TW201622751A - Application of using nucleic acid polysaccharide complex with innate immunity activity as antineoplastic drug - Google Patents

Abstract

The present invention provides an anticancer agent for use in a single dosage. Specifically, this invention provides an anticancer agent including a complex. The complex comprises: (a) deoxy oligonucleotide, which includes humanized K-type CpG deoxy oligonucleotide and poly-deoxy adenylate, wherein the poly-deoxy adenylate is configured at the 3' side of the humanized K-type CpG deoxy oligonucleotide; and (b) [beta]-1,3-glucan. Preferably, the complex is characterized in that: the anticancer agent is medicated when there is no cancer antigen. Furthermore, the anticancer agent is medicated in a manner of transferring to the reticuloendothelial system and/or lymph node. The reticuloendothelial system and/or lymph node may include tumors and macrophages. The reticuloendothelial system may include spleen and/or liver.

Description

Application of nucleic acid polysaccharide complex with immunostimulating activity as antitumor drug

The present invention relates to a novel cancer treatment.

A CpG ODN (CpG ODN, CpG Oligonucleotide) is a short (about 20 base pairs) single-stranded synthetic DNA (Deoxyribonucleic Acid) fragment containing an immunologically active CpG motif. It is a potent agonist of Toll receptor 9 (TLR9) and is used to activate dendritic cells (DCs, Dendritic Cells) and B cells to produce type I interferons (IFNs, Interferon). Type I) and inflammatory cytokines (Non-Patent Documents 1 and 2), including the Th1 type humoral and cellular immune response adjuvants including the cytotoxic T lymphocyte reaction (CTL) Action (Non-Patent Documents 3 and 4). Therefore, CpG ODN has been regarded as an immunotherapeutic agent which is likely to be effective against infection, cancer, asthma, and hay fever (Non-Patent Documents 2 and 5).

There are at least four types of CpG ODNs having different skeleton sequences and immunosynthesis characteristics (Non-Patent Document 6). Type D (also known as Type A) CpG ODN typically comprises a phosphodiester (PO, Phosphodiester) backbone, a Phosphorothioate (Poly G tail) and a palindrome of CpG. The phantom activates plasma-like DCs (pDCs, Plasmacytoid dendritic cells) to produce a large amount of IFN-α, but does not induce pDC maturation and B cell activation (Non-Patent Documents 7 and 8). The other three types of ODNs include the PS skeleton. K-type (also known as type B) CpG ODN typically contains a plurality of CpG motifs of non-palindromic structure, strongly activates B cells to produce IL-6, and activates pDCs to mature them. However, IFN-α is hardly produced (Non-Patent Documents 8 and 9). In recent years, the CpG ODNs of the C and P types developed by the industry contain one and two palindrome CpG sequences, respectively, both of which can activate B cells as K-type, and activate pDCs as D-type. However, C-type CpG ODN induces IFN-α production weaker than P-type CpG ODN (Non-Patent Documents 10-12). Patent Document 1 describes a large number of excellent K-type CpG ODNs.

It is revealed that D-type and P-type CpG ODNs respectively form a high-order structure: a Hoogsteen base pair which forms a parallel four-strand structure called G-tetrads (G-tetrazed), and Watson-Crick base pairs between the palindrome structure and the trans-palindrome, which are necessary for the production of strong IFN-α due to pDCs (non- Patent Documents 12 to 14). Such a high-order structure seems to be necessary for the localization in the early endosome or via TLR9, but these are affected by the diversity and precipitation of the product, and the result hinders its clinical application (Non-Patent Document 15) ). Therefore, generally only K-type and C-type CpG ODN can be used as an immunotherapeutic agent and a vaccine adjuvant for humans (Non-Patent Documents 16 and 17). Regarding the K-type CpG ODN, the immunogenicity of a vaccine targeting infectious diseases and cancer is improved in human clinical trials (Non-Patent Documents 6 and 16), but in order to achieve an optimal adjuvant effect, it is required The chemical and physical link between the antigen and the K-type CpG ODN. These results show that there are advantages and disadvantages of CpG ODNs of four types (K, D, P, and C), and it is expected to develop an "integration" that activates both B cells and pDCs without aggregation. All in one)" CpG ODN.

As a soluble β-1,3-glucan from SPizophyllum commune, SPG (Schizophyllan) is used as activating agent for radiation therapy for cervical cancer patients in Japan for the last 30 years. Licensed medicine (Non-Patent Document 18). Similarly, lentinan (LNT, Lentinan), which is a soluble β-1,3-glucan from shiitake mushroom, was approved in 1985, and it was used in combination with fluoropyrimidine-based agents for patients who were unable to relapse and have recurrence of gastric cancer. Use (Non-Patent Documents 19 and 20). Revealing that β-1,3-glucan will be combined with poly Deoxyadenosine (dA) forms a complex of a triple helix structure (Non-Patent Document 21).

Patent Documents 2 to 4 disclose the use of a water-soluble complex of β-1,3-glucan and a nucleic acid (gene) of Schizophyllum polysaccharide as a gene vector. It is described in these documents that by forming the complex, the antisense effect of the gene and the tolerance to the nucleic acid decomposing enzyme (nuclease) can be enhanced.

Patent Document 5 discloses that by using a polysaccharide having a β-1,3-bond as a carrier (transfection agent), it is possible to increase the inclusion of a CpG sequence and to replace a phosphodiester bond with a phosphorothioate bond or two. The role of immunostimulatory oligonucleotides for phosphorothioate linkages.

Patent Document 6 describes an immunostimulating complex comprising an immunostimulatory oligonucleotide and a β-1,3-peptide having a long-chain β-1,6-glucosidic side chain. Glucan.

The present inventors have previously revealed that a mouse and a humanized CpG ODN having a phosphodiester bond at the 5' end, which is complexed with SPG, enhances cytokine production, and It functions as an influenza vaccine adjuvant or a preventive or therapeutic agent for a Th2 cell-related disease (Non-Patent Documents 22 and 23, and Patent Document 7). When poly(dA) is added to the 5' end of each of K-type and D-type CpG to form a complex with SPG, the characteristics of K-type and D-type are maintained, respectively, and the activity thereof is enhanced. However, it is difficult to achieve high yields of CpG-SPG complexes that are more efficient, cost effective, and that are oriented to preclinical and clinical development. In recent years, it has been revealed that when CpG ODN is bonded to a poly(dA) having a phosphorothioate bond, the formation of the composite rises to nearly 100% (Non-Patent Document 24). However, a thorough test for optimizing the optimal humanized CpG sequence and using a factor to obtain "integration" activity of four types of CpG ODN has not been performed.

Patent Document 8 discloses a method for producing a ternary complex of an antigen/CpG oligonucleotide/β-1,3-glucan system.

Synthetic nucleic acid CpG oligodeoxynucleoside as a ligand for Toll receptor 9 (TLR9) Acid (CpG ODN) has strong natural immune activation ability, and it is expected as a vaccine adjuvant. Further, since it has antitumor activity when administered in a single dose, CpG ODN is also expected as an immunotherapeutic agent against cancer. However, although the previous CpG ODN has antitumor activity, it can only be exerted by direct administration to a tumor, and it is considered to be difficult to apply to the clinic. In fact, it is considered difficult to directly administer a drug to a tumor at an initial stage on a clinical site. In addition, surgical treatment is also required in the deep, and the obstacles are high.

Recently, the present inventors conducted development of a novel TLR9 ligand (K3-SPG) having a β-glucan-encapsulated CpG ODN using a polysaccharide (PCT Application (PCT/JP2014/074835)). K3-SPG does not form agglomerates, strongly activating natural immunity compared to the previous type of CpG ODN, and exhibits a strong adjuvant effect by experiments using mice. Furthermore, it was clarified that K3-SPG not only induced strong acquired immunity to mice but also cynomolgus macaques, and successfully overcome the difference in reactivity between mice and primates that were previously worried.

Thus, although the use of the CpG ODN as an adjuvant is highly anticipated, it is not clear whether it can be used alone as a medicine.

In the case of cancer treatment, cancer-associated antigens recognized by cytotoxic T cells have been identified since 1991 and reported (Non-Patent Document 25: van der Bruggen et al. Science (New York, NY) 254, 1643-1647 (1991)), a large number of cancer-associated antigens are identified at the molecular level, and clinical applications of cancer immunotherapy with such targets are achieved (Non-Patent Document 26: Jager, E., et al. The Journal of experimental Medicine 187, 265-270 (1998); Non-Patent Document 27: Jager, D. et al. Journal of clinical pathology 54, 669-674 (2001). Non-Patent Document 28: Imai, K., et al. British journal of cancer 104,300 -307 (2011); Non-Patent Document 29: Kang, X., et al. The Journal of Immunology 155, 1343-1348 (1995)). In particular, cancer immunotherapy, which is of concern, was first recognized by the US Food and Drug Administration (FDA) in April 2010 for patients with prostate cancer. A cancer vaccine Provenge which exhibits antigen using antigen of autologous peripheral blood (Non-Patent Document 30: Cancer vaccine approval could open floodgates. Nature medicine 16, 615-615 (2010); Non-Patent Document 31: Higano, CS, et al. Cancer 115, 3670-3679 (2009)). Thereafter, the inhibitory antibody Ipilimumab (ipilimumab) against cytotoxic T lymphocyte antigen 4 (CTLA-4), which is an inhibitory molecule of T lymphocyte activation, 2011 5 It is recognized in the United States for malignant melanoma patients in the United States (Non-Patent Document 32: Phan, GQ, et al. Proc. Natl. Acad. Sci. USA 100, 8372-8377 (2003); Non-Patent Document 33: Camacho, LH, Et al. Journal of clinical oncology: official journal of the American Society of Clinical Oncology 27, 1075-1081 (2009); Non-Patent Document 34: Hodi, FS, et al. New England Journal of Medicine 363, 711-723 (2010) . Further, nivolumab, a PD-1 (Programmed Cell Death-1) receptor inhibitor of an immunoreactive inhibitor, is in a clinical trial stage (Non-Patent Document 35: AZIJLI, K., et al. Anticancer) Research 34, 1493-1505 (2014); Non-Patent Document 36: Okazaki, T., et al. Nature Immunology 14, 1212-1218 (2013); Non-Patent Document 37: Ishida, Y., et al. The EMBO journal 11, 3887-3895 (1992); Non-Patent Document 38: Topalian, SL, et al. The New England journal of medicine 366, 2443-2454 (2012)).

For the formation of an environment in which dendritic cells effectively guide the anticancer effect, it is necessary to display a pattern molecule of natural immunity derived from the anticancer action of the inflammatory part (Non-Patent Document 39: Chiba, S., et al. Nature immunology 13, 832-842 (2012)). Although the above molecular group and the process associated therewith are not specific, the dendritic cells activate lymphocytes such as CD4, CD8, and NK (Non-Patent Document 40: Engelhardt, JJ, et al. Cancer cell 21, 402-417 (2012) ). Tumor infiltrating macrophages (Tumor Associated Macrophages) are the culprit of the inflammatory response. It is known (Non-Patent Document 41: Huang, Y., et al. Cancer cell 19, 1-2 (2011)). On the other hand, dendritic cells that function as a playmaker for anti-cancer immunity are also the same as the macrophage cells (Non-Patent Document 42: Huang, Y., et al. Proc. Natl. Acad) .Sci. USA 109, 17561-17566 (2012)). Although the method of converting these into anti-cancer directivity has not been found, it is believed that tumors have evaded immunity due to complicated factors. Both TAM and dendritic cells are governed by inflammation and pattern recognition responses (Non-Patent Document 43: Garaude, J., et al. Science translational medicine 4, 120ra 116 (2012); Non-Patent Document 44: Martinez-Pomares, L. Et al. Trends in immunology 33, 66-70 (2012)). Immunological effector cells and cancer cells are required to be attacked by cancer cells (Non-Patent Document 40: Engelhardt, JJ, et al. Cancer cell 21, 402-417 (2012); Non-Patent Document 45: Palucka, K. et al. Nature reviews. Cancer 12, 265-277 (2012)).

[Previous Technical Literature] [Patent Literature]

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[Patent Document 2] WO 01/034207 A1

[Patent Document 3] WO 02/072152 A1

[Patent Document 4] Japanese Patent Laid-Open Publication No. 2004-107272

[Patent Document 5] WO 2004/100965 A1

[Patent Document 6] Japanese Patent Laid-Open Publication No. 2007-70307

[Patent Document 7] Japanese Patent Laid-Open Publication No. 2008-100919

[Patent Document 8] Japanese Patent Laid-Open Publication No. 2010-174107

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[Non-Patent Document 19] Oba, K. et al., J. Individual patient based meta-analysis of lentinan for unresectable/recurrent gastric cancer. Anticancer Res., 2009, 29, 2739-2746.

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[Non-Patent Document 24] Minari, J., et al. Bioconjugate chemistry 22, 9-15 (2011).

[Non-Patent Document 25] van der Bruggen et al. Science (New York, N. Y.) 254, 1643-1647 (1991)

[Non-Patent Document 26] Jager, E., et al. The Journal of experimental medicine 187, 265-270 (1998)

[Non-Patent Document 27] Jager, D. et al. Journal of clinical pathology 54, 669-674 (2001).

[Non-Patent Document 28] Imai, K., et al. British journal of cancer 104, 300- 307 (2011)

[Non-Patent Document 29] Kang, X., et al. The Journal of Immunology 155, 1343-1348 (1995)

[Non-Patent Document 30] Cancer vaccine approval could open floodgates. Nature medicine 16, 615-615 (2010)

[Non-Patent Document 31] Higano, C. S., et al. Cancer 115, 3670-3679 (2009)

[Non-Patent Document 32] Phan, G. Q., et al. Proc. Natl. Acad. Sci. U. S. A. 100, 8372-8377 (2003)

[Non-Patent Document 33] Camacho, L. H., et al. Journal of clinical oncology: official journal of the American Society of Clinical Oncology 27, 1075-1081 (2009)

[Non-Patent Document 34] Hodi, F. S., et al. New England Journal of Medicine 363, 711-723 (2010)

[Non-Patent Document 35] AZIJLI, K., et al. Anticancer Research 34, 1493-1505 (2014)

[Non-Patent Document 36] Okazaki, T., et al. Nature immunology 14, 1212-1218 (2013)

[Non-Patent Document 37] Ishida, Y., et al. The EMBO journal 11, 3887-3895 (1992)

[Non-Patent Document 38] Topalian, S. L., et al. The New England journal of medicine 366, 2443-2454 (2012)

[Non-Patent Document 39] Chiba, S., et al. Nature immunology 13, 832-842 (2012)

[Non-Patent Document 40] Engelhardt, J. J., et al. Cancer cell 21, 402-417 (2012)

[Non-Patent Document 41] Huang, Y., et al. Cancer cell 19, 1-2 (2011)

[Non-Patent Document 42] Huang, Y., et al. Proc. Natl. Acad. Sci. U. S. A. 109, 17561-17566 (2012)

[Non-Patent Document 43] Garaude, J., et al. Science translational medicine 4, 120ra116 (2012)

[Non-Patent Document 44] Martinez-Pomares, L. et al. Trends in immunology 33, 66-70 (2012)

[Non-Patent Document 45] Palucka, K. et al. Nature reviews. Cancer 12, 265-277 (2012)

The present inventors conducted an effort to study, and as a result, a CpG-β glucan complex (for example, K3-SPG (using K3 and β-glucan as human type K CpG ODN) developed as an adjuvant was used. The complex was used as a single-dose antitumor drug, and as a result, K3-SPG confirmed tumor regression in a tumor-bearing mouse when intravenously administered without effect using the prior type CpG ODN (K3) (Fig. 2(A~B)), thereby completing the present invention. The present inventors have further confirmed that strong antitumor activity is also exhibited in a model of peritoneal vaccination which is a model closer to the clinical (Fig. 2g, m (Fig. 2B) The present inventors do not need to administer an antigen for this effect, and the effect is confirmed when administered in a single dose.

Further, the present inventors have used gene-deficient mice to show that, for the antitumor effect of K3-SPG, it is important that an acquired immune response, and type I interferon induced by a natural immune response ( IFN) and IL-12 (Fig. 6a, b, c (Fig. 6A)) are necessary. Further, the inventors of the present invention confirmed that CD45-negative tumor cells are concentrated in the spleen by intravenous administration of K3-SPG, thereby confirming that most of the cells are undergoing cell death (necrosis or apoptosis). )). When the mice were immunized with the CD45-negative cells, cell death of CD45-negative cells that were specifically concentrated in the spleen showed an important effect due to the strong anti-tumor effect (Fig. 6g, h, i, j (Fig. 6B). )). Further, the inventors of the present invention have also confirmed that activated K8 T cells accumulate in tumors by administration of K3-SPG, and it is confirmed that these cells are necessary for antitumor effects.

