'-tcgcaacgtttacgacggcgttcga (SEQ ID NO: 63) The oligonucleotide according to any one of (1) to (3), comprising a sequence motif selected from the group consisting of:

[0034] (5) The oligonucleotide according to any one of (1) to (4), wherein the internucleotide bonds of the oligonucleotide are partially or completely chemically modified.

[0035] (6) The oligonucleotide according to (5), wherein the chemically modified internucleotide bond is phosphorothioated.

[0036] (7) 5'-tCgcaacgtttgcgacgtcgttcgA-3' (SEQ ID NO: 16); 5'-tCgcaaCgtttgcgacgtcgttcgA-3' (SEQ ID NO: 17); 5'-tCgCaaCgtttgcgacgtcgttcgA-3' (SEQ ID NO: 18); 5'-tCgCaacgtttgCgaCgtcgttcgA-3' (SEQ ID NO: 19); 5'-tCgCaacgtttgCgaCgtcgttCgA-3' (SEQ ID NO: 20); 5'-tCgCaaCgtttgcgacgtCgttCgA-3' (SEQ ID NO: 21); 5'-tCgCaaCgtttgCgaCgtCgttCgA-3' (SEQ ID NO: 22); 5'-tCgCaaCgtttgcgacgtCggtCgA-3' (SEQ ID NO: 23); 5'-tCgCaaCgtttgcgacggCgctCgA-3' (SEQ ID NO: 24); 5'-tCgCaaCgtttgcgacggCgttCgA-3' (SEQ ID NO: 26); 5'-tCgCaaCgtttgcgacgcCgttCgA-3' (SEQ ID NO: 27); 5'-tCgCaaCgtttgcgacggCgtaCgA-3' (SEQ ID NO: 28); 5'-tCgCaaCgtttgcgacggCgtgCgA-3' (SEQ ID NO: 29); 5'-tCgCaaCgtttacgacgtCggtCgA-3' (SEQ ID NO: 30); 5'-tCgCaaCgtttacgacggCgctCgA-3' (SEQ ID NO: 31); 5'-tCgCaaCgtttacgacgtCgttCgA-3' (SEQ ID NO: 32); 5'-tCgCaaCgtttGcgacgtCggtCgA-3' (SEQ ID NO: 33); 5'-tCgCaaCgtttAcgacgtCggtCgA-3' (SEQ ID NO: 34); 5'-tCgCaaCgtttGcgacggCgctCgA-3' (SEQ ID NO: 35); 5'-tCgCaaCgtttAcgacggCgctCgA-3' (SEQ ID NO: 36); 5'-tCgCaaCgtttGcgacgtCgttCgA-3' (SEQ ID NO: 37); 5'-tCgCaaCgtttAcgacgtCgttCgA-3' (SEQ ID NO: 38); 5'-tCgCaaCgtttGcgacgtCggtCgG-3' (SEQ ID NO: 39); 5'-tCgCaaCgtttAcgacgtCggtCgG-3' (SEQ ID NO: 40); 5'-tCgCaaCgtttGcgacggCgctCgG-3' (SEQ ID NO: 41); 5'-tCgCaaCgtttAcgacggCgctCgG-3' (SEQ ID NO: 42); 5'-tCgCaaCgtttGcgacgtCgttCgG-3' (SEQ ID NO: 43); and 5'-tCgCaaCgtttAcgacgtCgttCgG-3' (SEQ ID NO: 44) (In the sequences, capital letters represent nucleosides without a 3'-modified internucleotide bond, and lowercase letters represent nucleosides with a 3'-phosphorothioated internucleotide bond.) 7. The oligonucleotide according to claim 6, comprising a partially phosphorothioated oligonucleotide stretch selected from the group consisting of:

[0037] (8) 5'-tCgCaaCgtttGcgacgtCggtCgA-3' (SEQ ID NO: 33); 5'-tCgCaaCgtttAcgacgtCggtCgA-3' (SEQ ID NO: 34); 5'-tCgCaaCgtttGcgacggCgctCgA-3' (SEQ ID NO: 35); and 5'-tCgCaaCgtttAcgacggCgctCgA-3' (SEQ ID NO: 36) (In the sequences, capital letters represent nucleosides without a 3'-modified internucleotide bond, and lowercase letters represent nucleosides with a 3'-phosphorothioate internucleotide bond.) 7. The oligonucleotide according to claim 6, comprising a partially phosphorothioated oligonucleotide stretch selected from the group consisting of:

[0038] (9) The oligonucleotide according to any one of (1) to (8), which is composed of DNA.

[0039] (10) The oligonucleotide according to any one of (1) to (9), further comprising one or more nucleotides deleted, substituted with one or more nucleotides, or added with one or more nucleotides.

[0040] (11) The oligonucleotide according to any one of (1) to (10), which is contained in an expression vector.

[0041] (12) The oligonucleotide according to any one of (1) to (10), which is cyclic.

[0042] (13) The oligonucleotide according to any one of (1) to (10), which is linear.

[0043] (14) The oligonucleotide according to (13), wherein the linear oligonucleotide has no phosphate at the 3' end.

[0044] (15) A double-stranded oligonucleotide comprising the oligonucleotide according to any one of (1) to (10) and its complementary strand oligonucleotide.

[0045] (16) The oligonucleotide according to any one of (1) to (15), which is bound to an active molecule.

[0046] (17) The oligonucleotide according to (16), wherein the active molecule is selected from the group consisting of (poly)peptides / antibodies and nucleic acids / oligonucleotides.

[0047] (18) The oligonucleotide according to (16) or (17), wherein the conjugation is performed via a linker.

[0048] (19) The oligonucleotide according to (18), wherein the linker is selected from the group consisting of glycerol, (S)-(-)-1,2,4-butanetriol, 1,3,5-pentanetriol, cis,cis-1,3,5-cyclohexanetriol, cistrans-1,3,5-cyclohexanetriol, 1,3,5-tris-(2-hydroxyethyl)isocyanurate, tetraethylene glycol, and hexaethylene glycol, diols such as 1,3-propanediol or dodecane-1,12-diol, cyclohexanediol, cholesterol, nitroindole, triethylene glycol, hexaethylene glycol, d-spacer, PEG-spacer, and alkyl linker.

[0049] (20) A pharmaceutical composition comprising a therapeutically effective amount of the oligonucleotide according to any one of (1) to (19) and a pharmaceutically acceptable carrier.

[0050] (21) A pharmaceutical composition comprising a therapeutically effective amount of the oligonucleotide according to any one of (1) to (19) for the prevention or treatment of a target disease or disorder, the target disease or disorder being any one selected from the group consisting of neoplasms, infectious diseases, Th2 / Th17-related diseases, primary immunodeficiency diseases, and post-traumatic stress disorder (PTSD).

[0051] (22) The pharmaceutical composition according to (21), wherein the target disease or disorder is a neoplasm selected from the group consisting of malignant neoplasms, epithelial neoplasms or hematopoietic tumors, sarcoma, mesothelioma, benign tumors, dysplasia, and metaplasia.

[0052] (23) The pharmaceutical composition according to (21), wherein the target disease or disorder is an infectious disease caused by a microorganism, including a virus, a bacterium, or a fungus.

[0053] (24) The pharmaceutical composition according to (21), wherein the target disease or disorder is a Th2 / Th17-associated disease selected from the group consisting of asthma, atopic disease, allergy, multiple sclerosis, inflammatory bowel disease including ulcerative colitis and Crohn's disease, cutaneous lichen planus, and Alzheimer's disease.

[0054] (25) The pharmaceutical composition according to (21), wherein the target disease or disorder is a primary immunodeficiency disease caused by IRAK4 deficiency, MyD88 deficiency, Unc93B deficiency, or a mutation in a TLR.

[0055] (26) The pharmaceutical composition according to (20) or (21), further comprising at least one active ingredient.

[0056] (27) The pharmaceutical composition according to (20) or (21), which is administered in combination with at least one active ingredient.

[0057] (28) The pharmaceutical composition according to (26) or (27), wherein the active ingredient is selected from the group consisting of anticancer drugs; molecular targeted drugs including tyrosine kinase inhibitors, angiogenesis inhibitors, and proteasome inhibitors; anticancer antibody drugs; cytokines; vaccines; antibacterial drugs; antifungal drugs; antiviral drugs; antiparasitic drugs; antibody drugs that neutralize toxins; agonists of other TLRs, and combinations thereof.

[0058] (29) The pharmaceutical composition according to (20) or (21), which is administered to a subject via an administration route selected from the group consisting of enteral administration, parenteral administration, topical administration, and inhalation.

[0059] (30) The pharmaceutical composition according to (29), wherein the subject is a human.

[0060] (31) The pharmaceutical composition according to (30), which is administered to the subject at a dose of 0.3 to 60 mg / day, preferably 1 to 30 mg / day, more preferably 2 to 8 mg / day.

[0061] (32) The pharmaceutical composition according to (20) or (21), which is administered before or after adoptive immune cell therapy or surgical treatment including radiation therapy, cryoablation, radiofrequency ablation, and photodynamic therapy (PDT).

[0062] (A1) Use of the oligonucleotide according to any one of (1) to (19) in the manufacture of a medicament for the treatment or prevention of a target disease or disorder selected from the group consisting of neoplasms, infections, Th2 / Th17-associated diseases, primary immunodeficiency diseases, and post-traumatic stress disorder (PTSD).

[0063] (A2) Use of the oligonucleotide according to any one of (1) to (19) in the manufacture of a medicament for modulating an immune response in a subject.

[0064] (A3) The use according to (A1), wherein the target disease or disorder is a neoplasm selected from the group consisting of malignant neoplasms, epithelial neoplasms or hematopoietic tumors, sarcomas, mesotheliomas, benign tumors, dysplasias and metaplasias.

[0065] (A4) The use according to (A1), wherein the target disease or disorder is an infectious disease caused by a virus, bacteria, or fungus.

[0066] (A5) The use according to (A1), wherein the target disease or disorder is a Th2 / Th17-associated disease selected from the group consisting of asthma, atopic disease, allergy, multiple sclerosis, inflammatory bowel disease including ulcerative colitis and Crohn's disease, lichen planus, and Alzheimer's disease.

[0067] (A6) The use according to (A1), wherein the target disease or disorder is a primary immunodeficiency disease caused by IRAK4 deficiency, MyD88 deficiency, Unc93B deficiency or a mutation in a TLR.

[0068] (A7) The use according to (A1) or (A2), wherein the medicament further comprises at least one active ingredient.

[0069] (A8) The use according to (A1) or (A2), wherein the drug is administered in combination with at least one active ingredient.

[0070] (A9) The use according to (A7) or (A8), wherein the active ingredient is selected from the group consisting of anticancer drugs; molecular targeted drugs including tyrosine kinase inhibitors, angiogenesis inhibitors, and proteasome inhibitors; anticancer antibody drugs; cytokines; vaccines; antibacterial drugs; antifungal drugs; antiviral drugs; antiparasitic drugs; antibody drugs that neutralize toxins; agonists of other TLRs, and combinations thereof.

[0071] (A10) The use according to (A1) or (A2), wherein the drug is administered to a subject via an administration route selected from the group consisting of enteral administration, parenteral administration, topical administration, and inhalation.

[0072] (A11) The use according to (A10), wherein the subject is a human.

[0073] (A12) The use according to (A11), wherein the drug is administered to the subject at 0.3 to 60 mg / day, preferably 1 to 30 mg / day, more preferably 2 to 8 mg / day.

[0074] (A13) The use according to (A1) or (A2), wherein the agent is administered before or after adoptive immune cell therapy or surgical procedures including radiation therapy, cryoablation, radiofrequency ablation and photodynamic therapy (PDT).

[0075] (B1) A method for treating or preventing a target disease or disorder in a subject, comprising administering to the subject the oligonucleotide according to any one of (1) to (19), wherein the target disease or disorder is any one selected from the group consisting of neoplasms, infections, Th2 / Th17-associated diseases, primary immunodeficiency diseases, and post-traumatic stress disorder (PTSD), and preferably, the target disease or disorder is treated or prevented in the subject by activating NF-kB with the oligonucleotide.

[0076] (B2) The method according to (B1), wherein the target disease or disorder is a neoplasm selected from the group consisting of malignant neoplasms, epithelial neoplasms or hematopoietic tumors, sarcomas, mesotheliomas, benign tumors, dysplasias and metaplasias.

[0077] (B3) The method according to (B1), wherein the target disease or disorder is an infectious disease caused by a microorganism, including a virus, a bacterium, or a fungus.

[0078] (B4) The method according to (B1), wherein the target disease or disorder is a Th2 / Th17-associated disease selected from the group consisting of asthma, atopic disease, allergy, multiple sclerosis, inflammatory bowel disease including ulcerative colitis and Crohn's disease, lichen planus, and Alzheimer's disease.

[0079] (B5) The method according to (B1), wherein the target disease or disorder is a primary immunodeficiency disease caused by IRAK4 deficiency, MyD88 deficiency, Unc93B deficiency, or a mutation in a TLR.

[0080] (B6) The method according to (B1), wherein the oligonucleotide is administered in combination with at least one active ingredient.

[0081] (B7) The method according to (B6), wherein the active ingredient is selected from the group consisting of anticancer drugs; molecular targeted drugs including tyrosine kinase inhibitors, angiogenesis inhibitors, and proteasome inhibitors; anticancer antibody drugs; cytokines; vaccines; antibacterial drugs; antifungal drugs; antiviral drugs; antiparasitic drugs; antibody drugs that neutralize toxins; agonists of other TLRs, and combinations thereof.

[0082] (B8) The method according to (B1), wherein the oligonucleotide is administered to the subject via a route of administration selected from the group consisting of enteral administration, parenteral administration, topical administration and inhalation.

[0083] (B9) The method according to (B8), wherein the subject is a human.

[0084] (B10) The method according to (B9), wherein the oligonucleotide is administered to the subject at a dose of 0.3 to 60 mg / day, preferably 1 to 30 mg / day, more preferably 2 to 8 mg / day.

[0085] (B11) The method according to (B1), further comprising a surgical procedure including adoptive immune cell therapy or radiation therapy, cryoablation, radiofrequency ablation and photodynamic therapy (PDT).

[0086] (C1) A method for stimulating an immune response in a subject, comprising administering to the subject a therapeutically effective amount of the oligonucleotide according to any one of (1) to (19) to induce inflammatory cytokines in the subject.

[0087] (C2) A method for redirecting a Th2-biased immune response in a subject to a Th1-biased immune response, the method comprising administering to the subject a therapeutically effective amount of the oligonucleotide according to any one of (1) to (19) to induce inflammatory cytokines in the subject.

