mRNA vaccine adjuvant

Abstract

The present invention provides an adjuvant comprising a B-type CpG oligodeoxynucleotide (CpG ODN), a modification thereof, or a complex thereof, to be administered together with an mRNA vaccine in which mRNA is encapsulated in particles. The adjuvant contains the B-type CpG ODN that is present independently of the particles that encapsulate mRNA. This adjuvant can enhance antigen-specific cytotoxic T cell (CTL) induction by mRNA vaccines.

Description

mRNA vaccine adjuvants

[0001] The present invention relates to an adjuvant that enhances the induction of antigen-specific cytotoxic T cells (CTLs) by mRNA vaccines, and more particularly to an adjuvant containing type B CpG oligodeoxynucleotides (CpG ODN).

[0002] Unlike live vaccines and inactivated vaccines, subunit vaccines and recombinant protein vaccines have a low ability to induce immune responses in the body, which can be a challenge in vaccine development. To improve the efficacy of these vaccines, adjuvants have been developed (Non-Patent Document 1).

[0003] Adjuvants approved for human use for subunit vaccines and recombinant protein vaccines include Alum (Aluminum Hydroxide, Aluminum Phosphate Salts), Squalene (MF59, AS03), Monophosphoryl Lipid A (AS04), Saponin (AS01), and CpG 1018 (CpG Oligodeoxynucleotide [CpG ODN]) (Non-Patent Documents 1 and 2).

[0004] Lipid nanoparticle (LNP)-mRNA vaccines, which have been put into practical use since the 2020s, are vaccines designed to use LNPs as intracellular internalization carriers, encapsulating mRNA encoding antigenic proteins, so that the mRNA is efficiently translocated into the cytoplasm. By administering the LNP-mRNA vaccine to a living body, the antigenic protein is expressed in the living body through translation from the mRNA, thereby eliciting an immune response against the antigenic protein. In other words, LNP-mRNA vaccines have a different mechanism of action from conventional vaccines that administer antigens themselves, attenuated antigens, or portions thereof (Patent Document 1, Patent Document 2, Non-Patent Document 3).

[0005] With the practical application of mRNA vaccines, research aimed at improving their efficacy is being promoted. For example, there are mRNA vaccines using a TLR7 / 8 agonist-binding lipid as an LNP component (Non-Patent Document 4), mRNA vaccines using Pam3, a lipopeptide with TLR1 / 2 activation activity, as an LNP component (Non-Patent Document 5), mRNA vaccines using Monophosphoryl Lipid A with TLR4 activation activity as an LNP component (Non-Patent Document 6), and mRNA vaccines using a lipid with STING activation activity as an LNP component (Non-Patent Document 7). In addition, new carriers with similar functions to LNPs have been reported, including a group of cationic polymers called Charge-Altering Releasable Transporters (hereinafter referred to as CART) (Non-Patent Document 8) and a group of polymers called Poly Beta-Aminoesters (hereinafter referred to as PBAE) (Non-Patent Document 9). Here, in CART, CpG ODNs (IMO-2055, SD101, and ODN 2395 are highly active, while type B CpG ODNs are lowly active) that have TLR9 activation activity are encapsulated in the carrier together with mRNA, and it is believed that there is a combined effect of mRNA and these CpG ODNs. Similarly, in PBAE, certain types of CpG ODNs are encapsulated together with mRNA, and a combined effect has been observed.

[0006] CpG ODNs are short (approximately 20 base pairs), single-stranded synthetic DNA fragments containing immunostimulatory CpG motifs and are potent agonists of TLR9 (Non-Patent Document 10). CpG ODNs activate plasmacytoid dendritic cells (pDCs) and B cells to produce type I interferon (IFN) and proinflammatory cytokines (Non-Patent Document 11). CpG ODNs act as adjuvants for Th1-type humoral and cellular immune responses, including cytotoxic T lymphocyte (CTL) responses, to recombinant protein vaccines (Non-Patent Document 12). There are several types of CpG ODNs with different backbone sequences and immunostimulatory properties (Non-Patent Document 13, Non-Patent Document 14, Non-Patent Document 15).

[0007] Type A CpG ODNs (also called type D, such as D35 CpG ODN and ODN 2216) typically have a structural characteristic of containing a single palindromic CpG motif along with a phosphorothioate poly-G tail, forming a higher-order structure. They activate pDCs to produce large amounts of IFN-α, but are unable to induce pDC maturation or B cell activation (Non-Patent Documents 16 and 17).

