JP2019163302A - Application of nucleic acid polysaccharide complex with immunostimulatory activity as antitumor agent - Google Patents

Abstract

To provide anticancer drugs used as a single agent.SOLUTION: Provided is an anticancer agent containing a complex comprising (a) an oligodeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylate, in which polydeoxyadenylic acid is located 3' of a humanized K-type CpG oligodeoxynucleotide, and (b) β-1,3-glucan. Preferably, the anticancer agent is administered without a cancer antigen.SELECTED DRAWING: None

Description

  The present invention relates to a novel cancer treatment.

  CpG oligonucleotides (CpG ODN) are short (approximately 20 base pairs), single-stranded synthetic DNA fragments containing immunostimulatory CpG motifs that are potent agonists of Toll-like receptor 9 (TLR9) And activates dendritic cells (DCs) and B cells to produce type I interferons (IFNs) and inflammatory cytokines (Non-Patent Documents 1 and 2), and causes cytotoxic T lymphocyte (CTL) reaction. It acts as an adjuvant for Th1 type humoral and cellular immune responses (Non-Patent Documents 3 and 4). Therefore, CpG ODN has been regarded as a potential immunotherapeutic agent for infectious diseases, cancer, asthma and hay fever (Non-patent Documents 2 and 5).

  There are at least four types of CpG ODN with different skeletal sequences and immunostimulatory properties (Non-Patent Document 6). D-type (also called A-type) CpG ODNs typically contain one palindromic CpG motif with a phosphodiester (PO) backbone and a phosphorothioate (PS) poly G tail, and plasmacytoid DCs (pDCs) Is activated to produce a large amount of IFN-α, but cannot induce pDC maturation or B cell activation (Non-patent Documents 7 and 8). The other three types of ODN consist of a PS skeleton. K-type (also called B-type) CpG ODN typically contains multiple CpG motifs in a non-palindrome structure that strongly activates B cells to produce IL-6 and activates pDCs However, IFN-α is hardly produced (Non-patent Documents 8 and 9). Recently developed C-type and P-type CpG ODNs contain one and two palindromic CpG sequences, both of which activate B cells like K type and like D type Although pDCs can be activated, C-type CpG ODN induces IFN-α production weaker than P-type CpG ODN (Non-patent Documents 10-12). Patent Document 1 describes many excellent K-type CpG ODNs.

  D-type and P-type CpG ODNs are Hoogsteen base pairs that form parallel four-stranded structures called G-tetrads, and Watson-Crick base pairs between cis and trans palindrome sites, These are required for strong IFN-α production by pDCs (Non-patent Documents 12-14). Such higher-order structures appear to be necessary for localization to early endosomes and TLR9-mediated signaling, but these are affected by product polymorphism and precipitation, thus preventing its clinical application (Non-patent document 15). Therefore, only K-type and C-type CpG ODN are generally available as human immunotherapeutic agents and vaccine adjuvants (Non-patent Documents 16 and 17). K-type CpG ODN enhances the immunogenicity of vaccines targeting infectious diseases and cancer in human clinical trials (Non-Patent Documents 6 and 16), but for optimal adjuvant effect, antigen-K-type CpG Chemical and physical linkage between ODN is required. These results show that the four types (K, D, P, and C) of CpG ODN have advantages and disadvantages and activate both B cells and pDCs without aggregation. The development of “all-in-one” CpG ODN is expected.

  Schizophyllum (SPG), a soluble β-1,3-glucan derived from Schizophyllum commune, has been approved in Japan for the last 30 years as a radiation therapy stimulant in patients with cervical cancer. Patent Document 18). Similarly, Lentinan (LNT), a soluble β-1,3-glucan from Shiitake mushroom, was approved in 1985 and is used in combination with fluoropyrimidines for patients with inoperable and recurrent gastric cancer. (Non-Patent Documents 19 and 20). It has been shown that β-1,3-glucan forms a complex of triple helix structure with polydeoxyadenylic acid (dA) (Non-patent Document 21).

  Patent Documents 2 to 4 disclose the use of a water-soluble complex of β-1,3-glucan containing schizophyllan and a nucleic acid (gene) as a gene carrier. These documents describe that the antisense action of a gene and the resistance action to a nucleolytic enzyme (nuclease) are enhanced by forming the complex.

  In Patent Document 5, by using a polysaccharide having β-1,3-linkage as a carrier (transfection agent), a CpG sequence is contained, and a phosphodiester bond is substituted with a phosphorothioate bond or a phosphorodithioate bond. It is disclosed that the action of immunostimulatory oligonucleotides is enhanced.

  Patent Document 6 describes an immunostimulatory complex comprising an immunostimulatory oligonucleotide and β-1,3-glucan having a long β-1,6-glucoside-binding side chain. Has been.

  The present inventors have previously reported that mouse and humanized CpG ODN linked to poly (dA) having a phosphodiester bond at the 5 ′ end complexed with SPG enhanced cytokine production and influenza vaccine. It has been shown to act as an adjuvant or a preventive or therapeutic agent for Th2 cell-related diseases (Non-patent Documents 22 and 23, Patent Document 7). When poly (dA) was added to the 5 'end of CpG of each of K-type and D-type to form a complex with SPG, the activity was enhanced while maintaining the characteristics of K-type and D-type, respectively. However, it has been difficult to achieve high yields of CpG-SPG conjugates for more effective and cost effective preclinical and clinical development. In recent years, it has been shown that when poly (dA) having a phosphorothioate bond is linked to CpG ODN, the complex formation increases to almost 100% (Non-patent Document 24). However, no thorough testing has been done to identify optimal humanized CpG sequences and optimize factors to obtain the “all-in-one” activity of the four types of CpG ODN.

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

  Synthetic nucleic acid CpG oligodeoxynucleotide (CpG ODN) which is a ligand of Toll-like receptor 9 (TLR9) has strong innate immunity activation ability and is expected as a vaccine adjuvant. In addition, since CpG ODN has antitumor activity when administered as a single agent, CpG ODN is also expected as an immunotherapy agent for cancer. However, although conventional CpG ODN has antitumor activity, it can only exert its effects by being administered directly to the tumor, and it has been considered difficult to apply to clinical practice. In fact, it may be difficult to administer a drug directly to a tumor at an early stage in clinical practice. In addition, surgical treatment is required in the deep part, and the hurdle is high.

  Recently, the present inventors have developed a new TLR9 ligand (K3-SPG) in which CpG ODN is wrapped with a polysaccharide beta-glucan (PCT application (PCT / JP2014 / 074835))). K3-SPG has been shown by experiments using mice to activate innate immunity more strongly than conventional CpG ODN without creating aggregates and at the same time a strong adjuvant effect. Furthermore, K3-SPG was found to induce strong acquired immunity not only in mice, but also in cynomolgus monkeys, and was able to overcome the difference in reactivity between mice and primates, which had been feared so far.

  Thus, although this CpG ODN is expected to be used as an adjuvant, it is unclear whether it can be used alone as a medicine.

  Regarding cancer treatment, since cancer-related antigens recognized by cytotoxic T cells were identified and reported in 1991 (Non-patent Document 25 = van der Bruggen et al. Science (New York, NY) 254, 1643-1647 (1991)), a large number of cancer-related antigens have been identified at the molecular level, and clinical application of cancer immunotherapy targeting them has been achieved (Non-patent Document 26 = Jager, E., 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)). Cancer immunotherapy, which has received particular attention, is a cancer vaccine that uses antigen-presenting cells of autologous peripheral blood that was first approved by the US Food and Drug Administration (FDA) in April 2010 for prostate cancer patients. (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)). Subsequently, the inhibitory antibody ipilimumab against cytotoxic T lymphocyte antigen 4 (CTLA-4), an inhibitory molecule of T lymphocyte activation, was approved in May 2011 for patients with malignant melanoma. (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)). Furthermore, nivolumab, which is a PD-1 (programmed cell death 1) receptor inhibitor of an immune response 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)).

  The formation of an environment in which dendritic cells effectively lead to anticancer effectors requires innate immunity pattern molecules that exhibit anticancer effects in the inflamed area (Non-patent Document 39 = Chiba, S., et. al. Nature immunology 13, 832-842 (2012)). Although the above-mentioned molecular group and processes involved in this are not specified, 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 (major associated macrophages: TAM) are known as the culprits of inflammatory reactions (Non-patent Document 41 = Huang, Y., et al. Cancer cell 19, 1-2 (2011)). On the other hand, dendritic cells that function as a player for anti-cancer immunity are also the same bone marrow cells as macrophages (Non-patent Document 42 = Huang, Y., et al. Proc. Natl. Acad. Sci. USA 109, 17561-17566 (2012)). Although no method has been found to turn them into anti-cancer-directed, tumors are thought to have avoided immunity due to complex 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, 120ra116 (2012); Non-Patent Document 44 = Martinez-Pomares , L. et al. Trends in immunology 33, 66-70 (2012)). The contact of immunological effector cells with cancer cells is indispensable for the attack of 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)).

US 8,030,285 B2 WO 01/034207 A1 WO 02/072152 A1 JP 2004-107272 A WO 2004/100965 A1 JP 2007-70307 A JP 2008-100919 JP 2010-174107 A

Hemmi, H., et al. Nature 408, 740-745 (2000). Krieg, A.M.Nature reviews.Drug discovery 5, 471-484 (2006). Brazolot Millan, C.L., et al., Proceedings of the National Academy of Sciences of the United States of America 95, 15553-15558 (1998). Chu, R.S., et al., The Journal of experimental medicine 186, 1623-1631 (1997). Klinman, D.M.Nature reviews.Immunology 4, 249-258 (2004). Vollmer, J. & Krieg, A.M.Advanced drug delivery reviews 61, 195-204 (2009). Krug, A., et al. European journal of immunology 31, 2154-2163 (2001). Verthelyi, D., et al., Journal of immunology 166, 2372-2377 (2001). Hartmann, G. & Krieg, A.M.Journal of immunology 164, 944-953 (2000). Hartmann, G., et al. European journal of immunology 33,1633-1641 (2003). Marshall, J.D., et al. Journal of leukocyte biology 73, 781-792 (2003). Samulowitz, U., et al. Oligonucleotides 20, 93-101 (2010). Kerkmann, M., et al. The Journal of Biological Chemistry 280, 8086-8093 (2005). Klein, D.C., et al., Ultramicroscopy 110, 689-693 (2010). Puig, M., et al. Nucleic acids research 34, 6488-6495 (2006). Bode, C., et al., Expert review of vaccines 10, 499-511 (2011). McHutchison, J.G., et al. Hepatology 46, 1341-1349 (2007). Okamura, K., et al. Cancer 58, 865-872 (1986). Oba, K. et al., J. Individual patient based meta-analysis of lentinan for unresectable / recurrent gastric cancer.Anticancer Res., 2009, 29, 2739-2746. Nakano, H. et al., Hepato-Gastroenterol., 1999, 46, 2662-2668. Sakurai, K., et al., Biomacromolecules 2, 641-650 (2001). Shimada, N., et al. Bioconjugate chemistry 18, 1280-1286 (2007). Koyama, S., et al. Science translational medicine 2, 25ra24 (2010). Minari, J., et al. Bioconjugate chemistry 22, 9-15 (2011). van der Bruggen et al. Science (New York, N.Y.) 254, 1643-1647 (1991) Jager, E., et al. The Journal of experimental medicine 187, 265-270 (1998) Jager, D. et al. Journal of clinical pathology 54, 669-674 (2001). Imai, K., et al. British journal of cancer 104, 300-307 (2011) Kang, X., et al. The Journal of Immunology 155, 1343-1348 (1995) Cancer vaccine approval could open floodgates. Nature medicine 16, 615-615 (2010) Higano, C.S., et al. Cancer 115, 3670-3679 (2009) Phan, G.Q., et al. Proc. Natl. Acad. Sci. U. S. A. 100, 8372-8377 (2003) Camacho, L.H., et al. Journal of clinical oncology: official journal of the American Society of Clinical Oncology 27, 1075-1081 (2009) Hodi, F.S., et al. New England Journal of Medicine 363, 711-723 (2010) AZIJLI, K., et al. Anticancer Research 34, 1493-1505 (2014) Okazaki, T., et al. Nature immunology 14, 1212-1218 (2013) Ishida, Y., et al. The EMBO journal 11, 3887-3895 (1992) Topalian, S.L., et al. The New England journal of medicine 366, 2443-2454 (2012) Chiba, S., et al. Nature immunology 13, 832-842 (2012) Engelhardt, J.J., et al. Cancer cell 21, 402-417 (2012) Huang, Y., et al. Cancer cell 19, 1-2 (2011) Huang, Y., et al. Proc. Natl. Acad. Sci. U. S. A. 109, 17561-17566 (2012) Garaude, J., et al. Science translational medicine 4, 120ra116 (2012) Martinez-Pomares, L. et al. Trends in immunology 33, 66-70 (2012) Palucka, K. et al. Nature reviews. Cancer 12, 265-277 (2012)

  As a result of diligent research, the present inventors have developed a CpG-β glucan complex (for example, K3-SPG (a complex of human K-type CpG ODN K3 and beta glucan) as a single agent. When used as an anti-tumor agent in the case of intravenous administration, which was not effective with conventional CpG ODN (K3), K3-SPG confirmed the regression of the mass in tumor-bearing mice (FIG. 2 (A to 2)). B)), the present inventors have further demonstrated that the antiperitoneal activity is also demonstrated in a peritoneal dissemination model, which is a more clinical model (FIGS. 2g and m). 2B)) The present inventors did not require antigen administration for this effect, and confirmed the effect by administration of a single agent.