[0088] (C3) The method described in (C1) or (C2), wherein the inflammatory cytokine is selected from the group consisting of IL-6, TNF-α, IFN-γ, and IL-12.

[0089] (D1) The oligonucleotide according to any one of (1) to (19) for use in the prevention or treatment of a target disease or disorder selected from the group consisting of neoplasms, infectious diseases, Th2 / Th17-related diseases, primary immunodeficiency diseases, and post-traumatic stress disorder (PTSD).

[0090] (D2) The oligonucleotide for use according to (D1), wherein the target disease or disorder is a neoplasm selected from the group consisting of malignant neoplasms, epithelial neoplasms or hematopoietic tumors, sarcomas, mesotheliomas, benign tumors, dysplasias and metaplasias.

[0091] (D3) The oligonucleotide for use according to (D1), wherein the target disease or disorder is an infectious disease caused by a microorganism, including a virus, a bacterium, or a fungus.

[0092] (D4) The oligonucleotide for use according to (D1), wherein the target disease or disorder is a Th2 / Th17-associated disease selected from the group consisting of asthma, atopic disease, allergy, multiple sclerosis, inflammatory bowel disease including ulcerative colitis and Crohn's disease, lichen planus, and Alzheimer's disease.

[0093] (D5) The oligonucleotide for use according to (D1), wherein the target disease or disorder is a primary immunodeficiency disease caused by IRAK4 deficiency, MyD88 deficiency, Unc93B deficiency or a mutation in a TLR.

[0094] (D6) An oligonucleotide for use according to (D1), administered in combination with at least one active ingredient.

[0095] (D7) The oligonucleotide for use according to (D6), wherein the active ingredient is selected from the group consisting of tyrosine kinase inhibitors, angiogenesis inhibitors, molecular targeted drugs including proteasome inhibitors, anti-cancer antibody drugs and cytokines, and combinations thereof.

[0096] (D8) The oligonucleotide for use according to (D1), which is administered to a subject via an administration route selected from the group consisting of enteral administration, parenteral administration, topical administration and inhalation.

[0097] (D9) The oligonucleotide for use according to (D8), wherein the subject is a human.

[0098] (D10) The oligonucleotide for use according to (D9), which is administered to a subject at 0.3 to 60 mg / day, preferably 1 to 30 mg / day, more preferably 2 to 8 mg / day.

[0099] (D11) The oligonucleotide for use according to (D1), administered before or after adoptive immune cell therapy or surgical procedures including radiation therapy, cryoablation, radiofrequency ablation and photodynamic therapy (PDT).

[0100] (E1) Use of the oligonucleotide according to any one of (1) to (19) for the treatment or prevention of a target disease or disorder selected from the group consisting of neoplasms, infections, Th2 / Th17-related diseases, primary immunodeficiency diseases, and post-traumatic stress disorder (PTSD).

[0101] (F1) An in vivo or in vitro agent for regulating immune responses, comprising an effective amount of the oligonucleotide according to any one of (1) to (19). [Effects of the Invention]

[0102] The present invention can provide novel CpG oligonucleotides and therapeutic uses of the oligonucleotides. [Brief explanation of the drawings]

[0103] [Figure 1] Activation of NF-kB and production of proinflammatory cytokines by TLR agonists in a human plasmacytoid dendritic cell (pDC) line. The CAL-1 / NF-kB-GFP cell line was designed to monitor the activity of the NF-kB transcription factor in a cellular assay. The human pDC cell line, CAL-1, was transfected with a vector encoding a GFP reporter gene driven by the NF-kB consensus transcription response element. [Figure 1A] CAL-1 / NF-kB-GFP cells were stimulated with 1 μM (micromolar) of the oligodeoxynucleotides (ODN) of the present invention or a positive control for 6 hours. GFP expression induced by TLR9 agonists is shown by dot plots. [Figure 1B]Graph showing the ratio of TLR9 activation activity of ODN in CAL-1 / NF-kB-GFP cells. CAL-1 / NF-kB-GFP cells were stimulated with 0.3 μM of the ODN of the present invention or a positive control for 6 hours. The ratio of GFP-positive cells in the sample containing CpG2395 was set to 100%. The activity of each ODN was calculated by comparing the ratio of GFP-positive cells with the ratio of CpG2395-positive cells. [Figure 1C] CAL-1 / NF-kB-GFP cells were stimulated with 1.0 μM of the ODN of the present invention for 6 hours. Although the activity of A003#delA was slightly weaker than that of A003, it was still much stronger than that of the standard TLR9 agonist CpG2395 (see Figure 1A). The TLR9 activation activity of A003#endG and A003 was at the same level, indicating that the 3' nucleotide is not important for activity. [Figure 1D] CAL-1 / NF-kB-GFP cells were stimulated with 1 μM ODN for 6 hours, and the culture supernatant was collected. Cytokine production was assessed by ELISA. [Figure 1E] CAL-1 / NF-kB-GFP cells were stimulated with 1.0 μM ODN for 6 hours. GFP expression was examined. Compared to each other, A003 and A013 had similar activity, indicating that the base changes in these oligonucleotides do not affect activity. [Figure 1F] CAL-1 / NF-kB-GFP cells were stimulated with 1.0 μM ODN for 6 hours. Cytokine production in the culture supernatant was assessed by ELISA (Figure 1F). Compared to each other, the activities of A003 and A013 were similar, indicating that the base changes in this oligonucleotide do not affect activity. [Figure 1G] CAL-1 / NF-kB-GFP cells were stimulated with 1.0 μM ODN for 6 hours, and GFP expression was examined. Each set of A001 / A011, A002 / A012, A003 / A013, or A004 / A014 showed the same activity as each other, indicating that the base changes in each set of these ODNs do not affect activity. [Figure 2] Activation of human TLR7 and TLR8 [Figure 2A] HEK blue® TLR7 cells were stimulated with TLR agonists for 24 hours. As shown, the TLR7 agonists Gardiquimod (GQ) and CL264 activated TLR7 and produced positive signals. In contrast, 0.3 μM of the standard TLR9 agonist CpG2395 and the ODNs A001 to A004 of the present invention were unable to activate the TLR7 signaling pathway. [Figure 2B] HEK-Blue® TLR8 cells were stimulated with ODN for 24 hours. As shown in the figure, the TLR8 agonists TL8-506 and CL075 activated TLR8 and produced positive signals. In contrast, CpG2395 and A001-A004 (0.3 μM) were unable to activate the TLR8 signaling pathway. [Figure 3] NF-kB activation and proinflammatory cytokine production [Figure 3A] CAL-1 / NF-kB-GFP cells were stimulated with 0.3 μM of the ODN of the present invention for 6 hours. CaaCg is important for the activation of human TLR9. [Figure 3B] CAL-1 / NF-kB-GFP cells were stimulated with 0.3 μM or 0.1 μM of the ODN of the present invention for 6 hours. The percentage of GFP-positive cells is shown. [Figure 3C] CAL-1 / NF-kB-GFP cells were stimulated with 0.3 μM or 0.1 μM of the present invention's ODN for 6 hours. A303, A603 and A703 showed higher activity than other tested ODNs, suggesting that the presence of CaaCg is more important than the number of Cg. [Figure 3D] CAL-1 / NF-kB-GFP cells were stimulated with 0.3 μM ODN for 6 hours, and the culture supernatants were collected. Cytokine production was assessed by ELISA. A403 and A503 clearly showed lower levels of cytokine production-inducing activity, suggesting that CaaCg is important for optimal TLR9 stimulation. [Figure 3E]CAL-1 / NF-kB-GFP cells were stimulated with 0.3 μM of the ODN of the present invention for 6 hours. In this figure, the GFP-positive cell rate of CpG2395 was set to 100%. The activity of each ODN was calculated by comparing the GFP-positive cell rate with the GFP-positive cell rate of CpG2395. DV093 and DV094 showed lower TLR9 activity than A003, suggesting that the specific caacg motif present in A003 (but not randomly positioned caacg motifs) is important for TLR9 activity. [Figure 3F] CAL-1 / NF-kB-GFP cells were stimulated with 0.3 μM ODN for 6 hours. The GFP-positive cell ratio of CpG2395 was set as 100%. The activity of each ODN was calculated by comparing the GFP-positive cell ratio with that of CpG2395. Note that the structural change from the caacg stretch to the CaaCg stretch due to the change in internucleotide bond in DV093 and DV094 did not enhance TLR9 activity. [Figure 4] Non-essential nucleotides in the 3' region of the ODN of the present invention [Figure 4A] CAL-1 / NF-kB-GFP cells were stimulated with 0.3 μM ODN for 6 hours. [Figure 4B] CAL-1 / NF-kB-GFP cells were stimulated with 0.3 μM ODN for 6 hours. The percentage of GFP-positive cells (%) is shown. The standard TLR9 agonist CpG2006 showed much weaker activity than A601, A602, A603, A604, A605, A606, and A607. [Figure 5] Activation of human TLR7 and TLR8 [Figure 5A] HEK-Blue® TLR7 cells were stimulated with ODN or a positive control for 24 hours. The TLR7 agonist CL264 activated TLR7 and produced a positive signal. In contrast, CpG2395, A601, A602, and A603 (0.3 μM) were unable to activate the TLR7 signaling pathway. [Figure 5B]HEK-Blue® TLR8 cells were stimulated with ODN or positive control for 24 hours. TLR8 agonist CL075 activated TLR8 and produced a positive signal. In contrast, CpG2395, A601, A602 and A603 (0.3 μM) were unable to activate the TLR8 signaling pathway. [Figure 6] The slight effect of internucleotide bond changes and base changes in the central region of the ODN of the present invention on TLR9 activation activity. [Figure 6A] CAL-1 / NF-kB-GFP cells were stimulated with A601, A601G, A602, or A602G (0.3 μM) for 3 hours. [Figure 6B] CAL-1 / NF-kB-GFP cells were stimulated with A601G, A611A, A602G, or A612A (0.3 μM) for 3 h. [Figure 6C] CAL-1 / NF-kB-GFP cells were stimulated with 0.3 μM ODN for 3 hours. The percentage of GFP-positive cells is shown. [Figure 7] Activation of human TLR7 and TLR8 [Figure 7A] A. HEK-Blue® TLR7 cells were stimulated with TLR agonists for 24 hours. The TLR7 agonist CL264 activated TLR7 and produced a positive signal. In contrast, CpG2395, A601G, A611A, A602G, and A612A (0.3 μM) failed to activate the TLR7 signaling pathway. [Figure 7B] B. HEK-Blue® TLR8 cells were stimulated with TLR agonists for 24 hours. The TLR8 agonist CL075 activated TLR8 and produced a positive signal. In contrast, CpG2395, A601G, A611A, A602G, and A612A (0.3 μM) failed to activate the TLR8 signaling pathway. [Figure 8] Cytokine production-inducing activity in human B cells or human PBMCs [Figure 8A]HAL-01 cells were stimulated with 1 μM of the ODN of the present invention for 24 hours. Cells were stained with anti-CD40 antibody (Ab) and anti-CD86 Ab. The induction of CD40 and CD86 expression was evaluated using a flow cytometer. [Figure 8B] Human PBMCs were stimulated with the ODN of the present invention (0.1 μM) for 24 hours, and cell proliferation was evaluated by WST-1 assay. [Figure 8C] Human PBMCs were stimulated with the ODN of the present invention (0.3 μM) for 24 hours, and the culture supernatant was collected. Cytokine production was evaluated by ELISA. [Figure 9] Agonist activity on mouse cells [Figure 9A] Mouse splenocytes were stimulated with the ODN of the present invention for 24 hours, and the supernatant was collected. Cytokine production was evaluated by ELISA. [Figure 9B] Mouse splenocytes were stimulated with the ODN of the present invention for 48 hours. Cell proliferation was evaluated by WST-1 assay. [Figure 9C] Mouse splenocytes were stimulated with the ODN of the present invention for 48 hours.Cell proliferation was evaluated by WST-1 assay.A603 showed stronger activity than the standard TLR9 agonists CpG2395 and CpG2006. [Figure 10] In vivo antitumor activity [Figure 10A] CT26 cells were inoculated into the right flank, and the mice were maintained without treatment for 2 weeks. Tumor volume was measured, and the mice were divided into 3 groups based on tumor volume. On the day of grouping (day 0), the ODN of the present invention or PBS was administered to the peritumoral area, and this was repeated on day 2. The ODN of the present invention was administered twice in total during the test. The tumor volume of each group of mice was measured every 2 days. [Figure 10B] Tumor volume for each mouse on each day. [Figure 10C] Average weight of mice in each group during the study. No significant weight loss was observed in any group. [Figure 11] Induction of antitumor immune memory [Figure 11A]CT26 cells were inoculated into the right flank, and the mice were maintained without treatment for 2 weeks. Tumor volume was measured, and the mice were divided into 3 groups based on tumor volume. On the day of grouping (day 0), administration of the ODN of the present invention or PBS was started, and repeated on day 2. The ODN of the present invention was administered twice in total during the test. The tumor volume of each group of mice was measured every 2 days. [Figure 11B] On the 14th day, CT26 cells are re-inoculated into the left flank of each group of mice, and mice are maintained without treatment.The tumor volume of the left flank is measured up to 14 days after re-inoculation with CT26.It should be noted that the mice injected with ODN of the present invention reject the re-inoculated tumor without further treatment for 2 weeks, which indicates that ODN of the present invention induces the memory of anti-tumor immunity. [Figure 12] In vivo efficacy of lung metastasis model To induce lung metastasis of CT26 cells, BALB / c mice were intravenously injected with cell suspension (5x105 cells / mouse) through the tail vein (day 0). On day 1, the administration of the ODN of the present invention was started (subcutaneous injection into the skin of the back, 40 μg / 50 μl / mouse). On day 5, the same dose of ODN was administered again. On day 18, mice were sacrificed. Lung weight was measured, and metastatic tumor nodules were counted in each lung of mice. [Figure 13] Systemic effects of tumor metastasis [Figure 13A] To induce lung metastasis of CT26 cells, BALB / c mice were intravenously injected with a cell suspension (5 x 105 cells / mouse) via the tail vein (day 0). A602 administration began the day after tumor inoculation (day 1). Two administration routes were tested: subcutaneous (SC) injection into the dorsal skin and intradermal (ID) injection into the base of the ear (25 μg / mouse). The same dose was administered on days 3 and 5. On day 16, the mice were sacrificed, and the number of metastatic tumor nodules in each lung was counted (graph on the right). Photo: Isolated lung of a test mouse on day 16. [Figure 13B]To induce liver metastasis of CT26 cells, the cell suspension was injected into the spleen (1 x 10 cells / mouse, day 0). On day 2, mice were divided into three groups based on body weight, and A602 administration was initiated. Two administration routes were tested in this study: SC injection into the dorsal skin and ID injection into the base of the ear (12.5 μg / mouse). The same dose was administered on days 5, 8, and 12. On day 20, the mice were sacrificed, and the number of metastatic tumor nodules in each liver of the mice was counted. [Figure 14] Elimination of B-ALL cells by activated PBMCs [Figure 14A] Human PBMC was co-cultured with human B-ALL cells RCH-ACV together with the ODN of the present invention (0.1 μM) for 3 days, and all cells were stained with CD19 antibody and CD138 antibody and then analyzed by flow cytometry. [Figure 14B] The percentage of RCH-ACV population in the sample compared to the unstimulated sample, which is taken as 100%, is shown. [Figure 15] Elimination of colon cancer cells by activated PBMCs [Figure 15A] Human PBMC (5x105) was co-cultured with human colon cancer cells COLO205 together with the ODN of the present invention (0.1 μM) for 3 days, and all cells were analyzed by staining with CD45 antibody and CD24 antibody. [Figure 15B] The percentage of COLO205 population in the sample compared to the unstimulated sample, which is taken as 100%, is shown. [Figure 16] Effects on immune cells [Figure 16A] Human PBMCs and mouse splenocytes were stimulated with the ODN of the present invention (0.15 μM) for 24 hours, and cell proliferation was evaluated by WST-1 assay. [Figure 16B] Human PBMCs were co-cultured with cancer cells (RCH-ACV or COLO205) and the ODN of the present invention (0.1 μM) for 3 days.The elimination of cancer cells by human PBMCs was evaluated.The percentage of cancer cells in the sample is shown, compared with the unstimulated sample, which is set as 100%. DETAILED DESCRIPTION OF THE INVENTION