[0008] B-type (also called K-type) CpG ODNs (such as K3 CpG ODN, ODN 2006 [ODN 7909, PF-3,512,676], ODN 1018, and ODN 1826) are CpG ODNs that lack phosphodiester bonds, contain one or more CpG motifs with a phosphorothioate backbone, and lack a poly-G tail, and are believed to lack intermolecular higher-order structures. Their functional properties include potently activating B cells to produce IL-6 and activating and maturing pDCs, but producing very little IFN-α (Non-Patent Document 14, Non-Patent Document 17). Palindromic sequences in the base sequence of CpG ODN are believed to affect the formation of higher-order structures, and when ODNs were synthesized in which palindromic sequences of different lengths were introduced into the sequence of B-type CpG ODN and their ability to induce INF-α production in pDCs was evaluated, it was reported that the ability to induce INF-α production was enhanced in proportion to the length of the introduced palindromic sequence (Non-Patent Document 13).It has also been pointed out that in B-type CpG ODN, the sequence consisting of TCG is the motif recognized by human TLR9 (Non-Patent Document 18).

[0009] C-type (ODN 2395, SD101, etc.) and P-type CpG ODNs typically have a phosphorothioate backbone, contain one or two palindromic CpG motifs, and have the structural characteristic of forming an intermolecular higher-order structure. Both activate B cells like type B, and activate pDC like type D, strongly inducing IFN-α production (Non-Patent Document 19, Non-Patent Document 20).

[0010] Furthermore, next-generation CpG ODNs (IMO-2125, IMO-2055, etc.) have been developed based on the prospects of sustained IFN-α production and antitumor effects as a single agent (Non-Patent Document 21).

[0011] It is known that K3 CpG ODN, which has polydeoxyadenylic acid with a phosphodiester bond at its 3' end, efficiently forms complexes with the β-1,3-glucans lentinan (LNT) and schizophyllan (SPG) (Non-Patent Document 22). The complexes, K3-LNT and K3-SPG, have been found to have stronger immunostimulatory activity, such as inducing IFN-α production, than K3, and are potent adjuvants for protein vaccines (Patent Documents 3, 4, and Non-Patent Document 23).

[0012] Furthermore, a complex of lipid and CpG has been reported as a technique for increasing the delivery of vaccine adjuvants to lymph nodes (Patent Document 5).

[0013] International Publication No. 2021-095838 International Publication No. 2021-251453 International Publication No. 2015-041318 International Publication No. 2022-102694 International Publication No. 2013-151771

[0014] Luchner M., et al. Pharmaceutics. 2021;13(2).Pogostin BH., et al. Bioengineering (Basel). 2021 Oct 24;8(11):155.Barbier AJ., et al. Nat Biotechnol. 2022;40(6):840-54.Han X., et al. Nat Nanotechnol. 2023;18(9):1105-14.Lee K, Kim SY., et al. Biomater Sci. 2020;8(4):1101-5.Pan L., et al. J Control Release. 2023;357:133-48.Miao L., et al. Nat Biotechnol. 2019;37(10):1174-85.Haabeth OAW., et al. ACS Cent Sci. 2021;7(7):1191-204.Ben-Akiva E., et al. Proc Natl Acad Sci US A. 2023;120(26):e2301606120.Krieg AM., et al. Nature. 1995;374(6522):546-9.Krieg AM. Nat Rev Drug Discov. 2006 Jun;5(6):471-84.Brazolot Millan CL., et al. Proc Natl Acad Sci US A. 1998 Dec 22;95(26):15553-8.Vollmer J., et al. Adv Drug Deliver Rev. 2009 Mar 28;61(3):195-204.Krieg AM. Annu Rev Immunol. 2002;20:709-60.Bode J., et al. Expert Rev Vaccines. 2011;10(4):499-511.Krug A. Eur J Immunol. 2001 Jul;31(7):2154-63.Verthelyi D. J Immunol. 2001 Feb 15;166(4):2372-7.Stefan Bauer, et al., PNAS. 2001;98 (16) 9237-9242.Hartmann G.Eur J Immunol. 2003 Jun;33(6):1633-41.Samulowitz U., et al. Oligonucleotides. 2010;20(2):93-101.Jin Y., et al, Expert Rev Anticancer Ther. 2021;21(8):841-51.Sakurai K., et al, Biomacromolecules. 2001 Fall;2(3):641-50.Kobiyama K., et al. Proc Natl Acad Sci US A. 2014;111(8):3086-91.Yu D., et al. Nucleic Acids Res. 2002 Oct 15;30(20):4460-9.

[0015] An object of the present invention is to provide an adjuvant that enhances CTL induction by an mRNA vaccine, particularly an adjuvant containing type B CpG oligodeoxynucleotide (CpG ODN).

