RU2846775C1 - Novel tlr9 agonists - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed are: a single-chain oligonucleotide-agonist of Toll-like receptor 9 (TLR9), characterized in that the oligonucleotide contains a sequence motif selected from the group, a double-stranded oligonucleotide that can bind to TLR9, a conjugate for the treatment or prevention of a target disease or disorder selected from the group consisting of neoplasm, infection, Th2/Th17 mediated disease, primary immunodeficiency disease and post-traumatic stress disorder (PTSD), and therapeutic use thereof. Also provided is a pharmaceutical composition for preventing or treating a target disease or disorder, an agent for activating TLR9 in an individual and a method of treating or preventing a target disease or disorder in an individual.

EFFECT: invention can be used to activate or modulate immunity in an individual.

35 cl, 41 dwg, 7 tbl, 13 ex

Description

[FIELD OF TECHNOLOGY]

[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 a novel oligonucleotide or oligonucleotide derivative thereof that can bind to a nucleic acid receptor, including TLR9. Furthermore, the present invention relates to a pharmaceutical composition comprising such an oligonucleotide. Furthermore, the present invention relates to a method for the prevention or treatment of a target disease, including a malignant tumor or an infectious disease, using such an oligonucleotide.

[State of the art]

[0003]

TLR (toll-like receptor) is a family of receptors that recognize pathogen-associated molecular patterns (PAMPs) and are located on the surface of the cell membrane or endosomal membrane. TLR activation leads to the secretion of type I interferons and inflammatory cytokines. Ten types of TLRs are known in humans, from TLR1 to TLR10. Among them, TLR3, TLR7, TLR8, and TLR9 are located in endosomes and primarily detect nucleic acids. While TLR3 primarily recognizes double-stranded RNA and synthetic RNAs called poly(I:C), which are similar to viral RNA, TLR7 and TLR8 primarily recognize single-stranded RNA. TLR9, on the other hand, primarily recognizes single-stranded DNA. In humans, TLR9 is expressed in plasmacytoid dendritic cells (pDC), B cells, eosinophils, basophils, macrophages, and NK cells (Roda, J.M., et al., J Immunol, 2005. 175(3): p.1619-27; Liu, M., et al., Nat Immunol, 2019. 20(3): p.265-275; Hemmi, H., et al., Nature, 2000. 408(6813): p.740-5).

[0004]

TLR9 agonists activate pDCs and B cells, promoting their proliferation, production of inflammatory cytokines and antibodies, antigen presentation, and expression of costimulatory and MHC molecules. Stimulation with TLR9 agonists also increases chemokine receptor expression in pDCs and B cells, increasing resistance to apoptosis and cytokine production, including Th1-inducing chemokines such as macrophage inflammatory protein 1 (MIP1) and interferon-inducible protein 10 kDa (IP10). Memory B cells, in particular, differentiate into plasma cells, which secrete antibodies dependent solely on TLR9 activation. An example of a TLR9 agonist is CpG DNA (CpG). CpG DNA contains unmethylated cytosine and guanine, which was originally discovered as an important motif constituting pathogen-associated molecular patterns (PAMPs) in bacterial DNA (Krieg, A.M., Nat Rev Drug Discov, 2006. 5(6): p.471-84).

[0005]

CpG oligodeoxynucleotides (ODN) are reported to be divided into four classes based on structure and function. Class A ODN, also known as class D ODN, have a phosphorothioate backbone of several nucleotides at the 3' end and several nucleotides at the 5' end. Class A ODN contain palindromic sequences including CpG that can form a stem-loop structure. The ends of class A ODN contain poly-G sequences that can form parallel quadruplex structures called G tetrads (Puig M. et al., Nucleic Acids Res. 2006, 34(22): p.6488-95). Class A ODN strongly promote interferon-alpha (IFN-α) production from pDCs but only weakly activate B cells. Class B ODN, also known as class K ODN, have one or more CpG sequence(s) and a backbone in which most nucleotides are phosphorothioate. Class B ODN highly induce B-cell proliferation, promote IgM antibody secretion from B cells, and induce pDC differentiation and maturation. Class C ODN have a phosphorothioate backbone. Class C ODN have a palindromic sequence with a CpG at the 3' end, and the palindromic sequence can undergo intramolecular hybridization to form a hairpin structure. Class C ODN promote IFN-α production from pDC and IL-6 production from B cells. Thus, class C ODN have similar functions to both class A and class B ODN. Class P ODN have two palindromic sequences. Class P ODN can undergo intermolecular hybridization to form concatemers at the palindrome at the 5' end or intramolecular hybridization to form a hairpin structure at the GC-rich 3' end. Class P ODN highly induce type I IFN production (Scheiermann, J. et al., Vaccine, 2014. 32(48): p.6377-89).

[0006]

The mechanisms by which CpG-ODNs activate TLR9-expressing cells are described below. Initially, internalized CpG-ODNs bind to TLR9 in the endosome. Following CpG-ODN binding to TLR9, the adaptor protein MyD88, which binds to the cytosolic side of TLR9, is activated by phosphorylation. Activated MyD88 induces the transcription of cytokines, including type I interferons (IFNs), through the activation of transcription factors such as IRF3, 5, and 7. Activated MyD88 also signals through the nuclear factor-kappa B (NF-κB) cascade; the signal downstream of MyD88 leads to the ubiquitination of IB (inhibitor B or inhibitor of kappa beta) and induces the degradation of IB, which activates NF-κB. Upon activation, NF-κB binds to NF-κB promoters and activates the expression of target genes. This results in the induction of production of inflammatory cytokines such as interleukin-1 beta (IL-1β), TNF-alpha (TNF-α) and interleukin-6 (IL-6).

[0007]

Since CpG-ODNs have the above-mentioned biological activity of activating TLR9, the administration of CpG-ODNs is suitable for the treatment and/or prevention of several diseases or disorders in the following manner.

[0008]

<Antinumber activity (antitumor activity)>

CpG-ODNs can affect the tumor microenvironment (™E) and convert a cold tumor into a hot tumor ("Warming "Cold" Melanoma with TLR9 Agonists", Cancer Discov, 2018. 8(6): p.670). ™E is an environment constructed by tumor cells and non-tumor cells surrounding tumors, such as immune cells, fibroblasts, and vascular cells. ™E status significantly influences tumor progression. A cold tumor is a tumor that contains immunosuppressive cells such as tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs), which make up the immunosuppressive ™E, and only a few activated tumor-infiltrating lymphocytes (TILs). A cold tumor is often resistant to malignant tumor treatments, including immunotherapy. A hot tumor is a tumor that contains antitumor immune cells, including TILs and M1 macrophages, which constitute the immunologically activated ™E. A hot tumor usually responds to malignant tumor treatment. For example, it has been reported that intratumoral administration of CpG-ODN leads to an increase in T cells, pDCs, and NK cells, a decrease in Tregs, and a suppression of MDSCs (Shirota, H., et al., Vaccines (Basel), 2015. 3(2): p.390-407). Furthermore, TLR9 agonists have been described to retrain protumor M2-like macrophages to induce the polarization of antitumor M1-like macrophages (Mantovani, A., et al., J Exp Med, 2015. 212(4): p.435-45).

CpG-ODNs have also been reported to enhance the antitumor activity of macrophages aimed at engulfing malignant cells that express the "don't eat me" signal, such as CD47 (Liu, M., et al., Nat Immunol, 2019. 20(3): p.265-275).

[0009]

CpG-ODNs can also suppress the immunosuppressive nature of monocytic (CD11b+, Ly6G-, Ly6C ^high ) MDSCs, such as suppressive activity on T cell function (Shirota, Y., et al., J Immunol, 2012. 188(4): p.1592-9).

[0010]

<Prevention or therapy of Th2- or Th17-mediated diseases>

CpG-ODNs are suitable for the effective prevention or treatment of Th2- or Th17-mediated diseases. Conventional CpG-ODNs, through their immunomodulatory effects, have been shown to modulate the balance between the immune response of activating Th1 cells and Tregs, resulting in the suppression of Th2 and Th17 cells. It is believed that inducing differentiation into Th1 cells by CpG-ODNs leads to a decrease in Th2 cell levels, since Th1 cells and Th2 cells reciprocally inhibit each other's differentiation into Th2 or Th1 cells, respectively.

[0011]

Recently, numerous factors have been described that modulate the balance between Th17 cells and Tregs. Examples of such factors include downstream T-cell receptor signaling, costimulatory molecules, cytokines, metabolic cascades, and the intestinal microbiota (Lee, G.R., Int J Mol Sci, 2018. 19(3): p.730).

It has been reported that the TLR4 agonist LPS induces Th17 cell differentiation, and the TLR2 agonist peptide glycan moderately induces Th17 and Th1 cell differentiation. Meanwhile, CpG-ODNs have been reported to preferentially induce Th1 cell differentiation through IFN-α production (Shi, G., et al., J Immunol, 2013. 191(1): p.415-23).

[0012]

The feasibility of immune balance-supporting therapies has been demonstrated in vivo . For example, when CpG-ODNs were administered to ulcerative colitis model mice and patients, Th17 cell counts and IL-17 and IL-6 production were decreased, while Tregs and IL-10 production increased, resulting in improved symptoms (Schmitt, H et al., J Crohns Colitis, 2018, 12(Suppl1): p.S003). CpG supplementation was also reported to reduce EAE symptoms in mice induced with complete Freund's adjuvant, demonstrating the effect of CpG in reducing Th17-mediated pathogenesis (Tigno-Aranjuez, JT, et al., J Immunol, 2009, 183(9): p.5654-61).

[0013]

CpG-ODNs have also been suggested to be effective against asthma and atopic diseases, which are Th17-mediated diseases, based on the observation that CpG-ODNs induced the production of indoleamine 2,3-dioxygenase (IDO) from dendritic cells and the activation and proliferation of Tregs (Kline, J.N., et al., Drug News Perspective, 2008. 21(8): p.434-9).

[0014]

<Combination therapy>

Furthermore, it is also described that CpG-ODNs are suitable for combination therapy because CpG-ODNs exhibit immune modulating activity as described above. Previous reports have described combination with therapies including (i) vaccines; (ii) antibody drugs; (iii) conventional chemotherapy; (iv) molecular targeted drugs; (v) surgery; (vi) cytokines; (vii) adoptive immune cell therapies (also known as adoptive cell transfer (ACT)); and (viii) nucleic acid receptor agonists.

[0015]

Combination with "(i) vaccines"

CpG-ODNs can enhance the immunogenicity of vaccines. For example, CpG-ODNs can be administered together with a cancer vaccine, such as a melanoma antigen-based vaccine (Scheiermann, J. et al., D.M., Vaccine, 2014. 32(48): p.6377-89; Shirota, H., et al., Vaccines (Basel), 2015. 3(2): p.390-407), as well as with vaccines against viruses, such as cytomegalovirus, malaria virus, anthrax virus, and influenza virus (Scheiermann, J. et al., D.M., Vaccine, 2014. 32(48): p.6377-89). Heplisav-B®, a hepatitis B vaccine containing CpG-ODN and hepatitis B antigens, has been approved by the FDA.[0016]

Combination with "(ii) antibody drugs" (such as immune checkpoint inhibitors (CPIs) and cytotoxic antibodies)

CPIs are drugs that modify the immunosuppressive status 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 with Keytruda (pembrolizumab) was investigated in a clinical trial in advanced melanoma (Ribas, A., et al., Cancer Discov, 2018. 8(10): p.1250-1257). A clinical trial of the combined use of Yervoy (ipilimumab) was conducted for the treatment of advanced solid tumors (Reilley, M., et al., Journal of Clinical Oncology, 2019. 37(15_suppl): p.TPS2669-TPS2669). Interestingly, the combination of CpG-ODN and CPIs was suggested to be effective for patients who are resistant to standard treatment. For example, it has been reported that the combination of CpG-ODN and CPI exhibits antitumor activity even though patients are resistant to CPIs such as anti-PD-1 antibodies (Wang, S., et al., Proc Natl Acad Sci U S A, 2016. 113(46): p.E7240-E7249). Cytotoxic antibodies are drugs that 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 described (Hiramatsu, K., et al., Cancer Sci, 2015. 106(10): p.1474-8). For example, a combination of CpG-ODN with Rituxan (rituximab) was investigated in a clinical trial in non-Hodgkin's lymphoma (Friedberg, J.W., et al., Blood, 2005. 105(2): p.489-95). A combination of a CpG oligonucleotide, poly(I:C), and a cytotoxic antibody was also shown to have antitumor activity even when patients were resistant to a cytotoxic antibody such as Herceptin (trastuzumab) (Charlebois, R., et al., Cancer Res, 2017. 77(2): p.312-319).

[0017]

Combination with "(iii) conventional chemotherapy"

Conventional chemotherapeutic drugs consist of chemicals that inhibit the proliferation of rapidly growing tumor cells and kill them. For example, a combination of a platinum-based drug, a taxane-based drug, and a CpG-ODN was studied in a clinical trial (Krieg, A.M., J Clin Invest, 2007. 117(5): 1184-94).

[0018]

Combination with "(iv) molecular targeted drugs"

Conventional molecular targeted therapies typically consist of small molecules that bind to specific target molecules and regulate their functions. Examples of target molecules include those that induce carcinogenesis, are associated with driver mutations, or are involved in tumor cell homeostasis. For example, synergy between CpG and bortezomib in multiple myeloma has been described in a preclinical setting and concluded to be feasible for testing at this clinical stage (Ray, A., et al., Leukemia, 2014. 28(8): 1716-24).

[0019]

Combination with (v) surgical treatment (including radiotherapy, cryoablation or radiofrequency ablation)

Administration of CpG-ODN after surgical resection has been reported to improve survival (Weigel, B.J., et al., Clin Cancer Res, 2003. 9(8): p.3105-14). Radiation therapy is a treatment method that damages the DNA of proliferating tumor cells and destroys them using radiation. Cryoablation is a treatment method that freezes and destroys tumor cells. Radiofrequency ablation is a treatment method that coagulates tumor cells using heat generated by radio frequencies and destroys tumor cells. CpG-ODNs are considered effective in inducing immunity against tumor antigens released from dead cells killed by this treatment (Jahrsdörfer, B. et al., G.J., Update Cancer Ther, 2008. 3(1): p.27-32).

[0020]

Combination with "(vi) cytokines"

Examples of cytokines include IFN-α and IL-18, which activate NK cells or dendritic cells. A combination of IL-18 and CpG-ODN was described to induce apoptosis of malignant B cells, and these B cells secreted granzyme, which then killed neighboring malignant B cells (Jahrsdörfer, B. et al., Update Cancer Ther, 2008. 3(1): pp. 27-32).

[0021]

Combination with "(vii) adoptive immune cell therapy"

Adoptive immune cell therapy is a type of therapy that involves the administration of immune cells modified ex vivo ; examples of such therapy include CAR-T therapy using genetically modified T cells transduced with TCRs against specific cancer antigens and immune cells such as TILs or dendritic cells, either from patients or donors, stimulated ex vivo to acquire antitumor effects (Xu, L., et al., Clin Dev Immunol, 2010. 2010: p.410893).

[0022]

Combination with "(viii) nucleic acid receptor agonists"

The combination with a TLR7/8 agonist has been described to show synergistic effects in clinical trials (Shirota, H., et al., Vaccines (Basel), 2015. 3(2): p.390-407). The synergistic effect between a TLR9 agonist such as CpG and a stimulator of interferon genes (STING) agonist has also been described to enhance Th1-biased immune responses such as the production of antigen-specific IgG and IFN-γ in PBMCs, as well as cytotoxic CD8(+) T cell responses (Temizoz B., et al, Eur J Immunol, 2015, 45(4): p.1159-69).

[0023]

<Mechanisms underlying combination therapy involving CpG administration>

Some of the treatments and drugs described above have been shown to induce immunogenic cell death (ICD) and have been investigated in combination with CpG-ODNs. ICD is a type of cell death where scaffolds or dying cells killed by ICD induce a strong immune response through the secretion of DAMPs; the molecules that constitute DAMPs have been described to be, for example, calreticulin, high-mobility group box (HMGB), heat shock protein, or ATP. The presentation of increased amounts of such DAMPs has been observed compared to normal cell death. (Bedognetti, D., et al., J Immunother Cancer, 2019. 7(1): p.131). Some chemotherapeutic drugs and some molecular targeted drugs are known as ICD inducers; Such ICD inducers include doxorubicin, mitoxantrone, oxaliplatin, and bortezomib. Radiation therapy and photodynamic therapy (PDT) are also known as ICD inducers. (Galluzzi, L., et al., Nat Rev Immunol, 2017. 17(2): 97-111)

[0024]

Several treatments or drugs that suppress or eliminate Tregs have been studied in combination with the CpG-ODNs described above. For example, CpG-ODNs synergistically enhance the immune response following Tregs depletion by an anti-CD25 antibody (Jarry, U., et al., J Neuroimmunol, 2014. 267(1-2): 35-42).