[0104] Preferred Embodiments Unless otherwise stated, all terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular forms "a," "an," and "the" include plural referents unless the context dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context dictates otherwise. The term "few" as used herein means a number of 2 to 3. The term "several" as used herein means a number of 2 to 6. In the case of conflict, the present specification, including explanations of terms, will control. Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting. Treat, treating, or treatment shall have the same meaning, regardless of grammar. Similarly, prevent, preventing, or prevention shall have the same meaning, regardless of grammar.

[0105] "Nucleotide": Nucleotides constitute nucleic acid molecules such as DNA, RNA, and chimeric molecules of DNA and RNA. Nucleotides can consist of a base, a phosphate, and a sugar. Nucleotides can be phosphate esters of nucleosides. "Nucleoside": A nucleoside can consist of a base and a sugar molecule and is a component of a nucleotide. Nucleosides can include deoxyadenosine, deoxyguanosine, thymidine, deoxycytidine, adenosine, guanosine, uridine, and cytidine. "Oligonucleotide": An oligonucleotide is a polymer / oligomer of nucleotides linked by internucleotide bonds. The conformation of an oligonucleotide can be linear or cyclic. "Base": A base is one of the components of nucleotides and nucleosides. Natural bases include two groups: purine bases such as guanine and adenine, and pyrimidine bases such as thymine, cytosine, and uracil. "Sugar": A sugar is one of the components of nucleotides and nucleosides. Generally, the sugar in DNA is deoxyribose, and the sugar in RNA is ribose. "Internucleotide linkage": Internucleotide linkage is intended to mean the bond between two adjacent nucleotides of a nucleic acid molecule.

[0106] The parts of nucleotides, nucleosides, bases and sugars, may be replaced or modified with other molecules called their analogs. For example, nucleotides may further include unnatural artificial nucleotides such as PNA; bases may further include unnatural bases such as hypoxanthine (i.e., inosine as a nucleoside); and sugars may include unnatural artificial structures such as 2'-4' bridges in locked nucleic acids.

[0107] In this application, a "(sequence) motif" is defined solely on the basis of "bases", and a "(sequence) stretch" is defined on the basis of "bases", "sugars" and "internucleotide linkages".

[0108] The oligonucleotides of the present invention may be composed of deoxyribonucleic acids with a deoxyribose backbone, ribonucleic acids with a ribose backbone, or mixtures thereof; the sugar backbone may also be replaced with synthetic molecules such as locked nucleic acids (LNA), bridged nucleic acids (BNA), morpholinos, or peptide nucleic acids (PNA). The oligonucleotides of the invention may contain chemically modified nucleosides and / or modified internucleotide linkages to enhance one or more properties, such as nuclease resistance or pharmacokinetics.

[0109] Chemically modified nucleosides can include, but are not limited to, those with modified bases or related structures such as: 8-halogen (bromo, chloro, fluoro, iodo)-, 8-amino-, 8-thiol-, 8-thioalkyl-, 8-hydroxyl-, 8-aza-, 8-oxo- and other 8-substituted purines; 5-halogen (bromo, chloro, fluoro, iodo)-, 5-difluoromethyl-, 5-trifluoromethyl-, 5-hydroxy, 5-carboxy-, 5-hydroxymethyl-, 5-bromovinyl-, 5-formyl-, 5-aza-, 5-alkynyl-5-propynyl-, 5-(C1-C6)-alkyl-, 5-(C2-C6)-alkenyl-, 5-(C2-C6)-alkynyl-, and other 5-substituted pyrimidines; 5,6-dihydroxy-5,6-dihydrothymine, N6-methyl-adenine; N4-ethylcytosine, N4-alkylcytosine and other N4-substituted-cytosines; 2-mercapto-cytosine, iso-cytosine, pseudo-isocytosine, 4-thio-uracil, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 2-aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine, 6-thioguanine, N2-methyl-guanine, N2-dimethylguanine and other N2-substituted guanines. Modified nucleobases may also be bases substituted with other heterocycles, such as 7-deaza-adenine, 8-aza-7-deaza-adenine, 7-deaza-guanine, 2-aminopyridine and 2-pyridone. Modified nucleobases also contain additional fused rings, examples of such nucleobases are N6-ethenoadenosine, N4-ethenocytidine, N2-ethenoguanosine, and other related derivatives.

[0110] Internucleotide bond is a covalent backbone bond between nucleotides in oligo / polynucleotide. Generally, natural internucleotide bond is a phosphodiester structure, but various modifications have been reported to affect the chemical or physical properties of these molecules. The chemical modifications in the modified internucleotide bond that can be used in the oligonucleotide of the present invention include the following bond structures of natural phosphodiester structure: phosphorothioate, phosphorodithioate, methyl phosphonate, methyl phosphorothioate, ethyl phosphate, phosphonoacetate, α-hydroxybenzyl phosphate, isopropyl-phenoxyacetate, boranophosphate, phosphonocarboxylate, α-hydroxybenzyl phosphonate, phosphate-(C1-C21)-O-alkyl ester, phosphate-[(C6-C12)aryl-(C1-C21)-O-alkyl] ester, phospho Includes conversion to triesters, phosphotriesters, amines, ethers, acetals, thioethers, thioacetals, phosphoramidates, siloxanes, carbonates, carboxymethyl esters, acetamidates, carbamates, ureas, thioureas, sulfonamides, or sulfonylureas, carbonates, carboxymethyl, amides, ethylene oxide linkers, sulfonates, thioformacetals, formacetals, oximes, methyleneimino, methyleneaminocarbonyl, methylenemethylimino (MMI), methylenehydrazo, methylenedimethylhydrazo (MDH), and methyleneoxymethylimino.

[0111] The oligonucleotides of the present invention may be, but are not limited to, single-stranded deoxyribonucleic acid, single-stranded ribonucleic acid, or chimeric molecules thereof. The oligonucleotides of the present invention can be single-stranded, double-stranded, or a hybrid of single and double strands. They can also form circular structures by connecting the 5' and 3' ends of a single molecule, and two or more oligonucleotides of the present invention can be linked at either the 5' or 3' ends covalently or via a unique linker to form tandemly linked elongated or multivalent structures. Oligonucleotides can be annealed intermolecularly or intramolecularly to form elongated structures, multimeric structures, or concatemers. These oligonucleotides can be linked to other molecules to form multimers. Oligonucleotides of the present invention can be linked to other oligonucleotides. Oligonucleotides of the present invention can be included in expression vectors, including plasmid vectors and viral vectors.

[0112] The oligonucleotides of the invention may contain CpG motifs, ie, the unmethylated dinucleotides cytosine and guanine.

[0113] Oligonucleotides of the invention may comprise the common sequence motifs tcgcaacgttt (SEQ ID NO: 1) and cgacg, preferably tcgcaacgttt-n-cgacg-n-cg-nn-cg (SEQ ID NO: 2), or more preferably tcgcaacgttt-r-cgacg-k-cg-bd-cg (SEQ ID NO: 3), where each of a, t, c, or g represents a base corresponding to adenine, thymine / uracil, cytosine, or guanine, respectively; n represents any base; r represents adenine or guanine; k represents guanine or thymine / uracil, respectively; b represents cytosine, guanine, or thymine / uracil; and d represents adenine, guanine, or thymine / uracil. Oligonucleotides of the invention can have an r (adenine or guanine) 3' to the motif to form tcgcaacgttt-r-cgacg-k-cg-bn-cg-r (SEQ ID NO: 4).

[0114] The oligonucleotides of the invention may contain phosphorothioated internucleotide linkages, each of which may contain a stereogenic α-phosphorus atom to give rise to R or S diastereomers. The phosphorothioated internucleotide bond can also comprise a phosphorodithioate structure. The oligonucleotide of the present invention may preferably comprise a partially phosphorothioated stretch CaaCg characteristic of the 5' region of the oligonucleotide, which has partially phosphorothioated internucleotide bonds or phosphodiester internucleotide bonds, wherein capital letters represent nucleosides with unstable 3' internucleotide bonds, such as phosphodiester bonds, without non-natural chemical modifications, and lowercase letters represent nucleosides with stable 3' internucleotide bonds, such as phosphorothioate bonds.

[0115] Partially phosphorothioated forms of the oligonucleotides of the invention may preferably comprise partially phosphorothioated stretches such as tCgCaaCgttt-n-cgacg-n-Cg-nn-Cg (SEQ ID NO: 5), where the lower case letters represent nucleosides having a 3' internucleotide linkage that is a stable structure, such as a phosphorothioate linkage.

[0116] The above motifs or stretches must be present side by side in the same oligonucleotide; these motifs may be separated in the middle by one or more insertions of nucleotides, preferably one or two nucleotides inserted at each position. The above motifs may be mutated, preferably one or two nucleotides mutated within each motif. The motifs may be partially deleted at their ends by up to two bases.

[0117] In one embodiment, the oligonucleotide of the present invention may be selected from oligonucleotides having the following sequence motifs shown in Table 1, wherein the internucleotide linkages may be selected from natural (e.g., phosphodiester) or synthetic (e.g., phosphorothioated) or mixtures thereof.

[0118] [Table 1]

[0119] In another embodiment, the oligonucleotides of the present invention may be fully phosphorothioated at the internucleotide linkages. Examples of oligonucleotides of the present invention are shown in Table 2 below.

[0120] [Table 2]

[0121] The uppercase letters in the above sequences represent nucleosides or nucleosides with a phosphodiester bond as the 3' internucleotide linkage, and the lowercase letters represent nucleosides with a phosphorothioate at the 3' internucleotide linkage.

[0122] In another embodiment, the oligonucleotides of the present invention may have partially phosphorothioated internucleotide linkages. The oligonucleotides of the invention may comprise the sequence motif caacg, preferably the partially phosphorothioated stretch caaCg, Caacg or CaaCg, more preferably the partially phosphorothioated stretch CaaCg. Examples of such oligonucleotides are shown in Table 3 as follows:

[0123] [Table 3]

[0124] The uppercase letters in the above sequences represent nucleosides or nucleosides with a phosphodiester bond as the 3' internucleotide linkage, and the lowercase letters represent nucleosides with a phosphorothioate at the 3' internucleotide linkage.

[0125] The oligonucleotides of the present invention can bind to TLR9 and have activity such as enhancing downstream signaling of the receptor. Alternatively, TLR9 activation activity may be evaluated by, but is not limited to, the following phenomena (Patent Document 1, Patent Document 2): (i) the strength of promoter activity of the NF-kB binding oligonucleotide, expressed as the level of promoter-driven GFP expression in cells, such as CAL-1 / NF-kB-GFP; (ii) the level of cytokine production from target cells; and (iii) the expression level of marker proteins, such as costimulatory molecules including CD40, CD80 or CD86, on target cells, such as antigen-presenting cells;

[0126] Target cell activation can be assessed by the enhancement of the surrogate indicators described above. Enhancement of such markers is measured by an increase in levels compared to normal conditions or conditions without an activating stimulus, including the addition of a ligand.

[0127] Such activity of the oligonucleotides of the present invention can be assayed in cultured cells such as peripheral blood mononuclear cells (PBMCs) or established and immortalized cell lines, including HAL-1 and RCH-ACV as B cell surrogates, and CAL-1 (Non-Patent Document 31), Gen2.2 / Gen3 (Non-Patent Document 32), or PMDC05 (Non-Patent Document 33) as plasmacytoid dendritic cell surrogates.

[0128] <Target diseases> The oligonucleotide of the present invention can be used to prevent or treat the target disease or disorder associated with immune response in subjects, or regulate immune response.The examples of target disease or disorder are neoplasm, infectious disease, Th2 or Th17-related disease, including the disease that occurs when Th2 or Th17 is activated, primary immunodeficiency disease, or post-traumatic stress disorder (PTSD).

[0129] The neoplasms include (malignant) tumors, such as carcinomas (including malignant neoplasms, epithelial neoplasms, intraepithelial neoplasias, and hematopoietic tumors), sarcomas and mesotheliomas, benign tumors, dysplasias, and metaplasias. The malignant tumors include, but are not limited to, lung cancer (small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, gastric cancer, esophageal cancer, pancreatic cancer, liver cancer, biliary tract cancer, bile duct cancer, kidney cancer, renal pelvis and ureter cancer, adrenal cancer, bladder cancer, testicular cancer, prostate cancer, penile cancer, thyroid cancer, uterine cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, melanoma, squamous cell carcinoma, neuroblastoma, oral cancer, acute lymphocytic leukemia, acute myeloid leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic myeloid leukemia, malignant lymphoma, and multiple myeloma.