Therefore, the development of CpG ODN, which is expected to exert an antitumor effect when administered systemically, is also strongly exerted on cancer tumors that have hitherto been difficult to treat. Further, since CpG ODN exerts an antitumor effect under the condition of no antigen, it can be expected to be used as a single agent.

To date, CpG ODN has been shown to be a single agent (Pratesi, G., et al. Cancer research 65, 6388-6393 (2005); Manegold, C., et al. Annals of oncology: official journal of the European Society For Medical Oncology/ESMO 23, 72-77 (2012); Kim, YH, et al. Blood 119, 355-363 (2012); Hirsh, V., et al. Journal of clinical oncology: official journal of the American Society of Clinical Oncology 29, 2667-2674 (2011); Weber, JS, et al. Cancer 115, 3944-3954 (2009)) or cancer vaccine adjuvant (Reed, SG, Nature medicine 19, 1597-1608 (2013); Perret, R., et al. Cancer research 73, 6597-6608 (2013); Mbow, ML, et al. Current opinion in immunology 22, 411-416 (2010); Duthie, MS, et al. Immunological reviews 239, 178-196 (2011) ) Promising drugs. However, as for the treatment of anticancer agents utilizing CpG-ODN to date, tumor growth can be inhibited only when injected into a tumor (Schettini, J., et al. Cancer immunology, immunotherapy: CII 61, 2055-2065 (2012); Lin, AY, et al. PLoS One 8, e63550 (2013); Ishii, KJ, et al. Clinical cancer research: an official journal of the American Association for Cancer Research 9, 6516-6522 ( 2003); Lou, Y., et al. Journal of immunotherapy (Hagerstown, Md.: 1997) 34, 279-288 (2011); Auf, G., Clinical cancer research: an official journal of the American Association for Cancer Research 7, 3540-3543 (2001); Nierkens, S., et al. PLoS One 4, e8368 (2009); Heckelsmiller, K., et al. Journal of immunology 169, 3892-3899 (2002)). The present inventors have developed a nanoparticle-like TLR9 agonist called K3-SPG, which comprises Schizophyllum polysaccharide (SPG; β-glucan) and B/K-type CpG (K3) complex. It was shown that the K3-SPG system functions as a vaccine adjuvant stronger than K3 itself (with the induction of strong IFN-α). In the present example, the inventors of the present invention further investigated the potential of single-agent immunotherapy for K3-SPG of cancer (without using further tumor peptides and antigens), and as a result, it was found that the above-described effects can be obtained, thereby completing this invention. Accordingly, the present invention is representatively provided as follows.

(anti-cancer agent single agent)

(1) An anticancer agent comprising a complex comprising: (a) an oligodeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenosine And polydeoxyadenosine is disposed on the 3' side of the humanized K-type CpG oligodeoxynucleotide; and (b) β-1,3-glucan.

(2) The anticancer agent according to the above item (1), wherein the anticancer agent is administered in the absence of a cancer antigen.

(3) The anticancer agent according to the above item (1) or (2), wherein the anticancer agent is administered by delivery to the reticuloendothelial system and/or lymph nodes.

(4) The anticancer agent according to the above item (3), wherein the reticuloendothelial system and/or lymph nodes comprise tumors and macrophages.

(5) The anticancer agent according to the above item (3) or (4), wherein the reticuloendothelial system comprises a spleen and/or a liver.

(6) The anticancer agent according to any one of the items (1) to (5), wherein the anticancer agent is administered in the absence of a cancer antigen.

The anticancer agent according to any one of the items (2) to (6), wherein the administration includes systemic administration.

(8) The anticancer agent according to the above item (7), wherein the systemic administration is selected from the group consisting of intravenous administration, intraperitoneal administration, oral administration, subcutaneous administration, intramuscular administration, and And swollen Intratumoral administration.

(9) The anticancer agent according to any one of items 1 to 8, wherein the oligodeoxynucleotide is selected from K3 (SEQ ID NO: 1), K3-dA ₄₀ (SEQ ID NO: 2), dA Groups consisting of _40- K3 (SEQ ID NO: 3), K3-dA20 (SEQ ID NO: 4), K3-dA25 (SEQ ID NO: 5), K3-dA30 (SEQ ID NO: 6), and K3-dA35 (SEQ ID NO: 12→7) in.

(10) The anticancer agent according to any one of items 1 to 9, wherein the β-1,3-glucan is selected from the group consisting of Schizophyllum polysaccharide (SPG), lentinan, and scleroglucan ( Scleroglucan), a group of Curdlan, Pachyman, Grifolan, and Laminaran.

The anticancer agent according to any one of items 1 to 10, wherein the complex is K3-SPG.

(reticular endothelial system (including spleen and / or liver) and / or lymph node aggregating agent)

(12) A composition for accumulating dead cells of cancer in a spleen, comprising a complex comprising: (a) an oligodeoxynucleotide comprising a humanized K-type CpG oligomeric deoxygenation Nucleotide and polydeoxyadenylate, and polydeoxyadenosine is disposed on the 3' side of humanized K-type CpG oligodeoxynucleotides; and (b) β-1,3-gluco Glycans.

(13) The composition according to the above item (12), wherein the oligodeoxynucleotide is selected from the group consisting of K3 (SEQ ID NO: 1), K3-dA ₄₀ (SEQ ID NO: 2), and dA _{40 -} K3 ( Among the groups consisting of SEQ ID NO: 3), K3-dA20 (SEQ ID NO: 4), K3-dA25 (SEQ ID NO: 5), K3-dA30 (SEQ ID NO: 6), and K3-dA35 (SEQ ID NO: 7).

The composition according to the above item (12) or (13), wherein the β-1,3-glucan is selected from the group consisting of Schizophyllum polysaccharide (SPG), lentinan, scleroglucan, Among the group consisting of Cardland polysaccharide, Lycium barbarum polysaccharide, Grifola frondosa polysaccharide and Laminaria.

The composition according to any one of the items (12) to (14), wherein the composite is K3-SPG.

The composition according to any one of the above items 12 to 15, wherein the reticuloendothelial system and/or lymph nodes comprise tumors and macrophages.

The composition according to any one of the items (12) to (16), wherein the reticuloendothelial system comprises a spleen and/or a liver.

The composition of any one of the above items (12) to (17), wherein the administration comprises systemic administration.

(19) The composition according to the above item (18), wherein the systemic administration is selected from the group consisting of intravenous administration, intraperitoneal administration, oral administration, subcutaneous administration, intramuscular administration, and Intratumoral administration.

<Composition for performance or performance promotion of interleukin 12 (IL12) and/or interferon (IFN) γ >

(20) A composition for promoting the expression or expression of interleukin 12 (IL12) and/or interferon (IFN) γ, comprising: (a) an oligodeoxynucleotide comprising humanization K-type CpG oligodeoxynucleotides and polydeoxyadenylate, and polydeoxyadenosine is disposed on the 3' side of humanized K-type CpG oligodeoxynucleotides; and (b) --1,3-glucan.

(21) The composition according to the above item (20), wherein the oligodeoxynucleotide is K3 (SEQ ID NO: 1), K3-dA ₄₀ (SEQ ID NO: 2), and dA _{40 -} K3 (SEQ ID NO: 3), K3-dA20 (SEQ ID NO: 4), K3-dA25 (SEQ ID NO: 5), K3-dA30 (SEQ ID NO: 6), and K3-dA35 (SEQ ID NO: 7).

The composition according to the above item (20) or (21), wherein the β-1,3-glucan is selected from the group consisting of Schizophyllum polysaccharide (SPG), lentinan, scleroglucan, Among the group consisting of Cardland polysaccharide, Lycium barbarum polysaccharide, Grifola frondosa polysaccharide and Laminaria.

The composition according to any one of the above items (20) to (22), wherein the composite is K3-SPG.

In the present invention, with respect to one or more of the above features, it is intended to In addition, it may be further provided in combination. Further embodiments and advantages of the present invention will be appreciated by those skilled in the art by reading the following detailed description as needed.

The use of the K3-SPG of the present invention as an antitumor agent exerts a potent antitumor effect when administered systemically by a previous CpG ODN. Therefore, it is also considered very useful from a clinical point of view. Moreover, since a sufficient effect (natural immune response) is confirmed even for human cells, the possibility of application to humans is also high. According to the findings of the present inventors and the like, compared with the CpG ODN used in clinical trials so far, in addition to having a very strong natural immune activation ability, it also has a strong antitumor effect, so K3-SPG can It is expected as a useful immunotherapeutic drug. Further, it is considered that it can be applied to various cancers by inducing cell death by inducing cell death without administering an antigen. Based on these results, K3-SPG has the possibility of being a natural immune-activated antitumor drug that does not require an antigen.

Figure 1 shows a method of complexing SPG with CpG ODN.

2A to 2B show that systemic injection of antigen-free nanoparticle-like CpG (K3-SPG) can be applied to various established tumor models including a pancreatic cancer peritoneal vaccination model. Figure 2A shows a~i. On day 0, C57BL/6 mice were inoculated subcutaneously (subcutaneously) with EG7 cells, and on days 7, 9 and 11 PBS (a~c), K3 (30 μg) (d~f), or K3 -SPG (10 μg) (g~i) is administered to the id (intradermal) (area surrounding the tumor) (a, d, g), intraduct (it, intratumoral) (b, e, h), or vein Internal (iv, Intravenous) (c, f, i). Tumor size was determined for 23 days (n=4). Each curve represents each mouse. The arrows indicate the period of treatment.

2A to 2B show that systemic injection of CpG (K3-SPG) containing no antigen-like nanoparticles can be applied to various established tumor models including a pancreatic cancer peritoneal vaccination model. Figure 2B shows j~n. (j~l) Inoculation of B16 cells on C57BL/6 mice on day 0, B16F10 cells or MC38 cells. The B16 population was treated with i.v. or i.t. on days 10, 12 and 14 using K3-SPG. The B16F10 population was treated with i.v. or i.t. on days 7, 9, and 11 using K3-SPG. The MC38 population was treated with i.v. or i.t. on days 14, 16 and 18 using K3-SPG. The error bar indicates the mean + SEM (Standard error of the mean) (n = 4). * p < 0.05 (t assay). (m) Pan02 cells were intraperitoneally injected into C57BL/6 mice on day 0 and treated with i.v. on days 11, 13 and 15 using K3 or K3-SPG or PBS (control). The weight of the tumor (g) indicates the 21st day. * p < 0.05 (t assay). (n) C02BL/6 mice were intraperitoneally injected with Pan02 on day 0 and treated three times with i.v. using K3 or K3-SPG or PBS. Indicates survival rate (%) (n=8). * p < 0.05 (logarithmic level check).

Fig. 3 is obtained by comparing the results of systemic administration of K3-SPG with other controls and K3. It has been shown that systemic administration of K3-SPG can be a cancer immunotherapy agent that does not require an antigen. After EG7, which is a tumor cell line, was transplanted to mice, K3 and K3-SPG were administered intravenously three times (days 7, 9, and 11). Tumor size was determined from day 7 after transplantation of tumor cells.

Figure 4 shows that K3-SPG targets phagocytic cells (Phagocytes) in the tumor microenvironment. (a~c) C57BL/6 mice were inoculated with EG7 on day 0, and PBS (control), ALEXA 647-K3 (30 μg), or ALEXA 647-K3-SPG (10 μg) was administered iv on day 12. One hour after the administration, the mice were analyzed using an in vivo imaging system (IVIS), and the image measured by relative fluorescence was converted into the physical unit of surface radiance (photon/sec). /cm ² /sr). The white arrow indicates the tumor inoculation area (a). (b, c) staining frozen sections of the tumor from Fig. 6a (Fig. 6A) with anti-CD3e antibody (red, EG7 staining) and Hoechst 33258 (blue, nuclear staining), followed by analysis using a fluorescence microscope (scale bar) , 100μm). The white arrow indicates a fluorescent positive area. (d~g) Inoculate EG7 with C57BL/6 mice on day 0, and combine Alexa 647-K3, Alexa 647-K3-SPG, or FITC-SPG with dextran-PE on day 12 Cast. (d~f) One hour after the injection, frozen sections of the tumor were analyzed by a fluorescence microscope (scale bar, 100 μm). (g) Count green, red or fused cells (10 fields from 3 tumors). Error bars indicate the mean +SD (Standard deviation). The asterisk indicates a significant difference in the number of fused cells injected with K3. (h) C57BL/6 mice (n=3 or 4) were inoculated with EG7 on day 0, and clodronate liposomes or control liposomes were administered iv on day 5. Mice were injected with PBS (control) or K3-SPG on days 7, 9, and 11. Error bars indicate the mean + SEM. The arrows indicate the period of treatment. * p < 0.05 (t assay).

Figure 5 shows that F4/80 positive cells in tumors are depleted by clodronate liposomes. C57BL/6 mice were inoculated with EG7 on day 0, clodronate liposome (a) or control liposome (b) was administered iv on day 5, and then Alexa 647-K3-SPG was utilized on day 7. Perform iv processing. After 1 hour of treatment, frozen sections of the tumor were stained with anti-F4/80 antibody (red) and Hoechst 33258 (blue), and thereafter analyzed by a fluorescence microscope (scale bar, 100 μm).

Figures 6A-6B show that both IL-12 and IFN are important for tumor shrinkage and the potential role of such immunogenic cell death. Figure 6A shows a~f. (a~c) On day 0, Il12p40 heterogene knockout mice (a), Ifnar2 heterogene knockout mice (b), and Il12p40-Ifnar2 gene double knockout mice (c) sc inoculated EG7 cells The mice were subjected to iv treatment with K3-SPG on days 7, 9, and 11. Error bars indicate mean + SEM (n = 4). The arrows indicate the period of treatment. * p < 0.05 (t assay). (d, f) On day 0, Rag2 heterogeneity and gene knockout and Il12p40-Ifnar2 gene double knockout mice were inoculated with EG7 cells and performed 3 times with K3-SPG (days 7, 9 and 11 with black arrows), 6 Times (7th, 9th, 11th, 14th, 16th, 18th day, grey arrow), or 0 (control) iv treatment. (e) The enlarged view shows the 4th to the 21st day.

Figures 6A-6B show that both IL-12 and IFN are important for tumor shrinkage and the potential role of such immunogenic cell death. Fig. 6B shows g~k. (g) C57BL/6 mice and Il12p40-Ifnar2 gene double knockout mice were inoculated with EG7 cells on day 0, and mice were treated with K3-SPG on days 7, 9, and 11 for iv treatment, followed by On the 12th day, he was executed. The spleen cells were recovered, stained with an anti-CD45 antibody, and thereafter analyzed by a flow cytometer. (h) The scatter plot represents a subpopulation of CD45 negative cells. Error bars indicate the mean + SEM. * p < 0.05 (t assay). (i) For staining of dead cells, CD45-negative subpopulations were stained with Hoechst 33342 and PI (propidium iodide, propidium iodide), and then analyzed by flow cytometry. The histograms represent a subset of apoptotic cells, necrotic cells, and CD45-negative viable cells. Error bars indicate the average +SD (n=3). * p < 0.05 (t assay). (j) C57BL/6 mice were immunized with PBS or CD45-negative cells. Seven days after the immunization, the mice were sc-inoculated with EG7 cells on day 0, and the tumor size (n=3) was determined 25 days thereafter. Error bars indicate the mean + SEM. * p < 0.05 (t assay). (k) by the histogram and scatter paper showing FIG. 25 days, respectively, tumor volume and OVA (Ovalbumin, ovalbumin)) _{257 to 264} specific tetramer + number of CD8T cells. * p < 0.05 (t assay).

Figure 7 shows the detection of IFN-[beta] in the tumor microenvironment. (a) IFN-[beta]GFP (Green Fluorescent Protein) mice were inoculated with EG7 on day 0 and treated with i.d. or i.v. on days 7, 9 and 11 using K3-SPG. Twelve days after the inoculation, the tumor was recovered, and the frozen sections were stained with anti-CD11b antibody, anti-CD169 antibody, anti-F4/80 antibody, anti-MARCO antibody (red), and Hoechst 33258 (blue), followed by fluorescence microscopy. Analysis (scale bar, 100 μm). (b) Counting IFN-β positive cells (10 fields were selected from 3 tumors). Error bars indicate the average + SD. * p < 0.05 (t assay).

