A complex of beta-glucan and nucleic acid with controlled particle size.

Abstract

The present invention provides a complex of a β(1→3) glucan and a nucleic acid, said complex having a controlled particle size. Further, the present invention provides a complex of a β(1→3) glucan and a nucleic acid, said complex having a controlled particle size.

Description

[Technical Field]

[0001] This invention relates to a complex formed by beta-glucan and nucleic acid, with controlled particle size. [Background technology]

[0002] CpG oligonucleotides (CpG ODNs) are single-stranded synthetic DNA molecules of approximately 20 nucleotides that contain an immunostimulatory CpG motif. Toll-like receptor 9 (TLR9) has been identified as the host receptor for CpG ODNs, and via TLR9, innate immunity is activated, inducing type I interferons (hereinafter referred to as type I IFNs) and inflammatory cytokines (Non-Patent Literature 1, 2). TLR9 is expressed in the endosomes of plasmacytoid dendritic cells (hereinafter referred to as pDCs) and B cells, and CpG ODNs taken up by pDCs and B cells interact with TLR9 present in the endosomes, thereby activating innate immunity (Non-Patent Literature 2).

[0003] There is a desire to develop new immunotherapies for the prevention or treatment of diseases such as infectious diseases, cancer, asthma, and hay fever by utilizing type I IFNs and inflammatory cytokines induced by CpG ODN (Non-Patent Literature 2, 3). An example of a drug containing CpG ODN that is already in practical use is the hepatitis B vaccine Heplisav-B(R). Heplisav-B(R) is a vaccine to prevent hepatitis B virus (hereinafter referred to as HBV) infection, and CpG ODN is added as a vaccine adjuvant. Since Heplisav-B(R) has been shown to potentially exhibit higher efficacy than the existing aluminum adjuvant-containing hepatitis B vaccine Engerix-B(R), it has been suggested that CpG ODN may have higher vaccine adjuvant activity than the aluminum adjuvant most commonly used in vaccines (Non-Patent Literature 4). Clinical trials utilizing the anticancer activity of CpG ODN are also underway, and development as a treatment for metastatic melanoma is progressing (Non-Patent Literature 5).

[0004] There are at least four types of CpG ODNs, each with different skeletal arrangements and immunostimulatory properties (Non-Patent Literature 6). CpG ODNs, called type A or type D, typically contain a single palindromic CpG motif along with a phosphodiester (PO) skeleton and a phosphorothioate (PS) poly-G tail. They activate pDCs to produce large amounts of IFN-α, but do not induce pDC maturation or B cell activation (Non-Patent Literature 7, 8). CpG ODNs, called type B or type K, consist of a PS skeleton and most contain multiple non-palindromic CpG motifs. They activate B cells to produce inflammatory cytokines such as IL-6 and activate pDCs to mature, but do not induce IFN-α production (Non-Patent Literature 8, 9). Patent Literature 1 describes numerous type K CpG ODNs. Other types of CpG ODNs, type C and type P, contain one and two palindromic CpG sequences, respectively. Both can activate B cells like type K and pDCs like type D, but type C CpG ODNs induce IFN-α production more weakly than type P CpG ODNs (Non-Patent Literature 10-12).

[0005] D-type and P-type CpG ODNs form higher-order structures of Hougsteen base pairs that form parallel quadruple-stranded structures called G-tetrads, and Watson-Crick base pairs between cis-palindromic and trans-palindromic structural sites. These structures are necessary for potent IFN-α production by pDCs (Non-Patent Literature 12-14). While these higher-order structures are thought to be necessary for localization to early endosomes and signal transduction via TLR9, they are also thought to cause polymorphism and aggregation of CpG ODNs, hindering the clinical application of D-type and P-type CpG ODNs (Non-Patent Literature 15). On the other hand, K-type and C-type CpG ODNs do not exhibit manufacturing hurdles such as aggregation and are considered usable as immunotherapeutic agents and vaccine adjuvants for humans (Non-Patent Literature 16 and 17). Furthermore, K-type CpG ODNs have been reported to enhance the immunogenicity of vaccines targeting infectious diseases and cancer in human clinical trials (Non-Patent Literature 6, 16).

[0006] Schizophyllan (hereinafter referred to as SPG), a soluble β(1→3) glucan derived from Schizophyllum commune, has a history of clinical use in Japan as an activator for radiotherapy in patients with cervical cancer (Non-Patent Literature 18). Similarly, lentinan (hereinafter referred to as LNT), a soluble β(1→3) glucan derived from shiitake mushrooms, is a drug approved in 1985 and has been used in combination with fluoropyrimidine drugs for patients with inoperable and recurrent gastric cancer (Non-Patent Literature 19, 20). β(1→3) glucans have been shown to form a triple-helix complex with polydeoxyadenosine (hereinafter referred to as poly(dA)) (Non-Patent Literature 21).

[0007] Patent documents 2 to 4 disclose the use of a water-soluble complex of β(1→3) glucan containing SPG and nucleic acid (gene) as a gene carrier. These documents state that the formation of this complex enhances the antisense effect and resistance to nucleases of the gene.

[0008] Patent Document 5 discloses that the effects of immunostimulant oligonucleotides containing a CpG sequence and in which the phosphate diester bond is replaced with a phosphorothioate bond or a phosphorodithioate bond can be enhanced by using a polysaccharide having a β(1→3) bond as a carrier (transfection agent).

[0009] Patent document 6 describes an immunostimulatory complex characterized by comprising an immunostimulatory oligonucleotide and a β(1→3) glucan having long-chain β(1→6) glucoside bond side chains.

[0010] It has been shown that mice in which poly(dA) having a phosphate diester bond is linked to the 3' end of CpG ODN complexed with SPG, and humanized CpG ODN, enhance cytokine production and act as an influenza vaccine adjuvant or as a preventive or therapeutic agent for helper T cell type 2 (hereinafter referred to as Th2)-related diseases (Non-Patent Documents 22, 23, Patent Document 7). Furthermore, it has been shown that linking poly(dA) having a phosphorothioate bond to the 3' end of CpG ODN increases complex formation to almost 100% (Non-Patent Document 24).

[0011] When a complex of a CpG ODN (K3) with 40 dA nucleotides ligated to the 3' end (hereinafter referred to as K3-dA40) and SPG (hereinafter referred to as K3-SPG) was applied to human peripheral blood mononuclear cells (hPBMCs), IFN-α production not observed with K3 alone was detected, and under conditions of low nucleic acid treatment concentration, the IFN-α production level was higher compared to D, P, and C type CpG ODNs (Non-Patent Literature 25). Furthermore, in a mouse model, it was revealed that the induction of IFN-α production by K3-SPG, and the enhanced induction activity of cytotoxic T cells (CTLs) and helper T cell type 1 (hereinafter referred to as Th1) when K3-SPG was used as a vaccine adjuvant, are dependent on type I IFNs mediated by TLR9 (Non-Patent Literature 25). Furthermore, evaluations of the anticancer activity of K3-SPG alone using various mouse cancer transplantation models revealed that intravenously administered K3-SPG exhibited higher anticancer activity than K3 (Non-Patent Literature 26). The anticancer activity of K3-SPG in mouse cancer transplantation models has also been suggested to be dependent on type I IFNs, and possibly also dependent on IL-12, which is important for CTL induction (Non-Patent Literature 26).

[0012] Patent document 8 discloses a method for producing an antigen / CpG oligonucleotide / β(1→3) glucan ternary complex.

[0013] The complex formed by β(1→3) glucans (BG) and CpG ODN nucleic acids (hereinafter referred to as BG-CpG) activates innate immunity in the body and has physiological activity such as inducing stronger inflammatory responses compared to CpG ODN alone (Non-Patent Literature 25). In other words, BG-CpG is considered a potentially effective immunotherapy agent for target diseases for which clinical application of CpG ODN alone is expected, such as infectious diseases, cancer, and allergic diseases such as asthma and hay fever (Non-Patent Literature 2, 3, 25, 26).

[0014] Recently, the application of CpG ODN to cancer immunotherapy has attracted attention (Non-Patent Literature 27). As an example of reported clinical applications, a nucleic acid preparation in which CpG ODN was encapsulated in virus-like nanoparticles (VLPs) modified with melanoma cancer antigen CTL epitopes induced melanoma cancer antigen-specific CTLs in 14 out of 22 patients with stage II to IV melanoma (Non-Patent Literature 27). Furthermore, in non-clinical models, the anticancer activity of nanoparticle-formulated CpG ODN has been reported against T-cell lymphoma, liver cancer, colorectal cancer, and glioma (Non-Patent Literature 28-31). From these reports, it is suggested that there may be a correlation between the particle size of CpG ODN preparations and their anticancer activity, and BG-CpG in nanoparticle form is considered useful for cancer immunotherapy.

[0015] An important indicator of BG-CpG's physiological activity is the induction of IFN-α production, a type I IFN (Non-Patent Literature 25). In particular, an in vitro evaluation system that measures the level of IFN-α production induction using hPBMCs, which suggests the pharmacological efficacy of BG-CpG in clinical settings, is superior in quantitative accuracy and data stability compared to pharmacological efficacy evaluation systems using living non-clinical cancer transplant models (Non-Patent Literature 25, 26). Furthermore, regarding in vitro pharmacological efficacy evaluation in non-clinical mouse models, it is considered possible to predict anti-cancer activity in cancer transplant models using living mice by measuring IFN-α and IFN-γ production, which correlate with anti-cancer activity in living mice (Non-Patent Literature 25, 26). On the other hand, the cancer growth inhibitory activity of BG-CpG needs to be clarified through evaluation using mouse transplant models (Non-Patent Literature 26). Representative mouse cancer transplant models include the B16 cancer subcutaneous transplant model, a melanoma model, and the Pan-02 cancer peritoneal transplant model, a peritoneal dissemination model of pancreatic cancer (Non-Patent Literature 26).

[0016] As described above, the usefulness of BG-CpG in the pharmaceutical field, particularly in cancer immunotherapy and vaccine adjuvants, has been confirmed. For practical application, it is desirable to reduce manufacturing costs by reducing the dosage due to improved activity of BG-CpG, and to establish detailed manufacturing methods and quality evaluation methods. The inventors focused on particle size as one of the factors determining the effectiveness of BG-CpG. As is well known to those skilled in the art, BG-CpG used for this application has been confirmed to have an average particle size of several tens to several hundreds of nanometers in an aqueous medium (Patent Documents 9-14). Pharmaceuticals with such a size are called nanomedicines, and they are attracting attention as a form of pharmaceutical with different characteristics and functions from chemically synthesized pharmaceuticals or biopharmaceuticals manufactured using cell culture, etc. In recent years, guidelines for development have been successively published (Non-Patent Documents 32-34). These documents state that it is necessary to optimize various characteristics in order to obtain the quality that a pharmaceutical should possess while maximizing the effectiveness of nanomedicines. Among these, particle size is considered to be one of the particularly important factors determining the characteristics of nanomedicines.

[0017] Regarding the relationship between the particle size of BG-CpG and its pharmacological activity, the following reports exist. Patent Document 10 discloses a range of 10 nm to 100 nm as the normal average particle size for the application in question, and 20 nm to 50 nm as the preferred average particle size. It also discloses that the average particle size of BG-CpG (K3-SPG) actually used in the test was 30 nm. On the other hand, Patent Document 11, which investigated the relationship between the particle size of BG-CpG and its pharmacological activity using in vitro cell irritancy as a basic indicator, discloses that the preferred radius of inertia range for BG-CpG (a complex in which the nucleic acid is D35-dA40 and the beta-glucan is SPG) is 20 nm to 200 nm. In detail, as a result of comparing the pharmacological activity of the complex having a radius of inertia size from 5 nm to 200 nm, a radius of inertia range of 20 nm to 150 nm is indicated as a preferred range. More specifically, the following facts 1)-3) regarding the dependence of the bioactivity of the complex on the radius of inertia are disclosed: 1) The complex with a radius of inertia of 5 nm to 10 nm exhibits significantly low bioactivity; 2) The complex with a radius of inertia of 150 nm to 200 nm exhibits slightly low bioactivity; and 3) There is no significant difference in the bioactivity of the complex in the range of 20 nm to 150 nm.