[0130] The sarcomas include, but are not limited to, osteosarcoma, bone cyst, aneurysmal bone cyst, osteoid osteoma, chondrosarcoma, poorly differentiated spherical / spindle cell tumor (including Ewing's sarcoma), hemangioendothelioma, angiosarcoma, fibrosarcoma / myofibrosarcoma, chordoma, adamantinoma, liposarcoma, leiomyosarcoma, malignant peripheral nerve sheath tumor, rhabdomyosarcoma, synovial sarcoma, malignant solitary fibrous tumor, atypical lipoid tumor, dermatofibrosarcoma protuberans, malignant solitary fibrous tumor, inflammatory myofibroblastic tumor, low-grade myofibroblastic sarcoma, fibrosarcoma (including adult and sclerosing epithelioid tumor), myxofibrosarcoma ... and sarcoma. Included are malignant fibromyxoid sarcoma, soft tissue giant cell tumor, malignant glomus tumor, rhabdomyosarcoma, hemangioendothelioma, soft tissue angiosarcoma, extraskeletal osteosarcoma, gastrointestinal stromal tumor (gist), malignant peripheral nerve sheath tumor, malignant Triton tumor, malignant granular cell tumor, malignant ossifying fibromyxoma, stromal sarcoma, myoepithelial carcinoma, malignant phosphaturic mesenchymoma, epithelioid sarcoma, alveolar soft part sarcoma, clear cell sarcoma of soft tissue, extraskeletal myxoid chondrosarcoma, extraskeletal Ewing's sarcoma, desmoplastic small round cell tumor, extrarenal rhabdomyoid tumor, perivascular epithelioid cell tumor, hemangiointimal sarcoma, pleomorphic sarcoma, and round cell sarcoma. The mesothelioma includes, but is not limited to, pericardial mesothelioma, peritoneal mesothelioma, and pleural mesothelioma.

[0131] Such dysplasias include, but are not limited to, myelodysplastic syndromes and cervical dysplasia.

[0132] The oligonucleotides of the present invention are effective against target diseases based on immune activation against malignant cells. When used alone, they are expected to be more effective when used against highly immunogenic neoplasms. Highly immunogenic neoplasms include, but are not limited to, cancer cells that express neoantigens, which are produced by genetic mutations that occur during the tumorigenesis stage, including those with mismatch repair (dMMR) deficiency or microsatellite instability-high (MSI-H) (Non-Patent Document 34; Non-Patent Document 35).

[0133] The oligonucleotides of the present invention can activate pDCs through activation of TLR9. Activated pDCs further activate various other immune cells through interferon. Therefore, the oligonucleotides of the present invention can be used to prevent or treat various infectious diseases caused by microorganisms, including viruses, bacteria, and fungi.

[0134] Viruses that cause infectious diseases include, but are not limited to, molluscum contagiosum virus, herpes simplex virus (HSV), varicella virus, varicella-zoster virus, rotavirus, human papillomavirus, cytomegalovirus (CMV), poliovirus, coxsackievirus, rhinovirus, rubella virus, measles virus, influenza virus, mumps virus, respiratory syncytial (RS) virus, hepatitis virus, and human immunodeficiency virus (HIV). Some viruses, such as CMV, HIV, influenza virus, HSV, hepatitis B virus (HBV), or hepatitis C virus (HCV), have been reported to directly activate host immunity through TLR9 binding (Non-Patent Document 36).

[0135] Bacteria that cause infectious diseases are classified into Gram-negative and Gram-positive bacteria, and the present invention can be applied to both groups of bacteria. At least, it has been reported that TLR9 is necessary for the activation of innate immunity against Gram-negative bacteria (Non-Patent Document 37). Gram-negative bacteria include, but are not limited to, Neisseria gonorrhea, Neisseria meningitides, Hemophilus parainfluenzae, Escherichia coli, Pseudomonas aeruginosa, Chlamydia trachomatis, and Yersinia pestis, Moraxella catarrhalis, Haemophilus ducreyi, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Citrobacter spp., Salmonella spp., Salmonella enterica subsp. enterica serovar Typhi, Salmonella enterica subsp. enterica serovar Paratyphi A, Salmonella enterica subsp.enterica serovar Paratyphi B, Salmonella Typhimurium, Salmonella enterica serovar Enteritidis, Shigella dysenteriae, Shigella frexneri, Shigella sonnei, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter, Serratia, Hafnia, Proteus, Morganella, Providencia, Yersinia, Campylobacter, Vibrio cholera, Vibrio parahaemolyticus These include the genera Acinetobacter, Pseudomonas, Xanthomonas, Acinetobacter, Flavobacterium, Brucella, Legionella, Veillonella, Bacteroides, and Fusobacterium.

[0136] Infectious diseases caused by fungi include, but are not limited to, cryptococcosis, candidiasis, aspergillosis, pneumonia caused by Pneumocystis carinii, athlete's foot caused by Trichophyton, and skin infections caused by Tinea versicolor. It has been reported that DNA or RNA derived from fungi is actually recognized by mammalian TLR9 (Non-Patent Document 38; Non-Patent Document 39).

[0137] Th2 / Th17-associated diseases are diseases caused by the suppression of Th1 and the activation of Th2 and / or Th17, including, but not limited to, asthma, atopic diseases (dermatitis, eczema), allergies, multiple sclerosis, inflammatory bowel diseases including ulcerative colitis and Crohn's disease, cutaneous lichen planus, and Alzheimer's disease.

[0138] Primary immunodeficiency disease is a disorder in which a part of immune system is congenitally missing or does not function properly, and it is known that patients with these diseases are particularly susceptible to infection.The oligonucleotide of the present invention is particularly effective in patients who lack functional virus TLR-related system, such as patients with IRAK4 deficiency, MyD88 deficiency, Unc93B deficiency or TLR mutation, but it is fully expected that the effectiveness of the oligonucleotide of the present invention is not limited to these diseases, because it is expected to activate immunity widely against infectious diseases.

[0139] Post-traumatic stress disorder (PTSD) is one of the diseases that has been reported to be improved by TLR9 agonists, and this disease is also considered to be one of the target diseases (Non-Patent Document 40).

[0140] The oligonucleotides of the present invention can be used as one of the active ingredients of a pharmaceutical composition administered to patients suffering from the above-mentioned target diseases or disorders, but the oligonucleotides of the present invention can also be applied to subjects for the purpose of preventing the above-mentioned target diseases.

[0141] The oligonucleotide of the present invention can be connected to other active molecules possibly through a specific linker.For example, the conjugation of pharmaceutical compounds for co-administration can facilitate co-administration, and lipid conjugation or PEGylation can improve tissue distribution or pharmacokinetics.It has been reported that PEGylation of oligonucleotides can reduce renal clearance, thereby prolonging their in vivo duration in subjects.

[0142] The oligonucleotides of the present invention can also be conjugated to some organic compounds, such as vitamin E (α-tocopherol) or vitamin D, for the purpose of improving pharmacokinetics, such as in vivo half-life or cellular absorption (Non-Patent Document 41).

[0143] To connect the oligonucleotides of the present invention to other organic or inorganic moieties, organic chemical linkers can be used, including, but not limited to, glycerol, (S)-(-)-1,2,4-butanetriol, 1,3,5-pentanetriol, cis,cis-1,3,5-cyclohexanetriol, cistrans-1,3,5-cyclohexanetriol, 1,3,5-tris-(2-hydroxyethyl)isocyanurate, tetraethylene glycol, and hexaethylene glycol, diols (e.g., 1,3-propanediol or dodecane-1,12-diol, cyclohexanediol), cholesterol, nitroindole, triethylene glycol, hexaethylene glycol, d-spacers, PEG-spacers, and alkyl linkers. The linker moiety can also be composed of amino acids, nucleotides, and / or their derivatives.

[0144] Linkers can also be used to form non-covalent bonds, including, but not limited to, biotin-avidin, SPB (succinimidyl-[4-(psoralen / psoralen-8-yloxy)]-butyrate) which intercalates into nucleic acids, nucleic acids that are complementary in sequence and bind to each other, and protein-protein interactions such as coiled-coil structures.

[0145] In one embodiment, the oligonucleotides of the present invention can be used in combination with each other, with other oligonucleotides having a similar mechanism of action, or with other organic compounds such as (poly)peptides / antibodies or nucleic acids / oligonucleotides, or inorganic compounds such as cytotoxic agents, which are often used to improve or modify the physical properties of the drug. Such combinations can be made with or without covalent bonds.

[0146] The oligonucleotides of the present invention can be obtained from existing nucleic acid sources (e.g., genome or cDNA), but are preferably synthetic. The oligonucleotides of the present invention can be synthesized using various commercially available nucleic acid synthesizers. These oligonucleotides are called synthetic oligonucleotides.

[0147] The oligonucleotides of the present invention may be administered alone or in combination with one or more other active ingredients, either simultaneously or sequentially. The oligonucleotides of the present invention may also be used simultaneously or sequentially with one or more other therapeutic modalities.

[0148] The oligonucleotide of the present invention can be administered in combination with conventional anticancer drugs or drugs for alleviating various symptoms associated with tumor formation.Examples of conventional anticancer drugs include but are not limited to antibiotics such as anthracyclines or mitoxantrone; platinum; alkylating agents; antimetabolites; hormonal therapy drugs; plant alkaloids such as vinca alkaloids; taxanes such as paclitaxel or docetaxel; and topoisomerase inhibitors such as irinotecan or etoposide.

[0149] The oligonucleotides of the present invention can also be administered to a subject in need thereof in combination with other molecularly targeted drugs that target biological characteristics of cancer cells, including tyrosine kinase inhibitors, angiogenesis inhibitors, or proteasome inhibitors. Examples of molecularly targeted drugs include, but are not limited to, everolimus (Afinitor®), tamoxifen (Nolvadex®), toremifene (Fareston®), fulvestrant (Faslodex®), anastrozole (Arimidex®), exemestane (Aromasin®), lapatinib (Tykerb®), letrozole (Femara®), palbociclib (Ibrance®), ribozyme (Ribosin®), and ribozyme inhibitors. Clib (Kisqali®), neratinib maleate (Nerlynx®), abemaciclib (Verzenio®), olaparib (Lynparza®), regorafenib (Stivarga®), imatinib mesylate (Gleevec®), lanreotide acetate (Somatuline Depot®), sunitinib (Sutent®), sorafenib (Nexavar®), pazopanib (Votrient®), temsirolimus (Torisel®), Registered trademark), axitinib (Inlyta®), cabozantinib (Cabometyx®), lenvatinib mesylate (Lenvima®), tretinoin (Vesanoid®), dasatinib (Sprycel®), nilotinib (Tasigna®), bosutinib (Bosulif®), ibrutinib (Imbruvica®), idelalisib (Zydelig®), venetoclax (Venclexta®), ponatinib hydrochloride (A Iclusig (registered trademark), midostaurin (Ridapt (registered trademark)), enasidenib mesylate (Idohyfa (registered trademark)), inotuzumab ozogamicin (Besponsa (registered trademark)), tisagenlecleucel (Kymriah (registered trademark)), ivosidenib (Tibsovo (registered trademark)), duvelisib (Copictra (registered trademark)), sorafenib (Nexavar (registered trademark)), crizotinib (Xalkori (registered trademark)), erlotinib (Tarceva (registered trademark)), gefitinib (Iressa (registered trademark)),Afatinib dimaleate (Giotrif®), ceritinib (LDK378 / Zykadia®), osimertinib (Tagrisso®), alectinib (Alecensa®), bringatinib (Alumbrig®), trametinib (Mekinist®), dabrafenib (Tafinlar®), dacomitinib (Vizinpro®), denileukin diftitox (Ontak®), vorinostat (Zolinza®), romidepsin (Istodax®), bexarotene (Targretin®), bortezomib (Velcade®), pralatrexate (Folotin®), Siltuximab (Sylvant®), idelalisi (Zydelig®), belinstat (Veleodaq®), copanlisib hydrochloride (Alicopa®), acalabrutinib (Calquence®), duvelisib (Copictra®), carfilzomib (Kyprolis®), panobistat (Farydak®), ixazomib citrate (Ninlaro®), ruxolitinib phosphate (Jakafi®), cabazitaxel (Jevtana®), enzalutamide (Xtandi®), abiraterone acetate (Zambezi®), Itiga®), radium-223 dichloride (Xofigo®), apalutamide (Erleida®), vismodegib (Elivege®), sonidegib (Odomzo®), vemurafenib (Zelboraf®), cobimetinib (Cotellic®), alitretinoin (Panretin®), encorafenib (Braftovi®), binimetinib (Mektovi®), cemiplimab-rwlc (Libtayo®), alitretinoin (Panretin®), and vandetanib (Caprelsa®).

[0150] The oligonucleotide of the present invention can also be administered to the subject in combination with anti-cancer antibody drugs.The combined administration with ODN of the present invention is expected to synergistically enhance the active antibody drugs, particularly those that utilize immune system to attack cancer cells.Examples of such antibody drugs include, but are not limited to: (i) Single-type antibodies: trastuzumab (Herceptin®), alemtuzumab (Campath®), bevacizumab (Avastin®), pertuzumab (Perjeta®), cetuximab (Erbitux®), panitumumab (Vectibix®), necitumumab (Portraza®), dinutuximab (Unituxin®), ramucirumab (Cyramza®), olaratumab (Raltruvo®), ipilimumab (Yervoy®), nivolumab (Opdivo®), (R)), pembrolizumab (Keytruda®), atezolizumab (Tecentriq®), denosumab (Xgeva®), durvalumab (Imfinzi®), avelumab (Bavencio®), ibritumomab tiuxetan (Zevalin®), brentuximab vedotin (Adcetris®), obinutuzumab (Gazyva®), mogamulizumab-kpkc (Potelizio®), daratumumab (Darazalex®), elotuzumab (Empliciti®), rucaparib camsylate (Rubraca®), niraparib tosylate monohydrate (Zejula®), antibody drugs including anti-OX40; (ii) Bispecific antibodies: antibody drugs including blinatumomab (Blincyto®); (iii) antibody-drug conjugates (ADCs): antibody drugs including ibritumomab tiuxetan (Zevalin®), brentuximab vedotin (Adcetris®), Ado-trastuzumab emetacin (Kadcyla®), and gemtuzumab ozogamicin (Mylotarg®); and (iv) ziv-aflibercept (Zaltrap®) Examples include:

[0151] The oligonucleotides of the invention may also be administered in combination with cytokines such as GM-CSF, IFN-α, IFN-β or IFN-γ, which are already used in standard cases of cancer therapy.

[0152] The oligonucleotides of the present invention may also be used in combination with radiation therapy, radiofrequency ablation, surgical procedures including cryoablation (Non-Patent Document 42), or photodynamic therapy (PDT) after administration of a photosensitizer. In particular, considering that PDT has been reported to upregulate anti-cancer immunity (Non-Patent Document 43), combined administration of PDT with the oligonucleotide of the present invention is expected to show synergistic efficacy. The oligonucleotides of the present invention may be administered as active ingredients in adjuvant or neoadjuvant therapy (Non-Patent Document 44).

