CpG寡聚核苷酸(CpG ODN，CpG Oligonucleotide)係含有免疫賦活性之CpG模體之較短(約20個鹼基對)之單鏈之合成DNA(Deoxyribonucleic Acid，去氧核糖核酸)片段，且係類Toll受體9(TLR9，Toll-like receptor 9)之強力之促效劑，並且係作為將樹狀細胞(DCs，Dendritic Cells)及B細胞活化，產生I型干擾素(IFNs，Interferon type I)及炎症性細胞激素(非專利文獻1、2)，包括細胞毒殺性T淋巴球(CTL，Cytotoxic T Lymphocyte)反應在內之Th1型之體液性及細胞性免疫反應之佐劑而發揮作用(非專利文獻3、4)。因此，CpG ODN開始被視為針對感染症、癌、哮喘及花粉症具有可能性之免疫治療劑(非專利文獻2、5)。
存在骨架序列及免疫賦活特性分別不同之至少4個類型之CpG ODN(非專利文獻6)。D型(亦稱為A型)CpG ODN典型而言包含磷酸二酯(PO，Phosphodiester)骨架、硫代磷酸酯(PS，Phosphorothioate)聚G尾(Poly G tail)及1個迴文結構之CpG模體，將類漿細胞DCs(pDCs，Plasmacytoid dendritic cells)活化而產生大量IFN-α，但無法誘導pDC成熟化及B細胞活化(非專利文獻7、8)。其他3個類型之ODN包含PS骨架。K型(亦稱為B型)CpG ODN典型而言含有非迴文結構之複數個CpG模體，強力地將B細胞活化而產生IL-6，將pDCs活化而使之成熟化，但幾乎不產生IFN-α(非專利文獻8、9)。近年來，業界開發出之C型及P型之CpG ODN分別含有1個及2個迴文結構CpG序列，兩者均可如K型般使B細胞活化，且如D型般使pDCs活化，但與P型CpG ODN相比，C型CpG ODN更弱地誘導IFN-α產生(非專利文獻10-12)。於專利文獻1中，記載有大量優異之K型CpG ODN。
揭示有D型及P型CpG ODN分別形成如下高次結構：形成稱為G-tetrads(G-四聯體)之平行四鏈結構之胡格斯丁鹼基對(Hoogsteen base pair)、及順式迴文結構部位與反式迴文結構部位間之華特生-克里克(Watson-Crick)鹼基對，該等對於因pDCs所引起之強力之IFN-α產生而言為必須(非專利文獻12～14)。此種高次結構似乎為向早期內體(early endosome)中之定位或經由TLR9之訊息傳遞所必須，但該等受到產物之多樣性及沈澱之影響，結果妨礙其臨床應用(非專利文獻15)。因此，通常僅K型及C型CpG ODN可用作人類用之免疫治療劑及疫苗佐劑(非專利文獻16及17)。關於K型CpG ODN，於人類臨床試驗中係提高以感染症及癌為標靶之疫苗之免疫原性(immunogenicity)(非專利文獻6、16)，但為了實現最佳之佐劑效果，需要抗原與K型CpG ODN間之化學性及物理性之連結。該等結果顯示，4個類型(K、D、P、及C)之CpG ODN存在優點及缺點，而期待開發出在不進行聚集之情況下將B細胞及pDCs兩者活化之「一體化(all in one)」之CpG ODN。
作為來自裂褶菌(Schizophyllum commune)之可溶性β-1,3-葡聚糖之裂褶菌多糖(SPG(Schizophyllan))係作為針對宮頸癌患者之放射線療法之賦活藥而於最近30年於日本被許可之醫藥(非專利文獻18)。同樣地，作為來自香菇之可溶性β-1,3-葡聚糖之香菇多糖(LNT，Lentinan)係於1985年被認可之醫藥，藉由與氟嘧啶系藥劑併用而對無法手術及胃癌復發患者使用(非專利文獻19、20)。揭示有β-1,3-葡聚糖會與聚去氧腺苷酸(dA)形成三股螺旋結構之複合體(非專利文獻21)。
專利文獻2～4中揭示有，包含裂褶菌多糖之β-1,3-葡聚糖與核酸(基因)之水溶性複合體作為基因載體之用途。該等文獻中記載有藉由形成該複合體，可提高基因之反義作用及對核酸分解酵素(核酸酶)之耐性作用。
專利文獻5中揭示有，藉由將具有β-1,3-鍵之多糖類用作載體(轉染劑)，可提高包含CpG序列且將磷酸二酯鍵取代為硫代磷酸酯鍵或二硫代磷酸酯鍵之免疫刺激性寡聚核苷酸之作用。
專利文獻6中記載有一種免疫刺激性複合體，其特徵在於：包含免疫刺激性寡聚核苷酸、及具有長鏈之β-1,6-葡糖苷鍵側鏈之β-1,3-葡聚糖。
本發明者等人先前揭示有：與SPG進行複合體化而成之連結有於5'末端具有磷酸二酯鍵之聚(dA)的小鼠及人類化之CpG ODN會增強細胞激素產生，而作為流行性感冒疫苗佐劑或Th2細胞相關疾病之預防或治療劑發揮作用(非專利文獻22、23、專利文獻7)。若於K型及D型之各自CpG之5'末端附加聚(dA)，與SPG形成複合體，則分別維持K型及D型之特性，並且其活性得以增強。然而，難以實現更有效、成本效益(cost effectiveness)較高、且面向臨床前期及臨床開發之CpG-SPG複合體之高產率。近年來，揭示有若使CpG ODN與具有硫代磷酸酯鍵之聚(dA)進行連結，則複合體之形成上升至將近100%(非專利文獻24)。然而，尚未進行用於對最佳之人類化CpG序列進行鑑定，使用以獲得4個類型之CpG ODN之「一體化」活性的因子進行最佳化之周密試驗。
專利文獻8中揭示有，抗原/CpG寡聚核苷酸/β-1,3-葡聚糖系之三元複合體之製造方法。
作為類Toll受體9(TLR9)之配位子的合成核酸CpG寡聚去氧核苷酸(CpG ODN)具有較強之自然免疫活化能力，而作為疫苗佐劑備受期待。又，由於在以單劑形式投予時具有抗腫瘤活性，故而CpG ODN亦作為針對癌之免疫治療劑而備受期待。然而，先前之CpG ODN雖然具有抗腫瘤活性，但僅可藉由向腫瘤直接投予才會發揮效果，認為難以應用於臨床。實際上，認為難以於臨床現場向初始階段之腫瘤直接投予藥劑。又，於深部亦需要外科處理，其障礙較高。
最近，本發明者等人進行有利用多糖之β-葡聚糖包裹CpG ODN之新穎TLR9之配位子(K3-SPG)之開發(PCT申請(PCT/JP2014/074835)))。K3-SPG不會形成凝聚塊，與先前型之CpG ODN相比，強力地將自然免疫活化，同時藉由使用小鼠之實驗而顯示出較強之佐劑效果。進而明確K3-SPG不僅對小鼠，亦對食蟹獼猴誘導較強之後天性免疫(acquired immunity)，成功克服了以往所擔憂之小鼠與靈長類體內之反應性之差異。
如此，該CpG ODN作為佐劑之用途雖然備受期待，但尚不明確是否可單獨用作醫藥。
就癌治療而言，自1991年鑑定出細胞毒殺性T細胞所識別之癌相關抗原並進行報告以來(非專利文獻25：van der Bruggen et al. Science (New York, N. Y.) 254, 1643-1647 (1991))，非常多之癌相關抗原以分子水平得以鑑定，而實現以該等作為標靶之癌免疫療法之臨床應用(非專利文獻26：Jager, E., et al. The Journal of experimental medicine 187, 265-270 (1998)；非專利文獻27：Jager, D. et al. Journal of clinical pathology 54, 669-674 (2001).；非專利文獻28：Imai, K., et al. British journal of cancer 104, 300-307 (2011)；非專利文獻29：Kang, X., et al. The Journal of Immunology 155, 1343-1348 (1995))。尤其是令人關注之癌免疫療法，係於2010年4月針對攝護腺癌患者，首次受到美國食品及藥物管理局(FDA，Food and Drug Administration)之認可，使用自體末梢血液之抗原呈現細胞之癌疫苗Provenge(非專利文獻30：Cancer vaccine approval could open floodgates. Nature medicine 16, 615-615 (2010)；非專利文獻31：Higano, C. S., et al. Cancer 115, 3670-3679 (2009))。其後，關於針對作為T淋巴細胞之活化之抑制性分子的細胞毒殺性T淋巴球抗原4(CTLA-4，Cytotoxic T Lymphocyte Antigen 4)之抑制性抗體伊匹單抗(ipilimumab)，2011年5月於美國針對惡性黑色素瘤患者被認可(非專利文獻32：Phan, G. Q., et al. Proc. Natl. Acad. Sci. U. S. A. 100, 8372-8377 (2003)；非專利文獻33：Camacho, L. H., et al. Journal of clinical oncology: official journal of the American Society of Clinical Oncology 27, 1075-1081 (2009)；非專利文獻34：Hodi, F. S., et al. New England Journal of Medicine 363, 711-723 (2010))。進而，作為免疫反應抑制因子之PD-1(Programed Cell Death-1，計畫性細胞死亡1)受體抑制劑之nivolumab處於臨床試驗階段(非專利文獻35：AZIJLI, K., et al. Anticancer Research 34, 1493-1505 (2014)；非專利文獻36：Okazaki, T., et al. Nature immunology 14, 1212-1218 (2013)；非專利文獻37：Ishida, Y., et al. The EMBO journal 11, 3887-3895 (1992)；非專利文獻38：Topalian, S. L., et al. The New England journal of medicine 366, 2443-2454 (2012))。
對於樹狀細胞有效地引導出抗癌效應之環境之形成，需要顯示出於炎症部之抗癌作用的自然免疫之模式分子(pattern molecule)(非專利文獻39：Chiba, S., et al. Nature immunology 13, 832-842 (2012))。上述分子群及與其相關之過程雖未經特定，但樹狀細胞會將CD4、CD8、及NK等淋巴球活化(非專利文獻40：Engelhardt, J. J., et al. Cancer cell 21, 402-417 (2012))。腫瘤浸潤巨噬細胞(主要相關巨噬細胞(TAM，Tumor Associated Macrophages))係作為炎症反應之元兇而為人所知(非專利文獻41：Huang, Y., et al. Cancer cell 19, 1-2 (2011))。另一方面，作為抗癌免疫之主導者(playmaker)而發揮功能之樹狀細胞亦為與巨噬細胞相同之骨髄細胞(非專利文獻42：Huang, Y., et al. Proc. Natl. Acad. Sci. U. S. A. 109, 17561-17566 (2012))。雖然將該等轉變為抗癌指向性之方法尚未找到，但認為腫瘤已因複雜之因素而逃避免疫。TAM及樹狀細胞之兩者係由炎症及模式識別應答支配(非專利文獻43：Garaude, J., et al. Science translational medicine 4, 120ra116 (2012)；非專利文獻44：Martinez-Pomares, L. et al. Trends in immunology 33, 66-70 (2012))。免疫學之效應細胞與癌細胞接觸係癌細胞之攻擊所必須(非專利文獻40：Engelhardt, J. J., et al. Cancer cell 21, 402-417 (2012)；非專利文獻45：Palucka, K. et al. Nature reviews. Cancer 12, 265-277 (2012))。
[先前技術文獻]
[專利文獻]
[專利文獻1] US 8,030,285 B2
[專利文獻2] WO 01/034207 A1
[專利文獻3] WO 02/072152 A1
[專利文獻4]日本專利特開2004-107272號公報
[專利文獻5] WO 2004/100965 A1
[專利文獻6]日本專利特開2007-70307號公報
[專利文獻7]日本專利特開2008-100919號公報
[專利文獻8]日本專利特開2010-174107號公報
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[非專利文獻19] Oba, K. et al., J. Individual patient based meta-analysis of lentinan for unresectable/recurrent gastric cancer. Anticancer Res., 2009, 29, 2739-2746.
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[非專利文獻21] Sakurai, K., et al., Biomacromolecules 2, 641-650 (2001).
[非專利文獻22] Shimada, N., et al. Bioconjugate chemistry 18, 1280-1286 (2007).
[非專利文獻23] Koyama, S., et al. Science translational medicine 2, 25ra24 (2010).
[非專利文獻24] Minari, J., et al. Bioconjugate chemistry 22, 9-15 (2011).
[非專利文獻25] van der Bruggen et al. Science (New York, N. Y.) 254, 1643-1647 (1991)
[非專利文獻26] Jager, E., et al. The Journal of experimental medicine 187, 265-270 (1998)
[非專利文獻27] Jager, D. et al. Journal of clinical pathology 54, 669-674 (2001).
[非專利文獻28] Imai, K., et al. British journal of cancer 104, 300-307 (2011)
[非專利文獻29] Kang, X., et al. The Journal of Immunology 155, 1343-1348 (1995)
[非專利文獻30] Cancer vaccine approval could open floodgates. Nature medicine 16, 615-615 (2010)
[非專利文獻31] Higano, C. S., et al. Cancer 115, 3670-3679 (2009)
[非專利文獻32] Phan, G. Q., et al. Proc. Natl. Acad. Sci. U. S. A. 100, 8372-8377 (2003)
[非專利文獻33] Camacho, L. H., et al. Journal of clinical oncology: official journal of the American Society of Clinical Oncology 27, 1075-1081 (2009)
[非專利文獻34] Hodi, F. S., et al. New England Journal of Medicine 363, 711-723 (2010)
[非專利文獻35] AZIJLI, K., et al. Anticancer Research 34, 1493-1505 (2014)
[非專利文獻36] Okazaki, T., et al. Nature immunology 14, 1212-1218 (2013)
[非專利文獻37] Ishida, Y., et al. The EMBO journal 11, 3887-3895 (1992)
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[非專利文獻40] Engelhardt, J. J., et al. Cancer cell 21, 402-417 (2012)
[非專利文獻41] Huang, Y., et al. Cancer cell 19, 1-2 (2011)
[非專利文獻42] Huang, Y., et al. Proc. Natl. Acad. Sci. U. S. A. 109, 17561-17566 (2012)
[非專利文獻43] Garaude, J., et al. Science translational medicine 4, 120ra116 (2012)
[非專利文獻44] Martinez-Pomares, L. et al. Trends in immunology 33, 66-70 (2012)
[非專利文獻45] Palucka, K. et al. Nature reviews. Cancer 12, 265-277 (2012)CpG ODN (CpG ODN, CpG Oligonucleotide) is a short (about 20 base pairs) single-stranded synthetic DNA (Deoxyribonucleic Acid) fragment containing immunoactive CpG motifs, It is a powerful agonist of Toll-like receptor 9 (TLR9, Toll-like receptor 9), and is used to activate dendritic cells (DCs, Dendritic Cells) and B cells to produce type I interferons (IFNs, Interferon Type I) and inflammatory cytokines (Non-patent Documents 1 and 2), including the cytotoxic T lymphocyte (CTL, Cytotoxic T Lymphocyte) reaction, including Th1 type humoral and cellular immune response adjuvants Function (Non-Patent Documents 3 and 4). Therefore, CpG ODN is beginning to be considered as an immunotherapeutic agent against infections, cancer, asthma, and hay fever (Non-Patent Documents 2 and 5).
There are at least four types of CpG ODNs with different backbone sequences and immune activation properties (Non-Patent Document 6). Type D (also known as type A) CpG ODN typically contains a phosphodiester (PO, Phosphodiester) skeleton, phosphorothioate (PS, Phosphorothioate) poly G tail (Poly G tail) and a palindrome structure CpG The phantom activates plasma-like cell DCs (pDCs, Plasmacytoid dendritic cells) to produce a large amount of IFN-α, but fails to induce pDC maturation and B cell activation (Non-Patent Documents 7 and 8). The other three types of ODN include PS skeleton. K-type (also known as B-type) CpG ODN typically contains multiple CpG motifs of non-palindrome structure, which strongly activates B cells to produce IL-6 and activates pDCs to mature them, but hardly IFN-α is produced (Non-Patent Documents 8 and 9). In recent years, CpG ODNs of type C and type P developed by the industry contain one and two palindrome CpG sequences, both of which can activate B cells like K type and pDCs like D type. However, C-type CpG ODN induces IFN-α production more weakly than P-type CpG ODN (Non-Patent Documents 10-12). Patent Document 1 describes a large number of excellent K-type CpG ODNs.
It is revealed that D-type and P-type CpG ODNs respectively form the following higher-order structures: forming a Hoogsteen base pair (Hoogsteen base pair) of parallel four-strand structure called G-tetrads (G-quadruplex), and cis The Watson-Crick base pair between the palindrome structural part and the trans-palindrome structural part, these are necessary for the strong IFN-α production caused by pDCs (not Patent Literature 12-14). This higher-order structure seems to be necessary for positioning in the early endosome or the transmission of information via TLR9, but these are affected by the diversity of products and Shen Dian, which hinders its clinical application (Non-Patent Document 15 ). Therefore, generally only K-type and C-type CpG ODNs can be used as immunotherapeutics and vaccine adjuvants for humans (Non-Patent Documents 16 and 17). Regarding K-type CpG ODN, it is necessary to improve the immunogenicity of vaccines targeting infectious diseases and cancer in human clinical trials (Non-Patent Documents 6 and 16), but in order to achieve the best adjuvant effect, it is necessary The chemical and physical connection between antigen and K-type CpG ODN. These results show that the 4 types (K, D, P, and C) of CpG ODN have advantages and disadvantages, and it is expected to develop a "integration ("integration ( all in one)" CpG ODN.
As a soluble β-1,3-glucan-soluble Schizophyllan polysaccharide (SPG (Schizophyllan)) from Schizophyllum commune, it has been used as an active agent for radiotherapy for cervical cancer patients in Japan in the past 30 years Licensed medicine (Non-Patent Document 18). Similarly, Lentinan (LNT, Lentinan), which is soluble β-1,3-glucan from Lentinula edodes, is a recognized medicine in 1985. It is used in combination with fluoropyrimidine-based agents in patients with inoperable and gastric cancer relapse Use (Non-Patent Documents 19 and 20). It was revealed that β-1,3-glucan and polydeoxyadenylic acid (dA) form a triple-helix structure complex (Non-Patent Document 21).
Patent Documents 2 to 4 disclose the use of a water-soluble complex of β-1,3-glucan and nucleic acid (gene) containing Schizophyllum polysaccharide as a gene carrier. These documents describe that by forming this complex, the antisense effect of genes and the resistance to nucleolytic enzymes (nucleases) can be improved.
Patent Document 5 discloses that by using a polysaccharide having a β-1,3-bond as a carrier (transfection agent), it is possible to increase the CpG sequence and replace the phosphodiester bond with a phosphorothioate bond or di The role of phosphorothioate linkage immunostimulatory oligonucleotides.
Patent Document 6 describes an immunostimulatory complex, which is characterized by comprising an immunostimulatory oligonucleotide and β-1,3- having a long-chain β-1,6-glucosidic bond side chain Dextran.
The present inventors have previously revealed that mice and humanized CpG ODN which are formed by complexing with SPG and linked with a poly(dA) having a phosphodiester bond at the 5′ end enhance cytokine production, and It acts as an adjuvant for influenza vaccine or as a preventive or therapeutic agent for Th2 cell-related diseases (Non-Patent Documents 22 and 23, Patent Document 7). If poly (dA) is added to the 5'ends of the respective CpGs of K-type and D-type to form a complex with SPG, the characteristics of K-type and D-type are maintained, and their activities are enhanced. However, it is difficult to achieve a high-yield CpG-SPG complex that is more effective, has higher cost effectiveness, and is oriented toward preclinical and clinical development. In recent years, it has been revealed that if CpG ODN is linked to poly(dA) having a phosphorothioate bond, the formation of the complex increases to nearly 100% (Non-Patent Document 24). However, no detailed tests have been conducted to identify the optimal humanized CpG sequences, using factors to obtain the "integrated" activity of the 4 types of CpG ODN.
Patent Document 8 discloses a method for producing a ternary complex of antigen/CpG oligonucleotide/β-1,3-glucan.
The synthetic nucleic acid CpG oligodeoxynucleotide (CpG ODN), which is a ligand for Toll-like receptor 9 (TLR9), has a strong natural immune activation ability, and is expected as a vaccine adjuvant. In addition, since it has antitumor activity when administered as a single dose, CpG ODN is also expected as an immunotherapeutic agent against cancer. However, although the previous CpG ODN has anti-tumor activity, it can only exert its effect by direct administration to the tumor, and it is considered difficult to apply to clinical practice. In fact, it is considered difficult to directly administer the drug to the initial stage tumor at the clinical site. In addition, surgical treatment is also required in the deep part, and its obstacles are high.
Recently, the present inventors have developed a novel TLR9 ligand (K3-SPG) that encapsulates CpG ODN with β-glucan of polysaccharide (PCT application (PCT/JP2014/074835))). K3-SPG does not form agglomerates. Compared with the previous type of CpG ODN, it strongly activates natural immunity, and shows a stronger adjuvant effect through experiments using mice. It was further clarified that K3-SPG not only induced strong immunity to mice but also to cynomolgus macaques, successfully overcoming the difference in reactivity between mice and primates in the past.
As such, although the use of the CpG ODN as an adjuvant is highly anticipated, it is not yet clear whether it can be used alone as a medicine.
For cancer treatment, since the cancer-associated antigen recognized by cytotoxic T cells was 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-associated antigens have been identified at the molecular level, and the clinical application of cancer immunotherapy using these as targets has been achieved (Non-Patent Document 26: Jager, E., et al. The Journal of experimental medicine 187, 265-270 (1998); non-patent document 27: Jager, D. et al. Journal of clinical pathology 54, 669-674 (2001).; non-patent document 28: Imai, K., et al. British journal of cancer 104, 300-307 (2011); Non-Patent Document 29: Kang, X., et al. The Journal of Immunology 155, 1343-1348 (1995)). In particular, the cancer immunotherapy, which was of concern, was the first to be approved by the US Food and Drug Administration (FDA, Food and Drug Administration) in April 2010 for patients with prostate cancer. Provenge (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, regarding the inhibitory antibody ipilimumab (CTLA-4, Cytotoxic T Lymphocyte Antigen 4), which is an inhibitory molecule for activation of T lymphocytes, ipilimumab (2011) Recognized in the United States 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 Literature 34: Hodi, FS, et al. New England Journal of Medicine 363, 711-723 (2010 )). Furthermore, nivolumab, a PD-1 (Programed Cell Death-1) receptor inhibitor that is an immune response inhibitor, is in clinical trials (Non-Patent Document 35: AZIJLI, K., et al. Anticancer Research 34, 1493-1505 (2014); Non-patent document 36: Okazaki, T., et al. Nature immunology 14, 1212-1218 (2013); Non-patent document 37: Ishida, Y., et al. The EMBO journal 11, 3887-3895 (1992); Non-Patent Document 38: Topalian, SL, et al. The New England journal of medicine 366, 2443-2454 (2012)).
For the formation of an environment in which dendritic cells effectively guide the anticancer effect, it is necessary to show a pattern molecule of natural immunity due to the anticancer effect of the inflammatory part (Non-Patent Document 39: Chiba, S., et al. Nature immunology 13, 832-842 (2012)). Although the above molecular groups and the processes related to them are not specified, dendritic cells will 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 (mainly associated macrophages (TAM, Tumor Associated Macrophages)) are known as the culprit of the inflammatory response (Non-Patent Document 41: Huang, Y., et al. Cancer cell 19, 1- 2 (2011)). On the other hand, the dendritic cells functioning as the leader of anti-cancer immunity (playmaker) are also bone marrow cells similar to macrophages (Non-Patent Document 42: Huang, Y., et al. Proc. Natl. Acad . Sci. USA 109, 17561-17566 (2012)). Although there is no way to turn these into anti-cancer directivity, it is believed that the tumor has escaped immunity due to complex factors. Both TAM and dendritic cells are dominated by inflammation and pattern recognition response (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)). Immunological effector cells are in contact with cancer cells to attack 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)).
[Prior Technical Literature]
[Patent Literature]
[Patent Literature 1] US 8,030,285 B2
[Patent Document 2] WO 01/034207 A1
[Patent Literature 3] WO 02/072152 A1
[Patent Document 4] Japanese Patent Laid-Open No. 2004-107272
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[Patent Document 6] Japanese Patent Laid-Open No. 2007-70307
[Patent Document 7] Japanese Patent Laid-Open No. 2008-100919
[Patent Document 8] Japanese Patent Laid-Open No. 2010-174107
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[Non-Patent Document 2] Krieg, A. M. Nature reviews. Drug discovery 5, 471-484 (2006).
[Non-Patent Document 3] Brazolot Millan, C. L., et al., Proceedings of the National Academy of Sciences of the United States of America 95, 15553-15558 (1998).
[Non-Patent Document 4] Chu, R. S., et al., The Journal of experimental medicine 186, 1623-1631 (1997).
[Non-Patent Literature 5] Klinman, D. M. Nature reviews. Immunology 4, 249-258 (2004).
[Non-Patent Document 6] Vollmer, J. & Krieg, A. M. Advanced drug delivery reviews 61, 195-204 (2009).
[Non-Patent Document 7] Krug, A., et al. European journal of immunology 31, 2154-2163 (2001).