[0018] Here, the reasons why complexes with small radii of inertia have low activity include the fact that complex formation efficiency is not actually sufficient for small complexes, and that small molecules are not easily taken up by cells. Here, although there are differences in the measurement principles, if doubling the radius of inertia is roughly converted to the average particle diameter, then the average particle diameter of the complex is preferably in the range of 40 nm to 300 nm, and it has been disclosed that the pharmacological effects of the complex do not differ significantly within this range. On the other hand, regarding the molecular weight of beta-glucan as a component of BG-CpG and methods for controlling the particle diameter of BG-CpG, Patent Document 11 shows that when the nucleic acid is D35-dA40, a series of BG-CpGs with different average particle diameters in the range of 10 nm or less to about 200 nm can be obtained by compounding with SPGs of various molecular weights. Furthermore, Patent Document 12 discloses a method for producing SPG with a molecular weight of 450,000 (as a trimer), but the effect of the molecular weight of SPG on the particle diameter of the complex is not discussed. Patent document 13 shows that beta-glucan should have a weight-average molecular weight of 2000 or more, and that SPG with a molecular weight of 450,000 (as a trimer) was used as a component of BG-CpG. However, it does not discuss the effect of the molecular weight of SPG on the particle size of the composite.

[0019] Based on the above, research has been conducted on the relationship between the particle size of BG-CpG and its pharmacological activity in the average particle size range of several nanometers to several hundred nanometers. It has been disclosed that 1) BG-CpG with an average particle size of 20 nm or less or 300 nm or more has lower activity compared to BG-CpG with an average particle size of 20 nm to 300 nm, and 2) although the particle size dependence of the physiological activity of BG-CpG in the average particle size range of 20 nm to 300 nm is not clear, the range in which it is usually applied is 10 nm to 100 nm, and the preferred average particle size range is 20 nm to 50 nm. Furthermore, it is evident that BG-CpG with an average particle size of 30 nm is being actively used as an anticancer agent or vaccine adjuvant. [Prior art documents] [Patent Documents]

[0020] [Patent Document 1] US 8,030,285 B2 [Patent Document 2] WO 01 / 034207 A1 [Patent Document 3] WO 02 / 072152 A1 [Patent Document 4] Japanese Unexamined Patent Application Publication No. 2004 - 107272 [Patent Document 5] WO 2004 / 100965 A1 [Patent Document 6] Japanese Unexamined Patent Application Publication No. 2007 - 70307 [Patent Document 7] Japanese Unexamined Patent Application Publication No. 2008 - 100919 [Patent Document 8] Japanese Unexamined Patent Application Publication No. 2010 - 174107 [Patent Document 9] WO2016 / 103531 [Patent Document 10] WO2015 / 041318 [Patent Document 11] WO2016 / 098832 [Patent Document 12] WO2004 / 100965 [Patent Document 13] WO2001 / 034207 [Patent Document 14] Japanese Patent No. 5605793 [Non - Patent Document]

[0021] [Non - Patent Document 1] Hemmi, H., et al. A Toll - like receptor recognizes bacterial DNA. Nature 408, 740 - 745 (2000). [Non - Patent Document 2] Krieg, AM Therapeutic potential of Toll-like receptor 9 activation. Nature reviews. Drug discovery 5, 471-484 (2006). [Non-Patent Document 3] Klinman, DM Immunotherapeutic uses of CpG oligodeoxynucleotides. Nature reviews. Immunology 4, 249-258 (2004). [Non-Patent Document 4] Schillie, S., et al. Recommendations of the Advisory Committee on Immunization Practices for Use of a Hepatitis B Vaccine with a Novel Adjuvant. MMWR Morb Mortal Wkly Rep. 2018 Apr 20; 67(15): 455-458. [Non-Patent Document 5] Ribas, A., et al. SD-101 in Combination with Pembrolizumab in Advanced Melanoma: Results of a Phase Ib, Multicenter Study. Cancer Discov. 2018 Oct;8(10):1250-1257. [Non-Patent Document 6] Vollmer, J. & Krieg, AM Immunotherapeutic applications of CpG oligodeoxynucleotide TLR9 agonists. Advanced drug delivery reviews 61, 195-204 (2009). [Non-Patent Document 7] Krug, A., et al. Identification of CpG oligonucleotide sequences with high induction of IFN-alpha / beta in plasmacytoid dendritic cells. European journal of immunology 31, 2154-2163 (2001). [Non-Patent Document 8] Verthelyi, D., Ishii, K.J., Gursel, M., Takeshita, F. & Klinman, D.M. Human peripheral blood cells differentially recognize and respond to two distinct CPG motifs. Journal of immunology 166, 2372-2377 (2001). [Non-Patent Document 9] Hartmann, G. & Krieg, A.M. Mechanism and function of a newly identified CpG DNA motif in human primary B cells. Journal of immunology 164, 944-953 (2000). [Non-Patent Document 10] Hartmann, G., et al. Rational design of new CpG oligonucleotides that combine B cell activation with high IFN-alpha induction in plasmacytoid dendritic cells. European journal of immunology 33, 1633-1641 (2003). [Non-Patent Document 11] Marshall, J.D., et al. Identification of a novel CpG DNA class and motif that optimally stimulate B cell and plasmacytoid dendritic cell functions. Journal of leukocyte biology 73, 781-792 (2003). [Non-Patent Document 12] Samulowitz, U., et al. A novel class of immune-stimulatory CpG oligodeoxynucleotides unifies high potency in type I interferon induction with preferred structural properties. Oligonucleotides 20, 93-101 (2010). [Non-Patent Document 13] Kerkmann, M., et al. Spontaneous formation of nucleic acid-based nanoparticles is responsible for high interferon-alpha induction by CpG-A in plasmacytoid dendritic cells. The Journal of biological chemistry 280, 8086-8093 (2005). [Non-Patent Document 14] Klein, D.C., Latz, E., Espevik, T. & Stokke, B.T. Higher order structure of short immunostimulatory oligonucleotides studied by atomic force microscopy. Ultramicroscopy 110, 689-693 (2010). [Non-Patent Document 15] Puig, M., et al. Use of thermolytic protective groups to prevent G-tetrad formation in CpG ODN type D: structural studies and immunomodulatory activity in primates. Nucleic acids research 34, 6488-6495 (2006). [Non-Patent Document 16] Bode, C., Zhao, G., Steinhagen, F., Kinjo, T. & Klinman, DM CpG DNA as a vaccine adjuvant. Expert review of vaccines 10, 499-511 (2011). [Non-Patent Document 17] McHutchison, JG, et al. Phase 1B, randomized, double-blind, dose-escalation trial of CPG 10101 in patients with chronic hepatitis C virus. Hepatology 46, 1341-1349 (2007). [Non-Patent Document 18] Okamura, K., et al. Clinical evaluation of schizophyllan combined with irradiation in patients with cervical cancer. A randomized controlled study. Cancer 58, 865-872 (1986). [Non-Patent Document 19] Oba, K.; Kobayashi, M.; Matsui, T.; Kodera, Y.; Sakamoto, J. Individual patient based meta-analysis of lentinan for unresectable / recurrent gastric cancer. Anticancer Res., 2009, 29, 2739-2746.

Non-Patent Document 20

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Non-Patent Document 28

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Non-Patent Document 31

[0022] BG-CpG has a characteristic structure in which beta-glucan and nucleic acid are loosely assembled by non-covalent bonds, but there is still insufficient knowledge regarding the relationship between the particle size of BG-CpG and its pharmacological activity. Therefore, in order to obtain pharmaceuticals consisting of beta-glucan and nucleic acid complexes such as BG-CpG, it is necessary to investigate in detail the effect of particle size on pharmacological activity. An object of the present invention is to find a range of suitable particle sizes for beta-glucan and nucleic acid complexes that has not been previously known. Furthermore, an object of the present invention is to establish a robust manufacturing method and analytical method for producing beta-glucan and nucleic acid complexes such as BG-CpG with particle size controlled within a suitable range. [Means for solving the problem]

[0023] The inventors conducted diligent research and manufactured a series of BG-CpG with progressively different average particle sizes. As a result, the inventors found that it is possible to manufacture BG-CpG with an average particle size strictly controlled within the range of approximately 20 nm to 180 nm, preferably approximately 20 nm to 130 nm, or approximately 40 nm to 180 nm, more preferably approximately 80 nm to 130 nm, or approximately 130 nm to 180 nm. The inventors then investigated the anticancer effects of BG-CpG with average particle sizes in the range of 20 nm to 130 nm or 40 nm to 180 nm and found that the effect increases as the average particle size increases.

[0024] Therefore, the present invention encompasses the following: [1] A complex of β(1→3) glucan and nucleic acid with controlled particle size. [2] The composite according to [1], characterized in that the average particle diameter is 80 nm to 130 nm. [3] The composite according to [1], characterized in that the average particle diameter is 130 nm to 180 nm. [4] The complex according to [1] or [2], characterized in that the β(1→3) glucan is schizophyllan. [5] The complex according to [1] or [3], characterized in that the β(1→3) glucan is lentinan. [6] The complex according to any one of [1] to [5], characterized in that the nucleic acid is an oligonucleotide containing a K-type CpG oligonucleotide. [7] The complex according to any one of [1] to [6], characterized in that the nucleic acid is a nucleic acid in which polydeoxyadenosine is linked to the 3' end of a K-type CpG oligonucleotide. [8] The complex according to any one of [1] to [7], wherein the nucleic acid comprises a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylic acid, the nucleotide sequence represented by SEQ ID NO: 1, wherein polydexiadenosine is ligated to the 3' end of the humanized K-type CpG oligodeoxynucleotide, and some or all of the phosphate diester bonds of the oligodeoxynucleotide are substituted by phosphorothioate bonds. [9] A pharmaceutical composition comprising the complex described in any one of items [1] to [8].

[10] The pharmaceutical composition according to [9] for the prevention or treatment of viral infections, cancer, allergic diseases, intracellular parasitic protozoa, or bacterial infections.

[11] The pharmaceutical composition according to

[10] for the prevention or treatment of viral infections.

[12] The pharmaceutical composition according to

[11] , wherein the viral infection is RSV or influenza virus infection.

[13] An immunostimulant containing the complex described in any one of items [1] to [8].

[14] A vaccine adjuvant, an immunostimulant,

[13] .

[15] (a) A complex described in any one of items [1] to [8], or an immunostimulant described in

[13] or

[14] , and (b) Antigen A pharmaceutical composition containing the following:

[16] The composition according to

[15] for inducing an immune response to the antigen.

[17] The composition according to

[16] , wherein the antigen is an antigen derived from a pathogen.

[18] The composition described in

[17] for the prevention or treatment of infectious diseases caused by pathogens.

[19] The composition described in

[18] , wherein the pathogen is a virus.

[20] The composition according to

[19] , wherein the virus is RSV or influenza virus. [twenty one] The composition according to

[16] , wherein the antigen is a cancer-derived antigen. [twenty two] The composition according to

[21] , for the prevention or treatment of cancer. [twenty three] (a) A complex described in any one of items [1] to [8], or a pharmaceutical composition described in any one of items [9],

[10] ,

[15] ,

[16] ,

[21] , or

[22] , and (b) anticancer drugs A pharmaceutical composition containing the following: [twenty four] The composition according to

[23] , wherein the anticancer agent is an immune checkpoint inhibitor. [twenty five] The composition according to

[24] , wherein the immune checkpoint inhibitor is one of a PD1 inhibitor, a PDL-1 inhibitor, and a CTLA-4 inhibitor.