Figure 8 shows the detection of IL12-p40 in the tumor microenvironment. (a) C57BL/6 mice were inoculated with EG7 on day 0 and treated with i.d. or i.v. on days 7, 9 and 11 using K3-SPG. Twelve days after the inoculation, the tumors were recovered, and frozen sections were stained with anti-IL12-p40 antibody (red) and Hoechst 33258 (blue), and thereafter analyzed by a fluorescence microscope (scale bar, 100 μm). (b) IL12-p40 positive cells were counted (10 fields were selected from 3 tumors). Error bars indicate the average + SD. * p < 0.05 (t assay).

Figure 9 shows that CD45-negative cell lines are derived from tumor cells, but not from host cells. EG mice were inoculated with GFP mouse s.c. on day 0, and treated with K3-SPG on day 7, 9 and 11 for i.v. treatment, and then sacrificed on day 12. Splenocytes were recovered and stained with an anti-CD45 antibody, after which the cells were analyzed by flow cytometry.

Figures 10A-10B show that K3-SPG-induced tumor shrinkage requires both natural immune responses and adaptive immunity including Il12, type 1 IFN, Batf3, CD8 ⁺ DC, and potent cytotoxic T cells infiltrating tumors. Answer. Fig. 10A shows a to c. C57BL/6 knockout mice (a), Batf3 heterogeneity and Batf3 knockout mice (b) were inoculated with EG7 cells on day 0 and treated with K3-SPG on days 7, 9 and 11 (black) arrow). (a) CD8 depleted antibody (200 μg/mouse) was administered on days 6 and 13. Error bars indicate mean + SEM (n = 4). * p < 0.05 (t assay). The arrows indicate the period of treatment. (c) C57BL/6 mice were inoculated with EG7 on day 0 and K3-SPG was treated by id or iv on days 7, 9 and 11. Twelve days after the inoculation, the tumors were recovered, and frozen sections were stained with anti-CD8β antibody (red) and Hoechst 33258 (blue), and thereafter analyzed by a fluorescence microscope (scale bar, 100 μm). CD8β positive cells were counted (10 fields were selected from 3 tumors). Error bars indicate the average + SD. * p < 0.05 (t assay).

Figures 10A-10B show that K3-SPG-induced tumor shrinkage requires both natural immune responses and adaptive immunity including Il12, type 1 IFN, Batf3, CD8 ⁺ DC, and potent cytotoxic T cells infiltrating tumors. Answer. Fig. 10B shows d~e. (d) C57BL/6 (WT (wild-type, wild type)) mice and Il12p40-Ifnar2 gene double knockout (DKO, Double knockout) mice were inoculated with EG7 cells on day 0, and on days 7, 9 and The iv treatment was performed using K3-SPG for 11 days. On the 14th day, CD8α ⁺ T cells derived from tumor-bearing mice injected with either K3-SPG or PBS were stained and transplanted (iv) using Xenolight DiR (registered trademark), and thereafter, Mice were analyzed using IVIS for 15 days. (I, II) Transplanted mice: WT mice bearing EG7 treated with K3-SPG. Mice were transplanted with K3-SPG-treated CD8α ⁺ T cells (I) or transplanted with untreated CD8α ⁺ T cells (II). (e) (I, II) transplanted mice: untreated WT mice bearing EG7 (I) and DKO mice treated with K3-SPG for iv treatment. Mice were transplanted with K3-SPG-treated CD8α+ T cells (I, II).

Figure 11 shows a schematic diagram of the experimental system. WT mice and Il12p40-Ifnar2 DKO mice were inoculated with EG7 cells on day 0 and treated with K3-SPG or PBS on days 7, 9 and 11 for iv treatment. CD8α ⁺ T cells were purified from the spleens of these mice on day 14, labeled with Xenolight DiR (registered trademark), and then transplanted to another treated with K3-SPG (days 7, 9 and 11) with EG7 The mice (after 14 days of inoculation) were analyzed on the 15th day by IVIS on the distribution of CD8 T cells labeled with Xenolight DiR (registered trademark).

Figure 12 shows the strategy of K3-SPG processing. K3-SPG targets the tumor microenvironment via blood flow. Further, K3-SPG is activated by phagocytic cells as a target. In the tumor microenvironment, IFN and IL-12 were induced by K3-SPG treatment. The antigen is then released via lymphatic flow and blood flow. The presentation of this antigen induces potent tumor-specific CTL.

Hereinafter, the best mode will be disclosed, and the present invention will be described. Throughout the specification, the singular expressions are to be understood as including the concept of the plural forms, unless otherwise specified. Therefore, the singular articles (for example, in the case of English, "a", "an", "the", etc.) are understood to include the concept of plural forms. Further, the terms used in the present specification are to be understood as being used based on the meanings commonly used in the field unless otherwise specified. Therefore, unless otherwise defined, all the specific terms and technical terms used in the specification have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In the event of a conflict, the present specification, including definitions, will control.

In the following, the definitions and/or basic skills of the terms specifically used in this specification are appropriately explained. Content.

The present invention provides an oligodeoxynucleotide comprising a K-type CpG oligodeoxynucleotide and polydeoxyadenylate (dA) (hereinafter, referred to as an oligodeoxynucleotide of the present invention). The oligodeoxynucleotide of the present invention comprises a modification in which a phosphodiester bond is modified (for example, a part or all of a phosphodiester bond is substituted by a phosphorothioate bond). The oligodeoxynucleotides of the invention comprise a pharmaceutically acceptable salt.

In the present specification, "CpG oligonucleotide (residue)" or "CpG oligodeoxynucleotide (residue)", "CpG ODN (residue)" or only "CpG (residue)" "polynucleotide, preferably an oligonucleotide, comprising at least one unmethylated CG dinucleotide sequence which can be used interchangeably, with or without the term "residue" at the end, meaning the same. An oligonucleotide comprising at least one CpG motif can comprise a plurality of CpG motifs. As used in this specification, the term "CpG motif" refers to an unmethylated dinucleotide of an oligonucleotide comprising a cytosine nucleotide followed by a guanosine nucleotide. section. 5-methylcytosine can also be used in place of cytosine. Further, polydeoxyadenylate has the same meaning as polydeoxyadenylate (residue). The term "residue" refers to a partial structure of a compound having a larger molecular weight. In the present specification, "CpG oligodeoxynucleotide (CpG ODN)" means an independent molecule or a compound of a larger molecular weight. Part of the structure, as long as it is a practitioner, can be easily understood by the context. The terms "polydeoxyadenylate" and the like relating to other partial structures contained in the oligodeoxynucleotide of the present invention are also the same.

CpG Oligonucleotide (CpG ODN) is a short (about 20 base pair) single-stranded synthetic DNA fragment containing an immunologically active CpG motif, and is a potent Toll receptor 9 (TLR9) An agonist that activates dendritic cells (DCs) and B cells to produce type I interferons (IFNs) and inflammatory cytokines (Hemmi, H., et al. Nature 408, 740-745 (2000); Krieg , AMNature reviews. Drug discovery 5, 471-484 (2006).), including the cytotoxic T lymphocyte (CTL) reaction, including the Th1 type of humoral and cellular immune response adjuvant (Brazolot Millan, CL, Weeratna, R., Krieg, AM, Siegrist, CA & Davis, HL Proceedings of the National Academy of Sciences of the United States of America 95, 15553-15558 (1998).; Chu, RS, Targoni, OS, Krieg, AM , Lehmann, PV & Harding, CV The Journal of experimental medicine 186, 1623-1631 (1997)). Therefore, CpG ODN is considered to be an immunotherapeutic agent for the possibility of infection, cancer, asthma and hay fever (Krieg, AMNature reviews. Drug discovery 5, 471-484 (2006); Klinman, DMNature reviews. Immunology 4, 249-258 (2004)).

CpG oligodeoxynucleotide (CpG ODN) is a single-stranded DNA containing an immunologically active unmethylated CpG motif and is an agonist of TLR9. CpG ODN has four types of K-type (also known as B-type), D-type (also known as type A), C-type and P-type, which have different skeleton sequences and immunosynthesis characteristics (Advanced drug delivery reviews 61, 195-204 ( 2009)). The oligodeoxynucleotides of the invention comprise K-type CpG ODNs thereof.

The K-type CpG ODN system typically contains a plurality of unmethylated CpG motifs in a non-palindromic structure that activates B cells to produce IL-6, but has IFN that induces little plasmacytoid dendritic cells (pDCs). - CpG ODN of the structural and functional properties produced by α. The so-called unmethylated CpG model system comprises at least one short nucleotide sequence of the cytosine (C)-guanine (G) sequence, which means that the cytosine in the cytosine-guanine sequence is 5 Methylated. In the following description, CpG means unmethylated CpG unless otherwise specified. Therefore, the oligodeoxynucleotide of the present invention has an immunostimulating activity specific to K-type CpG ODN by including a K-type CpG ODN (for example, activation of B cells (preferably human B cells) to produce IL- 6 activity). A large number of humanized K-type CpG ODNs are known in the art (Journal of Immunology 166, 2372-2377 (2001); Journal of Immunology 164, 944-953 (2000); US 8,030, 285 B2).

The K-type CpG ODN contained in the oligodeoxynucleotide of the present invention is preferably humanized. By "humanization" is meant the agonist activity against human TLR9. Therefore, the oligodeoxynucleotide of the present invention comprising a humanized K-type CpG ODN has an immunostimulating activity specific to a K-type CpG ODN (for example, activation of human B cells to produce IL-6). The K-type CpG ODN which can be suitably used in the present invention is a length of 10 nucleotides or more, and contains the formula: [Chemical Formula 1] 5'N ₁ N ₂ N ₃ T-CpG-WN ₄ N ₅ N ₆ 3 '

(wherein, the central CpG motif is unmethylated, W is A or T, and N ₁ , N ₂ , N ₃ , N ₄ , N ₅ and N ₆ may also be any nucleotides) Glycosidic acid sequence.

In one embodiment, the K-type CpG ODN of the present invention has a length of 10 nucleotides or more and comprises a nucleotide sequence of the above formula. In the above formula, the CpG motif (TCpGW) having four bases in the center may be contained in 10 nucleotides, and is not necessarily required to be located between N ₃ and N ₄ in the above formula. Further, in the above formula, N ₁ , N ₂ , N ₃ , N ₄ , N ₅ and N ₆ may be any nucleotide, N ₁ and N ₂ , N ₂ and N ₃ , N ₃ and N ₄ , N ₄ And N ₅ and at least one (preferably one) combination of N ₅ and N ₆ may also be a 2-base CpG motif. In the case where the above four base CpG motif is not located between N ₃ and N ₄ , in the above formula, any two consecutive bases in the center of 4 bases (4 to 7 bases) For the CpG motif, the other 2 bases can also be any nucleotide.

The K-type CpG ODN which can be more suitably used in the present invention comprises a non-palindromic structure containing one or a plurality of CpG motifs. A K-type CpG ODN which can be suitably used includes a non-palindromic structure containing one or a plurality of CpG motifs.

The humanized K-type CpG ODN is typically characterized by a CpG motif comprising four bases of TCGA or TCGT. Further, in many cases, 2 or 3 of the 4 base CpG motifs are contained in one humanized K-type CpG ODN. Therefore, in a preferred embodiment, the present invention The K-type CpG ODN contained in the oligodeoxynucleotide of the present invention contains at least one, more preferably 2 or more, still more preferably 2 or 3 CpG motifs comprising 4 bases of TCGA or TCGT. In the case where the K-type CpG ODN has 2 or 3 4 base CpG motifs, the 4 base CpG motifs may be the same or different. Among them, there is no particular limitation as long as it has an agonist activity against human TLR9.

More preferably, the K-type CpG ODN contained in the oligodeoxynucleotide of the present invention comprises the nucleotide sequence represented by SEQ ID NO: 1.

Regarding the length of the K-type CpG ODN, there is no particular limitation as long as the oligodeoxynucleotide of the present invention has immunostimulating activity (for example, activation of B cells (preferably human B cells) to produce IL-6) Preferably, it is less than 100 nucleotides in length (e.g., 10 to 75 nucleotides in length). The length of the K-type CpG ODN is preferably less than 50 nucleotides in length (e.g., 10 to 40 nucleotides in length). The length of the K-type CpG ODN is further preferably 30 nucleotides or less (e.g., 10 to 25 nucleotides in length). The length of the K-type CpG ODN is preferably 12 to 25 nucleotides in length.

The length of polydeoxyadenosine (dA) is sufficient for forming a triple helix structure together with a β-1,3-glucan (preferably lentinan or schizophyllum) chain. The length is not particularly limited, and is generally 20 nucleotides or longer, preferably 40 nucleotides or longer, and more preferably 60 nucleotides or longer from the viewpoint of forming a stable triple helix structure. . Since the longer the polydA, the more stable the triple helix structure with β-1,3-glucan, there is no theoretical limit, but if it is too long, it will be length when synthesizing oligodeoxynucleotides. The aspect is uneven, and therefore it is usually 100 nucleotides or less, preferably 80 or less. On the other hand, in addition to the formation of the above stable triple helix structure, the amount of the oligodeoxynucleotide of the present invention bonded to the unit amount of β-1,3-glucan is increased and the synthesis oligomerization is avoided. The length of polydA is preferably 20 to 60 nucleotides in length from the viewpoint of length unevenness and complexation efficiency in deoxynucleotides (specifically, 20, 21, 22, 23, 24, 25). ,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43, 44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59 or 60 nucleotides in length), more preferably 30 to 50 nucleotides Length (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides in length) 18 , preferably 30 to 45 nucleotides in length (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 nucleotides in length) . Especially in the case of a length of 30 nucleotides or more, a good compounding efficiency is exhibited. The oligodeoxynucleotide of the present invention has an activity of forming a triple helix structure together with two Schizophyllan polysaccharide chains by including polydA. Further, there is also a case where polydeoxyadenosine is expressed as "poly(dA)" or "poly(dA)".

The oligodeoxynucleotide of the present invention may further comprise a plurality of K-type CpG ODNs and/or polyd-A, preferably one of K-type CpG ODN and poly-dA, preferably containing One K-type CpG ODN and one poly-dA.

Examples of the sequence of the exemplary CpG include K3 CpG (SEQ ID NO: 5'-atcgactctcgagcgttctc-3'), but are not limited thereto.

The oligodeoxynucleotide of the present invention is characterized in that the polydA line is disposed on the 3' side of the K-type CpG ODN. It is considered that the anti-cancer effect of the complex of the present invention (described in detail below) may be enhanced by this arrangement, but it is not limited thereto, and may be combined with any of them as an anticancer agent.

The K-type CpG ODN and the poly-dA may be linked by a direct covalent bond or may be linked via a gap subsequence. The gap subsequence refers to a nucleotide sequence comprising one or more nucleotides inserted between two closely related constituent elements. Regarding the length of the spacer sequence, as long as the complex of the present invention has immunostimulating activity (preferably, activation of B cells to produce IL-6 activity and activation of dendritic cells to produce IFN-α activity), no Particularly, it is usually 1 to 10 nucleotides in length, preferably 1 to 5 nucleotides in length, more preferably 1 to 3 nucleotides in length. Preferably, the K-type CpG ODN and the poly-dA are linked by a direct covalent bond.

The oligodeoxynucleotide of the present invention has K-type CpG ODN, poly-dA and any In addition to the spacer sequence, an additional nucleotide sequence may be provided at the 5' end and/or the 3' end. Regarding the length of the additional nucleotide sequence, the complex of the present invention has immunostimulating activity (preferably, activation of B cells to produce IL-6 activity, and activation of dendritic cells to produce IFN-α activity) Further, it is not particularly limited and is usually 1 to 10 nucleotides in length, preferably 1 to 5 nucleotides in length, more preferably 1 to 3 nucleotides in length.

In a preferred aspect, the oligodeoxynucleotides of the invention do not contain such additional nucleotide sequences at the 5' end and/or the 3' end. That is, the oligodeoxynucleotide of the present invention preferably comprises a K-type CpG ODN, a polydA, and an arbitrary spacer sequence, and further preferably contains a K-type CpG ODN and a polydA.

In an optimal aspect, the oligodeoxynucleotide of the present invention comprises a K-type CpG ODN (specifically, for example, an oligodeoxynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1) and a poly dA, a K-type CpG ODN is located at the 5' end of the oligodeoxynucleotide, and a polydA is located at the 3' end. Specifically, the 3' end of the oligodeoxynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 is 20 to 60 nucleotides in length (more preferably 30 to 50 nucleosides). Acid length (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides in length) , preferably 30 to 45 nucleotides in length (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 nucleotides in length) An oligomeric deoxynucleotide of polydA, for example, an oligodeoxynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 2 or 9-12.