[Non-Patent Document 8] Verthelyi, D., et al., Journal of immunology 166, 2372-2377 (2001).
[Non-Patent Document 9] Hartmann, G. & Krieg, A. M. Journal of immunology 164, 944-953 (2000).
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[Non-Patent Document 16] Bode, C., et al., Expert review of vaccines 10, 499-511 (2011).
[Non-Patent Document 17] McHutchison, J. G., et al. Hepatology 46, 1341-1349 (2007).
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[Non-Patent Document 22] Shimada, N., et al. Bioconjugate chemistry 18, 1280-1286 (2007).
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[Non-Patent Document 25] van der Bruggen et al. Science (New York, N. Y.) 254, 1643-1647 (1991)
[Non-Patent Document 26] Jager, E., et al. The Journal of experimental medicine 187, 265-270 (1998)
[Non-Patent Literature 27] Jager, D. et al. Journal of clinical pathology 54, 669-674 (2001).
[Non-Patent Document 28] Imai, K., et al. British journal of cancer 104, 300-307 (2011)
[Non-Patent Document 29] Kang, X., et al. The Journal of Immunology 155, 1343-1348 (1995)
[Non-Patent Document 30] Cancer vaccine approval could open floodgates. Nature medicine 16, 615-615 (2010)
[Non-Patent Document 31] Higano, C. S., et al. Cancer 115, 3670-3679 (2009)
[Non-Patent Document 32] Phan, G. Q., et al. Proc. Natl. Acad. Sci. U. S. A. 100, 8372-8377 (2003)
[Non-Patent Literature 33] Camacho, L. H., et al. Journal of clinical oncology: official journal of the American Society of Clinical Oncology 27, 1075-1081 (2009)
[Non-Patent Document 34] Hodi, F. S., et al. New England Journal of Medicine 363, 711-723 (2010)
[Non-Patent Document 35] AZIJLI, K., et al. Anticancer Research 34, 1493-1505 (2014)
[Non-Patent Document 36] Okazaki, T., et al. Nature immunology 14, 1212-1218 (2013)
[Non-Patent Literature 37] Ishida, Y., et al. The EMBO journal 11, 3887-3895 (1992)
[Non-Patent Document 38] Topalian, S. L., et al. The New England journal of medicine 366, 2443-2454 (2012)
[Non-Patent Document 39] Chiba, S., et al. Nature immunology 13, 832-842 (2012)
[Non-Patent Document 40] Engelhardt, J. J., et al. Cancer cell 21, 402-417 (2012)
[Non-Patent Literature 41] Huang, Y., et al. Cancer cell 19, 1-2 (2011)
[Non-Patent Document 42] Huang, Y., et al. Proc. Natl. Acad. Sci. U. S. A. 109, 17561-17566 (2012)
[Non-Patent Document 43] Garaude, J., et al. Science translational medicine 4, 120ra116 (2012)
[Non-Patent Document 44] Martinez-Pomares, L. et al. Trends in immunology 33, 66-70 (2012)
[Non-Patent Document 45] Palucka, K. et al. Nature reviews. Cancer 12, 265-277 (2012)
以下，一面揭示最佳形態，一面對本發明進行說明。於本說明書之全文中，單數形式之表述只要未特別說明，則應理解為亦包括其複數形式之概念。因此，單數形式之冠詞(例如於英語之情形時，為「a」、「an」、「the」等)只要未特別說明，則應理解為亦包括其複數形式之概念。又，本說明書中所使用之用語只要未特別說明，則應理解為基於該領域中通常使用之含義而使用。因此，此外只要未加以定義，則本說明書中所使用之所有專門用語及科學技術用語具有與本發明之所屬領域之從業者通常理解者相同之含義。於發生矛盾之情形時，以本說明書(包括定義)為準。
以下，適當說明本說明書中特別使用之用語之定義及/或基本技術內容。
本發明提供一種包含K型CpG寡聚去氧核苷酸及聚去氧腺苷酸(dA)之寡聚去氧核苷酸(以下，稱為本發明之寡聚去氧核苷酸)。於本發明之寡聚去氧核苷酸中包含磷酸二酯鍵經修飾(例如一部分或全部磷酸二酯鍵經硫代磷酸酯鍵取代)者。本發明之寡聚去氧核苷酸包含藥學上可容許之鹽。
於本說明書中，所謂「CpG寡聚核苷酸(殘基)」或「CpG寡聚去氧核苷酸(殘基)」、「CpG ODN(殘基)」或僅「CpG(殘基)」係指包含可用於交換使用且至少1個未經甲基化之CG二核苷酸序列之多核苷酸、較佳為寡聚核苷酸，無論有無末尾之用語「殘基」，含義均相同。包含至少1個CpG模體之寡聚核苷酸可包含複數個CpG模體。於本說明書中使用之情形時，所謂用語「CpG模體」係指包含胞嘧啶核苷酸及其後之鳥苷核苷酸的寡聚核苷酸之未經甲基化之二核苷酸部分。亦可使用5-甲基胞嘧啶代替胞嘧啶。進而，聚去氧腺苷酸與聚去氧腺苷酸(殘基)含義相同。用語「殘基」係指更大分子量之化合物之部分結構，於本說明書中，「CpG寡聚去氧核苷酸(CpG ODN)」意指獨立之分子、或意指更大分子量之化合物之部分結構，只要為從業者，則可由上下文容易地理解。關於「聚去氧腺苷酸」等與本發明之寡聚去氧核苷酸中所含之其他部分結構相關之用語亦相同。
CpG寡聚核苷酸(CpG ODN)係含有免疫賦活性之CpG模體的較短(約20個鹼基對)之單鏈之合成DNA片段，且係類Toll受體9(TLR9)之強力之促效劑，係作為將樹狀細胞(DCs)及B細胞活化，產生I型干擾素(IFNs)及炎症性細胞激素(Hemmi， H., et al. Nature 408, 740-745(2000); Krieg, A. M. Nature reviews. Drug discovery 5, 471-484(2006).)，包括細胞毒殺性T淋巴球(CTL)反應在內之Th1型之體液性及細胞性免疫反應之佐劑而發揮作用(Brazolot Millan, C. L., Weeratna, R., Krieg, A. M., Siegrist, C. A. ＆ Davis, H. L. Proceedings of the National Academy of Sciences of the United States of America 95, 15553-15558(1998).; Chu, R.S., Targoni, O.S., Krieg, A. M., Lehmann, P. V. ＆ Harding, C. V. The Journal of experimental medicine 186, 1623-1631(1997))。因此，CpG ODN被視為對感染症、癌、哮喘及花粉症具有可能性之免疫治療劑(Krieg, A. M. Nature reviews. Drug discovery 5, 471-484(2006); Klinman, D. M. Nature reviews. Immunology 4, 249-258(2004))。
CpG寡聚去氧核苷酸(CpG ODN)係含有免疫賦活性之非甲基化CpG模體之單鏈DNA，為TLR9之促效劑。CpG ODN存在骨架序列及免疫賦活特性分別不同之K型(亦稱為B型)、D型(亦稱為A型)、C型及P型之4個類型(Advanced drug delivery reviews 61, 195-204 (2009))。本發明之寡聚去氧核苷酸包含該等中之K型CpG ODN。
K型CpG ODN係典型而言含有非迴文結構之複數個非甲基化CpG模體，將B細胞活化而產生IL-6，但具有幾乎不誘導類漿細胞樹狀細胞(pDCs)之IFN-α產生之結構及功能特性之CpG ODN。所謂非甲基化CpG模體係包含至少1個胞嘧啶(C)-鳥嘌呤(G)序列之較短之核苷酸序列，係指該胞嘧啶-鳥嘌呤序列中之胞嘧啶之5位未經甲基化者。再者，於以下之說明中，所謂CpG，只要未特別說明，則係指非甲基化CpG。因此，本發明之寡聚去氧核苷酸藉由包含K型CpG ODN，而具有對K型CpG ODN特有之免疫賦活活性(例如將B細胞(較佳為人類B細胞)活化而產生IL-6之活性)。於該技術領域中，已知有大量人類化K型CpG ODN(Journal of immunology 166, 2372-2377 (2001); Journal of immunology 164, 944-953 (2000); US 8,030,285 B2)。
本發明之寡聚去氧核苷酸中所含之K型CpG ODN較佳為經人類化。所謂「人類化」係指具有針對人類TLR9之促效劑活性。因此，包含人類化K型CpG ODN之本發明之寡聚去氧核苷酸對人類具有對K型CpG ODN特有之免疫賦活活性(例如將人類B細胞活化而產生IL-6之活性)。於本發明中可適宜地使用之K型CpG ODN為10個核苷酸以上之長度，且包含式：
[化1]
(式中，中央之CpG模體未經甲基化，W為A或T，N1
、N2
、N3
、N4
、N5
及N6
亦可為任何核苷酸)所表示之核苷酸序列。
於一實施形態中，本發明之K型CpG ODN係具有10個核苷酸以上之長度且包含上述式之核苷酸序列。其中，上述式中，中央之4個鹼基之CpG模體(TCpGW)只要包含在10個核苷酸中即可，並非必須於上述式中位於N3
及N4
之間。又，上述式中，N1
、N2
、N3
、N4
、N5
及N6
可為任何核苷酸，N1
及N2
、N2
及N3
、N3
及N4
、N4
及N5
、以及N5
及N6
之至少任一個(較佳為一個)組合亦可為2個鹼基之CpG模體。於上述4個鹼基之CpG模體未位於N3
及N4
間之情形時，上述式中，中央之4個鹼基(第4～7個鹼基)中之連續之任兩個鹼基為CpG模體，其他2個鹼基亦可為任何核苷酸。
本發明中可更適宜地使用之K型CpG ODN包含含有1個或複數個CpG模體(motif)之非迴文結構。可更適宜地使用之K型CpG ODN包含含有1個或複數個CpG模體之非迴文結構。
人類化K型CpG ODN通常係以包含TCGA或TCGT之4個鹼基之CpG模體作為特徵。又，多數情況下，於1個人類化K型CpG ODN中包含2或3個該4個鹼基之CpG模體。因此，於較佳實施形態中，本發明之寡聚去氧核苷酸中所含之K型CpG ODN包含至少1個、更佳為2以上、進而較佳為2或3個包含TCGA或TCGT之4個鹼基之CpG模體。於該K型CpG ODN具有2或3個之4個鹼基之CpG模體之情形時，該等4個鹼基之CpG模體可相同亦可不同。其中，只要具有針對人類TLR9之促效劑活性，則無特別限定。
本發明之寡聚去氧核苷酸中所含之K型CpG ODN更佳為包含序列編號1所表示之核苷酸序列。
關於K型CpG ODN之長度，只要本發明之寡聚去氧核苷酸具有免疫賦活活性(例如將B細胞(較佳為人類B細胞)活化而產生IL-6之活性)，則無特別限定，較佳為100個核苷酸長度以下(例如10～75個核苷酸長度)。K型CpG ODN之長度更佳為50個核苷酸長度以下(例如10～40核苷酸長度)。K型CpG ODN之長度進而較佳為30個核苷酸長度以下(例如10～25核苷酸長度)。K型CpG ODN之長度最佳為12～25核苷酸長度。
關於聚去氧腺苷酸(dA)之長度，只要為對於與β-1,3-葡聚糖(較佳為香菇多糖、或裂褶菌多糖)鏈一併形成三股螺旋結構而言充分之長度，則無特別限定，就形成穩定之三股螺旋結構之觀點而言，通常為20個核苷酸長度以上，較佳為40個核苷酸長度以上，更佳為60個核苷酸長度以上。由於聚dA越長越會與β-1,3-葡聚糖形成穩定之三股螺旋結構，故而理論上並無上限，但若過長，則於合成寡聚去氧核苷酸時會於長度方面產生不均，故而通常為100個核苷酸長度以下，較佳為80個以下。另一方面，除了形成上述穩定之三股螺旋結構以外，就增大與每單位量之β-1,3-葡聚糖鍵結之本發明之寡聚去氧核苷酸量且避免合成寡聚去氧核苷酸時之長度不均、複合化效率之觀點而言，聚dA之長度較佳為20～60個核苷酸長度(具體而言為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或60個核苷酸長度)，更佳為30～50個核苷酸長度(30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50個核苷酸長度)18，最佳為30～45個核苷酸長度(30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45個核苷酸長度)。尤其為30個核苷酸長度以上之情形時，顯示出良好之複合化效率。本發明之寡聚去氧核苷酸藉由包含聚dA，而具有與2條裂褶菌多糖鏈一併形成三股螺旋結構之活性。再者，亦存在將聚去氧腺苷酸表述為「聚(dA)」或「poly(dA)」之情形。
於1分子之本發明之寡聚去氧核苷酸中，亦可含有複數個K型CpG ODN及/或聚dA，較佳為含有K型CpG ODN及聚dA各1個，最佳為包含K型CpG ODN及聚dA各1個。
作為例示性之CpG之序列，可列舉K3 CpG(序列編號1：5'-atcgactctcgagcgttctc-3')等，但並不限定於此。
本發明之寡聚去氧核苷酸之特徵在於：聚dA係配置於K型CpG ODN之3'側。認為藉由該配置，本發明之複合體(詳細情況如下所述)之抗癌作用亦有可能增強，但並不限定於該等，亦可作為抗癌劑而與任一者結合。
K型CpG ODN與聚dA可藉由直接共價鍵進行連結，亦可經由間隙子序列進行連結。所謂間隙子序列係指***至2個接近之構成要素間之包含1個以上核苷酸之核苷酸序列。關於間隙子序列之長度，只要本發明之複合體具有免疫賦活活性(較佳為將B細胞活化而產生IL-6之活性、及將樹狀細胞活化而產生IFN-α之活性)，則無特別限定，通常為1～10個核苷酸長度，較佳為1～5個核苷酸長度，更佳為1～3個核苷酸長度。最佳為K型CpG ODN與聚dA藉由直接共價鍵進行連結。
本發明之寡聚去氧核苷酸除具有K型CpG ODN、聚dA及任意之間隙子序列以外，亦可於其5'末端及/或3'末端具有附加核苷酸序列。關於該附加核苷酸序列之長度，只要本發明之複合體具有免疫賦活活性(較佳為將B細胞活化而產生IL-6之活性、及將樹狀細胞活化而產生IFN-α之活性)，則無特別限定，通常為1～10個核苷酸長度，較佳為1～5個核苷酸長度，更佳為1～3個核苷酸長度。
於較佳態樣中，本發明之寡聚去氧核苷酸不含此種5'末端及/或3'末端之附加核苷酸序列。即，本發明之寡聚去氧核苷酸較佳為包含K型CpG ODN、聚dA及任意之間隙子序列，進而較佳為包含K型CpG ODN及聚dA。
於最佳態樣中，本發明之寡聚去氧核苷酸包含K型CpG ODN(具體而言，例如包含序列編號1所表示之核苷酸序列之寡聚去氧核苷酸)及聚dA，K型CpG ODN位於該寡聚去氧核苷酸之5'末端，聚dA位於3'末端。具體而言，係於包含序列編號1所表示之核苷酸序列之寡聚去氧核苷酸之3'末端鍵結有20～60個核苷酸長度(更佳為30～50個核苷酸長度(30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50個核苷酸長度)，最佳為30～45個核苷酸長度(30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45個核苷酸長度))之聚dA之寡聚去氧核苷酸，例如係包含序列編號2、或9～12所表示之核苷酸序列的寡聚去氧核苷酸。
本發明之寡聚去氧核苷酸之總長度通常為30～200個核苷酸長度，較佳為35～100個核苷酸長度，更佳為40～80個核苷酸長度(具體而言，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或80個核苷酸長度)，更佳為50～70個核苷酸長度(具體而言，50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70個核苷酸長度)，最佳為50～65個核苷酸長度(具體而言，50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65個核苷酸長度)。本發明之寡聚去氧核苷酸亦能以對活體內(in vivo)之分解(例如因外或內核酸酶所引起之分解)呈現抗性之方式適當地進行修飾。較佳為該改型包含硫代磷酸酯修飾或二硫代磷酸酯修飾。即，本發明之寡聚去氧核苷酸中之磷酸二酯鍵之一部分或全部被取代為硫代磷酸酯鍵或二硫代磷酸酯鍵。
較佳為本發明之寡聚去氧核苷酸包含磷酸二酯鍵之修飾，更佳為磷酸二酯鍵之修飾為硫代磷酸酯鍵(即，如WO 95/26204中所記載般，非交聯氧原子中之1個被取代為硫原子)。即，本發明之寡聚去氧核苷酸中之磷酸二酯鍵之一部分或全部被取代為硫代磷酸酯鍵。
關於本發明之寡聚去氧核苷酸，較佳為於K型CpG ODN中包含利用硫代磷酸酯鍵、或二硫代磷酸酯鍵之修飾，更佳為該K型CpG ODN之磷酸二酯鍵之全部被取代為硫代磷酸酯鍵。又，關於本發明之寡聚去氧核苷酸，較佳為於聚dA中包含硫代磷酸酯鍵、或二硫代磷酸酯鍵，更佳為該聚dA之磷酸二酯鍵之全部被取代為硫代磷酸酯鍵。進而較佳為本發明之包含人類化K型CpG寡聚去氧核苷酸及聚去氧腺苷酸之寡聚去氧核苷酸的磷酸二酯鍵之全部被取代為硫代磷酸酯鍵。最佳為本發明之寡聚去氧核苷酸係於人類化K型CpG寡聚去氧核苷酸(例如序列編號1)之3'末端鍵結有20～60個核苷酸長度(更佳為30～50個核苷酸長度(30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50個核苷酸長度)，最佳為30～45個核苷酸長度(30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45個核苷酸長度))之聚dA者，且該寡聚去氧核苷酸中所含之所有磷酸二酯鍵被取代為硫代磷酸酯鍵。其原因在於，藉由硫代磷酸酯鍵，對於本發明之寡聚去氧核苷酸，不僅期待針對分解之抗性，亦期待免疫賦活活性(例如使pDC活化而產生IFN-α之活性)之增強、及CpG-β-1,3-葡聚糖複合體之高產率、以及抗癌活性之增強。再者，本說明書中之硫代磷酸酯鍵與硫代磷酸酯骨架含義相同，磷酸二酯鍵與磷酸骨架含義相同。於本發明之寡聚去氧核苷酸中，包括上述寡聚去氧核苷酸之所有藥學上可容許之鹽類、酯、或此種酯之鹽類。
作為本發明之寡聚去氧核苷酸之藥學上可容許之鹽類，可適宜地列舉：鈉鹽、鉀鹽、鋰鹽之類的鹼金屬鹽，鈣鹽、鎂鹽之類的鹼土金屬鹽，鋁鹽、鐵鹽、鋅鹽、銅鹽、鎳鹽、鈷鹽等金屬鹽；銨鹽之類的無機鹽、第三辛基胺鹽、二苄基胺鹽、嗎啉鹽、葡糖胺鹽、苯基甘胺酸烷基酯鹽、乙二胺鹽、N-甲基葡糖胺鹽、胍鹽、二乙基胺鹽、三乙基胺鹽、二環己基胺鹽、N,N'-二苄基乙二胺鹽、氯普魯卡因鹽、普魯卡因鹽、二乙醇胺鹽、N-苄基-苯乙基胺鹽、哌鹽、四甲基銨鹽、三(羥基甲基)胺基甲烷鹽之類的有機鹽等胺鹽；氫氟酸鹽、鹽酸鹽、氫溴酸鹽、氫碘酸鹽之類的氫鹵酸鹽，硝酸鹽、過氯酸鹽、硫酸鹽、磷酸鹽等無機酸鹽；甲磺酸鹽、三氟甲磺酸鹽、乙磺酸鹽之類的低級烷磺酸鹽，苯磺酸鹽、對甲苯磺酸鹽之類的芳基磺酸鹽，乙酸鹽、蘋果酸鹽、反丁烯二酸鹽、琥珀酸鹽、檸檬酸鹽、酒石酸鹽、草酸鹽、順丁烯二酸鹽等有機酸鹽；及甘胺酸鹽、離胺酸鹽、精胺酸鹽、鳥胺酸鹽、麩胺酸鹽、天冬胺酸鹽之類的胺基酸鹽。
本發明之寡聚去氧核苷酸可為單鏈、雙鏈、三鏈之任一形態，較佳為單鏈。
本發明之寡聚去氧核苷酸較佳為進行單離。所謂「單離」係指進行去除目標成分以外之因子的操作，而脫離天然存在狀態。「所單離之寡聚去氧核苷酸」之純度(目標寡聚去氧核苷酸重量於評價對象物之總重量中所占之百分率)通常為70%以上，較佳為80%以上，更佳為90%以上，進而較佳為99%以上。
本發明之寡聚去氧核苷酸由於具有優異之免疫賦活活性(例如將B細胞(較佳為人類B細胞)活化而產生IL-6之活性)，故而作為免疫賦活劑等有用。進而，本發明之寡聚去氧核苷酸由於具有與2條β-1,3-葡聚糖(較佳為裂褶菌多糖、香菇多糖或硬葡聚糖)一併形成三股螺旋結構之性質，故而對本發明之複合體之製備有用。
本發明提供一種含有上述本發明之寡聚去氧核苷酸及β-1,3-葡聚糖之複合體(以下，稱為本發明之複合體)。
上述本發明之寡聚去氧核苷酸由於包含K型CpG ODN，故而其單獨發揮出對K型CpG ODN特有之免疫賦活活性(例如將B細胞(較佳為人類B細胞)活化而產生IL-6之活性)，缺乏對D型CpG ODN特有之免疫賦活活性(例如將類漿細胞樹狀細胞活化而產生IFN-α之活性)。然而，令人驚訝的是，藉由與β-1,3-葡聚糖(較佳為香菇多糖、裂褶菌多糖)形成複合體，無需D型CpG ODN之序列，而獲得對D型CpG ODN特有之免疫賦活活性(例如將類漿細胞樹狀細胞活化而產生IFN-α之活性)。即，本發明之複合體具有對K型CpG ODN特有之免疫賦活活性(例如將B細胞(較佳為人類B細胞)活化而產生IL-6之活性)、及對D型CpG ODN特有之免疫賦活活性(例如將類漿細胞樹狀細胞(較佳為人類類漿細胞樹狀細胞)活化而產生IFN-α之活性)之兩者。作為本發明中所使用之β-1,3-葡聚糖，可列舉：裂褶菌多糖、硬葡聚糖、卡德蘭多糖、茯苓多糖、灰樹花多糖、香菇多糖、昆布糖等。β-1,3-葡聚糖較佳為如裂褶菌多糖、香菇多糖或硬葡聚糖般含有大量1,6-葡萄糖苷支鏈(側鏈率33～40%)之β-1,3-葡聚糖，更佳為裂褶菌多糖。
香菇多糖(LNT)係源自香菇之公知之β-1,3-1,6-葡聚糖，分子式為(C6
H10
O5
)n
，分子量約為30～70萬。幾乎不溶於水、甲醇、乙醇(95)、或丙酮，但會溶解於作為極性有機溶劑之DMSO(dimethylsulfoxide，二甲基亞碸)或氫氧化鈉水溶液。
香菇多糖具有活化巨噬細胞、殺手T細胞、自然殺手(Natural Killer)細胞及抗體依賴性巨噬細胞介導之細胞毒殺作用(ADMC，Antibody dependent monocyte cytotoxicity)活性之增強作用(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)。於動物實驗中，對同系腫瘤及自體腫瘤，藉由與化學治療劑之併用投予，確認到腫瘤增生抑制作用以及延長壽命效果。又，藉由香菇多糖之單獨投予，亦確認到腫瘤增生抑制作用以及延長壽命效果。於臨床試驗中，針對無法手術或胃癌復發患者，藉由與替加氟經口投予之併用，確認到存活時間之延長(醫藥品評估表「香菇多糖靜脈注射用1 mg「Ajinomoto」」)，而於日本得到認可。因香菇多糖之單獨投予所產生之效果目前尚未確認。
裂褶菌多糖(SPG)係源自裂褶菌之公知之可溶性β-葡聚糖。SPG包含β-(1→3)-D-葡聚糖之主鏈、及每3個葡萄糖為1個之β-(1→6)-D-葡糖基側鏈(Tabata, K., Ito, W., Kojima, T., Kawabata, S. and Misaki A.,「Carbohydr. Res.」, 1981, 89, 1, p.121-135)。SPG作為針對婦科癌之免疫增強法之肌內注射製劑臨床藥，有20年以上之使用實績(清水, 陳, 荷見, 增淵，「Biotherapy」, 1990, 4, p.1390 長谷川, 「Oncology and Chemotherapy(腫瘤學及化療)」, 1992, 8, p.225)，且確認到於活體內之安全性(Theresa, M. McIntire and David, A. Brant, 「J. Am. Chem. Soc.」, 1998, 120, p.6909)。
於本說明書中，所謂「複合體」係指藉由複數個分子經由靜電鍵、凡得瓦鍵、氫鍵、疏水***互作用等非共價鍵或共價鍵進行聚集而獲得之產物。
本發明之複合體較佳為三股螺旋結構狀。於較佳態樣中，於形成該三股螺旋結構之3條鏈中，2條為β-1,3-葡聚糖鏈，1條為上述本發明之寡聚去氧核苷酸中之聚去氧腺苷酸之鏈。再者，該複合體亦可包含一部分未形成三股螺旋結構之部分。
關於本發明之複合體中之寡聚去氧核苷酸與β-1,3-葡聚糖之組成比，可根據寡聚去氧核苷酸中之聚去氧腺苷酸之鏈長、及β-1,3-葡聚糖之長度等而變化。例如，於β-1,3-葡聚糖鏈與聚去氧腺苷酸之鏈之長度相等之情形時，2條β-1,3-葡聚糖鏈與1條本發明之寡聚去氧核苷酸可進行聚集而形成三股螺旋結構。通常，相對於β-1,3-葡聚糖鏈，聚去氧腺苷酸之鏈長較短，因此複數個本發明之寡聚去氧核苷酸可針對2條β-1,3-葡聚糖鏈，經由聚去氧腺苷酸進行聚集，而形成三股螺旋結構(參照圖1)。
本發明之複合體係含有人類化K型CpG ODN及β-1,3-葡聚糖(例如香菇多糖、裂褶菌多糖、硬葡聚糖、卡德蘭多糖、茯苓多糖、灰樹花多糖、昆布糖)之複合體，較佳為包含人類化K型CpG ODN及β-1,3-葡聚糖(例如香菇多糖、裂褶菌多糖、硬葡聚糖)之複合體。更佳為包含於含有序列編號1所表示之核苷酸序列之寡聚去氧核苷酸之3'側鍵結有20～60個核苷酸長度(具體而言，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或60個核苷酸長度)之聚去氧腺苷酸且磷酸二酯鍵之全部被取代為硫代磷酸酯鍵的寡聚去氧核苷酸、及β-1,3-葡聚糖(例如香菇多糖、裂褶菌多糖)之複合體(例如K3-dA20～60-LNT、K3-dA20～60-SPG)，進而較佳為包含於含有序列編號1所表示之核苷酸序列之寡聚去氧核苷酸之3'側鍵結有30～50個核苷酸長度(具體而言，30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50個核苷酸長度)之聚去氧腺苷酸且磷酸二酯鍵之全部被取代為硫代磷酸酯鍵的寡聚去氧核苷酸、及β-1,3-葡聚糖(例如香菇多糖、裂褶菌多糖)之複合體(例如K3-dA30～50-LNT、K3-dA30～50-SPG)，最佳為包含於含有序列編號1所表示之核苷酸序列之寡聚去氧核苷酸之3'側鍵結有30～45個核苷酸長度(具體而言，30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45個核苷酸長度)之聚去氧腺苷酸且磷酸二酯鍵之全部被取代為硫代磷酸酯鍵之寡聚去氧核苷酸、及β-1,3-葡聚糖(例如香菇多糖、裂褶菌多糖)之複合體(K3-dA30～45-LNT、K3-dA30～45-SPG)。
關於本發明之複合體之製備方法，可與非專利文獻21～24、或日本專利特開2008-100919號公報中所記載之條件相同地進行。即，使原本為天然且以三股螺旋結構之形式存在之β-1,3-葡聚糖溶解於非質子性有機極性溶劑(二甲基亞碸(DMSO)、乙腈、丙酮等)或鹼性水溶液(氫氧化鈉、氫氧化鉀、氨、氫氧化鈣等)中而解開為單鏈。將以上述方式獲得之單鏈之β-1,3-葡聚糖的溶液與本發明之寡聚去氧核苷酸之溶液(水溶液、中性附近之pH值之緩衝水溶液、或酸性之緩衝水溶液，較佳為水溶液或中性附近之pH值之緩衝水溶液)加以混合，視需要再次將pH值調整至中性附近後，保持適當時間，例如於5℃下保持一夜。其結果為，2條β-1,3-葡聚糖鏈與該寡聚去氧核苷酸中之聚dA鏈形成三股螺旋結構，藉此形成本發明之複合體。藉由對所產生之複合體利用尺寸排除層析法進行純化、超濾、透析等，可去除未形成複合體之寡聚去氧核苷酸。又，藉由對所產生之複合體利用陰離子交換層析法進行純化，可去除未形成複合體之β-1,3-葡聚糖。藉由上述方法，可對複合體進行適當純化。
本發明之複合體之形成例如可藉由測定利用CD(Circular Dichroism，圓偏振光二色性)光譜之構象變化、利用尺寸排除層析法之UV(Ultra Violet，紫外線)吸收位移、凝膠電泳、微晶片電泳、毛細管電泳而進行確認，但並不限定於此。
關於本發明之寡聚去氧核苷酸與β-1,3-葡聚糖之混合比，可考慮聚dA鏈之長度等而適當設定，通常莫耳比(SPG/ODN)為0.02～2.0，較佳為0.1～0.5。於進一步之態樣中，莫耳比(β-1,3-葡聚糖(LNT等)/ODN)例如為0.005～1.0，較佳為0.020～0.25。
以CpG-ODN與LNT複合體為例對本發明之複合體之製備方法進行說明。使LNT溶解於0.05～2 N、較佳為0.1～1.5 N之鹼性水溶液(例如0.25 N氫氧化鈉水溶液)中，於1℃～40℃下放置10小時～4天(例如於室溫下放置一晩)，而製備單鏈之LNT水溶液(例如50 mg/ml之LNT水溶液)。以莫耳比(LNT/ODN)0.005～1.0將上述LNT水溶液與另外製備之CpG水溶液(例如100 μM之CpG水溶液)加以混合，繼而向上述LNT水溶液中添加酸性之緩衝水溶液(例如NaH2
PO4
)而加以中和，於1～40℃下維持6小時～4天(例如於4℃下維持一晩)，藉此結束複合化。再者，為了上述複合化，亦可最後添加LNT水溶液並加以混合。複合體之形成例如可藉由如下方法確認，即，使用尺寸排除層析法，針對CpG ODN向高分子量側之位移，監測240～280 nm(例如260 nm)下之吸收。
於一態樣中，本發明之複合體呈現出桿狀粒子之形態。粒徑係與藉由使用作材料之β-1,3-葡聚糖(例如裂褶菌多糖)為天然且呈現三股螺旋結構而形成之粒子之直徑同等，通常平均粒徑為10～100 nm，較佳為20～50 nm。關於該粒徑，可將複合體溶解於水中，使用馬爾文儀器Zeta分級器(Malvern Instruments Zeta Sizer)於80℃之條件下藉由動態光散射法進行計測。
本發明之複合體較佳為加以單離。「所單離之複合體」之純度(目標複合體重量於評價對象物之總重量中所占之百分率)通常為70%以上，較佳為80%以上，更佳為90%以上，進而較佳為99%以上。
進而，本發明之複合體除具有抗癌活性以外，亦具有優異之免疫賦活活性，尤其是具有對K型CpG ODN特有之免疫賦活活性(例如將B細胞(較佳為人類B細胞)活化而產生IL-6之活性)、及對D型CpG ODN特有之免疫賦活活性(例如將類漿細胞樹狀細胞(較佳為人類類漿細胞樹狀細胞)活化而產生IFN-α之活性)之兩者，故而作為免疫賦活劑等亦可賦予效果，因此有利。例如，關於包含K型CpG ODN(例如序列編號2、11、12與SPG之複合體及包含K型CpG ODN(例如序列編號2)與SPG之複合體(K3-SPG)，作為發揮出炎症應答誘導能力(pan-IFN-a、IL-6等)、病毒接種個體中之血清中抗原特異性IgG抗體效價(Total IgG、IgG2c等)之增強作用、病毒接種個體中之抗原特異性細胞激素產生能力(IFN-γ、IL2等)、針對病毒之感染防禦效果者亦有利。
(醫藥組合物)
本發明提供一種包含上述本發明之寡聚去氧核苷酸或上述本發明之複合體之醫藥組合物。本發明之醫藥組合物可藉由依據慣用方法將上述本發明之寡聚去氧核苷酸或上述本發明之複合體製劑化而獲得。本發明之醫藥組合物包含本發明之寡聚去氧核苷酸或複合體與藥理學上可容許之載體。又，本醫藥組合物亦可進而包含抗原。此種醫藥組合物係製成適於經口或非經口投予之劑型而提供。
作為用於非經口投予之組合物，例如可使用注射劑、栓劑等，注射劑亦可包含靜脈注射劑、皮下注射劑、皮內注射劑、肌肉注射劑、點滴注射劑等劑型。此種注射劑可根據公知之方法而製備。作為注射劑之製備方法，例如可藉由使上述本發明之寡聚去氧核苷酸或複合體溶解或懸浮於通常注射劑中所使用之無菌水性溶劑中而製備。作為注射用之水性溶劑，例如可使用蒸餾水；生理鹽水；磷酸緩衝液、碳酸緩衝液、三羥甲基胺基甲烷緩衝液、乙酸緩衝液等緩衝液等。此種水性溶劑之pH值可列舉5～10，較佳為6～8。所製備之注射液較佳為填充至適當之安瓿(ampoule)中。
又，亦可藉由對本發明之寡聚去氧核苷酸或複合體之懸浮液實施真空乾燥、冷凍乾燥等處理，而製備本發明之寡聚去氧核苷酸或複合體之粉末製劑。可藉由將本發明之寡聚去氧核苷酸或複合體於粉末狀態下保存，且於使用時將該粉末於注射用之水性溶劑中分散並供於使用。
關於醫藥組合物中之本發明之寡聚去氧核苷酸或複合體之含量，通常為醫藥組合物整體之約0.1～100重量%，較佳為約1～99重量%，進而較佳為約10～90重量%左右。
本發明之醫藥組合物可單獨含有本發明之寡聚去氧核苷酸或複合體作為有效成分，亦可與其他有效成分組合而含有本發明之寡聚去氧核苷酸或複合體。
(醫藥用途)
發現本發明之寡聚去氧核苷酸及複合體單獨具有抗癌作用。可認為此種效果與作為佐劑而開發之本發明之特性相比，為無法預料之效果。因此，提供如下之抗癌劑，其無需迄今為止之作為佐劑之使用方法、即與癌抗原一併投予，並且不限定於特定癌種類，作為通用之抗癌劑而對身體適度地發揮作用。又，當然亦具有免疫賦活活性，因此亦期待針對其他疾病之免疫賦活活性，且亦期待對體力較弱之癌患者具有協同效應。
由於本發明除具有抗癌作用以外，亦具有優異之免疫賦活活性，故而本發明之寡聚去氧核苷酸、複合體及醫藥組合物可用作免疫賦活劑。藉由向哺乳動物(人類等靈長類、小鼠等嚙齒類等)投予本發明之寡聚去氧核苷酸、複合體或醫藥組合物，可引起該哺乳動物之免疫反應。尤其是本發明之複合體具有D型CpG ODN之活性特性，刺激末梢血液單核球，而產生大量I型干擾素(Pan-IFN-α、IFN-α2等)及II型干擾素(IFN-γ)之兩者，故而作為I型干擾素產生誘導劑、II型干擾素產生誘導劑、I型及II型干擾素產生誘導劑有用。由於誘導產生I型及II型干擾素之兩者，故而本發明之複合體及含有其之醫藥組合物對於I型及II型干擾素之任一者或兩者有效之疾病之預防或治療有用。
作為醫藥用途之實現方法，例如可藉由向癌之患者或有可能罹患癌之人類投予(a)本發明之寡聚去氧核苷酸、或包含本發明之複合體之組合物，使接受該投予之對象之細胞毒殺性T淋巴球(CTL)抗原特異性地活化，而直接(作為單劑效果)對癌進行預防、治療。
於本說明書中，所謂「被實驗體(者)」係指成為本發明之診斷或檢測、或治療等之對象之對象(例如人類等生物或自生物提取之細胞、血液、血清等)。
於本說明書中，「藥劑」、「劑」或「因子」(任一者於英語中均相當於agent)廣義上可交換使用，只要可達成所欲實現之目的，則亦可為任何物質或其他要素(例如光、輻射能、熱、電等能量)。作為此種物質，例如可列舉：蛋白質、多肽、寡肽、肽、多核苷酸、寡聚核苷酸、核苷酸、核酸(例如包括cDNA(Complementary Deoxyribonucleic Acid，互補去氧核糖核酸)、基因組DNA之類的DNA、mRNA(Messenger Ribonucleic Acid，信使核糖核酸)之類的RNA(Ribonucleic Acid，核糖核酸))、多糖、寡糖、脂質、有機低分子(例如激素、配位子、訊息傳遞物質、有機低分子、藉由組合化學(combinatorial chemistry)而合成之分子、可用作醫藥品之低分子(例如低分子配位子等)等)、該等之複合分子，但並不限定於該等。作為對多核苷酸具有特異性之因子，代表性而言可列舉：對該多核苷酸之序列具有一定之序列同源性(例如70%以上之序列一致性(sequence identity))且具有互補性之多核苷酸、結合於啟動子區域之轉錄因子之類的多肽等，但並不限定於該等。作為對多肽具有特異性之因子，代表性而言可列舉對該多肽特異性地進行指向之抗體或其衍生物或其類似物(例如單鏈抗體)、該多肽為受體或配位子之情形時之特異性之配位子或受體，於該多肽為酵素之情形時，可列舉其受質等，但並不限定於該等。
於本說明書中，所謂「治療」係指針對某疾病或障礙(例如癌、過敏)，於成為此種狀態之情形時，防止此種疾病或障礙之惡化，較佳為維持現狀，更佳為使之減輕，進而較佳為使之消退，包括可發揮出患者之疾病、或者疾病所伴有之1個以上症狀之症狀改善效果或預防效果的情況。存在將事前進行診斷而進行適當之治療稱為「伴隨治療(companion treatment)」，將用於其之診斷藥稱為「伴隨診斷藥」之情形。
於本說明書中，所謂「治療藥(劑)」廣義上係指可對目標狀態(例如癌、過敏等疾病等)進行治療之所有藥劑。於本發明之一實施形態中，「治療藥」亦可為包含有效成分、及藥理學上容許之1種或者其以上之載體之醫藥組合物。醫藥組合物例如可將有效成分與上述載體混合，藉由製劑學之技術領域中已知之任意方法而製造。又，治療藥只要為用於治療者，則使用形態並無限定，可為有效成分單獨，亦可為有效成分與任意成分之混合物。又，上述載體之形狀並無特別限定，例如亦可為固體或液體(例如緩衝液)。再者，癌、過敏等之治療藥包括用於預防癌、過敏等之藥物(預防藥)、或癌、過敏等之抑制劑。
於本說明書中，所謂「預防」係指針對某疾病或障礙(例如過敏)，在成為此種狀態前阻止成為此種狀態。可使用本發明之藥劑進行診斷，視需要使用本發明之藥劑例如進行過敏等之預防，或採取用於預防之對策。
於本說明書中，所謂「預防藥(劑)」廣義上係指可對目標狀態(例如過敏等疾病等)進行預防之所有藥劑。
於本說明書中，所謂「套組」通常係指提供應分成2個以上之子部分而提供之部分(例如檢驗藥、診斷藥、治療藥、抗體、標記、說明書等)之單元。為了穩定性等，不應進行混合而提供，於以提供如較佳為於即將使用前進行混合而使用之組合物為目標時，該套組之形態較佳。較佳為，此種套組具備記載如何使用所提供之部分(例如檢驗藥、診斷藥、治療藥)、或應如何處理試劑之指示書或說明書，此情況較有利。於本說明書中，於套組係以試劑套組之形式使用之情形時，套組中通常包含記載有檢驗藥、診斷藥、治療藥、抗體等之用法等之指示書等。
於本說明書中，「指示書」係記載有對醫師或其他使用者說明使用本發明之方法者。該指示書記載有本發明之檢測方法、診斷藥之用法、或指示投予醫藥等之內容。又，於指示書中，亦可記載有指示經口投予、向食道之投予(例如利用注射等)作為投予部位之內容。該指示書係根據實施本發明之國家之監督政府機構(例如，於日本為厚生勞動省，於美國為食品及藥物管理局(FDA)等)所規定之格式而製作，明確記載有由其監督政府機構所認可之要點。指示書係所謂隨附文件(package insert(藥品說明書))，通常係以紙媒體提供，但並不限定於此，例如亦能以電子媒體(例如網際網路中所提供之首頁、電子郵件)之類的形態提供。
(較佳實施形態之態樣)
以下，對本發明之較佳實施形態進行說明。以下所提供之實施形態係為了更佳地理解本發明而提供者，可理解本發明之範圍不應限定於以下之記載。因此，明確從業者可參考本說明書中之記載，而於本發明之範圍內進行適當改變。又，可理解本發明之以下之實施形態可單獨使用或可將該等組合而使用。
＜單劑形態＞
於一態樣中，本發明提供一種包含複合體之抗癌劑，該複合體包含：(a)寡聚去氧核苷酸，其係包含人類化K型CpG寡聚去氧核苷酸及聚去氧腺苷酸者，並且聚去氧腺苷酸係配置於人類化K型CpG寡聚去氧核苷酸之3'側；及(b)β-1,3-葡聚糖。於本發明中，發現本發明之複合體本身係作為抗癌劑而發揮作用。先前，本發明者等人僅發現該複合體可用作佐劑並提出申請，未預測可直接以單劑之形式用作抗癌劑。因此，就於無癌抗原之情況下使用之觀點而言，可認為帶來無法預料之效果。
於一實施形態中，本發明之抗癌劑之特徵在於：於無癌抗原之情況下進行投予。
於另一實施態樣中，本發明之抗癌劑之特徵在於：其係以傳遞至網狀內皮系統及/或淋巴結之方式進行投予。較佳為上述網狀內皮系統及/或淋巴結包含腫瘤及巨噬細胞。例示性地上述網狀內皮系統包含脾臟及/或肝臟。因此，本發明之抗癌劑之特徵在於：其係以傳遞至包含腫瘤及巨噬細胞之網狀內皮系統臟器(脾臟、肝臟等)及/或淋巴結之方式進行投予。不希望被理論所束縛，揭示本發明之複合體被傳遞至腫瘤及巨噬細胞，並於此處將癌之死細胞募集(recruit)至網狀內皮系統臟器(脾臟、肝臟等)內。藉此，認為可進一步驅逐身體內之癌細胞。因此，本發明並非使用特定癌抗原作為佐劑而針對特定之癌，就可殺傷存在於身體內之任意癌細胞而可提供前所未有之抗癌劑之觀點而言，可認為帶來顯著之效果。
因此，於更佳實施形態中，本發明之上述抗癌劑之特徵在於：其係於無癌抗原之情況下，以傳遞至腫瘤及巨噬細胞之方式進行投予。
此種傳遞方法可使用任意方法，例如上述投予可列舉全身性投予，但並不限定於此。較佳為全身性投予，作為全身性投予，可列舉：靜脈內投予、腹腔內投予、經口投予、皮下投予、肌內投予等。
於一實施形態中，本發明中所使用之寡聚去氧核苷酸可列舉：K3(序列編號1)、K3-dA40
(序列編號2)、dA40
-K3(序列編號3)、K3-dA20(序列編號4)、K3-dA25(序列編號5)、K3-dA30(序列編號6)及K3-dA35(序列編號7)等，但並不限定於該等。
於一實施形態中，本發明中所使用之β-1,3-葡聚糖亦可為裂褶菌多糖(SPG)、香菇多糖、硬葡聚糖、卡德蘭多糖、茯苓多糖、灰樹花多糖、昆布糖等。
於較佳實施形態中，本發明之複合體為K3-SPG或其類似物。此處，所謂類似物，可列舉於CpG側結構與K3類似者、於β葡聚糖側結構與SPG類似者等，但並不限定於該等。
再者，由於抗癌作用係利用各種機制，故而用於使癌之死細胞集聚於脾臟等用途並不容易想到。尤其是於投予全身性時，不會想到用於集聚於腫瘤，使死亡之腫瘤細胞集聚於脾臟等組織之用途。又，介白素12(IL12)及/或干擾素(IFN)α之表現或表現促進效果亦利用與抗癌作用不同之機制，介白素12(IL12)及/或干擾素(IFN)α之表現或表現促進於抗癌以外亦可發揮，故而並非容易相互想到者。因此，可認為本發明之CpG-β葡聚糖複合體之各用途(抗癌用途(以單劑之形式)、使癌之死細胞集聚於脾臟之用途、用於介白素12(IL12)及/或干擾素(IFN)α之表現或表現促進之用途)處於不認為可容易相互想到之關係。
＜網狀內皮系統(包括脾臟及/或肝臟)及/或淋巴結集聚劑＞
於另一態樣中，本發明提供一種包含複合體，且用於使癌之死細胞集聚於網狀內皮系統(包含脾臟及/或肝臟)及/或淋巴結之組合物，且該複合體包含：(a)寡聚去氧核苷酸，其係包含人類化K型CpG寡聚去氧核苷酸及聚去氧腺苷酸者，並且聚去氧腺苷酸係配置於人類化K型CpG寡聚去氧核苷酸之3'側；及(b)β-1,3-葡聚糖。不希望被理論所束縛，發現本發明之複合體可使癌之死細胞集聚於網狀內皮系統(包含脾臟及/或肝臟)及/或淋巴結。如實施例中所例證，證實利用K3-SPG之類的本發明之複合體所進行之處理係以依賴於IL12p40及IFN-I之兩者之態樣而誘發腫瘤細胞死亡。先前未預測到複合體具有此種作用，於該意義上可認為實現無法預料之作用效果。即，CpG係經腫瘤微環境中之吞噬細胞進行標靶化者。若癌之死細胞集聚於網狀內皮系統(包含脾臟及/或肝臟)及/或淋巴結，則其後，所釋出之腫瘤死細胞誘發針對複數個腫瘤抗原之抗腫瘤CTL，位於身體內之癌細胞亦可如被霰彈槍攻擊般被殺傷而根治。不希望被理論所束縛，產生腫瘤微環境中之IL12及IFN-I細胞激素之兩者雖然不認為係K3-SPG單劑治療所必須，但較重要。
於一實施形態中，本發明中所使用之寡聚去氧核苷酸係選自由K3(序列編號1)、K3-dA40
(序列編號2)、dA40