[0016] The present inventors have found that adjuvants that enhance the immune response of conventional subunit vaccines or recombinant protein vaccines do not necessarily sufficiently enhance CTL induction by mRNA vaccines, and in some cases may even attenuate it. Through further investigation, the present inventors have found that B-type CpG ODN (K3 CpG ODN, ODN 1018, ODN 2006), K3-LNT, K3-SPG, and Lipid-K3 enhance CTL induction by LNP-mRNA vaccines, and that K3-lentinan (K3-LNT) enhances the antitumor activity of LNP-mRNA vaccines, thereby completing the present invention. Specifically, for LNP-mRNA encapsulating mRNA encoding human papillomavirus type 16 (HPV16) E6 and E7, AH1-A5 (a sequence in which the fifth amino acid of AH1, a 9-mer CTL epitope sequence contained in gp70, is substituted with A), or gp70 AH1-A5 (the full-length gp70 sequence in which the AH1 sequence contained in gp70 is substituted with the AH1-A5 sequence), the researchers found that type B CpG ODN (K3 CpG ODN, ODN 1018, ODN 2006), K3-LNT, and K3-SPG have excellent effects in enhancing CTL induction as adjuvants that enhance the induction of MHC Class I epitope-specific CTLs contained in the proteins encoded by the respective mRNAs. Furthermore, it was found that co-administration of K3-LNT with LNP-mRNA gp70 AH1-A5 enhanced the inhibitory effect of LNP-mRNA gp70 AH1-A5 on the proliferation of gp70-expressing CT26 tumor cells.

[0017] That is, the present invention is as follows: [1] An adjuvant containing a type-B CpG oligodeoxynucleotide (CpG ODN), a modified form thereof, or a complex thereof for administration in combination with an mRNA vaccine in which mRNA encoding an antigen is encapsulated in particles, the adjuvant existing independently from the particles encapsulating the mRNA. [2] The adjuvant according to [1], in which the type-B CpG ODN portion in the type-B CpG ODN, a modified form thereof, or a complex thereof has all phosphodiester bonds replaced with phosphorothioate bonds and contains one or more unmethylated CpG motifs. Here, the type-B CpG ODN portion has a nucleotide sequence that is unlikely to adopt a higher-order structure, and the functional properties of type-B CpG ODN are preferably at least one of the following: the activity of activating human B cells to produce IL-6; the activity of promoting the maturation of pDCs when acting on them; and the activity of hardly inducing IFN-α production in pDCs when acting on them; more preferably, any two of these, and even more preferably, all three of these. The type-B CpG ODN portion preferably does not have a palindromic sequence of a certain base length or more (preferably, does not have a palindromic sequence longer than 10 bases, more preferably, does not have a palindromic sequence longer than 8 bases, and even more preferably, does not have a palindromic sequence longer than 6 bases). [3] The adjuvant according to [1] or [2], wherein the length of the type-B CpG ODN portion in the type-B CpG ODN, its modified form, or its complex is 100 nucleotides or less. [4] The adjuvant according to any one of [1] to [3], characterized in that the adjuvant enhances the ability to induce cellular immunity specific to an antigen encoded by the mRNA. [5] The adjuvant according to any one of [1] to [4], wherein the type B CpG ODN portion in the type B CpG ODN, its modified form, or its complex contains a sequence consisting of TCG. [6] The adjuvant according to any one of [1] to [5], wherein the mRNA vaccine is an infectious disease vaccine or a cancer vaccine.[7] The adjuvant according to any one of [1] to [6], wherein the type-B CpG ODN, its modified product, or its complex comprises one selected from the group consisting of nucleic acid sequences of SEQ ID NOs: 1 to 6 and 13. [8] The adjuvant according to any one of [1] to [6], wherein the type-B CpG ODN, its modified product, or its complex comprises K3 CpG ODN, K3-dA40, K3 CpG ODN analog, ODN 2006, ODN 1018, ODN 1826, or ODN 1668. [9] The adjuvant according to any one of [1] to [8], wherein the type-B CpG ODN complex is a complex of type-B CpG ODN or its modified product with β(1→3) glucan.

[10] The adjuvant according to [9], wherein the β(1→3) glucan is lentinan or schizophyllan.

[11] The adjuvant according to any one of [1] to [8], wherein the modified B-type CpG ODN is a lipid-modified form.

[12] The adjuvant according to any one of [1] to

[11] , wherein in the mRNA vaccine, the particles encapsulating the mRNA comprise lipid nanoparticles (LNPs).

[13] The adjuvant according to any one of [1] to

[12] , wherein the mRNA comprises at least one modified nucleotide.

[14] The adjuvant according to

[13] , wherein the modified nucleotide comprises at least one pyrimidine nucleotide substituted at position 5 and / or pseudouridine optionally substituted at position 1.

[15] The adjuvant according to

[13] , wherein the modified nucleotide comprises at least one nucleotide selected from the group consisting of 5-methylcytidine, 5-methoxyuridine, 5-methyluridine, pseudouridine, and 1-alkylpseudouridine.