[0025]

Therefore, the CpG-ODN can be administered alone or in combination with at least one active ingredient for the following pharmaceutical purposes: (i) immunostimulatory effects for the prevention and treatment of neoplasms, including malignant tumors (e.g., metastatic solid malignant tumor, melanoma, cutaneous T-cell lymphoma, chronic lymphocytic leukemia, etc.) and infectious diseases, (ii) immunomodulatory effects for the prevention or treatment of immune-mediated diseases, such as Th2 or Th17-mediated diseases, including some types of autoimmune diseases and allergic diseases, and (iii) modulating the responsiveness of TLR7/9-expressing tumor cells to anticancer drugs and immune cells.

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[0026]

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[0027]

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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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[Essence of the invention]

[Technical problems]

[0028]

The present invention relates to novel TLR agonists and their therapeutic applications.

[Problem solution]

[0029]

The inventors discovered for the first time that

(1) the oligonucleotides of the present invention can be used to activate or modulate immunity in an individual; and

(2) The oligonucleotides of the present invention are particularly useful for treating an individual suffering from diseases including a tumor, a microorganism infection, a primary immunodeficiency disease and a Th2/Th17-mediated disease, or for preventing such 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'.

(2) The oligonucleotide according to (1), wherein the oligonucleotide comprises a nucleotide sequence motif 5'-tcgcaacgttt-n-cgacg-n-cg-nn-cg-3' (SEQ ID NO:2), where n is any base.

(3) The oligonucleotide according to (1) or (2), wherein the total number of nucleotides is from 20 to 25, preferably from 21 to 24, more preferably from 22 to 23.

(4) An oligonucleotide according to any one of (1) to (3), wherein the oligonucleotide comprises a sequence motif selected from the group consisting of the following:

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).

(5) An oligonucleotide according to any of (1) to (4), wherein the internucleotide linkage(s) in the oligonucleotide is partially or fully chemically modified.

(6) An oligonucleotide according to (5), wherein the chemically modified internucleotide linkage is phosphorothioate.

(7) The oligonucleotide according to (6), wherein the oligonucleotide comprises a partially phosphorothioate oligonucleotide region selected from the group consisting of

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);

where the capital letter indicates a nucleoside without a modified internucleotide linkage on the 3' side, and the small letter indicates a nucleoside with a phosphorothioate internucleotide linkage on the 3' side.

(8) The oligonucleotide according to (6), wherein the oligonucleotide comprises a partially phosphorothioate oligonucleotide region selected from the group consisting of,

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),

where the capital letters indicate the nucleoside without a modified internucleotide linkage on the 3' side, and the lowercase letter indicates the nucleoside with a phosphorothioate internucleotide linkage on the 3' side.

(9) An oligonucleotide according to any one of (1) to (8), wherein the oligonucleotide is composed of DNA.

(10) An oligonucleotide according to any one of (1) to (9), wherein the oligonucleotide further has one or more nucleotide(s) deleted, substituted, or added.

(11) An oligonucleotide according to any one of (1) to (10), wherein the oligonucleotide is contained in an expression vector.

(12) An oligonucleotide according to any one of (1) to (10), wherein the oligonucleotide is circular.

(13) An oligonucleotide according to any one of (1) to (10), wherein the oligonucleotide is linear.

(14) An oligonucleotide according to (13), where the 3'-end of the linear oligonucleotide does not have a phosphoric acid.

(15) A double-stranded oligonucleotide comprising an oligonucleotide according to any one of (1) to (10) and an oligonucleotide complementary thereto.

(16) An oligonucleotide according to any of (1) to (15), which is conjugated to active molecules.

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

(18) An oligonucleotide according to (16) or (17), wherein conjugation is achieved via a linker.

(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, cis, trans-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, nitrodiol, triethylene glycol, hexaethylene glycol, d-spacer, PEG-spacer and alkyl linker.

[0031]

(20) A pharmaceutical composition comprising a therapeutically effective amount of an oligonucleotide according to any of (1) to (19) and a pharmaceutically acceptable carrier.

[0032]

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

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

(23) The pharmaceutical composition according to (21), wherein the target disease or disorder is an infectious disease caused by microorganisms including viruses, bacteria or fungi.

(24) The pharmaceutical composition according to (21), wherein the target disease or disorder is a Th2/Th17-mediated 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.

(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 TLR mutations.

(26) The pharmaceutical composition according to (20) or (21), wherein the composition further comprises at least one active ingredient.

(27) A pharmaceutical composition according to (20) or (21), wherein the composition is administered together with at least one active ingredient.

(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 agents; antifungal agents; antiviral agents; antiparasitic drugs; antibody drugs for neutralizing a toxin; agonists of other TLRs and combinations thereof.

(29) The pharmaceutical composition according to (20) or (21), wherein the composition is administered to an individual by a dosing method selected from the group consisting of enteral administration, parenteral administration, topical administration and inhalation.

(30) The pharmaceutical composition according to (29), wherein the individual is a human.

(31) The pharmaceutical composition according to (30), wherein the composition is administered to an individual at a dose of 0.3-60 mg/day, preferably 1-30 mg/day, more preferably 2-8 mg/day.

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

[0033]

(A1) Use of the oligonucleotides according to any of (1) to (19) for the manufacture of a medicament for the treatment or prevention of a target disease or disorder selected from the group consisting of a neoplasm, an infection, a Th2/Th17-mediated disease, a primary immunodeficiency disease, and post-traumatic stress disorder (PTSD).

(A2) Use of the oligonucleotides according to any of (1) to (19) for the manufacture of a medicament for modulating an immune response in an individual.

(A3) The use according to (A1), wherein the target disease or disorder is a neoplasm selected from the group consisting of a malignant neoplasm, an epithelial neoplasm or a tumor of a hematopoietic organ, sarcoma, mesothelioma, a benign tumor, dysplasia and metaplasia.

(A4) Use according to (A1), where the target disease or disorder is an infectious disease caused by microorganisms, including viruses, bacteria or fungi.

(A5) The use according to (A1), wherein the target disease or disorder is a Th2/Th17-mediated 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.

(A6) Use according to (A1), where the target disease or disorder is a primary immunodeficiency disease caused by IRAK4 deficiency, MyD88 deficiency, Unc93B deficiency, or TLR mutations.

(A7) Use according to (A1) or (A2), wherein the medicinal product additionally contains at least one active ingredient.

(A8) Use according to (A1) or (A2), wherein the medicinal product is administered together with at least one active ingredient.

(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 agents; antifungal agents; antiviral agents; antiparasitic drugs; antibody drugs for neutralizing a toxin; agonists of other TLRs and combinations thereof.

(A10) Use according to (A1) or (A2), wherein the medicinal product is administered to the individual by a dosing route selected from the group consisting of enteral administration, parenteral administration, topical administration and inhalation.

(A11) Use according to (A10), where the individual is a human being.

(A12) Use according to (A11), wherein the medicinal product is administered to the individual at a dose of 0.3-60 mg/day, preferably 1-30 mg/day, more preferably 2-8 mg/day.

(A13) Use according to (A1) or (A2), where the medicinal product is administered before or after adoptive immune cell therapy or surgical treatment, including radiation therapy, cryoablation, radiofrequency ablation and photodynamic therapy (PDT).

[0034]

(B1) A method for treating or preventing a target disease or disorder in an individual, comprising administering to the individual an oligonucleotide according to any one of (1) to (19), wherein the target disease or disorder is a disease or disorder selected from the group consisting of a neoplasm, an infection, a Th2/Th17-mediated disease, a primary immunodeficiency disease, and post-traumatic stress disorder (PTSD); preferably, wherein the target disease or disorder is treated or prevented by activating NF-B by the oligonucleotide in the individual.

(B2) The method according to (B1), wherein the target disease or disorder is a neoplasm selected from the group consisting of a malignant neoplasm, an epithelial neoplasm or a tumor of a hematopoietic organ, a sarcoma, a mesothelioma, a benign tumor, a dysplasia, and a metaplasia.

(B3) The method according to (B1), wherein the target disease or disorder is an infectious disease caused by microorganisms, including viruses, bacteria, or fungi.

(B4) The method according to (B1), wherein the target disease or disorder is a Th2/Th17-mediated 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.

(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 TLR mutations.

(B6) The method according to (B1), wherein the oligonucleotide is administered together with at least one active ingredient.

(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 agents; antifungal agents; antiviral agents; antiparasitic drugs; antibody drugs for neutralizing a toxin; agonists of other TLRs and combinations thereof.

(B8) The method according to (B1), wherein the oligonucleotide is administered to the individual by a dosing method selected from the group consisting of enteral administration, parenteral administration, topical administration and inhalation.

(B9) The method according to (B8), where the individual is a human being.

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

(B11) The method according to (B1), further comprising the step of adoptive therapy with immune cells or surgical treatment, including radiation therapy, cryoablation, radiofrequency ablation and photodynamic therapy (PDT).

[0035]

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

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

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

[0036]

(D1) An oligonucleotide according to any of (1) to (19) for use in the prevention or treatment of a target disease or disorder, wherein the target disease or disorder is a disease or disorder selected from the group consisting of a neoplasm, an infectious disease, a Th2/Th17-mediated disease, a primary immunodeficiency disease, and post-traumatic stress disorder (PTSD).

(D2) An oligonucleotide for use according to (D1), wherein the target disease or disorder is a neoplasm selected from the group consisting of a malignant neoplasm, an epithelial neoplasm or a tumor of a hematopoietic organ, a sarcoma, a mesothelioma, a benign tumor, a dysplasia and a metaplasia.

(D3) An oligonucleotide for use according to (D1), wherein the target disease or disorder is an infectious disease caused by microorganisms, including viruses, bacteria or fungi.

(D4) An oligonucleotide for use according to (D1), wherein the target disease or disorder is a Th2/Th17-mediated 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.

(D5) An 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 TLR mutations.

(D6) An oligonucleotide for use according to (D1), wherein the oligonucleotide is administered together with at least one active ingredient.

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

(D8) An oligonucleotide for use according to (D1), wherein the oligonucleotide is administered to an individual by a dosing route selected from the group consisting of enteral administration, parenteral administration, topical administration and inhalation.

(D9) An oligonucleotide for use according to (D8), wherein the individual is a human.

(D10) An oligonucleotide for use according to (D9), wherein the oligonucleotide is administered to an individual at a dose of 0.3-60 mg/day, preferably 1-30 mg/day, more preferably 2-8 mg/day.

(D11) An oligonucleotide for use according to (D1), wherein the oligonucleotide is administered before or after adoptive immune cell therapy or surgical treatment, including radiation therapy, cryoablation, radiofrequency ablation and photodynamic therapy (PDT).

[0037]

(E1) Use of an oligonucleotide according to any of (1) to (19) for the treatment or prevention of a target disease or disorder selected from the group consisting of a neoplasm, an infectious disease, a Th2/Th17-mediated disease, a primary immunodeficiency disease, and post-traumatic stress disorder (PTSD).

[0038]

(F1) An agent for modulating an immune response in vivo or in vitro , comprising an effective amount of an oligonucleotide according to any one of (1) to (19).

[Advantageous effects of the invention]

[0039]

The present invention can provide novel CpG oligonucleotides and therapeutic uses of the oligonucleotides.

[Brief description of the drawings]

[0040]

[FIG. 1] NF-κB activation and proinflammatory cytokine production by TLR agonists in human plasmacytoid dendritic cells (pDC) cell line.

The CAL-1/NF-κB-GFP cell line was created to monitor NF-κB transcription factor activity in cell-based assays. A vector encoding a GFP reporter gene under the control of a consensus transcriptional element for the NF-κB response was transfected into the human pDC CAL-1 cell line.

A. CAL-1/NF-B-GFP cells were stimulated with oligodeoxynucleotides (ODN) of the present invention or a positive control for 6 hours at 1 μM (micromol/L). GFP expression induced by TLR9 agonists is shown in dot plots.

B. The graph shows the ratio of CAL-1/NF-B-GFP cells activated through TLR9 by ODN. CAL-1/NF-B-GFP cells were stimulated with ODN of the present invention or positive controls for 6 hours at a dose of 0.3 μM. The proportion of GFP-positive cells in the sample with CpG2395 was set to 100%. The activity of each ODN was calculated by comparing the ratio of GFP-positive cells to cells with CpG2395.

C. CAL-1/NF-B-GFP cells were stimulated with the ODN of the present invention for 6 hours at 1.0 μM. The activity of A003#delA was slightly weaker than that of A003, but the activity was still significantly higher than that of one of the authentic TLR9 agonists, CpG2395 (see FIG. 1A). The TLR9 activation activity of A003#endG and A003 was similar, indicating that the 3'-nucleoside is not essential for the activity.

D. CAL-1/NF-κB-GFP cells were stimulated with 1 μM ODN for 6 hours, and the culture supernatants were collected. Cytokine production was assessed by ELISA.

E, F. CAL-1/NF-B-GFP cells were stimulated with 1.0 μM ODN for 6 h. GFP expression was examined (FIG. 1E), and cytokine production in culture supernatants was assessed by ELISA (FIG. 1F). The activities of A003 and A013 were virtually identical when compared, indicating that the base change in the oligonucleotide does not affect the activity.

G. CAL-1/NF-B-GFP cells were stimulated with 1.0 μM ODN for 6 h, and GFP expression was examined. Each set of A001/A011, A002/A012, A003/A013, or A004/A014 showed similar activity, indicating that changing the base in each ODN set does not affect activity.

[0041]

[FIG. 2] Activation of human TLR7 and TLR8

A. HEK blue™ TLR7 cells were stimulated with TLR agonists for 24 hours. As shown, the TLR7 agonists gardiquimod (GQ) and CL264 activated TLR7 and provided positive signals. In contrast, the authentic TLR9 agonist CpG2395 and the ODN of the present invention, A001-A004, at 0.3 μM failed to activate the TLR7 signaling cascade.

B. HEK blue™ TLR8 cells were stimulated with ODN for 24 hours. As shown in FIG. , the TLR8 agonists TL8-506 and CL075 activated TLR8 and produced positive signals. In contrast, CpG2395 and A001-A004 (0.3 μM) failed to activate the TLR8 signaling cascade.

[0042]

[FIG. 3] NF-B activation and production of proinflammatory cytokines

A. CAL-1/NF-B-GFP cells were stimulated with the ODN of the present invention for 6 hours at 0.3 μM. CaaCg is important for the activation of human TLR9.

B. CAL-1/NF-B-GFP cells were stimulated with the ODN of the present invention for 6 hours at 0.3 μM or 0.1 μM. The proportions of GFP-positive cells are shown (shown in %).

C. CAL-1/NF-B-GFP cells were stimulated with the ODN of the present invention for 6 hours at 0.3 μM or 0.1 μM. A303, A603, and A703 demonstrated higher activity than the other ODN tested, indicating that the presence of CaaCg is more important than the amount of Cg.

D. CAL-1/NF-κB-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 cells clearly demonstrated lower levels of cytokine production induction activity, indicating that CaaCg is essential for optimal TLR9 stimulation.

E. CAL-1/NF-B-GFP cells were stimulated with the ODN of the present invention for 6 hours at 0.3 μM. The proportion of GFP-positive cells for CpG2395 is set as 100% in this figure. The activity of each ODN was calculated from the proportion of GFP-positive cells compared to CpG2395. DV093 and DV094 demonstrated lower activity against TLR9 than A003, indicating that the specific caacg motif localized in A003, but not the randomly positioned caacg motif, is important for TLR9 activity.

F. CAL-1/NF-B-GFP cells were stimulated with ODN at 0.3 μM for 6 hours. The proportion of GFP-positive cells for CpG2395 was set to 100%. The activity of each ODN was calculated from the proportion of GFP-positive cells compared to CpG2395. It should be noted that structural modification of the caacg region to the CaaCg region in DV093 and DV094 by altering internucleotide linkages does not enhance TLR9 activity.

[0043]

[FIG. 4] Optional nucleotides in the 3' region of the ODN of the present invention

A, B. CAL-1/NF-κB-GFP cells were stimulated with 0.3 μM ODN for 6 h. The proportion of GFP-positive cells (%) is shown in (B). The authentic TLR9 agonist CpG2006 showed significantly weaker activity compared to A601, A602, A603, A604, A605, A606, and A607 (B).

[0044]

[FIG. 5] Activation of human TLR7 and TLR8

A. HEK blue™ TLR7 cells were stimulated with ODN or a positive control for 24 hours. The TLR7 agonist CL264 activated TLR7 and generated a positive signal. In contrast, CpG2395, A601, A602, and A603 (0.3 μM) failed to activate the TLR7 signaling cascade.