-K3(序列編號3)、K3-dA20(序列編號4)、K3-dA25(序列編號5)、K3-dA30(序列編號6)及K3-dA35(序列編號7)所組成之群中。
於另一實施態樣中，本發明中所使用之β-1,3-葡聚糖係選自由裂褶菌多糖(SPG)、硬葡聚糖、卡德蘭多糖、茯苓多糖、灰樹花多糖及昆布糖所組成之群中。
於較佳實施形態中，本發明之複合體為K3-SPG。
於一實施形態中，成為本發明之組合物之對象的網狀內皮系統及/或淋巴結包含腫瘤及巨噬細胞。例示性地上述網狀內皮系統包含脾臟及/或肝臟。因此，本發明之組合物之特徵在於：其係以傳遞至包含腫瘤及巨噬細胞之網狀內皮系統臟器(脾臟、肝臟等)及/或淋巴結之方式進行投予。不希望被理論所束縛，揭示本發明之複合體被傳遞至腫瘤及巨噬細胞，並在此將癌之死細胞募集至網狀內皮系統臟器(脾臟、肝臟等)。藉此，認為可進一步驅逐身體內之癌細胞。因此，本發明並非使用特定癌抗原作為佐劑而針對特定之癌，就可殺傷存在於身體內之任意癌細胞而可提供前所未有之抗癌劑之觀點而言，可認為帶來顯著之效果。
因此，於更佳實施形態中，本發明之上述抗癌劑之特徵在於：其係於無癌抗原之情況下，以傳遞至腫瘤及巨噬細胞之方式進行投予。
此種傳遞方法可使用任意方法，例如上述投予可列舉全身性投予，但並不限定於此。較佳為全身性投予，作為全身性投予，可列舉：靜脈內投予、腹腔內投予、經口投予、皮下投予、肌內投予等。
＜IL12及/或IFN表現促進劑＞
進而，於另一態樣中，本發明提供一種用於介白素12(IL12)及/或干擾素(IFN)γ之表現或表現促進之組合物，其包含：(a)寡聚去氧核苷酸，其係包含人類化K型CpG寡聚去氧核苷酸及聚去氧腺苷酸者，並且聚去氧腺苷酸係配置於人類化K型CpG寡聚去氧核苷酸之3'側；及(b)β-1,3-葡聚糖。產生腫瘤微環境中之IL12及IFN-I細胞激素之兩者係K3-SPG單劑治療中之重要作用效果，此種效果除作為抗癌劑之作用以外，於其他用途中亦較重要。作為此種處理之對象，可列舉癌、此外之病毒等慢性感染症疾病、病毒感染預防等，但並不限定於該等。
於一實施形態中，本發明中所使用之寡聚去氧核苷酸係選自由K3(序列編號1)、K3-dA40
(序列編號2)、dA40
-K3(序列編號3)、K3-dA20(序列編號4)、K3-dA25(序列編號5)、K3-dA30(序列編號6)及K3-dA35(序列編號7)所組成之群中。
於另一實施態樣中，本發明中所使用之β-1,3-葡聚糖係選自由裂褶菌多糖(SPG)、硬葡聚糖、卡德蘭多糖、茯苓多糖、灰樹花多糖及昆布糖所組成之群中。
於較佳實施形態中，本發明之複合體為K3-SPG。
(醫藥品、劑型等)
本發明係以上述各種形態之醫藥(治療藥或預防藥)之形式提供。
治療藥之投予途徑較佳為使用在治療時有效者，例如亦可為靜脈內、皮下、肌內、腹腔內、或經口投予等。作為投予形態，例如亦可為注射劑、膠囊劑、錠劑、顆粒劑等。於投予本發明之成分之情形時，有效的是製成注射劑而使用。注射用之水溶液例如亦可於小玻璃瓶、或不鏽鋼容器中保存。又，注射用之水溶液例如亦可調配生理鹽水、糖(例如海藻糖)、NaCl、或NaOH等。又，治療藥例如亦可調配緩衝劑(例如磷酸鹽緩衝液)、穩定劑等。
通常，本發明之組合物、醫藥、治療劑、預防劑等包含治療有效量之治療劑或有效成分、及藥學上可容許之載體或者賦形劑。於本說明書中，「藥學上可容許」係指於用於動物、並且更詳細而言人體之經政府之監督政府機構許可、或藥典或其他通常所認可之藥典中被列舉。於本說明書中，所使用之「載體」係指與治療劑一併投予之稀釋劑、佐劑、賦形劑、或媒劑。此種載體亦可為無菌液體、例如水及油，包含石油、動物、植物或合成起源者，並無限定，包含花生油、大豆油、礦物油、芝麻油等。於經口投予醫藥之情形時，水為較佳之載體。於將醫藥組合物靜脈內投予之情形時，生理鹽水及水性葡萄糖為較佳之載體。較佳為將生理鹽水溶液、以及水性葡萄糖及甘油溶液用作可注射之溶液之液體載體。於適當之賦形劑中，包含輕質無水矽酸、結晶纖維素、甘露醇、澱粉、葡萄糖、乳糖、蔗糖、明膠、麥芽、米、小麥粉、白堊、矽膠、硬脂酸鈉、單硬脂酸甘油、滑石、氯化鈉、脫脂粉乳、甘油、丙烯、乙二醇、水、乙醇、羧甲基纖維素鈣、羧甲基纖維素鈉、羥丙基纖維素、羥丙基甲基纖維素、聚乙烯基縮醛(二乙基胺基)乙酸酯、聚乙烯基吡咯啶酮、明膠、中鏈脂肪酸三甘油酯、聚氧乙烯氫化蓖麻油60、白糖、羧甲基纖維素、玉米澱粉、無機鹽等。於較理想之情形時，組合物亦可含有少量濕潤劑或乳化劑、或pH值緩衝劑。該等組合物亦可採用溶液、懸浮物、乳膠、錠劑、丸劑、膠囊、粉末、緩釋調配物等形態。亦可使用傳統之結合劑及載體、例如三酸甘油酯，調配組合物而製成栓劑。經口調配物亦可包含醫藥等級之甘露醇、乳糖、澱粉、硬脂酸鎂、糖精鈉、纖維素、碳酸鎂等標準載體。適當之載體之例係記載於E. W. Martin, Remington's Pharmaceutical Sciences (Mark Publishing Company, Easton, U. S. A)中。關於此種組合物，為了提供向患者適當地投予之形態，含有適當量之載體及治療有效量之治療劑、較佳為純化型者。調配物必須適合投予方式。除該等以外，例如亦可含有界面活性劑、賦形劑、著色料、著香料、防腐劑、穩定劑、緩衝劑、懸浮劑、等張劑、結合劑、崩解劑、潤滑劑、流動性促進劑、矯味劑等。
於本發明之一實施形態中，「鹽」例如包含由任意酸性(例如羧基)基所形成之陰離子鹽、或由任意鹼性(例如胺基)基所形成之陽離子鹽。鹽類包含無機鹽或有機鹽，例如包含Berge et al., J. Pharm. Sci., 1977, 66, 1-19中所記載之鹽。又，例如可列舉：金屬鹽、銨鹽、與有機鹼之鹽、與無機酸之鹽、與有機酸之鹽等。於本發明之一實施形態中，「溶劑合物」係由溶質及溶劑所形成之化合物。關於溶劑合物，例如可參照J. Honiget al., The Van Nostrand Chemist's Dictionary P650 (1953)。若溶劑為水，則所形成之溶劑合物為水合物。該溶劑較佳為不阻礙溶質之生物活性者。作為此種較佳溶劑之例，並無特別限定，可列舉水、或各種緩衝液。於本發明之一實施形態中，「化學修飾」例如可列舉：利用PEG(Polyethylene Glycol，聚乙二醇)或者其衍生物所進行之修飾、螢光素修飾、或生物素修飾等。
於將本發明製成醫藥而投予之情形時，已知有各種傳遞(Delivery)系統，並且亦可使用此種系統，向適當之部位(例如食道)投予本發明之治療劑，此種系統中例如有於脂質體、微小粒子、及微小膠囊中之囊封；可表現治療劑(例如多肽)之重組細胞之使用，受體所介導之內噬作用(endocytosis)之使用；治療核酸作為反轉錄病毒載體或其他載體之一部分之構建等。導入法包括皮內、肌內、腹腔內、靜脈內、皮下、鼻內、硬膜外、及經口途徑，但不限定於該等。亦可藉由適宜途徑之任一種，例如藉由注入、藉由團注(bolus injection)、藉由通過上皮或皮膚黏膜內層(例如口腔黏膜、直腸黏膜及腸黏膜等)之吸收而投予醫藥，視需要可使用霧劑，使用吸入器或噴霧器，並且亦可與其他生物學活性劑一併投予。投予亦可為全身性或局部。於本發明用於癌之情形時，進而可藉由向癌(病變部)直接注入等適當途徑之任一種而進行投予。
於較佳實施形態中，可依據公知之方法，以適合向人類之投予之醫藥組合物之形式調配組合物。此種組合物可藉由注射而投予。用於注射投予之組合物代表性而言為無菌等張水性緩衝劑中之溶液。又，於必要時，組合物亦可包含助溶劑及緩解注射部位之疼痛之利多卡因等局部麻醉劑。通常，分別供給成分或於單位劑量投藥器中一併混合而供給，例如可於顯示活性劑之量之安瓿或藥囊(sachet)等密封容器中，以冷凍乾燥粉末或不含水之濃縮物之形式供給。欲藉由注入組合物而進行投予之情形時，亦可使用含有無菌藥劑等級之水或生理鹽水之注入器而進行分配。於欲藉由注射組合物而進行投予之情形時，亦可於投予前，以可混合成分之方式提供注射用之無菌水或生理鹽水之安瓿。
亦可於中性型或鹽型或其他前驅藥(例如酯等)中調配本發明之組合物、醫藥、治療劑、預防劑。藥學上可容許之鹽包括與源自鹽酸、磷酸、乙酸、草酸、酒石酸等之游離型之羧基一併形成者，與源自異丙基胺、三乙基胺、2-乙基胺基乙醇、組胺酸、普魯卡因等者等之游離型之胺基一併形成者，以及源自鈉、鉀、銨、鈣、及氫氧化鐵等者。
對於特定障礙或狀態之治療有效之本發明之治療劑之量可根據障礙或狀態之性質而變化，熟知本技藝者可基於本說明書之記載根據標準臨床技術而確定。進而，視情形亦可使用活體外分析，而輔助最佳投藥量範圍之鑑定。又，欲用於調配物中之準確之用量亦可根據投予途徑、及疾病或障礙之重大性而變化，故而應依據主治醫師之判斷及各患者之狀況而確定。但是，投予量並無特別限定，例如可為每1次0.001、1、5、10、15、100、或1000 mg/kg體重，亦可為該等任兩個值之範圍內。投予間隔並無特別限定，例如可每1、7、14、21、或28天投予1或2次，亦可每該等任兩個值之範圍投予1或2次。關於投予量、投予間隔、投予方法，亦可根據患者之年齡或體重、症狀、對象臟器等而進行適當選擇。又，治療藥較佳為包含治療有效量、或發揮所需作用之有效量之有效成分。於惡性腫瘤標記物於投予後顯著減少之情形時，亦可判斷為具有治療效果。有效用量可根據由活體外或動物模型試驗系統獲得之用量-反應曲線而進行推定。
於本發明之一實施形態中，「患者」或「被實驗體」包括人類、或除人類以外之哺乳動物(例如小鼠、豚鼠、倉鼠、大鼠、老鼠、兔、豬、綿羊、山羊、牛、馬、貓、狗、狨猿(marmoset)、猴、或黑猩猩(chimpanzee)等之1種以上)。
本發明之醫藥組合物或治療劑或者預防劑可以套組之形式提供。
於特定實施形態中，本發明提供一種藥劑包或套組，其包含填充有本發明之組合物或醫藥之1種以上之成分的1個以上之容器。視情形亦可於此種容器上，以由控制醫藥或生物學製品之製造、使用或銷售之政府機關所規定之形式附帶顯示表明政府機關許可用於人類投予之製造、使用或銷售之資訊。
於特定實施形態中，可藉由脂質體、微小粒子、或微小膠囊而投予包含本發明之成分之醫藥組合物。於本發明之多樣化態樣中，亦有使用此種組合物而於實現本發明之成分之緩釋方面有用之可能性。
本發明之治療藥、預防藥等作為醫藥等之製劑化順序係於該領域中公知，例如記載於日本藥典、美國藥典、其他國家之藥典等中。因此，只要有本說明書之記載，則從業者可在不過度進行實驗之情況下決定應使用之量等實施形態。
(一般技術)
本說明書中可使用之分子生物學方法、生物化學方法、微生物學方法係於該領域中周知且慣用者，例如係記載於Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor及其3rd Ed. (2001); Ausubel, F. M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F. M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, M. A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F. M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F. M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M. A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F. M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, J. J. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press、增刊實驗醫學「基因導入＆表現解析實驗法」羊土社、1997等中，且於本說明書中係以參考之形式引用該等之相關部分(可為全部)。
關於用於製作人工合成之基因的DNA合成技術及核酸化學，例如記載於Gait, M. J. (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, M. J. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, R. L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman & Hall; Shabarova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G. M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G. T. (I996). Bioconjugate Techniques, Academic Press等中，且於本說明書中係以參考之形式引用該等之相關部分。
例如，於本說明書中，亦可藉由該領域中已知之標準法，例如藉由使用自動化DNA合成裝置(由Biosearch、Applied Biosystems等所銷售者等)，而合成本發明之寡聚核苷酸。例如亦可藉由Stein等人(Stein et al., 1988, Nucl. Acids Res. 16: 3209)之方法，而合成硫代磷酸-寡聚核苷酸，亦可藉由使用調節孔玻璃聚合物支持體(Sarinet al., 1988, Proc. Natl. Acad. Sci. USA 85: 7448-7451)等，而製備膦酸甲酯-寡聚核苷酸。
於本說明書中，「或」係於可採用文章中所列舉之事項之「至少1個以上」之時使用。「或者」亦相同。於本說明書中，於明確記載為「兩個值之範圍內」之情形時，該範圍亦包括兩個值本身。
於本說明書中，關於所引用之科學文獻、專利、專利申請等參考文獻，其所有內容係以與分別具體地記載者相同之程度，以參考之形式引用至本說明書中。
以上，為了使本發明易於理解，揭示較佳實施形態而進行了說明。以下，基於實施例對本發明進行說明，但上述說明及以下之實施例係僅基於例示之目的而提供，並非為了限定本發明而提供。因此，關於本發明之範圍，並不限定於本說明書中具體地記載之實施形態及實施例，而僅受申請專利範圍所限定。
[實施例]
以下，記載實施例。必要時，關於以下之實施例中所使用之動物之處理，所有動物實驗係依據日本醫藥基盤研究所及大阪大學動物設施之機關指南而實施。又，基於赫爾辛基(Helsinki)宣言而進行。試劑類具體而言係使用實施例中所記載之製品，但亦可利用其他製造商(Sigma-Aldrich、和光純藥、Nacalai、R&D Systems、USCN Life Science INC等)之同等品代替使用。
(製造實施例)
以下之CpG ODNs係Genedesign股份有限公司所合成(下劃線表示硫代磷酸酯鍵)。
特別是，除上述K3-dA40 (序列編號2)外，記載關於K3-dA35 (序列編號7)、K3-dA30 (序列編號6)、K3-dA25 (序列編號5)及K3-dA20 (序列編號4)之合成(表2)。
(上述序列中之s表示核苷間之磷酸二酯鍵被取代為硫代磷酸酯鍵)
本寡聚去氧核苷酸係使用作為常法之固相亞磷醯胺法(Nucleic Acids in Chemistry and Biology, 3. Chemical synthesis (1990) ed. G. Michael Blackburn and Michael J. Gait. Oxford University Press)而進行合成。
卵白蛋白(OVA)係自生化學工業股份有限公司購買。DQ-OVA、Alexa488-OVA、CFSE、及Lipofectamine 2000係自Invitrogen購買。Hoechst 33258、酵母聚糖(zymosan)及卡德蘭多糖係自SIGMA購買。Zymosan-Depleted係自Invivogen購買。氯屈膦酸鹽脂質體係自FormuMax購買。流行性感冒裂解產物疫苗、福馬林不活化全病毒(WIV)、及純化流行性感冒病毒(H1N1)係如先前所記載般製備(Koyama, S., et al., Science translational medicine 2, 25ra24 (2010))。
CpG ODN與SPG之複合體化(製造實施例圖1)
將7.22 mg之K3-dA40溶解於水(3.7 mL)中。將SPG(三井製糖)15 mg溶解於0.25 N之NaOH(1 mL)中。將1 mL之330 mM之NaH2
PO4
添加至DNA溶液中，繼而將SPG溶液添加至DNA/NaH2
PO4
溶液中，並於4℃下維持一晩，藉此結束複合體化。莫耳比(MSPG/MDNA)係固定為0.27。關於複合體之形成，利用微晶片電泳裝置(SHIMADZU：MultiNA)所確認之複合體之形成係藉由使用尺寸排除層析法，監測260 nm下之吸收而確認CpG ODN之向高分子量側之位移(系統：Agilent 1100系列，管柱(Column)：將Asahipak GF7M-HQ(Shodex)2根進行連結，流速：0.8 mL/min，緩衝液：10 mM之EDTA(Ethylenediamine Tetraacetic Acid，乙二胺四乙酸)PBS(Phosphate Buffer Solution，磷酸鹽緩衝液)，pH值7.4，溫度：40℃)。
(為了用於實施例之準備)
於以下之實施例中，揭示可進行如下全身性之單劑治療，該全身性之單劑治療係利用誘發較強之腫瘤縮小之以腫瘤微環境中之巨噬細胞作為標靶之奈米粒子狀TLR9促效劑而進行。
(材料及方法)
以下，對本實施例中所使用之試劑、材料、動物、細胞及其方法進行說明。於各實施例中亦適當進行補充說明。
(動物及試劑)
自Nihon CLEA購買6週齡之雌性C57BL/6J小鼠。自Jackson Laboratory購買Il12p40缺損小鼠及Batf3缺損小鼠。Ifnar2缺損小鼠、Myd88缺損小鼠及Dectin-1缺損小鼠係如上述所記載(Kobiyama, K., et al. Proc. Natl. Acad. Sci. U. S. A. 111, 3086-3091 (2014))。依據醫藥基盤研究所之機關指南進行所有動物實驗。K3係藉由Gene Design(基因設計)而合成。自生化學工業購買卵白蛋白(OVA)。
(細胞株)
EL4及表現OVA之EL4(EG7)為C57BL/6J小鼠之胸腺瘤細胞株，係自ATCC購買。自Japanese Collection of Research Bioresourses(日本細胞庫)購買B16(黑色素瘤)。自理研細胞庫購買B16F10(黑色素瘤)，MC38(結腸癌)係由F. JAMES Primus博士提供。自Jackson's Laboratory購買Pan02(胰臟癌)。於完全RPMI(追加有10%(v/v)胎牛血清(FBS，Fetal Bovine Serum)、青黴素、及鏈黴素之RPMI 1640)中培養EL4、EG7、MC38、及Pan02。於完全DMEM(Dulbecco Modified Eagle Medium，杜貝克改良伊格爾培養基)(追加有10%(v/v)胎牛血清(FBS)、青黴素、及鏈黴素之DMEM)中培養B16及B16F10。
(腫瘤實驗及治療方法)
將EG7、EL4、B16、B16F10、及MC38細胞(於10%受質膠/PBS中以5×106
細胞/mL計為100 μl)(s.c)接種至小鼠之右側腹部之皮下。對於腫瘤尺寸，測定腫瘤之長度(L)、寬度(W)、及高度(H)，將腫瘤體積(V)根據V＝L×W×H而計算。藉由腫瘤內注射(i.t.)而直接注射至腫瘤部位。CpG治療係於腫瘤體積達到約100 mm3
後開始，該時期為EG7及B16F10之接種7天後、B16之接種10天後、以及MC38之接種14天後。每隔1天利用K3(30 μg)或K3-SPG(10 μg)對帶有腫瘤之小鼠進行3次處理。
(Pan02之腹膜接種模型)
於Pan02之腹膜接種模型中，向腹腔內注射1×106
個Pan02細胞(於PBS中以1×107
細胞/mL計為100 μl)。於接種11天後開始CpG治療，於第21天自小鼠之腹膜摘除所有腫瘤小結節，其後測定該等之重量(g)。CpG治療中之投予量如上所述。
(活體內成像實驗)
為了評價K3及K3-SPG之存在，於第0天對C57BL/6小鼠s.c.接種EG7，於第12天i.v.投予PBS(對照)、Alexa 647-K3(30 μg)、或Alexa 647-K3-SPG(10 μg)。投予1小時後，利用IVIS(註冊商標)螢光成像系統(Lumina Imaging System)及分析軟體(Ver.2.6，Xenogen)對小鼠進行分析，將藉由相對螢光測得之影像轉換為表面放射亮度之物理單位(光子/sec/cm2
/sr)。為了檢測於活體內經標記之CD8+
T細胞，於第14天自於第7、9、11天利用K3-SPG進行處理、或未經處理之帶有EG7之C57BL/6小鼠或Il12p40-Ifnar2基因雙剔除小鼠回收脾細胞。於脾細胞之懸浮後，利用ACK溶解緩衝液(150 mM之NH4
Cl、10 mM之KHCO3
、0.1 mM之Na2
EDTA)將紅血球溶解，並將細胞維持於完全RPMI中。藉由MACS(Magnetic Activated Cell Sorting，磁性激活細胞分選)(Miltenyi Biotec)對CD8α+
T細胞進行分選。藉由陰性選擇之方法而分選CD8α+
T細胞。其後，利用Xenolight DiR(註冊商標)對所分選之CD8α+
T細胞進行染色。於第14天(於第0天接種EG7，於7、9、第11天利用K3-SPG進行i.v.處理、或未經處理之C57BL/6小鼠或Il12p40-Ifnar2基因雙剔除小鼠)將經染色之CD8α+
T細胞移植至受移植小鼠。移植經染色之細胞24小時後，利用IVIS(註冊商標)螢光成像系統(Lumina Imaging System)(Ver.2.6)對小鼠進行分析。將目標區域彙集於腫瘤區域，利用活動影像軟體(Living Image Software)(Ver.2.6、Xenogen)對螢光強度進行分析。
(免疫組織化學)
對C57BL/6J小鼠(6-8週齡，雌，CLEA Japan)，自尾靜脈i.v.注射Alexa 647-K3(30 μg)、Alexa 647-K3-SPG(10 μg)、及葡聚糖-PE(20 μg)。注射後1小時後將腫瘤回收，利用4%(w/v)多聚甲醛將冷凍切片固定10分鐘，利用Hoechst 33258及抗CD3e抗體、抗CD8β抗體進行染色。使用Olympus IX81系統對細胞進行拍攝。利用MetaMorph而分析影像資料。
(耗竭實驗)
為了使吞噬細胞(樹狀細胞及巨噬細胞)耗竭，於接種EG7 5天後對C57BL/6小鼠i.v.注射氯屈膦酸鹽脂質體或對照脂質體(100 nm)(片山化學)。為了使CD8+
T細胞耗竭，於接種EG7 6天後及13天後向尾靜脈i.v.注射200 μg之抗CD8α抗體。
(脾細胞之分析)
於第14天自於第7、9、11天利用K3-SPG進行i.v.處理、或未經處理之帶有EG7之C57BL/6小鼠或Il12p40-Ifnar2基因雙剔除小鼠回收脾細胞。於製備脾細胞後，利用ACK溶解緩衝液將紅血球溶解，並於完全RPMI中維持細胞。利用H-2Kb
OVA四聚物(MBL)、抗CD8α抗體(KT15)、抗TCRβ抗體(H57-597)、抗CD62L抗體(MEL-14)、及抗CD44抗體(IM7)、以及7-胺基放線菌素D(7AAD，7-Aminoactinomycin D)對脾細胞進行染色。利用流式細胞儀確定OVA四聚物+
CD44+
CD8α+
TCRβ+
之細胞數。關於其他實驗，將所製備之脾細胞與抗CD45抗體、抗CD3e抗體、抗CD8α抗體、及抗CD11a抗體一併培養，其後利用流式細胞儀進行分析。
(CD45陰性細胞之分析及免疫化)
於第12天自於第7、9、11天利用K3-SPG進行i.v.處理、或未經處理之帶有EG7之C57BL/6小鼠或Il12p40-Ifnar2基因雙剔除小鼠回收脾細胞。於製備脾細胞後，利用ACK溶解緩衝液將紅血球溶解，並於完全RPMI中維持細胞。利用抗CD45抗體(APC)對脾細胞進行染色，利用流式細胞儀確定CD45-
細胞之數量。進而，利用PI及Hoechst 33342對凋亡細胞、壞死細胞、及CD45陰性活細胞之亞群進行染色，其後利用流式細胞儀進行分析。其次，自利用K3-SPG進行處理之帶有腫瘤之C57BL/6小鼠中，利用INFLUX(BD Bioscience)而分選CD45-
細胞。
(疫苗接種模型)
對C57BL/6小鼠於第-7天i.v.投予5×105
個CD45-
細胞。免疫化7天後，於第0天對小鼠s.c.接種5×105
個EG7細胞。
(細胞激素之測定)
使用R＆D之ELISA套組測定小鼠IL-12p40、小鼠IL-13、及人類IFNγ之量。
(統計分析)
將包括Mann-Whitney之U檢定、Student之t檢定或Bonferroni之多重比較檢定在內之單向變方分析(one-way analysis of variance)用於統計分析(﹡p＜0.05；﹡﹡p＜0.01；﹡﹡﹡p＜0.001)。使用GraphPad Prism software(La Jolla、CA、USA)進行統計分析。
(實施例1：關於K3-SPG之靜脈內注射，即便完全未添加腫瘤抗原，亦會誘發較強之腫瘤生長之抑制)
於本實施例中，藉由K3-SPG之靜脈內注射，證明即便完全未添加腫瘤抗原，亦會誘發較強之腫瘤生長之抑制。
(利用EG7(表現OVA之小鼠胸腺瘤細胞株)模型之實驗)
於第0天對C57BL/6小鼠於右側腹部接種EG7(表現OVA之小鼠胸腺瘤細胞株)，經由尾基部附近之皮下(i.d.)投予、腫瘤內(i.t.)投予、或靜脈內(i.v.)投予之3種不同途徑，利用PBS、及等莫耳量之K3(30 μg)或K3-SPG(10 μg)進行3次處理(接種7、9、11天後)。每2～3天測定腫瘤尺寸直至第23天為止。
(結果)
結果示於圖2(A～B)以下。於PBS之群(對照)中，腫瘤生長經由任一投予途徑均未受到抑制(圖2a、b、c(圖2A))。於K3處理中，腫瘤縮小僅於i.t.中觀察到，於其他途徑中未觀察到(圖2d、e、f(圖2A))。於K3-SPG處理中，藉由i.t.及i.v.之兩者觀察到較強之腫瘤縮小，i.d.投予未顯示出對腫瘤生長之影響(圖2g、h、i(圖2A))。將比較對照、K3及K3-SPG而表示之圖示於圖(圖2a、d、g(圖2A))中。
於先前技術中，由於針對癌之全身性CpG ODN治療中之大部分嘗試並未成功(Lou, Y., et al. Journal of immunotherapy (Hagerstown, Md.: 1997) 34, 279-288 (2011); Nierkens, S., et al. PLoS One 4, e8368 (2009))，故而無法預測利用K3-SPG之i.v.單劑治療可較強地抑制腫瘤生長之事實，於該方面證明本發明產生了無法預料之效果。
(利用其他腫瘤細胞株之實驗)
為了調查該K3-SPG全身性單劑治療之潛能，其他腫瘤細胞株亦藉由在EG7模型中所使用之同樣之實驗方法進行試驗。
(結果)
K3-SPG之靜脈內投予亦抑制黑色素瘤(B16及B16F10)及結腸癌(MC38)之生長(圖2j、k、l(圖2B))。本發明者等人進而藉由製作臨床惡性度更高之腫瘤接種性模型而進行試驗。將小鼠胰臟腫瘤株、Pan02(1×106
細胞)接種至腹腔內(亦稱為「i.p.」)，其後，於接種11天後開始利用K3或K3-SPG之治療(每隔1天進行3次)。於第21天處死所有小鼠，評價腹腔之腫瘤之總重量(圖2m(圖2B))。關於腫瘤生長，於K3-SPG i.v.處理群中顯著地得到抑制，但於K3 i.p.及K3-SPG i.p.處理群中，未受到抑制(圖2m(圖2B))。與此相應地，於K3-SPG i.v.處理群中觀察到顯著之存活之延長，但於K3 i.v.群中未觀察到(圖2n(圖2B))。該等結果證明，K3-SPG之全身性i.v.投予係針對大部分不同之癌之有前景之單劑治療，進而亦無需任一腫瘤肽及抗原。
(實施例2：K3-SPG係於腫瘤微環境中以吞噬細胞作為標靶)
其次，本發明者等人闡明了K3-SPG之腫瘤微環境之機制。
K3-SPG形成約30 nm之尺寸之奈米粒子(Kobiyama, K., et al. Proc. Natl. Acad. Sci. U. S. A. 111, 3086-3091 (2014))。本發明者等人假設K3-SPG係經由及至腫瘤之藥物傳遞系統而發揮功能(Na, J.H., et al. Journal of controlled release: official journal of the Controlled Release Society 163, 2-9 (2012); Petros, R. A. et al. Nat Rev Drug Discov 9, 615-627 (2010); Pante, N. et al. Molecular biology of the cell 13, 425-434 (2002); Davis, M. E., et al. Nat Rev Drug Discov 7, 771-782 (2008); Farokhzad, O. C. et al. ACS Nano 3, 16-20 (2009))。
(螢光標記成像)
為了對活體內之分佈進行試驗，將K3及K3-SPG進行螢光標記。對帶有EG7腫瘤之小鼠i.v.注射PBS、Alexa647-K3(30 μg)、或Alexa647-K3-SPG(10 μg)，其後利用活體內成像系統(IVIS)對螢光之分佈進行試驗。
將結果示於圖4以下。
IVIS成像表明，於i.v.投予1小時後，並非K3蓄積於腫瘤部位，而是K3-SPG蓄積於腫瘤部位(圖4a)。觀察到腫瘤中之K3-SPG之蓄積係與CpG單劑治療之腫瘤縮小之有效性密切相關(圖2)。於免疫組織化學(IHC，Immunology Histology Chemistry)試驗中，本發明者等人未能於腫瘤微環境中檢測出Alexa647-K3(圖4b)。另一方面，Alexa647-K3-SPG於腫瘤區域中被觀察到(圖4c)。本發明者等人於24小時後未能檢測到IHC中之Alexa647訊號。EG7細胞於其表面上表現CD3e(其原因在於EG7係源自胸腺瘤細胞株)，K3-SPG未聚集於CD3e，此顯示出K3-SPG係被非腫瘤細胞取入。奈米粒子係選擇被巨噬細胞及樹狀細胞(DC)等吞噬細胞取入，該等細胞可於活體內藉由TRITC(Tetramethyl Rhodamine Iso-thiocyanate，四甲基異硫氰酸羅丹明)-葡聚糖進行標記。因此，本發明者將經螢光染色之K3、K3-SPG、或SPG及TRITC-葡聚糖進行靜脈內注射，對利用IHC之該等之共存進行試驗(圖4d、e、f)。i.v.注射1小時後，葡聚糖於全部試樣之腫瘤區域中被觀察到(圖4d、e、f)，此顯示出腫瘤微環境包含吞噬細胞。與先前之結果一致，Alexa647-K3於腫瘤內未被觀察到(圖4d)。於腫瘤內觀察到之約50%之Alexa647-K3-SPG及FITC-SPG與TRITC-葡聚糖陽性細胞共存(圖4e、f、g)，此顯示出於腫瘤微環境中K3-SPG被吞噬細胞取入。一部分K3-SPG未與葡聚糖聚集，本發明者等人推測藉由增強血管透過性及增強滲透滯留(EPR，Enhanced permeability and retention)效應，該等被動地蓄積於腫瘤組織內之空間。為了試驗吞噬細胞對K3-SPG i.v.處理之重要性，本發明者等人將氯屈膦酸鹽脂質體進行靜脈內注射。本發明者等人並未使用通常之200～300 nm之脂質體，而是使用100 nm之氯屈膦酸鹽脂質體，並將其注射，藉此使腫瘤內之吞噬細胞耗竭(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))，藉由該注射，腫瘤中之F4/80陽性細胞於2天內幾乎耗竭(圖5)。於第5天(最初之K3-SPG處理之2天前)對帶有腫瘤之小鼠注射氯屈膦酸鹽脂質體，或不注射，與圖2(A～B)相同地利用K3-SPG對小鼠進行處理。於先注射氯屈膦酸鹽脂質體之情形時，顯著地抵消K3-SPG介導腫瘤生長之抑制(p＜0.05)(圖4h)，另一方面，氯屈膦酸鹽脂質體之注射本身與PBS處理小鼠相比，未對腫瘤生長產生影響。該等結果顯示，K3-SPG係以腫瘤微環境中之吞噬細胞作為標靶，K3-SPG之抗腫瘤效果大部分依賴於K3-SPG向腫瘤微環境中之吞噬細胞中之導入。
(實施例3：產生腫瘤微環境中之IL12及IFN-I細胞激素之兩者對K3-SPG單劑治療較重要)
其次，於本實施例中，本發明者等人對K3-SPG單劑治療之成功所需之因子進行試驗。
揭示有IL-12及IFN-I等細胞激素係包含K3-SPG(Kobiyama, K., et al. Proc. Natl. Acad. Sci. U. S. A. 111, 3086-3091 (2014))之CpG ODN(Krieg, A. M., et al. Journal of immunology 161, 2428-2434 (1998); Klinman, D. M., et al. Immunity 11, 123-129 (1999); Ishii, K. J., et al. Current opinion in molecular therapeutics 6, 166-174 (2004))之重要免疫刺激因子。因此，本發明者等人針對IL-12及IFN-I對於利用K3-SPG之治療之腫瘤縮小是否為必須進行了試驗。
於第0天對Il12p40及IFNAR2缺損小鼠皮下接種EG7細胞，如圖2(A～B)般利用PBS或K3-SPG(10 μg)進行3次i.v.處理。其後，觀察K3-SPG對腫瘤縮小之影響。
(結果)
將結果示於圖6(A～B)以下。K3-SPG對腫瘤縮小之影響部分地依賴於IL-12p40及IFN-I訊號傳遞(圖6a、b(圖6A))。又，本發明者等人對IL12p40及IFNAR2之雙剔除(DKO)小鼠進行試驗，發現K3-SPG之影響於DKO小鼠中被完全抑制(圖6c(圖6A))。IFN-β及IL-12p40亦因IHC染色而於腫瘤內被檢測到(圖7、圖8)。該等資料顯示，腫瘤內之IL12p40及IFN-I細胞激素之兩者之分泌對K3-SPG介導性腫瘤抑制較重要。
又，本發明者等人對完全缺損T細胞及B細胞介導性之適應免疫應答之Rag2小鼠進行試驗。得知關於Rag2小鼠，即便進行K3-SPG處理，亦無法控制所有腫瘤生長(圖6d(圖6A))，但本發明者等人於利用K3-SPG之3次處理之期間，成功地部分控制rag2缺損小鼠之腫瘤生長(圖6f(圖6A))。為了確認該觀察，本發明者等人製作rag2小鼠之6次處理群(第7、9、11天及第14、16、18天)，並於rag2小鼠之該實驗方法中，發現更明顯之腫瘤控制(圖6f(圖6A))。頗具意味的是，IL12p40及IFNAR2 DKO小鼠即便藉由該範圍較寬之處理實驗方法，亦對K3-SPG單劑治療完全無應答(圖6e(圖6A))。該等資料顯示，K3-SPG之治療會誘發腫瘤內IL-12p40及IFN-I之兩者，其結果為，產生對腫瘤之自然免疫應答及適應免疫應答之兩者。
(實施例4：K3-SPG處理於依賴於IL12p40及IFN-I之兩者之態樣中，會誘發腫瘤細胞死亡)
於本實施例中，證明K3-SPG處理於依賴於IL12p40及IFN-I之兩者之態樣中，會誘發腫瘤細胞死亡。
由於觀察到於rag2小鼠中觀察到之不依賴於適應免疫之部分腫瘤生長之抑制、及IL12p40及IFNAR2 DKO小鼠中之不依賴於適應免疫之部分腫瘤生長之完全抑制，故而本發明者等人對範圍較寬之K3-SPG處理中之腫瘤-宿主交互作用進行試驗。
本發明者等人發現，於第12天摘除之脾臟(利用K3-SPG之3次處理之第二天)與經PBS處理之脾臟相比，包含大量CD45陰性細胞(圖6g(圖6B))。頗具意味的是，該等CD45陰性細胞於IL12p40及IFNAR2 DKO小鼠中顯著地減少(圖6g、h(圖6B))。本發明者等人分選該等CD45陰性細胞。該尺寸及形態充分顯示該等係源自腫瘤細胞。藉由對GFP小鼠之EG7接種實驗，進一步確認該等CD45陰性細胞亦為GFP陰性，該情況顯示該等細胞係源自腫瘤細胞(圖9)。由於EG7細胞未表現CD45，故而CD45為陰性之情況亦支持該假設。關於Hoechst及PI染色，脾臟中之大部分CD45陰性細胞係包含凋亡及壞死之兩者之特徵的死細胞(圖6i(圖6B))。該等資料顯示，因K3-SPG而成為標靶之腫瘤吞噬細胞於腫瘤微環境中分泌IL-12p40及IFN-I，該等細胞激素會誘發腫瘤細胞死亡，並循環地釋出該等，而最終被脾臟捕捉到。
(實施例5：所釋出之腫瘤死細胞誘發針對複數個腫瘤抗原之抗腫瘤CTL)
於本實施例中，證明所釋出之腫瘤死細胞誘發針對複數個腫瘤抗原之抗腫瘤CTL。
為了對在經K3-SPG處理之小鼠之脾臟中發現之該等CD45陰性細胞之免疫原性進行試驗，本發明者等人分選該等細胞，作為免疫化而對未處理小鼠進行靜脈內注射。其後，於投予經分選之細胞7天後，對經免疫化之小鼠移植EG7腫瘤細胞。CD45陰性細胞免疫化小鼠對EG7腫瘤之增生顯著地進行防禦(圖6j(圖6B))。頗具意味的是，對照小鼠及免疫化小鼠中之OVA257四聚物陽性細胞(圖6k(圖6B)中之紅點)顯示出不與腫瘤尺寸相關(圖6k(圖6B)之柱體)，且關於因CD45陰性細胞所引起之免疫化，相較於單純之OVA257抗原決定基，會誘發針對EG7腫瘤之更有效之進一步之免疫應答(圖6k(圖6B))。該等結果顯示，K3-SPG單劑治療會誘發依賴於IL-12及IFN-I之兩者之腫瘤細胞死亡，該腫瘤死細胞係作為用於抗腫瘤免疫應答之有效之免疫原而發揮功能。
(CD8T細胞係對於K3-SPG介導性之腫瘤縮小而言重要之效應物)
Rag2小鼠之結果顯示，K3-SPG之腫瘤抑制效果亦依賴於適應免疫應答。因此，本發明者等人對K3-SPG治療所需之CD8T細胞進行試驗。顯示活體內之CD8 T細胞之耗竭顯著地抑制K3-SPG之抗腫瘤效果(圖10a(圖10A))，CD8T細胞係該K3-SPG治療中之重要效應細胞。又，顯示利用K3-SPG之腫瘤縮小亦依賴於Batf3(缺損交叉呈現(cross presentation)CD8α+
DC)(圖10b(圖10A))，且K3-SPG單劑治療亦增強CD8α+
DC介導***叉呈現。本發明者等人觀察到CD8T細胞之腫瘤浸潤與腫瘤生長間之明顯之相關性。CD8T細胞於K3-SPG i.v.群中係蓄積於腫瘤區域中，但於i.d.群中未蓄積(圖10c(圖10A))。
最後，本發明者等人對為了使該等CD8T細胞進入至腫瘤區域而必須者進行試驗。於第0天對WT小鼠及Il12p40-Ifnar2 DKO小鼠接種EG7細胞，並於第7、9、及11天利用K3-SPG或PBS進行i.v.處理。於第14天，自該等小鼠之脾臟中純化CD8α+
T細胞，利用Xenolight DiR(註冊商標)進行染色，並移植至經K3-SPG處理之(第7、9、及11天)其他帶有EG7之小鼠(接種14天後)，其後藉由IVIS於第15天分析利用Xenolight DiR(註冊商標)進行標記之CD8T細胞之分佈(圖11)。於第15天，關於源自帶有未處理腫瘤之供體小鼠之CD8T細胞，即便利用K3-SPG進行處理，亦未蓄積於WT受移植小鼠之腫瘤部位(圖10d(圖10B)、II)。另一方面，源自帶有利用K3-SPG進行處理之腫瘤之供體小鼠之CD8T細胞於受移植小鼠之腫瘤部位被檢測到(圖10d(圖10B)、I)，顯示K3-SPG單劑治療誘發可向腫瘤微環境移動並進行浸潤之抗腫瘤CD8T細胞。該等活體內活化CD8T細胞可進入至DKO受移植小鼠之腫瘤微環境(圖10e(圖10B))。即便IL-12及IFN-I對因全身性之K3-SPG單劑治療所產生之自然免疫及CD8T細胞之誘發較重要，其結果亦顯示，若CD8T細胞於K3-SPG治療中經活化，則腫瘤微環境中之IL-12及IFN-I細胞激素之分泌對於CD8T細胞之腫瘤浸潤而言未必必須。總之，該等結果顯示，腫瘤特異性CD8T細胞之活化對於向腫瘤之浸潤而言充分。令人驚訝的是，該等CD8T細胞之浸潤不依賴於腫瘤微環境中之細胞激素之產生。
(探討)
本發明者等人揭示了新穎癌免疫療法之可能性。其係CpG經腫瘤微環境中之吞噬細胞標靶化之新穎治療(圖12)。經由TLR9之刺激，CpG誘發免疫細胞之免疫應答，尤其是將巨噬細胞及DC活化(Klinman, D. M., et al. Immunity 11, 123-129 (1999); Ishii, K. J., et al. Current opinion in molecular therapeutics 6, 166-174 (2004))。該活化對抗癌免疫應答非常重要。於先前之報告中，必須直接向腫瘤內投予CpG，但若為SPG與CpG之複合體，則即便於附加有DDS(Drug Delivery System，藥物傳遞系統)功能之全身投予中，亦顯示出與腫瘤內投予同等或者其以上之有效性(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, K. J., et al. Current opinion in molecular therapeutics 6, 166-174 (2004))，本發明者等人解決了該課題。因奈米粒子之形成所產生之SPG與CpG之複合體(Kobiyama, K., et al. Proc. Natl. Acad. Sci. U. S. A. 111, 3086-3091 (2014))可於活體內穩定化。得知該效果能以腫瘤環境作為標靶，TLR9免疫活性細胞被供於腫瘤環境中。關於由本發明者等人開發出之該新穎CpG，由於形成奈米粒子，故而被吞噬細胞吞噬。
其後，於腫瘤環境中，吞噬有該新穎CpG之吞噬細胞產生IFN及IL-12等細胞激素。非常重要的是該等細胞激素於腫瘤環境中得以誘發。於先前之報告中記載有以腫瘤環境作為直接標靶之IFNβ治療中，使樹狀細胞於腫瘤內移動，增加腫瘤內微環境內之抗原交叉呈現，藉此使CTL再活化。該等細胞激素會引起腫瘤細胞之細胞死亡。進而，本發明者等人發現該效果係由自然免疫之活化所引起。該細胞死亡承擔非常重要之作用。其成為自然免疫與適應免疫間之協作。藉由自腫瘤微環境釋出腫瘤細胞死亡，而誘發後天性免疫。該免疫原性腫瘤細胞死亡會誘發複數個細胞毒殺性T淋巴球。如上所述般活體內之腫瘤特異性誘發之CTL可對腫瘤進行應答而浸潤於腫瘤微環境。認為該抗腫瘤免疫系統可使用內因性抗原而應對作為癌免疫療法之障礙之免疫編輯(Immunoediting)。
K3-SPG單劑治療後之腫瘤細胞之循環可作為針對腫瘤之處理效果優異之生物標記物而發揮功能。
(實施例6：製劑例)
以下，揭示進行製劑化之情形時之組成
製劑例如係使7.22 mg之K3-dA40
(序列編號2)溶解於水(3.7 mL)中，並使SPG(15 mg)溶解於0.25 N之NaOH(1 mL)中。將1 mL之容積之330 mM NaH2
PO4
添加至DNA溶液中，繼而將SPG溶液添加至該DNA/NaH2
PO4
溶液中，並於4℃下維持一晩，而完成複合體化。莫耳比(MSPG
/MDNA
)可固定為0.27而製造。
製劑中所使用之藥劑可自Genedesign、invivogen、Wako等獲取。
如上所述，使用本發明之較佳實施形態對本發明進行了例示，但理解為本發明應僅藉由申請專利範圍對其範圍進行解釋。於本說明書中，關於所引用之專利、專利申請及文獻，理解為與將其內容本身具體地記載於本說明書中之情況同樣地，其內容應以對本說明書之參考之形式引用。
[產業上可之利用性]
根據本發明，可提供能以單劑之形式使用之新穎形態之抗癌劑。因此，本發明之複合體作為抗癌劑而於醫藥領域有用。
[序列表自由內容]
序列編號1：K3
序列編號2：K3-dA40
序列編號3：dA40
-K3
序列編號4：K3-dA20
序列編號5：K3-dA25
序列編號6：K3-dA30