[0153] The oligonucleotides of the present invention may also be used in combination with chimeric antigen receptor T (CAR-T) cell therapy (also known as adoptive cell transfer (ACT)), such as axicabtadine ciloreucel (Yescarta®) or tisagenlecleucel (Kymriah®), tumor infiltrating lymphocyte (TIL) therapy, dendritic cell therapy, or NK cell therapy. The ODNs of the present invention may also be used in combination with oncolytic virus therapy, which utilizes an artificially modified virus that dissolves tumor masses (Non-Patent Document 45; Non-Patent Document 46; Non-Patent Document 47; Non-Patent Document 48; Non-Patent Document 49).

[0154] The oligonucleotides of the present invention may be used in combination with therapeutic vaccines, which are expected to enhance the immune response necessary for therapy. Therapeutic vaccines are a type of vaccine used to treat existing diseases. These therapeutic vaccines include not only vaccines prepared using attenuated or inactivated microorganisms, but also ex vivo cellular vaccines prepared using cells treated ex vivo, and in vivo vaccines that activate immune responses, such as anti-cancer immunity, by activating target immune cells. Examples of ex vivo cellular vaccines include sipuleucel-T (Provenge), which is prepared from autologous or allogeneic dendritic cells, and GVAX, which is prepared by modifying autologous or allogeneic cancer cells. Loading of such dendritic cells with antigens can be achieved by contacting dendritic cells with antigens, including peptides, recombinant proteins, or tumor lysates, during ex vivo culture, or by fusing dendritic cells with cells expressing the antigen (e.g., cancer cells) (Non-Patent Document 50).

[0155] The oligonucleotides of the present invention may be used in combination with in vivo vaccines. The in vivo vaccines may include a delivery tool to target antigen-presenting cells (APCs) in combination with an antigen such as a cancer antigen or a viral antigen, and the target proteins for the delivery tool to APCs include cell surface proteins such as: Dendritic cells: DEC205, CD11c, DC-SIGN, mannose receptor, TLR, CD91 B cells: CD180, BCR, CD21, CD19 Plasmacytoid dendritic cells (pDC): CD32, CLEC12a, BDCA2, DCIR, TLR9 Antigens used in in vivo vaccines may include peptides or recombinant proteins, including cancer antigens, or viral antigens, as described below, and polynucleotides encoding the antigens can be used to directly express the antigen in such target APCs.

[0156] Prophylactic cancer vaccines include hepatitis B virus vaccines and human papillomavirus (HPV) vaccines, such as Gardasil® or Cervarix®. The oligonucleotides of the invention are also expected to enhance the prophylactic activity of commercially available or pre-marketed vaccines.

[0157] The oligonucleotide of the present invention can be used for the prevention or treatment of infectious diseases.These oligonucleotides can also be mixed into vaccine compositions as adjuvants.Otherwise, these oligonucleotides can be administered to subjects in combination with pharmaceutical compositions for the treatment of infectious diseases, including antibacterial drugs, antifungal drugs, antiviral drugs, antiparasitic drugs, vaccines, and antibody drugs for neutralizing toxins. Vaccines used to prevent or treat infectious diseases include the following categories: Live attenuated vaccines containing attenuated live microorganisms. Inactivated vaccines contain killed microorganisms and retain antigenicity. · Subunit vaccines contain only the antigens that most effectively stimulate the immune system. -Toxoid vaccines are used to neutralize toxins derived from microorganisms. Conjugate vaccines are a special type of subunit vaccine that can establish immunity against weakly immunogenic microorganisms by combining other more immunogenic subunits. Nucleic acid vaccines that induce somatic cells to produce antigens to achieve further in vivo immune induction. · Recombinant vector vaccines that carry the genetic information of recombinant antigens to stimulate immunity against target microorganisms. Examples of vaccines that can be used in combination with the oligonucleotides of the present invention include BCG vaccine, cholera vaccine, diphtheria vaccine, Haemophilus influenzae vaccine, hepatitis A vaccine, hepatitis B vaccine, human papillomavirus vaccine, pandemic H1N1 influenza vaccine, seasonal influenza vaccine, Japanese encephalitis vaccine, measles vaccine, mumps vaccine, meningococcal vaccine, pneumococcal vaccine, Bordetella pertussis vaccine, polio vaccine, rabies vaccine, rotavirus vaccine, rubella vaccine, tetanus toxoid vaccine, typhoid vaccine, and yellow fever vaccine.

[0158] Recently, there has been a strong demand for a class of drugs called immune checkpoint inhibitors (CPIs), which affect immune checkpoint molecules, a group of proteins that regulate immunity in order to stimulate the immunity required for the treatment of target diseases. The oligonucleotides of the present invention may be used in combination with CPIs to synergistically enhance the effectiveness of CPIs through the activation of antigen-presenting cells involved in the immune checkpoint process. Examples of target molecules of immune checkpoint inhibitors include PD-1, PD-L1, PD-L2, CD28, CD80, CD86, ICOS, B7RP1 (ICOSL), B7-H3 (CD276), B7-H4 (VTCN1), CD28H, B7-H5 VISTA, BTLA, HVEM, CD40L, CD40, OX40, OX40L, CD137, CD137L, CD27, CD70, TIM3, GAL9, GITR, GITRL, LAG-3, MHC-II, CD47, ADORA2A (adenosine A2A receptor), and adenosine (Non-Patent Document 51).

[0159] Therapies or drugs that suppress the activity of immunosuppressive immune cells, such as Tregs, tumor-associated macrophages (TAMs), or MDSCs, can also be used in combination therapy with the oligonucleotides of the present invention. Target molecules or pathways affected by such therapies or drugs include: Tregs - Cytotoxic T-lymphocyte antigen-4 (CTLA-4), TGF-beta (TGFβ), IL-10, IL-35, ICOS, and lymphocyte activation gene-3 (LAG-3), indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO), CD39, CD73, PI3K, Atg7, and Atg5 TAM - CCL2-CCR2 axis and CSF1 / CSF1 receptor (CSF1R) signaling MDSCs - PDE-5, COX-2, HDAC, STAT3, CCL2 / CCR2, VEGF-A / MET / TIE2 / VEGFR2 pathway, IL-8 / CXCR1 / 2, galectin-1 (Gal-1) is. Therapies or drugs that remove such immunosuppressive immune cells can also be used in combination with the oligonucleotides of the present invention. Examples of cell surface marker proteins that can be used to deplete such cells are: Tregs - CD25, CTLA-4, PD-1, ICOS, GITR, OX40, CD15s, CCR4, and CCR8 TAMs - CD206, legumain, scavenger receptor A, and CD52 (Non-patent document 52; Non-patent document 53; Non-patent document 54; Non-patent document 55).

[0160] The oligonucleotides of the present invention can be used in combination with antigenic proteins, peptides, glycoconjugates, or other organic substances to enhance specific antigenic responses to target diseases. Examples of these antigenic substances are: AFP, AKAP-4, ALK, androgen receptor, B7H3, BAGE, bcr-abl, BMLF1, BmpA, BmpB, BORIS, BRLF1, BZLF1, carbonic anhydrase IX, catalase B, CDC27, CDCA1, CDH3, CDK4, CEA, crf1, cyclin B1, CYP1B1, DEPDC1, EBNA1, EBNA-1, EGFRvIII, envelope glycoprotein D, EpCAM, EphA2, EphA3, ERG, ETV6-AML, FAP, Fos-related antigen 1, FOXM1, fucosyl GM1, GD2, GD3, Gel1, GloboH, GM3, gp100, GPC3, HA, HBV protein, HCV protein, HER2, hexon, HJURP, HMWMAA, HPV-16 E6, HPV-16 E7, HPV-18E7, surface Ig idiotype, IE-1, KIF20A, KOC1, KSV protein, large T antigen, small T antigen, LCK, legumain, LMP1, LMP2, MAD-CT-1, MAD-CT-2, MAGE, MAGE-3, MAGE-A1, MAGE-A4, MAM-A, melan-A / MART-1, MELK, MELOE-1 / 2, mesothelin, ML-IAP, MP1, MP2, M P65, MPHOSPH1, MUC1, mucin-1, MYCN, NA, NA17, NeuGcGM3, nonstructural protein NS4, nonstructural protein NS5, NY-BR-1, NY-ESO-1, ospA, ospB, ospC, OY-TES1, p53, Page4, PAP, PAX3, PAX5, PDGFR-β, penton, PLAC1, pmel17, pmp20, polysialic acid, pp65, PR AME, prostate-specific membrane antigen 10, prostein, proteinase 3 (PR1), PSA, PSCA, PSMA, Ras, RGS5, RhoC, RM2, RNF43, ROR1, sarcoma translocation breakpoint, SART3, Select, serine protease NS3, SHMP, sLe, SOD, sperm protein 17, SSX2, STEAP1, STn, survivin, TARP, Tax protein, telomerase, telomerase, TERT, Tie2, TM4SF5, Tn, TOMM34, triosephosphate isomerase, TRP1, TRP2, TTK, tyrosinase, tyrosinase-related protein 1, tyrosinase-related protein 2, URLC10, VEGFR2, viral capsid protein, viral core protein, viral nucleoprotein, WT1, XAGE1, α-actinin-4, and β-catenin. Neoantigens are also good candidates for use as antigenic substances; they are antigens that are formed de novo by in vivo processes such as mutagenesis during tumorigenesis or are produced from infectious xenobiotics and become recognized by the innate immune system.

[0161] Anticancer chemotherapeutic drugs, particularly drugs that induce immunogenic cell death (ICD), can be administered in combination with the oligonucleotides of the present invention to demonstrate synergistically enhanced efficacy. Examples of drugs that induce ICD are anthracyclines, including mitoxantrone, and platinum-based anticancer drugs, including oxaliplatin, cisplatin, carboplatin, nedaplatin, triplatin tetranitrate, picoplatin, and satraplatin (Non-Patent Document 56).

[0162] Other drugs known for their anti-cancer immune activating properties, such as indoleamine 2,3-dioxygenase (IDO) inhibitors (Non-Patent Document 57) or proteasome inhibitors, including bortezomib (Non-Patent Document 58; Non-Patent Document 59), can be administered in combination with the oligonucleotides of the present invention to demonstrate synergistic efficacy.

[0163] The oligonucleotides of the present invention can be administered in combination with other TLR9 agonists or agonists of other TLRs, such as TLR3, 4, 8, or 7. The oligonucleotides of the present invention can also be administered in combination with enhancers of intracellular nucleotide sensor signaling pathways, such as the cGAS-STING pathway or the RIG-I / MDA5 pathway (Non-Patent Document 60; Non-Patent Document 61), because these pathways contain common target molecules with TLR9, such as IFN-α or NF-kB, and are expected to exhibit synergistic effects when activated simultaneously. Examples of molecules that activate each receptor are as follows: TLR3 agonists: Lintatorimod, PolyC 3SBIO, Poly(I:C), Hiltonol TLR4 agonists: ALD046, CRX527, CRX675, G100, lipid A, GSK1795091, OM174, PGN007 TLR7 agonists: vesatolimod, VML600, 852A, NKTR262, TMX101, GS9620, RG7795, DSP0509, PF4878691, RG7854, RG7863, TMX202, TQA3334 TLR8 agonists: GS-9688, VTX2337 TLR9 agonists: Heplisub, SD-101, IMO2125, IMO2055, MGN1703, MGN1706, CPG 7909, Litenimod, AST008, DUK-CPG-001, Actilon, CMP001, DV281, Cobitolimod TLR9 and NOD2 agonist: MIS416 STING signaling pathway activator: ADU-S100 STING agonists: MK-1454, SB11285, IMSA101 RIG-I agonist: RGT100.

[0164] The oligonucleotide of the present invention can be administered in a delivery carrier or in the form of being linked to a carrier.Carriers include but are not limited to sterols (for example, cholesterol), cochleates, emulsomes, ISCOMs; lipids (for example, cationic lipids, anionic lipids), liposomes; ethylene glycol (PEG); polyglycolide-co-lactide (PGLA); microspheres; polymers (for example, carboxymethylcellulose (CMC), chitosan, mannitol, hydroxypropylmethylcellulose (HPMC)); live bacterial vectors (for example, Salmonella, Escherichia coli, Bacillus Calmette-Guerin, Shigella, Lactobacillus); live viral vectors (for example, vaccinia, adenovirus, herpes simplex), virosomes, virus-like particles.

[0165] The target subject to which the oligonucleotides of the invention are administered is preferably a human, although in one embodiment the subject may be a non-human animal such as a dog, cat, horse, pig, goat, sheep, cow, monkey, chicken, mouse, or rat.

[0166] "Therapeutically effective amount": A therapeutically effective amount of the oligonucleotide of the present invention is administered to a subject to treat or prevent a target disease or disorder. A "therapeutically effective amount" of one or more oligonucleotides refers to a sufficient amount of the oligonucleotide used to achieve the desired result of treating or preventing a disorder in a subject. The oligonucleotide of the present invention can be used in pure form or in a pharmaceutically acceptable carrier. Alternatively, the ODN of the present invention may be administered as a pharmaceutical composition. In the present invention, "amount" refers to a dose. Doses can be determined by standard techniques well known to those skilled in the art and may vary depending on factors including, but not limited to, the size of the subject and / or the overall health or severity of disease symptoms. The oligonucleotide of the present invention can be administered as a single treatment or a series of treatments. The subject dose of the oligonucleotide of the present invention for administration ranges from about 1 μg (microgram) to 10 g per administration. Preferably, the dose ranges from 0.1 mg to 5 g. More preferably, the dose ranges from 0.3 mg to 3 g. Most preferably, the dose ranges from 1 mg to 1 g. The therapeutically effective amount for a human subject can be estimated based on the amount suitable for a non-human animal by using the human equivalent dose (HED) or human equivalent concentration (HEC). The therapeutically effective amount for a human subject can be, but is not limited to, 0.3 to 60 mg / day, preferably 1 to 30 mg / day, and more preferably 2 to 8 mg / day.

[0167] "Route of administration": For clinical use, the oligonucleotides of the present invention can be administered alone or formulated as a pharmaceutical composition by any effective route effective to achieve the desired therapeutic result. The "route" of administering the oligonucleotides of the present invention refers to enteral administration, parenteral administration, and topical administration or inhalation. Enteral administration routes of the oligonucleotides of the present invention include oral, gastric, intestinal, and rectal routes. Parenteral routes include subcutaneous, intravenous, transdermal, intradermal, sublingual, intranasal, transmucosal, pulmonary, vaginal, aerosol, intraocular, intratracheal, intrarectal, intraspinal, intramuscular, intraarticular, intraperitoneal, intracardiac, intraosseous, intrathecal, intravitreal, inhalation, or topical administration. The topical administration route of the oligonucleotides of the present invention refers to the external application of the oligonucleotides to the epidermis, oral cavity, and ear, eye, and nose. Intratumoral administration is one of the administration routes commonly performed by injection of the test compound into the tumor or peritumoral area.