  Furthermore, the present inventors have found that the acquired immune response is important for the antitumor effect of K3-SPG and that the type I interferon (IFN) and IL-12 induced by the innate immune response are essential. Shown using mice (FIGS. 6a, b, c (FIG. 6A)). In addition, the present inventors confirmed that CD45-negative tumor cells were accumulated in the spleen by intravenous administration of K3-SPG, and many of these cells caused cell death (necrosis or apoptosis). I made it clear. When this CD45 negative cell was immunized to a mouse, it exerted a strong antitumor effect, and thus it was revealed that cell death of CD45 negative cells accumulated in the spleen seems to play an important role ( Fig. 6g, h, i, j (Fig. 6B)). In addition, the present inventors have confirmed that administration of K3-SPG causes accumulation of activated CD8 T cells in the tumor, and that these cells are essential for the antitumor effect. It was clear.

  For this reason, it is expected that CpG ODN that exhibits an antitumor effect in systemic administration will work strongly even in carcinomas that have been difficult to date. Furthermore, since CpG ODN exhibits an antitumor effect without an antigen, application as a single agent can also be expected.

  To date, CpG ODN has been treated as 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)) as a promising drug. However, treatment as an anticancer agent with CpG-ODN so far can suppress tumor growth only when injected into the 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 now developed a nanoparticulate TLR9 agonist called K3-SPG, which consists of schizophyllan (SPG; β-glucan) and a B / K type CpG (K3) complex, It was shown to function as a vaccine adjuvant stronger than K3 itself (with strong induction of IFN-α). In this example, the present inventors further examined the potential of K3-SPG single-agent immunotherapy for cancer (without using additional tumor peptides and antigens) and found that the above effects were obtained. As a result, the present invention was completed. Therefore, the present invention typically provides the following.

(Anticancer agent alone)
(1) (a) an oligodeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylic acid, wherein the polydeoxyadenylic acid is arranged on the 3 ′ side of the humanized K-type CpG oligodeoxynucleotide An oligodeoxynucleotide,
(B) An anticancer agent containing a complex containing β-1,3-glucan.
(2) The anticancer agent according to item (1), wherein the anticancer agent is administered without a cancer antigen.
(3) The anticancer agent according to item (1) or (2), wherein the anticancer agent is administered so as to be delivered to the reticuloendothelial system and / or lymph nodes.
(4) The anticancer agent according to item (3), wherein the reticuloendothelial system and / or lymph node includes a tumor and a phagocytic cell.
(5) The anticancer agent according to item (3) or (4), wherein the reticuloendothelial system includes spleen and / or liver.
(6) The anticancer agent according to any one of items (1) to (5), wherein the anticancer agent is administered without a cancer antigen.
(7) The anticancer agent according to any one of items (2) to (6), wherein the administration comprises systemic administration.
(8) The anticancer agent according to item (7), wherein the systemic administration is selected from intravenous administration, intraperitoneal administration, oral administration, subcutaneous administration, intramuscular administration, and intratumoral administration.
(9) The oligodeoxynucleotides are 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) The anticancer agent according to any one of items 1 to 8, which is selected from the group consisting of K3-dA30 (SEQ ID NO: 6) and K3-dA35 (SEQ ID NO: 12 → 7).
(10) The β-1,3-glucan is any one of items 1 to 9, wherein the β-1,3-glucan is selected from the group consisting of schizophyllan (SPG), lentinan, scleroglucan, curdlan, parkan, glyphoran, and laminaran. Anticancer drugs.
(11) The anticancer agent according to any one of Items 1 to 10, wherein the complex is K3-SPG.
(Reticuloendothelial system (including spleen and / or liver) and / or lymph node accumulating agent)
(12) (a) an oligodeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylic acid, wherein the polydeoxyadenylic acid is located 3 ′ of the humanized K-type CpG oligodeoxynucleotide An oligodeoxynucleotide,
(B) A composition for accumulating dead cancer cells in the spleen, comprising a complex containing β-1,3-glucan.
(13) The oligodeoxynucleotide 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) The composition according to item (12), selected from the group consisting of K3-dA30 (SEQ ID NO: 6) and K3-dA35 (SEQ ID NO: 7).
(14) The β-1,3-glucan is selected from the group consisting of schizophyllan (SPG), lentinan, scleroglucan, curdlan, parkan, glyphoran, and laminaran, according to item (12) or (13) Composition.
(15) The composition according to any one of items (12) to (14), wherein the complex is K3-SPG.
(16) The composition according to any one of items 12 to 15, wherein the reticuloendothelial system and / or lymph node comprises a tumor and a phagocytic cell.
(17) The composition according to any one of items (12) to (16), wherein the reticuloendothelial system includes a spleen and / or a liver.
(18) The composition according to any one of items (12) to (17), wherein the administration comprises systemic administration.
(19) The composition according to item (18), wherein the systemic administration is selected from intravenous administration, intraperitoneal administration, oral administration, subcutaneous administration, intramuscular administration, and intratumoral administration.
<Composition for expression or promotion of interleukin 12 (IL12) and / or interferon (IFN) γ>
(20) (a) an oligodeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylic acid, wherein the polydeoxyadenylic acid is located 3 ′ of the humanized K-type CpG oligodeoxynucleotide An oligodeoxynucleotide,
(B) A composition for expression or promotion of interleukin 12 (IL12) and / or interferon (IFN) γ, comprising β-1,3-glucan.
(21) The oligodeoxynucleotides are 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) The composition according to item (20), which is K3-dA30 (SEQ ID NO: 6) and K3-dA35 (SEQ ID NO: 7).
(22) The β-1,3-glucan is selected from the group consisting of schizophyllan (SPG), lentinan, scleroglucan, curdlan, parkan, glyphoran and laminaran, according to item (20) or (21) Composition.
(23) The composition according to any one of items (20) to (22), wherein the complex is K3-SPG.

  In the present invention, it is contemplated that one or more of the features described above may be provided in further combinations in addition to the express combinations. Still further embodiments and advantages of the invention will be appreciated by those skilled in the art upon reading and understanding the following detailed description as necessary.

  The application of K3-SPG according to the present invention as an antitumor agent can exert a strong antitumor effect in systemic administration that could not be overcome by conventional CpG ODN. Therefore, it is considered to be very useful from a clinical viewpoint. In addition, since sufficient effects (innate immune response) have been confirmed in human cells, there is a high possibility of application to humans. Our research results show that in addition to the very strong ability to activate innate immunity compared to CpG ODN used in clinical trials so far, K3- SPG can be expected as a useful immunotherapy drug. Furthermore, since the effect can be exerted by inducing cell death of tumor cells without requiring administration of an antigen, it can be applied to various carcinomas. From these results, K3-SPG has the potential as an innate immune activated antitumor drug that does not require an antigen.

FIG. 1 shows a method of complexing SPG with CpG ODN. FIG. 2 (AB) shows that systemic injection of antigen-free nanoparticulate CpG (K3-SPG) can be applied to many established tumor models including pancreatic cancer peritoneal dissemination models. Show. FIG. 2A shows ai. C57BL / 6 mice were treated with EG7 cells on day 0. c. Inoculate and on days 7, 9 and 11 PBS (ac), K3 (30 [mu] g) (df), or K3-SPG (10 [mu] g) (gi) intradermal (id) (Tumor area) (a, d, g), intratumoral (it) (b, e, h), or intravenous (iv) (c, f, i). Tumor size was measured for 23 days (n = 4). Each curve represents an individual mouse. The arrow indicates the time of treatment. FIG. 2 (AB) shows that systemic injection of antigen-free nanoparticulate CpG (K3-SPG) can be applied to many established tumor models including pancreatic cancer peritoneal dissemination models. Show. FIG. 2B shows j to n. (J to l) C57BL / 6 mice were inoculated with B16 cells, B16F10 cells or MC38 cells on day 0. The B16 inoculated group was challenged i.e. on days 10, 12, and 14 with K3-SPG. v. Or i. t. Treated. The B16F10 inoculated group was challenged i.e. on days 7, 9 and 11 with K3-SPG. v. Or i. t. Treated. MC38 inoculated groups were challenged i.p. on days 14, 16 and 18 with K3-SPG. v. Or i. t. Treated. Error bars represent mean + SEM (n = 4). * P <0.05 (t test). (M) C57BL / 6 mice were injected intraperitoneally with Pan02 cells on day 0 and on days 11, 13, and 15 with K3 or K3-SPG or PBS (control). v. Treated. Tumor weight (g) indicates day 21. * P <0.05 (t test). (N) C57BL / 6 mice were injected intraperitoneally with Pan02 on day 0 and i.p. 3 times with K3 or K3-SPG or PBS. v. Treated. The survival rate (%) is shown (n = 8). * P <0.05 (log rank test). FIG. 3 compares the results of systemic administration of K3-SPG with other controls, K3. It has been shown that systemic administration of K3-SPG can be a cancer immunotherapeutic agent that does not require an antigen. After transplanting EG7, which is a tumor cell line, into mice, K3 and K3-SPG were intravenously administered three times (7th, 9th and 11th days). Tumor size was measured from day 7 after transplantation of tumor cells. FIG. 4 shows that K3-SPG targets phagocytic cells within the tumor microenvironment. (Ac) C57BL / 6 mice were treated with EG7 on day 0. c. Inoculate PBS (control), ALEXA 647-K3 (30 μg), or ALEXA 647-K3-SPG (10 μg) i. v. Administered. One hour after dosing, the mice were analyzed with an in vivo fluorescence imaging system (IVIS) and the image measured with relative fluorescence was converted to physical units of surface radiance (photons / second / cm ² / sr). White arrows indicate the tumor inoculation area (a). (B, c) Cryosections of tumors from FIG. 6a (FIG. 6A) were stained with anti-CD3e antibody (red, EG7 staining) and Hoechst 33258 (blue, nuclear staining) and then analyzed with a fluorescence microscope (scale bar) , 100 μm). White arrows indicate fluorescence positive areas. (Dg) C57BL / 6 mice were treated with EG7 on day 0. c. Inoculate and received Alexa 647-K3, Alexa 647-K3-SPG, or FITC-SPG on day 12 with dextran-PE. v. Administered. (Df) One hour after injection, frozen sections of tumors were analyzed with a fluorescence microscope (scale bar, 100 μm). (G) Green, red or merged cells were counted (10 fields from each of 3 tumors). Error bars represent mean + SD. The asterisk indicates a significant difference from the number of merged cells injected with K3. (H) C57BL / 6 mice (n = 3 or 4) were treated with EG7 s. c. Inoculate and on day 5 clodronate liposomes or control liposomes i. v. Administered. Mice were injected on days 7, 9 and 11 with PBS (control) or K3-SPG. Error bars represent mean + SEM. The arrow indicates the time of treatment. * P <0.05 (t test). FIG. 5 shows that F4 / 80 positive cells in the tumor were depleted by clodronate liposomes. C57BL / 6 mice were inoculated with EG7 on day 0 and clodronate liposomes (a) or control liposomes (b) were injected i. v. And on day 7 with Alexa 647-K3-SPG i. v. Treated. One hour after treatment, frozen sections of tumors were stained with anti-F4 / 80 antibody (red) and Hoechst 33258 (blue) and then analyzed with a fluorescence microscope (scale bar, 100 μm). FIG. 6 (AB) shows that both IL-12 and IFN are important for their potential role in tumor shrinkage and their immunogenic cell death. 6A shows a through f. (Ac) Il12p40 hetero knockout mice (a), Ifnar2 hetero knockout mice (b), and Il12p40-Ifnar2 double knockout mice (c) were treated with EG7 cells on day 0. c. These mice were inoculated i.v. with K3-SPG on days 7, 9 and 11. v. Treated. Error bars represent mean + SEM (n = 4). The arrow indicates the time of treatment. * P <0.05 (t test). (D, f) Rag2 hetero and knockout and Il12p40-Ifnar2 double knockout mice were inoculated with EG7 cells on day 0, 3 times with K3-SPG (7th, 9th and 11th days, black arrows), 6 times (7, 9, 11, 14, 16, 18 days, gray arrows), or 0 times (control) i. v. Treated. (E) The enlarged view shows the fourth day to the 21st day. FIG. 6 (AB) shows that both IL-12 and IFN are important for their potential role in tumor shrinkage and their immunogenic cell death. FIG. 6B shows g to k. (G) C57BL / 6 mice and Il12p40-Ifnar2 double knockout mice were treated with EG7 cells on day 0. c. Inoculate and inoculate mice with K3-SPG on days 7, 9 and 11. v. Treated and then sacrificed on day 12. Splenocytes were collected and stained with anti-CD45 antibody, and then the cells were analyzed by flow cytometry. (H) Scatter plot shows CD45 negative cell population. Error bars represent mean + SEM. * P <0.05 (t test). (I) CD45 negative population was stained with Hoechst 33342 and PI for staining of dead cells and then analyzed by flow cytometry. The bar graph shows a population of apoptotic cells, necrotic cells, and live CD45 negative cells. Error bars represent mean + SD (n = 3). * P <0.05 (t test). (J) C57BL / 6 mice were immunized with PBS or CD45 negative cells. Seven days after immunization, mice were challenged with EG7 cells on day 0. c. Inoculated and tumor size was measured for the next 25 days (n = 3). Error bars represent mean + SEM. * P <0.05 (t test). (K) Tumor volume at day 25 and the number of OVA _257-264 specific tetramers + CD8 T cells are represented by bar graphs and scatter paper diagrams, respectively. * P <0.05 (t test). FIG. 7 shows that IFN-β was detected in the tumor microenvironment. (A) IFN-βGFP mice were inoculated with EG7 on day 0 and on days 7, 9, and 11 with K3-SPG. d. Or i. v. Treated. Twelve days after inoculation, tumors were collected and frozen sections were stained with anti-CD11b antibody, anti-CD169 antibody, anti-F4 / 80 antibody, anti-MARCO antibody (red) and Hoechst 33258 (blue) and then analyzed with a fluorescence microscope. (Scale bar, 100 μm). (B) IFN-β positive cells were counted (10 fields from each of 3 tumors). Error bars represent mean + SD. * P <0.05 (t test). FIG. 8 shows that IL12-p40 was detected in the tumor microenvironment. (A) C57BL / 6 mice were inoculated with EG7 on day 0 and on days 7, 9, and 11 with K3-SPG. d. Or i. v. Treated. Twelve days after inoculation, tumors were collected and frozen sections were stained with anti-IL12-p40 antibody (red) and Hoechst 33258 (blue) and then analyzed with a fluorescence microscope (scale bar, 100 μm). (B) IL12-p40 positive cells were counted (10 fields from each of 3 tumors). Error bars represent mean + SD. * P <0.05 (t test). FIG. 9 shows that CD45 negative cells are derived from tumor cells but not from host cells. GFP mice were treated with EG7 cells on day 0. c. The mice were inoculated i.v. K3-SPG on days 7, 9 and 11. v. Treated and sacrificed on day 12 thereafter. Splenocytes were collected and stained with anti-CD45 antibody, after which the cells were analyzed by flow cytometry. FIG. 10 (AB) shows that K3-SPG-induced tumor shrinkage is innate and adaptive immunity including Il12, type 1 IFN, Batf3, CD8 ⁺ DC, and potent cytotoxic T cells infiltrating the tumor. Indicates that both responses are required. FIG. 10A shows ac. C57BL / 6 knockout mice (a), as well as Batf3 heterozygous and Batf3 knockout mice (b), were inoculated with EG7 cells on day 0 and i.v. K3-SPG on days 7, 9, and 11. v. Treated (black arrow). (A) CD8 depleting antibody (200 μg / mouse) was administered on days 6 and 13. Error bars represent mean + SEM (n = 4). * P <0.05 (t test). The arrow indicates the time of treatment. (C) C57BL / 6 mice were inoculated with EG7 on day 0 and on days 7, 9 and 11 with K3-SPG i. d. Or i. v. Treated with. Twelve days after inoculation, tumors were collected and frozen sections were stained with anti-CD8β antibody (red) and Hoechst 33258 (blue) and then analyzed with a fluorescence microscope (scale bar, 100 μm). CD8β positive cells were counted (10 fields from each of 3 tumors). Error bars represent mean + SD. * P <0.05 (t test). FIG. 10 (AB) shows that K3-SPG-induced tumor shrinkage is innate and adaptive immunity including Il12, type 1 IFN, Batf3, CD8 ⁺ DC, and potent cytotoxic T cells infiltrating the tumor. Indicates that both responses are required. FIG. 10B shows de. (D) C57BL / 6 (WT) and Il12p40-Ifnar2 double knockout (DKO) mice were inoculated with EG7 cells on day 0 and i.p. K7-SPG on days 7, 9 and 11. v. Treated. CD8α ⁺ T cells from tumor-bearing mice injected with either K3-SPG or PBS were transferred on day 14 with Xenolight DiR® (iv) and then on day 15 Mice were analyzed by IVIS. (I, II) Recipient mice: i. v. Treated EG7 bearing WT mice. Mice are either transferred with K3-SPG treated CD8α ⁺ T cells (I) or untreated CD8α ⁺ T cells (II). (E) (I, II) Recipient mice: untreated EG7-bearing WT mice (I) and K3-SPG i. v. Treated DKO mice. Mice were transferred K8-SPG treated CD8α + T cells (I, II). FIG. 11 shows a schematic paper diagram of the experimental system. WT and Il12p40-Ifnar2 DKO mice are inoculated with EG7 cells on day 0 and on days 7, 9 and 11 with K3-SPG or PBS. v. Treated. On day 14, another CD8α ⁺ T cell was purified from the spleen of these mice, labeled with Xenlight DiR®, and treated with K3-SPG (days 7, 9 and 11). The distribution of CD8 T cells labeled with Xenright DiR® was analyzed by IVIS on day 15 after being transferred to EG7-bearing mice (14 days after inoculation). FIG. 12 shows the K3-SPG treatment strategy. K3-SPG targeted the tumor microenvironment via the bloodstream. K3-SPG targeted phagocytic cells and activated these cells. In the tumor microenvironment, IFN and IL-12 were induced by K3-SPG treatment. The antigen was then released via lymph flow and blood flow. The presentation of this antigen induced a strong tumor-specific CTL.