序列編號7：K3-dA35In the following, the best mode will be disclosed, and the invention will be described. Throughout this specification, expressions in the singular form should be understood to include the concept of the plural form unless otherwise specified. Therefore, articles in the singular form (for example, "a", "an", "the", etc. in the case of English) should be understood to include the concept of the plural form unless otherwise specified. In addition, the terms used in this specification are understood to be used based on the meanings generally used in the field unless otherwise specified. Therefore, unless otherwise defined, all technical terms and scientific and technical terms used in this specification have the same meaning as those generally understood by practitioners in the field to which the present invention belongs. In case of conflicts, this specification (including definitions) shall prevail. In the following, the definitions and/or basic technical contents of terms particularly used in this specification will be explained as appropriate. The present invention provides an oligodeoxynucleotide comprising K-type CpG oligodeoxynucleotide and polydeoxyadenosine (dA) (hereinafter, referred to as the oligodeoxynucleotide of the present invention). The oligodeoxynucleotides of the present invention include those in which phosphodiester bonds are modified (for example, part or all of the phosphodiester bonds are replaced by phosphorothioate bonds). The oligodeoxynucleotide of the present invention contains a pharmaceutically acceptable salt. In this specification, the so-called "CpG oligonucleotide (residue)" or "CpG oligodeoxynucleotide (residue)", "CpG ODN (residue)" or only "CpG (residue)""Refers to a polynucleotide, preferably an oligonucleotide, containing at least one unmethylated CG dinucleotide sequence that can be used interchangeably, with or without the term "residue" at the end the same. The oligonucleotide comprising at least one CpG motif can comprise a plurality of CpG motifs. When used in this specification, the term "CpG motif" refers to an unmethylated dinucleotide of an oligonucleotide containing cytosine nucleotides and subsequent guanosine nucleotides section. Instead of cytosine, 5-methylcytosine can also be used. Furthermore, polydeoxyadenylic acid has the same meaning as polydeoxyadenylic acid (residue). The term "residue" refers to a partial structure of a compound with a larger molecular weight. In this specification, "CpG oligodeoxynucleotide (CpG ODN)" means an independent molecule or a compound with a larger molecular weight. Part of the structure, as long as it is a practitioner, can be easily understood by the context. The terms related to "polydeoxyadenosine" and other parts of the structure contained in the oligodeoxynucleotide of the present invention are also the same. CpG oligonucleotide (CpG ODN) is a short (about 20 base pairs) single-stranded synthetic DNA fragment containing immunologically active CpG motifs, and is a powerful Toll-like receptor 9 (TLR9) The agonist is used to activate dendritic cells (DCs) and B cells to produce type I interferons (IFNs) and inflammatory cytokines (Hemmi, H., et al. Nature 408, 740-745 (2000) ; Krieg, AM Nature reviews. Drug discovery 5, 471-484 (2006).), including cytotoxic T lymphocyte (CTL) response to Th1 type humoral and cellular immune response adjuvant (Brazolot Millan, CL, Weeratna, R., Krieg, AM, Siegrist, CA & Davis, HL Proceedings of the National Academy of Sciences of the United States of America 95, 15553-15558 (1998).; Chu, RS, Targoni , OS, Krieg, AM, Lehmann, PV & Harding, CV The Journal of experimental medicine 186, 1623-1631 (1997)). Therefore, CpG ODN is regarded as an immunotherapeutic agent with the possibility of infection, cancer, asthma and hay fever (Krieg, AM Nature reviews. Drug discovery 5, 471-484 (2006); Klinman, DM Nature reviews. Immunology 4 , 249-258 (2004)). CpG oligodeoxynucleotide (CpG ODN) is a single-stranded DNA containing an immunologically active non-methylated CpG motif and is an agonist of TLR9. CpG ODN has four types of K-type (also known as B-type), D-type (also known as A-type), C-type and P-type with different backbone sequences and immune activation characteristics (Advanced drug delivery reviews 61, 195- 204 (2009)). The oligodeoxynucleotide of the present invention includes the K-type CpG ODN among these. K-type CpG ODN typically contains multiple non-methylated CpG motifs of non-palindrome structure, which activates B cells to produce IL-6, but has IFN that hardly induces plasmacytoid dendritic cells (pDCs) -CpG ODN of structural and functional properties produced by α. The so-called unmethylated CpG module system contains at least one shorter nucleotide sequence of cytosine (C)-guanine (G) sequence, which means that the 5-position of cytosine in the cytosine-guanine sequence is not Methylated. In the following description, CpG refers to unmethylated CpG unless otherwise specified. Therefore, the oligodeoxynucleotide of the present invention contains K-type CpG ODN and has an immunological activation activity unique to K-type CpG ODN (for example, activation of B cells (preferably human B cells) to produce IL- 6 activity). In this technical field, a large number of humanized K-type CpG ODNs are known (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. The so-called "humanization" refers to having agonist activity against human TLR9. Therefore, the oligodeoxynucleotides of the present invention containing humanized K-type CpG ODN have immunologically activating activity unique to K-type CpG ODN to humans (for example, the activity of activating human B cells to produce IL-6). The K-type CpG ODN that can be suitably used in the present invention is 10 nucleotides or more in length and includes the formula: [Chem 1] (In the formula, the central CpG motif is not methylated, W is A or T, N 1 , N 2 , N 3 , N 4 , N 5 And N 6 It can also be any nucleotide sequence represented by nucleotides. In one embodiment, the K-type CpG ODN of the present invention has a length of 10 nucleotides or more and includes the nucleotide sequence of the above formula. However, in the above formula, the central 4 base CpG motif (TCpGW) only needs to be included in 10 nucleotides, and it is not necessary to be located at N in the above formula 3 And N 4 between. Also, in the above formula, N 1 , N 2 , N 3 , N 4 , N 5 And N 6 Can be any nucleotide, N 1 And N 2 , N 2 And N 3 , N 3 And N 4 , N 4 And N 5 , And N 5 And N 6 At least any one (preferably one) combination may also be a 2 base CpG motif. The CpG motif at the above 4 bases is not located at N 3 And N 4 In the case of the above, in the above formula, any two consecutive bases of the 4 bases in the center (4th to 7th bases) are CpG motifs, and the other 2 bases may be any nucleotides . The K-type CpG ODN that can be more suitably used in the present invention includes a non-palindrome structure containing one or a plurality of CpG motifs. The K-type CpG ODN that can be used more suitably includes a non-palindrome structure containing one or a plurality of CpG motifs. The humanized K-type CpG ODN is usually characterized by a CpG motif containing 4 bases of TCGA or TCGT. In addition, in most cases, one humanized K-type CpG ODN contains 2 or 3 of these 4 base CpG motifs. Therefore, in a preferred embodiment, the K-type CpG ODN contained in the oligodeoxynucleotide of the present invention contains at least 1, more preferably 2 or more, and further preferably 2 or 3 include TCGA or TCGT The 4 base CpG motif. In the case where the K-type CpG ODN has 2 or 3 CpG motifs of 4 bases, the CpG motifs of these 4 bases may be the same or different. 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 preferably includes the nucleotide sequence represented by SEQ ID NO: 1. The length of the K-type CpG ODN is not particularly limited as long as the oligodeoxynucleotide of the present invention has immunological activation activity (for example, the activity of activating B cells (preferably human B cells) to produce IL-6). It is preferably 100 nucleotides or less in length (for example, 10 to 75 nucleotides in length). The length of the K-type CpG ODN is more preferably 50 nucleotides or less (for example, 10 to 40 nucleotides in length). The length of the K-type CpG ODN is preferably 30 nucleotides or less (for example, 10 to 25 nucleotides in length). The length of K-type CpG ODN is preferably 12-25 nucleotides in length. The length of polydeoxyadenosine (dA) is sufficient as long as it forms a triple-helix structure together with β-1,3-glucan (preferably lentinan or Schizophyllum polysaccharide) chain. The length is not particularly limited. From the viewpoint of forming a stable triple-helix structure, it is usually 20 nucleotides or longer, preferably 40 nucleotides or longer, and more preferably 60 nucleotides or longer. . The longer the poly dA, the more stable a triple helix structure with β-1,3-glucan, so there is no theoretical upper limit, but if it is too long, the length will be reduced when synthesizing oligodeoxynucleotides Since there is unevenness in the aspect, it is usually 100 nucleotides or less in length, preferably 80 or less. On the other hand, in addition to forming the above-mentioned stable three-strand helix structure, the amount of the oligodeoxynucleotide of the present invention bonded to each unit amount of β-1,3-glucan is increased and synthesis of oligomers is avoided From the viewpoint of uneven length of deoxynucleotides and compounding efficiency, the length of polydA 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 in length), more preferably 30-50 nucleotides in length (30, 31, 32, 33, 34, 35 , 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides in length) 18, preferably 30 to 45 nucleotides in length ( 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 nucleotides in length). Especially when the length is more than 30 nucleotides, it shows good recombination efficiency. The oligodeoxynucleotide of the present invention has an activity of forming a triple helix structure together with two Schizophyllum polysaccharide chains by including poly dA. Furthermore, there are also cases where polydeoxyadenylic acid is expressed as "poly(dA)" or "poly(dA)". In one molecule of the oligodeoxynucleotide of the present invention, a plurality of K-type CpG ODN and/or poly-dA may also be contained, preferably one K-type CpG ODN and one poly-dA each, most preferably including One K-type CpG ODN and one poly-dA each. Exemplary CpG sequences include K3 CpG (sequence number 1: 5'-atcgactctcgagcgttctc-3'), etc., but it is not limited thereto. The oligodeoxynucleotide of the present invention is characterized in that the polydA is disposed on the 3'side of the K-type CpG ODN. It is considered that with this configuration, the anticancer effect of the complex of the present invention (details will be described below) may be enhanced, but it is not limited to these, and may be combined with any one as an anticancer agent. The K-type CpG ODN and poly-dA can be linked by a direct covalent bond or via a gap subsequence. The so-called gap sequence refers to a nucleotide sequence including more than one nucleotide inserted between two close constituent elements. Regarding the length of the gap sequence, as long as the complex of the present invention has immunostimulating activity (preferably the activity of activating B cells to produce IL-6 and the activity of dendritic cells to produce IFN-α), there is no In particular, it is usually 1 to 10 nucleotides in length, preferably 1 to 5 nucleotides in length, and more preferably 1 to 3 nucleotides in length. The best is that the K-type CpG ODN and poly-dA are connected by a direct covalent bond. In addition to the K-type CpG ODN, polydA, and any interstitial sequences, the oligodeoxynucleotide of the present invention may also have additional nucleotide sequences at its 5'end and/or 3'end. Regarding the length of the additional nucleotide sequence, as long as the complex of the present invention has immunostimulating activity (preferably, the activity of activating B cells to produce IL-6 and the activity of dendritic cells to produce IFN-α) It is not particularly limited, and is usually 1 to 10 nucleotides in length, preferably 1 to 5 nucleotides in length, and more preferably 1 to 3 nucleotides in length. In a preferred aspect, the oligodeoxynucleotide of the present invention does not contain such additional nucleotide sequences at the 5'end and/or the 3'end. That is, the oligodeoxynucleotide of the present invention preferably includes K-type CpG ODN, poly-dA, and any spacer sequences, and more preferably includes K-type CpG ODN and poly-dA. In the best form, the oligodeoxynucleotide of the present invention includes K-type CpG ODN (specifically, for example, an oligodeoxynucleotide including the nucleotide sequence represented by SEQ ID NO. 1) and poly dA, K-type CpG ODN is located at the 5'end of the oligodeoxynucleotide, and poly dA is located at the 3'end. Specifically, the 3'end of the oligodeoxynucleotide containing the nucleotide sequence represented by SEQ ID NO: 1 has a length of 20 to 60 nucleotides (more preferably 30 to 50 nucleosides) Acid length (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides in length) , The best is 30 to 45 nucleotides in length (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 nucleotides in length) ) Of the polydA oligodeoxynucleotide, for example, an oligodeoxynucleotide containing the nucleotide sequence represented by SEQ ID NO. 2, or 9-12. The total length of the oligodeoxynucleotide of the present invention is usually 30 to 200 nucleotides in length, preferably 35 to 100 nucleotides in length, and more preferably 40 to 80 nucleotides in length (specifically Words, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 nucleotides in length), more preferably 50 to 70 nucleotides Length (specifically, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 nucleosides Acid length), preferably 50 to 65 nucleotides in length (specifically, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 nucleotides in length). The oligodeoxynucleotides of the present invention can also be suitably modified in such a way as to exhibit resistance to decomposition in vivo (for example, decomposition caused by external or internal nucleases). Preferably, the modification includes phosphorothioate modification or phosphorodithioate modification. That is, part or all of the phosphodiester bonds in the oligodeoxynucleotide of the present invention are replaced with phosphorothioate bonds or phosphorodithioate bonds. Preferably, the oligodeoxynucleotide of the present invention contains a modification of phosphodiester bonds, and more preferably the modification of phosphodiester bonds is phosphorothioate bonds (ie, as described in WO 95/26204, non- One of the cross-linked oxygen atoms is replaced with a sulfur atom). That is, part or all of the phosphodiester bonds in the oligodeoxynucleotide of the present invention are replaced with phosphorothioate bonds. With regard to the oligodeoxynucleotide of the present invention, it is preferable that the K-type CpG ODN includes a modification using a phosphorothioate bond or a phosphorodithioate bond, and it is more preferable that the K-type CpG ODN has a phosphate All of the ester bonds are replaced with phosphorothioate bonds. In addition, regarding the oligodeoxynucleotide of the present invention, it is preferable to include a phosphorothioate bond or a phosphorodithioate bond in the polydA, and it is more preferable that all the phosphodiester bonds of the polydA are Replaced by phosphorothioate bonds. It is further preferred that all of the phosphodiester bonds of the oligodeoxynucleotides of the humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylate are replaced with phosphorothioate bonds . Preferably, the oligodeoxynucleotide of the present invention is bound to the 3'end of a humanized K-type CpG oligodeoxynucleotide (for example, SEQ ID NO: 1) and has a length of 20 to 60 nucleotides (more Preferably, it is 30-50 nucleotides in length (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50 nucleotides in length), preferably 30 to 45 nucleotides in length (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 , 45 nucleotides in length))), and all the phosphodiester bonds contained in the oligodeoxynucleotide are replaced with phosphorothioate bonds. The reason for this is that the phosphorothioate bond not only anticipates resistance to decomposition, but also immunological activation activity (for example, the activity to generate IFN-α by activating pDC) for the oligodeoxynucleotide of the present invention. Enhancement, and high yield of CpG-β-1,3-glucan complex, and enhancement of anticancer activity. In this specification, the phosphorothioate bond has the same meaning as the phosphorothioate skeleton, and the phosphodiester bond has the same meaning as the phosphoric acid skeleton. The oligodeoxynucleotides of the present invention include all pharmaceutically acceptable salts, esters, or salts of such esters of the oligodeoxynucleotides described above. As the pharmaceutically acceptable salts of the oligodeoxynucleotide of the present invention, alkali metal salts such as sodium salts, potassium salts, and lithium salts, and alkaline earth metals such as calcium salts and magnesium salts can be suitably cited. Salt, aluminum salt, iron salt, zinc salt, copper salt, nickel salt, cobalt salt and other metal salts; inorganic salts such as ammonium salt, third octylamine salt, dibenzylamine salt, morpholine salt, glucose Amine salt, phenylglycine alkyl ester 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, piper Amine salts such as organic salts such as salts, tetramethylammonium salts, tris(hydroxymethyl)aminomethane salts; hydrohalides such as hydrofluoride, hydrochloride, hydrobromide, hydroiodide Salts, nitrates, perchlorates, sulfates, phosphates and other inorganic acid salts; methanesulfonate, trifluoromethanesulfonate, ethanesulfonate and other lower alkanesulfonates, benzenesulfonate , Arylsulfonates such as p-toluenesulfonate, acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleate And other organic acid salts; and amino acid salts such as glycinate, imidate, spermine, ornithate, glutamate, and aspartate. The oligodeoxynucleotide of the present invention may be in any form of single-stranded, double-stranded, or triple-stranded, preferably single-stranded. The oligodeoxynucleotide of the present invention is preferably isolated. The so-called "single separation" refers to the operation of removing the factors other than the target component, and leaving the natural state. The purity of the "isolated oligodeoxynucleotide" (the percentage of the weight of the target oligodeoxynucleotide in the total weight of the evaluation object) is usually 70% or more, preferably 80% or more , More preferably 90% or more, and further preferably 99% or more. The oligodeoxynucleotide of the present invention has excellent immune activating activity (for example, the activity of activating B cells (preferably human B cells) to produce IL-6), and therefore is useful as an immune activating agent and the like. Furthermore, the oligodeoxynucleotide of the present invention has a triple-helix structure together with two β-1,3-glucans (preferably Schizophyllum polysaccharide, lentinan or scleroglucan). The properties are therefore useful for the preparation of the composite of the present invention. The present invention provides a complex containing the above-described oligodeoxynucleotide of the present invention and β-1,3-glucan (hereinafter, referred to as the complex of the present invention). Since the oligodeoxynucleotide of the present invention contains K-type CpG ODN, it alone exhibits immunological activation activity unique to K-type CpG ODN (for example, activation of B cells (preferably human B cells) to produce IL -6 activity), lacking the immuno-activating activity unique to D-type CpG ODN (for example, the activity of activating plasma-like dendritic cells to produce IFN-α). However, surprisingly, by forming a complex with β-1,3-glucan (preferably lentinan, Schizophyllum polysaccharide), the sequence of D-type CpG ODN is not required, and the D-type CpG is obtained. ODN-specific immunostimulating activity (for example, the activity of activating plasma-like dendritic cells to produce IFN-α). That is, the complex of the present invention has an immunological activation activity unique to K-type CpG ODN (for example, the activity of activating B cells (preferably human B cells) to produce IL-6), and an immunity unique to D-type CpG ODN Both activating activities (for example, activation of plasmacytoid dendritic cells (preferably human plasmacytoid dendritic cells) to produce IFN-α). Examples of β-1,3-glucan used in the present invention include Schizophyllum polysaccharide, scleroglucan, cardlan polysaccharide, Poria cocos polysaccharide, Grifola frondosa polysaccharide, lentinan, laminin, and the like. The β-1,3-glucan is preferably β-1, such as Schizophyllum polysaccharide, Lentinan or Scleroglucan, which contains a large amount of 1,6-glucoside branches (side chain ratio 33 to 40%). 