The total length of the oligodeoxynucleotides of the present invention is usually 30 to 200 nucleotides in length, preferably 35 to 100 nucleotides in length, more preferably 40 to 80 nucleotides in length (specifically , 40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 nucleotides in length), more preferably 50 to 70 nucleotides Length (specifically, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 nucleosides Acid length), optimally 50 to 65 nucleotides in length (specifically , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 nucleotides in length). The oligodeoxynucleotides of the present invention can also be suitably modified in such a manner as to exhibit resistance to in vivo decomposition (e.g., decomposition by external or internal nuclease). Preferably, the modification comprises a phosphorothioate modification or a phosphorodithioate modification. That is, part or all of the phosphodiester bond in the oligodeoxynucleotide of the present invention is substituted with a phosphorothioate bond or a phosphorodithioate bond.

Preferably, the oligodeoxynucleotide of the present invention comprises a modification of a phosphodiester bond, more preferably a modification of a phosphodiester bond to a phosphorothioate linkage (i.e., as described in WO 95/26204, One of the crosslinked oxygen atoms is substituted with a sulfur atom). That is, part or all of the phosphodiester bond in the oligodeoxynucleotide of the present invention is substituted with a phosphorothioate bond.

With respect to the oligodeoxynucleotide of the present invention, it is preferred to include a modification using a phosphorothioate bond or a phosphorodithioate bond in the K-type CpG ODN, and more preferably a phosphoric acid of the K-type CpG ODN. All of the ester linkages are substituted with phosphorothioate linkages. Further, in the oligodeoxynucleotide of the present invention, it is preferred to include a phosphorothioate bond or a phosphorodithioate bond in the polydA, and more preferably all of the phosphodiester bond of the polydA is Substituted as a phosphorothioate bond. Further preferably, all of the phosphodiester bonds of the present invention comprising a humanized K-type CpG oligodeoxynucleotide and a polydeoxyadenosine oligodeoxynucleotide are substituted with a phosphorothioate bond. . Preferably, the oligodeoxynucleotide of the invention is 20 to 60 nucleotides in length at the 3' end of a humanized K-type CpG oligodeoxynucleotide (eg, SEQ ID NO: 1) (more Good 30 to 50 nucleotides in length (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50 nucleotides in length), optimally 30 to 45 nucleotides in length (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 , 45 nucleotides in length)), and all of the phosphodiester bonds contained in the oligodeoxynucleotide are substituted with phosphorothioate linkages. The reason for this is that, by the phosphorothioate bond, not only the resistance against decomposition but also the immunostimulating activity (for example, activation of pDC to produce IFN-α activity) is expected for the oligodeoxynucleotide of the present invention. Enhancement, and CpG-β-1,3-glucan complex High yield, and enhanced anticancer activity. Further, the phosphorothioate bond in the present specification has the same meaning as the phosphorothioate skeleton, and the phosphodiester bond has the same meaning as the phosphate skeleton. The oligodeoxynucleotides of the present invention include all pharmaceutically acceptable salts, esters, or salts of such esters of the above oligodeoxynucleotides.

The pharmaceutically acceptable salt of the oligodeoxynucleotide of the present invention may, for example, be an alkali metal salt such as a sodium salt, a potassium salt or a lithium salt, or an alkaline earth metal such as a calcium salt or a magnesium salt. a metal salt such as a salt, an aluminum salt, an iron salt, a zinc salt, a copper salt, a nickel salt or a cobalt salt; an inorganic salt such as an ammonium salt, a third octylamine salt, a dibenzylamine salt, a morpholine salt, or a glucose salt Amine salt, alkyl phenylglycine, ethylenediamine salt, N-methylglucamine salt, sulfonium salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine An amine salt such as an organic salt such as a salt, a tetramethylammonium salt or a tris(hydroxymethyl)aminomethane salt; a hydrohalide such as a hydrofluoric acid salt, a hydrochloride salt, a hydrobromide salt or a hydroiodide salt; Mineral acid salts such as acid salts, nitrates, perchlorates, sulfates, phosphates; lower alkane sulfonates such as methanesulfonate, triflate, ethanesulfonate, and besylate An aryl sulfonate such as p-toluenesulfonate, acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleate An organic acid salt; and an amine acid salt such as a glycinate, an aminate, a arginine, an alanate, a glutamate or an aspartate.

The oligodeoxynucleotide of the present invention may be in the form of any of a single chain, a double chain or a triple chain, and is preferably a single chain.

The oligodeoxynucleotides of the invention are preferably isolated. By "single separation" is meant the operation of removing factors other than the target component, and leaving the natural state. The purity of the "oligo-deoxynucleotide to be isolated" (the percentage of the target oligodeoxynucleotide to the total weight of the object to be evaluated) is usually 70% or more, preferably 80% or more. More preferably, it is 90% or more, and further preferably 99% or more.

The oligodeoxynucleotide of the present invention has excellent immunostimulating activity (for example, Since B cells (preferably human B cells are activated to produce IL-6 activity), they are useful as an immunostimulating agent or the like. Further, the oligodeoxynucleotide of the present invention has a triple helix structure together with two β-1,3-glucans (preferably, schizophyllum, lentinan or scleroglucan). The nature is therefore useful for the preparation of the composite of the invention.

The present invention provides a complex comprising the above-described oligodeoxynucleotide of the present invention and β-1,3-glucan (hereinafter referred to as a complex of the present invention).

Since the oligodeoxynucleotide of the present invention contains K-type CpG ODN, it exerts an immunostimulating activity specific to K-type CpG ODN alone (for example, activation of B cells (preferably human B cells) to produce IL) -6 activity), lack of immunostimulating activity specific to D-type CpG ODN (for example, activation of plasma-like dendritic cells to produce IFN-α activity). Surprisingly, however, by forming a complex with β-1,3-glucan (preferably lentinan, Schizophyllum polysaccharide), the D-type CpG is obtained without the sequence of the D-type CpG ODN. ODN-specific immunostimulating activity (for example, activation of plasmacytoid dendritic cells to produce IFN-α activity). That is, the complex of the present invention has an immunostimulating activity specific to K-type CpG ODN (for example, activation of B cells (preferably human B cells) to produce IL-6), and immunity specific to D-type CpG ODN. Activating activity (e.g., activation of plasmacytoid dendritic cells (preferably human plasmacytoid dendritic cells) to produce IFN-[alpha] activity). Examples of the β-1,3-glucan used in the present invention include Schizophyllum polysaccharide, scleroglucan, Cardland polysaccharide, Lycium barbarum polysaccharide, Grifola frondosa polysaccharide, Lentinus edodes polysaccharide, and lengua sugar. The β-1,3-glucan is preferably β-1 containing a large amount of 1,6-glucoside branch (side chain ratio 33 to 40%) like schizophyllum polysaccharide, lentinan or scleroglucan. 3-glucan, more preferably Schizophyllum polysaccharide.

Lentinus edodes polysaccharide (LNT) is a well-known β-1,3-1,6-glucan derived from shiitake mushroom, and has a molecular formula of (C ₆ H ₁₀ O ₅ ) _n and a molecular weight of about 300,000 to 700,000. It is hardly soluble in water, methanol, ethanol (95), or acetone, but dissolves in DMSO (dimethylsulfoxide, dimethyl sulfoxide) or aqueous sodium hydroxide as a polar organic solvent.

Lentinus edodes polysaccharides have activated macrophages, killer T cells, natural killers (Natural Killer) Cell and antibody-dependent macrophage-mediated cytotoxicity (ADMC) enhancement (Hamuro, J., et al.: Immunology, 39, 551-559, 1980, Hamuro, J. , et al.: Int. J. Immunopharmacol., 2, 171, 1980, Herlyn, D., et al.: Gann, 76, 37-42, 1985). In the animal experiment, the tumors of the same type and the autologous tumors were combined with the chemotherapeutic agent to confirm the tumor growth inhibition effect and prolong the life effect. Further, by the separate administration of lentinan, the tumor growth inhibition effect and the life extension effect were also confirmed. In clinical trials, patients with inoperable or recurrent gastric cancer were confirmed to have an extended survival time by oral administration with tegafur (medical evaluation form "1 mg "Ajinomoto" for intravenous injection of lentinan"). It is recognized in Japan. The effect produced by the separate administration of lentinan has not been confirmed yet.

Schizophyllum polysaccharide (SPG) is derived from the well-known soluble β-glucan of Schizophyllum. SPG contains the main chain of β-(1→3)-D-glucan and one β-(1→6)-D-glucosyl side chain for every three glucoses (Tabata, K., Ito) , W., Kojima, T., Kawabata, S. and Misaki A., "Carbohydr. Res.", 1981, 89, 1, p. 121-135). SPG is a clinical drug for intramuscular injection of an immunoenhancement method for gynecological cancer, and has been used for more than 20 years (Shimizu, Chen, Hoji, Zengyuan, "Biotherapy", 1990, 4, p. 1390 Hasegawa, "Oncology And Chemotherapy (1992, 8, p. 225), and confirmed in vivo safety (Theresa, M. McIntire and David, A. Brant, "J. Am. Chem. Soc. , 1998, 120, p. 6909).

In the present specification, the term "complex" refers to a product obtained by agglomeration of a plurality of molecules via a non-covalent bond or a covalent bond such as an electrostatic bond, a vanadium bond, a hydrogen bond, or a hydrophobic interaction.

The composite of the present invention is preferably in the form of a triple helix. In a preferred embodiment, among the three chains forming the triple helix structure, two are β-1,3-glucan chains, and one is agglomerated in the above oligodeoxynucleotides of the present invention. A chain of deoxyadenosine. Furthermore, the complex can also be packaged Contains a portion of the portion that does not form a triple helix.

The composition ratio of the oligodeoxynucleotide to the β-1,3-glucan in the complex of the present invention may be based on the chain length of the polydeoxyadenosine in the oligodeoxynucleotide, And the length of β-1,3-glucan changes and the like. For example, when the length of the β-1,3-glucan chain is equal to the length of the polydeoxyadenylate chain, two β-1,3-glucan chains are separated from one of the oligomers of the present invention. Oxynucleotides can aggregate to form a triple helix structure. Generally, the chain length of polydeoxyadenosine is relatively short relative to the β-1,3-glucan chain, and thus a plurality of oligodeoxynucleotides of the present invention can be targeted to two β-1,3- The glucan chains are aggregated via polydeoxyadenosine to form a triple helix structure (see Fig. 1).

The composite system of the invention comprises humanized K-type CpG ODN and β-1,3-glucan (for example, lentinan, schizophyllum polysaccharide, scleroglucan, cadmium polysaccharide, lycium polysaccharide, ash tree flower polysaccharide, The complex of laminarin is preferably a complex comprising a humanized K-type CpG ODN and a β-1,3-glucan (for example, lentinan, schizophyllum polysaccharide, scleroglucan). More preferably, the 3' side linkage of the oligodeoxynucleotide contained in the nucleotide sequence represented by SEQ ID NO: 1 is 20 to 60 nucleotides in length (specifically, 20, 21, 22, 23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47, 48,49,50,51,52,53,54,55,56,57,58,59 or 60 nucleotides in length of polydeoxyadenosine and all of the phosphodiester bonds are replaced by thio Phosphate-bonded oligodeoxynucleotides and complexes of β-1,3-glucan (eg, lentinan, Schizophyllum polysaccharide) (eg, K3-dA20~60-LNT, K3-dA20~60) -SPG), and further preferably the 3' side linkage of the oligodeoxynucleotide contained in the nucleotide sequence represented by SEQ ID NO: 1 is 30 to 50 nucleotides in length (specifically, 30 , 31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50 nucleotides in length) A complex of an adenylate and a phosphodiester bond substituted with a phosphorothioate linkage oligodeoxynucleotide and a β-1,3-glucan (eg, lentinan, Schizophyllum polysaccharide) (eg K3-dA30~50-LNT, K3-dA30~50-SPG), preferably included in the sequence number 1 Nucleotide sequence The 3' side linkage of the oligodeoxynucleotides is 30 to 45 nucleotides in length (specifically, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 , 41, 42, 43, 44, 45 nucleotides in length of polydeoxyadenylate and all of the phosphodiester bonds are substituted with phosphorothioate linkages of oligodeoxynucleotides, and β- A complex of 1,3-glucan (for example, lentinan, Schizophyllum polysaccharide) (K3-dA30~45-LNT, K3-dA30~45-SPG).

The preparation method of the composite of the present invention can be carried out in the same manner as described in Non-Patent Documents 21 to 24 or JP-A-2008-100919. That is, the β-1,3-glucan which is originally natural and exists in the form of a triple helix structure is dissolved in an aprotic organic polar solvent (dimethylammonium (DMSO), acetonitrile, acetone, etc.) or alkaline. The aqueous solution (sodium hydroxide, potassium hydroxide, ammonia, calcium hydroxide, etc.) is unwound into a single chain. A solution of the single-chain β-1,3-glucan obtained in the above manner and a solution of the oligodeoxynucleotide of the present invention (aqueous solution, pH-buffered aqueous solution near neutral, or acidic buffer) The aqueous solution, preferably an aqueous solution or a buffered aqueous solution having a pH near neutral, is mixed, and if necessary, the pH is adjusted to near neutral again, and then maintained for a suitable period of time, for example, at 5 ° C overnight. As a result, two β-1,3-glucan chains form a triple helix structure with the poly dA chain in the oligodeoxynucleotide, thereby forming a complex of the present invention. The oligodeoxynucleotide which does not form a complex can be removed by purification, ultrafiltration, dialysis, or the like by size exclusion chromatography on the resulting complex. Further, by purifying the resulting complex by anion exchange chromatography, the β-1,3-glucan which does not form a complex can be removed. The complex can be appropriately purified by the above method.

The formation of the composite of the present invention can be carried out, for example, by measuring a conformational change using a CD (Circular Dichroism) spectrum, UV (Ultra Violet) absorption displacement using size exclusion chromatography, gel electrophoresis, It is confirmed by microchip electrophoresis or capillary electrophoresis, but is not limited thereto.

The mixing ratio of the oligodeoxynucleotide of the present invention and β-1,3-glucan can be appropriately set in consideration of the length of the polydA chain, etc., and usually the molar ratio (SPG/ODN) is 0.02 to 2.0. , It is preferably 0.1 to 0.5. In a further aspect, the molar ratio (β-1,3-glucan (LNT, etc.)/ODN) is, for example, 0.005 to 1.0, preferably 0.020 to 0.25.

The preparation method of the composite of the present invention will be described by taking CpG-ODN and LNT complex as an example. LNT is dissolved in 0.05~2N, preferably 0.1~1.5N alkaline aqueous solution (such as 0.25N sodium hydroxide aqueous solution), and placed at 1 °C ~ 40 °C for 10 hours to 4 days (for example, placed at room temperature) One night), a single-chain LNT aqueous solution (for example, a 50 mg/ml LNT aqueous solution) was prepared. Mixing the above LNT aqueous solution with a separately prepared CpG aqueous solution (for example, 100 μM CpG aqueous solution) at a molar ratio (LNT/ODN) of 0.005 to 1.0, and then adding an acidic buffered aqueous solution (for example, NaH ₂ PO ₄ ) to the above LNT aqueous solution. Neutralization is carried out for 6 hours to 4 days at 1 to 40 ° C (for example, one night at 4 ° C), thereby ending the complexation. Further, for the above-mentioned compounding, an LNT aqueous solution may be finally added and mixed. The formation of the complex can be confirmed, for example, by using a size exclusion chromatography to monitor the absorption at a wavelength of 240 to 280 nm (for example, 260 nm) for the displacement of the CpG ODN to the high molecular weight side.

In one aspect, the composite of the present invention exhibits the morphology of rod-shaped particles. The particle size is the same as the diameter of the particles formed by using the β-1,3-glucan (for example, Schizophyllum polysaccharide) as a material and exhibiting a triple helix structure, and the average particle diameter is usually 10 to 100 nm. It is preferably 20 to 50 nm. Regarding the particle diameter, the composite was dissolved in water and measured by a dynamic light scattering method using a Malvern Instruments Zeta Sizer at 80 °C.

The composite of the present invention is preferably isolated. The purity of the "complexed body to be separated" (the percentage of the weight of the target complex in the total weight of the object to be evaluated) is usually 70% or more, preferably 80% or more, more preferably 90% or more, and further Good is over 99%.