  The present invention will be described below with reference to the best mode. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. Thus, it should be understood that singular articles (eg, “a”, “an”, “the”, etc. in the case of English) also include the plural concept unless otherwise stated. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

  Hereinafter, definitions of terms particularly used in the present specification and / or basic technical contents will be described as appropriate.

  The present invention provides oligodeoxynucleotides (hereinafter referred to as oligodeoxynucleotides of the present invention) containing K-type CpG oligodeoxynucleotides and polydeoxyadenylic acid (dA). The oligodeoxynucleotides of the present invention include those in which phosphodiester bonds are modified (for example, some or all of the phosphodiester bonds are replaced by phosphorothioate bonds). The oligodeoxynucleotides of the present invention include pharmaceutically acceptable salts.

  As used herein, “CpG oligonucleotide (residue)” or “CpG oligodeoxynucleotide (residue)”, “CpG ODN (residue)” or simply “CpG (residue)” are used interchangeably. Refers to a polynucleotide, preferably an oligonucleotide, comprising at least one unmethylated CG dinucleotide sequence, which is synonymous with or without the term “residue” at the end. An oligonucleotide comprising at least one CpG motif can comprise multiple CpG motifs. As used herein, the phrase “CpG motif” refers to an unmethylated dinucleotide portion of an oligonucleotide comprising a cytosine nucleotide followed by a guanosine nucleotide. 5-methylcytosine can also be used in place of cytosine. Furthermore, polydeoxyadenylic acid and polydeoxyadenosine acid (residue) are synonymous. The term “residue” refers to a substructure of a higher molecular weight compound, but in this specification “CpG oligodeoxynucleotide (CpG ODN)” refers to an independent molecule or a higher molecular weight compound. The meaning of the partial structure is easily understood by those skilled in the art from the context. The same applies to terms relating to other partial structures contained in the oligodeoxynucleotide of the present invention such as “polydeoxyadenylic acid”.

  CpG oligonucleotide (CpG ODN) is a short (about 20 base pair), single-stranded synthetic DNA fragment containing an immunostimulatory CpG motif, and is a potent agonist of Toll-like receptor 9 (TLR9) And 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 , AM. Nature reviews. Drug discovery 5, 471-484 (2006).), Acting as an adjuvant for Th1-type humoral and cellular immune responses, including cytotoxic T lymphocyte (CTL) responses (Brazolot Millan , CL., Weeratna, R., Krieg, AM., Siegrist, CA & Davis, HL Proceedings of the National Academy of Sciences of the United States of America 95, 15553-1 5558 (1998); Chu, RS, Targoni, OS, Krieg, AM, Lehmann, PV & Harding, CV The Journal of experimental medicine 186, 1623-1631 (1997)) . CpG ODN has therefore been regarded as a potential immunotherapeutic agent for infections, cancer, asthma and hay fever (Krieg, AM. Nature reviews. Drug discovery 5, 471-484 (2006); Klinman , D. M. Nature reviews. Immunology 4, 249-258 (2004)).

  CpG oligodeoxynucleotide (CpG ODN) is a single-stranded DNA containing an immunostimulatory unmethylated CpG motif and is an agonist of TLR9. There are four types of CpG ODN, K type (also called B type), D type (also called A type), C type and P type, which have different skeletal sequences and immunostimulatory properties (Advanced drug delivery reviews 61 , 195-204 (2009)). Among these, the oligodeoxynucleotide of the present invention contains K-type CpG ODN.

K-type CpG ODNs contain multiple unmethylated CpG motifs, typically non-palindromic, and activate B cells to produce IL-6, but plasmacytoid dendritic cells (pDCs) It is a CpG ODN having structural and functional properties that hardly induce IFN-α production. An unmethylated CpG motif refers to a short nucleotide sequence containing at least one cytosine (C) -guanine (G) sequence, wherein the cytosine 5-position in the cytosine-guanine sequence is not methylated. In the following description, CpG means unmethylated CpG unless otherwise specified. Therefore, the oligodeoxynucleotide of the present invention contains K-type CpG ODN, thereby activating the immunostimulatory activity peculiar to K-type CpG ODN (for example, B cells (preferably human B cells) and IL-6 Activity). Many 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. “Humanized” means having agonist activity against human TLR9. Therefore, the oligodeoxynucleotide of the present invention containing humanized K-type CpG ODN has an immunostimulatory activity specific to K-type CpG ODN (for example, an activity to activate human B cells to produce IL-6). Have The K-type CpG ODN suitably used in the present invention has a length of 10 nucleotides or more and has the formula:

(In the formula, the central CpG motif is not methylated, W is A or T, N1,
N2, N3, N4, N5 and N6 may be any nucleotide).

  In one embodiment, the K-type CpG ODN of the present invention is 10 nucleotides or more in length and comprises a nucleotide sequence of the above formula. However, in the above formula, the central 4-base CpG motif (TCpGW) may be contained in 10 nucleotides and does not necessarily need to be located between N3 and N4 in the above formula. In the above formula, N1, N2, N3, N4, N5 and N6 may be any nucleotide, and N1 and N2, N2 and N3, N3 and N4, N4 and N5, and at least one of N5 and N6 One (preferably one) combination may be a two-base CpG motif. When the 4-base CpG motif is not located between N3 and N4, in the above formula, any two consecutive bases in the central 4 bases (4th to 7th bases) are CpG motifs, The two bases can be any nucleotide.

  The K-type CpG ODN more preferably used in the present invention contains a non-palindrome structure containing one or more CpG motifs. Further preferably used K-type CpG ODN has a non-palindrome structure containing one or more CpG motifs.

  Humanized K-type CpG ODN is generally characterized by a 4-base CpG motif consisting of TCGA or TCGT. In many cases, two or three of these 4-base CpG motifs are contained in one humanized K-type CpG ODN. Therefore, in a preferred embodiment, the K-type CpG ODN contained in the oligodeoxynucleotide of the present invention has at least one, more preferably two or more, more preferably two or three, a 4-base CpG motif consisting of TCGA or TCGT. including. When the K-type CpG ODN has 2 or 3 4-base CpG motifs, these 4-base CpG motifs may be the same or different. However, there is no particular limitation as long as it has agonist activity against human TLR9.

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

  The length of K-type CpG ODN is particularly limited as long as the oligodeoxynucleotide of the present invention has immunostimulatory activity (for example, the activity of activating B cells (preferably human B cells) to produce IL-6). Although not preferred, it is preferably no more than 100 nucleotides long (eg, 10-75 nucleotides long). The length of K-type CpG ODN is more preferably 50 nucleotides or less (eg, 10-40 nucleotides). The length of K-type CpG ODN is more preferably 30 nucleotides or less (eg, 10-25 nucleotides). The length of K-type CpG ODN is most preferably 12-25 nucleotides long.

  The length of polydeoxyadenylic acid (dA) is particularly limited as long as it is long enough to form a triple helical structure with a β-1,3-glucan (preferably lentinan or schizophyllan) chain. However, from the viewpoint of forming a stable triple helix structure, it is usually 20 nucleotides or more, preferably 40 nucleotides or more, more preferably 60 nucleotides or more. Poly dA forms a stable triple helix structure with β-1,3-glucan the longer it is, so there is theoretically no upper limit, but if it is too long, the length at the time of oligodeoxynucleotide synthesis will vary. Is usually 100 nucleotides or less, preferably 80 or less. On the other hand, in addition to the formation of the stable triple helix structure, the amount of the oligodeoxynucleotide of the present invention bound per unit amount of β-1,3-glucan is increased, and the length variation during the synthesis of the oligodeoxynucleotide From the viewpoint of avoidance of the complexation efficiency, the length of the poly dA is preferably 20 to 60 nucleotides (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 long), more preferably 30-50 nucleotides long (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, most preferably 30-45 nucleotides in length (30, 31, 32, 33, 34, 35, 36, 37, 38 39, 40, 41, 42, 43, 44, 45 nucleotides long). In particular, when the length is 30 nucleotides or more, good conjugation efficiency is exhibited. By including poly dA, the oligodeoxynucleotide of the present invention has an activity of forming a triple helical structure with two schizophyllan chains. In addition, polydeoxyadenylic acid may be expressed as “poly (dA)” or “poly (dA)”.

  One molecule of the oligodeoxynucleotide of the present invention may contain a plurality of K-type CpG ODN and / or poly dA, but preferably contains one K-type CpG ODN and one poly dA, Most preferably, K-type CpG ODN and poly dA are composed of one by one.

  Exemplary CpG sequences include, but are not limited to, K3 CpG (SEQ ID NO: 1 = 5'-atcgactctcgagcgttctc-3 ').

  The oligodeoxynucleotide of the present invention is characterized in that poly dA is arranged on the 3 'side of K-type CpG ODN. With this arrangement, it is considered that the complex of the present invention (details will be described below) may also enhance the anticancer activity, but the present invention is not limited thereto. You may let them.

  K-type CpG ODN and poly dA may be linked directly by a covalent bond or via a spacer sequence. By spacer sequence is meant a nucleotide sequence comprising one or more nucleotides inserted between two adjacent components. The length of the spacer sequence is such that the complex of the present invention has an immunostimulatory activity (preferably an activity to activate B cells to produce IL-6 and an activity to activate dendritic cells to produce IFN-α). Although it is not specifically limited as long as it has, it is 1-10 nucleotide length normally, Preferably it is 1-5 nucleotide length, More preferably, it is 1-3 nucleotide length. Most preferably, K-type CpG ODN and poly dA are linked by a direct covalent bond.

  The oligodeoxynucleotide of the present invention may have an additional nucleotide sequence at its 5 'end and / or 3' end in addition to K-type CpG ODN, poly dA and an optional spacer sequence. The length of the additional nucleotide sequence indicates that the complex of the present invention has an immunostimulatory activity (preferably an activity that activates B cells to produce IL-6, and a dendritic cell that produces IFN-α. The activity is not particularly limited as long as it has an activity of 1 to 10 nucleotides, preferably 1 to 5 nucleotides, and more preferably 1 to 3 nucleotides.