[16] The adjuvant according to

[13] , wherein the modified nucleotide comprises at least one selected from the group consisting of 5-methylcytidine, 5-methyluridine, and 1-methylpseudouridine.

[17] The adjuvant according to

[12] , wherein the LNP comprises an amphipathic lipid, a sterol, an ionizable lipid, and a PEG-lipid.

[18] The adjuvant according to

[17] , wherein the ionizable lipid is selected from ALC-0315, SM-102, and (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-diene-7,29-diyl diacetate.

[19] The adjuvant according to

[17] or

[18] , wherein the lipid composition of the amphipathic lipid, sterols, ionizable lipid, and PEG lipid constituting the LNP is, in molar amounts, 10 to 22.5% amphipathic lipid, 15 to 55% sterols, 40 to 65% ionizable lipid, and 1 to 5% PEG lipid.

[20] A method for enhancing the induction of antigen-specific cytotoxic T cells by an mRNA vaccine, comprising administering an adjuvant containing a type-B CpG oligodeoxynucleotide (CpG ODN), a modified form thereof, or a complex thereof to a subject who will receive or has received an mRNA vaccine in which an mRNA encoding an antigen is encapsulated in particles, wherein the adjuvant exists independently of the particles encapsulating the mRNA.

[21] The method according to

[20] , in which the adjuvant and the mRNA vaccine are administered in combination.

[22] A method for treating a disease treatable by the antigen, comprising administering an mRNA vaccine in which an mRNA encoding an antigen is encapsulated in particles and an adjuvant containing a type-B CpG oligodeoxynucleotide (CpG ODN), a modified form thereof, or a complex thereof to a subject suffering from the disease, wherein the adjuvant exists independently of the particles encapsulating the mRNA.

[23] The method according to

[22] , wherein the antigen is an infectious disease antigen or a cancer antigen, and the disease is an infectious disease or cancer.

[24] An adjuvant comprising a B-type CpG oligodeoxynucleotide (CpG ODN), a modified form thereof, or a complex thereof, for use in enhancing the induction of antigen-specific cytotoxic T cells by an mRNA vaccine in which mRNA encoding the antigen is encapsulated in particles, wherein the adjuvant exists independently of the particles encapsulating the mRNA.

[25] An adjuvant comprising a type-B CpG oligodeoxynucleotide (CpG ODN), a modified form thereof, or a complex thereof, used to treat a disease treatable by an antigen using an mRNA vaccine in which mRNA encoding the antigen is encapsulated in particles, wherein the adjuvant exists independently of the particles encapsulating the mRNA.

[26] The adjuvant according to

[25] , wherein the antigen is an infectious disease antigen or a cancer antigen, and the disease is an infectious disease or cancer.

[27] Use of a type-B CpG oligodeoxynucleotide (CpG ODN), a modified form thereof, or a complex thereof for producing an adjuvant containing the CpG ODN, a modified form thereof, or a complex thereof, for administration in combination with an mRNA vaccine in which mRNA encoding the antigen is encapsulated in particles, wherein the adjuvant exists independently of the particles encapsulating the mRNA.

[28] The use according to

[27] , wherein the antigen is an infectious disease antigen or a cancer antigen, and the vaccine is used to treat an infectious disease and / or cancer.

[29] A pharmaceutical composition comprising an mRNA vaccine in which mRNA encoding an antigen is encapsulated in particles, and an adjuvant containing a type B CpG oligodeoxynucleotide (CpG ODN), a modified form thereof, or a complex thereof, wherein the adjuvant exists independently of the particles encapsulating the mRNA.

[30] The pharmaceutical composition according to

[29] , wherein the antigen is an infectious disease antigen or a cancer antigen, for treating an infectious disease or cancer.

[0018] The present invention provides adjuvants that enhance CTL induction by mRNA vaccines (hereinafter also referred to as "adjuvants of the present invention"). Specifically, B-type CpG oligodeoxynucleotides (CpG ODNs), complexes of B-type CpG ODNs with β(1→3) glucans, and lipid-modified B-type CpG ODNs are provided as adjuvants that enhance CTL induction by mRNA vaccines. Furthermore, since the enhancement of antigen-specific CTL induction was observed even when mRNA encoding only the CD8 epitope sequence was used as the mRNA encoding the antigen protein, shortening the length of the mRNA used in vaccines is expected to reduce vaccine production costs, facilitate production, enhance efficacy per unit mRNA amount, enable the production of safer vaccines, and reduce the administration volume of vaccines by reducing the particle size when encapsulated in LNPs.