[0168] "Pharmaceutical composition": A pharmaceutical composition is intended to mean a composition comprising a therapeutically effective amount of an oligonucleotide of the present invention, with or without a pharmaceutically acceptable carrier. A pharmaceutical composition may contain one or more oligonucleotides of the present invention. These compositions include, but are not limited to, aqueous or saline solutions, granules, aerosols, pellets, granules, powders, tablets, coated tablets, orally dissolving / disintegrating tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops, and other pharmaceutical compositions suitable for use in various drug delivery systems. These compositions can be administered parenterally, orally, rectally, intravaginally, intraperitoneally, topically (in the form of powders, ointments, gels, drops, or transdermal patches), bucally, or as an oral or nasal spray. In all cases, the compositions must be sterile and stable under the conditions of production and storage and must be preserved against microbial contamination. Pharmaceutical compositions of the present invention for parenteral injection include pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions immediately prior to use. Oligonucleotides of the present invention can be suspended in an aqueous carrier, for example, in an isotonic buffer solution at a pH of about 3.0 to about 8.0, preferably about 3.5 to about 7.4, 3.5 to 6.0, or 3.5 to about 5.0. Buffer solutions include sodium citrate-citric acid and sodium phosphate-phosphate, and sodium acetate-acetic acid buffers. For oral administration, compositions can be formulated with edible carriers to form powdered tablets, pills, orally dissolving / disintegrating tablets, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like. For solid compositions, conventional non-toxic solid carriers can include pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. For buccal administration, compositions can be in the form of tablets or lozenges in the conventional format. For inhalation, the compositions can be delivered in the form of a pressurized pack, an aerosol spray from a nebulizer, or a dry powder, as selected by one skilled in the art. In some cases, the oligonucleotides of the present invention are also suitably administered in sustained-release systems to prolong the effect of the oligonucleotides of the present invention.The oligonucleotide of the present invention can be used as a liquid suspension of crystalline or amorphous material with low water solubility to slow down the release of oligonucleotide.Alternatively, the delayed release of parenteral administration dosage form of oligonucleotide can be achieved by dissolving or suspending oligonucleotide in hydrophobic material (for example, acceptable oil vehicle).Injection depot form can be made by entrapping oligonucleotide in liposome or microemulsion or other biodegradable semipermeable polymer matrix, for example, polylactide-polyglycolide, polyorthoester and polyanhydride. [Example]

[0169] The present invention will be described in more detail in the following examples. However, the present invention is not limited to these examples. In these examples, unless otherwise specified, experiments were carried out using commercially available kits and reagents according to the accompanying protocols. Those skilled in the art will recognize that the oligonucleotides of the present invention can be easily applied to treat target diseases, including cancer and infectious diseases. Hereinafter, the present invention will be illustrated by the following non-limiting examples.

[0170] material and method: [Oligo-deoxynucleotide (ODN)] Single-stranded oligodeoxynucleotides (ODNs) were synthesized at Hokkaido System Science Co., Ltd. using the conventional phosphoramidite method without a phosphate group at the 3' end, and the purity and identity were confirmed by the manufacturer. In the following examples, nucleosides with a phosphorothioate (PS) internucleotide linkage at the 3' end are written in lowercase, while those or oligonucleosides with a phosphodiester (PO) internucleotide linkage at the 3' end are written in uppercase. All synthetic ODNs were first dissolved in pyrogen-free distilled water and further diluted with pyrogen-free reagent for testing.

[0171] [10% FBS-RPMI complete medium] RPMI 1640 medium was supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillin-streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, and 50 mM 2-mercaptoethanol, and the medium was further filtered through a 0.45 μm syringe filter.

[0172] [CAL-1 cells] CAL-1 cells (human plasmacytoid dendritic cell line; Non-Patent Document 60; Patent Document 2) were cultured in 10% FBS-RPMI complete medium at 37°C under humidified atmosphere and 5% CO2 conditions. CAL-1 cells become confluent within 2 days after 1:5 subculture, but these cells are subcultured before reaching confluence for the following tests. Suspension culture dishes are used to culture CAL-1 cells.

[0173] [Detection of TLR9 activation by GFP induction via NF-kB activation] The level of transcriptional activity of the NF-kB promoter induced by the TLR9 signaling pathway was monitored as an indicator of the activity level of TLR9 signaling. A CAL-1 / NF-kB-GFP cell line was established to monitor the activity of the NF-kB transcription factor in a cell-based assay (Patent Document 1). To establish the CAL-1 / NF-kB-GFP cell line, a vector encoding a GFP reporter gene driven by an NF-kB consensus transcription response element was transfected into CAL-1 cells by electroporation. The transfected cells were further selected with Zeocin. Stable transfectants were generated by single-cell cloning of the selected transfectants. GFP expression induced by the TLR9 agonist CpG2395 (5'-tcgtcgttttcggcgcgcgccG-3', SEQ ID NO: 45) was confirmed. Briefly, CAL-1 / NF-kB-GFP cells (1 x 10 5NF-kB-GFP / CAL-1 cells (1000kJ / well) were seeded into 96-well flat-bottom plates and cultured with or without CpG2395. These cells were incubated at 37°C in a 5% CO2 humidified incubator for 6 hours. The GFP expression level of the cells was assessed using a flow cytometer (FACS Calibur, BD Bioscience Co., Ltd.). The percentage of GFP-positive cells was analyzed as an indicator of the level of TLR9 signaling activity. The established NF-kB-GFP / CAL-1 cells were used in each assay.

[0174] [Detection of TLR7 activation] TLR7 activity was measured as the enzymatic activity of secreted embryonic alkaline phosphatase (SEAP) in HEK-Blue® TLR7 cells (Invivogen). Cells were incubated with the indicated oligonucleotides or TLR7 agonists, such as 1 μg / ml of gardquimod (GQ) or 1 μg / ml of CL264, for 24 hours at 37°C in a 5% CO2 humidified incubator, and induced SEAP was measured by the level of absorbance at 655 nm resulting from cleavage of the substrate (HEK-Blue® detection, Invivogen).

[0175] [Detection of TLR8 activation] TLR8 activity was measured as the enzymatic activity of secreted placental alkaline phosphatase (SEAP) in HEK-Blue® TLR8 cells (Invivogen). Cells were incubated with the indicated oligonucleotides or TLR7 agonists, e.g., 200 ng / ml TL8-506 or 5 μg / ml CL075, for 24 hours at 37°C in a 5% CO2 humidified incubator, and induced SEAP was measured by the level of absorbance at 655 nm resulting from cleavage of the substrate (HEK-Blue® detection, Invivogen).

[0176] [Detection of cytokine production] Cytokine levels were measured by ELISA using the following kits: human IFN-α (eBioscience), human IL-6, human TNF-α (Thermo Fisher Scientific), human IL-12p40 (BioLegend), mouse IFN-α (PBL), mouse IL-6, mouse TNF-α, and mouse IL-12p40 (Thermo Fisher Scientific) according to the manufacturer's protocol.

[0177] [Isolation of human PBMCs] Peripheral blood was collected from healthy volunteers. An equal volume of RPMI medium was added to the blood and mixed thoroughly. Human PBMCs were purified by centrifugation on Histopaque®. Briefly, Histopaque®-1077 (SIGMA) was placed in a centrifuge tube, and an equal volume of the blood / RPMI mixture was placed on the Histopaque®. The tube was centrifuged at 1800 rpm (700 x g) for 20 minutes. The interphase was collected into another tube with a pipette, and 10% FBS-RPMI complete medium was added. The mixture was centrifuged at 1800 rpm (700 x g) for 10 minutes, and the supernatant was removed. The cell pellet was treated with 2 ml of distilled water to lyse red blood cells, and 20 ml of 10% FBS-RPMI complete medium was immediately added to the tube. After washing twice with medium, the cells were suspended in medium and counted. Freshly isolated PBMCs were used for each assay.

[0178] [Preparation of mouse splenocytes] Spleens were harvested from mice and placed in a dish filled with RPMI medium. Using a nylon mesh, the spleens were disrupted using the rubber tip of a syringe plunger (2.5 ml syringe). The splenocytes contained in the medium were transferred to a 50 ml Falcon tube by filtering through a 70 μm strainer. After centrifugation at 1500 x g for 5 minutes, the cells were washed with 1x DPBS. The cells were treated with ACK lysis buffer to lyse red blood cells. 1 ml of ACK lysis buffer was added to the cell pellet, and the tube was pipetted 20 times with a P1000 pipette. The tube was then placed on ice for 2 minutes. 20 ml of RPMI complete medium containing 10% FBS was immediately added to the tube. After washing twice with medium, the cells were resuspended in the same medium and counted. These splenocytes were used for further studies.

[0179] Example 1 The fully phosphorothioated ODN of the present invention has adjuvant activity. The fully phosphorothioated ODNs used in this study are listed in Table 4 below. Lower case letters indicate that the nucleoside is modified with a phosphorothioate at the 3' internucleotide linkage, and upper case letters indicate that the nucleoside is unmodified at the 3' (no phosphodiester linkage).

[0180] [Table 4]

[0181] [Analysis of immunostimulatory activity of ODN] The ODNs shown in Table 4 were incubated with CAL-1 / NF-kB-GFP cells at the indicated concentrations (0.1 μM or 0.3 μM) for 6 hours. The activation of NF-kB by these ODNs was evaluated based on the percentage of GFP-positive cells analyzed by a flow cytometer (FACS Calibur, BD Bioscience Co., Ltd.). Data was analyzed using FlowJo® ver 10 (FlowJo® LLC).

[0182] As shown in Figure 1A, GFP expression was induced in CAL-1 / NF-kB-GFP cells by stimulation with CpG2395, a standard TLR9 agonist, indicating that NF-kB activation was induced by TLR9 activation by CpG2395. The fully phosphorothioated (PS) A001, A002, A003, and A004 ODNs of the present invention were shown to have stronger TLR9 activation activity than CpG2395. Furthermore, as shown in Figure 1B, the ODNs of the present invention exhibited stronger TLR9 activity than other known CpG ODNs, such as CpG685, M362, and D60-1. Given that the level of TLR9 activation induced by ODN is independent of nucleic acid length, this difference in agonistic activity was thought to be due to a specific sequence.

[0183] Many previous reports have discussed the importance of the gtcgtt sequence in CpG ODN for optimal activation of human TLR9 (Non-Patent Document 62, Non-Patent Document 63), and CpG2395 and CpG685 (5'-tcgtcgacgtcgttcgttctC-3'; SEQ ID NO: 46) contain a single gtcgtt sequence. Previous reports have also claimed that the tcg sequence at the 5' end of CpG ODN is crucial for human TLR9 activation (Non-Patent Document 64, Non-Patent Document 65). Conventional CpG ODNs, i.e., CpG2395, CpG685, and M362 (5'-tcgtcgtcgttcgaacgacgttgaT-3'; SEQ ID NO: 47), follow this rule and contain the tcgt sequence at the 5' end as a TLR9 activation motif. In the ODN of the present invention, only A003 has one gtcgtt sequence, and the others do not have gtcgtt sequence, and all ODN of the present invention do not have tcgt sequence at the 5' end.However, all ODN of the present invention showed stronger activation of human TLR9 than conventional CpG ODN such as CpG2395, CpG685 and M362.

[0184] As mentioned above, in humans, the 5'-tcgt and "gtcgtt" motifs in standard CpG ODN are known as TLR9 activation motifs (Non-Patent Document 66). However, these motifs in the ODN of the present invention have been shown to only slightly contribute to overall activity, suggesting that there are other stretches / motifs in the ODN of the present invention that are optimal for human TLR9. As shown in Figures 1C and 1D, the activity of A003 and A003#endG was the same. Although the activity of A003#delA was slightly reduced by changing the 3'-terminal base, its activity was still much stronger than that of the conventional CpG-ODN, CpG2395 (shown in Figure 1A). This indicates that the 3'-terminal nucleotide is not essential for activity.

[0185] In addition, as shown in Figures 1E, 1F and 1G, each ODN set, A001 and A011; A002 and A012; A003 and A013; and A004 and A014, exhibited comparable levels of activity relative to others in the same set, indicating that the substitution of the indicated nucleotides in these ODNs does not significantly alter the activity of the ODNs. Considering these factors, mutation of the 12th, 18th, 21st and 25th bases at the 5' end of ODN does not appear to reduce the stimulatory activity of ODN. The ODN of the present invention has been shown to have a core structure of 5'-tcgcaacgttt-n-cgacg-n-cg-nn-cg-3' (SEQ ID NO: 2) with TLR9 activation activity.

[0186] Example 2 The oligonucleotides of the present invention primarily activate TLR9. [Activation of human TLR7 and TLR8 by ODN] HEK blue® TLR7 cells were stimulated with TLR agonists for 24 hours. As shown in Figure 2A, TLR7 agonist gardikimod (GQ) and CL264 activate TLR7 and produce positive signal.In contrast, standard TLR9 agonist CpG2395 and ODN of the present invention cannot activate TLR7 signaling pathway. In addition, HEK blue TLR8 cells were stimulated with TLR agonists for 24 hours.As shown in Figure 2B, TLR8 agonists TL8-506 and CL075 activated TLR8 and produced positive signals.In contrast, standard TLR9 agonist CpG2395 and ODN of the present invention could not activate TLR8 signaling pathway.

[0187] Example 3 Characteristic nucleotide stretches with partial phosphorothioation of the oligonucleotides of the invention The existence of a minimal stretch of the oligonucleotides of the present invention that enhances TLR9 agonistic activity was investigated.

[0188] [ODN] As listed in Table 5, single-stranded ODNs having internucleotide linkages with partial phosphorothioation were produced.

[0189] [Table 5]

[0190] A lowercase letter indicates that the nucleoside is modified with a phosphorothioate at the 3' internucleotide linkage, and an uppercase letter indicates that the nucleoside has a phosphodiester internucleotide linkage at the 3' or is unmodified (no phosphodiester linkage).