[26] A method for producing the complex described in any one of the following [1] to [8], utilizing the molecular weight characteristics of β(1→3) glucan.

[27] A method for treating or preventing viral infections, cancer, allergic diseases, intracellular parasitic protozoa, or bacterial infections, comprising administering an effective amount of the pharmaceutical composition described in [9] to a subject.

[28] The treatment or prevention method described in

[27] , wherein the viral infection is RSV or influenza virus infection.

[29] A method for treating or preventing a viral infection, cancer, allergic disease, intracellular parasitic protozoan infection, or bacterial infection, comprising administering an effective amount of the pharmaceutical composition described in

[15] to a subject.

[30] The treatment or prevention method described in

[29] , wherein the viral infection is RSV or influenza virus infection.

[31] A method for treating or preventing cancer, as described in

[27] or

[29] , characterized by being used in combination with other anticancer drugs.

[32] The treatment or prevention method described in

[31] , wherein the anticancer drug is an immune checkpoint inhibitor.

[33] The treatment or prevention method according to

[32] , wherein the immune checkpoint inhibitor is one of a PD1 inhibitor, a PDL-1 inhibitor, or a CTLA-4 inhibitor. [Effects of the Invention]

[0025] According to the present invention, by selecting or controlling the molecular weight characteristics of beta-glucan using a highly practical manufacturing method and conditions, BG-CpG is provided in which the average particle size is strictly controlled within the range of 20 nm to 180 nm, preferably 20 nm to 130 nm, or 40 nm to 180 nm, more preferably 80 nm to 130 nm, or 130 nm to 180 nm. Therefore, the optimal particle size can be selected for use as a therapeutic agent or preventive agent, or as one of the components thereof, such as in cancer immunotherapy. Since the BG-CpG of the present invention shows remarkable efficacy with a smaller dose compared to conventional BG-CpG, it may be applicable to diseases that were difficult to treat or prevent with conventional BG-CpG. [Brief explanation of the drawing]

[0026] [Figure 1] Figure 1 is a chart of the 1H-NMR spectra of the prepared mSPG and SPG standard samples. [Figure 2] Figure 2 is a chart of the 13C-NMR spectra of the prepared mSPG and SPG standard samples. [Figure 3] Figure 3 shows the molecular weight calibration curve for pullulan. [Figure 4]Figure 4A is a graph showing the results of the evaluation of the ability of K3-SPG to induce IFN-α production from human PBMCs. Figure 4A shows the AUC values ​​of IFN-α production levels when hPBMCs were stimulated with K3-SPG at concentrations of 0, 0.75, 2, 6, and 20 μg / mL. Figure 4B is a graph showing the results of the evaluation of the ability of mouse splenocytes to induce IFN-γ production. Figure 4B shows the AUC values ​​of IFN-γ induction levels when mouse splenocytes were stimulated with K3-SPG at concentrations of 0, 0.75, 2, 6, and 20 μg / mL. The lot numbers of the test substances used in Figure 4 are as follows. ·KAInv3701:20170613-01 ·KAmSP001a:20170613-02 ·KAmSP002a:20170613-03 ·KASPG0070:20170613-04 ·KASPG0071:20170613-05 ·KASPG0095:20170926-07. To the right of the graph in Figure 4A, the DLS (nm) evaluation results for the samples of each lot number are shown. [Figure 5] Figure 5 shows the results of evaluating the correlation between the AUC value of IFN-α production level and the average particle size using DLS, as evaluated by Spearman's test, when hPBMCs were stimulated with BG-CpG at CpG ODN concentrations of 0, 0.75, 2, 6, and 20. [Figure 6] Figure 6 shows the in vivo evaluation of the pharmacological effects of K3-SPG. The upper part of Figure 6 shows the protocol for this in vivo evaluation experiment. The lower part of Figure 6 shows a graph showing the results of measuring tumor size over time in the K3-SPG, K3, and PBS (control) groups according to that protocol. In the graph in the lower part of Figure 6, the horizontal axis shows the number of days since subcutaneous transplantation of B16, and the vertical axis shows the tumor size in the K3-SPG, K3, and PBS administration groups. [Figure 7] Figure 7 shows the relationship between the alkali treatment time when lentinan is alkali-treated under predetermined conditions and the average particle size of BG-CpG containing lentinan as a component. [Figure 8]Figure 8 is a graph showing the results of evaluating the ability of K3-LNT with various average particle sizes to induce IFN-γ production from mouse splenocytes. It shows the total IFN-γ induction levels when mouse splenocytes were stimulated with K3-LNT at concentrations of 2 μg / mL and 10 μg / mL. [Figure 9] Figure 9 shows the in vivo evaluation of the pharmacological effects of K3-LNT. 2.5 × 10⁵ B16 tumor cells were transplanted subcutaneously into C57BL / 6 mice (transplantation day is designated as day 0), and K3-LNT was administered intratumorally a total of three times at 7, 9, and 11 days. For comparison, physiological saline (saline) was administered. The horizontal axis shows the number of days since subcutaneous transplantation of B16, and the vertical axis shows the tumor size in the K3-LNT and physiological saline administration groups. The lot numbers of the test substances used in Figure 9 are as follows: ·KALNT0706:20201106-07 ·KALNT0710:20201106-04 ·KALNT0709:20201106-03 ·KALNT0712:20201106-06. [Figure 10] Figure 10 shows the in vivo evaluation of the pharmacological effects of K3-LNT, similar to Figure 9. However, the number of transplanted tumor cells was set to 5 × 10⁵. For comparison, physiological saline (saline) and K3 were administered. The lot numbers of the test substances used in Figure 10 are as follows: ·KALNT0710:20201106-04·KALNT0709:20201106-03. [Modes for carrying out the invention]

[0027] 1. Complex of β(1→3) glucan with controlled particle size and nucleic acid The present invention provides a complex of β(1→3) glucan and nucleic acid with controlled particle size. Through diligent research, the inventors discovered that the anticancer activity of the complex is significantly higher when the average particle size of the β(1→3) glucan and nucleic acid complex is in the range of 20 nm to 180 nm, preferably 20 nm to 130 nm, or 40 nm to 180 nm, more preferably 80 nm to 130 nm, or about 130 nm to 180 nm, thus completing the present invention. Therefore, BG-CpG with a controlled average particle size in such a range can be used as a therapeutic or prophylactic agent for cancer immunotherapy and the like.

[0028] The β(1→3) glucans used in the present invention are not particularly limited as long as they have the property of forming complexes with nucleic acids, but examples include β(1→3) glucans such as schizophyllan (SPG), lentinan (LNT), curdlan, scleroglucan, parkyman, glyphorane, and laminan. It has already been reported that they have the property of forming complexes with nucleic acids (WO2004 / 100965 and Japanese Patent No. 4850512, immunostimulants). SPG and LNT are particularly suitable β(1→3) glucans that can be used in the present invention.

[0029] The complex of β(1→3) glucan and nucleic acid in the present invention is not limited to this, but is preferably a complex formed by β(1→3) glucan (BG) and nucleic acid containing CpG ODN (BG-CpG). More preferably, the complex in the present invention is a complex of SPG and CpG ODN (K3-dA40) obtained by affixing 40 dA bases to the 3' end of K-type CpG ODN (K3) (K3-SPG), or a complex of LNT and CpG ODN (K3-dA40) obtained by affixing 40 dA bases to the 3' end of K-type CpG ODN (K3) (K3-LNT).

[0030] As will be explained in detail in section 4 below, SPG can be obtained by selecting a strain of Schizophyllum commune with high SPG production capacity, culturing it under appropriate culture conditions to efficiently secrete SPG into the culture medium, and then purifying the medium. SPG can be used as is, but it is also used after controlling its molecular weight to an appropriate level as needed. The method for controlling the molecular weight of beta-glucan will also be explained in detail in section 2 below.

[0031] In this invention, "a complex with controlled particle size" means a complex manufactured using beta-glucan of an appropriate molecular weight as a component in order to set the average particle size of the beta-glucan and nucleic acid complex within a desired range. Here, the molecular weight of the beta-glucan does not need to be specified. Furthermore, in this invention, "strict control" means controlling the average particle size of the complex to approximately 10 nm to 50 nm or a narrower interval. Such strict control makes it possible to determine the optimal average particle size range for beta-glucan and nucleic acid complexes for various pharmaceutical applications. Furthermore, such strict control makes it possible to manufacture a beta-glucan and nucleic acid complex having an average particle size that exhibits the maximum pharmacological effect.

[0032] WO2016 / 098832 describes the production of complexes with a nucleic acid called d(A)40-D35 using a series of SPGs with molecular weights ranging from approximately 10,000 to 5,400,000, and discloses the relationship between their radius of inertia and physiological activity. According to this document, the controllable range for the radius of inertia is 5 nm to 8 nm, 8 nm to 10 nm, 20 nm to 70 nm, 60 nm to 100 nm, 100 nm to 150 nm, and 150 nm to 200 nm, with a controllable range of 50 nm or more for radii of inertia above 20 nm. Converting this to particle size, the controllable range is approximately 100 nm or more, which cannot be said to be strict control. Furthermore, the strength of the pharmacological effect of these complexes is limited to a three-stage semi-qualitative evaluation, and no difference in pharmacological effect is observed in the range of radii of inertia from 8 nm to 150 nm.

[0033] On the other hand, the prior art described above (Miyamoto N., et al., Bioconjugate Chemistry 2017, 28, 565-573) attempts to increase the particle size of a complex of beta-glucan and nucleic acid (referred to as CpG-SPG) with SPG and CpG-dA40 as components. Here, a nucleic acid having a sequence complementary to CpG (cCpG-dA40) was prepared, and a complex of this with SPG (cCpG-SPG) was manufactured. Next, by mixing CpG-SPG and cCpG-SPG, the CpG and cCpG in both complexes were linked by non-covalent bonds, crosslinking the complexes and generating a structure with a larger particle size (referred to as CL-CpG nanogel). While the radii of inertia of CpG-SPG and cCpG-SPG were approximately 10 nm, the radii of inertia of CL-CpG nanogel was approximately 150 nm. The radii of inertia of CL-CpG nanogel corresponds to an average particle diameter of approximately 300 nm, confirming that it has a stronger adjuvant effect compared to CpG-SPG.

[0034] As described above, while the CL-CpG nanogel of this prior art succeeds in increasing the particle size compared to CpG-SPG, it cannot be said that the particle size is precisely controlled. In other words, the average particle size around 300 nm is not precisely controlled, and it cannot be said that the average particle size of the beta-glucan and nucleic acid complex has been optimized.

[0035] As described above, while prior art has succeeded in increasing the average particle size to approximately 300 nm, it has not been possible to precisely control the average particle size of the complex, that is, to intervals of approximately 10 nm to 50 nm or smaller. The inventors considered that due to this lack of precise particle size control, the beta-glucan and nucleic acid complex may not be able to exert the remarkable pharmacological effects it is supposed to have. In other words, they considered it important to develop a technology that enables the production of a beta-glucan and nucleic acid complex having a precise average particle size, thereby identifying the average particle size that exerts the maximum pharmacological effect.

[0036] The molecular weight of beta-glucan produced and contained can vary depending on the bacterial strain and variety. Therefore, it is possible to produce beta-glucan with a large molecular weight by selecting the optimal strain and culture / growth conditions. Furthermore, since beta-glucan, being a natural polysaccharide, has a wide molecular weight distribution, even if the average molecular weight is small, it contains components with large molecular weights. By fractionating and purifying these components, it is possible to obtain beta-glucan with a large molecular weight. Moreover, it is known that the molecular weight can be increased by chemically or physically crosslinking polysaccharides with a predetermined molecular weight (Basu A., et al., Bioconjugate Chemistry 2015, 26, 1396-1412; Pawar HA, et al., Biology and Medicine (Aligarh) 2015, 6, 224).