[0019] Figure 1 shows the effect of various adjuvants on CTL induction by LNP-mRNA vaccines. Experiments were conducted with 4 mice per group, with open circles representing data from individual mice and open bars representing the mean. Figure 2 shows the adjuvant effect of various CpG ODN (0.1-1 μg) on ​​CTL induction by LNP-mRNA vaccines. Experiments were conducted with 4 mice per group, with open circles representing data from individual mice and open bars representing the mean. Figure 3 shows the adjuvant effect of various CpG ODN (1-10 μg) on ​​CTL induction by LNP-mRNA vaccines. Experiments were conducted with 4 mice per group, with open circles representing data from individual mice and open bars representing the mean. Figure 4 shows the adjuvant effect of K3-LNT or K3-SPG on CTL induction by LNP-mRNA vaccines. Experiments were conducted with 4 mice per group, with open circles representing data from individual mice and open bars representing the mean. Figure 1 shows a comparison of the adjuvant effect of K3 or K3 encapsulated in LNPs (LNP-K3) on CTL induction by an LNP-mRNA vaccine. Experiments were conducted with four mice per group, with open circles representing data from individual mice and open bars representing the mean. Figure 2 shows a comparison of the adjuvant effect of K3-LNT on CTL induction by an LNP-mRNA vaccine administered intradermally (i.d.) or intramuscularly (i.m.). Experiments were conducted with four mice per group, with open circles representing data from individual mice and open bars representing the mean. Figure 3 shows the adjuvant effect of K3-LNT on CTL induction by an LNP-mRNA vaccine composed of different modified nucleotides. Experiments were conducted with four mice per group, with open circles representing data from individual mice and open bars representing the mean. Figure 4 shows the adjuvant effect of K3-LNT on CTL induction by an LNP-mRNA vaccine composed of different cationic lipids. Experiments were performed on 4 mice per group. Open circles represent data from individual mice, and open bars represent the mean. Figure 1 shows the adjuvant effect of K3 CpG ODN or K3-LNT on CTL induction by LNP-mRNA vaccines consisting of different lipid ratios. Experiments were performed on 4 mice per group. Open circles represent data from individual mice, and open bars represent the mean. Figure 1 shows the adjuvant effect of K3-LNT on CTL induction by LNP-mRNA gp70 AH1-A5. Experiments were performed on 4 mice per group. Open circles represent data from individual mice, and open bars represent the mean. Figure 1 shows the tumor volume when CT26 tumor cells were transplanted into the non-immunized group (vehicle-administered group). Each broken line represents the tumor volume of each individual (8 mice).Fifteen days after CT26 tumor cell transplantation, all mice reached the humane endpoint. This figure shows the tumor volume when CT26 tumor cells were transplanted into the K3-LNT alone-administered group. Each line represents the tumor volume of an individual mouse (eight mice). Eighteen days after CT26 tumor cell transplantation, all mice reached the humane endpoint. This figure shows the tumor volume when CT26 tumor cells were transplanted into the LNP(1)-mRNA gp70 AH1-A5 (5MeC, 5MeU)-administered group. Each line represents the tumor volume of an individual mouse (eight mice). Three mice did not develop tumors. This figure shows the tumor volume when CT26 tumor cells were transplanted into the K3-LNT-administered group with LNP(1)-mRNA gp70 AH1-A5 (5MeC, 5MeU) co-administered group. Each line represents the tumor volume of an individual mouse (eight mice). Four mice did not develop tumors. The survival rates of each group in Figures 11A-D are shown. The adjuvant effect of K3 CpG ODN on CTL induction by LNP(1)-mRNA AH1-A5 (5MeC, 5MeU) or LNP(1)-mRNA gp70 AH1-A5 (5MeC, 5MeU), encoding only the CD8 epitope, is shown. Experiments were performed with four mice (vehicle-administered group) or six mice (LNP(1)-mRNA AH1-A5 (5MeC, 5MeU)-administered group or LNP(1)-mRNA gp70 AH1-A5 (5MeC, 5MeU)-administered group). Open circles represent data from individual mice, and open bars represent average values. The sequence information for the B-type CpG ODNs mentioned herein is shown. This shows the effect of various lipid-modified K3 (0.1-1 μg / mouse) on CTL induction by an LNP-mRNA vaccine (1 μg / mouse) in Example 13. Experiments were performed with 4 mice per group, with open circles representing data from individual mice and open bars representing average values.

[0020] 1. Adjuvant: In the present invention, an adjuvant refers to an auxiliary agent that promotes the immune response to a vaccine. When administered to a living body in combination with a vaccine containing an antigen or mRNA encoding an antigen, it enhances the immune response to the antigen. In particular, in the present invention, an adjuvant refers to an auxiliary agent that enhances the induction of CTLs specific to the antigen encoded by the mRNA. The antigen encoded by the mRNA can be, for example, a peptide, polypeptide, or protein.