Further, the complex of the present invention has excellent immunostimulating activity in addition to anticancer activity, and particularly has an immunostimulating activity specific to K-type CpG ODN (for example, activation of B cells (preferably human B cells). Produces IL-6 activity), and D-type CpG ODN-specific immunostimulating activity (for example, activation of plasma cell-like dendritic cells (preferably human plasma cell dendritic cells) to produce IFN-α activity) can also be imparted as an immunostimulating agent or the like. Therefore, it is advantageous. For example, a complex comprising a K-type CpG ODN (for example, a complex of SEQ ID Nos. 2, 11, 12 and SPG and a complex comprising K-type CpG ODN (for example, SEQ ID NO: 2) and SPG (K3-SPG) serves as an inflammatory response. Inducibility (pan-IFN-a, IL-6, etc.), enhancement of antigen-specific IgG antibody titers (Total IgG, IgG2c, etc.) in serum of virus-inoculated individuals, antigen-specific cytokines in virus-inoculated individuals The ability to produce (IFN-γ, IL2, etc.) and the protective effect against the infection of the virus is also advantageous.

(pharmaceutical composition)

The present invention provides a pharmaceutical composition comprising the above-described oligodeoxynucleotide of the present invention or the above-described complex of the present invention. The pharmaceutical composition of the present invention can be obtained by formulating the above-described oligodeoxynucleotide of the present invention or the above-described complex of the present invention in accordance with a conventional method. The pharmaceutical composition of the present invention comprises the oligodeoxynucleotide or complex of the present invention and a pharmacologically acceptable carrier. Further, the pharmaceutical composition may further comprise an antigen. Such pharmaceutical compositions are provided in a dosage form suitable for oral or parenteral administration.

As the composition for parenteral administration, for example, an injection, a suppository or the like can be used, and the injection can also be administered in the form of an intravenous injection, a subcutaneous injection, an intradermal injection, an intramuscular injection, a drip injection or the like. Such an injection can be prepared according to a known method. The preparation method of the injection can be prepared, for example, by dissolving or suspending the above-described oligodeoxynucleotide or complex of the present invention in a sterile aqueous solvent used in a usual injection. As the aqueous solvent for injection, for example, distilled water, physiological saline, a buffer solution such as a phosphate buffer solution, a carbonate buffer solution, a trishydroxymethylaminomethane buffer solution, or an acetate buffer solution can be used. The pH of such an aqueous solvent may be 5 to 10, preferably 6 to 8. The prepared injection is preferably filled into a suitable ampoule.

Also, by the suspension of the oligodeoxynucleotide or complex of the present invention A powder preparation of the oligodeoxynucleotide or complex of the present invention is prepared by vacuum drying, freeze drying or the like. The oligodeoxynucleotide or complex of the present invention can be stored in a powder state, and the powder is dispersed in an aqueous solvent for injection and used for use at the time of use.

The content of the oligodeoxynucleotide or complex of the present invention in the pharmaceutical composition is usually from about 0.1 to 100% by weight, preferably from about 1 to 99% by weight, based on the total amount of the pharmaceutical composition, and more preferably About 10 to 90% by weight.

The pharmaceutical composition of the present invention may contain the oligodeoxynucleotide or complex of the present invention as an active ingredient alone, or may contain the oligodeoxynucleotide or complex of the present invention in combination with other active ingredients.

(medical use)

The oligodeoxynucleotides and complexes of the present invention were found to have anticancer effects alone. It is considered that such an effect is an unpredictable effect as compared with the characteristics of the present invention developed as an adjuvant. Therefore, the following anticancer agent is provided, and it is not required to be administered together with a cancer antigen, and is not limited to a specific cancer type, and is appropriately used as a general anticancer agent for the body. effect. Further, of course, it also has immunostimulating activity, and therefore it is expected to have an immunostimulating activity against other diseases, and it is also expected to have a synergistic effect on cancer patients with weak physical strength.

Since the present invention has excellent immunostimulating activity in addition to an anticancer effect, the oligodeoxynucleotides, complexes and pharmaceutical compositions of the present invention can be used as an immunostimulating agent. The mammalian immune response can be caused by administering the oligodeoxynucleotide, complex or pharmaceutical composition of the present invention to a mammal (such as a primate such as a human or a rodent such as a mouse). In particular, the complex of the present invention has the active property of D-type CpG ODN, which stimulates peripheral blood mononuclear cells, and produces a large amount of type I interferon (Pan-IFN-α, IFN-α2, etc.) and type II interferon (IFN- Both of γ) are useful as type I interferon production inducers, type II interferon production inducers, and type I and type II interferon production inducers. Due to the induction of type I and In the case of both type II interferons, the complex of the present invention and the pharmaceutical composition containing the same are useful for the prevention or treatment of a disease in which either or both of type I and type II interferons are effective.

As a method for achieving medical use, for example, by administering to a cancer patient or a human having cancer, (a) the oligodeoxynucleotide of the present invention or the composition comprising the complex of the present invention, The cytotoxic T lymphocyte (CTL) antigen of the subject to be administered is specifically activated, and the cancer is directly (as a single agent) for prevention and treatment of cancer.

In the present specification, the term "subject" means a subject to be diagnosed or detected, or treated, or the like (for example, a human or the like, a biologically extracted cell, blood, serum, or the like).

In this specification, "pharmaceutical", "agent" or "factor" (any of which is equivalent to an agent in English) is used interchangeably in the broadest sense, and may be any substance or as long as it achieves the desired purpose. Other elements (such as light, radiant energy, heat, electricity, etc.). Examples of such a substance include proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, and nucleic acids (for example, including cDNA (Complementary Deoxyribonucleic Acid), genome). DNA such as DNA, RNA (Ribonucleic Acid, RNA), polysaccharides, oligosaccharides, lipids, organic low molecules (eg hormones, ligands, signaling substances) An organic low molecule, a molecule synthesized by combinatorial chemistry, a low molecule (for example, a low molecular ligand, etc.) which can be used as a pharmaceutical, or the like, but is not limited thereto. Wait. As a factor specific to a polynucleotide, a representative sequence may have a certain sequence homology (for example, 70% or more sequence identity) and complementarity to the sequence of the polynucleotide. The polynucleotide, a polypeptide such as a transcription factor that binds to a promoter region, and the like are not limited thereto. As a factor specific to a polypeptide, an antibody or a derivative thereof or an analog thereof (for example, a single-chain antibody) to which the polypeptide is specifically directed, which is a receptor or a ligand, can be exemplified. Specificity in the situation In the case where the polypeptide is an enzyme, the position or the receptor may be referred to as a substrate, but is not limited thereto.

In the present specification, the term "treatment" refers to a disease or disorder (for example, cancer or allergy), and in the case of such a state, it is preferable to maintain the current situation, and it is preferable to maintain the current situation. It is preferable to reduce it, and it is preferable to dissipate it, and it is a case where the symptom improvement effect or the preventive effect of one or more symptoms accompanying a disease, or a disease can be exhibited. There is a case where a diagnosis is performed in advance and an appropriate treatment is referred to as "companion treatment", and a diagnostic drug used therein is referred to as a "companion diagnostic drug".

In the present specification, the term "therapeutic drug (agent)" refers broadly to all agents that can treat a target state (for example, cancer, allergy, etc.). In one embodiment of the present invention, the "therapeutic agent" may be a pharmaceutical composition comprising an active ingredient and one or more carriers which are pharmacologically acceptable. The pharmaceutical composition can be prepared, for example, by mixing the active ingredient with the above carrier, by any method known in the art of pharmacy. Further, the therapeutic agent is not limited as long as it is used for the treatment, and may be an active ingredient alone or a mixture of an active ingredient and an optional ingredient. Further, the shape of the carrier is not particularly limited, and may be, for example, a solid or a liquid (for example, a buffer). Further, therapeutic drugs such as cancer and allergy include inhibitors for preventing cancer, allergies, and the like (prophylactic drugs), or cancers, allergies, and the like.

In the present specification, "prevention" refers to a state in which a disease or disorder (for example, an allergy) is prevented from becoming such a state. The drug of the present invention can be used for diagnosis, and the agent of the present invention can be used, for example, for prevention of allergy or the like, or for prevention.

In the present specification, the term "prophylactic agent (agent)" refers broadly to all agents that can prevent a target state (for example, a disease such as allergies).

In this specification, the term "set" usually means that the provision should be divided into two or more children. Some of the units provided (eg, test drugs, diagnostic drugs, therapeutic drugs, antibodies, markers, instructions, etc.). For stability and the like, it should not be provided by mixing, and the form of the kit is preferred in order to provide a composition which is preferably used for mixing just before use. Preferably, such a kit is advantageous in that it contains instructions or instructions on how to use the supplied portion (eg, test drug, diagnostic drug, therapeutic drug), or how the reagent should be handled. In the present specification, when the kit is used in the form of a reagent kit, the kit usually includes instructions such as the usage of the test drug, the diagnostic drug, the therapeutic drug, the antibody, and the like.

In the present specification, the "instruction" describes a method for explaining the use of the present invention to a physician or other user. The instruction book describes the detection method of the present invention, the usage of the diagnostic drug, or the content of the administration of the medicine or the like. Further, in the instruction book, the content of the injection site for the oral administration or the administration to the esophagus (for example, by injection) may be described. The instructions are produced in accordance with the format prescribed by the government's supervisory government agency (for example, the Ministry of Health, Labour and Welfare in Japan, the Food and Drug Administration (FDA) in the United States, etc.), and are clearly documented by it. Key points recognized by government agencies. The instruction form is a package insert (package insert), which is usually provided by paper media, but is not limited thereto. For example, it can also be electronic media (such as the home page provided by the Internet, email). Such forms are provided.

(the aspect of the preferred embodiment)

Hereinafter, preferred embodiments of the present invention will be described. The embodiments provided below are intended to provide a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is to be understood that the practitioner can refer to the description in the specification and make appropriate changes within the scope of the invention. Further, it is to be understood that the following embodiments of the invention may be used alone or in combination.

<single dose form>

In one aspect, the present invention provides an anticancer agent comprising a complex, the complex Including: (a) an oligodeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylate, and a polydeoxyadenosine system disposed in a humanized K a 3' side of a type CpG oligodeoxynucleotide; and (b) a β-1,3-glucan. In the present invention, it has been found that the complex of the present invention itself functions as an anticancer agent. Previously, the inventors of the present invention have only found that the complex can be used as an adjuvant and make an application, and it is not predicted that it can be directly used as an anticancer agent in the form of a single dose. Therefore, from the viewpoint of use in the case of a cancer-free antigen, it is considered to have an unpredictable effect.

In one embodiment, the anticancer agent of the present invention is characterized in that it is administered in the absence of a cancer antigen.

In another embodiment, the anticancer agent of the present invention is characterized in that it is administered in a manner to be delivered to the reticuloendothelial system and/or lymph nodes. Preferably, the reticuloendothelial system and/or lymph nodes comprise tumors and macrophages. Illustratively the above-described reticuloendothelial system comprises the spleen and/or the liver. Therefore, the anticancer agent of the present invention is characterized in that it is administered in such a manner as to be transmitted to a reticuloendothelial organ (spleen, liver, etc.) and/or lymph nodes containing tumors and macrophages. Without wishing to be bound by theory, it is disclosed that the complex of the present invention is delivered to tumors and macrophages, and the dead cells of the cancer are recruited here to the reticuloendothelial organs (spleen, liver, etc.). Therefore, it is considered that the cancer cells in the body can be further expelled. Therefore, the present invention can be considered to have a remarkable effect from the viewpoint of killing any cancer cells existing in the body and providing an unprecedented anticancer agent without using a specific cancer antigen as an adjuvant.

Therefore, in a further preferred embodiment, the anticancer agent of the present invention is characterized in that it is administered to a tumor and a macrophage in the case of a cancer-free antigen.

Any method can be used for such a delivery method. For example, the above administration can be administered systemically, but is not limited thereto. Systemic administration is preferred. Examples of systemic administration include intravenous administration, intraperitoneal administration, oral administration, subcutaneous administration, intramuscular administration, and the like. In one embodiment, the oligodeoxynucleotides used in the present invention may be K3 (SEQ ID NO: 1), K3-dA ₄₀ (SEQ ID NO: 2), dA _{40 -} K3 (SEQ ID NO: 3), and K3. -dA20 (SEQ ID NO: 4), K3-dA25 (SEQ ID NO: 5), K3-dA30 (SEQ ID NO: 6), and K3-dA35 (SEQ ID NO: 7), etc., but is not limited thereto.

In one embodiment, the β-1,3-glucan used in the present invention may also be Schizophyllum polysaccharide (SPG), lentinan, scleroglucan, cadmium, lycium polysaccharide, ash tree Flower polysaccharide, kelp sugar, etc.

In a preferred embodiment, the complex of the invention is K3-SPG or an analogue thereof. Here, the analog may be, for example, a CpG side structure similar to K3, a β glucan side structure similar to SPG, and the like, but is not limited thereto.

Further, since the anticancer action utilizes various mechanisms, it is not easy to think that it is used for accumulating dead cells of cancer in the spleen. In particular, when systemicity is administered, it is not thought of being used for accumulating tumors and accumulating dead tumor cells in tissues such as the spleen. Moreover, the expression or performance-promoting effect of interleukin 12 (IL12) and/or interferon (IFN) alpha also utilizes a mechanism different from that of anticancer, interleukin 12 (IL12) and/or interferon (IFN) alpha. The performance or performance is promoted in addition to anti-cancer, so it is not easy to think of each other. Therefore, it is considered that each of the uses of the CpG-β-glucan complex of the present invention (anti-cancer use (in the form of a single dose), use of a dead cell of a cancer in the spleen, and use of interleukin 12 (IL12) And/or the use of interferon (IFN) alpha for performance or performance promotion) is in a relationship that is not considered to be easily conceivable.

<The reticuloendothelial system (including the spleen and / or liver) and / or lymph node accumulation agent>

In another aspect, the present invention provides a composition comprising a complex and for collecting dead cells of cancer in a reticuloendothelial system (including spleen and/or liver) and/or lymph nodes, and the complex comprises : (a) an oligodeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and a polydeoxyadenylate, and a polydeoxyadenosine is disposed in a humanized K-type a 3' side of a CpG oligodeoxynucleotide; and (b) a beta-1,3-glucan. Don't want to be theory Under the constraint, it was found that the complex of the present invention can accumulate dead cells of cancer in the reticuloendothelial system (including the spleen and/or liver) and/or lymph nodes. As exemplified in the examples, it was confirmed that the treatment with the complex of the present invention such as K3-SPG induced tumor cell death in a manner dependent on both of IL12p40 and IFN-I. It has not been previously predicted that the composite has such an effect, and in this sense it can be considered to achieve an unpredictable effect. That is, the CpG is targeted by phagocytic cells in the tumor microenvironment. If the dead cells of cancer accumulate in the reticuloendothelial system (including the spleen and/or liver) and/or lymph nodes, then the released tumor cells induce anti-tumor CTL against a plurality of tumor antigens, located in the body. Cancer cells can also be killed by being attacked by shotguns. Without wishing to be bound by theory, the production of both IL12 and IFN-I cytokines in the tumor microenvironment is not considered essential for the single-dose treatment of K3-SPG, but is important.

In one embodiment, the oligodeoxynucleotide used in the present invention is selected from the group consisting of K3 (SEQ ID NO: 1), K3-dA ₄₀ (SEQ ID NO: 2), dA _{40 -} K3 (SEQ ID NO: 3), and K3. -dA20 (SEQ ID NO: 4), K3-dA25 (SEQ ID NO: 5), K3-dA30 (SEQ ID NO: 6), and K3-dA35 (SEQ ID NO: 7).

In another embodiment, the β-1,3-glucan used in the present invention is selected from the group consisting of Schizophyllum polysaccharide (SPG), scleroglucan, cadmium, lycium polysaccharide, ash tree flower A group consisting of polysaccharides and laminaria.

In a preferred embodiment, the composite of the present invention is K3-SPG.

In one embodiment, the reticuloendothelial system and/or lymph nodes that are the subject of the composition of the present invention comprise tumors and macrophages. Illustratively the above-described reticuloendothelial system comprises the spleen and/or the liver. Accordingly, the composition of the present invention is characterized in that it is administered in such a manner as to be delivered to a reticular endothelial system organ (spleen, liver, etc.) and/or lymph nodes containing tumors and macrophages. Without wishing to be bound by theory, it is revealed that the complex of the present invention is delivered to tumors and macrophages, and the dead cells of the cancer are recruited to the organs of the reticuloendothelial system (spleen, liver, etc.). Therefore, it is considered that the cancer cells in the body can be further expelled. therefore, The present invention can be considered to have a remarkable effect from the viewpoint of killing any cancer cells existing in the body and providing an unprecedented anticancer agent without using a specific cancer antigen as an adjuvant.