[0037] These methods make it possible to increase the molecular weight of polysaccharides to infinity, that is, to the point where they become insoluble as a gel. In other words, by appropriately applying these methods, the molecular weight of beta-glucan can be increased to infinity. Furthermore, by using the molecular weight control conditions disclosed in this invention, it is also possible to reduce the molecular weight after obtaining and producing high molecular weight beta-glucan. As already mentioned, prior art (WO2016 / 098832 and Miyamoto N., et al., Bioconjugate Chemistry 2017, 28, 565-573) has demonstrated the usefulness of a complex of beta-glucan with a radius of inertia of 150 nm and nucleic acid. By preparing high molecular weight beta-glucan using one of the above methods, it has been shown that a complex of beta-glucan with an average particle size of about 300 nm and nucleic acid is pharmaceutically useful. However, in this prior art, the particle size is not strictly controlled, that is, the average particle size cannot be controlled to approximately 10 nm to 50 nm or at narrower intervals, so an average particle size of 300 nm cannot be said to be optimal.

[0038] In this way, BG-CpG can be produced, consisting of SPG obtained in this manner and nucleic acid (preferably K3-dA40, which has been confirmed to be useful as a cancer immunotherapy agent), with a strictly controlled particle size. Surprisingly, in a pharmacological evaluation system intended for cancer immunotherapy, the inventors found that BG-CpG with an average particle size in the range of 80 nm to 130 nm, or 130 nm to 180 nm, exhibited significantly higher pharmacological effects. As already mentioned, the average particle size of BG-CpG, which has a proven track record in conventional research, is around 30 nm, or 20 nm to 50 nm, and the average particle size discovered by the inventors is outside that range.

[0039] In the examples described herein, the efficacy of BG-CpG with strictly controlled particle size was evaluated as a cancer immunotherapy agent using state-of-the-art in vitro and in vivo pharmacological evaluation systems that fully considered the suitability for clinical use. As a result, the inventors found that there is a strong correlation between the particle size of BG-CpG and its pharmacological activity, and that BG-CpG with an average particle size in the range of 80 nm to 130 nm, or 130 nm to 180 nm, exhibits significantly higher pharmacological activity.

[0040] As shown in the examples below, the optimal particle size of BG-CpG for pharmacological activity (anti-cancer activity) was determined using two in vitro evaluation systems: one using hPBMCs to evaluate the IFN-α inducing ability of BG-CpG, and another using mouse spleen cells to evaluate the IFN-γ inducing ability of BG-CpG. These in vitro evaluation systems are considered to have higher data stability and quantitative accuracy compared to mouse cancer transplantation models, which exhibit greater inter-experimental variability. Furthermore, the IFN-α induction level of BG-CpG showed a maximum effect (Emax) (so-called bell shape) at medium doses. To improve quantitative accuracy, the IFN-α inducing ability of BG-CpG was calculated using the area under the curve (AUC) in the examples below. Similarly, with IFN-γ, the amount of BG-CpG added was set to two levels, 2 μg / mL and 10 μg / mL. The amount of IFN-γ produced after 24 hours of incubation was measured for each level, and the sum of these values ​​was used as an indicator of the strength of the pharmacological effect. As a result, in the range of 20 nm to 130 nm or 40 nm to 180 nm, BG-CpG showed a stronger pharmacological effect as the average particle size increased. The average particle size of BG-CpG that exhibited the greatest pharmacological effect was discovered for the first time through the strict control of particle size in this invention.

[0041] Furthermore, in the following examples, the anticancer activity of BG-CpG, which was narrowed down by in vitro evaluation, was evaluated using a model in which B16 melanoma cancer was transplanted into mice (Kitahata, Y., et al. Circulating nano-particulate TLR9 agonist scouts out tumor microenvironment to release immunogenic dead tumor cells. Oncotarget 7, 48860-48869 (2016)). As a result, the anticancer activity of BG-CpG was observed in vivo. As described above, it was found that the BG-CpG of the present invention, in which the particle size is strictly controlled, exhibits high anticancer activity.

[0042] 2. A pharmaceutical composition containing a complex of β(1→3) glucan with controlled particle size and nucleic acid. Based on its mechanism of action, the BG-CpG of the present invention can be used as a therapeutic agent or preventive agent / vaccine for various diseases. The present invention provides a pharmaceutical composition containing the complex of the present invention described above. Prophylactic or therapeutic indications for specific diseases are determined, and the efficacy and safety are predicted or verified through exploratory research, non-clinical trials, and clinical trials before commercialization. At any stage of this drug development process, the particle size of the BG-CpG of the present invention is optimized or an acceptable range is determined. Generally, in the exploratory research stage, the particle size is optimized or an acceptable range is determined based on its in vitro or in vivo activity. Furthermore, in safety tests during non-clinical trials, the validity of the optimized particle size or the particle size range set as the acceptable range is verified from a safety perspective.

[0043] The pharmaceutical composition of the present invention can be obtained by formulating the complex of the present invention according to conventional methods. The pharmaceutical composition of the present invention comprises the complex of the present invention and a pharmacokinetically acceptable carrier. The pharmaceutical composition may further contain an antigen. Such a pharmaceutical composition is provided in a dosage form suitable for oral or parenteral administration.

[0044] For parenteral administration, for example, injectable preparations, suppositories, etc., can be used, and injectable preparations may include dosage forms such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, and drip infusion injection. Such injectable preparations can be prepared according to known methods. For example, the injectable preparation can be prepared by dissolving or suspending the complex of the present invention in a sterile aqueous solvent commonly used for injectable preparations. As aqueous solvents for injection, for example, distilled water; physiological saline; buffers such as phosphate buffer, carbonate buffer, Tris buffer, and acetate buffer can be used. The pH of such aqueous solvents is typically 5 to 10, preferably 6 to 8. The prepared injectable solution is preferably filled into a suitable ampoule.

[0045] Furthermore, a powder formulation of the composite of the present invention can be prepared by subjecting a suspension of the composite to treatments such as vacuum drying or freeze-drying. The composite of the present invention can be stored in powder form and used by dispersing the powder in an aqueous solvent for injection when needed.

[0046] The content of the complex of the present invention in the pharmaceutical composition is usually about 0.1 to 100% by weight of the total pharmaceutical composition, preferably about 1 to 99% by weight, and more preferably about 10 to 90% by weight.

[0047] The pharmaceutical composition of the present invention may contain the complex of the present invention alone as an active ingredient, or it may contain the complex of the present invention in combination with other active ingredients.

[0048] 3. Pharmaceutical Uses Since the complex of the present invention has excellent immunostimulatory activity, the complex and pharmaceutical composition of the present invention can be used as immunostimulants. By administering the complex or pharmaceutical composition of the present invention to mammals (primates such as humans, rodents such as mice, etc.), an immune response can be induced in the mammals. In particular, the complex of the present invention has the activity properties of type D CpG ODN and stimulates peripheral blood mononuclear cells to produce large amounts of both type I interferon (Pan-IFN-α, IFN-α2, etc.) and type II interferon (IFN-γ), making it useful as a type I interferon production inducer, a type II interferon production inducer, and a type I and type II interferon production inducer. Since it induces the production of both type I and type II interferon, the complex of the present invention and the pharmaceutical composition containing it are useful for the prevention or treatment of diseases in which either or both type I and type II interferon are effective. Diseases for which type I interferon is effective include viral infections (e.g., hepatitis C virus (HCV), herpesvirus, papillomavirus, respiratory syncytial virus, influenza virus, etc.) and cancer. Diseases for which type II interferon is effective include allergic diseases and infections caused by intracellular parasitic protozoa (e.g., Leishmania) and bacteria (e.g., Listeria, Mycobacterium tuberculosis, etc.). For acute viral infections, including respiratory syncytial virus and influenza virus, both type I and type II interferon enhance the immune response related to viral elimination; therefore, the complex of the present invention and the pharmaceutical composition containing it can be expected to be effective against acute viral infections.

[0049] The tumors that can be treated with the complex of the present invention and the pharmaceutical composition containing it are not particularly limited, but are preferably pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, kidney cancer, breast cancer, uterine cancer (e.g., cervical cancer), ovarian cancer, lung cancer, thyroid cancer, skin cancer (e.g., melanoma), head and neck cancer, sarcoma, prostate cancer, bladder cancer, brain tumor, gastrointestinal stromal tumor (GIST), leukemia (e.g., acute myeloid leukemia (AML), chronic myeloid leukemia (CML), or chronic lymphocytic leukemia (CLL) or acute lymphoblastic leukemia (ALL)), lymphoma or malignant lymphoma (e.g., T lymphoma (including adult T-cell leukemia), B-cell lymphoma, non-Hodgkin lymphoma, or diffuse large B-cell lymphoma (DLBCL)). )), more preferably, cancers that are highly sensitive to IFN-α and IFN-γ, which are cytokines potently induced by the complex of the present invention and pharmaceutical compositions containing the same (Non-Patent Literature 26 and Martin-Hijaro, L. and Sainz Jr., B. 2020: The interactions between cancer stem cells and the innate interferon signaling pathway. Frontiers in immunology 11, Article 526.), include melanoma, T lymphoma, colorectal cancer, pancreatic cancer, chronic myeloid leukemia, liver cancer, cervical cancer, ovarian cancer, bladder cancer, adult T-cell leukemia, and more preferably, melanoma, T lymphoma (including adult T-cell leukemia), colorectal cancer, and pancreatic cancer.

[0050] Furthermore, the complex of the present invention possesses potent vaccine adjuvant activity, and when administered to mammals together with an antigen, it can strongly induce an immune response against the antigen. Therefore, the present invention also provides (a) the complex of the present invention and (b) a composition for inducing an immune response against the antigen, comprising the antigen. The complex of the present invention strongly induces both humoral immune responses (antigen-specific antibody production) and cellular immune responses (antigen-specific CTL induction) against the antigen. Therefore, the complex of the present invention and pharmaceutical compositions containing it are useful as vaccine adjuvants. In this specification, "adjuvant" refers to an adjuvant that promotes an immune response, meaning a substance that, when administered to a living organism together with an antigen, nonspecifically enhances the immune response to that antigen.

[0051] The antigen is not particularly limited as long as it is antigenic to the target mammal (primates such as humans, rodents such as mice, etc.) and can be recognized as an antigen by antibodies or cytotoxic T lymphocytes (CTLs, CD8+ T cells). Any substance that can act as an antigen (proteins, peptides, nucleic acids, lipids, carbohydrates, and modified versions of the said substances (for example, modified versions with the introduction of one or more amino acid deletions, substitutions, and / or additions (hereinafter referred to as mutations, etc.)) can be used. Antigens derived from pathogens such as protozoa, fungi, bacteria, and viruses, as well as antigens related to cancer or specific diseases, can also be used.

[0052] In this specification, "antigen A is derived from pathogen X" means that antigen A is included as a component of pathogen X. For example, if antigen A is a polypeptide, it means that the amino acid sequence of that polypeptide is present in the amino acid sequence of the protein encoded by the genome of pathogen X.

[0053] Antigens derived from pathogens include the pathogen itself or a part thereof, the pathogen itself or a part thereof that has been inactivated or weakened, or modified forms thereof into which mutations have been introduced.

[0054] When an antigen derived from a pathogen is used as the antigen, an immune response to that antigen is induced, and a mechanism is established to immunologically eliminate the pathogen containing that antigen from the body. Therefore, (a) the complex of the present invention and (b) a composition for inducing an immune response to the antigen, which contains an antigen derived from a pathogen, are useful for the prevention or treatment of the pathogen.

[0055] The complex of the present invention potently induces both humoral immune responses (antigen-specific antibody production) and cellular immune responses (antigen-specific CTL induction) against antigens. Therefore, antigens derived from intracellular infectious pathogens (viruses, protozoa, fungi, bacteria, etc.) known to be recognized by cytotoxic T lymphocytes, and antigens associated with cancerous cells (e.g., tumor antigens) are suitably used as antigens.