[0021] The adjuvant exists independently from the vaccine in which the mRNA encoding the antigen is encapsulated in particles. "Existing independently" means that the particles encapsulating the mRNA and the adjuvant outside the particles may exist in the same solution, or the solution of the particles encapsulating the mRNA and the solution of the adjuvant may exist separately. Furthermore, "existing independently" does not exclude the case where the adjuvant is partially encapsulated in the particles. For example, the particles encapsulating the mRNA and a part of the adjuvant and the adjuvant outside the particles may exist in the same solution, or the solution of the particles encapsulating the mRNA and a part of the adjuvant and the solution of the adjuvant may exist separately.

[0022] The term "administered in combination" is not particularly limited as long as it is a method that can enhance the CTL induction of the vaccine. For example, when the vaccine and adjuvant are dissolved in the same solution, the solutions may be administered simultaneously using the same administration method. Specifically, when the vaccine and adjuvant are dissolved in the same solution, they may be administered simultaneously by an administration method such as intramuscular administration. In this case, the solution containing the vaccine and adjuvant may be one in which both are dissolved in advance, or one obtained by preparing a freeze-dried mixture of the two by dissolving them just before use.

[0023] For example, a vaccine solution and an adjuvant solution may be mixed and administered simultaneously by the same administration method. Specifically, a vaccine solution and an adjuvant solution may be mixed and the resulting solution may be administered simultaneously by an administration method such as intramuscular administration. In this case, the vaccine solution and the adjuvant solution may be pre-dissolved, or may be prepared by dissolving a freeze-dried vaccine and adjuvant immediately before use.

[0024] Alternatively, for example, a method can be employed in which the vaccine solution and the adjuvant solution are not mixed, but are administered consecutively with the administration of the vaccine solution, before and / or after the administration of the vaccine solution. Specifically, a vaccine solution and an adjuvant solution may be prepared separately, and the adjuvant may be administered consecutively with the administration of the vaccine, before and / or after the administration of the vaccine, for example, by intramuscular administration. In this case, the vaccine solution and the adjuvant solution may be pre-dissolved, or may be prepared by dissolving a freeze-dried vaccine and adjuvant immediately before use.

[0025] When the vaccine solution and adjuvant solution are administered without being mixed, the vaccine solution and the adjuvant solution can be administered by different administration methods. For example, the vaccine solution can be administered intramuscularly and the adjuvant solution can be administered subcutaneously. However, since it is preferable that the adjuvant is present near the site where CTL induction occurs against the antigen protein translated from mRNA, it is preferable that the vaccine and adjuvant are administered simultaneously by the same administration method.

[0026] 2. Type B CpG Oligodeoxynucleotide (Type B CpG ODN) CpG oligodeoxynucleotide (CpG ODN) is a single-stranded DNA containing unmethylated CpG motifs with immunostimulatory activity and is a TLR9 agonist. There are four types of CpG ODN: Type B (also called Type K), Type A (also called Type D), Type C, and Type P, each of which differs in backbone sequence and immunostimulatory properties (Non-Patent Document 13). The oligodeoxynucleotide used in the adjuvant of the present invention (hereinafter also referred to as the oligodeoxynucleotide of the present invention) is a deoxyoligonucleotide having structural characteristics that are classified as Type B CpG ODN. The oligonucleotides used in the present invention may also contain nucleotides (e.g., polydeoxyadenylic acid) that are not directly related to the properties of B-type CpG ODN. However, in this specification, the portion important for the properties of B-type CpG may be referred to as the B-type CpG ODN or the B-type CpG ODN portion.

[0027] The nucleotides constituting the Type B CpG ODN are natural or chemically modified non-natural deoxyribonucleotides. Natural bases are 2'-deoxyadenosine, thymidine, 2'-deoxyguanosine, and 2'-deoxycytidine. Each base may be natural or modified, for example, by methylation at the 5-position, but the base contained in the non-methylated CpG motif is natural.

[0028] The structural characteristics of Type B CpG ODN are that it has a phosphorothioate backbone, contains one or more unmethylated CpG motifs, does not have a poly-G tail, and has a base sequence that is unlikely to form an intermolecular higher-order structure. In the present invention, Type B CpG ODN may have one or more phosphorothioate bonds as internucleotide bonds constituting it, and the proportion of such bonds may be, for example, 50% or more, preferably 70% or more, more preferably 90% or more, and even more preferably 95% or more. It is more preferable that substantially all internucleotide bonds are phosphorothioate bonds. Here, "substantially" means 95% or more, preferably 98% or more, and more preferably 100%.

[0029] Furthermore, Type B CpG ODN has one or more unmethylated CpG motifs, which are short nucleotide sequences containing at least one cytosine (C)-guanine (G) sequence, in which the 5-position of cytosine in the cytosine-guanine sequence is not methylated.