3-glucan, more preferably Schizophyllum polysaccharide. Lentinan (LNT) is derived from the well-known β-1,3-1,6-glucan of Lentinula edodes, and its molecular formula is (C 6 H 10 O 5 ) n The molecular weight is about 300,000 to 700,000. Almost insoluble in water, methanol, ethanol (95), or acetone, but soluble in DMSO (dimethylsulfoxide, dimethyl sulfoxide) or aqueous sodium hydroxide solution as a polar organic solvent. Lentinan can enhance the activity of activated macrophages, killer T cells, natural killer cells and antibody-dependent macrophage-mediated cytotoxicity (ADMC, Antibody dependent monocyte cytotoxicity) (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, homologous tumors and autologous tumors were administered in combination with chemotherapeutic agents to confirm tumor hyperplasia inhibition and life extension effects. In addition, by administering lentinan alone, the tumor hyperplasia inhibitory effect and life extension effect were also confirmed. In clinical trials, for patients with inoperable or relapsed gastric cancer, by oral administration with tegafur, it was confirmed that the survival time was prolonged (medicine evaluation table "Lushman polysaccharide intravenous injection 1 mg "Ajinomoto") , And was recognized in Japan. The effect of the separate administration of lentinan has not been confirmed. Schizophyllum polysaccharide (SPG) is a well-known soluble β-glucan derived from Schizophyllum. SPG contains the main chain of β-(1→3)-D-glucan and one β-(1→6)-D-glucosyl side chain (Tabata, K., Ito , W., Kojima, T., Kawabata, S. and Misaki A., "Carbohydr. Res.", 1981, 89, 1, p. 121-135). SPG has been used as a clinical medicine for intramuscular injection of immunoenhancement method for gynecological cancer, and it has been used for more than 20 years (Qingshui, Chen, Hejian, Zengyuan, "Biotherapy", 1990, 4, p.1390 Hasegawa, "Oncology and Chemotherapy", 1992, 8, p. 225), and confirmed the safety in vivo (Theresa, M. McIntire and David, A. Brant, "J. Am. Chem. Soc. ", 1998, 120, p. 6909). In this specification, the so-called "complex" refers to a product obtained by aggregating a plurality of molecules through non-covalent bonds or covalent bonds such as electrostatic bonds, Van der Waals bonds, hydrogen bonds, hydrophobic interactions, and the like. The composite of the present invention is preferably in the shape of a triple-helix structure. In a preferred aspect, among the three chains forming the triple-helix structure, two are β-1,3-glucan chains, and one is the polymer in the oligodeoxynucleotide of the present invention described above Deoxyadenylate chain. Furthermore, the composite body may also include a part that does not form a triple helix structure. The composition ratio of oligodeoxynucleotide to β-1,3-glucan in the complex of the present invention can be determined according to the chain length of polydeoxyadenylic acid in oligodeoxynucleotide, And the length of β-1,3-glucan varies. For example, when the length of β-1,3-glucan chain and polydeoxyadenylate chain are equal, 2 β-1,3-glucan chains and 1 oligomeric Oxynucleotides can aggregate to form a triple helix structure. Generally, the chain length of polydeoxyadenylate is shorter than that of β-1,3-glucan chain, so a plurality of oligodeoxynucleotides of the present invention can target 2 β-1,3- The dextran chain aggregates via polydeoxyadenylic acid to form a triple-helix structure (see FIG. 1). The composite system of the present invention contains humanized K-type CpG ODN and β-1,3-glucan (such as lentinan, Schizophyllum polysaccharide, scleroglucan, cardlan polysaccharide, poria cocos, polysaccharide from Grifola frondosa, The complex of laminose) is preferably a complex containing humanized K-type CpG ODN and β-1,3-glucan (such as lentinan, Schizophyllan polysaccharide, and hard glucan). More preferably, the oligodeoxynucleotide contained in the nucleotide sequence represented by SEQ ID NO. 1 has a length of 20 to 60 nucleotides at 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 all of the phosphodiester bonds are replaced by thio Phosphate-bonded oligodeoxynucleotides and complexes of β-1,3-glucans (such as Lentinan, Schizophyllum polysaccharides) (such as K3-dA20～60-LNT, K3-dA20～60 -SPG), and it is more preferable that the oligodeoxynucleotide containing the nucleotide sequence represented by SEQ ID NO: 1 has a length of 30 to 50 nucleotides (specifically, 30 , 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides in length) A complex of oligodeoxynucleotides with adenylate and all phosphodiester bonds replaced by phosphorothioate bonds, and β-1,3-glucan (e.g. lentinan, Schizophyllan polysaccharide) (E.g. K3-dA30-50-LNT, K3-dA30-50-SPG), it is best to include the oligodeoxynucleotide containing the nucleotide sequence represented by SEQ ID No. 30 to 45 nucleotides in length (specifically, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 nucleotides in length) Oligodeoxynucleotides with polydeoxyadenylic acid and all phosphodiester bonds replaced by phosphorothioate bonds, and β-1,3-glucan (e.g. lentinan, Schizophyllan polysaccharide) ) Complex (K3-dA30～45-LNT, K3-dA30～45-SPG). The method for preparing the composite of the present invention can be carried out under the same conditions as described in Non-Patent Documents 21 to 24 or Japanese Patent Laid-Open No. 2008-100919. That is, β-1,3-glucan which is originally natural and exists in the form of a triple-helix structure is dissolved in an aprotic organic polar solvent (dimethyl sulfoxide (DMSO), acetonitrile, acetone, etc.) or alkaline In the aqueous solution (sodium hydroxide, potassium hydroxide, ammonia, calcium hydroxide, etc.) and untied into single chain. The solution of the single-stranded β-1,3-glucan obtained in the above manner and the solution of the oligodeoxynucleotide of the present invention (aqueous solution, buffer solution with a pH value near neutral, or acidic buffer) An aqueous solution, preferably an aqueous solution or a buffered aqueous solution with a pH value near neutral) is mixed, and if necessary, the pH value is adjusted to near neutral again, and then maintained for an appropriate time, for example, at 5°C overnight. As a result, the two β-1,3-glucan chains and the polydA chain in the oligodeoxynucleotide form a triple helix structure, thereby forming the complex of the present invention. By purification, ultrafiltration, dialysis, etc. of the resulting complex using size exclusion chromatography, oligodeoxynucleotides that have not formed a complex can be removed. Furthermore, by purifying the produced complex by anion exchange chromatography, β-1,3-glucan which has not formed a complex can be removed. By the above method, the complex can be appropriately purified. The formation of the complex of the present invention can be performed, for example, by measuring conformational changes using CD (Circular Dichroism) spectrum, UV (Ultra Violet) absorption shift using size exclusion chromatography, gel electrophoresis, It is confirmed by microchip electrophoresis and capillary electrophoresis, but it is not limited to this. The mixing ratio of the oligodeoxynucleotide and β-1,3-glucan of the present invention can be appropriately set in consideration of the length of the polydA chain, etc., and the molar ratio (SPG/ODN) is usually 0.02 to 2.0 , Preferably 0.1 to 0.5. In a further aspect, the molar ratio (β-1,3-glucan (LNT, etc.)/ODN) is, for example, 0.005 to 1.0, preferably 0.020 to 0.25. Taking the CpG-ODN and LNT complex as an example, the preparation method of the complex of the present invention will be described. Dissolve LNT in an alkaline aqueous solution (for example, 0.25 N sodium hydroxide aqueous solution) of 0.05 to 2 N, preferably 0.1 to 1.5 N, and stand at 1 to 40°C for 10 to 4 days (for example, at room temperature) After a while, a single-stranded LNT aqueous solution (for example, 50 mg/ml aqueous LNT solution) was prepared. Mix the above LNT aqueous solution with a separately prepared CpG aqueous solution (for example, 100 μM CpG aqueous solution) at a molar ratio (LNT/ODN) of 0.005 to 1.0, and then add an acidic buffered aqueous solution (such as NaH) to the above LNT aqueous solution 2 PO 4 ) And neutralized, maintained at 1 to 40 ° C for 6 hours to 4 days (for example, maintained at 4 ° C overnight), thereby ending the compounding. In addition, for the above compounding, an LNT aqueous solution may be finally added and mixed. The formation of the complex can be confirmed by, for example, the method of using size exclusion chromatography to monitor the absorption at 240 to 280 nm (eg, 260 nm) for the displacement of CpG ODN to the high molecular weight side. In one aspect, the composite of the present invention takes the form of rod-shaped particles. The particle size is equal to the diameter of particles formed by using β-1,3-glucan (such as Schizophyllan polysaccharide) which is used as a material and has a triple-helix structure, and the average particle size is usually 10 to 100 nm , Preferably 20-50 nm. Regarding the particle size, the complex can be dissolved in water and measured by a dynamic light scattering method using a Malvern Instruments Zeta Sizer at 80°C. The composite of the present invention is preferably isolated. The purity of the "separated complex" (the percentage of the weight of the target complex in the total weight of the evaluation object) is usually 70% or more, preferably 80% or more, more preferably 90% or more, and more Better than 99%. Furthermore, in addition to having anticancer activity, the complex of the present invention also has excellent immune activating activity, in particular, has a unique immune activating activity for K-type CpG ODN (for example, activation of B cells (preferably human B cells) and (IL-6 production activity), and immuno-activating activity unique to D-type CpG ODN (e.g., activation of plasmacytoid dendritic cells (preferably human plasmacytoid dendritic cells) to produce IFN-α)) Both are advantageous because they can also give effects as an immune activating agent. For example, regarding the complex containing K-type CpG ODN (for example, sequence numbers 2, 11, 12 and SPG) and the complex (K3-SPG) containing K-type CpG ODN (for example sequence number 2) and SPG, as an inflammatory response Inducibility (pan-IFN-a, IL-6, etc.), enhancement of antigen-specific IgG antibody titers (Total IgG, IgG2c, etc.) in serum of virus-vaccinated individuals, antigen-specific cytokines in virus-vaccinated individuals The ability to produce (IFN-γ, IL2, etc.) and the defense against virus infection are also advantageous. (Pharmaceutical composition) The present invention provides an oligodeoxynucleotide containing the above-mentioned present invention or a complex of the above-mentioned present invention Pharmaceutical composition. 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 a conventional method. The pharmaceutical composition of the present invention includes the Oligodeoxynucleotides or complexes and pharmacologically acceptable carriers. Furthermore, the pharmaceutical composition may further contain an antigen. Such a pharmaceutical composition is made suitable for oral or parenteral administration Provided as a dosage form. As a composition for parenteral administration, for example, injections, suppositories, etc. may be used, and injections may also include dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, and drip injections. Such injections may be It is prepared according to a well-known method. As an injection preparation method, for example, it can be prepared by dissolving or suspending the above-mentioned oligodeoxynucleotide or complex of the present invention in a sterile aqueous solvent generally used in injections. Aqueous solvents for injection, for example, distilled water; physiological saline; buffers such as phosphate buffer, carbonate buffer, trishydroxymethylaminomethane buffer, and acetate buffer, etc. The pH of such aqueous solvents can be exemplified by 5 ～10, preferably 6～8. The prepared injection is preferably filled into an appropriate ampoule. Alternatively, the suspension of the oligodeoxynucleotide or complex of the present invention can also be used Carry out vacuum drying, freeze drying and other treatments to prepare the powder preparation of the oligodeoxynucleotide or complex of the present invention. The oligodeoxynucleotide or complex of the present invention can be stored in a powder state , And when used, the powder is dispersed in an aqueous solvent for injection and used. The content of the oligodeoxynucleotide or complex of the present invention in the pharmaceutical composition is usually the whole of the pharmaceutical composition About 0.1 to 100% by weight, preferably about 1 to 99% by weight, and more preferably about 10 to 90% by weight. The pharmaceutical composition of the present invention may contain the oligodeoxynucleotide of the present invention alone or complex As an active ingredient, it can also be combined with other active ingredients to contain the oligodeoxynucleotide or complex of the present invention. (medical use) The oligodeoxynucleotide and complex of the present invention have been found to have anticancer properties alone It is considered that this effect is unpredictable compared with the characteristics of the present invention developed as an adjuvant. Therefore, the following anticancer agents are provided, which do not require The method of use as an adjuvant, that is, it is administered together with a cancer antigen, and is not limited to a specific type of cancer, as a general-purpose anti-cancer agent, it works moderately on the body. In addition, of course, it also has immune activating activity, so it is also expected to have immune activating activity against other diseases, and is also expected to have a synergistic effect on cancer patients with weaker physical strength. In addition to the anti-cancer effect, the present invention also has excellent immune activating activity. Therefore, the oligodeoxynucleotide, complex and pharmaceutical composition of the present invention can be used as an immune activating agent. By administering the oligodeoxynucleotide, complex, or pharmaceutical composition of the present invention to a mammal (primates such as humans, rodents such as mice, etc.), an immune response of the mammal can be caused. In particular, the complex of the present invention has the activity characteristics of D-type CpG ODN, stimulates peripheral blood mononuclear cells, and produces a large amount of type I interferon (Pan-IFN-α, IFN-α2, etc.) and type II interferon (IFN- Both γ) are useful as type I interferon production inducers, type II interferon production inducers, type I and type II interferon production inducers. Since both type I and type II interferons are induced, the complex of the present invention and the pharmaceutical composition containing the same are useful for the prevention or treatment of diseases in which either or both type I and type II interferons are effective . As a method for realizing medical use, for example, by administering (a) the oligodeoxynucleotide of the present invention or a composition containing the complex of the present invention to a cancer patient or a human who may suffer from cancer, The cytotoxic T lymphocyte (CTL) antigen of the subject receiving the administration is specifically activated, and directly (as a single-dose effect) prevent or treat cancer. In this specification, the "subject" refers to a subject that becomes the subject of diagnosis, detection, or treatment of the present invention (for example, organisms such as humans or cells extracted from organisms, blood, serum, etc.). In this specification, "medicine", "agent" or "factor" (either of which is equivalent to an agent in English) can be used interchangeably in a broad sense, as long as the desired purpose can be achieved, it can be any substance or Other elements (such as light, radiant energy, heat, electricity and other energy). Examples of such substances include proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, and nucleic acids (for example, including cDNA (Complementary Deoxyribonucleic Acid)), genomes DNA such as DNA, mRNA (Messenger Ribonucleic Acid, messenger ribonucleic acid) RNA (Ribonucleic Acid, ribonucleic acid)), polysaccharides, oligosaccharides, lipids, organic low-molecules (eg hormones, ligands, messaging substances) , Organic low-molecules, molecules synthesized by combinatorial chemistry, low-molecules that can be used as pharmaceuticals (such as low-molecular ligands, etc.), etc., but they are not limited to these Wait. As a factor specific to the polynucleotide, representatively include: the sequence of the polynucleotide has a certain sequence homology (for example, 70% or more sequence identity) and has complementarity Polynucleotides, polypeptides such as transcription factors bound to the promoter region, etc., but not limited to these. As a factor specific for a polypeptide, typically, an antibody or derivative or analog thereof (for example, a single chain antibody) that specifically targets the polypeptide, the polypeptide is a receptor or a ligand In the case of specific ligands or receptors, when the polypeptide is an enzyme, its substrate may be cited, but it is not limited to these. In this specification, the so-called "treatment" refers to a disease or disorder (such as cancer, allergies), when it becomes such a state, to prevent the deterioration of the disease or disorder, it is better to maintain the status quo, more preferably It is alleviated, and it is preferable that it subsides, including the case where the symptom-improving effect or prevention effect of the patient's disease or one or more symptoms associated