[0056] Examples of intracellular infectious viruses, though not particularly limited, include respiratory syncytial virus (RSV), influenza virus, parainfluenza virus, hepatitis C virus (HCV), hepatitis A virus (HAV), hepatitis B virus (HBV), Ebola virus, cytomegalovirus, adenovirus, poliovirus, Japanese encephalitis virus, measles virus, mumps virus, rubella virus, rabies virus, yellow fever virus, varicella-zoster virus, hantavirus, dengue virus, norovirus, rotavirus, parvovirus, coronavirus, distemper virus, adult T-cell leukemia virus (HTLV-1), human immunodeficiency virus (HIV), herpesvirus, and papillomavirus. Examples of intracellular infectious bacteria include mycoplasma. Examples of intracellular infectious protozoa include malaria parasites and schistosomiasis. The intracellular infectious pathogen is preferably a virus (specifically, RSV or influenza virus, etc.).

[0057] Antigens associated with cancerous cells include proteins, glycans, peptides, and variants (deletions, substitutions, and / or additions) or modifications thereof that are specifically expressed in cancerous cells.

[0058] Since the complex of the present invention potently induces both type I and type II interferons, in one embodiment, a virus that causes acute viral infections in which both type I and type II interferons are effective (e.g., RSV, influenza virus) is selected as the virus.

[0059] For example, by administering (a) the complex of the present invention and (b) a composition for inducing an immune response to a pathogen or cancer-derived antigen to a patient with a pathogen-related infection or cancer, or a person who may be susceptible to such an infection or cancer, cytotoxic T lymphocytes (CTLs) in the administered subject are activated in an antigen-specific manner, inducing antigen-specific antibody production, that is, by inducing a protective immune response in warm-blooded animals (preferably humans), the infection or cancer can be prevented or treated. In other words, the composition is useful as a vaccine for the prevention or treatment of the above-mentioned infections, cancer, and other diseases.

[0060] Furthermore, since the complex of the present invention can potently induce both humoral immune responses (antigen-specific antibody production) and cellular immune responses (antigen-specific CTL induction) against antigens, it is possible to use either surface antigens or internal antigens of pathogens or cancer cells as antigens, and it is also desirable to use a mixture of surface antigens and internal antigens.

[0061] A composition for inducing an immune response to an antigen, comprising (a) the complex of the present invention and (b) the antigen, can be prepared in accordance with the pharmaceutical composition of the present invention described above.

[0062] The complex or pharmaceutical composition of the present invention can be administered simultaneously with or individually with other pharmaceuticals. For example, the complex or pharmaceutical composition of the present invention may be administered after administering other pharmaceuticals, or other pharmaceuticals may be administered after administering such complex or pharmaceutical composition, or the complex or pharmaceutical composition and other pharmaceuticals may be administered simultaneously. Other pharmaceuticals include chemotherapeutic agents, radiotherapy agents and various anticancer agents, and pharmaceutical compositions containing further agents in addition to the complex or pharmaceutical composition of the present invention are also included in the present invention.

[0063] TLR-9 agonists exhibit remarkable antitumor effects when used in combination with immune checkpoint inhibitors (Adamus, T. and Kortylewski, M., The revival of CpG pligonucleotide-based cancer immunotherapies, Contenp. Oncol. (Pozn.) 21 (1A), 56-60 (2017).). Immune checkpoint inhibitors include PD1 inhibitors, PD-L1 inhibitors, and CTLA-4 inhibitors, such as anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-CTLA-4 antibodies, respectively (Bagchi, S., Yuan, R., and Engleman, EG, Immune checkpoint inhibitors for the treatment of cancer: clinical impact and mechanism of response and resistance, Ann. Rev. Pathol. 16, 223-249 (2021).). The combined efficacy of a TLR-9 agonist called IMO-2125 and an anti-PD-1 antibody has been validated in an animal model of pancreatic cancer (Carbone C., et al., Intratumoral injection of TLR-9 agonist promotes an immunopermissive microenvironment transition and causes cooperative antitumor activity in combination with anti-PD1 in pancreatic cancer, J. Immunother. Cancer 9 2021). Therefore, the combined use of the TLR-9 agonist conjugate or the pharmaceutical composition of the present invention with an immune checkpoint inhibitor may be a useful therapeutic method.

[0064] 4. Method for producing a complex of β(1→3) glucan and nucleic acid having an average particle size within a specific range. The present invention also provides a method for producing a complex of β(1→3) glucan and nucleic acid having an average particle size within a specific range. In this invention, we have succeeded in producing BG-CpG with strictly controlled particle size using raw materials with a known source and a feasible manufacturing method.

[0065] As already mentioned, SPG is a β(1→3) glucan produced by strains of Schizophyllum commune, and several such strains are known. The inventors diligently investigated the SPG production of different Schizophyllum commune strains and found that productivity was particularly high in certain strains. Therefore, obtaining SPG using a Schizophyllum commune strain with high SPG productivity is a preferred embodiment of the present invention. Similarly, lentinan (LNT) is a β(1→3) glucan contained in the fruiting bodies of shiitake mushrooms, and extracting LNT from shiitake fruiting bodies is a preferred embodiment of the present invention.

[0066] (1) Method for producing beta-glucan using the Suehirotake strain To produce beta-glucan using Schizophyllum sieboldii strains, etc., methods well known to those skilled in the art (Kikumoto, Shoichi et al.. Studies on polysaccharides produced by Schizophyllum sieboldii. Agricultural Chemistry 44, 337-342 (1970); Mizuno, Takashi & Kawai, Masayoshi, eds. Chemistry and Biochemistry of Mushrooms. Academic Publishing Center (1992)) are used, in which the Schizophyllum sieboldii strain is cultured in a medium in which the strain can grow, and SPG is produced in the extracellular medium. As the medium, a conventional medium containing nutrients (carbon source, nitrogen source, inorganic salts) that strains belonging to Schizophyllum sieboldii can normally utilize, and further containing organic nutrients as needed, can be used, but a culture solution containing sugars as a carbon source is preferred. Various synthetic media, semi-synthetic media, and natural media can all be used as these media.

[0067] As the carbon source mentioned above, sugars are preferred, and examples of such sugars include monosaccharides such as glucose, fructose, mannose, galactose, xylose, and arabinose; disaccharides such as sucrose, maltose, lactose, and trehalose; oligosaccharides such as fructooligosaccharides and xylooligosaccharides; and polysaccharides such as dextrin and starch. These can be used individually or in combination. Among these, it is preferable to use hexacarbons such as glucose and fructose, disaccharides such as sucrose and lactose, starch and dextrin, or polysaccharides such as hydrolysates of these carbohydrates as the main carbon source. In addition, beet juice, sugarcane juice, fruit juice including citrus fruits, or juices of these fruits to which sugar has been added can also be used. Other carbon sources such as alcohols such as glycerol and ethylene glycol, sugar alcohols such as mannitol, sorbitol, and erythritol, and organic acids can be used as appropriate. These carbon sources may be added as needed during cultivation. For example, feeding sugars such as glucose into the culture medium as appropriate can relatively increase the production rate and amount of beta-glucan produced.

[0068] As the nitrogen sources mentioned above, organic nitrogen sources such as peptone, meat extract, soy flour, casein, amino acids, malt extract, corn steep liquor, casein hydrolysate, yeast extract, and urea, and inorganic nitrogen sources such as sodium nitrate, ammonium sulfate, ammonia gas, and ammonia water can be used individually or in combination.

[0069] Examples of the inorganic salts used include sodium chloride, potassium chloride, calcium carbonate, magnesium sulfate, sodium phosphate, potassium phosphate, cobalt chloride, and heavy metal salts. Vitamins may also be added as needed. If foaming occurs during cultivation, various known defoaming agents may be added to the culture medium as appropriate.

[0070] There are no particular restrictions on the culture conditions for the strain of the present invention, and they can be appropriately selected within a range in which the strain can grow well. Typically, it is preferable to culture at pH 5.0 to pH 8.5 and 20°C to 35°C for about 5 to 14 days, but these culture conditions can be appropriately changed depending on the type and characteristics of the strain used, external conditions, etc., and the optimal conditions can be selected. Furthermore, the amount of strain inoculated into the culture medium is preferably 1% for flask culture, and 1% (v / v) to 10% (v / v) of seed culture solution to the main culture medium for platinum loop culture and scale-up, but this is not limited to cases where cultivation is substantially possible.

[0071] The bacterial strain of the present invention is cultured under aerobic conditions such as aeration, stirring, and shaking. The culture time is preferably continued until the desired beta-glucan production concentration is reached, which is usually 5 to 14 days. Alternatively, beta-glucan may be continuously produced by continuously adding sugars, which are substrates for beta-glucan, and culture medium components.

[0072] After culturing as described above, the beta-glucan secreted and produced outside the bacterial cells is separated and collected from the culture medium according to conventional methods. Specifically, various known purification methods can be selected and combined, such as separating and removing solid matter such as bacterial cells from the culture medium by centrifugation or filtration, or removing impurities and salts using activated carbon, ion exchange resin, etc. Furthermore, separation and purification can be achieved by using methods such as adsorption and elution to hydrophobic resins, solvent precipitation using ethanol, methanol, ethyl acetate, n-butanol, etc., column chromatography or thin-layer chromatography using silica gel, or preparative high-performance liquid chromatography using reversed-phase columns, either alone or in appropriate combinations, and sometimes repeatedly.

[0073] Furthermore, the bacterial cells may be sterilized before or after separating the beta-glucan secreted and produced outside the bacterial cells using the method described above. The sterilization temperature is not particularly limited as long as it is a temperature at which the bacterial cells are killed, but for example, it can be 90°C or higher, or 121°C under pressure. The sterilization time can be set to any time, but 15 to 60 minutes is preferred.

[0074] (2) Method for producing beta-glucan using shiitake mushroom fruiting bodies Lentinan is a beta-glucan found in the fruiting bodies of shiitake mushrooms. Therefore, lentinan can be obtained by extracting and purifying it from shiitake fruiting bodies. In the production of lentinan according to the present invention, shiitake fruiting bodies can be cultivated by methods such as log cultivation or substrate cultivation, according to methods well known to those skilled in the art (Kiyoshi Omori and Hiroshi Koide. Complete Guide to Mushroom Cultivation. Agricultural, Forestry and Fisheries Culture Association (2001); Edited by the Editorial Committee of the Complete Guide to Latest Biotechnology. Mushroom Propagation and Breeding. Agricultural Books (1992)) or novel methods, and lentinan can be extracted from said fruiting bodies.

[0075] Log cultivation is a method of cultivating wood-decaying fungi, primarily using sawtooth oak, Japanese oak, and Mongolian oak. It involves inoculating logs that have been felled and dried with fungal spores. After inoculation, growth management is carried out, and the shiitake mushrooms that grow are harvested.

[0076] Mushroom cultivation using a substrate involves mixing one or more types of wood-based materials such as broadleaf tree sawdust, chip dust, or chips with one or more types of crushed grains or legumes such as bran or rice bran, adding water to adjust the moisture content, and then pressurizing, shaping, bagging, sterilizing, and cooling the substrate according to conventional methods. Shiitake mushroom spawn is then inoculated into this substrate, and after cultivating it for a certain period, the substrate is managed for growth using either conventional cultivation (a cultivation method in which the substrate is removed from the bag and mushrooms grow from the entire substrate) or top-surface cultivation (a cultivation method in which only the top of the substrate is exposed and mushrooms grow only from the top of the substrate), and the resulting shiitake mushrooms are harvested.

[0077] Lentinan can be extracted and purified by methods well known to those skilled in the art (Chihara et al. Fractionation and purification of the polysaccharides with marked antitumor activity, especially lentinan, from Lentinus edodes (Berk.) Sing. Cancer Research 30, 2776-2781 (1970); Goro Chihara. Cancer and Immunoenhancement. Kodansha (1980)). Alternatively, lentinan can be extracted and purified by novel, unknown methods.