B. HEK blue™ TLR8 cells were stimulated with ODN or a positive control for 24 hours. The TLR8 agonist CL075 activated TLR8 and produced a positive signal. In contrast, CpG2395, A601, A602, and A603 (0.3 μM) failed to activate the TLR8 signaling cascade.

[0045]

[FIG. 6] Negligible effect on TLR9 activation activity through changes in internucleotide linkages and changes in the bases in the middle of ODN.

A. CAL-1/NF-B-GFP cells were stimulated with A601, A601G, A602, or A602G (0.3 μM) for 3 h.

B. CAL-1/NF-B-GFP cells were stimulated with A601G, A611A, A602G, or A612A (0.3 μM) for 3 h.

C. CAL-1/NF-B-GFP cells were stimulated with 0.3 μM ODN for 3 h. The percentage of GFP-positive cells (%) is shown.

[0046]

[FIG. 7] Activation of human TLR7 and TLR8

A. HEK blue™ TLR7 cells were stimulated with TLR agonists for 24 hours. The TLR7 agonist CL264 activated TLR7 and generated a positive signal. In contrast, CpG2395, A601G, A611A, A602G, and A612A (0.3 μM) failed to activate the TLR7 signaling cascade.

B. HEK blue™ TLR8 cells were stimulated with TLR agonists for 24 hours. The TLR8 agonist CL075 activated TLR8 and generated a positive signal. In contrast, CpG2395, A601G, A611A, A602G, and A612A (0.3 μM) failed to activate the TLR8 signaling cascade.

[0047]

[FIG. 8] Cytokine production induction activity in human B cells or human PBMCs

A. HAL-01 cells were stimulated with the ODN of the present invention for 24 hours at 1 μM. The cells were stained with an antibody (Ab) against CD40 and an Ab against CD86. The induction of CD40 and CD86 expression was assessed using a flow cytometer.

B. Human PBMCs were stimulated with ODN of the present invention (0.1 μM) for 24 hours and cell proliferation was assessed by WST-1 assay.

Human PBMC were stimulated with the ODN of the present invention (0.3 μM) for 24 hours, and the culture supernatants were separated. Cytokine production was assessed by ELISA.

[0048]

[FIG. 9] Agonist activity against mouse cells

A. Mouse splenocytes were stimulated with the ODN of the present invention for 24 hours, and supernatants were obtained. Cytokine production was assessed by ELISA.

B, C. Mouse splenocytes were stimulated with ODN of the present invention for 48 hours. Cell proliferation was assessed using the WST-1 assay. A601, A602, and A603 demonstrated higher activity than the authentic TLR9 agonists CpG2395 and CpG2006.

[0049]

[FIG. 10] Antitumor activity in vivo

A. CT26 cells were inoculated into the right flank, and mice were left untreated for two weeks. Tumor volumes were measured, and mice were divided into three groups based on tumor volumes. Administration of the ODN according to the invention or PBS to the peritumoral areas was performed on the day of group assignment (day 0) and repeated on day 2. The ODN according to the invention was administered a total of two times during the study. Tumor volumes in each group were measured every two days.

B. Tumor volume of each mouse each day.

C. Average body weight of mice in each group during the trial. No significant decrease in body weight was observed in any of the groups.

[0050]

[FIG. 11] Induction of antitumor immunity memory

A. CT26 cells were inoculated into the right flank, and mice were left untreated for two weeks. Tumor volumes were measured, and mice were divided into three groups based on tumor volumes. Administration of the ODN according to the invention or PBS to the peritumoral areas was performed on the day of group assignment (day 0) and repeated on day 2. The ODN according to the invention was administered a total of two times during the study. Tumor volumes in each group were measured every two days.

B. CT26 cells were reinoculated into the left flank of each group of mice on day 14, after which the mice were left untreated. Tumor volumes on the left flank were measured for up to 14 days after reinoculation with CT26. It should be noted that mice treated with the ODN of the present invention rejected the recurrent tumor without any additional treatment within 2 weeks, indicating that the ODN of the present invention induced antitumor immunity memory.

[0051]

[FIG. 12] In vivo efficacy in a lung metastasis model

To induce CT26 cell metastasis in the lungs, the cell suspension was injected intravenously into BALB/c mice (5× ¹⁰⁵ cells/mouse) via the tail vein (day 0). On day 1, administration of the ODN according to the present invention was started (subcutaneous injection into the back skin, 40 μg/50 μl/mouse). ODN at the same dose was re-administered on day 5. On day 18, the mice were sacrificed. Lung weight was measured, and metastatic tumor nodules in each mouse lung were counted.

[0052]

[FIG. 13] Systemic effect against tumor metastasis

A. To induce lung metastasis of CT26 cells, cell suspensions were injected intravenously into BALB/c mice (5× ¹⁰⁵ cells/mouse) via the tail veins (day 0). On the next day after tumor inoculation (day 1), A602 administration was started. Two administration routes were tested. One was a subcutaneous (s.c.) injection into the back skin, and the other was an intradermal (i.d.) injection into the base of the ear (25 μg/mouse). Administration at the same dose was performed on days 3 and 5. On day 16, mice were sacrificed, and metastatic tumor nodules in each lung were counted (graph on the right). Photographs: removed lungs of tested mice on day 16.

B. To induce liver metastasis of CT26 cells, cell suspensions were injected into the spleen (1× ¹⁰⁵ cells/mouse, day 0). On day 2, mice were divided into three groups based on body weight, and A602 administration began. This study tested two administration routes: subcutaneous injection into the skin of the back and intradermal injection into the base of the ear (12.5 μg/mouse). Administration at the same dose was also performed on days 5, 8, and 12. On day 20, mice were sacrificed, and metastatic tumor nodules in the liver from each mouse were counted.

[0053]

[FIG. 14] Killing of B-ALL cells by activated PBMCs

A. Human PBMCs were cocultured 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 analyzed by flow cytometry after staining with antibodies against CD19 and CD138.

B. RCH-ACV population proportions are shown compared to the unstimulated sample, which was set to 100%.

[0054]

[FIG. 15] Killing of colon carcinoma cells by activated PBMCs

A. Human PBMC (5×10 ⁵ ) were cocultured with human colon carcinoma cells, COLO205, together with the ODN of the present invention (0.1 μM) for 3 days, and all cells were analyzed by staining with antibodies against CD45 and CD24.

B. The proportions of COLO205 populations in samples are shown compared to the unstimulated sample, which was set to 100%.

[0055]

[FIG. 16] Effect on immune cells

A. Human PBMC and mouse splenocytes were stimulated with the ODN of the present invention (0.15 μM) for 24 hours and cell proliferation was assessed by the WST-1 assay.

B. Human PBMCs were cocultured with malignant cells (RCH-ACV or COLO205) together with the ODN of the present invention (0.1 μM) for 3 days. The killing of malignant cells by human PBMCs was assessed. The proportions of malignant cells in the samples are presented compared to the unstimulated sample, the values of which were set to 100%.

[Preferred Embodiment]

[0056]

Unless otherwise indicated, all terms in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms in the singular form include plurals of the referenced entities unless the context clearly indicates otherwise. Similarly, the word "or" is understood to include "and" unless the context clearly indicates otherwise. The term "few" means a number from 2 to 3 in this specification. The term "several" means a number from 2 to 6 in this specification. In case of conflict, the present specification, including explanations of terms, controls. Furthermore, materials, methods, and examples are illustrative only and are not intended to be limiting. "Treat," "treating," or "treatment" have the same meaning regardless of grammar. Similarly, "prevent," "preventing," or "prevention" have the same meaning regardless of grammar.

[0057]

"Nucleotide": A nucleotide is a component of a nucleic acid molecule, such as DNA, RNA, and DNA-RNA chimeric molecules. A nucleotide can consist of a base, phosphoric acid, and sugar. A nucleotide can also be a phosphate ester of a nucleoside.

Nucleoside: A nucleoside can consist of a base and a sugar molecule and is a component of a nucleotide. Nucleosides can include deoxyadenosine, deoxyguanosine, deoxythymidine, 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 circular.

"Base": A base is one of the components of a nucleotide and a nucleoside. Naturally occurring bases include two groups: purine bases, such as guanine and adenine, and pyrimidine bases, such as thymine, cytosine, and uracil.

Sugar: Sugar is one of the components of nucleotides and nucleosides. Essentially, the sugar found in DNA is deoxyribose, and the sugar found in RNA is ribose.

"Internucleotide linkage": An internucleotide linkage refers to a link between two adjacent nucleotides of a nucleic acid molecule.

[0058]

A portion of a nucleotide, nucleoside, base, or sugar may be replaced or modified by another molecule, called an analog. For example, a nucleotide may additionally include a non-natural artificial nucleotide, such as PNA; a base may further include a non-natural base, such as hypoxanthine (i.e., inosine as a nucleoside); and a sugar may include a non-natural element, such as a 2'-4' bridge in a locked nucleic acid.

[0059]

In this application, "motif (sequence)" is indicated only in terms of "base", while "region (sequence)" is indicated in terms of "base", "sugar", and "internucleotide linkage".

[0060]

The oligonucleotide of the present invention can be composed of deoxyribonucleic acids with a deoxyribose backbone, ribonucleic acids with a ribose backbone, or a mixture thereof; the sugar backbones can also be replaced by synthetic molecules such as a locked nucleic acid (LNA), a bridged nucleic acid (BNA), a morpholino, or a peptide nucleic acid (PNA).

The oligonucleotide of the present invention may contain chemically modified nucleosides and/or modified internucleotide linkages to enhance one or more properties, such as nuclease resistance or pharmacokinetics.

[0061]

Chemically modified nucleosides may contain, but are not limited to, modified bases as follows or modified bases of a related structure:

8-halogen (bromine, chlorine, fluorine, iodine)-, 8-amino-, 8-thiol-, 8-thioalkyl-, 8-hydroxyl-, 8-aza-, 8-oxo- and other 8-substituted purines;

5-halogen (bromine, chlorine, fluorine, iodine)-, 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-methyladenine; N4-ethylcytosine, N4-alkylcytosine and other N4-substituted cytosine;

2-mercaptocytosine, isocytosine, pseudo-isocytosine, 4-thiouracil, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 2-aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine, 6-thioguanine, N2-methylguanine, N2-dimethylguanine and other N2-substituted guanines.

Modified nucleotide bases can also be bases replaced by other heterocycles, such as 7-deazaadenine, 8-aza-7-deazaadenine, 7-deazaguanine, 2-aminopyridine, and 2-pyridone.

Modified nucleotide bases may also contain additional fused rings, examples of such nucleotide bases are N6-ethenoadenosine, N4-ethenocytidine, N2-ethenoguanosine and other related derivatives.

[0062]

Internucleotide linkages are covalent backbone bonds between nucleotides in oligonucleotides and polynucleotides. Natural internucleotide linkages typically have a phosphodiester structure, but various modifications have been described that affect the chemical or physical properties of the molecules. Chemical modification in modified internucleotide linkages that can be used in the oligonucleotide of the present invention includes converting the natural phosphodiester structure into the following linkage structures: phosphorothioate, phosphorodithioate, methylphosphonate, methylphosphorothioate, ethyl phosphate, phosphonoacetate, alpha-hydroxybenzyl phosphate, isopropylphenoxyacetate, boranophosphate, phosphonocarboxylate, alpha-hydroxybenzylphosphonate, phosphate-(C1-C21)-O-alkyl ester, phosphate-[(C6-C12)aryl-(C1-C21)-O-alkyl] ester, phosphotriester, phosphotriesteramide, ether, acetal, thioether, thioacetal, phosphoramidate, siloxane, carbonate, carboxymethyl ether, acetamidate, carbamate, urea, thiourea, sulfonamide or sulfonylurea, carbonate, carboxymethyl, amide, ethylene oxide linker, sulfonate, thioformacetal, formacetal, oxime, methyleneimino, methyleneaminocarbonyl, methylenemethylimino (MMI), methylenehydrazo, methylene dimethylhydrazo (MDH) and methyleneoxymethylimino.

[0063]

The oligonucleotide of the present invention may be, but is not limited to, a single-stranded deoxyribonucleic acid, a single-stranded ribonucleic acid, or a chimeric molecule thereof.

The oligonucleotide of the present invention may be either a single strand or a double strand, or a hybrid of a single and a double strand. It may also form a ring structure by connecting the 5' and 3' ends of a single molecule; several oligonucleotides of the present invention may be connected by covalent bonds or by separate linkers either at the 5' end or at the 3' end, to form tandemly connected elongated structures or multivalent structures. Oligonucleotides may undergo intermolecular or intramolecular hybridization to form elongated structures, multimeric structures or concatemers. Oligonucleotides may be linked to other molecules to form a multimer. The oligonucleotide of the present invention may be linked to other oligonucleotides. The oligonucleotide of the present invention may be included in an expression vector, including plasmid vectors and viral vectors.

[0064]

The oligonucleotide of the present invention may comprise a CpG motif, namely unmethylated dinucleotides, cytosine and guanine.

[0065]

The oligonucleotide of the present invention may comprise 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); wherein 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.

The oligonucleotide of the present invention may have r (adenine or guanine) at the 3' motif, forming tcgcaacgttt-r-cgacg-k-cg-b n-cg-r (SEQ ID NO:4).

[0066]

The oligonucleotide of the present invention may contain phosphorothioate internucleotide linkages. Each of the phosphorothioate internucleotide linkages may contain a stereogenic α-phosphorus atom, forming an R- or S-diastereomer.

Phosphorothioate internucleotide linkages may also contain phosphorodithioate structures. The oligonucleotide of the present invention preferably has a characteristic partial phosphorothioate site CaaCg in the 5' regions of oligonucleotides with partial phosphorothioate or phosphodiester internucleotide linkages, where a capital letter denotes a nucleoside with an unstabilized 3' internucleotide linkage, such as a phosphodiester linkage without unnatural chemical modification, and a lowercase letter denotes a nucleotide with a 3' internucleotide linkage that is a stabilized structure, such as a phosphorothioate linkage.

[0067]

The partially phosphorothioate version of the oligonucleotide of the present invention may preferably comprise a partially phosphorothioate region such as tCgCaaCgttt-n-cgacg-n-Cg-nn-C-g (SEQ ID NO:5), where the lowercase letter denotes a nucleoside with a 3' internucleotide linkage that is a stabilized structure such as a phosphorothioate linkage.

[0068]

The above mentioned motifs and regions must be present consecutively in one oligonucleotide; the motifs may be separated in the middle by insertion(s) of nucleotide(s), however, preferably, nucleotide(s) are inserted at each position in the amount of 1 or 2. The motifs mentioned above may be mutated, however, preferably, nucleotide(s) are mutated in each motif in the amount of 1 or 2. The motifs may be partially deleted at the ends in the amount of up to 2 bases.

[0069]

In one embodiment, the oligonucleotide of the present invention may be selected from oligonucleotides with the following sequence motifs presented in Table 1, wherein the internucleotide linkages may be selected from natural (e.g., phosphodiester) or synthetic (e.g., phosphorothioate), or a mixture thereof:

[Table 1]

[0070]

In another embodiment, the oligonucleotides of the present invention may be fully phosphorothioate in the internucleotide linkages. Examples of oligonucleotides of the present invention are presented in Table 2 below:

[Table 2]

In the sequences above, capital letters indicate a nucleoside or a nucleoside with a phosphodiester linkage as the 3' internucleotide linkage, and lowercase letters indicate a nucleoside with a phosphorothioate linkage as the 3' internucleotide linkage.

[0071]

In another embodiment, the oligonucleotides of the present invention may have partially phosphorothioate internucleotide linkages.

The oligonucleotides of the present invention may comprise a caacg sequence motif, preferably a partial phosphorothioate region of caaCg, Caacg or CaaCg, more preferably a partial phosphorothioate region of CaaCg.

Examples of such oligonucleotides are presented in Table 3 below:

[Table 3]

Capital letters in the sequences above denote a nucleoside or nucleotide with a phosphodiester linkage as the 3' internucleotide linkage, and lowercase letters denote a nucleoside with a phosphorothioate linkage as the 3' internucleotide linkage.

[0072]

The oligonucleotides of the present invention can bind to TLR9 and have an activity of enhancing signal transduction downstream of the receptor.

TLR9 activation activity can be assessed by the following events as surrogate indicators, but not limited to (WO2014082254A, JP5011520B):

(i) the potency of the NF-B binding oligonucleotide at the promoter, described as the levels of GFP expression controlled by the promoter in the cell, such as CAL-1/NF-B-GFP;

(ii) levels of cytokine production from cells; and

(iii) expression levels of marker proteins such as costimulatory molecules, including CD40, CD80 or CD86, in a target cell such as antigen-presenting cells.