Therefore, in a further preferred embodiment, the anticancer agent of the present invention is characterized in that it is administered to a tumor and a macrophage in the case of a cancer-free antigen.

Any method can be used for such a delivery method. For example, the above administration can be administered systemically, but is not limited thereto. Systemic administration is preferred. Examples of systemic administration include intravenous administration, intraperitoneal administration, oral administration, subcutaneous administration, intramuscular administration, and the like.

<IL12 and / or IFN performance enhancer>

Further, in another aspect, the present invention provides a composition for promoting the performance or performance of interleukin 12 (IL12) and/or interferon (IFN) gamma comprising: (a) oligomeric deoxygenation Nucleotide comprising humanized K-type CpG oligodeoxynucleotides and polydeoxyadenylate, and polydeoxyadenosine is disposed in humanized K-type CpG oligodeoxynucleotides 3' side; and (b) β-1,3-glucan. The production of IL12 and IFN-I cytokines in the tumor microenvironment is an important effect in the single-agent treatment of K3-SPG. In addition to its role as an anticancer agent, this effect is also important in other uses. Examples of such a treatment include chronic infectious diseases such as cancer and viruses, and prevention of viral infection, and the like, but are not limited thereto.

In one embodiment, the oligodeoxynucleotide used in the present invention is selected from the group consisting of K3 (SEQ ID NO: 1), K3-dA ₄₀ (SEQ ID NO: 2), dA _{40 -} K3 (SEQ ID NO: 3), and K3. -dA20 (SEQ ID NO: 4), K3-dA25 (SEQ ID NO: 5), K3-dA30 (SEQ ID NO: 6), and K3-dA35 (SEQ ID NO: 7).

In another embodiment, the β-1,3-glucan used in the present invention is selected from the group consisting of Schizophyllum polysaccharide (SPG), scleroglucan, cadmium, lycium polysaccharide, ash tree flower A group consisting of polysaccharides and laminaria.

In a preferred embodiment, the composite of the present invention is K3-SPG.

(pharmaceuticals, dosage forms, etc.)

The present invention is provided in the form of a pharmaceutical (therapeutic or prophylactic) of the above various forms.

The route of administration of the therapeutic agent is preferably such that it is effective at the time of treatment, and may be, for example, intravenous, subcutaneous, intramuscular, intraperitoneal, or oral administration. The administration form may, for example, be an injection, a capsule, a tablet, a granule or the like. In the case of administering the component of the present invention, it is effective to use it as an injection. The aqueous solution for injection can also be stored, for example, in a small glass bottle or a stainless steel container. Further, for the aqueous solution for injection, for example, physiological saline, sugar (for example, trehalose), NaCl, or NaOH or the like may be formulated. Further, as the therapeutic agent, for example, a buffer (for example, a phosphate buffer), a stabilizer, or the like may be formulated.

In general, the compositions, medicaments, therapeutic agents, prophylactic agents and the like of the present invention comprise a therapeutically effective amount of a therapeutic or active ingredient, and a pharmaceutically acceptable carrier or excipient. In the present specification, "pharmaceutically acceptable" means listed in the Pharmacopoeia of the government, and more specifically, the government's supervisory government agency, or pharmacopoeia or other commonly recognized pharmacopoeia. As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle that is administered with a therapeutic agent. Such carriers may also be sterile liquids, such as water and oil, including petroleum, animal, plant or synthetic origin, and are not limited, and include peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier for oral administration of medicine. In the case where the pharmaceutical composition is administered intravenously, physiological saline and aqueous glucose are preferred carriers. Preferably, a physiological saline solution, and aqueous dextrose and glycerol solutions are used as the liquid carrier for the injectable solution. In suitable excipients, including light anhydrous citric acid, crystalline cellulose, mannitol, starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, white peony, tannin, sodium stearate, single Stearic acid glycerin, talc, sodium chloride, skim milk, glycerin, propylene, ethylene glycol, water, ethanol, calcium carboxymethylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethyl Cellulose, polyvinyl acetal (diethylamino) acetate, polyvinylpyrrolidone, gelatin, medium chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, white sugar, carboxymethyl fiber Vegetarian, corn starch, Inorganic salts, etc. In a preferred embodiment, the composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The compositions may also be in the form of solutions, suspensions, emulsions, lozenges, pills, capsules, powders, sustained release formulations, and the like. A suppository can also be prepared by using a conventional binder and a carrier such as triglyceride to formulate the composition. Oral formulations may also contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable carriers are described in E. W. Martin, Remington's Pharmaceutical Sciences (Mark Publishing Company, Easton, U.S.A.). Such a composition contains an appropriate amount of a carrier and a therapeutically effective amount of a therapeutic agent, preferably a purified one, in order to provide a form suitable for administration to a patient. Formulations must be suitable for administration. In addition to these, for example, a surfactant, an excipient, a coloring agent, a flavoring agent, a preservative, a stabilizer, a buffer, a suspending agent, an isotonic agent, a binder, a disintegrant, a lubricant, and a flow may be contained. Sexual promoters, flavoring agents, etc.

In one embodiment of the present invention, the "salt" includes, for example, an anionic salt formed of any acidic (e.g., carboxyl group) group or a cationic salt formed of any basic (e.g., amino group) group. The salts include inorganic salts or organic salts, for example, the salts described in Berge et al., J. Pharm. Sci., 1977, 66, 1-19. Further, examples thereof include a metal salt, an ammonium salt, a salt with an organic base, a salt with an inorganic acid, and a salt with an organic acid. In one embodiment of the present invention, the "solvate" is a compound formed from a solute and a solvent. For the solvate, for example, J. Honiget al., The Van Nostrand Chemist's Dictionary P650 (1953) can be referred to. If the solvent is water, the solvate formed is a hydrate. The solvent is preferably one which does not inhibit the biological activity of the solute. Examples of such a preferred solvent are not particularly limited, and examples thereof include water or various buffer solutions. In one embodiment of the present invention, the "chemical modification" may, for example, be a modification using PEG (Polyethylene Glycol) or a derivative thereof, a luciferin modification, or a biotin modification.

In the case where the present invention is administered as a medicine, various delivery systems are known, and such a system can also be used to cast a suitable site (for example, an esophagus). The therapeutic agent of the present invention, for example, encapsulated in liposomes, microparticles, and microcapsules; the use of recombinant cells which can express therapeutic agents (e.g., polypeptides), receptor-mediated endocytosis Use of endocytosis; construction of a therapeutic nucleic acid as part of a retroviral vector or other vector, and the like. The introduction method includes intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes, but is not limited thereto. It can also be administered by any of a suitable route, for example, by infusion, by bolus injection, by absorption through the epithelium or the inner layer of the mucous membrane of the skin (eg, oral mucosa, rectal mucosa, and intestinal mucosa, etc.). For medical use, an aerosol may be used as needed, using an inhaler or a nebulizer, and may be administered together with other biologically active agents. Administration can also be systemic or topical. In the case where the present invention is used for cancer, it can be administered by any of appropriate routes such as direct injection into a cancer (lesion).

In a preferred embodiment, the composition can be formulated in a form suitable for pharmaceutical administration to humans in accordance with known methods. Such a composition can be administered by injection. Compositions for injection administration are representatively solutions in sterile isotonic aqueous buffers. Further, if necessary, the composition may also contain a co-solvent and a local anesthetic such as lidocaine which relieves pain at the injection site. Usually, the components are separately supplied or supplied in a unit dose applicator, for example, in a sealed container such as an ampoule or a sachet showing the amount of the active agent, or a lyophilized powder or a non-aqueous concentrate. Form supply. In the case where administration is to be carried out by injecting the composition, it may be dispensed using an injector containing sterile pharmaceutical grade water or physiological saline. In the case where administration is to be carried out by injecting the composition, ampoules for sterile water for injection or physiological saline may be supplied as a mixable ingredient before administration.

The compositions, medicaments, therapeutic agents, and prophylactic agents of the present invention may also be formulated in a neutral or salt form or other prodrugs (e.g., esters, etc.). The pharmaceutically acceptable salt includes a combination with a free carboxyl group derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and is derived from isopropylamine, triethylamine, 2-ethylaminoethanol. , histidine, procaine, etc. The isolated amine groups are formed together, and are derived from sodium, potassium, ammonium, calcium, and iron hydroxide.

The amount of the therapeutic agent of the present invention that is effective for the treatment of a particular disorder or condition may vary depending on the nature of the disorder or condition, and those skilled in the art will be able to determine based on standard clinical techniques based on the description of this specification. Further, in vitro analysis may be used depending on the situation, and the identification of the optimal dosage range may be assisted. Further, the precise amount to be used in the formulation may also vary depending on the route of administration, and the severity of the disease or disorder, and should be determined based on the judgment of the attending physician and the condition of each patient. However, the amount of administration is not particularly limited, and may be, for example, 0.001, 1, 5, 10, 15, 100, or 1000 mg/kg body weight per one time, or may be within the range of any of these two values. The administration interval is not particularly limited. For example, it may be administered once or twice every 1, 7, 14, 21, or 28 days, or may be administered once or twice for each of the two values. The administration amount, the administration interval, and the administration method can be appropriately selected depending on the age, body weight, symptoms, target organs, and the like of the patient. Further, the therapeutic agent is preferably an effective ingredient comprising a therapeutically effective amount or an effective amount to exert a desired effect. When the marker of a malignant tumor is significantly reduced after administration, it can also be judged to have a therapeutic effect. The effective amount can be estimated based on the amount-response curve obtained from an in vitro or animal model test system.

In one embodiment of the present invention, the "patient" or "subject" includes humans or mammals other than humans (eg, mice, guinea pigs, hamsters, rats, mice, rabbits, pigs, sheep, goats, One or more of a cow, a horse, a cat, a dog, a marmoset, a monkey, or a chimpanzee.

The pharmaceutical composition or therapeutic or prophylactic agent of the present invention can be provided in the form of a kit.

In a specific embodiment, the present invention provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the composition of the present invention or a medicine. Depending on the circumstances, it may also be displayed on such a container in the form prescribed by the government agency that controls the manufacture, use or sale of the medical or biological product, indicating that the government agency permits the use of human investment. Information on the manufacture, use or sale.

In a specific embodiment, the pharmaceutical composition comprising the component of the present invention can be administered by liposome, fine particles or microcapsules. In the diverse aspects of the invention, there is also the possibility of using such compositions to achieve a sustained release of the ingredients of the invention.

The formulation of the therapeutic or prophylactic agents of the present invention as medicines and the like is well known in the art, and is described, for example, in the Japanese Pharmacopoeia, the United States Pharmacopoeia, and other national pharmacopoeias. Therefore, as long as the description of the present specification is made, the practitioner can determine an embodiment such as the amount to be used without excessive experimentation.

(general technology)

Molecular biological methods, biochemical methods, and microbiological methods that can be used in the present specification are well known and used in the art, for example, in Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, FM (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, FM (1989). Short Protocols in Molecular Biology: A Compendium of Methods From Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, MA (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, FM (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, FM (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, MAet al. (1995). PCR Strategies, Academic Press; Ausubel, FM (1999). Short Proto Cols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, J. J. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, Supplementary Laboratory Medicine, "Gene Introduction and Expression Analysis Experiments", Yangtu, 1997, etc., and in the present specification Reference the relevant parts of the information (may be all).

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

For example, in the present specification, the oligonucleotide of the present invention can also be synthesized by standard methods known in the art, for example, by using an automated DNA synthesis device (sold by Biosearch, Applied Biosystems, etc.). . For example, a phosphorothioate-oligonucleotide can also be synthesized by the method of Stein et al. (Stein et al., 1988, Nucl. Acids Res. 16: 3209), or by using a tuned glass polymer. A methylphosphonate-oligonucleotide was prepared by a support (Sarinet al., 1988, Proc. Natl. Acad. Sci. USA 85: 7448-7451) and the like.

In this manual, "or" is used when "at least one or more" of the items listed in the article can be used. "Or" is the same. In the present specification, when clearly stated as "in the range of two values", the range also includes the two values themselves.

In this specification, reference to the cited scientific literature, patents, patent applications, etc. The contents of the documents are to be referred to in the specification in the form of reference to the same extent as those specifically described.

Hereinabove, in order to make the present invention easy to understand, a preferred embodiment has been described. The invention is described below by way of examples, but the description and the examples below are provided for the purpose of illustration only, and are not intended to limit the invention. Therefore, the scope of the present invention is not limited to the embodiments and examples specifically described in the specification, and is only limited by the scope of the claims.

[Examples]

Hereinafter, examples will be described. When necessary, regarding the treatment of the animals used in the following examples, all animal experiments were carried out in accordance with the guidelines of the Japan Pharmaceutical Research Institute and the Osaka University Animal Facilities. Also, based on the Helsinki Declaration. Specifically, the reagents described in the examples are used, but they may be replaced by other manufacturers (Sigma-Aldrich, Wako Pure Chemical Industries, Nacalai, R&D Systems, USCN Life Science INC, etc.).

(Manufacturing Example)

The following CpG ODNs were synthesized by Genedesign, Inc. (underlined for phosphorothioate linkages).

(sequence number 2); (SEQ ID NO: 3); Alexa 488-labeled K3; Alexa 488-labeled K3-dA40; Alexa 647-labeled K3;

(sequence number 2 of the sequence listing)

(sequence number 7 of the sequence listing)

(sequence number 6 of the sequence listing)

(sequence number 5 of the sequence listing)

(sequence number 4 of the sequence listing)

(s in the above sequence indicates that the phosphodiester bond between the nucleosides is substituted with a phosphorothioate bond)

This oligodeoxynucleotide is used as a conventional solid phase phosphatide method (Nucleic Acids in Chemistry and Biology, 3. Chemical synthesis (1990) ed. G. Michael Blackburn and Michael J. Gait. Oxford University Synthesis).

Ovalbumin (OVA) was purchased from Zisheng Chemical Industry Co., Ltd. DQ-OVA, Alexa488-OVA, CFSE, and Lipofectamine 2000 were purchased from Invitrogen. Hoechst 33258, zymosan and delamal are purchased from SIGMA. Zymosan-Depleted is purchased from Invivogen. The clodronate lipid system was purchased from FormuMax. The influenza lysate vaccine, the formalin-inactivated whole virus (WIV), and the purified influenza virus (H1N1) were prepared as previously described (Koyama, S., et al., Science translational medicine 2, 25ra24 ( 2010)).

Complexation of CpG ODN and SPG (Manufacturing Example Figure 1)

7.22 mg of K3-dA40 was dissolved in water (3.7 mL). SPG (Mitsui Sugar) 15 mg was dissolved in 0.25 N NaOH (1 mL). 1 mL of 330 mM NaH ₂ PO _{4 was} added to the DNA solution, and then the SPG solution was added to the DNA/NaH ₂ PO ₄ solution and maintained at 4 ° C overnight, thereby ending the complexation. The molar ratio (MSPG/MDNA) was fixed at 0.27. Regarding the formation of the composite, the formation of the composite confirmed by the microchip electrophoresis apparatus (SHIMADZU: MultiNA) was confirmed by using the size exclusion chromatography to monitor the absorption at 260 nm to confirm the displacement of the CpG ODN toward the high molecular weight side ( System: Agilent 1100 Series, Column: Two Asahipak GF7M-HQ (Shodex) were linked at a flow rate of 0.8 mL/min. Buffer: 10 mM EDTA (Ethylenediamine Tetraacetic Acid, ED) PBS (Phosphate Buffer Solution, phosphate buffer), pH 7.4, temperature: 40 ° C).

(for the preparation of the examples)

In the following examples, it is disclosed that a systemic single-agent treatment using a nanoparticle that targets a macrophage in a tumor microenvironment that induces a stronger tumor shrinkage is used as a single-agent treatment. The TLR9 agonist is carried out.

(Materials and methods)

Hereinafter, the reagents, materials, animals, cells, and methods used in the examples will be described. Supplementary explanations are also appropriately made in the respective embodiments.

(animals and reagents)

Female C57BL/6J mice, 6 weeks old, were purchased from Nihon CLEA. Il12p40 deficient mice and Batf3 deficient mice were purchased from the Jackson Laboratory. Ifnar2 deficient mice, Myd88 deficient mice, and Dectin-1 deficient mice are as described above (Kobiyama, K., et al. Proc. Natl. Acad. Sci. U.S.A. 111, 3086-3091 (2014)). All animal experiments were performed in accordance with the guidelines of the institution of the Institute of Pharmaceutical Sciences. K3 is synthesized by Gene Design. The autogenic chemical industry purchases ovalbumin (OVA).