  In preferred embodiments, the oligodeoxynucleotides of the invention do not include such additional 5 'and / or 3' terminal additional nucleotide sequences. That is, the oligodeoxynucleotide of the present invention preferably comprises K-type CpG ODN, poly dA and an optional spacer sequence, and more preferably comprises K-type CpG ODN and poly dA.

  In a most preferred embodiment, the oligodeoxynucleotide of the present invention consists of K-type CpG ODN (specifically, for example, an oligodeoxynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1) and poly dA, and K-type CpG ODN Is located at the 5 ′ end of the oligodeoxynucleotide and poly dA is located at the 3 ′ end. Specifically, the oligodeoxynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1 has a length of 20 to 60 nucleotides (more preferably 30 to 50 nucleotides (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides long, most preferably 30-45 nucleotides long (30, 31, 32 , 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 nucleotides long)), which is an oligodeoxynucleotide to which, for example, SEQ ID NO: 2 or 9 It is an oligodeoxynucleotide consisting of the nucleotide sequence represented by ~ 12.

  The total length of the oligodeoxynucleotide of the present invention is usually 30 to 200 nucleotides, preferably 35 to 100 nucleotides, more preferably 40 to 80 nucleotides (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 long), more preferably 50 to 70 nucleotides long (specifically, 50, 51, 52, 53, 54, 55 , 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 nucleotides long), most preferably 50 to 65 nucleotides long (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 may be suitably modified to be resistant to in vivo degradation (eg, degradation by exo or endonuclease). 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 by a phosphorothioate bond or a phosphorodithioate bond.

  Preferably, the oligodeoxynucleotide of the invention comprises a modification of a phosphodiester bond, more preferably the modification of a phosphodiester bond is non-phosphorothioate linkage (ie, as described in WO 95/26204). One of the bridging oxygen atoms is replaced by a sulfur atom). That is, part or all of the phosphodiester bond in the oligodeoxynucleotide of the present invention is replaced by a phosphorothioate bond.

  The oligodeoxynucleotide of the present invention preferably contains a modification by phosphorothioate linkage or phosphorodithioate linkage in K-type CpG ODN, more preferably all of the phosphodiester linkage of K-type CpG ODN Substituted with a phosphorothioate linkage. In addition, the oligodeoxynucleotide of the present invention preferably contains a phosphorothioate bond or a phosphorodithioate bond in poly dA, and more preferably, all of the phosphodiester bond of poly dA is substituted with a phosphorothioate bond. Is done. More preferably, all of the phosphodiester linkages of oligodeoxynucleotides, including humanized K-type CpG oligodeoxynucleotides and polydeoxyadenylates of the invention, are replaced with phosphorothioate linkages. Most preferably, the oligodeoxynucleotide of the present invention is 20-60 nucleotides long (more preferably 30-50 nucleotides long (30,30)) at the 3 ′ end of a humanized K-type CpG oligodeoxynucleotide (eg, SEQ ID NO: 1). 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides long), most preferably 30-45 An oligodeoxynucleotide having a nucleotide length (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 nucleotides)) , All phosphodiester bonds contained in the oligodeoxynucleotide are substituted with phosphorothioate bonds. By the phosphorothioate bond, in the oligodeoxynucleotide of the present invention, not only the resistance to degradation but also the immunostimulatory activity (for example, the activity of activating pDC to produce IFN-α), and CpG-β-1, This is because high yield of 3-glucan complex and enhancement of anticancer activity are expected. In the present specification, the phosphorothioate bond is synonymous with the phosphorothioate skeleton, and the phosphate diester bond is synonymous with the phosphate skeleton. The oligodeoxynucleotides of the present invention include any pharmaceutically acceptable salts, esters, or salts of such esters of the above oligodeoxynucleotides.

  The pharmaceutically acceptable salts of the oligodeoxynucleotide of the present invention are preferably alkali metal salts such as sodium salt, potassium salt and lithium salt, alkaline earth metal salts such as calcium salt and magnesium salt, aluminum Metal salts such as salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts; inorganic salts such as ammonium salts, t-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl esters Salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine Salt, piperazine salt, Amine salts such as organic salts such as tramethylammonium salt and tris (hydroxymethyl) aminomethane salt; Halogenated hydrogen halides such as hydrofluoride, hydrochloride, hydrobromide, hydroiodide Inorganic acid salts such as acid salts, nitrates, perchlorates, sulfates, phosphates; lower alkane sulfonates such as methanesulfonate, trifluoromethanesulfonate, ethanesulfonate, benzenesulfonate Organic acids such as aryl sulfonates such as p-toluenesulfonate, acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleate And amino acid salts such as glycine salt, lysine salt, arginine salt, ornithine salt, glutamate, aspartate.

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

  The oligodeoxynucleotide of the present invention is preferably isolated. “Isolated” means that an operation to remove factors other than the target component has been performed, and that the naturally occurring state has been removed. The purity of the “isolated oligodeoxynucleotide” (percentage of the desired oligodeoxynucleotide weight in the total weight of the evaluation object) is usually 70% or more, preferably 80% or more, more preferably 90% or more, More preferably, it is 99% or more.

  The oligodeoxynucleotide of the present invention has excellent immunostimulatory activity (for example, the activity of activating B cells (preferably human B cells) to produce IL-6), and thus is useful as an immunostimulator or the like. . Furthermore, since the oligodeoxynucleotide of the present invention has a property of forming a triple helix structure with two β-1,3-glucans (preferably schizophyllan, lentinan or scleroglucan), Useful for preparation.

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

  Since the above-mentioned oligodeoxynucleotide of the present invention contains K-type CpG ODN, it alone activates an immunostimulatory activity peculiar to K-type CpG ODN (for example, B cells (preferably human B cells) to activate IL -6), and the immunostimulatory activity unique to D-type CpG ODN (for example, the activity of activating plasmacytoid dendritic cells to produce IFN-α) is poor. Surprisingly, however, by forming a complex with β-1,3-glucan (preferably lentinan, schizophyllan), the immunity specific to D-type CpG ODN is not required without requiring the sequence of D-type CpG ODN. An activation activity (for example, an activity of activating plasmacytoid dendritic cells to produce IFN-α) is obtained. That is, the complex of the present invention has an immunostimulatory activity peculiar to K-type CpG ODN (for example, an activity for activating B cells (preferably human B cells) to produce IL-6) and D-type CpG ODN. Specific immunostimulatory activity (for example, the activity of activating plasmacytoid dendritic cells (preferably human plasmacytoid dendritic cells) to produce IFN-α). Examples of β-1,3-glucan used in the present invention include schizophyllan, scleroglucan, curdlan, parkan, glyphoran, lentinan, laminaran and the like. The β-1,3-glucan is preferably rich in 1,6-glucopyranoside branches (side chain ratio: 33 to 40%), such as schizophyllan, lentinan or scleroglucan. More preferred is schizophyllan.

  Lentinan (LNT) is a known β-1,3-1,6-glucan derived from shiitake mushroom, has a molecular formula of (C6H10O5) n, and a molecular weight of about 300 to 700,000. It hardly dissolves in water, methanol, ethanol (95), or acetone, but dissolves in polar organic solvents such as DMSO and aqueous sodium hydroxide.

  Lentinan has an effect of enhancing activated macrophages, killer T cells, natural killer cells and antibody-dependent macrophage-mediated cytotoxicity (ADMC) activity (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 animal experiments, tumor growth suppression and life-prolonging effects have been observed for syngeneic tumors and autologous tumors by combined administration with chemotherapeutic agents. In addition, administration of lentinan alone has been shown to suppress tumor growth and prolong life. In clinical trials, patients with inoperable or recurrent gastric cancer have an extended survival period when combined with oral administration of tegafur (pharmaceutical interview form “1 mg lentinan intravenous Ajinomoto”) and has been approved in Japan. . The effect of single administration of lentinan has not been confirmed so far.

  Schizophyllan (SPG) is a known soluble β-glucan derived from Shirohirotake. SPG consists of a β- (1 → 3) -D-glucan main chain and one β- (1 → 6) -D-glucosyl side chain for each three glucoses (Tabata, K., Ito , W., Kojima, T., Kawabata, S. and Misaki A., “Carbohydr. Res.”, 1981, 89, 1, p. 121-135). SPG has been used for more than 20 years as a clinical drug for intramuscular injection of immunity enhancement for gynecological cancer (Shimizu, Chen, Kumi, Masumi, “Biotherapy”, 1990, 4, p. 1390 Hasegawa, “Oncology” and Chemotherapy ", 1992, 8, p.225), there are confirmed in vivo safety (Theresa, M. McIntire and David, A. Brant," J. Am. Chem. Soc. ", 1998, 120, p.6909).

  As used herein, “complex” refers to a product obtained by association of a plurality of molecules through non-covalent or covalent bonds such as electrostatic bonds, van der Waals bonds, hydrogen bonds, and hydrophobic interactions. Means.

  The complex of the present invention preferably has a triple helical structure. In a preferred embodiment, two of the three strands forming the triple helix structure are β-1,3-glucan chains, and one is the polydeoxyadenylic acid in the oligodeoxynucleotide of the present invention. Is a chain. Note that the complex may partially include a portion that does not form a triple helical structure.

  The composition ratio of oligodeoxynucleotide and β-1,3-glucan in the complex of the present invention depends on the chain length of polydeoxyadenylic acid in the oligodeoxynucleotide, the length of β-1,3-glucan, etc. Can change. For example, when the β-1,3-glucan chain and the polydeoxyadenylic acid chain have the same length, two β-1,3-glucan chains and one oligodeoxynucleotide of the present invention are used. Can associate to form a triple helix structure. In general, since the chain length of polydeoxyadenylic acid is shorter than that of β-1,3-glucan chains, a plurality of oligodeoxy compounds of the present invention are used for two β-1,3-glucan chains. Nucleotides can associate through polydeoxyadenylate to form a triple helix structure (see FIG. 1).

  The complex of the present invention is a complex containing humanized K-type CpG ODN and β-1,3-glucan (eg, lentinan, schizophyllan, scleroglucan, curdlan, perchiman, glyphoran, laminaran), preferably Is a complex consisting of humanized K-type CpG ODN and β-1,3-glucan (eg, lentinan, schizophyllan, scleroglucan). More preferably, the oligodeoxynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1 has a length of 20 to 60 nucleotides on the 3 ′ side (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), and oligodeoxynucleotides in which all of the phosphodiester bonds are replaced with phosphorothioate bonds, and β-1, It is a complex (eg, K3-dA20-60-LNT, K3-dA20-60-SPG) composed of 3-glucan (eg, lentinan, schizophyllan), more preferably from the nucleotide sequence represented by SEQ ID NO: 1. 30 to 50 nucleotides (specifically 30, 31, 32, 33, 34, 35, 36, 37, 38, 3 ′ on the oligodeoxynucleotide (39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides long) polydeoxyadenylic acid and all phosphoric diester bonds are replaced with phosphorothioate bonds It is a complex (eg, K3-dA30-50-LNT, K3-dA30-50-SPG) composed of deoxynucleotide and β-1,3-glucan (eg, lentinan, schizophyllan), most preferably SEQ ID NO: 30 to 45 nucleotides on the 3 ′ side of the oligodeoxynucleotide consisting of the nucleotide sequence represented by 1 (specifically, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 nucleotides) of polydeoxyadenylic acid and oligodeoxynucleotides in which all of the phosphodiester bonds are replaced with phosphorothioate bonds, and β-1,3-glucans (eg, From lentinan, schizophyllan) A complex (K3-dA30~45-LNT, K3-dA30~45-SPG).

  Regarding the preparation method of the complex of the present invention, it can be carried out under the same conditions as described in Non-Patent Documents 21 to 24 and JP-A-2008-100919. That is, β-1,3-glucan, which originally exists as a triple helix structure in nature, is converted into an aprotic organic polar solvent (dimethyl sulfoxide (DMSO), acetonitrile, acetone, etc.) or an alkaline aqueous solution (sodium hydroxide, potassium hydroxide). , Ammonia, calcium hydroxide, etc.) The solution of the single-stranded β-1,3-glucan thus obtained and the oligodeoxynucleotide solution of the present invention (aqueous solution, buffer solution having a pH near neutral, or acidic buffer solution, preferably Aqueous solution or a buffered aqueous solution having a pH near neutral), and if necessary, adjust the pH again to near neutral and hold for an appropriate time, for example, at 5 ° C. overnight. As a result, the complex of the present invention is formed by the two β-1,3-glucan chains and the poly dA chain in the oligodeoxynucleotide forming a triple helical structure. By performing purification by size exclusion chromatography, ultrafiltration, dialysis and the like on the produced complex, oligodeoxynucleotides not formed in the complex can be removed. In addition, by purifying the produced complex by anion exchange chromatography, β-1,3-glucan having no complex formed can be removed. By the above method, the complex can be appropriately purified.

  The complex of the present invention can be formed by measuring, for example, conformational change by CD (circular dichroism) spectrum, UV absorption shift by size exclusion chromatography, gel electrophoresis, microchip electrophoresis, capillary electrophoresis. Although it can confirm, it is not restricted to this.

  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 poly dA chain and the like, but usually the molar ratio (SPG / ODN) is 0.02 to 2.0, preferably 0.1 to 0.5. In a further embodiment, the molar ratio (β-1,3-glucan (such as LNT) / ODN) is for example 0.005-1.0, preferably 0.020-0.25.