[0073]

Target cell activation can be assessed by increasing the aforementioned surrogate markers. Increased levels of these markers can be assessed by measuring elevations compared to normal levels or levels without activating stimuli, including the addition of ligands.

[0074]

Such activity of the oligonucleotides of the present invention can be assayed in cultured cells such as peripheral blood mononuclear cells (PBMC) or derived and immunohistochemically treated cell lines. Such cell lines include HAL-1 and RCH-ACV instead of B cells, CAL-1 (Maeda, T., et al., Int J Hematol, 2005. 81(2): p.148-54), Gen2,2/Gen3 (Di Domizio, J., et al., Blood, 2009. 114(9): p.1794-802) or PMDC05 (Narita, M., et al., Acta Haematol, 2008. 120(2): p.91-9) instead of plasmacytoid dendritic cells.

[0075]

<Target diseases>

The oligonucleotide of the present invention can be used for the prevention or therapy of target diseases or disorders associated with the immune response, or for modulating the immune response in an individual. Examples of target diseases or disorders include neoplasms, infectious diseases, Th2- or Th17-mediated diseases, including diseases arising from Th2 or Th17 activation, primary immunodeficiency disease, or post-traumatic stress disorder (PTSD).

[0076]

The neoplasms mentioned include (malignant) tumors such as carcinomas (including malignant neoplasms, epithelial neoplasms, intraepithelial neoplasms and hematopoietic neoplasms), sarcomas and mesotheliomas, benign tumors, dysplasia and metaplasia. Said malignant tumors include, but are not limited to, malignant tumors such as lung cancer (small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, stomach cancer, esophageal cancer, pancreatic cancer, liver cancer, bile duct cancer, bile duct cancer, kidney cancer, pyelourethral 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 lymphoma, Hodgkin lymphoma, chronic myeloid leukemia, malignant lymphoma, and multiple myeloma.

[0077]

These sarcomas include, but are not limited to, osteosarcoma, osteocytoma, aneurysmal bone cyst, osteoid osteoma, chondrosarcoma, poorly differentiated round cell/spindle cell tumors (includes Ewing sarcoma), hemangioendothelioma, angiosarcoma, fibrosarcoma/myofibrosarcoma, chordoma, adamantinoma, liposarcoma, leiomyosarcoma, malignant peripheral nerve sheath tumor, rhabdomyosarcoma, synovial sarcoma, malignant solitary fibrous tumor, atypical lipomatous tumor, elevated dermatofibrosarcoma, malignant solitary fibrous tumor, inflammatory myofibroblastic tumor, poorly differentiated myofibroblastic sarcoma, fibrosarcoma (includes adult and sclerosing epithelioid types), myxofibrosarcoma, poorly differentiated fibromyxoid sarcoma, giant cell tumor of soft tissue, malignant glomus tumor, rhabdomyosarcoma, hemangioendothelioma, angiosarcoma of soft tissue, extraskeletal osteosarcoma, gastrointestinal stromal tumor (GIST), malignant peripheral nerve sheath tumor, malignant Triton tumor, malignant granular cell tumor, malignant ossifying fibromyxoid tumor, stromal sarcoma, myoepithelial carcinoma, malignant phosphaturic mesenchymal tumor, epithelioid sarcoma, alveolar sarcoma of soft tissue, clear cell sarcoma of soft tissue, extraskeletal myxoid chondrosarcoma, extraskeletal Ewing sarcoma, desmoplastic small cell round cell tumor, extrahepatic rhabdoid tumor, perivascular epithelioid cell tumor, intimal sarcoma, pleiomorphic sarcoma, and round cell sarcoma.

These mesotheliomas include, but are not limited to, pericardial mesothelioma, peritoneal mesothelioma, and pleural mesothelioma.

[0078]

These dysplasias include, but are not limited to, myelodysplastic syndrome and cervical dysplasia.

[0079]

The oligonucleotide of the present invention exhibits efficacy against target diseases based on activation of immunity against malignant cells. It is expected that when used alone, the efficacy will be higher when used against neoplasms with high immunogenicity. Neoplasms with high immunogenicity include, but are not limited to, malignant cells that express neoantigens that are formed due to gene mutations that occur during the stages of tumor formation; such malignancies include malignancies with deficient mismatch repair (dMMR) or high microsatellite instability (MSI-H) (Sargent, D.J., et al., J Clin Oncol, 2010. 28(20): p.3219-26; Passardi, A., et al., Int J Mol Sci, 2017. 18(6 ): E1324).

[0080]

The oligonucleotide of the present invention can activate pDCs via TLR9 activation. Activated pDCs then activate various other immune cells via interferons. Thus, the oligonucleotide of the present invention can be used to prevent or treat various infectious diseases caused by microorganisms, including viruses, bacteria, and fungi.

[0081]

Viruses that cause infectious diseases include, but are not limited to, molluscum contagiosum virus, herpes simplex virus (HSV), varicella-zoster virus, herpes 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). Among these viruses, some viruses, such as CMV, HIV, influenza virus, HSV, hepatitis B virus (HBV), or hepatitis C virus (HCV), have been described to activate host immunity directly by binding to TLR9 (Swiecki, M., et al., Immunol Rev, 2010. 234(1): p.142-62).

[0082]

Bacteria that cause infectious diseases are divided into gram-negative and gram-positive, but the present invention can be used with respect to bacteria from any group. It is described that TLR9 is necessary for at least the activation of innate immunity against gram-negative bacteria (Bhan, U., et al., J Immunol, 2007. 179(6): p.3937-46). 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, 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 cholerae, Vibrio parahaemolyticus, Pseudomonas, Xanthomonas, Acinetobacter, Flavobacterium, Brucella, Legionella, Veillonella, Bacteroides and Fusobacterium.

[0083]

Fungal infections include, but are not limited to, cryptococcosis, candidiasis, aspergillosis, Pneumocystis carinii pneumonia, trichophyton dermatophytosis, and Tinea versicolor cutaneous infection. In fact, fungal-derived DNA or RNA has been reported to be recognized by mammalian TLR9 (Kasperkovitz, P.V., et al., Infect Immun, 2011. 79(12): p.4858-67; Patin, E.C., et al., Semin Cell Dev Biol, 2019. 89: p.24-33).

[0084]

Th2/Th17-mediated diseases are diseases caused by suppression of Th1 and activation of Th2 and/or Th17; these diseases include, but are not limited to, asthma, atopic disease (dermatitis, eczema), allergy, multiple sclerosis, inflammatory bowel disease including ulcerative colitis and Crohn's disease, cutaneous lichen planus, and Alzheimer's disease.

[0085]

Primary immunodeficiency diseases are disorders in which a portion of the immune system is congenitally absent or does not function properly, and patients with such diseases are known to be particularly susceptible to infections. The oligonucleotides of the invention are expected to demonstrate efficacy, particularly in patients lacking functional TLR-related systems, such as patients with IRAK4 deficiency, MyD88 deficiency, Unc93B deficiency, or TLR mutations. However, the efficacy is not limited to such diseases, as the oligonucleotides of the invention are expected to activate immunity against infectious diseases more broadly.

[0086]

Post-traumatic stress disorder (PTSD) is one of the diseases that has been reported to be ameliorated by TLR9 agonist, and this disease is also considered as one of the target diseases (Zimmerman, G., et al., Transl Psychiatry, 2012. 2: p.e78).

[0087]

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 invention can also be used in an individual for prophylactic purposes against the above-mentioned target diseases.

[0088]

The oligonucleotides of the present invention can be linked to other active molecules, possibly via specific linkers. For example, linking pharmaceutical compounds for co-administration can facilitate co-administration; lipid linkage or PEGylation can improve tissue distribution or pharmacokinetics. PEGylation of oligonucleotides has been described to prolong in vivo residence time in an individual by reducing renal excretion.

[0089]

The oligonucleotides of the present invention can also be conjugated with certain organic compounds, such as vitamin E (α-tocopherol) or vitamin D, to improve pharmacokinetic parameters such as in vivo half-life or cellular uptake (Winkler, J., Ther Deliv, 2013. 4(7): p.791-809).

[0090]

To connect the oligonucleotides of the present invention to other organic or inorganic moieties, an organic chemical compound can be used as a linker; the linker includes, but is not limited to, glycerol, (S)-(-)-1,2,4-butanetriol, 1,3,5-pentanetriol, cis, cis-1,3,5-cyclohexanetriol, cis, trans-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. The linker portion can also be composed of amino acids, nucleotides and/or their derivatives.

[0091]

Linkers can also be used to provide non-covalent bonds. Linkers include, but are not limited to, biotin-avidin, SPB (succinimidyl-[4-(psoralen-8-yloxy)]butyrate), which intercalates into nucleic acid, nucleic acids whose sequences are complementary and bind to each other, and protein-protein interactions such as coiled-coil structures.

[0092]

In one embodiment, the oligonucleotides of the present invention can be used in combination with each other, with other oligonucleotides with a similar mechanism of function, with other organic compounds such as (poly)peptides/antibodies or nucleic acids/oligonucleotides, or with inorganic compounds such as cytotoxic agents, which are often used to improve or modify the physical properties of drugs. Such a combination can be obtained with or without covalent linkage.

[0093]

The oligonucleotides of the present invention can be derived from existing nucleic acid sources (e.g., genomic or cDNA), but are preferably synthetic. The oligonucleotides of the invention can be synthesized using various commercially available automated nucleic acid synthesis devices. These oligonucleotides are referred to as synthetic oligonucleotides.

[0094]

The oligonucleotides of the present invention may be administered alone or co-administered with one or more other ingredients, simultaneously or sequentially. The oligonucleotides of the present invention may also be used simultaneously or sequentially with one or more other therapeutic methods.

[0095]

The oligonucleotides of the present invention can be administered in conjunction with conventional anticancer drugs and therapies to alleviate various symptoms that occur as the tumor forms. Examples of conventional anticancer drugs include, but are not limited to: antibiotics such as anthracycline or mitoxantrone; platinum; alkylating agents; antimetabolites; hormonal therapy drugs; phytoalkaloids such as vinca alkaloids; taxanes such as paclitaxel or docetaxel; and topoisomerase inhibitors such as irinotecan or etoposide.

[0096]

The oligonucleotides of the present invention can also be co-administered to individuals in need thereof with other molecular therapeutic agents targeting the biological characteristics of malignant cells; molecular targeted therapeutic agents include tyrosine kinase inhibitors, angiogenesis inhibitors, or proteasome inhibitors. Examples of molecular targeted agents include, but are not limited to:

everolimus (Afinitor), tamoxifen (Nolvadex), toremifene (Fareston), fulvestrant (Faslodex), anastrozole (Arimidex), exemestane (Aromasin), lapatinib (Tykerb), lectrozole (Femara), palbociclib (Ibrance), ribociclib (Kisqali), neratinib maleate (Nerlinks), amemaciclib (Verzenio), olaparib (Lynparza), regorafenib (Stivarga), imatinib mesylate (Gleevec), lanreotide acetate (Somatulin Depot), sunitinib (Sutent), sorafenib (Nexavar), pazopanib (Votrient), temsirolimus (Torisel), axitinib (Inlyta), carbozantinib (Carbometyx), lenvatinib mesylate (Lenvima), tretinoin (Vesanoid), dasatinib (Spricel), nilotinib (Tasigna), bosutinib (Bosulif), ibrutinib (Imbruvica), idelalisib (Zidelig), venetoclax (Venclexta), ponatinib hydrochloride (Iclusig), midostaurin (Rydap), enesidenib mesylate (Idhifa), inotuzumab ozagomicin (Besponsa), tisagenlecleucel (Kymriah), ivosidenib (Tibsovo), develisib (Copictra), sorafenib (Nexavar), crizotinib (Xalkori), erlotinib (Tarceva), gefitinib (Iressa), afatinib dimaleate (Gilotrif), ceritinib (LDK378/Zykadia), osimertinib (Tagrisso), Alectinib (Alecensa), brigatinib (Alunbrig), trametinib (Mekinist), dabrafenib (Tafinlar), dacomitinib (Vizimpro), denileukin diftitox (Ontac), vorinostat (Zolinza), romidepsin (Istodax), bexarotene (Targretin), bortezomib (Velcade), pralatrexate (Folotin), siltuximab (Sylvant), idelalisib (Zidelig), belinostat (Beleodac), copanlisib hydrochloride (Aliquopa), acalabrutinib (Calcvens), carfilzomib (Kyprolis), panobinostat (Faridac), ixazomib citrate (Ninlaro), ruxolitinib phosphate (Yakafi), cabazitaxel (Eutana), enzalutamide (Xtandi), abiraterone acetate (Zytiga), radium 223 dichloride (Xofigo), apalutamide (Erleada), vismodegib (Erivedge), sonidegib (Odomzo), vemurafenib (Zelboraf), cobimetinib (Cotellic), alitretinoin (Panretin), encorafenib (Braftovi), binimetinib (Mektovi), cemiplimab-rwlc (Libitayo), alitretinoin (Panretin), and vandetanib (Caprelsa).

[0097]

The oligonucleotides of the present invention can also be co-administered to individuals with anti-cancer antibody therapeutics. Co-administration with the ODN of the present invention is expected to synergistically enhance the activity of antibody therapeutics, which, in particular, utilize the immune system to attack malignant cells. Examples of such antibody therapeutics include, but are not limited to:

(i) antibody-based medicinal products containing a single type of antibody: trastuzumab (Herceptin), alemtuzumab (Campath), bevacizumab (Avastin), pertuzumab (Perjeta), cetuximab (Erbitux), panitumumab (Vectibix), necitumumab (Portrazza), dinutuximab (Unituxin), raumcirumab (Cyramza), olaratumab (Lartruvo), ipilimumab (Yervoy), nivolumab (Opdivo), pembrolizumab (Keytruda), atezolizumab (Tecentriq), denosumab (Xgeva), durvalumab (Imfinzi), avelumab (Bavencio), ibritutomab tiuxetan (Zevalin), brentuximab vedotin (Adcetris), obinutuzumab (Gazyva), mogamulizumab-kpkc (Poteligeo), daratumumab (Darzalex), elotuzumab (Emplicity), rucaparib camsilate (Rubraca), niraparib tosylate monohydrate (Zeyula), anti-OX40 antibody;

(ii) antibody medicinal products containing a bispecific antibody: blinatumomab (Blincyto);

(iii) antibody-drug conjugate (ADC) containing ibritutomab tiuxetan (Zevalin), brentuximab vedotin (Adcetris), ado-trastuzumab emtansine (Kadcyla), gemtuzumab ozagomicin (Mylotarg); and

(iv) ziv-aflibercept (Zaltrap).

[0098]

The oligonucleotides of the present invention can also be co-administered with cytokines such as GM-CSF, IFN-α, IFN-β or IFN-γ, which are already used as standard anti-cancer drugs.

[0099]

The oligonucleotides of the present invention can be used in conjunction with surgical procedures including radiation therapy, radiofrequency ablation, cryoablation (Aarts, B.M., et al., Insights Imaging, 2019. 10(1): p.53) or photodynamic therapy (PDT) after administration of a photosensitizing agent.

It is expected that the combination with the administration of the oligonucleotides of the present invention will demonstrate synergistic efficacy, given that PDT in particular has been described to enhance anti-cancer immunity (Kleinovink, J. W. et al., Cancer Immunol Res, 2019, 5(10): p.832-838).

The oligonucleotides of the present invention can be administered as active ingredients in an adjuvant or neoadjuvant context (O'Donnell, J.S., et al., Clin Cancer Res, 2019, 25(19): p.5743-5751).

[0100]

The oligonucleotides of the present invention can also be used in combination with adoptive immune cell therapy (also known as adoptive cell transfer (ACT)), such as chimeric antigen receptor T-cell (CAR-T) therapy (axicabtagene ciloleucel (Escarta®) or tisagenleucel (Kymriah®)), tumor-infiltrating lymphocyte (TIL) therapy, dendritic cell therapy, or NK cell therapy. The ODN of the present invention can also be combined with oncolytic virus therapies that utilize an engineered virus to lyse tumor mass (Davola, ME, et al., Oncoimmunology, 2019. 8(6): p.e1581528; Sivanandam, V, et al., Mol Ther Oncolytics, 2019. 13: p.93-106; Raja, J, et al., J Immunother Cancer, 2018. 6(1): p.140; Martinez-Quintanilla, J, et al., J Clin Invest, 2019. 130: p.1407-1418; Harrington, K, et al., Nat Rev Drug Discov, 2019. 18(9): p.689-706).