[0078] Beta-glucan contained in shiitake mushroom fruiting bodies is produced from the fruiting bodies according to conventional methods. Specifically, it can be produced by selecting and combining various known purification methods, such as hot water extraction of the cut fruiting bodies and removal of impurities and salts using activated carbon, ion exchange resins, etc. Furthermore, separation and purification can be achieved by using, for example, adsorption and elution onto solvent-precipitating hydrophobic resins using ethanol, methanol, ethyl acetate, n-butanol, etc., column chromatography or thin-layer chromatography using silica gel, or preparative high-performance liquid chromatography using reversed-phase columns, either alone or in appropriate combinations, and sometimes repeatedly.

[0079] During or at the end of these manufacturing processes, washing with organic solvents or aqueous media is performed as necessary, and the target product is obtained by freeze-drying, vacuum drying, or other methods well known to those skilled in the art.

[0080] (3) Method for controlling the molecular weight of beta-glucan Methods for controlling the molecular weight of beta-glucans include, but are not limited to, enzymatic degradation, alkaline hydrolysis, acid hydrolysis, shear force by high-pressure homogenizers, and degradation by ultrasonic energy. Appropriate methods can be selected from these and used as needed (Toshio Miyazaki, ed.. Structure and Physiological Activity of Polysaccharides. Asakura Shoten (1990)).

[0081] (4) Method for determining the structure of beta-glucan The structure of the obtained beta-glucan is similar to that of ordinary organic compounds. 1 H-NMR, 13 This can be determined by selecting or combining methods such as 13C-NMR, infrared absorption spectroscopy, and elemental analysis.

[0082] When using dimethyl sulfoxide-d6 (DMSO-d6), a commonly used measurement solvent for measuring the nuclear magnetic resonance spectrum (hereinafter referred to as NMR) of beta-glucan, 1 In 1H-NMR, the hydroxyl group signal is also observed, giving a very broad spectrum. On the other hand, beta-glucan dissolves relatively more easily in 0.25 M deuterated sodium hydroxide / heavy water than in DMSO-d6. 1 In 1H NMR, the hydroxyl group signal is not observed, allowing for a clear identification of the glucan constituent units. When using 0.25 M deuterated sodium hydroxide / heavy water as the measurement solvent, beta-glucan tends to decompose at room temperature. Therefore, by optimizing the NMR spectrum measurement temperature conditions and the required sample volume, a spectrum suitable for analysis can be obtained.

[0083] Molecular weight and molecular size can be determined as absolute values, relative values ​​equivalent to standard substances with known molecular weights, or as a scale with a certain correlation, by methods well known to those skilled in the art, such as multi-angle light scattering, size exclusion chromatography (SEC), and dynamic light scattering. β(1→3) glucan (BG) is a chain-like molecule that exists in a state where three chains are intertwined in a neutral aqueous solution, but dissociates into a single chain in an alkaline solution (WO2015 / 041318, Oligonucleotide-containing complex with immunostimulatory activity and its uses; Patent No. 5605793, Method for preparing β-1,3-glucan / nucleic acid complex (Patent Application 2010-043592); Daijiro Tanohata, Yusuke Sanada, Shinichi Mochizuki, Hiroko Miyamoto, Kazuo Sakurai. Solution properties of nucleic acid / β-1,3-glucan aggregates. Journal of Polymer Science, Japan 72:37-47 (2015)). Using pullulan, which has a chain-like structure similar to BG, as a molecular weight standard, relative molecular weight can be estimated by size exclusion chromatography (SEC) under alkaline conditions. Furthermore, by using a multi-angle light scattering detector (MALLS) as a chromatography detector, it is possible to estimate the absolute molecular weight and molecular size by static light scattering (Wyatt, PJ Anal Chim Acta 1993, 272, 1; Podzimek, S. J Appl Polym Sci 1994, 54, 91).

[0084] (5) Method for producing nucleic acids Nucleic acids, such as K3-dA40, can be produced based on synthesis techniques and nucleic acid chemistry well known to those skilled in the art. Such manufacturing methods are described, for example, in (1) Gait, MJ (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press, (2) Gait, MJ (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press, (3) Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press, (4) Adams, R. Let al. (1992). The Biochemistry of the Nucleic Acids, Chapman & Hall, (5) Shabarova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press, and (6) Hermanson, GT (1996). Bioconjugate Techniques, Academic Press. The relevant parts of these documents are incorporated for reference. Furthermore, phosphorothioate oligonucleotides can also be synthesized by the method described in Stein et al., 1988, Nucl. Acids Res. 16:3209. Additionally, methylphosphonate oligonucleotides can be prepared using a porosity glass polymer support or the like by the method described in Sarin et al., 1988, Proc. Natl. Acad. Sci. USA 85:7448-7451.

[0085] (6) Method for producing BG-CpG As described above, BG-CpG can be produced using the beta-glucan and nucleic acid obtained as raw materials according to methods well known to those skilled in the art ((1) WO2016 / 103531 and PCT / JP2014 / 084772, Application of Nucleic Acid Polysaccharide Complexes Having Immunostimulatory Activity as Antitumor Drugs, (2) WO2015 / 041318, Oligonucleotide-containing Complex Having Immunostimulatory Activity and its Uses, (3) Japanese Patent No. 5605793, Method for Preparing β-1,3-Glucan / Nucleic Acid Complex (Japanese Patent Application No. 2010-043592)).

[0086] Specifically, considering that the stoichiometric ratio of the number of sugar units constituting the main chain of beta-glucan (hereinafter referred to as mG) to the number of bases of the nucleic acid (hereinafter referred to as dA when deoxyadenosine is used as the nucleic acid that interacts with beta-glucan) is 2:1, i.e., the mG / dA ratio is 2, the mixing ratio of one of the components is increased or decreased as appropriate depending on the purpose. Since beta-glucan usually forms a helical structure or aggregates, it is treated with alkali or dissolved in an aqueous medium containing N,N-dimethyl sulfoxide to form monomers before being used for complex formation. Alternatively, both components are made to coexist in these mediums, and then the system is neutralized or the organic solvent is diluted or removed to form the complex. Characterization of BG-CpG is performed to confirm or guarantee its structural characteristics and the general quality characteristics that the pharmaceutical should possess.

[0087] BG-CpG oligonucleotide complexes are stable and soluble in neutral aqueous solutions, making particle size measurement possible using dynamic light scattering (DLS). When laser light is shone on particles undergoing Brownian motion in a solution or suspension, the scattered light from the particles fluctuates according to the diffusion coefficient. Larger particles move slowly, so the fluctuations in scattered light intensity are gentle, while smaller particles move quickly, so the fluctuations in scattered light change rapidly. DLS detects these fluctuations in scattered light that reflect the diffusion coefficient, and measures the particle size using the Stokes-Einstein equation. The average particle size and particle size distribution obtained by this method are important factors that indicate the characteristics of colloidal dispersion formulations (17th edition of the Japanese Pharmacopoeia, Reference Information, "Method for Measuring Particle Size in Liquids by Dynamic Light Scattering," pp. 2346-2348). The DLS method includes photon correlation analysis and frequency analysis, depending on the method of analyzing the detected signal, and is applicable to particles with diameters ranging from a few nanometers to approximately 1 μm, or until the effect of sedimentation is observed. In the examples described herein, the photon correlation method was used, and the average particle diameter based on scattered light intensity was determined by cumulant analysis from the autocorrelation function of scattered light intensity (17th edition of the Japanese Pharmacopoeia, Reference Information, "Method for Measuring Particle Size in Liquids by Dynamic Light Scattering," pp. 2346-2348; ISO 22412: 2008 Particle size analysis - Dynamic light scattering (DLS)). Here, if the particle size distribution of the test substance is unimodal, the particle diameter at the peak top is the average particle diameter, and in this invention, the correlation between this average particle diameter and the pharmacological effect of BG-CpG was investigated. On the other hand, in cases where a bimodal or multimodal particle size distribution was present, the peak showing the highest light scattering intensity was selected, and the correlation between the particle size at the top of that peak, i.e., the average particle size of that peak, and the pharmacological effect of BG-CpG was investigated.

[0088] Next, the present invention will be described in more detail with reference to examples, test examples, and examples of pharmaceutical compositions, but the present invention is not limited to these. [Examples]

[0089] Example 1: Production of SPG by bacterial culture From 33 strains of Schizophyllum commune isolated from samples collected in Japan and held by the inventors, a strain with high SPG production capacity was selected. The Schizophyllum commune strains were grown sufficiently on a Potato Dextrose Agar (PDA) slant, and 1 / 4 of the fungal cells that grew on the surface of the PDA slant were homogenized with 5 mL of sterile water. This was then inoculated into a 500 mL Erlenmeyer flask containing 80 mL of pre-sterilized SPG medium (glucose 3.0%, yeast extract 0.3%, ammonium dihydrogen phosphate 0.15%, potassium dihydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.05%, (pre-sterilization pH 4.8)). The 500 mL Erlenmeyer flask inoculated with the fungal cells was cultured by shaking it using a rotary shaker at 210 rpm and 28°C for 7 days (168 hours).

[0090] After culturing was complete, 45 mL or 15 mL of the obtained culture solution was centrifuged (10,000 rpm, 10 min) to separate the culture solution into culture supernatant and bacterial cells. Methanol was added to 30 mL or 10 mL of the obtained culture supernatant to prepare a 35% methanol solution. The precipitate that formed upon methanol addition was centrifuged (10,000 rpm, 10 min) and the precipitate was collected. The collected precipitate was washed with 10 mL or 5 mL of anhydrous methanol and then centrifuged again. 1 mL to 3 mL of sterile water was added to the precipitate and allowed to stand for about 1 hour, after which it was freeze-dried to obtain the SPG extract.

[0091] Test Example 1: Evaluation of SPG productivity of various strains of *Schizophyllum commune*. The amount of SPG extract obtained in Example 1 was weighed. The productivity of SPG extract for each strain was evaluated using the SPG extract concentration in the culture supernatant, calculated from the amount of SPG extract and the amount of culture supernatant, as an indicator.

[0092] As shown in Table 1, strains F-19065, F-30213, F-82046, F-33504, F-15977, and F-15537 were confirmed to have high SPG productivity. Furthermore, strains F-19065, F-30213, F-82046, and F-33504 were confirmed to have extremely high SPG productivity.

[0093] [Table 1]

[0094] Example 2: Production of SPG by bacterial culture of a high-SPG-producing strain Two strains of Schizophyllum commune, F-82046 and F-30213, which showed high productivity in SPG production, were selected and decided to be used in SPG manufacturing.

[0095] In the same manner as in Example 1, one-quarter of the bacterial cells grown on the surface of the PDA slant were homogenized with 3 mL of sterile water and inoculated into one 500 mL Erlenmeyer flask containing 80 mL of pre-sterilized SPG medium. The 500 mL Erlenmeyer flask inoculated with the bacterial cells was shaken using a rotary shaker at 210 rpm and 28°C for 4 days to perform pre-culture. 5 mL of the pre-culture solution was homogenized and inoculated into a 500 mL Erlenmeyer flask containing 80 mL of SPG medium. The 500 mL Erlenmeyer flask inoculated with the pre-culture solution was shaken using a rotary shaker at 210 rpm and 28°C for 10 days to perform the main culture.

[0096] After the culture was complete, the entire culture medium was dispensed into a centrifuge tube and centrifuged (10,000 rpm, 10 minutes). Because the resulting supernatant was highly viscous, 1.5 to 1 times the volume of sterile water was added, and then the supernatant was filtered by suction to remove suspended cells. Methanol was added to the filtrate to a concentration of 35%, and after stirring, the mixture was allowed to stand overnight. The precipitated material was centrifuged (10,000 rpm, 10 minutes), and the precipitate was washed with methanol. Next, sterile water was added to the precipitate to allow it to permeate, and then freeze-dried. This freeze-dried material was used as the SPG extract.