[0030] The B-type CpG ODN contained in the oligodeoxynucleotide of the present invention is preferably humanized. "Humanized" means having agonistic activity against human TLR9. Therefore, the oligodeoxynucleotide of the present invention containing a humanized B-type CpG ODN has the immunostimulatory activity specific to B-type CpG ODN in humans (e.g., the activity of activating human B cells to produce IL-6). While activating B cells to produce IL-6, B-type CpG ODN has the functional property of promoting the maturation of plasmacytoid dendritic cells (pDCs) with little induction of IFN-α production by pDCs. Such functional properties are properties of the B-type CpG ODN moiety and may vary depending on the modified form or complex of the B-type CpG ODN. In the following description, CpG refers to unmethylated CpG unless otherwise specified. Therefore, the oligodeoxynucleotide of the present invention, by containing a type-B CpG ODN (type-B CpG ODN portion), has immunostimulatory activity specific to type-B (e.g., at least one, preferably any two, and more preferably all three of the following activities: activating B cells (preferably human B cells) to produce IL-6; promoting the maturation of pDCs when acting on them; and barely inducing IFN-α production by pDCs when acting on them). Many humanized type-B CpG ODNs are known in the art (Non-Patent Document 17; Journal of Immunology 164, 944-953 (2000); U.S. Pat. No. 8,030,285 B2). It has also been pointed out that a sequence consisting of TCG is a motif recognized by human TLR9 (Non-Patent Document 18: Stefan Bauer, et al., PNAS. 2001;98 (16) 9237-9242). Therefore, the type B CpG ODN (type B CpG ODN portion) contained in the oligodeoxynucleotide of the present invention may contain a sequence consisting of TCG.

[0031] The CpG motif sequence contained in the type-B CpG ODN of the present invention contains unmethylated cytosine-guanine and has activity recognized by human TLR9, and is preferably a motif sequence consisting of 6 bases, with two bases before and two bases after cg, more preferably a motif sequence consisting of ntcgnn, nacgnn, or ngcgnn (each n is independently a, t, g, or unmethylated c), and even more preferably a motif sequence consisting of atcgac, ctcgag, agcgtt, gtcgtt, aacgtt, ttcgag, or gacgtt. The CpG motif sequence employed in the present invention has 1, 2, 3, 4, or 5 phosphorothioate bonds as internucleotide bonds constituting the motif sequence, and preferably all internucleotide bonds are phosphorothioate bonds.

[0032] The type-B CpG ODN of the present invention has a base sequence that is unlikely to form a higher-order structure, thereby exhibiting a biological activity specific to type-B. As used herein, the term "higher-order structure" of a CpG ODN refers to the formation of a secondary and / or tertiary structure between molecules. The formation of a secondary and / or tertiary structure of a CpG ODN is thought to control the retention time and distribution of the CpG ODN in endolysosomes upon intracellular uptake. Type-A and type-C CpG ODNs, which are known to form higher-order structures, are localized in endolysosomal compartments different from type-B CpG ODNs and are believed to activate an intracellular signaling pathway mediated by IRF-7 from early endosomes, resulting in potent IFN-α induction. On the other hand, type-B CpG ODN is believed to stimulate signaling mediated by NFκB primarily from late endosomes, resulting in potent B cell activation. Factors that affect the formation of this higher-order structure include the length of palindromic sequences permitted in the base sequence of the CpG ODN. Non-Patent Document 13 reports that when palindromic sequences of different lengths were introduced into type-B CpG ODN and their ability to induce INF-α production was tested, the INF-α production induction ability was enhanced in proportion to the length of the introduced palindromic sequence. That is, even if the base sequence of the type-B CpG ODN employed in the present invention contains a palindromic sequence, the base sequence of the type-B CpG ODN is unlikely to adopt a higher-order structure and retains the functional properties of type-B (preferably, at least one, more preferably any two, and even more preferably all three of the activity of activating human B cells to produce IL-6, the activity of promoting the maturation of pDCs when acting on them, and the activity of barely inducing IFN-α production in pDCs when acting on them). The acceptable length of the palindromic sequence is preferably 10 bases or less, more preferably 8 bases or less, and even more preferably 6 bases or less. The palindromic sequence need not be present.

[0033] The base sequence of the type-B CpG ODN of the present invention comprises one to several (preferably 5 or less, more preferably 1, 2, or 3) identical or mutually independent CpG motif sequences, and is a sequence that is unlikely to form a higher-order structure. Preferably, the base sequence comprises a type-B CpG ODN listed in Table 1 in which one or several (5, 4, 3, or 2) bases other than the CpG motif sequence may be substituted, inserted, deleted, and / or added. The base sequence may or may not contain a palindromic sequence of 10 bases or less (more preferably 8 bases or less, even more preferably 6 bases or less), and is even more preferably a base sequence comprising the sequence of a type-B CpG ODN listed in Table 1 or a modified version thereof. Most preferably, the base sequence is a type-B CpG ODN listed in Table 1 or a modified version thereof.