[0101]

The oligonucleotides of the present invention can be used in combination with therapeutic vaccines that are expected to enhance the immunity required for therapy. Therapeutic vaccines are a class of vaccines used for the purpose of therapeutic treatment of existing diseases. Such therapeutic vaccines include not only vaccines produced using attenuated or detoxified microorganisms, but also ex vivo cellular vaccines produced using ex vivo- treated cells, and in vivo vaccines that activate immunity, such as anti-cancer immunity, by activating target immune cells. Ex vivo cellular vaccines include Sipuleucel-T (Provenge), which is produced from autologous or allogeneic dendritic cells, or GVAX, which is produced by modifying autologous or allogeneic malignant cells. Loading of such dendritic cells with antigens can be achieved by contacting antigens, including peptides, recombinant proteins, or tumor lysate, with dendritic cells in ex vivo culture, but can also be achieved by fusing cells expressing antigens (e.g., malignant cells) with dendritic cells (Hollingsworth, RE, et al., NPJ Vaccines, 2019. 4: p.7).

[0102]

The oligonucleotides of the present invention can be used in combination with a variety of in vivo vaccines. In vivo vaccines can include delivery vehicles to target antigen-presenting cells (APCs) in combination with antigens such as cancer antigens or viral antigens; target proteins for APC delivery vehicles include the following cell surface proteins:

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 for in vivo vaccines may include peptides or recombinant proteins containing cancer antigens, which are described below, or viral antigens; polynucleotides encoding the antigens can be used to direct the expression of antigens in such target APCs.

[0103]

Prophylactic vaccines against cancer include the hepatitis B virus vaccine and the human papillomavirus (HPV) vaccine, such as Gardasil or Cervarix. The oligonucleotides of the present invention are also expected to enhance the prophylactic activity of commercially available vaccines or vaccines in premarket stages.

[0104]

The oligonucleotides of the present invention can be used for the prevention or treatment of infectious diseases. The oligonucleotides can also be mixed with a vaccine composition as an adjuvant. Alternatively, the oligonucleotides can be administered to an individual in combination with a pharmaceutical composition for the treatment of infectious diseases, including antibacterial agents, antifungal agents, antiviral agents, antiparasitic agents, vaccines, and antibody-based toxin-neutralizing drugs.

Vaccines used to prevent or treat infectious diseases include vaccines from the following categories:

- live attenuated vaccines, which contain a weakened version of a live microorganism.

- inactivated vaccines that contain killed microbes but retain antigenicity.

- subunit vaccines, which contain only the antigens that most effectively stimulate the immune system.

- toxoid vaccines, which aim to detoxify toxins from microbes.

- conjugate vaccines, which are specialized types of subunit vaccines that allow the formation of immunity against microorganisms in individuals with weak immunity through conjugation with other subunits with higher immunogenicity.

- nucleic acid-based vaccines that cause the body's cells to produce antigens, providing additional immunization in vivo .

- vaccines based on recombinant vectors that contain genetic information of recombinant antigens to stimulate immunity against target microorganisms.

Examples of vaccines that can be used in combination with the oligonucleotide of the present invention are: BCG vaccine, cholera vaccine, diphtheria vaccine, Haemophilus influenzae vaccine, hepatitis A vaccine, hepatitis B vaccine, human papillomavirus vaccine, pandemic H1N1 influenza virus vaccine, seasonal influenza virus vaccine, Japanese encephalitis vaccine, measles virus vaccine, mumps virus vaccine, meningococcal vaccine, pneumococcal vaccine, pertussis vaccine, poliovirus vaccine, rabies virus vaccine, rotavirus vaccine, rubella vaccine, tetanus toxoid vaccine, typhoid vaccine and yellow fever vaccine.

[0105]

Recently, a class of drugs called immune checkpoint inhibitors (ICIs) has been intensively developed. These drugs target immune checkpoint molecules, a group of proteins that regulate immunity, to stimulate immunity necessary for the treatment of target diseases. The oligonucleotides of the present invention can be used with ICIs to synergistically enhance the efficacy of ICIs by activating antigen-presenting cells associated with immune checkpoint processes.

Examples of target molecules for 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 (Pardoll, D.M., Nat Rev Cancer, 2012. 12(4): p.252-64).

[0106]

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. Examples of target molecules or pathways affected by such therapies or drugs include:

- Treg - 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,

- MDSC - PDE-5, COX-2, HDAC, STAT3, CCL2/CCR2, VEGF-A/MET/TIE2/VEGFR2 cascade, IL-8/CXCR1/2, galectin-1 (Gal-1),

Therapies or drugs that deplete such immunosuppressive immune cells can also be combined with the oligonucleotides of the present invention. Examples of cell surface marker proteins that can be used to deplete such cells include the following:

Tregs - CD25, CTLA-4, PD-1, ICOS, GITR, OX40, CD15, CCR4 and CCR8,

TAM - CD206, legumain, scavenger receptor A and CD52,

(Ohue, Y., et al., Cancer Sci, 2019. 110(7): p.2080-2089; Yang, L., et al., J Hematol Oncol, 2017, 10(1): p. 58; Gabrilovich, D. I., Cancer Immunol Res, 2017, 5(1): p.3-8; Ding A.S. et al., Front Immunol, 2019, 10: p.1715)

[0107]

The oligonucleotides of the present invention can be used in conjunction with antigenic proteins, peptides, glycoconjugates, or other organic substances to enhance specific antigenic responses against target diseases. Examples of antigenic substances include the following:

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 proteins, HCV proteins, HER2, hexon, HJURP, HMWMAA, HPV-16 E6, HPV-16 E7, HPV-18 E7, surface Ig idiotype, IE-1, KIF20A, KOC1, KSV proteins, 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, MP65, MPHOSPH1, MUC1, mucin-1, MYCN, NA, NA17, NeuGcGM3, nonstructural proteins NS4, nonstructural proteins NS5, NY-BR-1, NY-ESO-1, ospA, ospB, ospC, OY-TES1, p53, Page4, PAP, PAX3, PAX5, PDGFR-beta, penton, PLAC1, pmel17, pmp20, polysialic acid, pp65, PRAME, prostate-specific membrane antigen 10, prostein, protease 3 (PR1), PSA, PSCA, PSMA, Ras, RGS5, RhoC, RM2, RNF43, ROR1, sarcoma translocation breakpoints, SART3, Select, serine protease NS3, SHMP, sLe, SOD, sperm protein 17, SSX2, STEAP1, STn, survivin, TARP, Tax protein, telomerase, telomerase, TERT, Tie 2, ™4SF5, Tn, TOMM34, triosephosphate isomerase, TRP1, TRP2, TTK, tyrosinase, tyrosinase-related protein 1, tyrosinase-related protein 2, URLC10, VEGFR2, viral capsid proteins, viral core protein, viral nucleoprotein, WT1, XAGE 1, alpha-actinin-4, and beta-catenin.

A neoantigen is a good candidate for use as an antigenic substance; a neoantigen is an antigen that is newly formed through an in vivo process such as mutagenesis that occurs during tumor formation, or is formed from infectious foreign substances that are recognized by the body's own immune system.

[0108]

Anticancer chemotherapeutic drugs, especially immunogenic cell death (ICD)-inducing drugs, can be co-administered with the oligonucleotides of the present invention to demonstrate synergistically enhanced efficacy. Examples of ICD-inducing drugs include: anthracyclines including mitoxantrone, platinum-based anticancer agents including oxaliplatin, cisplatin, carboplatin, nedaplatin, triplatin tetranitrate, picoplatin, satraplatin (Hato, S.V., et al., Clin Cancer Res, 2014. 20(11): p.2831-7).

[0109]

Other drugs that are known for their property of activating anti-cancer immunity, such as indoleamine 2,3-dioxygenase (IDO) inhibitors (Prendergast, G.C., et al., Cancer Res, 2017. 77(24): p.6795-6811) or proteasome inhibitors, including bortezomib (Spisek, R., et al., Blood, 2007. 109(11): p.4839-45; Chang, C.L., et al., J Immunol, 2012. 189(6): p.3209-20), can be co-administered with the oligonucleotides of the present invention so that they exhibit synergistic efficacy.

[0110]

The oligonucleotides of the present invention can be co-administered 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 used in combination with agents that enhance intracellular nucleotide-sensitive signaling pathways, such as the cGAS-STING pathway or the RIG-I/MDA5 pathway (Bode, C., et al., Eur J Immunol, 2016. 46(7): p.1615-21; Iurescia, S., et al., Front Immunol, 2018. 9: p.711), since these cascades share common target molecules with TLR9, such as IFN-α or NF-κB, and are expected to exhibit a synergistic effect when activated simultaneously. Examples of molecules that activate each receptor include the following:

TLR3 agonist: Rintatolimod, poly C 3SBIO, poly(I:C), hiltonol,

TLR4 agonist: ALD046, CRX527, CRX675, G100, lipid A, GSK1795091, OM174, PGN007,

TLR7 agonist: vestolimod, VML600, 852A, NKTR262, ™X101, GS9620, RG7795, DSP0509, PF4878691, RG7854, RG7863, ™X202, TQA3334,

TLR8 agonist: GS-9688, VTX 2337,

TLR9 agonist: heplisav, SD-101, IMO2125, IMO2055, MGN1703, MGN1706, CPG 7909, litenimod, AST008, DUK-CPG-001, actilon, CMP001, DV281, cobitolimod,

TLR9 and NOD2 agonist: MIS416,

STING signal transmission cascade activator: ADU-S100,

STING agonist: MK-1454, SB 11285, IMSA101,

RIG-I agonist: RGT 100.

[0111]

The oligonucleotides of the invention can be administered in/with a delivery carrier or in a form associated with a carrier. The carrier includes, but is not limited to, sterol (e.g., cholesterol), cochleates, emulsomes, ISCOM; lipid (e.g., cationic lipid, anionic lipid), liposomes; ethylene glycol (PEG); polyglycolide-lactide copolymer (PGLA); microspheres; polymers (e.g., carboxymethylcellulose (CMC), chitosan, mannitol, hydroxypropyl methylcellulose (HPMC)); live bacterial vectors (e.g., Salmonella, Escherichia coli, bacillus Calmette-Gurin, Shigella, Lactobacillus); live viral vectors (e.g., vaccinia virus, adenovirus, herpes simplex virus), virosomes, virus-like particles.

[0112]

The target individual for administration of the oligonucleotide of the present invention is preferably a human, however, in one embodiment, the individual may be a non-human animal such as a dog, cat, horse, pig, goat, sheep, cow, monkey, chicken, mouse or rat.

[0113]

"Therapeutically effective amount": For the treatment or prevention of a target disease or disorder, a therapeutically effective amount of the oligonucleotides of the present invention is administered to an individual. A "therapeutically effective amount" of one or more oligonucleotides means a sufficient amount of the oligonucleotides used to achieve the desired result of treatment or prevention of a disorder in an individual. The oligonucleotides of the present invention can be used in pure form or in pharmaceutically acceptable carriers. Alternatively, the ODN of the present invention can be administered as pharmaceutical compositions. "Amount" as used herein refers to a dose. The dose can be determined by standard methods well known to those skilled in the art and may depend on various factors, including, but not limited to, the size and/or general health of the individual or the severity of the disease symptom. Administration of the oligonucleotide of the invention can be carried out as a single administration or over a series of administrations. Doses of the oligonucleotide according to the invention for an individual per administration range from approximately 1 μg (microgram) to 10 g per administration. Preferably, the doses range from 0.1 mg to 5 g. More preferably, the doses range from 0.3 mg to 3 g. Most preferably, the doses range from 1 mg to 1 g.

A therapeutically effective amount for humans can be estimated based on the amount for suitable non-human animals using the human equivalent dose (HED) or human equivalent concentration (HEC). A therapeutically effective amount for humans may range from, but is not limited to, 0.3-60 mg/day, preferably 1-30 mg/day, and more preferably 2-8 mg/day.

[0114]

"Route of Administration": For clinical use, the oligonucleotide of the present invention can be administered alone or formulated into a pharmaceutical composition by any suitable route of administration that is effective in achieving the desired therapeutic result. "Route" of administration of the oligonucleotide of the present invention means enteral, parenteral, and local administration or inhalation. Enteral routes of administration of the oligonucleotide of the present invention include oral, gastric, intestinal, and rectal. Parenteral routes include subcutaneous, intravenous, transdermal, intradermal, sublingual, intranasal, transmucosal, pulmonary, vaginal, via aerosol, intraocular, intratracheal, intrarectal, intravertebral, intramuscular, intraarticular, intraperitoneal, intracardiac, intraosseous, intrathecal, intravitreal, inhalation, or topical routes of administration. Local administration of the oligonucleotide according to the invention involves applying the oligonucleotide topically to the epidermis, buccal cavity, and eye, ear, and nose. Intratumoral administration is one route of administration, typically performed by injecting the test compound(s) into the tumor or peritumoral region.

[0115]

"Pharmaceutical composition": A pharmaceutical composition means a composition comprising a therapeutically effective amount of an oligonucleotide of the present invention, with or without a pharmaceutically acceptable carrier. Pharmaceutical compositions may comprise one or more oligonucleotides of the invention. The composition includes, but is not limited to, aqueous or saline solutions, particles, aerosols, beads, 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. The compositions can be administered parenterally, orally, rectally, intravaginally, intraperitoneally, topically (in a dosage form such as powders, ointments, gels, drops or a transdermal patch), buccally or as an oral or nasal spray. In all cases, the composition must be sterile and stable under the conditions of manufacture and storage, and must be protected from microbial contamination. The pharmaceutical compositions of the present invention for parenteral injection include pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injection solutions or dispersions immediately before use. The oligonucleotide of the invention can be suspended in an aqueous carrier, for example, in an isotonic buffer solution, at a pH of from about 3.0 to about 8.0, preferably at a pH of from about 3.5 to about 7.4, from 3.5 to 6.0, or from 3.5 to about 5.0. The buffer solution includes sodium citrate-citric acid and sodium phosphate-phosphoric acid and sodium acetate-acetic acid buffers. For oral administration, the composition is formulated with edible carriers to form powders such as tablets, pills, orally dissolving/disintegrating tablets, dragees, capsules, liquids, gels, syrups, suspensions, suspensions, and the like. For a solid composition, a conventional non-toxic solid carrier may include pharmaceutical grade mannitol, lactose, starch, or magnesium stearate. For buccal administration, the composition is a conventional tablet or lozenge. For inhalation, the composition is an aerosol spray from a pressurized package, a nebulizer, or a dry powder, and can be selected by one skilled in the art. In some cases, to prolong the effect of the oligonucleotide of the invention, the oligonucleotide of the present invention is also suitably administered via sustained-release systems. The oligonucleotide of the present invention can be used in a liquid suspension of a crystalline or amorphous material with low solubility in water to slow the release of the oligonucleotide. Alternatively, delayed release of a parenterally administered oligonucleotide dosage form is achieved by dissolving or suspending the oligonucleotide in a hydrophobic material (such as an acceptable oil carrier). An injectable depot form is obtained by encapsulating the oligonucleotide in liposomes or microemulsions, or other biodegradable semipermeable polymer matrices, such as polylactide-polyglycolide, polyorthoesters, and polyanhydrides.

[Examples]

[0116]

The invention is described in more detail in the examples below. However, the invention is not limited to these examples. Experiments in these examples were conducted using commercially available kits and reagents in accordance with the attached protocols, unless otherwise indicated. Those skilled in the art will appreciate that the oligonucleotides of the present invention can readily be used to treat target diseases, including malignant tumors and infectious diseases. The present invention is further demonstrated by the following non-limiting examples.

[0117]

Materials and methods:

<Oligodeoxynucleotides (ODN)>

Single-stranded oligodeoxynucleotides (ODN) were synthesized at Hokkaido System Science Co., Ltd. using a conventional phosphoramidite method in a form lacking a phosphate at the 3' end, and purity and identity were confirmed by the manufacturer. In the examples below, nucleosides with phosphorothioate (PS) internucleotide linkages at the 3' end are shown in lowercase letters, while nucleosides and oligonucleosides with phosphodiester (PO) intranucleotide linkages at the 3' end are shown in capital letters. All synthesized ODN were first dissolved in distilled pyrogen-free water and further diluted using pyrogen-free assay reagents.

[0118]

<Complete medium 10% FBS-RPMI>

RPMI1640 medium is supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillin-streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, and 50 mM 2-mercaptoethanol. The medium is then filtered through a 0.45-μm syringe filter.

[0119]

<CAL-1 cells>

CAL-1 cells (a human plasmacytoid dendritic cell line; Maeda et al., Int J Hematol., 2005, 81, 148-54; JP5011520B) are cultured in a humidified atmosphere and 5% _CO2 at 37°C with 10% FBS-complete RPMI medium. CAL-1 cells confluent within 2 days after subculture to a 1:5 ratio; however, for the assays described below, cells are passaged before confluency. CAL-1 cells are cultured in a suspension culture dish.