[0097] The SPG extract was dissolved in a 1M sodium hydroxide aqueous solution at a concentration of 1 mg / mL and gently shaken at 5°C for 24 hours. The solution was then filtered through a 5 μm pore size omnipore membrane filter (Merck Millipore), followed by a 1 μm pore size filter. Twice the volume of methanol was added to the resulting filtrate to precipitate SPG. The precipitated SPG was washed several times with a methanol:water mixture of 3:1. This washing was repeated until the solution became neutral, followed by one wash each with methanol and acetone. Finally, the solution was dried at 40°C to obtain purified, uncontrolled SPG (massive SPG; hereinafter referred to as mSPG).

[0098] mSPG produced using the Schizophyllum sieboldii strain F-82046 was designated mSPG-1a, and SPG derived from F-30213 was designated mSPG-2a. In addition, two lots of SPG purchased as reagents (manufacturer's lot number SCZ-37-01A is designated iSPG-1 in this invention, and SCZ-36-1 is designated iSPG-2 in this invention) were used as standard samples, and these mSPGs 1 H-NMR and 13 1C-NMR spectra were obtained and compared. Here, the solvent used was 0.25 M sodium deuterated hydroxide / heavy water, and it was found that the optimal measurement temperature for achieving both a high signal-to-noise ratio and compound stability was 15°C. Measurements were performed at this temperature. As shown in Figures 1 and 2, the spectra of mSPG-1a and mSPG-2a were obtained. 1 H-NMR spectrum and 13 The 1C-NMR spectra all matched those of the SPG standard samples, and no significant signals originating from impurities were observed. Therefore, it was confirmed that the two lots of mSPG produced in this example possessed high purity.

[0099] Example 3: Production of SPG by bacterial culture using a large flask To produce large quantities of SPG, cultivation was performed using Ultra Yield Flask 2.5L (manufactured by Thomson Instrument Company). The Schizophyllum strain F-82046 was used as the Schizophyllum strain. The cultivation was performed in three stages: pre-culture, pre-culture, and main culture. For inoculation, one frozen seed of Schizophyllum strain F-82046 was used.

[0100] To prepare frozen seeds of the *Schizophyllum commune* strain F-82046, one-quarter of the *Schizophyllum commune* cells grown on the surface of a PDA slant were homogenized with 3 mL of sterile water and inoculated into one 500 mL Erlenmeyer flask containing 80 mL of pre-sterilized SPG medium. The 500 mL Erlenmeyer flask inoculated with the cells was cultured by shaking it with a rotary shaker at 210 rpm and 28°C for 3 days. 7 mL of pre-sterilized glycerol was added to 63 mL of the culture solution and mixed. The mixture was then dispensed into 1.8 mL portions into Cryotubes (Thermo Fisher Scientific) and frozen in a -80°C freezer for storage.

[0101] In the pre-preculture, one frozen seed was thawed at room temperature and the entire amount was inoculated into one 500 mL Erlenmeyer flask containing 80 mL of pre-sterilized SPG medium. The 500 mL Erlenmeyer flask inoculated with the bacteria was shaken using a rotary shaker at 210 rpm and 28°C for 3 days to perform the pre-preculture. In the main culture, 20 mL of the pre-preculture solution was homogenized and the entire amount was inoculated into a 2.5 L Ultra Yield Flask containing 1000 mL of pre-sterilized SPG medium. The 2.5 L flask was shaken using a rotary shaker at 130 rpm and 28°C for 3 days to perform the pre-culture. In the main culture, 10 mL of the pre-culture solution was inoculated into a 2.5 L Ultra Yield Flask containing 1000 mL of pre-sterilized SPG medium. This 2.5 L flask was shaken using a rotary shaker at 130 rpm and 28°C for 11 days to perform the main culture. Stable SPG production was obtained across multiple flasks, and the average SPG concentration in the culture medium was approximately 2,000 μg / mL. After culturing, distilled water was added to each flask to a volume of 1.5 L, and the mixture was autoclaved (121°C, 20 minutes). The sterilized culture solution was further diluted with an equal volume of distilled water, and the bacterial cells were removed using a nylon bag (pore size 150 μm). The mixture was then filtered by suction using filter paper (No. 2, Advantec). The obtained processed filtrate was mixed with methanol using a smooth flow pump (Q series, Takumina Co., Ltd.) to a methanol concentration of 30% to 35%, and then allowed to stand overnight to obtain a precipitate. The suspended fraction and gel-like suspended matter in the liquid were collected using tweezers, a ladle, and a nylon bag. The collected precipitate was centrifuged (8,000 rpm, 30 minutes), and the precipitate was washed with methanol. Next, sterile water was added to the precipitate to allow it to permeate, and then freeze-dried to obtain the SPG extract as a freeze-dried product (SPG-247: yield 10.4g).

[0102] 151 mg of this SPG extract (SPG-247) was dissolved in a 1 M sodium hydroxide aqueous solution at a concentration of 1 mg / mL and gently shaken at 5°C for 24 hours. The solution was then filtered through an Omnipore membrane filter with a pore size of 5 μm, followed by a pore size of 1 μm. Twice the volume of methanol was added to the resulting filtrate to precipitate SPG. The precipitated SPG was washed several times with a methanol:water mixture of 3:1. This washing was repeated until the solution became neutral, followed by one wash each with methanol and acetone. Finally, the solution was dried at 40°C to obtain mSPG (mSPG-19a: yield 125 mg).

[0103] Example 4: Molecular weight control of SPG by alkali treatment mSPG-1b was prepared using the SPG extract produced using the Schizophyllum strain F-82046 described in Example 2, in the same manner as mSPG-1a. Using either mSPG-1b or mSPG-1a obtained here as a raw material, molecular weight-controlled SPG was prepared as follows: mSPG was dissolved in a 5M aqueous sodium hydroxide solution at a concentration of 1 mg / mL. The solution was slowly shaken at a temperature of 28°C to 50°C for 8 to 72 hours. Twice the volume of methanol to the shaken solution was added to precipitate SPG. The precipitated SPG was washed several times with a methanol:water = 3:1 mixture. This washing was repeated until the solution became neutral, and then washed once each with methanol and acetone. Finally, it was dried at 40°C to obtain purified, molecular weight-controlled SPG. By varying the temperature and time within these ranges, SPG with various molecular sizes was produced. Table 2 shows the SPGs that were manufactured.

[0104] [Table 2]

[0105] Furthermore, it was confirmed that molecular weight-controlled SPG can be produced by using a 1M to 5M aqueous sodium hydroxide solution and shaking it under various conditions ranging from 17°C to 55°C and 48 to 96 hours.

[0106] Example 5: Molecular weight control of SPG using a high-pressure homogenizer A pressure homogenizer (Emulsiflex C-5 from Avestin or Microfluidizer M-110EH from Powrec) was used as the high-pressure homogenizer. The SPG extract obtained from culturing Schizophyllum commune strain F-82046 was dissolved in 30 mL of distilled water to a concentration of 1 mg / mL to 3 mg / mL and imprinted. The SPG solution was added to the flow path of the pressure homogenizer and the SPG solution was homogenized repeatedly while gradually increasing the emulsification pressure until the maximum emulsification pressure reached 15,000 psi to 25,000 psi. An equal volume of methanol was added to the treated SPG solution and allowed to stand for several hours, and the precipitated material was collected by centrifugation. After washing with methanol, a small amount of water was added to the precipitate to imprint it, and then it was freeze-dried. In this way, we were able to produce SPG (SPG-85: yield 44 mg, SPG-86: yield 49 mg, SPG-87: yield 38 mg) with controlled molecular weight using a method that does not involve alkaline treatment.

[0107] Test Example 2: Measurement of Molecular Weight The molecular weight of SPG was measured as a pullulan equivalent value using the following procedure. A 0.25 M sodium hydroxide aqueous solution was added to SPG as the test substance, and the mixture was stirred at 5°C for 1 hour to prepare the measurement sample. Pullulan standards (Shodex, molecular weights 2350 kD, 1220 kD, 642 kD, 337 kD, 194 kD, and 107 kD, respectively) were dissolved in purified water to prepare the measurement samples. A differential refractive index (RI) detector was connected to a size exclusion chromatography (SEC) and these measurement samples were analyzed (SEC-RI method). A molecular weight calibration curve was created from the retention time of the pullulan standards, and the molecular weight of the samples was estimated. The analytical conditions for the SEC-RI method are described below.

[0108] High-performance liquid chromatography system (injector, pump, column heater, RI detector): A1200 (Agilent), mobile phase: approx. 10 mM sodium hydroxide aqueous solution (pH 12), flow rate: 0.4 mL / min, columns: Asahipak GS-2G 7B (Shodex), Asahipak GS-620HQ (Shodex), Asahipak GS-320HQ (Shodex) connected in series in this order, column temperature: 40°C, sample concentration: approx. 2 mg / mL, sample injection volume: 100 μL, analysis time: 75 minutes.

[0109] The molecular weight calibration curve is shown in Figure 3. It was confirmed that molecular weight can be measured in the range of 107 kD to 2350 kD in terms of pullulan (a range of 5.03 to 6.37 as "Log(molecular weight)").

[0110] Next, Tables 3 and 4 show the molecular weights of SPGs calculated from the molecular weight calibration curve and the retention time of the molecular weight measurement samples. It was found that the molecular weight of SPGs could be controlled over a wide range, from about 50 kD to over 1000 kD, depending on the alkali treatment conditions. Furthermore, for SPGs before molecular weight control (mSPGs), the weight-average molecular weight could not be calculated because some of the peaks exceeded the molecular weight upper limit of the molecular weight calibration curve. However, this measurement confirmed that these mSPGs have larger molecular weights than the molecular weight-controlled SPGs, i.e., large molecular weights generally greater than 1000 kD. On the other hand, for some molecular weight-controlled SPGs and purchased SPGs (SPG-95, SPG-72, SPG-76, and iSPG-1), although some of the peaks were below the molecular weight lower limit of the molecular weight calibration curve, a distribution was observed, so the peak-top molecular weight and weight-average molecular weight were calculated for reference. It was confirmed that these SPGs have smaller molecular weights than the other SPGs, i.e., molecular weights generally less than 100 kD.

[0111] [Table 3]

[0112] [Table 4]

[0113] Example 6: Preparation of a complex of β(1→3) glucan and nucleic acid. SPG (mSPG, iSPG, or SPG with controlled molecular weight) was selected as the β(1→3) glucan, and K3-dA40 was selected as the nucleic acid. The complex of β(1→3) glucan and nucleic acid was prepared as follows: Several mg of SPG was weighed and dissolved in a 0.25 M sodium hydroxide aqueous solution to a concentration of 10 mg / mL, and allowed to stand at 4°C for 24 hours with occasional stirring. Distilled water was added to K3-dA40 (59 sodium salt) to prepare a 100 μM aqueous solution. Next, the above SPG alkaline solution was added to the K3-dA40 aqueous solution in a predetermined ratio so that the mG / dA ratio was mainly 2.5, and the SPG and K3-dA40 were complexed by stirring. Then, an equal amount of 330 mM sodium dihydrogen phosphate aqueous solution was added to neutralize the SPG alkaline solution, and K3-SPG was obtained by letting it stand overnight at 4°C.

[0114] In addition to those described in Example 2 or Table 2, the SPGs used were those shown in Table 5.

[0115] [Table 5]

[0116] Table 6 shows the K3-SPG produced.

[0117] [Table 6]

[0118] Test Example 3: Evaluation of the efficiency of complex formation between β(1→3) glucan and nucleic acids. The efficiency of complex formation between β(1→3) glucan and nucleic acid, prepared in Example 6, was evaluated by SEC. K3-SPG, prepared in Example 6 and obtained as a solution, was used as the measurement sample. For control analysis, an aqueous solution of K3-dA40 alone was prepared using a 0.25 M sodium hydroxide aqueous solution without dissolving SPG, following the procedure shown in Example 6, so that the concentration was the same as the theoretical concentration of total K3-dA40 in the complex sample. The SEC conditions are as follows.