[0191] [Analysis of TLR9 agonist activity of ODN] The agonistic activity of ODNs was analyzed by GFP expression in CAL-1 / NF-kB-GFP cells. As shown in Figure 3A, full PS A003 exhibited low activity at the tested concentrations over a 6-hour stimulation period. It has been reported that altering the internucleotide bond by a phosphodiester (PO) bond within the CG motif of standard full PS CpG ODN enhanced its activity in activating TLR9 (Non-Patent Document 67, Patent Document 3). Therefore, A103 and A203, which have a CG motif with a PO bond and retain the same sequence as A003, also exhibited enhanced activity. However, as shown in Figures 3A and 3B, an additional PO bond (A303) in the CA of the CAACG stretch of the ODN further upregulated activity. As can be seen from the left panel of Figure 3B (0.1 μM), A303 exhibited significantly stronger activity than A103 and A203. This suggests the importance of the PO bond (CaaCg) within the CAACG stretch for activity. As shown in Figure 3C, A403 and A503 showed reduced activity compared to A303, but both A403 and A503 had an increased number of CG motifs with PO bonds compared to A303. This indicates that, although the number of CG motifs with PO bonds in standard CpG ODNs has traditionally been considered important for their activity (Patent Document 3), the number of CG motifs with PO bonds in the ODNs of the present invention is not important for their activity. Importantly, both A403 and A503 lack the CaaCg stretch (two PO bonds within the CAACG motif). Although A603 and A503 have the same number of PO bonds in the CG motif, A603 showed better activity than A503. A603 maintained the CaaCg stretch as A303 and showed activity comparable to A303, suggesting that the CaaCg stretch is more important for optimal activity than an increased number of CG motifs with PO bonds. Although A703 has PO bond in all CG motifs, it shows less activity than A303 and A603 (stimulation at 0.1 μM), which suggests that increasing the number of CG motifs with PO bond is not essential for maximizing the activity of ODN of the present invention.The total number of CG motifs with PO bond is not important for optimizing its activity, which is not expected from the effect of Py (pyrimidine)-PO-Pu (purine) known so far (Patent Document 3).

[0192] As shown in Figure 3D, the importance of the CaaCg stretch was further confirmed by detecting the production of inflammatory cytokines induced by ODNs. A303 and A603 induced comparable levels of cytokines, and their production levels were higher than those of A403 and A503. The importance of the CaaCg stretch was examined using previously reported CpGs, DV093 and DV094 (Fig. 3E). Although both DV093 and DV094 contain the caacg motif in their sequences, their activity was much weaker than that of A003.

[0193] Interestingly, the CaaCg stretch (CAACG motif with complete phosphorothioation) in DV093C and DV094C failed to enhance their activity compared to the original DV093 or DV094. Altering the internucleotide linkages to change the caacg stretch to CaaCg in DV093 and DV094 did not improve activity, as shown in Figure 3. These data suggest that the presence of specific CaaCg stretches, such as those in the ODNs of the present invention (but not in randomly arranged CaaCg ODNs), has unique properties for enhancing TLR9 activity. Taken together, 5'-tCgCaaCg stretches (such as those in A303 and A603) may be present in the core structure of the ODNs of the present invention.

[0194] Example 4 Core structure of the ODN of the present invention [ODN] As listed in Table 6, single-stranded ODNs having internucleotide linkages with partial phosphorothioation were prepared.

[0195] [Table 6]

[0196] The activity of ODN was analyzed by GFP expression in CAL-1 / NF-kB-GFP cells.As shown in Figures 4A and 4B, all the tested ODNs with variations in the 18th, 21st and 22nd bases of the 3' region showed comparable activity levels, and their activity was much higher than that of standard CpG ODN such as CpG2006.This indicates that the 18th, 21st and 22nd bases of the 3' region of ODN are not essential for its activity. As mentioned above, the "gtcgtt" motif in standard CpG ODN is known as a human TLR9 activation motif (Wang, X., et al., Vaccine, 2008. 26(15): p.1893-901), and CpG2006 has three motifs in its sequence. Meanwhile, A603 is the only ODN tested by the present inventors that has one motif, and all tested ODNs, including A603, exhibited similar levels of activity, suggesting that the ODNs of the present invention may contain other motifs or stretches that are optimal for human TLR9 activity. Furthermore, our data show that the "gtcgtt" motif in the ODNs of the present invention only contributes slightly to overall activity as long as the Cg-nn-Cg stretch is maintained in the 3' region. The 3' end of the ODNs of the present invention requires a core structure of gn-Cg-nn-Cg.

[0197] As shown in Figures 5A and 5B, the ODN of the present invention and the standard TLR9 agonist CpG2395 were unable to activate both the TLR7 and TLR8 signaling pathways, suggesting that the ODN of the present invention was a TLR9 agonist.

[0198] Example 5 The partially dephosphorothioated ODN of the present invention does not have TLR7 and TLR8 activity [ODN] As listed in Table 7, single-stranded ODNs having internucleotide linkages with partial phosphorothioation were prepared.

[0199] [Table 7]

[0200] The activity of ODN was analyzed by GFP expression in CAL-1 / NF-kB-GFP cells. As shown in Figures 6A, 6B and 6C, all tested ODNs with base variations and / or internucleotide linkages at the 3'-side 12th position showed comparable levels of activity in activating NF-kB. This indicates that the 12th nucleotide tolerates mutations, and the internucleotide linkage at the 3'-side of this nucleotide can be either a PO linkage or a PS linkage.

[0201] As shown in Figures 7A and 7B, these ODNs and the standard TLR9 agonist CpG2395 were unable to activate both TLR7 and TLR8 signaling pathways, suggesting that these ODNs were TLR9 activators.

[0202] Considering Examples 1 to 5, the ODN of the present invention has a core sequence of 5'-tcgcaacgttt-n-cgacg-n-cg-nn-cg-3' (SEQ ID NO: 2). For maximum human TLR9 stimulating activity, the ODN preferably has a core structure of 5'-tCgCaaCgttt-n-cgacg-n-Cg-nn-Cg-3' (SEQ ID NO: 5). Furthermore, the ODNs of the present invention do not follow previously reported rules, and therefore are different from reported standard CpG ODNs and are a novel type of TLR9 agonist.

[0203] Example 6 Assessing the level of TLR9 stimulation in human cells [Stimulation of HAL-01 cells] HAL-01 cells, a human B-ALL cell line, were purchased from DSMZ (catalog ACC610). HAL-01 cells were maintained in 10% FBS-RPMI complete medium and stimulated with 1.0 μM ODN for 24 hours. These cells were stained with APC-conjugated anti-CD40 Ab (eBiosciences) and PE-conjugated anti-CD86 Ab (BD Pharmingen). Induction of CD40 and CD86 expression was assessed using a flow cytometer.

[0204] [Stimulation of human PBMCs] Prepared human PBMCs (5 × 10 5 Cells (200 μl) were stimulated with 0.1 μM or 0.3 μM ODN for 24 hours. Cell proliferation was assessed by WST-1 assay (Roche) according to the manufacturer's protocol. Culture supernatants were collected, and cytokine production was assessed by ELISA according to the manufacturer's protocol.

[0205] It is known that human B cells express TLR9, and therefore TLR9 agonists can activate human B cells.Human B-ALL cell line is stimulated with TLR9 agonists, and it has been demonstrated that the surface expression of costimulatory molecules such as CD40 and CD86 is upregulated by this stimulation.As shown in Figure 8A, ODN of the present invention activates HAL-01 cells, which is a B-ALL cell line, and this is shown by the upregulation of the surface expression of CD40 and CD86. It has been demonstrated that ODN of the present invention can induce the proliferation of human PBMC and the production of inflammatory cytokines.As shown in Figure 8B and 8C, ODN of the present invention can stimulate human PBMC, thereby inducing cell proliferation and the production of inflammatory cytokines such as IFN-α, IL-6 and IL-12.

[0206] Example 7 Assessing the level of TLR9 stimulation in mouse cells [Stimulation of mouse spleen cells (splenocytes)] The prepared splenocytes were stimulated with 0.03 μM ODN for 24 hours. The culture supernatant was collected, and cytokine production was evaluated by ELISA of the culture supernatant according to the manufacturer's protocol. The prepared splenocytes were stimulated with various concentrations of ODN for 24 hours. Cell proliferation was evaluated by WST-1 assay (Roche) according to the manufacturer's protocol.

[0207] It is known that TLR9 agonists can induce the production of inflammatory cytokines and the proliferation of mouse splenocytes.As shown in Figure 9A, the ODN of the present invention can induce the production of mouse inflammatory cytokines such as TNF-α and IL-12.In addition, as shown in Figures 9B and 9C, ODN induces the proliferation of mouse splenocytes.The activity of ODN is obviously higher than that of genuine TLR9 agonists CpG2395 and CpG2006.

[0208] The tested ODNs A601, A602 and A603 have the structure 5'-tCgCaaCgttt-n-cgacg-n-Cg-nn-Cg-3' (SEQ ID NO: 5) as the core structure, and these ODNs can activate mouse splenocytes and human PBMCs.This indicates that the defined PO interlinkage bond at precise positions, such as "tCgCaaCg" and "Cg-nn-Cg" structures, is important for optimal TLR9 activation in both mice and humans.

[0209] Example 8 In vivo antitumor efficacy of ODNs [CT26 cells] The BALB / c-derived mouse colon carcinoma cell line, CT26, was purchased from the American Type Culture Collection (ATCC, CRL-2638). CT26 cells were cultured in 10% FBS-RPMI complete medium at 37°C in a humidified atmosphere with 5% CO2.

[0210] [Inoculation of CT26 cells into mice] CT26 cells were harvested using a 0.25% trypsin-EDTA solution in PBS. After suspending in medium, the cells were passed through a 40 μm strainer and counted. The cell concentration in the suspension was 2 × 10 6 For inoculation into BALB / c mice, 100 μl of cell suspension was used per mouse (2 × 10 5 After shaving the back of the mice, 100 μl of the CT26 cell suspension was injected subcutaneously into the right flank of the mice with a 28-gauge needle. The mice were then cultured until the tumor volume reached approximately 150 mm3 The mice were maintained without treatment for 2 weeks until tumor volume reached 100 μg / kg. Tumor volume was measured every 2 or 3 days, and the mice were divided into 3 groups based on tumor volume. Tumor volume was calculated using the following formula: Tumor volume (mm 3 )=1 / 2(length x width 2 )

[0211] [ODN administration] ODN (40 μg / 50 μl / mouse) was administered around the tumor starting on the day of grouping (day 0) and repeated on day 2. The ODN of the present invention was administered twice in total during the test. The tumor volume and body weight of each group of mice were measured every 2 or 3 days. As shown in Figure 10, administration of the ODN of the present invention clearly induced tumor regression in mice. On day 11, the tumors of almost all mice were rejected by administration of ODN (Figures 10A and 10B). During the test, no weight loss was observed in any group of mice (Figure 10C). This indicates that ODN (A601 and A602) have antitumor activity and are almost non-toxic. Considering the fact that A601, A602 and A603 showed comparable activity in mouse splenocytes as shown in Figure 9B, A603 shows the same strong antitumor activity as A601 and A602. The ODN of the present invention has been proven to have antitumor activity.

[0212] Example 9 In vivo antitumor efficacy of ODNs (2) [Inoculation of CT26 cells and treatment with ODN] CT26 cells were inoculated into the right flank of BALB / c mice as described above. Mice were cultured until tumor volumes reached approximately 100 mm 3It was maintained without treatment for 2 weeks until it reached [a certain state]. The tumor volume was measured every 2 or 3 days, and the mice were divided into 3 groups based on the tumor volume. Administration of ODN (A601 and A602) (40 μg / 50 μl / mouse) around the tumor was started on the day of grouping (day 0), and repeated on day 2. The administration of the ODN of the present invention was performed a total of 2 times during the test. As a negative control, PBS was administered instead of the solution containing ODN. The tumor volume and body weight of the mice in each group were measured every 2 or 3 days until tumor rejection was confirmed (day 14). The tumor volume was calculated using the following formula: Tumor volume (mm 3 ) = 1 / 2 (length × width 2 )

[0213] [Re - inoculation of CT26 cells in treated mice] CT26 cells (2 × 10 5 ) were re - inoculated on the left flank of mice in which tumors were inoculated and tumor rejection was confirmed on day 14, and the mice were maintained without treatment. The tumor volume of the left flank of each mouse was measured every 2 or 3 days from 14 days after the re - inoculation of CT26 until 14 days later.

[0214] As shown in Figure 11A, the administration of the ODN of the present invention clearly induced tumor rejection in mice. Furthermore, the mice treated with ODN also rejected tumors similarly (Figure 11B). The tumors in the left flank of the mice not treated with the ODN of the present invention grew, but all the mice treated with ODN rejected the re - administered tumors without further treatment for 2 weeks (Figure 11B). This suggests that the ODN of the present invention induced and established the memory of anti - tumor immunity.

[0215] [[ID=​​​​​​​​Adjusted to cells / ml. To induce lung metastasis of CT26 cells, 200 μl of cell suspension was intravenously injected (I.V.) into BALB / c mice from the tail vein using a 28-gauge needle (5×10 5 cells / mouse) (day 0). On day 2, administration of ODN was initiated (subcutaneous injection into the skin of the back, 40 μg / 50 μl / mouse). The same dose was repeated on day 5. As a negative control, PBS was administered instead of the solution containing ODN. Measurement of body weight and observation of mouse behavior were performed three times a week after transplantation of CT26 cells. On day 18, the mice were sacrificed and lung weight was measured. Metastatic tumor nodules in the lungs of the mice were also counted.

[0216] As shown in Figure 12, the lung weight of mice in the PBS treatment group increased dramatically and many tumor nodules were observed. In contrast, no such increase in lung weight was observed in mice treated with the ODN of the present invention. Furthermore, only a few tumor nodules were observed in mice of the treatment group. This indicates that the ODN of the present invention can suppress the growth of metastatic cancer cells in the lung.

[0217] Example 11 In vivo efficacy of ODN with a changed administration route <CT26 lung metastasis model> Induction of lung metastasis using CT26 cells was performed as described above. On the day after CT26 cell injection, administration of A602, an ODN, was initiated. In this study, two administration routes were tested. One was subcutaneous (S.C.) injection into the skin of the back (25 μg / 50 μl / mouse), and the other was intradermal (I.D.) injection into the ear root (25 μg / 20 μl / mouse). The same dose was repeated on day 3 and day 5. Measurement of body weight and observation of mouse behavior were performed three times a week after transplantation of CT26 cells. On day 16, the mice were sacrificed and metastatic tumor nodules in each lung of the mice were counted.