[0120]

<Detection of TLR9 activation by GFP induction via NF-κB activation>

Transcriptional activity levels of the NF-κB promoter, which is induced by the TLR9 signaling cascade, were monitored as an indicator of TLR9 signaling activity levels. The CAL-1/NF-κB-GFP cell line was generated to monitor NF-κB transcription factor activity in cell-based assays (WO2014082254A). To generate the CAL-1/NF-κB-GFP cell line, CAL-1 cells were transfected by electroporation with a vector encoding a GFP reporter gene controlled by the NF-κB consensus transcriptional response element. Transfected cells were then 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-κB-GFP cells (1× ¹⁰⁵ /well) were seeded in a 96-well flat-bottom plate and cultured with or without CpG2395. Cells were incubated at 37°C in a humidified incubator with 5% _CO2 for 6 h. The GFP expression level in 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 TLR9 signaling activity levels. These resulting NF-κB-GFP/CAL-1 cells were used in each assay.

[0121]

<Detection of TLR7 activation>

TLR7 activity was determined as secreted embryonic alkaline phosphatase (SEAP) enzymatic activity in HEK-Blue™ TLR7 cells (Invivogen). Cells were incubated with the indicated oligonucleotides or TLR7 agonists, such as 1 μg (micrograms)/mL gardiquimod (GQ) or 1 μg/mL CL264, at 37°C in a humidified incubator with 5% _CO2 for 24 hours; induced SEAP was determined by absorbance levels at 655 nm as a result of cleavage of substrates (HEK-Blue™ Detection, Invivogen).

[0122]

<Detection of TLR8 activation>

TLR8 activity was determined as secreted embryonic alkaline phosphatase (SEAP) enzymatic activity in HEK-Blue™ TLR8 cells (Invivogen). Cells were incubated with the indicated oligonucleotides or TLR7 agonists, such as 200 ng/ml TL8-506 or 5 μg/ml CL075, for 24 hours at 37°C in a humidified incubator with 5% _CO2 for 24 hours; induced SEAP was determined by absorbance levels at 655 nm as a result of cleavage of substrates (HEK-Blue™ Detection, Invivogen).

[0123]

<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). Measurements were performed according to the manufacturers' protocols.

[0124]

<Isolation of human PBMC>

Peripheral blood was isolated from healthy volunteers. The same volume of RPMI medium was added to the blood and mixed well. Human PBMCs were purified by centrifugation using Histopaque™. Briefly, Histopaque-1077 (SIGMA) was placed in a centrifuge tube, and the same volume of the blood/RPMI mixture was added to Histopaque™. The tube was centrifuged for 20 minutes at 1800 rpm (700 × g). The white layer in the middle was pipetted into another tube, and complete 10% FBS-RPMI medium was added. The solution mixture was centrifuged for 10 minutes at 1800 rpm (700 × g), and the supernatant was discarded. The cell pellet was treated with 2 ml of distilled water to lyse the erythrocytes, and then 20 ml of complete 10% FBS-RPMI medium was immediately added to the tube. After washing twice with the medium, the cells were suspended in the medium and counted. Freshly isolated PBMCs were used for each analysis.

[0125]

<Obtaining mouse splenocytes>

The spleen was removed from the mouse and placed in a dish filled with RPMI medium. The spleen was minced through a nylon sieve using the rubber tip of a 2.5-mL syringe plunger. The spleen cells, included in the medium, were transferred to a 50-mL Falcon tube by filtration through a 70-μm sieve. After centrifugation at 1500×g for 5 minutes, the cells were washed with 1×DPBS. The cells were treated with ACK lysis buffer to lyse erythrocytes. One ml of ACK lysis buffer was added to the cell pellet and pipetted 20 times with a P1000 pipette, and then the tube was left to stand on ice for 2 minutes. Twenty ml of complete RPMI medium with 10% FBS was immediately added to the tube. After washing twice with medium, the cells were resuspended in the same medium and counted. The splenocytes were then used for further analysis.

[0126]

Example 1

The fully phosphorothioate ODNs of the present invention have adjuvant activity.

The fully phosphorothioate ODNs used in this test are listed in the following Table 4.

Lowercase letters indicate a nucleoside modified with a phosphorothioate at the 3'-side internucleotide linkage, and capital letters indicate a nucleoside that is unmodified (without a phosphodiester linkage) at the 3'-side.

[Table 4]

[0127]

<Analysis of the immunostimulatory activity of ODN>

The ODNs presented in Table 4 were incubated with CAL-1/NF-κB-GFP cells for 6 hours at the indicated concentration (0.1 μM or 0.3 μM). NF-κB activation by ODNs was assessed based on the percentage of GFP-positive cells analyzed using a flow cytometer (FACS Calibur, BD Bioscience Co., Ltd.). Data were analyzed using FlowJo™ ver 10 (FlowJo LLC).

[0128]

As shown in FIG. 1A, GFP expression was induced in CAL-1/NF-κB-GFP cells by stimulation with the authentic TLR9 agonist, CpG2395, indicating that NF-κB activation was induced by TLR9 activation by CpG2395. The ODN of the present invention, all phosphorothioate (PS) A001, A002, A003, and A004, were shown to have higher TLR9 activation activity compared to CpG2395. Furthermore, as demonstrated in FIG. 1B, the ODN of the present invention exhibited more pronounced TLR9 activity than other known CpG ODNs, such as CpG685, M362, and D60-1. Given that ODN-induced TLR9 activity levels were independent of nucleic acid length, sequence-specific differences in agonist activity were thought to underlie these differences.

[0129]

Since many previous reports have argued for the importance of the gtcgtt sequence in CpG ODNs for optimal activation of human TLR9 (Har™ann, G. et al., J Immunol, 2000. 164(2): p.944-53, Bauer, S., et al., Proc Natl Acad Sci U S A, 2001. 98(16): p.9237-42), CpG2395 and CpG685 (5'-tcgtcgacgtcgttcgttctC-3'; SEQ ID NO:46) share the same gtcgtt sequence. Previous reports have suggested that the tcgt sequence at the 5' end of CpG-ODN is essential for the activation of human TLR9 (Pohar, J., et al., J Immunol, 2017. 198(5): p.2093-2104; Ohto, U., et al., Immunity, 2018. 48(4): p.649-658). The commonly accepted CpG-ODNs, CpG2395, CpG685, and M362 (5'-tcgtcgtcgttcgaacgacgttgaT-3'; SEQ ID NO:47), have the tcgt sequence at the 5' end as the TLR9 activation motif, consistent with this rule.

Among the ODNs of the present invention, only A003 has one gtcgtt sequence, while the others lack the gtcgtt sequence, and all of the ODNs lack the tcgt sequence at the 5' end. However, all of the ODNs of the present invention demonstrated more pronounced activation of human TLR9 than conventional CpG ODNs such as CpG2395, CpG685, and M362.

[0130]

As described above, while the 5'-tcgt and "gtcgtt" motifs in authentic CpG ODNs are known as TLR9-activating motifs in humans (Wang, X., et al., Vaccine, 2008. 26(15): p.1893-901), these motifs in the ODN of the present invention were shown to have a negligible contribution to the overall activity, indicating that other optimal sites/motifs for human TLR9 exist in the ODN of the present invention.

As shown in FIGS. 1C and 1D, the activities of A003 and A003#endG were similar. While the activity of A003#delA was slightly reduced by the conversion of the base at the 3' end, the activity was still significantly higher than that of the conventional CpG-CpG-ODN2395 (shown in FIG. 1A). This indicates that the nucleotide at the 3' end is dispensable for activity.

[0131]

Furthermore, as shown in FIGS. 1E, 1F, and 1G, all of the ODN sets: A001 and A011; A002 and A012; A003 and A013; and A004 and A014, exhibited similar activity levels to each other, indicating that substitution of the indicated nucleotides in the ODN did not result in any significant changes in the ODN activity.

Taken together, mutations of bases at positions 12, 18, 21, and 25 from the 5' end of the ODN may not result in a decrease in the stimulatory activity of the ODN. The ODN of the invention have been shown to have 5'-tcgcaacgttt-n-cgacg-n-cg-nn-cg-3' (SEQ ID NO:2) as the core structure with TLR9 activating activity.

[0132]

Example 2

The oligonucleotides of the present invention significantly activate TLR9.

<Activation of human TLR7 and TLR8 by ODN>

HEK blue™ TLR7 cells were stimulated with TLR agonists for 24 h.

As shown in FIG. 2A, the TLR7 agonists gardiquimod (GQ) and CL264 activated TLR7 and produced positive signals. In contrast, the authentic TLR9 agonist CpG2395 and the ODN of the present invention failed to activate the TLR7 signaling cascade.

Furthermore, HEK blue™ TLR8 cells were stimulated with TLR agonists for 24 hours. As shown in FIG. 2B, the TLR8 agonists TL8-506 and CL075 activated TLR8 and produced positive signals. In contrast, the authentic TLR9 agonist CpG2395 and the ODN of the present invention failed to activate the TLR8 signaling cascade.

[0133]

Example 3

Characteristic nucleotide regions with partial phosphorothioate linkages in the oligonucleotides of the present invention

The existence of a minimal region of the oligonucleotides of the present invention that enhances the activity of a TLR9 agonist was studied.

[0134]

<ODN>

Single-stranded ODNs with internucleotide linkages with partial phosphorothioate linkages were prepared as shown in Table 5.

[Table 5]

Lowercase letters indicate that the nucleoside is modified with a phosphorothioate at the 3'-side internucleotide linkage, and capital letters indicate that the nucleoside has a phosphodiester internucleotide linkage or is unmodified (without a phosphodiester linkage) at the 3'-side.

[0135]

<Analysis of ODN activity as TLR9 agonists>

The agonist activity of ODN was analyzed by GFP expression in CAL-1/NF-B-GFP cells.

As shown in FIG. 3A, all-PS A003 exhibited low activity at the tested concentration when stimulated for 6 hours. As described, altering the internucleotide linkage via a phosphodiester (PO) linkage in the CG motif of authentic all-PS CpG ODNs enhanced TLR9 activation activity (Pohar, J., et al., Sci Rep, 2017. 7(1): p.14598., WO2004016805A), A103 and A203, which have a CG motif with a PO linkage and retain the same sequence as A003, demonstrated increased activity. However, an additional PO linkage on CA in the CAACG region of ODN (A303) further enhanced the activity, as shown in FIGS. 3A and 3B. As can be seen from the left panel of FIG. 3B (0.1 μM), A303 exhibited significantly higher activity than A103 and A203, indicating the importance of PO bonds in the CAACG (CaaCg) region for activity.

As shown in FIG. 3C, A403 and A503 exhibited reduced activity compared to A303, while both A403 and A503 have an increased number of PO-linked CG motifs relative to A303. This indicates that the number of PO-linked CG motifs in the ODN of the present invention is not important for activity, although the number of PO-linked CG motifs in authentic CpG ODNs has been previously thought to be important for activity (WO2004016805A). Importantly, both A403 and A503 lack a CaaCg site (2 PO bonds in the CAACG motif). While A603 and A503 have the same number of PO bonds in CG motifs, A603 exhibited better activity than A503. A603 retains the CaaCg site similar to A303 and demonstrated similar activity to A303, indicating the importance of the CaaCg site over increased amounts of PO-linked CG motifs for optimal activity. A703 has PO-linkages in all CG motifs, but it demonstrated lower activity than A303 and A603 (0.1 μM stimulation), indicating that increasing the amount of PO-linked CG motifs is not necessary for maximizing the activity of the ODN of the present invention. The total amounts of PO-linked CG motifs are negligible for activity optimization, which was not expected based on the previously reported Py (pyrimidine)-PO-Pu (purine) effect (WO2004016805A).

[0136]

As shown in FIG. 3D, the importance of the CaaCg region was further confirmed by detecting ODN-induced inflammatory cytokine production. A303 and A603 induced similar cytokine levels to each other, and the production levels were higher than those of A403 and A503.

The importance of the CaaCg site was examined for previously described CpGs, DV093 and DV094 (FIG. 3E). While both DV093 and DV094 have a caacg motif in their sequence, their activity was significantly lower than that of A003.

[0137]

Interestingly, the CaaCg site (CAACG motif with partial phosphorothioate linkages) in DV093C and DV094C failed to enhance their activity compared to the parent DV093 or DV094. An internucleotide linkage change replacing the caacg site with CaaCg in DV093 and DV094 did not enhance activity, as shown in FIG. 3F.

These data indicate that the presence of a specific CaaCg region, such as that in the ODN of the present invention, but not in ODN with randomly located CaaCg, has the unique property of enhancing TLR9 activity. Taken together, the 5'-tCgCaaCg region (such as that in A303 and A603) may be located in the core structure of the ODN of the present invention.

[0138]

Example 4

Central structures of ODNs of the present invention

<ODN>

Single-stranded ODNs with internucleotide linkages containing partial phosphorothioate linkages were prepared as described in Table 6.

[Table 6]

[0139]

ODN activity was analyzed by GFP expression in CAL-1/NF-B-GFP cells. As shown in FIGS. 4A and 4B, all tested ODNs that had base variations at positions 18, 21, and 22 in the 3' regions exhibited similar activity levels to each other, and their activity was significantly higher than that of authentic CpG ODNs, such as CpG2006. This indicates that the bases at positions 18, 21, and 22 in the 3' region of ODN are dispensable for activity.

As described above, the "gtcgtt" motifs in authentic CpG ODNs are known as the TLR9 activating motif for human (Wang, X., et al., Vaccine, 2008. 26(15): p.1893-901), and CpG2006 has three motifs in the sequence. Meanwhile, only A603 has one motif among the ODNs tested by the inventors, and all the tested ODNs, including A603, showed similar activity levels, indicating that there are other optimal motifs or sites for activity against human TLR9 in the ODNs of the present invention. Furthermore, the data obtained by the inventors indicate that the "gtcgtt" motif in the ODNs of the present invention has a negligible contribution to the overall activity, provided that they retain the Cg-nn-Cg site in their 3' regions. The 3'-end of the ODN of the present invention requires g-n-Cg-nn-Cg as the core structure.

[0140]

As shown in FIGS. 5A and 5B, the ODN of the present invention, as well as the authentic TLR9 agonist CpG2395, could not activate both TLR7 and TLR8 signaling cascades, indicating that the ODN of the present invention are TLR9 agonists.

[0141]

Example 5

The partially dephosphorylated ODN of the present invention lacks TLR7 and TLR8 activity.

<ODN>

Single-stranded ODNs with partial phosphorothioate internucleotide linkages were prepared as described in Table 7.

[Table 7]

[0142]

ODN activity was analyzed by GFP expression in CAL-1/NF-κB-GFP cells. As shown in FIGS. 6A, 6B, and 6C, all tested ODNs that had base variations and/or internucleotide linkages at the 3' side at position 12 demonstrated similar levels of activity in NF-κB activation. This indicates that the nucleotide at position 12 is mutation-permissive and the internucleotide linkage at the 3' side of the nucleotide can be either a PO or a PS linkage.

[0143]

As shown in FIGS. 7A and 7B, ODN, as well as the authentic TLR9 agonist CpG2395, could not activate either TLR7 or TLR8 signaling, indicating that ODN were activators of TLR9.

[0144]

Taken together, based on Examples 1-5, it was shown that the ODN of the invention have 5'-tcgcaacgttt-n-cgacg-n-cg-nn-cg-3' (SEQ ID NO:2) as the core sequence. For maximum human TLR9 stimulating activity, the ODN preferably have 5'-tCgCaaCgttt-n-cgacg-n-Cg-nn-Cg-3' (SEQ ID NO:5) as the core structure.

In addition, since the ODNs of the present invention do not conform to the previously described rules, the ODNs of the present invention are different from the described authentic CpG ODNs and are a new type of TLR9 agonists.

[0145]

Example 6

Evaluation of TLR9 stimulation levels in human cells

<Stimulation of HAL-01 cells>

The human B-ALL cell line, HAL-01 cells, were purchased from DSMZ (catalog #ACC610). HAL-01 cells were maintained with 10% FBS-RPMI complete medium and stimulated with ODN for 24 hours at a dose of 1.0 μM. 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.

[0146]

<Stimulation of human PBMC>

The resulting human PBMCs (5× ¹⁰⁵ cells/200 μl) were stimulated with ODN for 24 hours at a dose of 0.1 μM or 0.3 μM. Cell proliferation was assessed using the WST-1 assay (Roche) according to the manufacturer's protocols. Culture supernatants were separated, and cytokine production was assessed by ELISA according to the manufacturer's protocols.

[0147]

Human B cells are known to express TLR9, and thus, a TLR9 agonist can activate human B cells. It was demonstrated that the B-ALL cell line was stimulated by a TLR9 agonist, and the surface expression of costimulatory molecules such as CD40 and CD86 was increased upon stimulation. As shown in FIG. 8A, the ODN of the present invention activated the B-ALL cell line, HAL-01 cells, as demonstrated by increased surface expression of CD40 and CD86.