(cell strain)

EL4 and EL4 (EG7) expressing OVA are thymoma cell lines of C57BL/6J mice, purchased from ATCC. B16 (melanoma) was purchased from the Japanese Collection of Research Bioresourses. Self-scientific cell bank purchased B16F10 (melanoma), MC38 (colon cancer) was provided by Dr. F.JAMES Primus. Purchase Pan02 (pancreatic cancer) from Jackson's Laboratory. EL4, EG7, MC38, and Pan02 were cultured in complete RPMI (RPMI 1640 supplemented with 10% (v/v) fetal bovine serum (FBS, Fetal Bovine Serum), penicillin, and streptomycin). B16 and B16F10 were cultured in complete DMEM (Dulbecco Modified Eagle Medium) (DMEM supplemented with 10% (v/v) fetal bovine serum (FBS), penicillin, and streptomycin).

(tumor experiments and treatment methods)

EG7, EL4, B16, B16F10, and MC38 cells (100 μl in 5% recipient gel/PBS at 5 × 10 ⁶ cells/mL) (sc) were inoculated subcutaneously into the right abdomen of the mice. For tumor size, the length (L), width (W), and height (H) of the tumor were measured, and the tumor volume (V) was calculated from V = L × W × H. Direct injection into the tumor site by intratumoral injection (it). CpG treatment begins after the tumor volume reaches approximately 100 mm ³ , which is 7 days after inoculation of EG7 and B16F10, 10 days after inoculation of B16, and 14 days after inoculation of MC38. Tumor-bearing mice were treated 3 times with K3 (30 μg) or K3-SPG (10 μg) every other day.

(Pan02 peritoneal inoculation model)

In the peritoneal inoculation model of Pan02, 1 × 10 ⁶ Pan02 cells (100 μl in 1 × 10 ⁷ cells/mL in PBS) were intraperitoneally injected. CpG treatment was started 11 days after the inoculation, and all tumor nodules were removed from the peritoneum of the mice on day 21, after which the weight (g) was determined. The amount of administration in CpG treatment is as described above.

(In vivo imaging experiment)

To evaluate the presence of K3 and K3-SPG, C57BL/6 mice were inoculated with EG7 on day 0 and PBS (control), Alexa 647-K3 (30 μg), or Alexa 647-K3- on day 12 SPG (10 μg). One hour after the administration, the mice were analyzed using the IVIS (registered trademark) fluorescent imaging system (Lumina Imaging System) and the analysis software (Ver. 2.6, Xenogen), and the images measured by the relative fluorescence were converted into the surface. The physical unit of radiance (photons/sec/cm ² /sr). In order to detect labeled CD8 ⁺ T cells in vivo, C57BL/6 mice or Il12p40-Ifnar with EG7 were treated with K3-SPG on days 7, 9, and 11 on day 14, or untreated. Gene double knockout mice recover spleen cells. After suspension of the spleen cells, the red blood cells were lysed using ACK lysis buffer (150 mM NH ₄ Cl, 10 mM KHCO ₃ , 0.1 mM Na ₂ EDTA), and the cells were maintained in complete RPMI. CD8α ⁺ T cells were sorted by MACS (Magnetic Activated Cell Sorting) (Miltenyi Biotec). CD8α ⁺ T cells were sorted by a negative selection method. Thereafter, the sorted CD8α ⁺ T cells were stained with Xenolight DiR (registered trademark). On day 14 (EG7 inoculated on day 0, iv treated with K3-SPG on days 7, 9, and 11 or untreated C57BL/6 mice or Il12p40-Ifnar2 gene double knockout mice) The stained CD8α ⁺ T cells were transplanted into the transplanted mice. After transplanting the stained cells for 24 hours, the mice were analyzed using an IVIS (registered trademark) Lumin Imaging System (Ver. 2.6). The target area was collected in the tumor area, and the fluorescence intensity was analyzed using Living Image Software (Ver. 2.6, Xenogen).

(immunochemistry)

For C57BL/6J mice (6-8 weeks old, female, CLEA Japan), iv injection of Alexa 647-K3 (30 μg), Alexa 647-K3-SPG (10 μg), and dextran-PE (20 μg) from the tail vein ). Tumors were recovered 1 hour after the injection, and frozen sections were fixed with 4% (w/v) paraformaldehyde for 10 minutes, and stained with Hoechst 33258 and anti-CD3e antibody, anti-CD8β antibody. Cells were photographed using the Olympus IX81 system. Analyze image data using MetaMorph.

(depletion experiment)

To deplete phagocytic cells (dendritic cells and macrophages), C57BL/6 mice were injected iv with clodronate liposomes or control liposomes (100 nm) (Katayama Chemical) 5 days after inoculation of EG7. To deplete CD8 ⁺ T cells, 200 μg of anti-CD8α antibody was iv injected into the tail vein 6 days after inoculation and 13 days after inoculation of EG7.

(analysis of splenocytes)

Splenocytes were recovered on day 14, from the iv treatment with K3-SPG on day 7, 9, and 11 or the untreated C57BL/6 mouse or Il12p40-Ifnar2 gene double knockout mice bearing EG7. After preparation of spleen cells, red blood cells were solubilized using ACK lysis buffer and cells were maintained in complete RPMI. H-2K ^b OVA tetramer (MBL), anti-CD8α antibody (KT15), anti-TCRβ antibody (H57-597), anti-CD62L antibody (MEL-14), and anti-CD44 antibody (IM7), and 7-amine Splenocytes were stained with actinomycin D (7AAD, 7-Aminoactinomycin D). The number of cells of OVA tetramer ⁺ CD44 ⁺ CD8α ⁺ TCRβ ⁺ was determined by flow cytometry. For other experiments, the prepared spleen cells were cultured together with an anti-CD45 antibody, an anti-CD3e antibody, an anti-CD8α antibody, and an anti-CD11a antibody, and then analyzed by flow cytometry.

(analysis and immunization of CD45-negative cells)

Splenocytes were recovered on day 12 from VII treatment with K3-SPG, or untreated C57BL/6 mice with Il7 or Il12p40-Ifnar2 gene double knockout mice on days 7, 9, and 11. After preparation of spleen cells, red blood cells were solubilized using ACK lysis buffer and cells were maintained in complete RPMI. Splenocytes were stained with an anti-CD45 antibody (APC) and the number of CD45 ^- cells was determined by flow cytometry. Further, a subset of apoptotic cells, necrotic cells, and CD45-negative living cells were stained with PI and Hoechst 33342, and then analyzed by flow cytometry. Next, CD45 ^- cells were sorted by INFLUX (BD Bioscience) from tumor-bearing C57BL/6 mice treated with K3-SPG.

(vaccination model)

Of C57BL / 6 mice were administered iv on day -7 to 5 × 10 ⁵ th CD45 ^- cells. After 7 days of immunization, mice were inoculated with 5 x 10 ⁵ EG7 cells on day 0.

(Measurement of cytokines)

The amount of mouse IL-12p40, mouse IL-13, and human IFNy was determined using an R&D ELISA kit.

(Statistical Analysis)

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

(Example 1: Intravenous injection of K3-SPG, even if no tumor antigen was added at all, induced inhibition of strong tumor growth)

In the present example, by intravenous injection of K3-SPG, it was confirmed that even if no tumor antigen was added at all, the inhibition of strong tumor growth was induced.

(Experiment using EG7 (mouse thymoma cell line expressing OVA) model)

On day 0, C57BL/6 mice were inoculated with EG7 (a mouse thymoma cell line expressing OVA) in the right abdomen, administered subcutaneously (id), intratumoral (it), or intravenously near the base of the tail. (iv) Three different routes of administration were performed by PBS, and other molar amounts of K3 (30 μg) or K3-SPG (10 μg) for three treatments (after 7, 9, 11 days after inoculation). Tumor size was measured every 2 to 3 days until day 23.

(result)

The results are shown below in Fig. 2 (A to B). In the group of PBS (control), tumor growth was not inhibited by any of the administration routes (Fig. 2a, b, c (Fig. 2A)). In the K3 treatment, tumor shrinkage was only observed in i.t. and was not observed in other routes (Fig. 2d, e, f (Fig. 2A)). In the K3-SPG treatment, stronger tumor shrinkage was observed by both i.t. and i.v., i.d. administration did not show an effect on tumor growth (Fig. 2g, h, i (Fig. 2A)). Will compare, The diagrams represented by K3 and K3-SPG are shown in the figure (Fig. 2a, d, g (Fig. 2A)).

In the prior art, most of the attempts in the treatment of systemic CpG ODN against cancer have not been successful (Lou, Y., et al. Journal of immunotherapy (Hagerstown, Md.: 1997) 34, 279-288 (2011); Nierkens , S., et al. PLoS One 4, e8368 (2009)), it is therefore impossible to predict the fact that treatment with K-SPG iv alone can strongly inhibit tumor growth, and in this respect, the invention proves to be unpredictable. effect.

(Experiment using other tumor cell lines)

To investigate the potential of this K3-SPG systemic single-dose treatment, other tumor cell lines were also tested by the same experimental method used in the EG7 model.

(result)

Intravenous administration of K3-SPG also inhibited the growth of melanoma (B16 and B16F10) and colon cancer (MC38) (Fig. 2j, k, l (Fig. 2B)). The inventors of the present invention further conducted experiments by producing a tumor inoculation model having a higher clinical malignancy. A mouse pancreatic tumor strain, Pan02 (1×10 ⁶ cells) was inoculated into the abdominal cavity (also referred to as “ip”), and thereafter, treatment with K3 or K3-SPG was started 11 days after inoculation (every 1 3 times a day). All mice were sacrificed on day 21 and the total weight of the tumor in the abdominal cavity was evaluated (Fig. 2m (Fig. 2B)). Regarding tumor growth, inhibition was significantly inhibited in the K3-SPG iv treated group, but not in the K3 ip and K3-SPG ip treated groups (Fig. 2m (Fig. 2B)). Correspondingly, a significant increase in survival was observed in the K3-SPG iv treated population, but not in the K3 iv population (Fig. 2n (Fig. 2B)). These results demonstrate that systemic iv administration of K3-SPG is a promising single-agent treatment for most different cancers, and thus does not require any tumor peptides and antigens.

(Example 2: K3-SPG is based on phagocytic cells in the tumor microenvironment)

Next, the inventors and the like have elucidated the mechanism of the tumor microenvironment of K3-SPG.

K3-SPG forms nanoparticles of a size of about 30 nm (Kobiyama, K., et al. Proc. Natl. Acad. Sci. U.S.A. 111, 3086-3091 (2014)). The inventors of the present invention hypothesized that K3-SPG functions via a drug delivery system to tumors (Na, J.H., et al. Journal of controlled release: official journal of the Controlled Release Society 163, 2-9 (2012); Petros, RA et al. Nat Rev Drug Discov 9, 615-627 (2010); Pante, N. et al. Molecular biology of the cell 13, 425-434 (2002); Davis, ME, et al. Nat Rev Drug Discov 7, 771-782 (2008); Farokhzad, OC et al. ACS Nano 3, 16-20 (2009)).

(fluorescent marker imaging)

In order to test the distribution in vivo, K3 and K3-SPG were fluorescently labeled. The ig7 tumor-bearing mouse i.v. was injected with PBS, Alexa647-K3 (30 μg), or Alexa647-K3-SPG (10 μg), after which the distribution of fluorescence was tested using an in vivo imaging system (IVIS).

The results are shown below in FIG.

IVIS imaging showed that after 1 hour of i.v. administration, not K3 accumulates in the tumor site, but K3-SPG accumulates in the tumor site (Fig. 4a). It was observed that the accumulation of K3-SPG in tumors was closely related to the effectiveness of tumor shrinkage in CpG single-agent therapy (Fig. 2). In the IHC (Immunology Histology Chemistry) test, the inventors of the present invention failed to detect Alexa647-K3 in the tumor microenvironment (Fig. 4b). On the other hand, Alexa647-K3-SPG was observed in the tumor area (Fig. 4c). The inventors of the present invention failed to detect the Alexa647 signal in IHC after 24 hours. EG7 cells exhibited CD3e on their surface (because EG7 was derived from a thymoma cell line), and K3-SPG did not aggregate in CD3e, indicating that the K3-SPG line was taken up by non-tumor cells. The nanoparticle system is selected by phagocytic cells such as macrophages and dendritic cells (DC), which can be in vivo by TRITC (Tetramethyl Rhodamine Iso-thiocyanate, rhodamine tetramethyl isothiocyanate)- Glucan is labeled. Therefore, the present inventors performed intravenous injection of fluorescently stained K3, K3-SPG, or SPG and TRITC-dextran, and tested the coexistence of such IHC (Fig. 4d, e, f). After 1 hour of i.v. injection, dextran was observed in the tumor area of all samples (Fig. 4d, e, f), which showed that the tumor microenvironment contained phagocytic cells. One with the previous result As a result, Alexa647-K3 was not observed in the tumor (Fig. 4d). About 50% of Alexa647-K3-SPG and FITC-SPG observed in tumors coexisted with TRITC-glucan-positive cells (Fig. 4e, f, g), which showed that K3-SPG was phagocytized in the tumor microenvironment. The cells are taken in. A part of K3-SPG is not aggregated with dextran, and the inventors of the present invention presumed to passively accumulate in the space in the tumor tissue by enhancing the vascular permeability and enhancing the effect of enhanced permeability (EPR). To test the importance of phagocytic cells for K3-SPG i.v. treatment, the inventors of the present invention injected clodronate liposomes intravenously. The present inventors did not use the usual liposome of 200 to 300 nm, but used a 100 nm clodronate liposome and injected it to deplete phagocytic cells in the tumor (Pante, N.et). Al. Molecular biology of the cell 13, 425-434 (2002); Pante, N. et al. Molecular biology of the cell 13, 425-434 (2002)), by injection, F4/80 positive cells in tumors are in 2 days. Almost exhausted inside (Figure 5). Mice with tumors were injected with clodronate liposomes on day 5 (2 days prior to the initial K3-SPG treatment), or no injection, using K3-SPG in the same manner as in Figure 2 (A-B) The mice were treated. In the case of prior injection of clodronate liposomes, the inhibition of K3-SPG-mediated tumor growth was significantly offset (p<0.05) (Fig. 4h), on the other hand, the injection of clodronate liposome itself There was no effect on tumor growth compared to PBS treated mice. These results show that K3-SPG is targeted by phagocytic cells in the tumor microenvironment, and the anti-tumor effect of K3-SPG is largely dependent on the introduction of K3-SPG into phagocytic cells in the tumor microenvironment.

(Example 3: Production of both IL12 and IFN-I cytokines in the tumor microenvironment is important for the single-dose treatment of K3-SPG)

Next, in the present example, the inventors of the present invention tested the factors required for the success of the K3-SPG single-agent treatment.

It is revealed that a cytokine such as IL-12 and IFN-I contains K3-SPG (Kobiyama, K., et al. Proc. Natl. Acad. Sci. USA 111, 3086-3091 (2014)) CpG ODN (Krieg, AM, et al. Journal of immunology 161, 2428-2434 (1998); Klinman, D. M., et al. Immunity 11, 123-129 (1999); Ishii, K. J., et al. Current opinion in molecular therapeutics 6, 166-174 (2004)) Important immunostimulatory factors. Therefore, the inventors of the present invention have tested whether or not the reduction of tumors by treatment with K3-SPG by IL-12 and IFN-I is necessary.

Il12p40 and IFNAR2 deficient mice were subcutaneously inoculated with EG7 cells on day 0, and i.v. treatment was performed three times with PBS or K3-SPG (10 μg) as shown in Fig. 2 (A to B). Thereafter, the effect of K3-SPG on tumor shrinkage was observed.

(result)

The results are shown below in Fig. 6 (A to B). The effect of K3-SPG on tumor shrinkage is partially dependent on IL-12p40 and IFN-I signaling (Figures 6a, b (Figure 6A)). Further, the present inventors conducted experiments on double knockout (DKO) mice of IL12p40 and IFNAR2, and found that the effect of K3-SPG was completely inhibited in DKO mice (Fig. 6c (Fig. 6A)). IFN-β and IL-12p40 were also detected in tumors by IHC staining (Fig. 7 and Fig. 8). These data indicate that secretion of both IL12p40 and IFN-I cytokines in tumors is important for K3-SPG-mediated tumor suppression.