  The method for preparing the complex of the present invention will be described using CpG-ODN and LNT complex as an example. LNT is dissolved in an alkaline aqueous solution of 0.05 to 2N, preferably 0.1 to 1.5N (for example, 0.25N aqueous sodium hydroxide solution) and left at 1 ° C to 40 ° C for 10 hours to 4 days (for example, Allow to stand overnight at room temperature) to prepare a single stranded LNT aqueous solution (eg 50 mg / ml LNT aqueous solution). The LNT aqueous solution and a separately prepared CpG aqueous solution (for example, 100 μM CpG aqueous solution) are mixed at a molar ratio (LNT / ODN) of 0.005 to 1.0, and then an acidic buffered aqueous solution (for example, NaH2PO4) is added to the LNT aqueous solution. Neutralize and maintain at 1-40 ° C. for 6 hours to 4 days (eg, overnight at 4 ° C.) to complete conjugation. In addition, an LNT aqueous solution may be added and mixed at the end for the complexation. The formation of the complex can be confirmed, for example, by using size exclusion chromatography and monitoring the absorption of CpG ODN toward the high molecular weight side at 240 to 280 nm (for example, 260 nm).

  In one embodiment, the composite of the present invention takes the form of rod-like particles. The particle size is the same as the particle size of β-1,3-glucan (eg, schizophyllan) used as a material and forms a natural triple-helical structure, and the average particle size is usually 10-100 nm, preferably Is 20-50 nm. The particle diameter can be measured by a dynamic light scattering method by dissolving the complex in water and using a Malvern Instruments Zeta Sizer at 80 ° C.

  The complex of the present invention is preferably isolated. The purity of the “isolated complex” (percentage of the target complex weight in the total weight of the evaluation object) is usually 70% or more, preferably 80% or more, more preferably 90% or more, and still more preferably Is over 99%.

  Furthermore, the complex of the present invention has excellent immunostimulatory activity in addition to anticancer activity, and in particular, immunostimulatory activity peculiar to K-type CpG ODN (for example, B cells (preferably human B cells)) And IL-6 production activity) and immunostimulatory activity specific to D-type CpG ODN (eg, plasmacytoid dendritic cells (preferably human plasmacytoid dendritic cells) are activated to activate IFN- Since it has both of (activity which produces (alpha)), since an effect can be provided also as an immunostimulant etc., it may be advantageous. For example, a K-type CpG ODN (for example, a complex containing SEQ ID NOs: 2, 11, 12 and SPG and a K-type CpG ODN (for example, SEQ ID NO: 2) and SPG (K3-SPG) induce an inflammatory response. Potency (pan-IFN-a, IL-6, etc.), serum antigen-specific IgG antibody titer (total IgG, IgG2c, etc.) in virus-inoculated individuals, antigen-specific cytokine production ability (IFN- γ, IL2, etc.), and can also be advantageous as those that exert an infection protective effect against viruses.

(Pharmaceutical composition)
The present invention provides a pharmaceutical composition comprising the oligodeoxynucleotide of the present invention or the complex of the present invention. The pharmaceutical composition of the present invention can be obtained by formulating the oligodeoxynucleotide of the present invention or the complex of the present invention according to conventional means. The pharmaceutical composition of the present invention comprises the oligodeoxynucleotide or complex of the present invention and a pharmacologically acceptable carrier. The pharmaceutical composition may further contain an antigen. Such a pharmaceutical composition is provided as a dosage form suitable for oral or parenteral administration.

  As a composition for parenteral administration, for example, injections, suppositories and the like are used. Injections are dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, infusions, and the like. May be included. Such an injection can be prepared according to a known method. As a method for preparing an injection, it can be prepared, for example, by dissolving or suspending the oligodeoxynucleotide or complex of the present invention in a sterile aqueous solvent usually used for injection. Examples of the aqueous solvent for injection include distilled water; physiological saline; phosphate buffer, carbonate buffer, Tris buffer, acetate buffer, and other buffer solutions. The pH of such an aqueous solvent is 5 to 10, preferably 6 to 8. The prepared injection solution is preferably filled in a suitable ampoule.

  In addition, a powder formulation of the oligodeoxynucleotide or complex of the present invention can be prepared by subjecting the suspension of the oligodeoxynucleotide or complex of the present invention to a treatment such as vacuum drying or freeze drying. The oligodeoxynucleotide or complex of the present invention can be stored in a powder state, and can be used by dispersing the powder with an aqueous solvent for injection at the time of use.

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

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

(Medical use)
The oligodeoxynucleotides and conjugates of the present invention have been found to have anticancer activity alone. Such an effect can be said to be an unexpected effect from the characteristics of the present invention that have been developed as an adjuvant. Therefore, it can be used as an adjuvant so far, that is, it is not necessary to administer with a cancer antigen, and it is not limited to a specific cancer type. An anticancer agent that acts mildly on the body as an agent will be provided. In addition, since it also has immunostimulatory activity, it can also be expected to have immune activation activity against other diseases, and to have a synergistic effect on cancer patients with weak physical strength.

  Since the present invention has an excellent immunostimulatory activity in addition to the anticancer activity, the oligodeoxynucleotide, complex and pharmaceutical composition of the present invention can be used as an immunostimulator. By administering the oligodeoxynucleotide, complex or pharmaceutical composition of the present invention to a mammal (a primate such as a human, a rodent such as a mouse), an immune reaction in the mammal can be induced. In particular, the complex of the present invention has the activity characteristics of D-type CpG ODN and stimulates peripheral blood mononuclear cells to give type I interferons (Pan-IFN-α, IFN-α2, etc.) and type II interferons ( Since both IFN-γ) are produced in large quantities, they are useful as type I interferon production inducers, type II interferon production inducers, type I and type II interferon production inducers. Since it induces the production of both type I and type II interferons, the complex of the present invention and the pharmaceutical composition containing the same can prevent or prevent diseases in which one or both of type I and type II interferons are effective. Useful for treatment.

  As a method for realizing the pharmaceutical use, for example, (a) by administering the oligodeoxynucleotide of the present invention or the composition containing the complex of the present invention to a cancer patient or a human who may be affected by cancer, Cytotoxic T lymphocytes (CTL) in a subject who has received the administration can be antigen-specifically activated to prevent or treat cancer directly (as a single agent effect).

  In the present specification, the “subject (person)” refers to a subject to be diagnosed or detected or treated according to the present invention (for example, an organism such as a human or a cell, blood, serum, etc. removed from the organism). .

  In this specification, “drug”, “agent” or “factor” (both corresponding to “agent” in English) are used interchangeably in a broad sense, and so long as they can achieve their intended purpose. It may also be a substance or other element (eg energy such as light, radioactivity, heat, electricity). Such substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, DNA such as cDNA, genomic DNA, RNA such as mRNA), poly Saccharides, oligosaccharides, lipids, small organic molecules (for example, hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (for example, small molecule ligands, etc.)) , These complex molecules are included, but not limited thereto. Typically, a factor specific for a polynucleotide is a polynucleotide having complementarity with a certain sequence homology to the sequence of the polynucleotide (eg, 70% or more sequence identity), Examples include, but are not limited to, a polypeptide such as a transcription factor that binds to the promoter region. Factors specific for a polypeptide typically include an antibody specifically directed against the polypeptide or a derivative or analog thereof (eg, a single chain antibody), and the polypeptide is a receptor. Alternatively, specific ligands or receptors in the case of ligands, and substrates thereof when the polypeptide is an enzyme include, but are not limited to.

  As used herein, “treatment” refers to prevention of worsening of a disease or disorder when a disease or disorder (eg, cancer, allergy) occurs, preferably, maintaining the status quo. More preferably, it means reduction, more preferably elimination, including the ability to exert a symptom improving effect or a preventive effect on one or more symptoms associated with a patient's disease or disease. Diagnosing in advance and performing appropriate treatment is referred to as “companion treatment”, and the diagnostic agent therefor is sometimes referred to as “companion diagnostic agent”.

  As used herein, “therapeutic agent (agent)” refers to any drug that can treat a target condition (for example, diseases such as cancer and allergies). In one embodiment of the present invention, the “therapeutic agent” may be a pharmaceutical composition comprising an active ingredient and one or more pharmacologically acceptable carriers. The pharmaceutical composition can be produced by any method known in the technical field of pharmaceutics, for example, by mixing the active ingredient and the carrier. In addition, the form of use of the therapeutic agent is not limited as long as it is a substance used for treatment, and it may be an active ingredient alone or a mixture of an active ingredient and an arbitrary ingredient. The shape of the carrier is not particularly limited, and may be, for example, a solid or a liquid (for example, a buffer solution). The therapeutic agent for cancer, allergy and the like includes a drug (preventive agent) used for preventing cancer, allergy and the like, or a suppressor for cancer, allergy and the like.

  As used herein, “prevention” refers to preventing a certain disease or disorder (eg, allergy) from becoming such a condition before it becomes such a condition. Diagnosis can be performed using the drug of the present invention, and for example, allergies can be prevented using the drug of the present invention, or countermeasures for prevention can be taken as necessary.

  As used herein, the term “prophylactic agent (agent)” refers to any agent that can prevent a target condition (for example, a disease such as allergy) in a broad sense.

  In this specification, the “kit” is a unit provided with a portion to be provided (eg, a test agent, a diagnostic agent, a therapeutic agent, an antibody, a label, instructions, etc.) usually divided into two or more compartments. Say. This kit form is preferred when it is intended to provide a composition that should not be provided in admixture for stability or the like, but preferably used in admixture immediately before use. Such kits preferably include instructions or instructions that describe how to use the provided parts (eg, test agents, diagnostic agents, therapeutic agents, or how the reagents should be processed). In the present specification, when the kit is used as a reagent kit, the kit usually contains instructions including usage of test agents, diagnostic agents, therapeutic agents, antibodies, etc. Is included.

  In the present specification, the “instruction” describes a method for using the present invention for a doctor or other user. This instruction manual includes a word indicating that the detection method of the present invention, how to use a diagnostic agent, or administration of a medicine or the like is given. In addition, the instructions may include a word indicating that the administration site is oral or esophageal administration (for example, by injection). This instruction is prepared in accordance with the format prescribed by the national supervisory authority (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States, etc.) It is clearly stated that it has been received. The instructions are so-called package inserts and are usually provided in a paper medium, but are not limited thereto, and are in a form such as an electronic medium (for example, a homepage or an e-mail provided on the Internet). But it can be provided.

(Aspects of preferred embodiments)
Hereinafter, preferred embodiments of the present invention will be described. The embodiments provided below are provided for 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 obvious that those skilled in the art can make appropriate modifications within the scope of the present invention with reference to the description in the present specification. It will also be appreciated that the following embodiments of the invention may be used alone or in combination.

<Single agent form>
In one aspect, the invention provides an oligodeoxynucleotide comprising (a) a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylic acid, wherein the polydeoxyadenylic acid is a humanized K-type CpG oligodeoxynucleotide. Provided is an anticancer agent comprising a complex comprising oligodeoxynucleotides arranged on the 3 ′ side and (b) β-1,3-glucan. In the present invention, it was found that the complex itself of the present invention acts as an anticancer agent. Conventionally, the present inventors have only found out and applied for the use of this complex as an adjuvant, and have not anticipated that it can be directly used as an anticancer agent as a single agent. Therefore, it can be said that it produces an unexpected effect in that it is used without a cancer antigen.

  In one embodiment, the anticancer agent of the present invention is administered without a cancer antigen.

  In another embodiment, the anticancer agent of the present invention is administered such that it is delivered to the reticuloendothelial system and / or lymph nodes. Preferably, the reticuloendothelial system and / or lymph nodes comprise tumors and phagocytic cells. Illustratively, the reticuloendothelial system includes the spleen and / or liver. Therefore, the anticancer agent of the present invention is characterized in that it is administered so as to be delivered to reticuloendothelial organs (spleen, liver, etc.) and / or lymph nodes containing tumors and phagocytic cells. Without wishing to be bound by theory, the complexes of the present invention are delivered to tumors and phagocytic cells where they can recruit cancer dead cells to reticuloendothelial organs (spleen, liver, etc.). Indicated. Thereby, it is considered that cancer cells in the body can be further destroyed. Therefore, it can be said that the present invention can kill any cancer cell present in the body, not against a specific cancer using a specific cancer antigen as an adjuvant, It can be said that a remarkable effect is brought about in that an agent can be provided.

  Accordingly, in a more preferred embodiment, the anticancer agent of the present invention is administered so as to be delivered to tumors and phagocytic cells without a cancer antigen.

  As such a delivery technique, any technique can be used. For example, the administration can include systemic administration, but is not limited thereto. Systemic administration is preferred, and examples of systemic administration include intravenous administration, intraperitoneal administration, oral administration, subcutaneous administration, and intramuscular administration.

In one embodiment, the oligodeoxynucleotide used in the present invention is 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), K3-dA35 (SEQ ID NO: 7), and the like.

  In one embodiment, the β-1,3-glucan used in the present invention may be schizophyllan (SPG), lentinan, scleroglucan, curdlan, perchiman, glyforan, laminaran and the like.

  In a preferred embodiment, the complex of the invention is K3-SPG or an analogue thereof. Examples of the class itself include, but are not limited to, those having a structure similar to K3 on the CpG side and those having a structure similar to SPG on the β-glucan side.

  In addition, since an anticancer effect | action is based on various mechanisms, uses, such as accumulating the dead cell of cancer in a spleen, cannot be easily considered. In particular, in systemic administration, the use for accumulating in a tumor and accumulating dead tumor cells in tissues such as the spleen is not conceivable. In addition, the expression of interleukin 12 (IL12) and / or interferon (IFN) α or its promoting effect is also due to a mechanism different from the anticancer action, and interleukin 12 (IL12) and / or interferon (IFN) ) Since the expression of α or its promotion can be exerted other than anti-cancer, it is not easy to come up with each other. Therefore, each use of the CpG-β glucan complex of the present invention (anti-cancer use (single agent), use to accumulate cancer dead cells in the spleen, interleukin 12 (IL12) and / or interferon (IFN)) It can be said that there is a relationship that cannot be easily conceived with each other.