with the disease can be exerted. There are cases where the diagnosis is made beforehand and appropriate treatment is called "companion treatment", and the diagnostic drug used for it is called "companion treatment drug". In the present specification, the term "therapeutic agent (agent)" broadly refers to all agents that can treat a target state (such as cancer, allergies, etc.). In one embodiment of the present invention, the "therapeutic agent" may also be a pharmaceutical composition containing 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 an active ingredient with the above-mentioned carrier. In addition, as long as the therapeutic agent is used for treatment, the use form is not limited, and the active ingredient may be a single ingredient or a mixture of the active ingredient and any ingredient. In addition, the shape of the above-mentioned carrier is not particularly limited, and for example, it may be solid or liquid (for example, a buffer). In addition, therapeutic drugs for cancer and allergy include drugs (preventive drugs) for preventing cancer and allergy, or inhibitors for cancer and allergy. In this specification, the term "prevention" refers to the prevention of a certain disease or disorder (eg, allergy) before it becomes such a state. The agent of the present invention can be used for diagnosis, and if necessary, the agent of the present invention can be used to prevent allergies, for example, or to take preventive measures. In this specification, the term "preventive drug (agent)" broadly refers to all agents that can prevent a target state (such as allergies and other diseases). In this specification, the so-called "kit" usually refers to a unit that provides a part (such as test drugs, diagnostic drugs, therapeutic drugs, antibodies, labels, instructions, etc.) that should be divided into two or more sub-parts. For stability and the like, it should not be provided by mixing. When the aim is to provide a composition to be used for mixing immediately before use, the form of the kit is better. Preferably, such a kit has instructions or instructions that describe how to use the provided parts (eg test drugs, diagnostic drugs, therapeutic drugs), or how to handle the reagents, which is more advantageous. In this specification, when the kit is used in the form of a reagent kit, the kit usually contains instructions and the like describing the usage of test drugs, diagnostic drugs, therapeutic drugs, antibodies, etc. In this specification, "instructions" are written to explain to the doctor or other users the method of using the present invention. The instructions describe the detection method of the present invention, the usage of diagnostic drugs, or instructions for administration of medicine. In addition, the instructions may also include instructions for oral administration and administration to the esophagus (for example, by injection, etc.) as the administration site. This instruction is made in accordance with the format prescribed by the supervisory government agency of the country where the invention is implemented (for example, the Ministry of Health, Labour and Welfare in Japan, and the Food and Drug Administration (FDA) in the United States, etc.), and clearly states that the supervision Key points recognized by government agencies. The instruction book is a so-called attached document (package insert), which is usually provided in paper media, but it is not limited to this, for example, it can also be in electronic media (such as the homepage and e-mail provided on the Internet) Provided in such forms. (Form of preferred embodiment) Hereinafter, a preferred embodiment of the present invention will be described. The embodiments provided below are provided for a better understanding of the present invention, and it should be understood that the scope of the present invention should not be limited to the following description. Therefore, it is clear that the practitioner can refer to the description in this specification and make appropriate changes within the scope of the present invention. In addition, it is understood that the following embodiments of the present invention can be used alone or in combination. <Single dose form> In one aspect, the present invention provides an anticancer agent comprising a complex, the complex comprising: (a) an oligodeoxynucleotide, which comprises a humanized K-type CpG oligodeoxynucleotide Oxynucleotide and polydeoxyadenylate, and the polydeoxyadenylate is placed on the 3'side of the humanized K-type CpG oligodeoxynucleotide; and (b) β-1,3- Dextran. In the present invention, it was found that the complex of the present invention itself functions as an anticancer agent. Previously, the present inventors only found that the complex can be used as an adjuvant and submitted an application, but it is not predicted that it can be used directly as an anticancer agent in the form of a single dose. Therefore, from the viewpoint of use without cancer antigens, it can be considered to bring unpredictable effects. In one embodiment, the anticancer agent of the present invention is characterized in that it is administered without a cancer antigen. In another embodiment, the anticancer agent of the present invention is characterized in that it is administered by delivery to the reticuloendothelial system and/or lymph nodes. Preferably, the reticuloendothelial system and/or lymph nodes include tumors and macrophages. Exemplarily, the reticuloendothelial system includes the spleen and/or liver. Therefore, the anticancer agent of the present invention is characterized in that it is administered by delivery to organs (spleen, liver, etc.) and/or lymph nodes of the reticuloendothelial system including tumors and macrophages. Without wishing to be bound by theory, it is revealed that the complex of the present invention is delivered to tumors and macrophages, where cancer dead cells are recruited into organs of the reticuloendothelial system (spleen, liver, etc.). Based on this, it is believed that the cancer cells in the body can be further expelled. Therefore, the present invention does not use specific cancer antigens as adjuvants for specific cancers, and it can be considered to bring significant effects from the viewpoint that it can kill any cancer cells present in the body and provide an unprecedented anticancer agent. Therefore, in a more preferred embodiment, the above-mentioned anticancer agent of the present invention is characterized in that it is administered by way of delivery to tumors and macrophages without cancer antigens. Any method can be used for this delivery method. For example, the above-mentioned administration may include systemic administration, but it is not limited thereto. Systemic administration is preferred. 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 may include: K3 (SEQ ID NO: 1), K3-dA 40 (Serial number 2), dA 40 -K3 (sequence number 3), K3-dA20 (sequence number 4), K3-dA25 (sequence number 5), K3-dA30 (sequence number 6), K3-dA35 (sequence number 7), etc., but not limited to Such. In one embodiment, the β-1,3-glucan used in the present invention may also be Schizophyllum polysaccharide (SPG), lentinan, scleroglucan, cardlan polysaccharide, Poria polysaccharide, ash tree Flower polysaccharides, laminin etc. In a preferred embodiment, the complex of the present invention is K3-SPG or its analog. Here, the analogs include those similar to K3 on the CpG side and those similar to SPG on the β-glucan side, but are not limited thereto. In addition, since the anti-cancer effect uses various mechanisms, it is not easy to think of applications such as gathering dead cells of cancer in the spleen. Especially when administered systemically, it is not expected to be used to accumulate in tumors and accumulate dead tumor cells in tissues such as the spleen. In addition, the expression or expression promotion effect of interleukin 12 (IL12) and/or interferon (IFN) α also utilizes a mechanism different from the anticancer effect, interleukin 12 (IL12) and/or interferon (IFN) α The performance or performance promotion can also be played in addition to anti-cancer, so it is not easy to think of each other. Therefore, it can be considered that the CpG-β glucan complex of the present invention has various uses (anti-cancer use (in the form of a single dose), the use of accumulating dead cells of cancer in the spleen, and interleukin 12 (IL12) And/or interferon (IFN) alpha expression or expression-promoting use) are in a relationship that is not considered to be easily conceivable to each other. <Reticuloendothelial system (including spleen and/or liver) and/or lymph node aggregating agent> In another aspect, the present invention provides a complex comprising the dead cells of cancer in the reticuloendothelial system ( A composition comprising spleen and/or liver) and/or lymph nodes, and the complex comprises: (a) oligodeoxynucleotide, which comprises humanized K-type CpG oligodeoxynucleotide and polydeoxynucleotide Oxyadenosine, and polydeoxyadenylate is placed on the 3'side of humanized K-type CpG oligodeoxynucleotide; and (b) β-1,3-glucan. Without wishing to be bound by theory, it is found that the complex of the present invention can accumulate dead cells of cancer in the reticuloendothelial system (including spleen and/or liver) and/or lymph nodes. As exemplified in the examples, it was confirmed that the treatment using the complex of the present invention such as K3-SPG induces tumor cell death in a manner dependent on both IL12p40 and IFN-I. It has not been previously predicted that the complex has such an effect, and in this sense, it can be considered to achieve an unexpected effect. That is, CpG is targeted by phagocytic cells in the tumor microenvironment. If cancer dead cells accumulate in the reticuloendothelial system (including spleen and/or liver) and/or lymph nodes, then the released tumor dead cells induce anti-tumor CTLs against multiple tumor antigens, located in the body Cancer cells can also be cured by being killed like a shotgun. Without wishing to be bound by theory, the production of both IL12 and IFN-I cytokines in the tumor microenvironment is not considered necessary for K3-SPG single-agent therapy, but it is more important. In one embodiment, the oligodeoxynucleotide used in the present invention is selected from K3 (sequence number 1), K3-dA 40 (Serial number 2), dA 40 -In a group consisting of K3 (sequence number 3), K3-dA20 (sequence number 4), K3-dA25 (sequence number 5), K3-dA30 (sequence number 6), and K3-dA35 (sequence number 7). In another embodiment, the β-1,3-glucan used in the present invention is selected from the group consisting of Schizophyllum polysaccharide (SPG), scleroglucan, cardlan polysaccharide, Poria polysaccharide, Grifola frondosa In the group consisting of polysaccharides and laminin. In a preferred embodiment, the complex of the present invention is K3-SPG. In one embodiment, the reticuloendothelial system and/or lymph nodes that are the subject of the composition of the present invention include tumors and macrophages. Illustratively, the reticuloendothelial system includes the spleen and/or liver. Therefore, the composition of the present invention is characterized in that it is administered by delivery to organs of the reticuloendothelial system (spleen, liver, etc.) and/or lymph nodes including tumors and macrophages. Without wishing to be bound by theory, it is revealed that the complex of the present invention is delivered to tumors and macrophages, where cancer dead cells are recruited to organs of the reticuloendothelial system (spleen, liver, etc.). Based on this, it is believed that the cancer cells in the body can be further expelled. Therefore, the present invention does not use specific cancer antigens as adjuvants to target specific cancers, and can be considered to bring significant effects from the viewpoint that it can kill any cancer cells present in the body and provide an unprecedented anticancer agent. Therefore, in a more preferred embodiment, the above-mentioned anticancer agent of the present invention is characterized in that it is administered by way of delivery to tumors and macrophages without cancer antigens. Any method can be used for this delivery method. For example, the above-mentioned administration may include systemic administration, but it is not limited thereto. Systemic administration is preferred. Examples of systemic administration include intravenous administration, intraperitoneal administration, oral administration, subcutaneous administration, and intramuscular administration. <IL12 and/or IFN expression promoter> Further, in another aspect, the present invention provides a composition for expression or expression promotion of interleukin 12 (IL12) and/or interferon (IFN) γ, It contains: (a) oligodeoxynucleotide, which contains humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylate, and the polydeoxyadenylate system is configured for humanization The 3'side of the K-type CpG oligodeoxynucleotide; and (b) β-1,3-glucan. The production of both IL12 and IFN-I cytokines in the tumor microenvironment is an important effect in K3-SPG single-dose therapy. This effect is also important in other uses in addition to its role as an anticancer agent. Examples of such treatment include chronic infection diseases such as cancer and other viruses, prevention of viral infections, etc., but not limited to these. In one embodiment, the oligodeoxynucleotide used in the present invention is selected from K3 (sequence number 1), K3-dA 40 (Serial number 2), dA 40 -In a group consisting of K3 (sequence number 3), K3-dA20 (sequence number 4), K3-dA25 (sequence number 5), K3-dA30 (sequence number 6), and K3-dA35 (sequence number 7). In another embodiment, the β-1,3-glucan used in the present invention is selected from the group consisting of Schizophyllum polysaccharide (SPG), scleroglucan, cardlan polysaccharide, Poria polysaccharide, Grifola frondosa In the group consisting of polysaccharides and laminin. In a preferred embodiment, the complex of the present invention is K3-SPG. (Pharmaceuticals, Dosage Forms, etc.) The present invention is provided in the form of medicines (therapeutic or preventive medicines) in the various forms described above. The administration route of the therapeutic drug is preferably used when it is effective during treatment. For example, it can also be administered intravenously, subcutaneously, intramuscularly, intraperitoneally, orally. As the administration form, for example, injections, capsules, lozenges, granules and the like can also be used. When the ingredient of the present invention is administered, it is effective to use it as an injection. The aqueous solution for injection can also be stored in a small glass bottle or a stainless steel container, for example. In addition, for example, an aqueous solution for injection may also contain physiological saline, sugar (for example, trehalose), NaCl, or NaOH. In addition, for example, a therapeutic agent may be formulated with a buffer (for example, phosphate buffer), a stabilizer, and the like. Generally, the composition, medicine, therapeutic agent, prophylactic agent, etc. of the present invention contain a therapeutically effective amount of a therapeutic agent or active ingredient, and a pharmaceutically acceptable carrier or excipient. In this specification, "pharmaceutically acceptable" means listed in animals, and more specifically humans, approved by the government's supervising government agency, or pharmacopoeia or other generally recognized pharmacopoeia. In this specification, the "carrier" used refers to a diluent, adjuvant, excipient, or vehicle administered together with the therapeutic agent. Such a carrier may also be a sterile liquid, such as water and oil, including petroleum, animal, plant, or synthetic origin, without limitation, including peanut oil, soybean oil, mineral oil, sesame oil, and the like. In the case of oral administration to medicine, water is the preferred carrier. When the pharmaceutical composition is administered intravenously, physiological saline and aqueous glucose are preferred carriers. It is preferable to use physiological saline solution and aqueous glucose and glycerin solutions as liquid carriers for injectable solutions. In suitable excipients, including light anhydrous silicic acid, crystalline cellulose, mannitol, starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silicone, sodium stearate, mono Glycerol stearate, talc, sodium chloride, skim milk powder, glycerin, propylene, ethylene glycol, water, ethanol, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl alcohol Cellulose, polyvinyl acetal (diethylamino) acetate, polyvinyl pyrrolidone, gelatin, medium-chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, white sugar, carboxymethyl fiber Vegetarian, corn starch, inorganic salts, etc. In a more ideal situation, the composition may also contain a small amount of wetting or emulsifying agent, or pH buffering agent. Such compositions may also take the form of solutions, suspensions, latex, lozenges, pills, capsules, powders, sustained-release formulations and the like. Suppositories can also be prepared by using traditional binding agents and carriers, such as triglycerides, to formulate the composition. Oral formulations may also contain pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and other standard carriers. Examples of suitable carriers are described in EW Martin, Remington's Pharmaceutical Sciences (Mark Publishing Company, Easton, US A). The composition contains an appropriate amount of a carrier and a therapeutically effective amount of a therapeutic agent, preferably a purified type, in order to provide an appropriate form for administration to a patient. The formulation must be suitable for the mode of administration. In addition to these, for example, surfactants, excipients, colorants, perfumes, preservatives, stabilizers, buffers, suspending agents, isotonic agents, binding agents, disintegrating agents, lubricants, fluids Sex accelerators, flavoring agents, etc. In one embodiment of the present invention, the "salt" includes, for example, an anion salt formed by any acidic (eg carboxyl) group, or a cationic salt formed by any basic (eg amine) group. Salts include inorganic salts or organic salts, for example, those described in Berge et al., J. Pharm. Sci., 1977, 66, 1-19. In addition, for example, metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, etc. may be mentioned. In one embodiment of the present invention, the "solvate" is a compound formed by a solute and a solvent. For solvates, for example, refer to J. Honiget al., The Van Nostrand Chemist's Dictionary P650 (1953). If the solvent is water, the solvate formed is a hydrate. The solvent is preferably one that does not hinder the biological activity of the solute. Examples of such a preferable solvent are not particularly limited, and water or various buffers can be cited. In one embodiment of the present invention, the "chemical modification" may include, for example, modification using PEG (Polyethylene Glycol) or a derivative thereof, luciferin modification, or biotin modification. In the case where the present invention