[0119] High-performance liquid chromatography system (injector, pump, column heater, UV detector): A1100 (Agilent), mobile phase: 100 mM phosphate buffer (pH 7.4, nacalai tesuque, catalog number: 37244-35), flow rate: 0.5 mL / min, columns: Asahipak GF-1G 7B (Shodex), Asahipak GF-7M HQ (Shodex), Asahipak GF-1G 7M HQ (Shodex) connected in series in this order, column temperature: 40°C, detection wavelength: 260 nm, sample injection volume: 10 μL.

[0120] The compounding efficiency was calculated from the amount of K3-dA40 remaining after compounding relative to the amount of K3-dA40 before compounding. When all of the K3-dA40 is compounded and the remaining amount is 0, the compound formation efficiency is 100%.

[0121] Table 7 shows the measurement results of the complex formation efficiency of K3-SPG produced in Example 6. In all cases, the formation efficiency was 96% or higher, confirming that all purified SPG (before molecular weight control), SPG with controlled molecular weight, and purchased SPG form complexes with nucleic acids with very high efficiency using the K3-SPG production method of the present invention.

[0122] [Table 7]

[0123] Test Example 4: Evaluation of composite particle size using DLS The particle size of the complex (K3-SPG) produced in Example 6 was measured by the photon correlation method in the DLS method as the average particle size based on the scattered light intensity criterion. At this time, it was appropriately diluted with a phosphate buffer (8 g / L NaCl, 200 mg / L potassium dihydrogen phosphate, 1150 mg / L disodium hydrogen phosphate anhydrous.) so as to reach the minimum K3-SPG concentration at which detectable scattered light intensity could be obtained.

[0124] Table 8 shows the measurement results of the average particle size of K3-SPG produced in Example 6. It was confirmed that it could be precisely controlled within the range of 21.9 nm to 122.2 nm in terms of the average particle size.

[0125]

Table 8

[0126] Test Example 5: In vitro evaluation of the pharmacological effects of K3-SPG Using human PBMCs (Lonza. Catalog number: Cat#CC-2702 Lot#31468) and spleen cells prepared from C57BL / 6 mice, the ability to induce IFN-α production and the ability to induce IFN-γ production were evaluated respectively. Human PBMCs and mouse spleen cells were placed in a 96-well plate, 1×10 per well 6Cells were seeded at a number of cells. K3-SPG was added to the cells at concentrations of 0, 0.75, 2, 6, and 20 μg / mL (converted to K3-dA40 (59 sodium salt)), and hIFN-α in the culture supernatant after 24 hours was measured using a cytokine ELISA kit (PBL). The hIFN-α induction ability of K3-SPG was evaluated by calculating the area under the curve (AUC) of the hIFN-α induction level for the total K3-SPG concentration. Similarly, the IFN-γ induction ability of K3-SPG was evaluated. The results are shown in Figure 4. As shown in the table on the right side of Figure 4, KAmSP001a (average particle size: 121.1 nm) and KAmSP002a (average particle size: 122.2 nm) have large particle sizes, while KAInv3701 (average particle size: 31.3 nm) and KASPG0095 (average particle size: 21.9 nm) have small particle sizes. As shown in Figure 4, in both human frozen PBMCs and mouse spleen cells, K3-SPGs with large particle sizes had a higher ability to induce IFN-α and IFN-γ production compared to K3-SPGs with small particle sizes.

[0127] Test Example 6: In vitro evaluation of the pharmacological effects of the complex. The correlation between the IFN-α (hIFN-α) production level from human PBMCs treated with K3-SPG complex (Lonza, Cat#CC-2702 Lot#31468) and the average particle size of K3-SPG was evaluated. Human PBMCs were processed using a 96-well plate with a ratio of 1 × 10⁶ per well. 6 Cells were seeded in individual numbers, and each BG-CpG was added to the cells at CpG ODN concentrations of 0, 0.75, 2, 6, and 20 μg / ml. After 24 hours, the hIFN-α production level in the culture supernatant was measured using a cytokine ELISA kit (PBL). The hIFN-α induction ability of BG-CpG was evaluated by calculating the AUC value of the hIFN-α induction level for the total CpG concentration. The correlation between hIFN-α production level and average particle size was examined using the Spearman test. The correlation coefficient was 0.7789, indicating a positive correlation (Figure 5). In other words, it was confirmed that in the range of average particle size from 20 nm to 130 nm, the pharmacological effect was stronger as the particle size increased.

[0128] Test Example 7: In vivo evaluation of the pharmacological effects of K3-SPG C57BL / 6 mice were given 2 × 10⁶ B16 cancer cells, a melanoma cell line derived from C57BL / 6 mice. 5 Individual doses were subcutaneously inoculated (day 0), and on day 9, K3 (Gene Design Co., Ltd.) or K3-SPG (complex name: KAmSP001a) manufactured in Example 3 was administered intravenously at doses of 30 μg / head and 100 μg / head. K3 or K3-SPG was administered every other day for a total of three times, and tumor size was measured over time. As shown in Figure 6, a significant antitumor effect was observed in the group administered K3-SPG (triangle) compared to the group administered K3 (square). In other words, when the dose is converted to moles, the efficacy of K3-SPG, which has approximately three times the weight difference compared to K3, was found to be superior to that of K3.

[0129] Example 7: Production of BG-CpG in which beta-glucan is lentinan As described in the section on embodiments for carrying out the above invention, lentinan was manufactured according to a conventional method. Here, substrate cultivation was applied as the cultivation method for shiitake mushroom fruiting bodies, and purification was performed by selecting and combining known purification methods. Furthermore, the molecular weight was controlled and purification was performed using the method shown in Example 4. Using the lentinan thus obtained, BG-CpG was manufactured using the method shown in Example 6. Hereinafter, BG-CpG containing lentinan as a component will be referred to as K3-LNT. Table 9 shows the average particle size of the manufactured K3-LNT. Hereinafter, Table 9 also shows the molecular weight control conditions of the lentinan used for the manufacture of each K3-LNT. It was confirmed that the particle size of K3-LNT could be controlled by changing the application time of alkaline treatment under predetermined conditions for lentinan. As can be seen from Figure 7, which shows the relationship between the alkaline treatment time of lentinan and the average particle size of K3-LNT, it was found that the present invention makes it possible to manufacture K3-LNT with a strictly controlled average particle size in the range of 40 nm to 180 nm.

[0130] [Table 9]

[0131] Test Example 8: In vitro evaluation of the pharmacological effects of K3-LNT The IFN-γ production induction ability of K3-LNT was evaluated in the same manner as in Test Example 5 described above. However, mouse splenocytes were selected as the cell type, and the number of cells seeded per well of the 96-well plate was 1 × 10⁶. 7 The K3-LNT was added at two levels, 2 μg / mL and 10 μg / mL, and the amount of IFN-γ produced after 24 hours of incubation was measured for each level. The sum of these values ​​was used as an indicator of the strength of the pharmacological effect of K3-LNT. As a result, as shown in Figure 8, the correlation coefficient was 0.9081 in the range of average particle size from approximately 40 nm to 180 nm, confirming that the larger the particle size of K3-LNT, the stronger the pharmacological effect.

[0132] Study Example 9: In vivo evaluation of the pharmacological effects of K3-LNT The antitumor effect of K3-LNT was evaluated in the same manner as in Test Example 7 described above. However, the subcutaneous transplantation of B16 cancer cells into C57BL / 6 mice on day 0 of evaluation was 2.5 × 10⁶. 5 1 or 5.0 x 10 5 Each individual was treated with K3-LNT, administered every other day for a total of three doses starting from day 7. The K3-LNT was administered intratumorally at a dose of 50 μg / head. For comparison, the dose of K3, a nucleic acid, was 16.7 μg / head. As shown in Figure 9, K3-LNT with an average particle size of 70 nm to 175 nm was confirmed to have a potent antitumor effect. Furthermore, when some samples were compared with K3 in the same manner as in Test Example 7, as shown in Figure 10, K3-LNT with average particle sizes of 149 nm and 175 nm showed superiority compared to K3. [Industrial applicability]

[0133] The optimal particle size of BG-CpG was selected for use as a therapeutic or preventive agent in cancer immunotherapy. The BG-CpG of the present invention has an average particle size that is strictly controlled to be within the range of 20 nm to 180 nm, preferably 20 nm to 130 nm, or 40 nm to 180 nm, more preferably 80 nm to 130 nm, or 130 nm to 180 nm. Furthermore, since the BG-CpG of the present invention can achieve remarkable effects with a smaller dose compared to conventional BG-CpG, it can contribute to industrial benefits by being applicable to diseases that were difficult to treat or prevent with conventional BG-CpG, and by being manufactured at a lower cost than conventional BG-CpG.

[0134] This application is based on Japanese Patent Application No. 2020-189032, filed in Japan on November 12, 2020, and by reference herein, all its contents are incorporated herein.

Claims

1. A complex of β(1→3) glucan and nucleic acid with controlled particle size, A composite having an average particle size of 80 nm to 130 nm, in which β(1→3) glucan is schizophyllan.

2. The complex according to claim 1, characterized in that the nucleic acid is an oligonucleotide containing a K-type CpG oligonucleotide.

3. The complex according to claim 1 or claim 2, characterized in that the nucleic acid is a nucleic acid in which polydeoxyadenosine is linked to the 3' end of a K-type CpG oligonucleotide.

4. The complex according to any one of claims 1 to 3, wherein the nucleic acid comprises a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylic acid, the nucleotide sequence represented by Sequence ID No. 1, wherein polydeoxyadenosine is linked to the 3' end of the humanized K-type CpG oligodeoxynucleotide, and some or all of the phosphate diester bonds of the oligodeoxynucleotide are substituted with phosphorothioate bonds.

5. A pharmaceutical composition comprising the complex described in any one of claims 1 to 4.

6. The pharmaceutical composition according to claim 5 for the prevention or treatment of viral infections, cancer, allergic diseases, intracellular parasitic protozoa, or bacterial infections.

7. The pharmaceutical composition according to claim 6 for the prevention or treatment of a viral infection.

8. The pharmaceutical composition according to claim 7, wherein the viral infection is RSV or influenza virus infection.

9. An immunostimulant comprising the complex described in any one of claims 1 to 4.

10. An immunostimulant according to claim 9, which is a vaccine adjuvant.

11. (a) The complex according to any one of claims 1 to 4, or the immunostimulant according to claim 9 or claim 10, and (b) Antigen A pharmaceutical composition containing the above.

12. The composition according to claim 11 for inducing an immune response to the antigen.

13. The composition according to claim 12, wherein the antigen is an antigen derived from a pathogen.

14. The composition according to claim 13, for the prevention or treatment of infectious diseases caused by pathogens.

15. The composition according to claim 14, wherein the pathogen is a virus.

16. The composition according to claim 15, wherein the virus is RSV or influenza virus.

17. The composition according to claim 12, wherein the antigen is a cancer-derived antigen.

18. The composition according to claim 17, for the prevention or treatment of cancer.

19. (a) A complex according to any one of claims 1 to 4, or a pharmaceutical composition according to any one of claims 5, 6, 11, 12, 17, or 18, and (b) anticancer drugs A pharmaceutical composition containing the above.

20. The composition according to claim 19, wherein the anticancer agent is an immune checkpoint inhibitor.

21. The composition according to claim 20, wherein the immune checkpoint inhibitor is one of a PD1 inhibitor, a PDL-1 inhibitor, and a CTLA-4 inhibitor.

22. A method for producing the complex according to any one of claims 1 to 4, utilizing the molecular weight characteristics of β(1→3) glucan.

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