<Reticuloendothelial system (including spleen and / or liver) and / or lymph node accumulation agent>
In another aspect, the present invention provides an oligodeoxynucleotide comprising (a) a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylic acid, wherein the polydeoxyadenylic acid is a humanized K-type CpG oligodeoxynucleotide. 3'-side oligodeoxynucleotide and (b) β-1,3-glucan-containing complex, cancerous dead cells containing reticuloendothelial system (spleen and / or liver) And / or a composition for accumulation in lymph nodes. While not wishing to be bound by theory, it has been found that the complex of the present invention can accumulate cancer dead cells in the reticuloendothelial system (including spleen and / or liver) and / or lymph nodes. It was. As illustrated in the Examples, treatment with a complex of the invention such as K3-SPG has been demonstrated to induce tumor cell death in a manner that is dependent on both IL12p40 and IFN-I. It has not been conventionally expected that the complex has such an action, and in that sense, it can be said that an unexpected action and effect has been achieved. That is, CpG is targeted to phagocytic cells in the tumor microenvironment. Once cancer dead cells accumulate in the reticuloendothelial system (including spleen and / or liver) and / or lymph nodes, the released tumor dead cells then induce anti-tumor CTL against multiple tumor antigens. The cancer cells in the body can be killed and cured as if they were attacked with a shotgun. Without wishing to be bound by theory, it is important, although not necessarily essential for K3-SPG monotherapy, to produce both IL12 and IFN-I cytokines in the tumor microenvironment.

In one embodiment, the oligodeoxynucleotide used in the present invention is 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).

  In another embodiment, the β-1,3-glucan used in the present invention is selected from the group consisting of schizophyllan (SPG), scleroglucan, curdlan, perchiman, glyphoran and laminaran.

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

  In one embodiment, the reticuloendothelial system and / or lymph node targeted by the composition of the present invention comprises tumors and phagocytic cells. Illustratively, the reticuloendothelial system includes the spleen and / or liver. Accordingly, the composition of the present invention is characterized in that it is administered to be delivered to reticuloendothelial organs (spleen, liver, etc.) and / or lymph nodes containing tumors and phagocytic cells. Without wishing to be bound by theory, the complexes of the present invention are delivered to tumors and phagocytic cells where they can recruit cancer dead cells to reticuloendothelial organs (spleen, liver, etc.). Indicated. Thereby, it is considered that cancer cells in the body can be further destroyed. Therefore, it can be said that the present invention can kill any cancer cell present in the body, not against a specific cancer using a specific cancer antigen as an adjuvant, It can be said that a remarkable effect is brought about in that an agent can be provided.

  Accordingly, in a more preferred embodiment, the anticancer agent of the present invention is administered so as to be delivered to tumors and phagocytic cells without a cancer antigen.

  As such a delivery technique, any technique can be used. For example, the administration can include systemic administration, but is not limited thereto. Systemic administration is preferred, and examples of systemic administration include intravenous administration, intraperitoneal administration, oral administration, subcutaneous administration, and intramuscular administration.

<IL12 and / or IFN expression promoter>
In yet another aspect, the present invention provides (a) an oligodeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylic acid, wherein the polydeoxyadenylic acid is a humanized K-type CpG oligodeoxynucleotide. For expression or promotion of interleukin 12 (IL12) and / or interferon (IFN) γ, including oligodeoxynucleotides and (b) β-1,3-glucan, arranged 3 ′ of A composition is provided. Producing both IL12 and IFN-I cytokines in the tumor microenvironment is an important effect in K3-SPG monotherapy, such as acting as an anti-cancer agent and other It is also important in applications. Examples of such treatment include, but are not limited to, cancer, chronic infectious diseases such as viruses, and prevention of viral infection.

In one embodiment, the oligodeoxynucleotide used in the present invention is 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).

  In another embodiment, the β-1,3-glucan used in the present invention is selected from the group consisting of schizophyllan (SPG), scleroglucan, curdlan, perchiman, glyphoran and laminaran.

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

(Pharmaceuticals, dosage forms, etc.)
The present invention is provided as the above-mentioned various forms of medicaments (therapeutic or preventive).

  The administration route of the therapeutic agent is preferably one that is effective for treatment, and may be, for example, intravenous, subcutaneous, intramuscular, intraperitoneal, or oral administration. The administration form may be, for example, an injection, capsule, tablet, granule or the like. When administering the component of this invention, using as an injection is effective. Aqueous solutions for injection may be stored, for example, in vials or stainless steel containers. The aqueous solution for injection may contain, for example, physiological saline, sugar (for example, trehalose), NaCl, or NaOH. The therapeutic agent may contain, for example, a buffer (for example, phosphate buffer), a stabilizer and the like.

  In general, a composition, medicament, therapeutic agent, prophylactic agent, etc. of the present invention comprises a therapeutically effective amount of a therapeutic agent or active ingredient, and a pharmaceutically acceptable carrier or excipient. As used herein, “pharmaceutically acceptable” refers to a licensed or otherwise recognized pharmacopoeia of a government for use in animals, and more particularly in humans, by a government supervisory authority. It means that it is enumerated. As used herein, “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such carriers can be sterile liquids, such as water and oils, including but not limited to those of petroleum, animal, vegetable or synthetic origin, including but not limited to peanut oil, soybean oil, minerals Oil, sesame oil, etc. are included. Water is a preferred carrier when the drug is administered orally. Saline and aqueous dextrose are preferred carriers when the pharmaceutical composition is administered intravenously. Preferably, saline solutions and aqueous dextrose and glycerol solutions are used as liquid carriers for injectable solutions. Suitable excipients include light anhydrous silicic acid, crystalline cellulose, mannitol, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, chloride Sodium, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, carmellose calcium, carmellose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylacetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain fatty acid triglyceride, polyoxyethylene hardening Castor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganic salts and the like are included. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. It is also possible to formulate the composition as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations may also include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate. Examples of suitable carriers are E.I. W. Martin, Remington's Pharmaceutical Sciences (Mark Publishing Company, Easton, U.S.A). Such compositions contain a therapeutically effective amount of the therapeutic agent, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation must be suitable for the mode of administration. In addition to these, for example, surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffering agents, suspending agents, tonicity agents, binders, disintegrating agents, lubricants, fluidity Accelerators, flavoring agents and the like may be included.

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

  When the present invention is administered as a pharmaceutical, various delivery systems are known, and such systems can be used to administer the therapeutic agent of the present invention to an appropriate site (eg, esophagus). Such systems include, for example, encapsulation in liposomes, microparticles, and microcapsules: the use of recombinant cells capable of expressing therapeutic agents (eg, polypeptides), receptor-mediated endocytosis Use; such as the construction of therapeutic nucleic acids as part of a retroviral vector or other vector. Introduction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. It is also possible to administer the medicament by any suitable route, for example by injection, by bolus injection, by absorption through epithelial or dermal mucosal lining (eg oral, rectal and intestinal mucosa, etc.) Accordingly, an inhaler or nebulizer can be used with an aerosolizing agent and can be administered with other biologically active agents. Administration can be systemic or local. When the present invention is used for cancer, it can be administered by any appropriate route such as direct injection into cancer (lesion).

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

  It is also possible to mix the composition, medicament, therapeutic agent, or preventive agent of the present invention in a neutral or salt form or other prodrug (eg, ester). Pharmaceutically acceptable salts include those formed with free carboxyl groups derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine And those formed with free amine groups such as those derived from, and those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, and the like.

  The amount of the therapeutic agent of the invention that is effective in the treatment of a particular disorder or condition can vary depending on the nature of the disorder or condition, but can be determined by those skilled in the art by standard clinical techniques based on the description herein. In addition, in some cases, in vitro assays can be used to help identify optimal dosage ranges. The exact dose to be used in the formulation can also vary depending on the route of administration and the severity of the disease or disorder and should be determined according to the judgment of the attending physician and the circumstances of each patient. However, the dose is not particularly limited, and may be, for example, 0.001, 1, 5, 10, 15, 100, or 1000 mg / kg body weight per dose, and within the range of any two of them. Also good. The dosing interval is not particularly limited, for example, it may be administered once or twice per 1, 7, 14, 21, or 28 days, or once or twice per any two of these ranges Also good. The dose, administration interval, and administration method may be appropriately selected depending on the age, weight, symptoms, target organ, etc. of the patient. The therapeutic agent preferably contains a therapeutically effective amount or an effective amount of an active ingredient that exhibits a desired action. If the malignant tumor marker is significantly decreased after administration, it may be determined that there is a therapeutic effect. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

  In one embodiment of the present invention, a “patient” or “subject” is a human or non-human mammal (eg, mouse, guinea pig, hamster, rat, mouse, rabbit, pig, sheep, goat, cow, horse, Including one or more of a cat, dog, marmoset, monkey, or chimpanzee).

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

  In certain embodiments, the present invention provides a drug pack or kit comprising one or more containers filled with one or more components of a composition or medicament of the present invention. In some cases, associated with such containers, manufactured, used or sold for human administration by a government agency in a manner prescribed by the government agency that regulates the manufacture, use or sale of a pharmaceutical or biological product. It is also possible to indicate information indicating authorization.

  In certain embodiments, pharmaceutical compositions comprising the components of the present invention can be administered via liposomes, microparticles, or microcapsules. In various embodiments of the present invention, it may be useful to achieve sustained release of the components of the present invention using such compositions.

  Formulation procedures as pharmaceuticals such as therapeutic agents and prophylactic agents of the present invention are known in the art, and are described in, for example, the Japanese Pharmacopeia, the US Pharmacopeia, and the pharmacopoeia of other countries. Accordingly, those skilled in the art can determine the embodiment, such as the amount to be used, without undue experimentation as described herein.

(General technology)
Molecular biological techniques, biochemical techniques, and microbiological techniques used in this specification are well known and commonly used in the art, for example, 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). 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. Innis, MA et al. (1995) .PCR Str ategies, Academic Press; Ausubel, FM (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, JJ et al. (1999). PCR Applications: Protocols for It is described in Functional Genomics, Academic Press, a separate volume of experimental medicine "Gene Transfer & Expression Analysis Experimental Method" Yodosha, 1997, etc., and related portions (which may be all) are incorporated herein by reference. The

  For DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, see, for example, 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, GMetal. (1996) Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, GT (I996). Bioconjugate Techniques, Academic Press, etc. Which are incorporated herein by reference in the relevant part.

  For example, it is possible herein to synthesize the oligonucleotides of the invention by standard methods known in the art, for example, by using automated DNA synthesizers (such as those commercially available from Biosearch, Applied Biosystems, etc.). is there. For example, phosphorothioate oligonucleotides can be synthesized by the method of Stein et al. (Stein et al., 1988, Nucl. Acids Res. 16: 3209) or controlled pore glass polymer supports (Sarinet al., 1988, Proc. Natl. Acad. Sci. USA 85: 7448-7451) and the like can also be used to prepare methylphosphonate oligonucleotides.

  In this specification, “or” is used when “at least one or more” of the items listed in the text can be adopted. The same applies to “or”. In this specification, when “in the range of two values” is specified, the range includes the two values themselves.

  References such as scientific literature, patents and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as if each was specifically described.

  The present invention has been described with reference to the preferred embodiments for easy understanding. In the following, the present invention will be described based on examples, but the above description and the following examples are provided only for the purpose of illustration, not for the purpose of limiting the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described in the present specification, but is limited only by the scope of the claims.

  Examples are described below. When necessary, the animals used in the following examples were handled in accordance with institutional guidelines of the National Institute of Pharmaceutical Sciences and Osaka University animal facilities for all animal experiments. Also, based on the Declaration of Helsinki. Specifically, the reagents described in the Examples were used as reagents, but equivalent products from other manufacturers (Sigma-Aldrich, Wako Pure Chemicals, Nakarai, R & D Systems, USCN Life Science INC, etc.) can be substituted.

(Production Example)
The following CpG ODNs were synthesized by Gene Design Co., Ltd. (underlined indicates phosphorothioate linkage).

  In particular, in addition to K3-dA40 (SEQ ID NO: 2), K3-dA35 (SEQ ID NO: 7), K3-dA30 (SEQ ID NO: 6), K3-dA25 (SEQ ID NO: 5), and K3-dA20 (SEQ ID NO: 4) Is described (Table 2).

(S in the above sequence indicates that the phosphodiester bond between nucleosides is replaced with a phosphorothioate bond.)

  This oligodeoxynucleotide is prepared using a conventional solid phase phosphoramidite method (Nucleic Acids in Chemistry and Biology, 3. Chemical synthesis (1990) ed. G. Michael Blackburn and Michael J. Gait. Oxford University Press). Synthesized.

  Ovalbumin (OVA) was purchased from Seikagaku Corporation. DQ-OVA, Alexa488-OVA, CFSE, and Lipofectamine 2000 were purchased from Invitrogen. Hoechst33258, zymozan and curdlan were purchased from SIGMA. Zymosan-Depleted was purchased from Invivogen. Clodronate liposomes were purchased from FormuMax. Influenza split product vaccine, formalin inactivated whole virus (WIV), and 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 (Production Example Figure 1)
7.22 mg of K3-dA40 was dissolved in water (3.7 mL). 15 mg of SPG (Mitsui Sugar) was dissolved in 0.25N NaOH (1 mL). Complexation was completed by adding 1 mL of 330 mM NaH2PO4 to the DNA solution, then adding the SPG solution to the DNA / NaH2PO4 solution and maintaining at 4 ° C. overnight. The molar ratio (MSPG / MDNA) was fixed at 0.27. Complex formation was confirmed by a microchip electrophoresis apparatus (SHIMADZU: MultiNA). The formation of the complex should be monitored by absorption at 260 nm, using size exclusion chromatography, and shifting the high molecular weight side of CpG ODN. (System: Agilent 1100 series, Column: Asahipak GF7M-HQ (Shodex) 2 ligation, Flow rate: 0.8 mL / min, Buffer: 10 mM EDTA PBS, pH 7.4, Temperature: 40 ° C.).