[0218] <CT26 liver metastasis model> The cell suspension of CT26 cells was prepared as described above. This cell suspension was adjusted to a final concentration of 1.0×10 6For induction of liver metastasis of CT26 cells, 100 μl of the cell suspension was injected into the spleen as follows (1 × 10 5 Cells / mouse) (Day 0). Briefly, mice were anesthetized and the skin was shaved. An abdominal incision (0.5 cm) was made adjacent to the spleen (the left flank incision was approximately 2 cm left of the abdominal midline). The prepared CT26 cell suspension was injected into the spleen using a 30G needle and maintained in the spleen for 5 minutes after injection. To prevent bleeding, the blood vessels leading to the spleen were tightly ligated with sutures, and then the spleen was removed with sharp scissors. The peritoneum and skin were sutured, and the mice were warmed under a light source. The mice were monitored for recovery from anesthesia. On Day 2, the mice were divided into three groups based on their weight, and administration of the ODN A602 was initiated. Two administration routes were tested in this study. One was injected SC into the dorsal skin (12.5 μg / 50 μl / mouse), and the other was injected ID into the base of the ear (12.5 μg / 20 μl / mouse). The same dose was repeated on days 5, 8, and 12. Body weight and behavioral observations of the mice were performed three times one week after CT26 cell implantation. On day 20, the mice were sacrificed, and the number of metastatic tumor nodules in each liver of the mice was counted.

[0219] As shown in Figure 13A, many tumor nodules were found in the PBS treatment group.In contrast, only a few tumor nodules were found in both groups treated by SC or ID route.Interestingly, ID administration to the base of the ear showed better efficacy than SC administration.These results show that ODN of the present invention can block the growth of metastatic cancer cells in lungs and can achieve systemic efficacy. As shown in Figure 13B, PBS treatment group also showed severe liver metastasis.In contrast, in both groups treated by SC or ID route, no tumor nodules were found, which indicates that the ODN of the present invention can prevent the growth of metastatic cancer cells in the liver and achieve systemic efficacy. Taking these factors into consideration, the ODN of the present invention can systemically block and reject the growth of metastatic tumors.

[0220] Example 12 Activation of antitumor immunity in human PBMCs [Coculture of human B-ALL cells and human PBMCs] Prepared human PBMCs (5 × 10 5 cells) were incubated with ODN (0.1 μM) in 200 μl of 10% FBS-RPMI complete medium for 3 days, along with the human B-ALL cell line RCH-ACV (5 × 10 4 Human PBMCs were co-cultured with RCH-ACV cells (cells), and the co-cultured cells were harvested. Elimination of RCH-ACV by human PBMCs was assessed by the reduction of RCH-ACV cells identified by staining with APC-conjugated anti-CD19 Ab and FITC-conjugated anti-CD138 Ab. Human PBMCs alone and RCH-ACV alone were used as staining controls. The presence of CD19 and CD138 double-positive cells (RCH-ACV) was analyzed by flow cytometry. In Figure 14B, the presence of RCH-ACV in unstimulated PBMCs was designated as 100%.

[0221] [Co-culture of human colon cancer cells and human PBMCs] Prepared human PBMCs (5 × 10 5 Human colon cancer cells COLO205 (ATCC, CCL-222) (5 × 10 cells) were incubated with ODN (0.1 μM) in 200 μl of 10% FBS-RPMI complete medium for 3 days. 4 COLO205 cells were co-cultured with human PBMCs, and the co-cultured cells were harvested. Elimination of COLO205 by human PBMCs was assessed by the reduction of COLO205 cells, as identified by staining with APC-conjugated anti-CD24 Ab and FITC-conjugated anti-CD45 Ab. Human PBMCs alone and COLO205 alone were used as staining controls. The presence of CD24-positive / CD45-negative cells (COLO205) was analyzed using a flow cytometer. In Figure 15B, the presence of COLO205 in unstimulated PBMCs was designated as 100%.

[0222] As shown in Figure 14A, RCH-ACV cells were CD19 and CD138 double positive, and no CD19 and CD138 double positive cells were observed in the prepared human PBMC alone. When human PBMC and RCH-ACV were co-cultured under unstimulated conditions, 8.6% of RCH-ACV cells were detected. In the presence of A601, A602, and A603, only 0.2% to 0.3% of RCH-ACV cells were detected in the cultured cells, indicating that human PBMCs eliminated almost all RCH-ACV cells in response to the ODN of the present invention. As shown in Figure 14B, the effectiveness of the elimination of hematopoietic cancer cells was further confirmed.

[0223] As shown in Figure 15A, COLO205 cells were CD24 positive and CD45 negative, and such cells were not found in the prepared human PBMCs alone. When human PBMCs and COLO205 were co-cultured under unstimulated conditions (culture without ODN), 19.2% of COLO205 cells were detected. In the presence of A601, A602, and A603, only 0.5% to 0.9% of COLO205 cells were detected in the cultured cells, indicating that human PBMCs eliminated almost all COLO205 cells in response to the ODNs of the present invention. The effectiveness of elimination was further confirmed as shown in Figure 15B.

[0224] Taken together, these findings suggest that human PBMCs activated with the ODNs of the present invention can eliminate both hematological malignancies and solid tumors.

[0225] Example 13 Efficacy of ODNs other than A601, A602 and A603 Human PBMCs and mouse splenocytes were stimulated with the ODN of the present invention (0.15 μM) for 24 hours, and cell proliferation was evaluated by WST-1 assay. Human PBMC was co-cultured with cancer cells (RCH-ACV or COLO205) and the ODN of the present invention (0.1 μM) for 3 days as in Example 12.Examine the elimination of cancer cells by human PBMC.In Figure 16B, the existence of cancer cells in unstimulated PBMC is set as 100%.

[0226] As shown previously in Figure 6, when examined in CAL-1 / NF-kB-GFP cells, ODNs A601, A602, A601G, A611A, A602G and A612A all showed the same level of activity in TLR9 activation.To evaluate whether ODNs show the same characteristics in primary human PBMCs and mouse cells, ODNs A601G, A611A, A602G and A612A were evaluated in several assay systems previously tested with A601 and A602.As shown in Figure 16A, all ODNs of the present invention tested induced cell proliferation at the same level in both human PBMCs and mouse splenocytes.Furthermore, as shown in Figure 16B, all ODNs of the present invention tested induced significant elimination of cancer cells in the presence of human PBMCs. These results indicate that human PBMCs activated with ODNs A601G, A611A, A602G and A612A, like A601 and A602, can eliminate both hematological malignancies and solid tumors.

[0227] In summary, all of the ODNs of the present invention that showed activity levels equivalent to A601 or A602 in CAL-1 / NF-kB-GFP cells suggest that they induce the elimination of human cancer cells in human PBMCs and anti-tumor immune responses in mice. [Industrial Applicability]

[0228] The present invention provides novel oligonucleotides and their derivative oligonucleotides. In addition, the present invention provides pharmaceutical compositions containing oligonucleotides selected from the above oligonucleotides. The present invention also provides methods for treating target diseases by administering oligonucleotides selected from the above oligonucleotides.

Claims

1. A single-stranded oligonucleotide comprising the nucleotide sequence motif 5'-tcgcaacgttt-n-cgacg-n-cg-nn-cg-3' (SEQ ID NO: 2) (n represents any base), the total number of bases being 24 or 25.

2. 5′-tcgcaacgtttgcgacgtcggtcga (SEQ ID NO: 54); 5′-tcgcaacgtttgcgacggcgctcga (SEQ ID NO: 55); 5′-tcgcaacgtttgcgacgtcgttcga (SEQ ID NO: 56); 5′-tcgcaacgtttgcgacggcgttcga (SEQ ID NO: 57); 5′-tcgcaacgtttgcgacgtcgttcg (SEQ ID NO: 58); 5′-tcgcaacgtttgcgacgtcgttcgg (SEQ ID NO: 59); 5′-tcgcaacgtttacgacgtcggtcga (SEQ ID NO: 60); 5′-tcgcaacgtttacgacggcgctcga (SEQ ID NO: 61); 5'-tcgcaacgtttacgacgtcgttcga (SEQ ID NO: 62); and 5'-tcgcaacgtttacgacggcgttcga (SEQ ID NO: 63) 2. The oligonucleotide of claim 1, comprising a nucleotide sequence motif selected from the group consisting of:

3. 3. The oligonucleotide according to claim 1, wherein the internucleotide bonds of the oligonucleotide are partially or completely chemically modified.

4. The oligonucleotide of claim 3, wherein the chemically modified internucleotide bond is phosphorothioated.

5. 5′-tCgcaacgtttgcgacgtcgttcgA-3′ (SEQ ID NO: 16); 5′-tCgcaaCgtttgcgacgtcgttcgA-3′ (SEQ ID NO: 17); 5′-tCgCaaCgtttgcgacgtcgttcgA-3′ (SEQ ID NO: 18); 5′-tCgCaacgtttgCgaCgtcgttcgA-3′ (SEQ ID NO: 19); 5′-tCgCaacgtttgCgaCgtcgttCgA-3′ (SEQ ID NO: 20); 5′-tCgCaaCgtttgcgacgtCgttCgA-3′ (SEQ ID NO: 21); 5′-tCgCaaCgtttgCgaCgtCgttCgA-3′ (SEQ ID NO: 22); 5′-tCgCaaCgtttgcgacgtCggtCgA-3′ (SEQ ID NO: 23); 5′-tCgCaaCgtttgcgacggCgctCgA-3′ (SEQ ID NO: 24); 5′-tCgCaaCgtttgcgacggCgttCgA-3′ (SEQ ID NO: 26); 5′-tCgCaaCgtttgcgacgcCgttCgA-3′ (SEQ ID NO: 27); 5′-tCgCaaCgtttgcgacggCgtaCgA-3′ (SEQ ID NO: 28); 5′-tCgCaaCgtttgcgacggCgtgCgA-3′ (SEQ ID NO: 29); 5′-tCgCaaCgtttacgacgtCggtCgA-3′ (SEQ ID NO: 30); 5′-tCgCaaCgtttacgacggCgctCgA-3′ (SEQ ID NO: 31); 5′-tCgCaaCgtttacgacgtCgttCgA-3′ (SEQ ID NO: 32); 5′-tCgCaaCgtttGcgacgtCggtCgA-3′ (SEQ ID NO: 33); 5′-tCgCaaCgtttAcgacgtCggtCgA-3′ (SEQ ID NO: 34); 5′-tCgCaaCgtttGcgacggCgctCgA-3′ (SEQ ID NO: 35); 5′-tCgCaaCgtttAcgacggCgctCgA-3′ (SEQ ID NO: 36); 5′-tCgCaaCgtttGcgacgtCgttCgA-3′ (SEQ ID NO: 37); 5′-tCgCaaCgtttAcgacgtCgttCgA-3′ (SEQ ID NO: 38); 5′-tCgCaaCgtttGcgacgtCggtCgG-3′ (SEQ ID NO: 39); 5′-tCgCaaCgtttAcgacgtCggtCgG-3′ (SEQ ID NO: 40); 5′-tCgCaaCgtttGcgacggCgctCgG-3′ (SEQ ID NO: 41); 5′-tCgCaaCgtttAcgacggCgctCgG-3′ (SEQ ID NO: 42); 5'-tCgCaaCgtttGcgacgtCgttCgG-3' (SEQ ID NO:43); and 5'-tCgCaaCgtttAcgacgtCgttCgG-3' (SEQ ID NO: 44) (In the sequences, capital letters represent nucleosides without a 3'-modified internucleotide bond, and lowercase letters represent nucleosides with a 3'-phosphorothioated internucleotide bond.) 5. The oligonucleotide of claim 4, comprising a partially phosphorothioated oligonucleotide stretch selected from the group consisting of:

6. 5′-tCgCaaCgtttgcgacggCgctCgA-3′ (SEQ ID NO: 24); 5′-tCgCaaCgtttGcgacgtCggtCgA-3′ (SEQ ID NO: 33); 5′-tCgCaaCgtttAcgacgtCggtCgA-3′ (SEQ ID NO: 34); 5'-tCgCaaCgtttGcgacggCgctCgA-3' (SEQ ID NO: 35); and 5'-tCgCaaCgtttAcgacggCgctCgA-3' (SEQ ID NO: 36) (In the sequences, capital letters represent nucleosides without a 3'-modified internucleotide bond, and lowercase letters represent nucleosides with a 3'-phosphorothioated internucleotide bond.) 6. The oligonucleotide of claim 5, comprising a partially phosphorothioated oligonucleotide stretch selected from the group consisting of:

7. The oligonucleotide according to any one of claims 1 to 6, which is composed of DNA.

8. The oligonucleotide of any one of claims 1 to 7, which is contained in an expression vector, or which is circular or linear.

9. A double-stranded oligonucleotide comprising the oligonucleotide according to any one of claims 1 to 8 and its complementary strand oligonucleotide.

10. The oligonucleotide according to any one of claims 1 to 9, bound to at least one active molecule.

11. The oligonucleotide of claim 10, wherein the active molecule is selected from the group consisting of (poly)peptides / antibodies and nucleic acids / oligonucleotides.

12. A pharmaceutical composition comprising a therapeutically effective amount of the oligonucleotide according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier.

13. A pharmaceutical composition comprising a therapeutically effective amount of the oligonucleotide of any one of claims 1 to 11 for the prevention or treatment of a target disease or disorder, the target disease or disorder being any one selected from the group consisting of neoplasms, infectious diseases, Th2 / Th17-related diseases, primary immunodeficiency diseases, and post-traumatic stress disorder (PTSD).

14. A pharmaceutical composition comprising a therapeutically effective amount of an oligonucleotide according to any one of claims 1 to 11 for modulating an immune response in a subject.

15. the target disease or disorder is (a) a neoplasm selected from the group consisting of a malignant neoplasm, an epithelial neoplasm or a hematopoietic neoplasm, a sarcoma, a mesothelioma, a benign tumor, a dysplasia, and a metaplasia; (b) infectious diseases caused by microorganisms, including viruses, bacteria, and fungi; (c) a Th2 / Th17-associated disease selected from the group consisting of asthma, allergies, multiple sclerosis, inflammatory bowel disease including ulcerative colitis and Crohn's disease, cutaneous lichen planus, and Alzheimer's disease; or (d) a primary immunodeficiency disease caused by IRAK4 deficiency, MyD88 deficiency, Unc93B deficiency, or a mutation in a TLR; That is, The pharmaceutical composition of claim 13.

16. The pharmaceutical composition according to any one of claims 12 to 15, further comprising at least one active ingredient.

17. The pharmaceutical composition according to any one of claims 12 to 15, which is administered in combination with at least one active ingredient.

18. Use of the oligonucleotide of any one of claims 1 to 11 in the manufacture of a medicament for the treatment or prevention of any one selected from the group consisting of neoplasms, infections, Th2 / Th17-associated diseases, primary immunodeficiency diseases, and post-traumatic stress disorder (PTSD).

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