It was demonstrated that the ODN of the present invention can induce the proliferation of human PBMCs and the production of inflammatory cytokines. As shown in FIGS. 8B and 8C, the ODN of the present invention could stimulate human PBMCs, which induced cell proliferation and the production of inflammatory cytokines such as IFN-α, IL-6, and IL-12.

[0148]

Example 7

Evaluation of TLR9 stimulation levels in mouse cells

<Stimulation of mouse spleen cells (splenocytes)>

The resulting splenocytes were stimulated with ODN at a dose of 0.03 μM for 24 hours. Culture supernatants were separated, and cytokine production in the culture supernatants was assessed by ELISA according to the manufacturer's protocols.

The resulting splenocytes were stimulated with ODN at various concentrations for 24 hours. Cell proliferation was assessed using the WST-1 assay (Roche) according to the manufacturer's protocols.

[0149]

It is known that a TLR9 agonist can induce the production of inflammatory cytokines and the proliferation of mouse splenocytes. As shown in FIG. 9A, the ODN of the present invention could induce the production of inflammatory cytokines such as TNF-α and IL-12. Furthermore, as shown in FIGS. 9B and 9C, the ODN induced the proliferation of mouse splenocytes. The activity of ODN was apparently higher than that of the authentic TLR9 agonists CpG2395 and CpG2006.

[0150]

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 were able to activate mouse splenocytes as well as human PBMCs. This indicates that a specific internucleotide PO linkage at a specific position, such as in the “tCgCaaCg” and “Cg-nn-Cg” structures, is important for optimal TLR9 activation in both mice and humans.

[0151]

Example 8

Antitumor efficacy of ODN in vivo

<CT26 cells>

The BALB/c mouse-derived colon carcinoma cell line CT26 was purchased from the American Type Culture Collection (ATCC, CRL-2638). CT26 cells were cultured in complete medium with 10% FBS-RPMI in a humidified atmosphere under 5% CO2 at 37°C.

[0152]

<Inoculation of CT26 cells into mice>

CT26 cells were isolated using 0.25% trypsin-EDTA in PBS. After suspension in the medium, the cells were passed through a 40 μM mesh strainer and counted. The cell concentration in the suspension was adjusted to 2 × 10 ⁶ cells/mL. BALB/c mice were inoculated with 100 μL of the cell suspension per mouse (2 × 10 ⁵ cells/mouse). The mouse back was shaved, and 100 μL of the CT26 cell suspension was injected subcutaneously into the right flank of the mice using a 28-gauge needle. Mice were maintained without treatment for two weeks until the tumor volume reached approximately 150 mm ³ . Tumor volume was determined every 2 or 3 days, and mice were divided into three groups based on tumor volume.

The tumor volume was calculated using the following formula:

Tumor volume (mm ³ ) = 1/2 (length × width ² )

[0153]

<Introduction to ODN>

Administration of ODN (40 μg/50 μl/mouse) into the peritumoral region began on the day of group assignment (day 0) and was repeated on day 2. Administration of ODN according to the present invention was performed only twice during the study. Tumor volume and body weight of each mouse in each group were determined every two or three days.

As shown in FIG. 10, administration of the ODN of the present invention clearly induced tumor regression in mice. On the 11th day, tumors in almost all mice were eliminated by administration of ODN (FIGS. 10A and 10B). No weight loss was observed in any of the groups of mice during the test (FIG. 10C). This indicates that the ODN (A601 and A602) have antitumor activity and have little toxicity. Considering the fact that A601, A602 and A603 showed the same activity in mouse splenocytes as shown in FIG. 9B, A603 will show the same high antitumor activity as A601 and A602. It was shown that the ODN of the present invention have antitumor activity.

[0154]

Example 9

Antitumor efficacy of ODN in vivo (2)

<CT26 cell inoculation and ODN administration>

CT26 cells were inoculated into the right flank of BALB/c mice as described above. Mice were maintained without treatment for two weeks until the tumor volume reached approximately 100 ^mm3 . Tumor volume was determined every 2 or 3 days, and mice were divided into three groups based on tumor volume. Administration of ODN (A601 and A602) (40 μg/50 μl/mouse) into the peritumoral area began on the day of group allocation (day 0) and was repeated on day 2. Administration of ODN according to the present invention was performed a total of two times during the study. As a negative control, PBS was administered instead of the ODN-containing solution. Tumor volume and body weight of each mouse in each group were determined every two or three days until tumor eradication was confirmed (day 14).

The tumor volume was calculated using the following formula:

Tumor volume (mm ³ ) = 1/2 (length × width ² )

[0155]

<Re-inoculation of CT26 cells into treated mice>

CT26 cells (2× ¹⁰ ) were reinoculated into the left flank of tumor-inoculated mice on day 14, confirming tumor clearance. The mice were then left untreated. Tumor volume in the left flank of each mouse was determined every 2 or 3 days until day 14 after reinoculation with CT26.

[0156]

As shown in FIG. 11A, administration of the ODN of the present invention clearly induced tumor eradication in mice. Furthermore, tumor eradication also occurred in mice administered with the ODN (FIG. 11B). While the tumor in the left flank grew in mice without treatment with the ODN of the present invention, all mice administered with the ODN eradicated the reintroduced tumor without any additional treatment within 2 weeks (FIG. 11B). This indicates that the ODN of the present invention can induce and form anti-tumor immunity memory.

[0157]

Example 10

In vivo efficacy of ODN against lung metastases

<CT26 lung metastasis model>

A CT26 cell suspension was prepared as described above. The suspension was adjusted to a final concentration of 2.5× ¹⁰⁶ cells/mL. To induce lung metastasis in CT26 cells, 200 μL of the cell suspension were injected intravenously (i.v.) into BALB/c mice (5× ¹⁰⁵ cells/mouse) from the tail vein using a 28-gauge needle (day 0). On day 2, ODN administration began (subcutaneous injection into the back skin, 40 μg/50 μL/mouse). The same administration dose was repeated on day 5. As a negative control, aPBS was administered instead of the ODN-containing solution.

Body weight and behavioral monitoring of mice were performed three times a week after CT26 cell transplantation. On day 18, the mice were sacrificed and lung weights were determined. Metastatic tumor nodules in each lung were also counted.

[0158]

As shown in FIG. 12, the lung weight of mice in the PBS-administered group increased significantly, and numerous tumor nodules were observed. In contrast, no such increase in lung weight was observed in mice in the group administered with the ODN of the present invention. Furthermore, only a small number of tumor nodules were observed in mice in the treatment group. This indicates that the ODN of the present invention can suppress the growth of metastatic malignant cells in the lung.

[0159]

Example 11

In vivo efficacy of ODN studied with varying routes of administration

<CT26 lung metastasis model>

Induction of CT26 cell metastases in the lungs was performed as described above. The day after CT26 cell injection, ODN A602 administration began. Two routes of administration were tested in this study. One was a subcutaneous (s.c.) injection into the back skin (25 μg/50 μl/mouse), while the other was an intradermal (i.d.) injection into the base of the ear (25 μg/20 μl/mouse). Administration at the same dose was repeated on days 3 and 5. After CT26 cell transplantation, body weight was measured and behavior was observed three times a week. On day 16, mice were sacrificed, and metastatic tumor nodules in each mouse lung were counted.

[0160]

<CT26 liver metastasis model>

CT26 cell suspensions were prepared as described above. The cell suspension was adjusted to a final concentration of 1.0 × 10 ⁶ cells/mL. To induce liver metastasis of CT26 cells, 100 μL of the cell suspension was injected into the spleen (1 × 10 ⁵ cells/mouse) as described below (day 0). Briefly, mice were anesthetized and the skin was shaved. A 0.5 cm abdominal incision was created adjacent to the spleen (the incision on the left flank was approximately 2 cm left of the abdominal midline). The resulting CT26 cell suspension was injected into the spleen using a 30G needle, which was left in the spleen for 5 minutes after injection. To prevent bleeding, the splenic blood vessels were tightly ligated with surgical sutures, and then splenectomy was performed with sharp scissors. The peritoneum and skin were sutured, and the mouse was heated under light. Recovery from anesthesia was monitored and confirmed. On day 2, the mice were divided into three groups based on body weight, and ODN A602 administration began. Two administration routes were tested in this study. One was a subcutaneous injection into the skin of the back (12.5 μg/50 μl/mouse), and the other was an intradermal injection into the base of the ear (12.5 μg/20 μl/mouse). The same administration dose was repeated on days 5, 8, and 12. Body weight was measured and behavioral observations were performed three times a week after CT26 cell transplantation. On day 20, the mice were sacrificed, and metastatic tumor nodules in each mouse liver were counted.

[0161]

As shown in FIG. 13A, numerous tumor nodules were observed in the PBS administration group. In contrast, only small numbers of tumor nodules were observed in both the subcutaneous and intradermal administration groups. Interestingly, intradermal administration to the base of the ear demonstrated better efficacy than subcutaneous administration. These results indicate that the ODNs of the present invention can block the growth of metastatic malignant cells in the lung and can achieve systemic efficacy.

As shown in FIG. 13B, severe liver metastases were also observed in the PBS treatment group. In contrast, no tumor nodules were detected in either the subcutaneous or intradermal treatment groups, indicating that the ODNs of the present invention can prevent the growth of metastatic malignant cells in the liver and can achieve systemic efficacy.

Taken together, the ODNs of the present invention can systemically block and eliminate the growth of metastatic tumors.

[0162]

Example 12

Activation of antitumor immunity by human PBMCs

<Co-cultivation of human B-ALL cells and human PBMC>

The resulting human PBMCs (5 × 10 ⁵ cells) were cocultured with the human B-ALL cell line RCH-ACV (5 × 10 ⁴ cells) together with ODN (0.1 μM) in 200 μl of complete medium 10% FBS-RPMI for 3 days, and the cocultured cells were isolated. RCH-ACV elimination by human PBMCs was assessed by the reduction in RCH-ACV amounts determined 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 using a flow cytometer. The presence of RCH-ACV in unstimulated PBMCs is set as 100% in FIG. 14B.

[0163]

<Co-cultivation of human colon carcinoma cells and human PBMC>

The resulting human PBMCs (5× ¹⁰⁵ cells) were cocultured with human colon carcinoma cell line COLO205 (ATCC, CCL-222) (5× ¹⁰⁴ cells) together with ODN (0.1 μM) in 200 μl of complete medium 10% FBS-RPMI for 3 days, and the cocultured cells were isolated. COLO205 elimination by human PBMCs was assessed by the reduction in COLO205 cell levels determined 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 by flow cytometry. The presence of COLO205 in unstimulated PBMCs is set to 100% in FIG. 15B.

[0164]

As shown in FIG. 14A, RCH-ACV cells were double-positive for CD19 and CD138, while no double-positive cells for CD19 and CD138 were observed in the obtained human PBMC alone. 8.6% of RCH-ACV cells were detected when human PBMC and RCH-ACV were cocultured under conditions without stimulation. In the presence of A601, A602, and A603, only 0.2% to 0.3% of RCH-ACV were detected in the cultured cells, indicating that human PBMC eliminated virtually all of the RCH-ACV cells in response to the ODN of the present invention. The efficiency of eliminating hematological malignant cells was further confirmed as shown in FIG. 14B.

[0165]

As shown in FIG. 15A, COLO205 cells were positive for CD24 and negative for CD45, while such cells were not observed in the obtained human PBMC alone. 19.2% of COLO205 cells were detected when human PBMC and COLO205 were cocultured under conditions without stimulation (culture without ODN). In the presence of A601, A602, and A603, only 0.5% to 0.9% of COLO205 were detected in the cultured cells, indicating that human PBMC eliminated virtually all of the COLO205 cells in response to the ODN of the present invention. The elimination efficiency was further confirmed as shown in FIG. 15B.

[0166]

Taken together, it was suggested that the activated human PBMCs with ODN of the present invention can eliminate both hematological malignancies and solid tumors.

[0167]

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 assessed by the WST-1 assay.

PBMCs were cocultured with malignant cells (RCH-ACV or COLO205) together with the ODN of the present invention (0.1 μM) for 3 days, as performed in Example 12. The elimination of malignant cells by human PBMCs was studied. The existence of malignant cells in PBMCs without stimulation is taken as 100% in FIG. 16B.

[0168]

As previously shown in FIG. 6, all of the ODNs, A601, A602, A601G, A611A, A602G, and A612A, demonstrated similar levels of activity in activating TLR9 when tested in CAL-1/NF-B-GFP cells. To assess whether the ODNs exhibited the same features in primary human PBMCs and in mouse cells, the ODNs A601G, A611A, A602G, and A612A were evaluated in several assay systems in which A601 and A602 had previously been tested. As shown in FIG. 16A, all of the tested ODNs of the present invention induced cell proliferation at a similar level in both human PBMCs and mouse splenocytes. Furthermore, as shown in FIG. 16B, all of the tested ODNs of the present invention induced significant elimination of malignant cells in the presence of human PBMCs. These results indicate that activated human PBMCs with ODN A601G, A611A, A602G, and A612A can eliminate both hematological malignancies and solid tumors, as well as A601 and A602.

[0169]

Taken together, it is expected that all of the ODNs of the present invention that exhibited similar levels of activity to A601 or A602 in CAL-1/NF-κB-GFP cells will induce the elimination of human malignant cells by human PBMCs, as well as anti-tumor immune responses in mice.

[Industrial applicability]

[0170]

The present invention relates to novel oligonucleotides and their derivatives. Furthermore, the present invention relates to a pharmaceutical composition comprising an oligonucleotide(s) selected from said oligonucleotides. The present invention also relates to a method for treating target diseases by administering an oligonucleotide(s) selected from said oligonucleotides.

--->

SEQUENCE LIST:

<110> SBI Biotech Co., Ltd.

<120> New TLR9 agonists

<130> FP4645PCT

<150> JP 2020-002715

<151> 2020-01-10

<160> 63

<170> PatentIn version 3.5

<210> 1

<211> 11

<212> DNA

<213> Artificial sequence

<220>

<223> common sequence motif

<400> 1

tcgcaacgttt 11

<210> 2

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> common sequence motif

<220>

<221> additional feature

<222> (12)..(12)

<223> n means any base

<220>

<221> additional feature

<222> (18)..(18)

<223> n means any base

<220>

<221> additional feature

<222> (21)..(21)

<223> n means any base

<220>

<221> additional feature

<222> (22)..(22)

<223> n means any base

<400> 2

tcgcaacgtt tncgacgncg nncg 24

<210> 3

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> common sequence motif

<400> 3

tcgcaacgtt trcgacgkcg bdcg 24

<210> 4

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> common sequence motif

<220>

<221> additional feature

<222> (22)..(22)

<223> n means any base

<400> 4

tcgcaacgtt trcgacgkcg bncgr 25

<210> 5

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> widespread partially phosphorothioate site

<220>

<221> additional feature

<222> (12)..(12)

<223> n means any base

<220>

<221> additional feature

<222> (18)..(18)

<223> n means any base

<220>

<221> additional feature

<222> (21)..(21)

<223> n means any base

<220>

<221> additional feature

<222> (22)..(22)