(Preparation for use in examples)
In the examples below, it was shown that systemic monotherapy with nanoparticulate TLR9 agonists targeting phagocytic cells in a tumor microenvironment that induces strong tumor shrinkage is possible.

(Materials and methods)
Hereinafter, reagents, materials, animals, cells and methods used in this example will be described. The description will be supplemented in each embodiment as appropriate.

(Animals and reagents)
Six week old female C57BL / 6J mice were purchased from Nihon CLEA. Il12p40-deficient mice and Batf3-deficient mice were purchased from Jackson Laboratory. Ifnar2-deficient mice, Myd88-deficient mice and Dectin-1-deficient mice are as previously described (Kobiyama, K., et al. Proc. Natl. Acad. Sci. USA 111, 3086-3091 (2014)). All animal experiments were performed according to the Institute of Pharmaceutical Sciences Institute guidelines. K3 was synthesized by Gene Design. Ovalbumin (OVA) was purchased from Seikagaku.

(Cell line)
EL4 and OVA expressing EL4 (EG7) is a thymoma cell line of C57BL / 6J mice and was purchased from ATCC. B16 (melanoma) was purchased from the Japan Collection of Research Bioresources. B16F10 (melanoma) was purchased from Riken Cell Bank and MC38 (colon cancer) was Provided by Dr. JAMES Primus. Pan02 (pancreatic cancer) was purchased 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), penicillin, and streptomycin). B16 and B16F10 were cultured in complete DMEM (DMEM supplemented with 10% (v / v) fetal bovine serum (FBS), penicillin, and streptomycin).

(Tumor experiment and treatment method)
EG7, EL4, B16, B16F10, and MC38 cells (100 μl at 5 × 10 ⁶ cells / mL in 10% Matrigel / PBS) were inoculated subcutaneously (sc) in the right flank of mice. Tumor size was measured by tumor length (L), width (W), and height (H), and tumor volume (V) was calculated as V = L × W × H. Intratumoral injection (it) was injected directly into the tumor site. CpG treatment began when the tumor volume reached approximately 100 mm ³ , which was 7 days after EG7 and B16F10 inoculation, 10 days after B16 inoculation, and 14 days after MC38 inoculation. Tumor-bearing mice were treated 3 times every other day with K3 (30 μg) or K3-SPG (10 μg).

(Pan02 peritoneal seeding model)
In the Pan02 peritoneal seeding model, 1 × 10 ⁶ Pan02 cells (100 μl at 1 × 10 ⁷ cells / mL in PBS) were injected intraperitoneally. CpG treatment was started 11 days after inoculation and all tumor nodules were excised from the peritoneum of mice on day 21 and their weight (g) was then measured. The dosage for CpG treatment is as described above.

(In vivo imaging experiment)
To assess the localization of K3 and K3-SPG, C57BL / 6 mice were treated with EG7 at day 0. c. Inoculate and receive PBS (control), Alexa 647-K3 (30 μg), or Alexa 647-K3-SPG (10 μg) i. v. Administered. One hour after dosing, mice were analyzed with IVIS® Lumina Imaging System and analysis software (Ver. 2.6, Xenogen), and images measured with relative fluorescence were measured in physical units (photons / photon / surface radiance). Second / cm ² / sr). To detect CD8 ⁺ T cells labeled in vivo, splenocytes were treated with K3-SPG on days 7, 9, 11 or untreated, C57BL / 6 mice carrying EG7 Or harvested on day 14 from Il12p40-Ifnar2 double knockout mice. After spleen cell suspension, erythrocytes were lysed with ACK lysis buffer (150 mM NH ₄ Cl, 10 mM KHCO ₃ , 0.1 mM Na ₂ EDTA) and cells were maintained in complete RPMI. CD8α ⁺ T cells were sorted by MACS (Miltenyi Biotec). CD8α ⁺ T cells were sorted by the negative selection method. The sorted CD8α ⁺ T cells were then stained with Xenolight DiR®. Stained CD8α ⁺ T cells were injected or treated with recipient mice (EG7 on day 0, iv7 on days 0, and K3-SPG on days 7, 9, 11). C57BL / 6 mice or Il12p40-Ifnar2 double knockout mice). Twenty-four hours after transferring the stained cells, the mice were analyzed with the IVIS® Lumina Imaging System (Ver. 2.6). The region of interest was aggregated into the tumor region and the fluorescence intensity was analyzed with Living Image Software (Ver. 2.6, Xenogen).

(Immunohistochemistry)
C57BL / 6J mice (6-8 weeks old, female, CLEA Japan) were treated with Alexa 647-K3 (30 μg), Alexa 647-K3-SPG (10 μg), and dextran-PE (20 μg) i.e. from the tail vein. v. Injected. Tumors were collected 1 hour after injection and frozen sections were fixed with 4% (w / v) paraformaldehyde for 10 minutes and stained with anti-CD3e and anti-CD8β antibodies together with Hoechst 33258. Cells were photographed using an Olympus IX81 system. Image data was analyzed with MetaMorph.

(Depletion experiment)
In order to deplete phagocytic cells (dendritic cells and macrophages), C57BL / 6 mice were treated with clodronate liposomes or control liposomes (100 nm) (Katayama Chemical) i. v. Injected. To deplete CD8 ⁺ T cells, 200 μg of anti-CD8α antibody was administered i.v. to the tail vein 6 and 13 days after EG7 inoculation. v. Injected.

(Analysis of splenocytes)
Splenocytes were injected i.p. with K3-SPG on days 7, 9, 11. v. Recovered on day 14 from treated or untreated C57BL / 6 mice carrying EG7 or Il12p40-Ifnar2 double knockout mice. After preparation of splenocytes, erythrocytes were lysed with ACK lysis buffer and the cells were maintained with complete RPMI. Splenocytes were isolated from 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-amino. Stained with actinomycin D (7AAD). The cell number of OVA tetramer ⁺ CD44 ⁺ CD8α ⁺ TCRβ ⁺ was determined by flow cytometry. For other experiments, prepared splenocytes were incubated with anti-CD45 antibody, anti-CD3e antibody, anti-CD8α antibody, and anti-CD11a antibody and then analyzed by flow cytometry.

(Assay and immunization of CD45 negative cells)
Splenocytes were injected i.p. with K3-SPG on days 7, 9, 11. v. Harvested at day 12 from treated or untreated C57BL / 6 mice carrying EG7 or Il12p40-Ifnar2 double knockout mice. After preparation of splenocytes, erythrocytes were lysed with ACK lysis buffer and the cells were maintained in complete RPMI. Splenocytes were stained with anti-CD45 antibody (APC) and the number of CD45 ⁻ cells was determined by flow cytometry. In addition, populations of apoptotic cells, necrotic cells, and live CD45 negative 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)
C57BL / 6 mice were challenged with 5 × 10 ⁵ CD45 ⁻ cells on day −7. v. Administered. Seven days after immunization, mice were challenged with 5 × 10 ⁵ EG7 cells on day 0. c. Vaccinated.

(Measurement of cytokines)
Mouse IL-12p40, mouse IL-13, and human IFNγ levels were measured using an R & D ELISA kit.

(Statistical analysis)
One-way analysis of variance including Mann-Whitney 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 induces strong tumor growth inhibition without any additional tumor antigen)
In this example, it was demonstrated that intravenous injection of K3-SPG induces strong tumor growth inhibition without any additional tumor antigen.

(Experiment with EG7 (OVA expressing mouse thymoma cell line) model)
C57BL / 6 mice were inoculated with EG7 (OVA expressing mouse thymoma cell line) on the right flank on day 0, administered subcutaneously (id) near the tail base, intratumoral (it), Or three times with PBS and equimolar amounts of K3 (30 μg) or K3-SPG (10 μg) via three different routes of intravenous (iv) administration (7, 9, 11 of inoculation) days after). Tumor size was measured every 2-3 days until day 23.

(result)
The results are shown in FIG. In the PBS group (control), tumor growth was not inhibited via any route of administration (FIGS. 2a, b, c (FIG. 2A)). In K3 treatment, tumor shrinkage is i. t. Was observed only in, but not in other routes (FIGS. 2d, e, f (FIG. 2A)). In K3-SPG treatment, i. t. And i. v. Strong tumor shrinkage was observed in both cases, i. d. Administration showed no effect on tumor growth (Figure 2g, h, i (Figure 2A)). The figure which compared and showed control, K3, and K3-SPG is shown in a figure (FIG. 2a, d, g (FIG. 2A)).

  In the prior art, many attempts at systemic CpG ODN treatment for 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)), i.e. in K3-SPG. v. The fact that single agent treatment can strongly suppress tumor growth was unpredictable, and in this respect it was demonstrated that the present invention had an unexpected effect.

(Experiment with other tumor cell lines)
To investigate the potential of this K3-SPG systemic monotherapy, other tumor cell lines were also tested with similar protocols used in the EG7 model.

(result)
Intravenous administration of K3-SPG also suppressed the growth of melanoma (B16 and B16F10) and colon cancer (MC38) (Figure 2j, k, l (Figure 2B)). We tested by creating a tumor dissemination model with even higher clinical malignancy. Mouse pancreatic tumor line, Pan02 (1 × 10 ⁶ cells) is inoculated intraperitoneally (also referred to as “ip”) followed by K3 or K3-SPG treatment (3 times every other day) 11 days later. On day 21, all mice were sacrificed and the total weight of the abdominal tumor was assessed (FIG. 2m (FIG. 2B)). Tumor growth is determined by K3-SPG i. v. Although significantly suppressed in the treatment group, K3 i. p. And K3-SPG i. p. It was not suppressed in the treatment group (FIG. 2m (FIG. 2B)). In response, a significant prolongation of survival was observed in K3-SPG i. v. Although observed in the treatment group, K3 i. v. Not observed in the group (FIG. 2n (FIG. 2B)). These results indicate that the systemic i. v. It demonstrates that administration is a promising monotherapy for many different cancers and does not require any further tumor peptides and antigens.

(Example 2: K3-SPG targeted phagocytic cells in tumor microenvironment)
Next, the present inventors elucidated the mechanism of K3-SPG in the tumor microenvironment.

  K3-SPG forms nanoparticles with a size of about 30 nm (Kobiyama, K., et al. Proc. Natl. Acad. Sci. U. S. A. 111, 3086-3091 (2014)). The inventors hypothesized that K3-SPG functions via a drug delivery system to the tumor (Na, JH, 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); Farokzad, OC et al. ACS Nano 3, 16-20 (2009)).

(Fluorescent labeling imaging)
To test the distribution in vivo, K3 and K3-SPG were fluorescently labeled. EG7 tumor-bearing mice were given PBS, Alexa647-K3 (30 μg), or Alexa647-K3-SPG (10 μg) i. v. After injection, the distribution of fluorescence was examined by an in vivo imaging system (IVIS).

  The results are shown in FIG.

  IVIS imaging uses K3-SPG instead of K3, i. v. It was revealed that it accumulated at the tumor site 1 hour after administration (FIG. 4a). It was observed that the accumulation of K3-SPG in the tumor is well related to the effectiveness of CpG monotherapy treatment for tumor shrinkage (FIG. 2). In immunohistochemistry (IHC) studies, we were unable 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). We were unable to detect Alexa647 signal on IHC after 24 hours. EG7 cells express CD3e on their surface (because EG7 is derived from a thymoma cell line), but K3-SPG does not associate with CD3e, which is taken up by non-tumor cells. It showed that. The nanoparticles choose to be taken up by phagocytic cells such as macrophages and dendritic cells (DC), which can be labeled with TRITC-dextran in vivo. Therefore, the inventor injected TRITC-dextran intravenously with fluorescently stained K3, K3-SPG, or SPG and tested for their coexistence by IHC (FIGS. 4d, e, f). i. v. One hour after injection, dextran was observed in the tumor area of all samples (FIGS. 4d, e, f), indicating that the tumor microenvironment contains phagocytes. Consistent with previous results, Alexa647-K3 was not observed within the tumor (FIG. 4d). Approximately 50% of Alexa647-K3-SPG and FITC-SPG observed within the tumor coexist with TRITC-dextran positive cells (FIGS. 4e, f, g), indicating that K3-SPG is phagocytosed in the tumor microenvironment. It is taken up by cells. Some K3-SPGs do not associate with dextran and we speculate that they accumulated passively in the space within the tumor tissue via the vascular permeability and retention (EPR) effect. ing. K3-SPG i. v. To test the importance of phagocytes for treatment, we injected clodronate liposomes intravenously. We usually deplete phagocytes within the tumor by injecting 100 nm clodronate liposomes rather than 200-300 nm liposomes (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)), this injection caused almost no F4 / 80 positive cells in the tumor in 2 days. It was depleted (Figure 5). Tumor-bearing mice are injected with clodronate liposomes on day 5 (2 days before the first K3-SPG treatment) or not, and mice are treated with K3-SPG as in FIG. 2 (AB). Treated. The previous injection of clodronate liposomes significantly offset the suppression of K3-SPG-mediated tumor growth (p <0.05) (FIG. 4h), whereas the injection of clodronate liposomes itself is PBS There was no effect on tumor growth compared to treated mice. These results indicate that K3-SPG targets phagocytic cells in the tumor microenvironment, and the antitumor effect of K3-SPG is largely dependent on phagocytic cell uptake in the tumor microenvironment. Show.

(Example 3: Producing both IL12 and IFN-I cytokines in the tumor microenvironment is important for K3-SPG monotherapy)
Next, in this example, the inventors tested for factors required for successful K3-SPG monotherapy.