Further, the inventors of the present invention tested Rag2 mice which were completely deficient in T cells and B cell-mediated adaptive immune responses. It was found that with regard to Rag2 mice, even if K3-SPG treatment was performed, it was impossible to control all tumor growth (Fig. 6d (Fig. 6A)), but the inventors of the present invention successfully succeeded during the three treatments using K3-SPG. Tumor growth in rag2-deficient mice was controlled (Fig. 6f (Fig. 6A)). In order to confirm this observation, the inventors of the present invention made 6 treatment groups of rag2 mice (days 7, 9, and 14 and days 14, 16, and 18), and found that in the experimental method of rag2 mice, Obvious tumor control (Fig. 6f (Fig. 6A)). Significantly, IL12p40 and IFNAR2 DKO mice did not respond at all to K3-SPG single-agent treatments even with this wide range of treatment assays (Fig. 6e (Fig. 6A)). These data indicate that treatment with K3-SPG induces both intratumoral IL-12p40 and IFN-I, resulting in both a natural immune response to the tumor and an adaptive immune response.

(Example 4: K3-SPG treatment induces tumor cell death in a state dependent on both IL12p40 and IFN-I)

In this example, it was demonstrated that K3-SPG treatment induces tumor cell death in a state dependent on both IL12p40 and IFN-I.

The inventors of the present invention observed that the inhibition of growth of a part of the tumor which is not dependent on the immunization observed in the rag2 mouse, and the complete inhibition of the growth of the tumor in the IL12p40 and IFNAR2 DKO mice which are independent of the adaptation of the immunity are observed. Humans tested the tumor-host interaction in a wide range of K3-SPG treatments.

The present inventors have found that the spleen removed on the 12th day (the second day of treatment with K3-SPG) contains a large amount of CD45-negative cells as compared with the PBS-treated spleen (Fig. 6g (Fig. 6B)) . Significantly, these CD45-negative cells were significantly reduced in IL12p40 and IFNAR2 DKO mice (Fig. 6g, h (Fig. 6B)). The inventors and the like sorted the CD45-negative cells. This size and morphology fully demonstrate that these lines are derived from tumor cells. It was further confirmed by EG7 inoculation experiments on GFP mice that these CD45-negative cells were also GFP-negative, indicating that the cell lines were derived from tumor cells (Fig. 9). This hypothesis is also supported by the fact that EG7 cells do not exhibit CD45, and CD45 is negative. Regarding Hoechst and PI staining, most of the CD45-negative cell lines in the spleen contain dead cells characterized by both apoptosis and necrosis (Fig. 6i (Fig. 6B)). These data indicate that tumor phagocytic cells that are targeted by K3-SPG secrete IL-12p40 and IFN-I in the tumor microenvironment, which induce tumor cell death and circulate it, and It was eventually caught by the spleen.

(Example 5: Released tumor dead cells induce anti-tumor CTL against a plurality of tumor antigens)

In this example, it was demonstrated that the released tumor dead cells induced anti-tumor CTL against a plurality of tumor antigens.

In order to test the immunogenicity of the CD45-negative cells found in the spleens of K3-SPG-treated mice, the inventors of the present invention sorted the cells as immunologically. Untreated mice were injected intravenously. Thereafter, the immunized mice were transplanted with EG7 tumor cells 7 days after administration of the sorted cells. CD45-negative cell-immunized mice significantly defended the proliferation of EG7 tumors (Fig. 6j (Fig. 6B)). Significantly, OVA257 tetramer-positive cells in the control and immunized mice (red dots in Figure 6k (Figure 6B)) showed no correlation with tumor size (Figure 6k (Figure 6B) Regarding the immunization caused by CD45-negative cells, a more effective further immune response against EG7 tumors was induced compared to the simple OVA257 epitope (Fig. 6k (Fig. 6B)). These results show that K3-SPG single-agent treatment induces tumor cell death depending on both IL-12 and IFN-I, which functions as an effective immunogen for anti-tumor immune responses. .

(CD8T cell line is an important effector for K3-SPG-mediated tumor shrinkage)

The results of Rag2 mice show that the tumor suppressive effect of K3-SPG also depends on the adaptive immune response. Therefore, the present inventors conducted experiments on CD8 T cells required for K3-SPG treatment. Depletion of CD8 T cells in vivo was shown to significantly inhibit the anti-tumor effect of K3-SPG (Fig. 10a (Fig. 10A)), an important effector cell in the K3-SPG treatment. Furthermore, it was shown that tumor shrinkage using K3-SPG also relied on Batf3 (cross presentation CD8α ⁺ DC) (Fig. 10b (Fig. 10A)), and K3-SPG single-agent treatment also enhanced CD8α ⁺ DC-mediated Cross presentation. The inventors of the present invention observed a significant correlation between tumor infiltration of CD8 T cells and tumor growth. CD8 T cells accumulated in the tumor region in the K3-SPG iv group but did not accumulate in the id group (Fig. 10c (Fig. 10A)).

Finally, the inventors of the present invention conducted tests necessary for the entry of such CD8 T cells into the tumor region. WT mice and Il12p40-Ifnar2 DKO mice were inoculated with EG7 cells on day 0 and treated with K3-SPG or PBS on days 7, 9, and 11 for iv treatment. On day 14, CD8α ⁺ T cells were purified from the spleens of these mice, stained with Xenolight DiR (registered trademark), and transplanted to other bands treated with K3-SPG (days 7, 9, and 11). Mice with EG7 (after 14 days of inoculation), and then distribution of CD8 T cells labeled with Xenolight DiR (registered trademark) were analyzed by IVIS on day 15 (Fig. 11). On day 15, CD8 T cells derived from donor mice with untreated tumors did not accumulate in the tumor sites of WT-transplanted mice even after treatment with K3-SPG (Fig. 10d (Fig. 10B), II). On the other hand, CD8 T cells derived from donor mice bearing tumors treated with K3-SPG were detected in tumor sites of transplanted mice (Fig. 10d (Fig. 10B), I), showing K3-SPG A single dose of treatment induces anti-tumor CD8 T cells that can move to the tumor microenvironment and infiltrate. These in vivo activated CD8 T cells can enter the tumor microenvironment of DKO-transplanted mice (Fig. 10e (Fig. 10B)). Even though IL-12 and IFN-I are important for natural immunity and CD8 T cell induction by systemic K3-SPG single-agent treatment, the results also show that if CD8 T cells are activated in K3-SPG therapy, Secretion of IL-12 and IFN-I cytokines in the tumor microenvironment is not necessarily necessary for tumor infiltration of CD8 T cells. Taken together, these results show that activation of tumor-specific CD8 T cells is sufficient for tumor infiltration. Surprisingly, the infiltration of these CD8 T cells is independent of the production of cytokines in the tumor microenvironment.

(discussion)

The inventors and the like have revealed the possibility of novel cancer immunotherapy. It is a novel treatment of CpG targeted by phagocytic cells in the tumor microenvironment (Fig. 12). Through TLR9 stimulation, CpG induces immune responses to immune cells, especially macrophages and DCs (Klinman, DM, et al. Immunity 11, 123-129 (1999); Ishii, KJ, et al. Current opinion in molecular therapeutics 6,166-174 (2004)). This activation is very important in combating the cancer immune response. In the previous report, CpG must be administered directly into the tumor, but if it is a complex of SPG and CpG, it is displayed even in the systemic administration with the function of DDS (Drug Delivery System). Efficacy with intratumoral administration or above (Schettini, J., et al. Cancer immunology, immunotherapy: CII 61, 2055-2065 (2012); Lou, Y., et al. Journal of immunotherapy (Hagerstown, Md.: 1997) 34, 279-288 (2011); Nierkens, S., et al. PLoS One 4, e8368 (2009); Heckelsmiller, K., et al. Journal of immunology 169, 3892-3899 (2002); Ishii, KJ, et al. Current opinion in molecular therapeutics 6,166 -174 (2004)), the inventors of the present invention have solved this problem. A complex of SPG and CpG produced by the formation of nanoparticles (Kobiyama, K., et al. Proc. Natl. Acad. Sci. U.S.A. 111, 3086-3091 (2014)) can be stabilized in vivo. It is known that this effect can be targeted by the tumor environment, and TLR9 immunocompetent cells are supplied to the tumor environment. The novel CpG developed by the inventors of the present invention is engulfed by phagocytic cells due to the formation of nanoparticles.

Thereafter, in the tumor environment, phagocytic cells phagocytosed with the novel CpG produce cytokines such as IFN and IL-12. It is very important that these cytokines are induced in the tumor environment. In the previous report, in the treatment of IFNβ with the tumor environment as a direct target, the dendritic cells are moved within the tumor, and the antigen cross-presentation in the microenvironment within the tumor is increased, thereby re-activating the CTL. These cytokines cause cell death in tumor cells. Further, the inventors of the present invention found that the effect is caused by activation of natural immunity. This cell death takes on a very important role. It becomes a collaboration between natural immunity and adaptive immunity. Infertility is induced by the release of tumor cell death from the tumor microenvironment. The death of the immunogenic tumor cells induces a plurality of cellular toxic T lymphocytes. The tumor-specific induced CTL in vivo as described above can infiltrate the tumor microenvironment in response to the tumor. It is believed that the anti-tumor immune system can use immunological editing (Immunoediting) as a barrier to cancer immunotherapy using an endogenous antigen.

The circulation of tumor cells after a single dose of K3-SPG can function as a biomarker that is excellent in the treatment effect of tumors.

(Example 6: Formulation Example)

Hereinafter, the composition in the case of formulation is disclosed.

For example, 7.22 mg of K3-dA ₄₀ (SEQ ID NO: 2) was dissolved in water (3.7 mL), and SPG (15 mg) was dissolved in 0.25 N NaOH (1 mL). A 1 mL volume of 330 mM NaH ₂ PO _{4 was} added to the DNA solution, and then the SPG solution was added to the DNA/NaH ₂ PO ₄ solution, and maintained at 4 ° C overnight to complete the complexation. The molar ratio (M _SPG /M _DNA ) can be fixed at 0.27.

The agents used in the formulations are available from Genedesign, invivogen, Wako, and the like.

The present invention has been exemplified by the preferred embodiments of the present invention as described above, but it is understood that the scope of the invention should be construed only by the scope of the claims. In the present specification, the cited patents, patent applications, and documents are to be understood as being the same as the contents of the present disclosure.

[Industry availability]

According to the present invention, a novel form of an anticancer agent which can be used in the form of a single dose can be provided. Therefore, the complex of the present invention is useful as an anticancer agent in the field of medicine.

[sequence table free content]

Sequence number 1: K3

Sequence number 2: K3-dA ₄₀

Sequence number 3: dA ₄₀ -K3

Sequence number 4: K3-dA20

Sequence number 5: K3-dA25

Sequence number 6: K3-dA30

Sequence number 7: K3-dA35

<110> Independent Administrative Corporation Pharmaceutical Foundation Research Institute

<120> Application of nucleic acid polysaccharide complex with immunostimulating activity as antitumor drug

<130> NIB006PCT

<160> 7

<170> PatentIn version 3.5

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<211> 20

<212> DNA

<213> Artificial sequence

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<223> K3

<220>

<221> Phosphorothioate linkage

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<211> 60

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<220>

<223> K3-dA40

<220>

<221> Phosphorothioate linkage

<222> (1)..(59)

<400> 2

<210> 3

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> dA40-K3

<220>

<221> Phosphorothioate linkage

<222> (1)..(59)

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<210> 4

<211> 40

<212> DNA

<213> Artificial sequence

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<223> K3-dA20

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<221> Phosphorothioate linkage

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<221> Phosphorothioate linkage

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<210> 6

<211> 50

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<213> Artificial sequence

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<221> Phosphorothioate linkage

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Claims (23)

An anticancer agent comprising a complex comprising: (a) an oligodeoxynucleotide comprising humanized K-type CpG oligodeoxynucleotides and polydeoxyadenylate, and The polydeoxyadenosine is disposed on the 3' side of the humanized K-type CpG oligodeoxynucleotide; and (b) β-1,3-glucan. The anticancer agent according to claim 1, wherein the anticancer agent is administered in the absence of a cancer antigen. The anticancer agent according to claim 1 or 2, wherein the anticancer agent is administered by delivery to the reticuloendothelial system and/or lymph nodes. The anticancer agent according to claim 3, wherein the reticuloendothelial system and/or lymph nodes comprise tumors and macrophages. The anticancer agent according to claim 3 or 4, wherein the reticuloendothelial system comprises a spleen and/or a liver. The anticancer agent according to any one of claims 1 to 5, wherein the anticancer agent is administered in the absence of a cancer antigen. The anticancer agent according to any one of claims 2 to 6, wherein the above administration comprises systemic administration. The anticancer agent according to claim 7, wherein the systemic administration is selected from the group consisting of intravenous administration, intraperitoneal administration, oral administration, subcutaneous administration, intramuscular administration, and intratumoral administration. The anticancer agent according to any one of claims 1 to 8, wherein the oligodeoxynucleotide is selected from the group consisting of K3 (SEQ ID NO: 1), K3-dA ₄₀ (SEQ ID NO: 2), and dA _{40 -} K3 (sequence). No. 3), K3-dA20 (SEQ ID NO: 4), K3-dA25 (SEQ ID NO: 5), K3-dA30 (SEQ ID NO: 6), and K3-dA35 (SEQ ID NO: 7). The anticancer agent according to any one of claims 1 to 9, wherein the β-1,3-glucan is selected from the group consisting of Schizophyllum polysaccharide (SPG), lentinan, scleroglucan, and cadmium, It is composed of polysaccharides, ash tree flower polysaccharides and laminaria. The anticancer agent according to any one of claims 1 to 10, wherein the above complex is K3-SPG. A composition for collecting dead cells of cancer in a reticuloendothelial system and/or a lymph node, comprising a complex comprising: (a) an oligodeoxynucleotide comprising a humanized K-type CpG Oligodeoxynucleotides and polydeoxyadenylate, and polydeoxyadenosine is disposed on the 3' side of a humanized K-type CpG oligodeoxynucleotide; and (b) β-1 , 3-glucan. The composition of claim 12, wherein the oligodeoxynucleotide is selected from the group consisting of K3 (SEQ ID NO: 1), K3-dA ₄₀ (SEQ ID NO: 2), dA _{40 -} K3 (SEQ ID NO: 3), K3-dA20 (SEQ ID NO: 4), K3-dA25 (SEQ ID NO: 5), K3-dA30 (SEQ ID NO: 6), and K3-dA35 (SEQ ID NO: 7). The composition of claim 12 or 13, wherein the β-1,3-glucan is selected from the group consisting of Schizophyllum polysaccharide (SPG), lentinan, scleroglucan, cadmium, lycium polysaccharide, ash tree A group consisting of flower polysaccharides and laminaria. The composition of any one of claims 12 to 14, wherein the complex is K3-SPG. The composition of any one of claims 12 to 15, wherein the reticuloendothelial system and/or lymph nodes comprise tumors and macrophages. The composition of any one of claims 12 to 16, wherein the reticuloendothelial system comprises a spleen and/or a liver. The composition of any one of claims 12 to 17, wherein the administration comprises systemic administration. The composition of claim 18, wherein the systemic administration is selected from the group consisting of intravenous administration, intraperitoneal administration, oral administration, subcutaneous administration, intramuscular administration, and intratumoral administration. Investing. A composition for promoting the performance or expression of interleukin 12 (IL12) and/or interferon (IFN) gamma comprising: (a) an oligodeoxynucleotide comprising a humanized K-type CpG Oligodeoxynucleotides and polydeoxyadenylate, and polydeoxyadenosine is disposed on the 3' side of a humanized K-type CpG oligodeoxynucleotide; and (b) β-1 , 3-glucan. The composition of claim 20, wherein the oligodeoxynucleotide is K3 (SEQ ID NO: 1), K3-dA ₄₀ (SEQ ID NO: 2), dA _{40 -} K3 (SEQ ID NO: 3), K3-dA20 (sequence) No. 4), K3-dA25 (SEQ ID NO: 5), K3-dA30 (SEQ ID NO: 6), and K3-dA35 (SEQ ID NO: 7). The composition of claim 20 or 21, wherein the β-1,3-glucan is selected from the group consisting of Schizophyllum polysaccharide (SPG), lentinan, scleroglucan, cadmium, lycium polysaccharide, ash tree A group consisting of flower polysaccharides and laminaria. The composition of any one of claims 20 to 22, wherein the complex is K3-SPG.