<223> n means any base

<220>

<221> modified base

<222> (1)..(1)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (3)..(3)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (5)..(6)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (8)..(18)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (20)..(22)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 5

tcgcaacgtt tncgacgncg nncg 24

<210> 6

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A001

<220>

<221> modified base

<222> (1)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 6

tcgcaacgtt tgcgacgtcg gtcga 25

<210> 7

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A002

<220>

<221> modified base

<222> (1)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 7

tcgcaacgtt tgcgacggcg ctcga 25

<210> 8

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A003

<220>

<221> modified base

<222> (1)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 8

tcgcaacgtt tgcgacgtcg ttcga 25

<210> 9

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A004

<220>

<221> modified base

<222> (1)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 9

tcgcaacgtt tgcgacggcg ttcga 25

<210> 10

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> A003#delA

<220>

<221> modified base

<222> (1)..(23)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 10

tcgcaacgtt tgcgacgtcg ttcg 24

<210> 11

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A003#endG

<220>

<221> modified base

<222> (1)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 11

tcgcaacgtt tgcgacgtcg ttcgg 25

<210> 12

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A011

<220>

<221> modified base

<222> (1)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 12

tcgcaacgtt tacgacgtcg gtcga 25

<210> 13

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A012

<220>

<221> modified base

<222> (1)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 13

tcgcaacgtt tacgacggcg ctcga 25

<210> 14

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A013

<220>

<221> modified base

<222> (1)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 14

tcgcaacgtt tacgacgtcg ttcga 25

<210> 15

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A014

<220>

<221> modified base

<222> (1)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 15

tcgcaacgtt tacgacggcg ttcga 25

<210> 16

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A103

<220>

<221> modified base

<222> (1)..(1)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (3)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 16

tcgcaacgtt tgcgacgtcg ttcga 25

<210> 17

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A203

<220>

<221> modified base

<222> (1)..(1)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (3)..(6)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (8)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 17

tcgcaacgtt tgcgacgtcg ttcga 25

<210> 18

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A303

<220>

<221> modified base

<222> (1)..(1)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (3)..(3)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (5)..(6)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (8)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 18

tcgcaacgtt tgcgacgtcg ttcga 25

<210> 19

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A403

<220>

<221> modified base

<222> (1)..(1)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (3)..(3)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (5)..(12)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (14)..(15)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (17)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 19

tcgcaacgtt tgcgacgtcg ttcga 25

<210> 20

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A503

<220>

<221> modified base

<222> (1)..(1)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (3)..(3)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (5)..(12)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (14)..(15)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (17)..(22)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (24)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 20

tcgcaacgtt tgcgacgtcg ttcga 25

<210> 21

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A603

<220>

<221> modified base

<222> (1)..(1)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (3)..(3)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (5)..(6)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (8)..(18)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (20)..(22)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (24)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 21

tcgcaacgtt tgcgacgtcg ttcga 25

<210> 22

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A703

<220>

<221> modified base

<222> (1)..(1)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (3)..(3)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (5)..(6)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (8)..(12)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (14)..(15)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (17)..(18)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (20)..(22)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (24)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 22

tcgcaacgtt tgcgacgtcg ttcga 25

<210> 23

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A601

<220>

<221> modified base

<222> (1)..(1)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (3)..(3)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (5)..(6)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (8)..(18)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (20)..(22)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (24)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 23

tcgcaacgtt tgcgacgtcg gtcga 25

<210> 24

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A602

<220>

<221> modified base

<222> (1)..(1)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (3)..(3)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (5)..(6)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (8)..(18)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (20)..(22)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (24)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 24

tcgcaacgtt tgcgacggcg ctcga 25

<210> 25

<400> 25

000 25

<210> 26

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A604

<220>

<221> modified base

<222> (1)..(1)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (3)..(3)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (5)..(6)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (8)..(18)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (20)..(22)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (24)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 26

tcgcaacgtt tgcgacggcg ttcga 25

<210> 27

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A605

<220>

<221> modified base

<222> (1)..(1)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (3)..(3)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (5)..(6)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (8)..(18)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (20)..(22)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (24)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 27

tcgcaacgtt tgcgacgccg ttcga 25

<210> 28

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A606

<220>

<221> modified base

<222> (1)..(1)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (3)..(3)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (5)..(6)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (8)..(18)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (20)..(22)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (24)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 28

tcgcaacgtt tgcgacggcg tacga 25

<210> 29

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A607

<220>

<221> modified base

<222> (1)..(1)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (3)..(3)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (5)..(6)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (8)..(18)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (20)..(22)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (24)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 29

tcgcaacgtt tgcgacggcg tgcga 25

<210> 30

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A611

<220>

<221> modified base

<222> (1)..(1)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (3)..(3)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (5)..(6)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (8)..(18)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (20)..(22)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (24)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 30

tcgcaacgtt tacgacgtcg gtcga 25

<210> 31

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A612

<220>

<221> modified base

<222> (1)..(1)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (3)..(3)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (5)..(6)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (8)..(18)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (20)..(22)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (24)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 31

tcgcaacgtt tacgacggcg ctcga 25

<210> 32

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A613

<220>

<221> modified base

<222> (1)..(1)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (3)..(3)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (5)..(6)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (8)..(18)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (20)..(22)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (24)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 32

tcgcaacgtt tacgacgtcg ttcga 25

<210> 33

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A601G

<220>

<221> modified base

<222> (1)..(1)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (3)..(3)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (5)..(6)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (8)..(11)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (13)..(18)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (20)..(22)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (24)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 33

tcgcaacgtt tgcgacgtcg gtcga 25

<210> 34

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A611A

<220>

<221> modified base

<222> (1)..(1)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (3)..(3)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (5)..(6)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (8)..(11)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (13)..(18)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (20)..(22)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (24)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 34

tcgcaacgtt tacgacgtcg gtcga 25

<210> 35

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A602G

<220>

<221> modified base

<222> (1)..(1)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (3)..(3)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (5)..(6)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (8)..(11)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (13)..(18)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (20)..(22)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (24)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 35

tcgcaacgtt tgcgacggcg ctcga 25

<210> 36

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A612A

<220>

<221> modified base

<222> (1)..(1)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (3)..(3)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (5)..(6)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (8)..(11)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (13)..(18)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (20)..(22)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (24)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 36

tcgcaacgtt tacgacggcg ctcga 25

<210> 37

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A603G?

<220>

<221> modified base

<222> (1)..(1)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (3)..(3)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (5)..(6)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (8)..(11)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (13)..(18)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (20)..(22)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (24)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 37

tcgcaacgtt tgcgacgtcg ttcga 25

<210> 38

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A613A

<220>

<221> modified base

<222> (1)..(1)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (3)..(3)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (5)..(6)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (8)..(11)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (13)..(18)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (20)..(22)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (24)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 38

tcgcaacgtt tacgacgtcg ttcga 25

<210> 39

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A601GendG

<220>

<221> modified base

<222> (1)..(1)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (3)..(3)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (5)..(6)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (8)..(11)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (13)..(18)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (20)..(22)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (24)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 39

tcgcaacgtt tgcgacgtcg gtcgg 25

<210> 40

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A611AendG

<220>

<221> modified base

<222> (1)..(1)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (3)..(3)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (5)..(6)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (8)..(11)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (13)..(18)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (20)..(22)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (24)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 40

tcgcaacgtt tacgacgtcg gtcgg 25

<210> 41

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A602GendG

<220>

<221> modified base

<222> (1)..(1)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (3)..(3)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (5)..(6)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (8)..(11)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (13)..(18)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (20)..(22)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (24)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 41

tcgcaacgtt tgcgacggcg ctcgg 25

<210> 42

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A612AendG

<220>

<221> modified base

<222> (1)..(1)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (3)..(3)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (5)..(6)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (8)..(11)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (13)..(18)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (20)..(22)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (24)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 42

tcgcaacgtt tacgacggcg ctcgg 25

<210> 43

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A603GendG

<220>

<221> modified base

<222> (1)..(1)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (3)..(3)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (5)..(6)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (8)..(11)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (13)..(18)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (20)..(22)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (24)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 43

tcgcaacgtt tgcgacgtcg ttcgg 25

<210> 44

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> A613AendG

<220>

<221> modified base

<222> (1)..(1)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (3)..(3)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (5)..(6)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (8)..(11)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (13)..(18)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (20)..(22)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (24)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 44

tcgcaacgtt tacgacgtcg ttcgg 25

<210> 45

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> CpG2395

<220>

<221> modified base

<222> (1)..(21)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 45

tcgtcgtttt cggcgcgcgc cg 22

<210> 46

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> CpG685

<220>

<221> modified base

<222> (1)..(20)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 46

tcgtcgacgt cgttcgttct from 21

<210> 47

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> M362

<220>

<221> modified base

<222> (1)..(24)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 47

tcgtcgtcgt tcgaacgacg ttgat 25

<210> 48

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> D60-1

<220>

<221> modified base

<222> (1)..(29)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 48

tcgaacgttc gaacgttcga acgttcgaat 30

<210> 49

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223>DV093

<220>

<221> modified base

<222> (1)..(1)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (3)..(17)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 49

tcgtgcatcg atgcaacg 18

<210> 50

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> DV093C

<220>

<221> modified base

<222> (1)..(1)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (3)..(13)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (15)..(16)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 50

tcgtgcatcg atgcaacg 18

<210> 51

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> DV094

<220>

<221> modified base

<222> (1)..(17)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 51

aacaacaacg ttgttgtt 18

<210> 52

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> DV094C

<220>

<221> modified base

<222> (1)..(2)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (4)..(5)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (7)..(8)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<220>

<221> modified base

<222> (10)..(17)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 52

aacaacaacg ttgttgtt 18

<210> 53

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> CpG2006

<220>

<221> modified base

<222> (1)..(23)

<223> modified with phosphorothioate at the 3'-internucleotide linkage

<400> 53

tcgtcgtttt gtcgttttgt cgtt 24

<210> 54

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Sequence of the invention

<400> 54

tcgcaacgtt tgcgacgtcg gtcga 25

<210> 55

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Sequence of the invention

<400> 55

tcgcaacgtt tgcgacggcg ctcga 25

<210> 56

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Sequence of the invention

<400> 56

tcgcaacgtt tgcgacgtcg ttcga 25

<210> 57

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Sequence of the invention

<400> 57

tcgcaacgtt tgcgacggcg ttcga 25

<210> 58

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Sequence of the invention

<400> 58

tcgcaacgtt tgcgacgtcg ttcg 24

<210> 59

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Sequence of the invention

<400> 59

tcgcaacgtt tgcgacgtcg ttcgg 25

<210> 60

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Sequence of the invention

<400> 60

tcgcaacgtt tacgacgtcg gtcga 25

<210> 61

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Sequence of the invention

<400> 61

tcgcaacgtt tacgacggcg ctcga 25

<210> 62

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Sequence of the invention

<400> 62

tcgcaacgtt tacgacgtcg ttcga 25

<210> 63

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Sequence of the invention

<400> 63

tcgcaacgtt tacgacggcg ttcga 25

<---

Claims (80)

1. A single-chain oligonucleotide agonist of Toll-like receptor 9 (TLR9), characterized in that the oligonucleotide contains a sequence motif selected from the group consisting of the following: 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. A single-stranded TLR9 oligonucleotide agonist according to claim 1, wherein the internucleotide linkage(s) in the oligonucleotide is partially or fully chemically modified. 3. A single-stranded TLR9 oligonucleotide agonist according to claim 2, wherein the chemically modified internucleotide linkage is phosphorothioate. 4. A single-stranded TLR9 oligonucleotide agonist according to claim 3, characterized in that the oligonucleotide comprises a partially phosphorothioate oligonucleotide region selected from the group consisting of 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); where the capital letter denotes a nucleoside without a modified internucleotide linkage on the 3' side and the lowercase letter denotes a nucleoside with an internucleotide phosphorothioate linkage on the 3' side. 5. A single-stranded TLR9 oligonucleotide agonist according to claim 3, characterized in that the oligonucleotide comprises a partially phosphorothioate oligonucleotide region selected from the group consisting of 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), where the capital letter denotes a nucleoside without a modified internucleotide linkage at the 3' side, and the lowercase letter denotes a nucleoside with a phosphorothioate internucleotide linkage at the 3' side. 6. A single-stranded TLR9 oligonucleotide agonist according to any one of claims 1 to 5, characterized in that the oligonucleotide consists of DNA. 7. A single-stranded TLR9 oligonucleotide agonist according to any one of claims 1 to 6, characterized in that the oligonucleotide is contained in an expression vector. 8. A single-stranded TLR9 oligonucleotide agonist according to any one of claims 1 to 6, characterized in that the oligonucleotide is circular. 9. A single-stranded TLR9 oligonucleotide agonist according to any one of claims 1 to 6, characterized in that the oligonucleotide is linear. 10. A single-stranded TLR9 oligonucleotide agonist according to claim 9, characterized in that the 3’-end of the linear oligonucleotide does not have phosphoric acid. 11. A double-stranded oligonucleotide that can bind to TLR9, comprising a single-stranded TLR9 agonist oligonucleotide according to any one of claims 1 to 6 and an oligonucleotide chain complementary thereto. 12. A conjugate of a single-stranded TLR9 agonist oligonucleotide according to any one of claims 1 to 10 or a double-stranded oligonucleotide according to claim 11 and at least one active molecule for the treatment or prevention of a target disease or disorder selected from the group consisting of a neoplasm, an infection, a Th2/Th17-mediated disease, a primary immunodeficiency disease and post-traumatic stress disorder (PTSD). 13. The conjugate according to claim 12, wherein the active molecule is selected from the group consisting of (poly)peptides/antibodies and nucleic acids/oligonucleotides. 14. The conjugate according to claim 12 or 13, wherein the single-stranded oligonucleotide according to any one of claims 1-10 or the double-stranded oligonucleotide according to claim 11 is conjugated to at least one active molecule via a linker. 15. The conjugate of claim 14, 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, cis, trans-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. 16. A pharmaceutical composition for the prevention or treatment of a target disease or disorder, comprising a therapeutically effective amount of a single-stranded TLR9 agonist oligonucleotide according to any one of claims 1-10, a double-stranded polynucleotide according to claim 11, or a conjugate according to any one of claims 12-15, wherein the target disease or disorder is a disease or disorder selected from the group consisting of neoplasms, an infectious disease, a Th2/Th17-mediated disease, a primary immunodeficiency disease, and post-traumatic stress disorder (PTSD). 17. A means for activating TLR9 in an individual, comprising a therapeutically effective amount of a single-stranded TLR9 agonist oligonucleotide according to any one of claims 1-10, a double-stranded oligonucleotide according to claim 11, or a conjugate according to any one of claims 12-15. 18. The pharmaceutical composition of claim 16, wherein the target disease or disorder is a neoplasm selected from the group consisting of a malignant neoplasm, an epithelial neoplasm or a tumor of a hematopoietic organ, sarcoma, mesothelioma, a benign tumor, dysplasia and metaplasia. 19. The pharmaceutical composition of claim 16, wherein the target disease or disorder is an infectious disease caused by microorganisms including viruses, bacteria or fungi. 20. The pharmaceutical composition of claim 16, wherein the target disease or disorder is a Th2/Th17 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. 21. The pharmaceutical composition of claim 16, wherein the target disease or disorder is a primary immunodeficiency disease caused by IRAK4 deficiency, MyD88 deficiency, Unc93B deficiency, or TLR mutations. 22. The pharmaceutical composition according to claim 16, wherein the composition additionally contains at least one active ingredient. 23. The pharmaceutical composition of claim 16, wherein the composition is administered together with at least one active ingredient. 24. The pharmaceutical composition according to claim 22 or 23, 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 agents; antifungal agents; antiviral agents; antiparasitic drugs; antibody drugs for neutralizing a toxin; agonists of other TLRs and combinations thereof. 25. The pharmaceutical composition of claim 16, wherein the composition is administered to an individual by a dosing method selected from the group consisting of enteral administration, parenteral administration, topical administration, and inhalation. 26. The pharmaceutical composition according to claim 25, wherein the individual is a human. 27. The pharmaceutical composition of claim 26, wherein the composition is administered to an individual in an amount of 2 to 8 mg/day. 28. The use of a single-stranded TLR9 agonist oligonucleotide according to any one of claims 1-10, a double-stranded oligonucleotide according to claim 11, a conjugate according to any one of claims 12-15, or a pharmaceutical composition according to claim 16 for the manufacture of a medicament for the treatment or prevention of any one selected from the group consisting of neoplasms, infection, Th2/Th17-mediated disease, primary immunodeficiency disease, and post-traumatic stress disorder (PTSD). 29. The pharmaceutical composition of claim 16, wherein the composition is administered before or after adoptive immune cell therapy or surgical treatment, including radiation therapy, cryoablation, radiofrequency ablation and photodynamic therapy (PDT). 30. A method for treating or preventing a target disease or disorder in an individual, comprising administering to the individual a single-stranded TLR9 agonist oligonucleotide according to any one of claims 1-10, a double-stranded oligonucleotide according to claim 11, a conjugate according to any one of claims 12-15, or a pharmaceutical composition according to claim 16, wherein the target disease or disorder is a disease or disorder selected from the group consisting of a neoplasm, an infection, a Th2/Th17-mediated disease, a primary immunodeficiency disease, and post-traumatic stress disorder (PTSD); preferably, wherein the target disease or disorder is treated or prevented by activating NF-κB via the oligonucleotide in the individual. 31. The method of claim 30, wherein the target disease or disorder is a neoplasm selected from the group consisting of a malignant neoplasm, an epithelial neoplasm or a tumor of a hematopoietic organ, sarcoma, mesothelioma, a benign tumor, dysplasia and metaplasia. 32. The method of claim 30, wherein the target disease or disorder is an infectious disease caused by microorganisms including viruses, bacteria or fungi. 33. The method of claim 30, wherein the target disease or disorder is a Th2/Th17 mediated 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. 34. The method of claim 30, wherein the target disease or disorder is a primary immunodeficiency disease caused by IRAK4 deficiency, MyD88 deficiency, Unc93B deficiency, or TLR mutations. 35. The use of a single-stranded oligonucleotide according to any one of claims 1 to 10, a double-stranded oligonucleotide according to claim 11, a conjugate according to any one of claims 12 to 15, or a pharmaceutical composition according to claim 16 for the treatment or prevention of a target disease or disorder selected from the group consisting of a neoplasm, an infection, a Th2/Th17-mediated disease, a primary immunodeficiency disease, and post-traumatic stress disorder (PTSD).