  Cytokines such as IL-12 and IFN-I are CpG ODN (Krieg, AM) including K3-SPG (Kobiyama, K., et al. Proc. Natl. Acad. Sci. USA 111, 3086-3091 (2014)). , et al. Journal of immunology 161, 2428-2434 (1998); Klinman, DM, et al. Immunity 11, 123-129 (1999); Ishii, KJ, et al. Current opinion in molecular therapeutics 6, 166-174 (2004)) have been shown to be important immunostimulators. Therefore, we tested whether IL-12 and IFN-I are required for tumor reduction by treatment with K3-SPG.

Ll7p40 and IFNAR2-deficient mice were inoculated subcutaneously with EG7 cells on day 0 and i.p. three times with PBS or K3-SPG (10 [mu] g) as shown in FIG. v. Treated. Thereafter, the effect of K3-SPG on tumor shrinkage was observed.
(result)

  The results are shown in Fig. 6 (AB) and below. The effect of K3-SPG on tumor shrinkage was partially dependent on IL-12p40 and IFN-I signaling (FIGS. 6a, b (FIG. 6A)). We also tested IL12p40 and IFNAR2 double deficient (DKO) mice and found that the effects of K3-SPG were completely abrogated in DKO mice (FIG. 6c (FIG. 6A)). . IFN-β and IL-12p40 were also detected in the tumor by IHC staining (FIGS. 7 and 8). These data indicated that secretion of both IL12p40 and IFN-I cytokines within the tumor is important for K3-SPG-mediated tumor suppression.

  We also tested Rag2 mice that are completely deficient in T cell and B cell mediated adaptive immune responses. Although Rag2 mice were unable to control all tumor growth even with K3-SPG treatment (FIG. 6d (FIG. 6A)), we observed that during the three treatments with K3-SPG It was found that rag2-deficient mice were able to partially control tumor growth (FIG. 6f (FIG. 6A)). To confirm this observation, we created 6 treatment groups of rag2 mice (days 7, 9, 11 and 14, 16, 18), which is more evident in this protocol for rag2 mice. Tumor control was found (FIG. 6f (FIG. 6A)). Interestingly, IL12p40 and IFNAR2 DKO mice were completely unresponsive to K3-SPG monotherapy even in this broad treatment protocol (FIG. 6e (FIG. 6A)). These data indicated that K3-SPG treatment induced both IL-12p40 and IFN-I within the tumor, resulting in both an innate and adaptive immune response against the tumor. .

Example 4: K3-SPG treatment induces tumor cell death in a manner dependent on both IL12p40 and IFN-I
In this example, K3-SPG treatment was demonstrated to induce tumor cell death in a manner that is dependent on both IL12p40 and IFN-I.

  Due to the observed partial suppression of tumor growth independent of adaptive immunity observed in rag2 mice and its complete suppression in IL12p40 and IFNAR2 DKO mice, we have observed extensive tumors during K3-SPG treatment. -Host interaction was tested.

  We found that the spleen removed on day 12 (next day after 3 treatments with K3-SPG) contained a greater amount of CD45 negative cells compared to the spleen treated with PBS. (FIG. 6g (FIG. 6B)). Interestingly, these CD45 negative cells were markedly reduced in IL12p40 and IFNAR2 DKO mice (FIG. 6g, h (FIG. 6B)). We sorted these CD45 negative cells. This size and morphology strongly indicated that they were derived from tumor cells. EG7 inoculation experiments in GFP mice further confirmed that these CD45 negative cells were also GFP negative, indicating that these cells were derived from tumor cells (FIG. 9). Since EG7 cells do not express CD45, CD45 negative is also supporting this hypothesis. Hoechst and PI staining revealed that most CD45 negative cells in the spleen were dead cells consisting of both apoptotic and necrotic features (FIG. 6i (FIG. 6B)). These data indicate that tumor phagocytes targeted by K3-SPG secrete IL-12p40 and IFN-I in the tumor microenvironment, and these cytokines induce tumor cell death and release them into the circulation. It was shown that it was finally trapped in the spleen.

(Example 5: Released tumor dead cells induce anti-tumor CTL against multiple tumor antigens)
In this example, it was demonstrated that released tumor dead cells induce anti-tumor CTL against multiple tumor antigens.

  To test the immunogenicity of these CD45 negative cells found in the spleen of K3-SPG treated mice, we sorted these cells and injected intravenously into untreated mice as an immunization. did. The immunized mice were then transplanted with EG7 tumor cells 7 days after sorted cell administration. CD45 negative cell immunized mice were significantly protected against the growth of EG7 tumors (FIG. 6j (FIG. 6B)). Interestingly, OVA257 tetramer positive cells (red dots in FIG. 6k (FIG. 6B)) in control and immunized mice did not correlate with tumor size (FIG. 6k (FIG. 6B) bars) and were due to CD45 negative cells Immunization showed that it elicited a more effective additional immune response against the EG7 tumor, rather than just the OVA257 epitope (Figure 6k (Figure 6B)). These results indicate that K3-SPG monotherapy induces tumor cell death that is dependent on both IL-12 and IFN-I, which is an efficient immunogen for anti-tumor immune responses. As shown to function as.

(CD8 T cells are important effectors in K3-SPG-mediated tumor shrinkage)
The results of Rag2 mice showed that the tumor suppressive effect of K3-SPG is also dependent on the adaptive immune response. Therefore, we tested CD8 T cells required for K3-SPG treatment. In vivo CD8 T cell depletion markedly abrogated the antitumor effect of K3-SPG (FIG. 10a (FIG. 10A)), indicating that CD8 T cells are important effector cells in this K3-SPG treatment Yes. Tumor reduction by K3-SPG also depends on Batf3 (deficient in cross-presented CD8α ⁺ DC) (FIG. 10b (FIG. 10A)), and K3-SPG monotherapy is CD8α ⁺ DC-mediated crossover. It also shows that presentation has been enhanced. The inventors observed a clear link between tumor invasion of CD8 T cells and tumor growth. CD8 T cells are expressed as K3-SPG i. v. Accumulated in the tumor area in the group i. d. There was no accumulation in the group (FIG. 10c (FIG. 10A)).

Finally, we tested what is necessary for these CD8 T cells to enter the tumor area. WT and Il12p40-Ifnar2 DKO mice are inoculated with EG7 cells on day 0 and i.p. with K3-SPG or PBS on days 7, 9, and 11. v. Treated. On day 14, CD8α ⁺ T cells were purified from the spleens of these mice, stained with Xenlight DiR®, and K3-SPG treated (day 7, 9, and 11) another EG7 possessed The distribution of CD8 T cells labeled with Xenolight DiR® was analyzed by IVIS on day 15 (FIG. 11). On day 15, CD8 T cells from donor mice bearing untreated tumors did not accumulate at the tumor site of WT recipient mice even when treated with K3-SPG (FIG. 10d (FIG. 10B), II). On the other hand, CD8 T cells from donor mice bearing tumors treated with K3-SPG were detected at the tumor site of recipient mice (FIG. 10d (FIG. 10B), I), and K3-SPG monotherapy was We showed that we induced anti-tumor CD8 T cells that could migrate to and invade the tumor microenvironment. These in vivo activated CD8 T cells were able to enter the tumor microenvironment of DKO recipient mice (FIG. 10e (FIG. 10B)). Even though IL-12 and IFN-I are important for innate immunity and CD8 T cell induction by systemic K3-SPG monotherapy, this result indicates that CD8 T cells are activated by K3-SPG treatment Thus, it was shown that secretion of IL-12 and IFN-I cytokines in the tumor microenvironment is not necessarily required for tumor invasion of CD8 T cells. Taken together, these results indicated that tumor-specific CD8 T cell activation was sufficient for tumor invasion. Surprisingly, the infiltration of these CD8 T cells is independent of cytokine production in the tumor microenvironment.
(Discussion)

  The inventors have shown the potential for novel cancer immunotherapy. This is a new treatment where CpG is targeted to phagocytes in the tumor microenvironment (FIG. 12). Via stimulation of TLR9, CpG induces immune responses by immune cells, particularly activating 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 for the anti-cancer immune response. In previous reports, CpG had to be administered directly into the tumor, but the DPG function was added to the SPG and CpG complex, and systemic administration is as effective or better than intratumoral administration ( 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 present inventors have solved this problem. The complex of SPG and CpG due to the formation of nanoparticles (Kobiyama, K., et al. Proc. Natl. Acad. Sci. USA 111, 3086-3091 (2014)) can be stabilized in vivo. Met. It was found that this effect made it possible to target the tumor environment, and TLR9 immunocompetent cells were subjected to the tumor environment. This novel CpG developed by the inventors is phagocytosed by phagocytic cells to form nanoparticles.

  Thereafter, phagocytes phagocytosed with this novel CpG in the tumor environment produce cytokines such as IFN and IL-12. It is very important that these cytokines are induced in the tumor environment. Previous reports described that IFNβ treatment directly targeting the tumor environment reactivates CTL by moving dendritic cells into the tumor and increasing antigen cross-presentation within the tumor microenvironment. Has been. These cytokines cause tumor cell death. Furthermore, the present inventors have found that this effect is exerted by activation of innate immunity. This cell death plays a very important role. This was a link between innate and adaptive immunity. Tumor cell death is released from the tumor microenvironment, thereby inducing acquired immunity. This immunogenic tumor cell death induces multiple cytotoxic T lymphocytes. As described above, tumor-specific CTLs in vivo can invade the tumor microenvironment in response to tumors. This anti-tumor immune system may use endogenous antigens to address immune editing, a barrier to cancer immunotherapy.

  Tumor cell circulation after K3-SPG monotherapy can serve as an excellent biomarker for this treatment effect on tumors.

(Example 6: Formulation example)
Please tell me the composition when formulating. For example, 7.22 mg of K3-dA ₄₀ (SEQ ID NO: 2) is dissolved in water (3.7 mL), and SPG (15 mg) is dissolved in 0.25N 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 prepared by fixing at 0.27.

  The drugs used in the formulation can be obtained from Gene Design, Invivogen, Wako and others.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, it is understood that the scope of this invention should be construed only by the claims. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  The present invention provides a new form of an anticancer agent that can be used as a single agent. Therefore, the complex of the present invention is useful in the pharmaceutical field as an anticancer agent.

SEQ ID NO: 1 K3
SEQ ID NO: 2: K3-dA ₄₀
SEQ ID NO: _3: dA 40 -K3
SEQ ID NO: 4: K3-dA20
SEQ ID NO: 5: K3-dA25
SEQ ID NO: 6: K3-dA30
SEQ ID NO: 7: K3-dA35

Claims (23)

(A) an oligodeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylic acid, wherein the polydeoxyadenylic acid is located 3 ′ of the humanized K-type CpG oligodeoxynucleotide; Oligodeoxynucleotides;
(B) β-1,3-glucan
An anticancer agent containing a complex. The anticancer agent according to claim 1, wherein the anticancer agent is administered without a cancer antigen. The anticancer agent according to claim 1 or 2, wherein the anticancer agent is administered so as to be delivered to a reticuloendothelial system and / or a lymph node. The anticancer agent according to claim 3, wherein the reticuloendothelial system and / or lymph node includes tumor and phagocytic cells. The anticancer agent according to claim 3 or 4, wherein the reticuloendothelial system includes spleen and / or liver. The anticancer agent according to any one of claims 1 to 5, wherein the anticancer agent is administered without a cancer antigen. The anticancer agent according to any one of claims 2 to 6, wherein the administration comprises systemic administration. The anticancer agent according to claim 7, wherein the systemic administration is selected from intravenous administration, intraperitoneal administration, oral administration, subcutaneous administration, intramuscular administration, and intratumoral administration. The oligodeoxynucleotide is K3 (SEQ ID NO: 1), K3-dA. ₄₀ (SEQ ID NO: 2), dA ₄₀ -Selected from the group consisting of 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 anticancer agent according to any one of claims 1 to 8. 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 schizophyllan (SPG), lentinan, scleroglucan, curdlan, parkan, glyphoran, and laminaran. . The anticancer agent according to any one of claims 1 to 10, wherein the complex is K3-SPG. (A) an oligodeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylic acid, wherein the polydeoxyadenylic acid is located 3 ′ of the humanized K-type CpG oligodeoxynucleotide; Oligodeoxynucleotides;
(B) β-1,3-glucan
Containing complex
A composition for accumulating dead cells of cancer in the reticuloendothelial system and / or lymph nodes. The oligodeoxynucleotide is K3 (SEQ ID NO: 1), K3-dA. ₄₀ (SEQ ID NO: 2), dA ₄₀ -Selected from the group consisting of 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 claim 12. The composition according to claim 12 or 13, wherein the β-1,3-glucan is selected from the group consisting of schizophyllan (SPG), lentinan, scleroglucan, curdlan, parkan, glyphoran and laminaran. The composition according to any one of claims 12 to 14, wherein the complex is K3-SPG. 16. A composition according to any one of claims 12 to 15, wherein the reticuloendothelial system and / or lymph nodes comprise tumors and phagocytic cells. 17. A composition according to any one of claims 12 to 16, wherein the reticuloendothelial system comprises the spleen and / or liver. 18. A composition according to any one of claims 12 to 17, wherein the administration comprises systemic administration. 19. The composition of claim 18, wherein the systemic administration is selected from intravenous administration, intraperitoneal administration, oral administration, subcutaneous administration, intramuscular administration, and intratumoral administration. (A) an oligodeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylic acid, wherein the polydeoxyadenylic acid is located 3 ′ of the humanized K-type CpG oligodeoxynucleotide; Oligodeoxynucleotides;
(B) β-1,3-glucan
A composition for expression or promotion of interleukin 12 (IL12) and / or interferon (IFN) γ, comprising: The oligodeoxynucleotide is 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 as described. The composition according to claim 20 or 21, wherein the β-1,3-glucan is selected from the group consisting of schizophyllan (SPG), lentinan, scleroglucan, curdlan, parkan, glyphoran, and laminaran. The composition according to any one of claims 20 to 22, wherein the complex is K3-SPG.