is made into medicine and administered, various delivery systems are known, and such systems can also be used to administer the therapeutic agent of the present invention to an appropriate site (such as the esophagus). For example, there are encapsulation in liposomes, microparticles, and microcapsules; the use of recombinant cells that can express therapeutic agents (such as polypeptides), the use of receptor-mediated endocytosis; therapeutic nucleic acids Construction as part of retroviral vectors or other vectors. The introduction method includes intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes, but is not limited thereto. It can also be administered by any of the appropriate routes, for example, by injection, by bolus injection, or by absorption through the epithelium or the lining of the skin mucosa (eg, oral mucosa, rectal mucosa, intestinal mucosa, etc.) For medicine, aerosols, inhalers or nebulizers can be used if necessary, and they can also be administered together with other biologically active agents. Administration can also be systemic or local. When the present invention is applied to cancer, it can be administered by any suitable route such as direct injection into cancer (lesion). In a preferred embodiment, the composition can be formulated in the form of a pharmaceutical composition suitable for administration to humans according to known methods. Such a composition can be administered by injection. The composition for injection administration is typically a solution in sterile isotonic aqueous buffer. In addition, when necessary, the composition may also contain a cosolvent and a local anesthetic such as lidocaine to relieve pain at the injection site. Generally, the ingredients are supplied separately or mixed together in a unit dose applicator. For example, they can be freeze-dried powders or water-free concentrates in sealed containers such as ampoules or sachets showing the amount of active agent. Form supply. In the case where the composition is to be administered by injection, it can also be dispensed using an injector containing sterile pharmaceutical grade water or physiological saline. In the case where the composition is to be administered by injection, an ampoule of sterile water or physiological saline for injection may also be provided in a manner in which the ingredients can be mixed before administration. The composition, medicine, therapeutic agent, and prophylactic agent of the present invention can also be formulated in a neutral type, a salt type, or other prodrugs (such as esters). Pharmaceutically acceptable salts include those formed with free carboxyl groups derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those derived from isopropylamine, triethylamine, and 2-ethylaminoethanol , Histidine, procaine, etc., together with free amine groups, and those derived from sodium, potassium, ammonium, calcium, and iron hydroxide. The amount of the therapeutic agent of the present invention that is effective for the treatment of a specific disorder or state may vary according to the nature of the disorder or state, and those skilled in the art can determine it based on the standard clinical techniques based on the description in this specification. Furthermore, in-vitro analysis can be used as appropriate to assist in the identification of the optimal dose range. In addition, the exact amount to be used in the formulation may also vary according to the route of administration and the severity of the disease or disorder, so it should be determined according to the judgment of the attending physician and the condition of each patient. However, the amount administered is not particularly limited, and may be, for example, 0.001, 1, 5, 10, 15, 100, or 1000 mg/kg body weight per time, or may be within the range of any two of these values. The administration interval is not particularly limited. For example, it can be administered once or twice every 1, 7, 14, 21, or 28 days, or once or twice every such two ranges of values. The dosage, interval, and method of administration can also be appropriately selected according to the age or weight of the patient, symptoms, target organs, and the like. In addition, the therapeutic agent is preferably an effective ingredient containing a therapeutically effective amount or an effective amount to exert a desired effect. In the case where the malignant tumor marker significantly decreases after administration, it can also be judged to have a therapeutic effect. The effective dosage can be estimated based on the dosage-response curve obtained from the in vitro or animal model test system. In one embodiment of the present invention, "patient" or "subject" includes humans or mammals other than humans (such as mice, guinea pigs, hamsters, rats, mice, rabbits, pigs, sheep, goats, (One or more of cattle, horses, cats, dogs, marmosets, monkeys, chimpanzee, etc.) The pharmaceutical composition, therapeutic agent or prophylactic agent of the present invention can be provided in the form of a kit. In a specific embodiment, the present invention provides a pharmaceutical pack or kit including one or more containers filled with one or more components of the composition or medicine of the present invention. It may also be displayed on such containers, in a form prescribed by the government agency that controls the manufacture, use, or sale of pharmaceutical or biological products, with information indicating that the government agency permits the manufacture, use, or sale of humans. . In a specific embodiment, the pharmaceutical composition containing the components of the present invention can be administered by liposomes, microparticles, or microcapsules. Among the various aspects of the present invention, there is also the possibility of using such a composition to achieve the sustained release of the ingredients of the present invention. The preparation procedures of the therapeutic drugs, preventive drugs, etc. of the present invention as medicines are well known in this field, and are described in, for example, the Japanese Pharmacopoeia, the United States Pharmacopoeia, and the Pharmacopoeia of other countries. Therefore, as long as there is a description in this specification, practitioners can determine the amount of use and other embodiments without excessively conducting experiments. (General technique) Molecular biology methods, biochemical methods, and microbiological methods that can be used in this specification are well known and commonly used in this field, for example, they are described in Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, FM (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, FM (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, MA (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, FM (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, FM (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, MA et al. (1995). PCR Strategies, Academic Press; Ausubel, FM (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecul ar Biology, Wiley, and annual updates; Sninsky, JJ et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, Supplementary Experimental Medicine "Gene Introduction & Expression Analysis Experimental Method", Yangtu Society, 1997, etc. In this specification, the relevant parts (which may be all) are cited by reference. The DNA synthesis technology and nucleic acid chemistry used to make artificially synthesized genes are described in Gait, MJ (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, MJ (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, RL et al. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, GM et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, GT (I996). Bioconjugate Techniques, Academic Press, etc., and These relevant parts are cited in the form of references in this specification. For example, in this specification, the oligonucleotide of the present invention can also be synthesized by standard methods known in the art, for example, by using an automated DNA synthesis device (by vendors such as Biosearch, Applied Biosystems, etc.) . For example, the method of Stein et al. (Stein et al., 1988, Nucl. Acids Res. 16: 3209) can be used to synthesize phosphorothioate-oligonucleotides, or by using modified pore glass polymers. Support (Sarinet al., 1988, Proc. Natl. Acad. Sci. USA 85: 7448-7451), etc., to prepare methyl phosphonate-oligonucleotide. In this manual, "or" is used when "at least one or more" of the items listed in the article can be adopted. "Or" is the same. In this specification, when clearly stated as "within a range of two values", the range also includes the two values themselves. In this specification, all contents of cited scientific documents, patents, patent applications, etc. are cited to the specification by reference to the same extent as those described specifically. In the above, in order to make the present invention easy to understand, the preferred embodiments have been disclosed. Hereinafter, 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, and are not provided to limit the present invention. Therefore, the scope of the present invention is not limited to the embodiments and examples specifically described in this specification, but is only limited by the scope of the patent application. [Examples] Examples are described below. If necessary, all animal experiments will be carried out in accordance with the guidelines of the Japanese Pharmaceutical Fundamental Research Institute and the Osaka University Animal Facility. Also, based on the Declaration of Helsinki. The reagents specifically use the products described in the examples, but equivalent products of other manufacturers (Sigma-Aldrich, Wako Pure Chemicals, Nacalai, R&D Systems, USCN Life Science INC, etc.) can also be used instead. (Production Example) The following CpG ODNs were synthesized by Genedesign Co., Ltd. (underlined phosphorothioate bond). In particular, in addition to the above-mentioned K3-dA40 (sequence number 2), descriptions are made about K3-dA35 (sequence number 7), K3-dA30 (sequence number 6), K3-dA25 (sequence number 5), and K3-dA20 (sequence number 4) Synthesis (Table 2). (S in the above sequence indicates that the phosphodiester bond between the nucleosides is replaced by a phosphorothioate bond.) This oligodeoxynucleotide uses a solid-phase phosphoramidite method (Nucleic Acids in Chemistry) as a common method and Biology, 3. Chemical synthesis (1990) ed. G. Michael Blackburn and Michael J. Gait. Oxford University Press). Ovalbumin (OVA) was purchased from Shengsheng Chemical Industry Co., Ltd. DQ-OVA, Alexa488-OVA, CFSE, and Lipofectamine 2000 were purchased from Invitrogen. Hoechst 33258, zymosan and cadran polysaccharides were purchased from SIGMA. Zymosan-Depleted was purchased from Invivogen. The clodronate lipid system was purchased from FormuMax. Influenza lysate 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)). Complexization of CpG ODN and SPG (Manufacturing 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 0.25 N NaOH (1 mL). Combine 1 mL of 330 mM NaH 2 PO 4 Add to DNA solution, then add SPG solution to DNA/NaH 2 PO 4 The solution was maintained at 4°C overnight, thereby ending the complexation. The molar ratio (MSPG/MDNA) was fixed at 0.27. Regarding the formation of the complex, the formation of the complex confirmed by the microchip electrophoresis device (SHIMADZU: MultiNA) was confirmed by using size exclusion chromatography to monitor the absorption at 260 nm to confirm the shift of CpG ODN to the high molecular weight side (System: Agilent 1100 series, Column: Connect two Asahipak GF7M-HQ (Shodex), flow rate: 0.8 mL/min, buffer: 10 mM EDTA (Ethylenediamine Tetraacetic Acid, EDTA) ) PBS (Phosphate Buffer Solution, phosphate buffer), pH 7.4, temperature: 40°C). (For the preparation of the examples) In the following examples, it is revealed that the following systemic single-dose treatment can be performed. This systemic single-dose treatment utilizes the induction of strong tumor shrinkage to the giant tumor microenvironment. The phagocytes act as target nanoparticle TLR9 agonists. (Materials and methods) Hereinafter, the reagents, materials, animals, cells, and methods used in this example will be described. Supplementary explanations are also appropriately made in each embodiment. (Animals and reagents) 6-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 described above (Kobiyama, K., et al. Proc. Natl. Acad. Sci. USA 111, 3086-3091 (2014)). All animal experiments were conducted according to the guidelines of the Institute of Pharmaceutical Fundamental Research Institute. K3 is synthesized by Gene Design. Ovalbumin (OVA) is purchased from the self-generated chemical industry. (Cell line) EL4 and OVA expressing EL4 (EG7) are thymoma cell lines of C57BL/6J mice, purchased from ATCC. B16 (melanoma) was purchased from the Japanese Collection of Research Bioresourses (Japanese Cell Bank). B16F10 (melanoma) was purchased from Liyan Cell Bank, and MC38 (colon cancer) was provided by Dr. F. 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, Fetal Bovine Serum), penicillin, and streptomycin). B16 and B16F10 were cultured in complete DMEM (Dulbecco Modified Eagle Medium) (DMEM supplemented with 10% (v/v) fetal bovine serum (FBS), penicillin, and streptomycin). (Tumor experiment and treatment) EG7, EL4, B16, B16F10, and MC38 cells (in 10% gelatin/PBS at 5×10 6 Cells/mL (100 μl) (sc) were inoculated subcutaneously on the right abdomen of mice. For the tumor size, the length (L), width (W), and height (H) of the tumor were measured, and the tumor volume (V) was calculated according to V=L×W×H. Direct injection into the tumor site by intratumoral injection (it). CpG treatment is based on tumor volume reaching approximately 100 mm 3 After the start, the period is 7 days after the inoculation of EG7 and B16F10, 10 days after the inoculation of B16, and 14 days after the inoculation of MC38. Tumor-bearing mice were treated 3 times with K3 (30 μg) or K3-SPG (10 μg) every other day. (Pan02 peritoneal inoculation model) In the peritoneal inoculation model of Pan02, intraperitoneal injection of 1×10 6 Pan02 cells (1×10 in PBS 7 Cells/mL is 100 μl). CpG treatment was started 11 days after the inoculation, and all tumor nodules were removed from the peritoneum of the mice on the 21st day, and then the weight (g) of these was measured. The dosage of CpG treatment is as described above. (In vivo imaging experiment) To evaluate the presence of K3 and K3-SPG, C57BL/6 mice were sc-inoculated with EG7 on day 0, and iv (PBS) and Alexa 647-K3 (30 μg) were administered iv on day 12 , Or Alexa 647-K3-SPG (10 μg). One hour after the administration, the mice were analyzed using the IVIS (registered trademark) Lumina Imaging System (Lumina Imaging System) and analysis software (Ver.2.6, Xenogen), and the images measured by relative fluorescence were converted to the surface Physical unit of radiance (photon/sec/cm 2 /sr). To detect labeled CD8 in vivo + For T cells, splenocytes were recovered from C57BL/6 mice treated with K3-SPG or untreated C57BL/6 mice or Il12p40-Ifnar2 gene untreated mice treated with K3-SPG on days 7, 9, and 11 on day 14. After suspension of spleen cells, use ACK lysis buffer (150 mM NH 4 Cl, 10 mM KHCO 3 , 0.1 mM Na 2 EDTA) lyses red blood cells and maintains the cells in complete RPMI. By MACS (Magnetic Activated Cell Sorting) (Miltenyi Biotec) to CD8α + T cells are sorted. Sort CD8α by negative selection + T cells. Thereafter, the CD8α selected by Xenolight DiR (registered trademark) + T cells are stained. On day 14 (inoculated with EG7 on day 0, iv-treated with K3-SPG on days 7, 9, and 11, or untreated C57BL/6 mice or Il12p40-Ifnar2 gene double knockout mice) Stained CD8α + T cells were transplanted into transplanted mice. 24 hours after transplantation of the stained cells, the mice were analyzed using an IVIS (registered trademark) Lumina Imaging System (Ver. 2.6). The target area was collected in the tumor area, and the fluorescence intensity was analyzed using Living Image Software (Ver.2.6, Xenogen). (Immunohistochemistry) C57BL/6J mice (6-8 weeks old, female, CLEA Japan) were iv injected with Alexa 647-K3 (30 μg), Alexa 647-K3-SPG (10 μg) from the tail vein, and Dextran-PE (20 μg). Tumors were recovered 1 hour after injection, frozen sections were fixed with 4% (w/v) paraformaldehyde for 10 minutes, and stained with Hoechst 33258, anti-CD3e antibody, and anti-CD8β antibody. Cells were photographed using Olympus IX81 system. Use MetaMorph to analyze image data. (Exhaustion experiment) To deplete phagocytes (dendritic cells and macrophages), C57BL/6 mice were injected iv with clodronate liposomes or control liposomes (100 nm) (Katayama) 5 days after EG7 inoculation. Chemistry). To make CD8 + T cells were depleted and 200 μg of anti-CD8α antibody was injected iv into the tail vein 6 days and 13 days after EG7 inoculation. (Analysis of splenocytes) C57BL/6 mice with Il-3 or Il12p40-Ifnar2 gene knock-out mice treated with K3-SPG on day 14, 9 and 11 or untreated on day 14, 9 and 11 Recover spleen cells. After preparing spleen cells, erythrocytes were lysed using ACK lysis buffer and the cells were maintained in complete RPMI. Use 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-aminoactinomycin D (7AAD, 7-Aminoactinomycin D) Spleen cells were stained. Determination of OVA tetramer using flow cytometry + CD44 + CD8α + TCRβ + The number of cells. For other experiments, the prepared splenocytes were incubated with anti-CD45 antibody, anti-CD3e antibody, anti-CD8α antibody, and anti-CD11a antibody, and then analyzed by flow cytometry. (Analysis and immunization of CD45-negative cells) C57BL/6 mice with EG7 or Il12p40-Ifnar2 gene treated with K3-SPG for iv treatment on day 12, from day 7, 9, and 11 or untreated with EG7 Spleen cells were recovered from double-eliminated mice. After preparing spleen cells, erythrocytes were lysed using ACK lysis buffer and the cells were maintained in complete RPMI. Spleen cells were stained with anti-CD45 antibody (APC) and CD45 was determined using flow cytometry - The number of cells. Furthermore, PI and Hoechst 33342 were used to stain a subset of apoptotic cells, necrotic cells, and CD45-negative living cells, and then analyzed by flow cytometry. Next, CD45 was sorted using INFLUX (BD Bioscience) from tumor-bearing C57BL/6 mice treated with K3-SPG - cell. (Vaccination model) C57BL/6 mice were iv administered 5×10 on day -7 5 CD45 - cell. After 7 days of immunization, mice were sc-inoculated with 5×10 on day 0 5 EG7 cells. (Measurement of cytokines) The amount of mouse IL-12p40, mouse IL-13, and human IFNγ was measured using R&D ELISA kit. (Statistical analysis) One-way analysis of variance including Mann-Whitney's U test, Student's t test or Bonferroni's multiple comparison test is 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: Regarding the intravenous injection of K3-SPG, even if the tumor antigen is not added at all, it will induce a strong inhibition of tumor growth.) In this example, the intravenous injection of K3-SPG proved that even No addition of tumor antigens at all will also induce stronger inhibition of tumor growth. (Experiment using EG7 (ovarium thymoma cell line expressing OVA) model) On day 0, C57BL/6 mice were inoculated with EG7 (ovarium thymoma cell line expressing OVA) on the right abdomen through the base of the tail Three different routes of subcutaneous (id) administration, intratumoral (it) administration, or intravenous (iv) administration, using PBS, and equal molar amounts of K3 (30 μg) or K3-SPG (10 μg ) 3 treatments (7, 9, and 11 days after inoculation). The tumor size was measured every 2 to 3 days until the 23rd day. (Results) The results are shown below in Fig. 2 (A to B). In the PBS group (control), tumor growth was not inhibited by any route of administration (Figure 2a, b, c (Figure 2A)). In K3 treatment, tumor shrinkage was only observed in it, but not in other pathways (Figure 2d, e, f (Figure 2A)). In K3-SPG treatment, stronger tumor shrinkage was observed by both it and iv, and id administration did not show an effect on tumor growth (Figure 2g, h, i (Figure 2A)). The graphs showing comparison control, K3 and K3-SPG are shown in the figures (Figure 2a, d, g (Figure 2A)). In the prior art, most attempts in systemic CpG ODN treatment for cancer were unsuccessful (Lou, Y., et al. Journal of immunotherapy (Hagerstown, Md.: 1997) 34, 279-288 (2011) ; Nierkens, S., et al. PLoS One 4, e8368 (2009)), so it is impossible to predict the fact that K3-SPG iv single-dose treatment can strongly inhibit tumor growth, which proves that the present invention has failed Expected effect. (Experiment with other tumor cell lines) To investigate the potential of this K3-SPG systemic single-dose treatment, other tumor cell lines were also tested by the same experimental method used in the EG7 model. (Results) Intravenous administration of K3-SPG also inhibited the growth of melanoma (B16 and B16F10) and colon cancer (MC38) (Figure 2j, k, l (Figure 2B)). The present inventors further conducted experiments by making a tumor vaccination model with higher clinical malignancy. The mouse pancreas tumor strain, Pan02 (1×10 6 Cells are inoculated into the abdominal cavity (also called "ip"), and thereafter, treatment with K3 or K3-SPG is started 11 days after inoculation (3 times every other day). All mice were sacrificed on the 21st day, and the total weight of the abdominal tumor was evaluated (Figure 2m (Figure 2B)). Regarding tumor growth, it was significantly inhibited in the K3-SPG iv treatment group, but not inhibited in the K3 ip and K3-SPG ip treatment groups (FIG. 2m (FIG. 2B )). Accordingly, a significant prolongation of survival was observed in the K3-SPG iv treatment group, but not in the K3 iv group (Figure 2n (Figure 2B)). These results prove that the systemic iv administration of K3-SPG is a promising single-dose treatment for most different cancers, and thus does not require any tumor peptides and antigens. (Example 2: K3-SPG is targeted to phagocytic cells in the tumor microenvironment) Secondly, the inventors et al. clarified 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. USA 111, 3086-3091 (2014)). The inventors and others 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); Farokhzad, OC et al. ACS Nano 3, 16-20 (2009)). (Fluorescent imaging) In order to test the distribution in vivo, K3 and K3-SPG were fluorescently labeled. Mice bearing EG7 tumors were iv injected with PBS, Alexa647-K3 (30 μg), or Alexa647-K3-SPG (10 μg), and then the fluorescence distribution was tested using an in vivo imaging system (IVIS). The results are shown in Figure 4 below. IVIS imaging showed that after 1 hour of iv administration, K3 was not accumulated in the tumor site, but K3-SPG was accumulated in the tumor site (Figure 4a). It was observed that the accumulation of K3-SPG in the tumor is closely related to the effectiveness of CpG single-agent treatment for tumor shrinkage (Figure 2). In the immunohistochemistry (IHC, Immunology Histology Chemistry) test, the inventors and others failed to detect Alexa647-K3 in the tumor microenvironment (Figure 4b). On the other hand, Alexa647-K3-SPG was observed in the tumor area (Figure 4c). The inventors and others failed to detect the Alexa647 signal in IHC after 24 hours. EG7 cells express CD3e on their surface (the reason is that the EG7 line is derived from a thymoma cell line), and K3-SPG is not aggregated in CD3e, which shows that the K3-SPG line is taken in by non-tumor cells. Nanoparticles are selected to be taken in by phagocytic cells such as macrophages and dendritic cells (DC), which can be used in vivo by TRITC (Tetramethyl Rhodamine Iso-thiocyanate, tetramethylisothiocyanate rhodamine)- Dextran is labeled. Therefore, the inventors injected fluorescently stained K3, K3-SPG, or SPG and TRITC-dextran intravenously, and tested the coexistence of these using IHC (Fig. 4d, e, f). One hour after iv injection, dextran was observed in the tumor area of all samples (Figure 4d, e, f), which shows that the tumor microenvironment contains phagocytic cells. Consistent with previous results, Alexa647-K3 was not observed in the tumor (Figure 4d). About 50% of Alexa647-K3-SPG and FITC-SPG coexist with TRITC-dextran-positive cells (Figure 4e, f, g) observed in the tumor, which shows that K3-SPG is engulfed in the tumor microenvironment Cell intake. A part of K3-SPG does not aggregate with dextran, and the inventors speculate that by enhancing vascular permeability and enhanced permeability and retention (EPR) effect, these passively accumulate in the space within the tumor tissue. To test the importance of phagocytic cells for K3-SPG iv treatment, the inventors et al. injected clodronate liposomes intravenously. The inventors and others did not use liposomes of 200-300 nm in general, but used clodronate liposomes of 100 nm and injected them, thereby depleting the phagocytes in the tumor (Pante, N . et al. Molecular biology of the cell 13, 425-434 (2002); Pante, N. et al. Molecular biology of the cell 13, 425-434 (2002)), by this injection, F4/ 80 positive cells were almost exhausted within 2 days (Figure 5). On day 5 (2 days before the initial K3-SPG treatment), mice with tumors were injected with clodronate liposomes, or not, and K3-SPG was used in the same way as in Figure 2 (A-B) Treat the mice. When clodronate liposomes were injected first, it significantly counteracted the inhibition of tumor growth mediated by K3-SPG (p<0.05) (Figure 4h). On the other hand, the injection of clodronate liposomes itself Compared with PBS-treated mice, it had no effect on tumor growth. These results show that K3-SPG targets phagocytic cells in the tumor microenvironment, and the anti-tumor effect of K3-SPG mostly depends on the introduction of K3-SPG into phagocytic cells in the tumor microenvironment. (Example 3: The production of both IL12 and IFN-I cytokines in the tumor microenvironment is important for K3-SPG single-agent treatment) Secondly, in this example, the inventors et al. Factors required for the success of treatment are tested. It is revealed that IL-12 and IFN-I and other cytokines include C3-GG (Kobiyama, K., et al. Proc. Natl. Acad. Sci. USA 111, 3086-3091 (2014)) CpG ODN (Krieg, AM, et al. Journal of immunology 161, 2428-2434 (1998); Klinman, DM, et al. Immunity 11, 123-129 (1999); Ishii, KJ, et al. Current opinion in molecular therapeutics 6, 166- 174 (2004)) is an important immune stimulating factor. Therefore, the present inventors conducted tests on whether IL-12 and IFN-I are necessary for tumor shrinkage using K3-SPG. On day 0, Il12p40 and IFNAR2-deficient mice were subcutaneously inoculated with EG7 cells, and subjected to three iv treatments with PBS or K3-SPG (10 μg) as shown in FIG. 2 (A-B). Thereafter, the effect of K3-SPG on tumor shrinkage was observed. (Results) The results are shown below in Fig. 6 (A to B). The effect of K3-SPG on tumor shrinkage depends in part on IL-12p40 and IFN-I signaling (Figure 6a, b (Figure 6A)). Furthermore, the inventors of the present invention tested double-elimination (DKO) mice of IL12p40 and IFNAR2, and found that the effect of K3-SPG was completely suppressed in DKO mice (FIG. 6c (FIG. 6A)). IFN-β and IL-12p40 were also detected in the tumor due to IHC staining (Figure 7 and Figure 8). These data show that the secretion of both IL12p40 and IFN-I cytokines within the tumor is important for K3-SPG-mediated tumor suppression. In addition, the inventors of the present invention tested Rag2 mice that were completely deficient in T cell and B cell mediated immune responses. It was learned that even with K3-SPG treatment, Rag2 mice could not control the growth of all tumors (Figure 6d (Figure 6A)), but the inventors and others succeeded in partially during the three treatments using K3-SPG Control of tumor growth in rag2-deficient mice (Figure 6f (Figure 6A)). In order to confirm this observation, the present inventors made six treatment groups of rag2 mice (days 7, 9, 11 and 14, 16, 18), and found that in the experimental method of rag2 mice, Obvious tumor control (Figure 6f (Figure 6A)). Significantly, IL12p40 and IFNAR2 DKO mice did not respond to K3-SPG single-dose treatment even with this wide range of treatment experimental methods (Figure 6e (Figure 6A)). These data show that K3-SPG treatment induces both IL-12p40 and IFN-I in the tumor, with the result that both a natural immune response to the tumor and an adaptive immune response are generated. (Example 4: K3-SPG treatment in a state dependent on both IL12p40 and IFN-I will induce tumor cell death) In this example, it was demonstrated that K3-SPG treatment depends on IL12p40 and IFN-I In both cases, it will induce tumor cell death. Since the inhibition of tumor growth that was observed in rag2 mice independent of immune adaptation was observed, and the complete suppression of tumor growth in IL12p40 and IFNAR2 DKO mice that did not depend on immune adaptation, the inventors, etc. Humans have tested tumor-host interactions in a wider range of K3-SPG treatments. The inventors found that the spleen removed on the 12th day (the second day of the third treatment with K3-SPG) contained a large number of CD45 negative cells compared to the spleen treated with PBS (Figure 6g (Figure 6B)) . Interestingly, these CD45-negative cells were significantly reduced in IL12p40 and IFNAR2 DKO mice (Figure 6g, h (Figure 6B)). The inventors of the present invention sorted the CD45 negative cells. The size and morphology fully show that these lines are derived from tumor cells. Through EG7 inoculation experiments on GFP mice, it was further confirmed that the CD45 negative cells were also GFP negative, which indicates that the cell lines were derived from tumor cells (FIG. 9 ). Since EG7 cells do not express CD45, the fact that CD45 is negative also supports this hypothesis. Regarding Hoechst and PI staining, most of the CD45-negative cell lines in the spleen contained dead cells characterized by both apoptosis and necrosis (Figure 6i (Figure 6B)). These data show that tumor phagocytes that are targeted by K3-SPG secrete IL-12p40 and IFN-I in the tumor microenvironment. These cytokines can induce tumor cell death and release these cyclically, and Eventually caught by the spleen. (Example 5: The released tumor dead cells induce anti-tumor CTL against multiple tumor antigens) In this example, it was demonstrated that the released tumor dead cells induced anti-tumor CTL against multiple tumor antigens. In order to test the immunogenicity of the CD45-negative cells found in the spleens of K3-SPG-treated mice, the inventors sorted these cells and immunized untreated mice as an immunization Internal injection. Thereafter, 7 days after the administration of the sorted cells, EG7 tumor cells were transplanted to the immunized mice. CD45-negative cell immunized mice significantly defended against the proliferation of EG7 tumors (Figure 6j (Figure 6B)). Significantly, OVA257 tetramer positive cells in control and immunized mice (red dots in Figure 6k (Figure 6B)) showed cylinders that were not related to tumor size (Figure 6k (Figure 6B) ), and regarding the immuneization caused by CD45 negative cells, compared with the simple OVA257 epitope, it will induce a more effective further immune response against EG7 tumors (Figure 6k (Figure 6B)). These results show that K3-SPG single-dose treatment induces the death of tumor cells dependent on both IL-12 and IFN-I, which function as an effective immunogen for anti-tumor immune response . (CD8T cell line is an important effector for K3-SPG-mediated tumor shrinkage) The results of Rag2 mice show that the tumor suppressive effect of K3-SPG also depends on adapting to the immune response. Therefore, the present inventors tested CD8 T cells required for K3-SPG treatment. It is shown that the depletion of CD8 T cells in vivo significantly inhibits the antitumor effect of K3-SPG (Figure 10a (Figure 10A)), and CD8 T cells are important effector cells in this K3-SPG treatment. In addition, it was shown that tumor shrinkage using K3-SPG also relies on Batf3 (cross presentation CD8α + DC) (Figure 10b (Figure 10A)), and K3-SPG single-dose treatment also enhanced CD8α + DC-mediated cross-presentation. The inventors observed a significant correlation between tumor infiltration of CD8 T cells and tumor growth. CD8 T cells accumulate in the tumor area in the K3-SPG iv group, but not in the id group (Figure 10c (Figure 10A)). Finally, the inventors of the present invention conducted experiments on those who are necessary to allow the CD8 T cells to enter the tumor area. On day 0, WT mice and Il12p40-Ifnar2 DKO mice were inoculated with EG7 cells, and were treated with K3-SPG or PBS on days 7, 9, and 11 for iv treatment. On day 14, CD8α was purified from the spleens of these mice + T cells were stained with Xenolight DiR (registered trademark) and transplanted into K3-SPG-treated (days 7, 9, and 11) other EG7-bearing mice (14 days after inoculation), followed by IVIS analyzed the distribution of CD8T cells labeled with Xenolight DiR (registered trademark) on day 15 (Figure 11). On day 15, CD8 T cells derived from donor mice with untreated tumors were not accumulated in the tumor site of WT transplanted mice even when treated with K3-SPG (Figure 10d (Figure 10B), II). On the other hand, CD8T cells derived from donor mice with tumors treated with K3-SPG were detected at the tumor site of the transplanted mice (Figure 10d (Figure 10B), I), showing K3-SPG Single-dose treatment induces anti-tumor CD8 T cells that can move to the tumor microenvironment and infiltrate. These activated CD8 T cells in vivo can enter the tumor microenvironment of DKO transplanted mice (Figure 10e (Figure 10B)). Even though IL-12 and IFN-I are more important for the induction of natural immunity and CD8 T cells produced by systemic K3-SPG single-dose treatment, the results also show that if CD8 T cells are activated during K3-SPG treatment, then The secretion of IL-12 and IFN-I cytokines in the tumor microenvironment is not necessary for tumor infiltration of CD8 T cells. Taken together, these results show that activation of tumor-specific CD8 T cells is sufficient for infiltration into tumors. Surprisingly, the infiltration of these CD8 T cells does not depend on the production of cytokines in the tumor microenvironment. (Discussion) The inventors and others have revealed the possibility of novel cancer immunotherapy. It is a novel treatment of CpG targeted by phagocytic cells in the tumor microenvironment (Figure 12). Through the stimulation of TLR9, CpG induces immune responses of immune cells, especially the activation of macrophages and DC (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 the previous report, CpG must be administered directly into the tumor, but if it is a complex of SPG and CpG, even if it is administered in the whole body with the DDS (Drug Delivery System, drug delivery system) function, it also shows Effectiveness equal to or higher than that of 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 inventors and others have solved this problem. The complex of SPG and CpG (Kobiyama, K., et al. Proc. Natl. Acad. Sci. USA 111, 3086-3091 (2014)) produced by the formation of nanoparticles can be stabilized in vivo. It is known that this effect can target the tumor environment, and TLR9 immunocompetent cells are supplied to the tumor environment. The novel CpG developed by the present inventors and others was engulfed by phagocytes because of the formation of nanoparticles. Subsequently, in the tumor environment, phagocytic cells phagocytosing the novel CpG produce cytokines such as IFN and IL-12. It is very important that these cytokines are induced in the tumor environment. In the previous report, it was described that in IFNβ treatment with the tumor environment as a direct target, dendritic cells move within the tumor, increasing the cross-presentation of antigens within the tumor microenvironment, thereby reactivating CTL. These cytokines can cause cell death of tumor cells. Furthermore, the present inventors found that this effect is caused by the activation of natural immunity. This cell death plays a very important role. It becomes a collaboration between natural immunity and adaptive immunity. By releasing tumor cell death from the tumor microenvironment, it induces acquired immunity. The death of the immunogenic tumor cells induces multiple cytotoxic T lymphocytes. CTLs specifically induced by tumors in vivo as described above can respond to tumors and infiltrate the tumor microenvironment. It is believed that the anti-tumor immune system can use endogenous antigens to cope with immune editing (Immunoediting), which is a barrier to cancer immunotherapy. The K3-SPG single-dose treatment of tumor cell circulation can function as a biomarker with excellent tumor treatment effect. (Example 6: Formulation example) The following is a description of the composition of the formulation when it is formulated. For example, 7.22 mg of K3-dA is used 40 (Serial number 2) Dissolve in water (3.7 mL), and dissolve SPG (15 mg) in 0.25 N NaOH (1 mL). Combine 330 mM NaH with a volume of 1 mL 2 PO 4 Add to DNA solution, then add SPG solution to the DNA/NaH 2 PO 4 The solution was maintained at 4°C overnight, and the complexation was completed. Morbi (M SPG /M DNA ) Can be fixed to 0.27. The medicament used in the preparation can be obtained from Genedesign, Invivogen, Wako, etc. As described above, the present invention has been exemplified using preferred embodiments of the present invention, but it is understood that the scope of the present invention should be interpreted only by the scope of patent application. In this specification, the cited patents, patent applications, and documents are understood to be the same as the case where the content itself is specifically described in this specification, and its content should be cited as a reference to this specification. [Industrial Applicability] According to the present invention, a novel form of anticancer agent that can be used as a single dose can be provided. Therefore, the complex of the present invention is useful as an anticancer agent in the medical field. [Free content of the sequence listing] Sequence number 1: K3 Sequence number 2: K3-dA 40 Serial number 3: dA 40 -K3 sequence number 4: K3-dA20 sequence number 5: K3-dA25 sequence number 6: K3-dA30 sequence number 7: K3-dA35