[0034] The length of the B-type CpG ODN is preferably 100 nucleotides or less (e.g., 10-75 nucleotides). The length of the B-type CpG ODN is more preferably 50 nucleotides or less (e.g., 10-40 nucleotides). The length of the B-type CpG ODN is even more preferably 30 nucleotides or less (e.g., 10-25 nucleotides). The length of the B-type CpG ODN is most preferably 12-25 nucleotides. Note that the above description refers to the length of the B-type CpG ODN portion, and in the case of a modified form in which another nucleic acid unit such as polydeoxyadenylic acid is linked, the length corresponds to the length of the B-type CpG ODN portion contained therein.

[0035] 3. Adjuvants Comprising Type B CpG ODN The present invention relates to adjuvants comprising type B CpG ODN. "Comprising type B CpG ODN" refers to any embodiment as long as it contains a type B CpG ODN portion. For example, the type B CpG ODN may be present alone, or the type B CpG ODN portion may be modified (modified form), or the type B CpG ODN or its modified form may be conjugated (conjugate). The type B CpG ODN, its modified form, or conjugate contained in the adjuvant of the present invention may be in the free form or in the form of a pharmaceutically acceptable salt, ester, or salt of such an ester. In this specification, such esters or salts of esters are also included in the term "pharmaceutically acceptable salt."

[0036] In the present invention, the "pharmaceutically acceptable salt" preferably includes metal salts such as alkali metal salts (e.g., sodium salt, potassium salt, and lithium salt), alkaline earth metal salts (e.g., calcium salt, and magnesium salt), aluminum salt, iron salt, zinc salt, copper salt, nickel salt, and cobalt salt; inorganic salts (e.g., ammonium salt), t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, and the like. inorganic acid salts such as hydrohalogenated salts such as hydrofluoride, hydrochloride, hydrobromide and hydroiodide, nitrate, perchlorate, sulfate and phosphate; organic acid salts such as lower alkanesulfonates such as methanesulfonate, trifluoromethanesulfonate and ethanesulfonate, arylsulfonates such as benzenesulfonate and p-toluenesulfonate, acetate, malate, fumarate, succinate, citrate, tartrate, oxalate and maleate; and amino acid salts such as glycine salt, lysine salt, arginine salt, ornithine salt, glutamate and aspartate.

[0037] (1) When Type B CpG ODN is Included Alone in the Adjuvant: The above-mentioned Type B CpG ODN can be administered in combination with an mRNA vaccine without any particular limitation, and can enhance CTL induction. For example, the Type B CpG ODNs listed in Table 1 (K3 CpG ODN, K3 CpG ODN analog, ODN2006, ODN1018, ODN1826, and ODN1668) were confirmed in the Examples to enhance CTL induction. Note that K3 CpG ODN is also referred to as K3, and K3 CpG ODN analog is also referred to as K3 analog.

[0038] [In the table, a, t, g, and c represent 2'-deoxyadenosine, thymidine, 2'-deoxyguanosine, and 2'-deoxycytidine, respectively.]

[0039] (2) When Type B CpG ODN Forms a Modified Form and is Included in the Adjuvant Type B CpG ODN may form a modified form (hereinafter also referred to as the modified form of the present invention). The modified form of the present invention is not particularly limited as long as it is a modified form in which the Type B CpG ODN (the Type B CpG ODN portion) is chemically modified. Examples of the form of modification include a modified form in which the Type B CpG ODN is bound to a lipid such as a fatty acid or cholesterol (lipid-modified form: also referred to as lipid-conjugated form; see Patent Document 5, etc.), and a modified form in which another nucleic acid unit is further bound.

[0040] One embodiment of the lipid-modified type B CpG ODN used as an adjuvant of the present invention is one in which a lipid moiety is bound to the 5'-end or 3'-end via a linker containing a phosphate group.

[0041] In the present invention, the lipid moiety means a structural unit containing a linear or branched long-chain aliphatic hydrocarbon. The lipid moiety is preferably a linear or branched C １０ ~C ３０ The lipid moiety is preferably a linear chain. The lipid moiety is preferably a C １２ ~C ２５ is an alkyl or alkenyl group, more preferably C １５~C ２０ is an alkyl or alkenyl group. １５ ~C ２０ Specific examples of the alkyl group include linear alkyl groups such as pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, and icosyl groups; isopentadecyl, isohexadecyl, isoheptadecyl, isooctadecyl, isononadecyl, isoicosyl, sec-pentadecyl, sec-hexadecyl, sec-heptadecyl, and sec- Examples of branched alkyl groups include a tert-octadecyl group, a sec-nonadecyl group, a sec-icosyl group, a t-pentadecyl group, a t-hexadecyl group, a tert-heptadecyl group, a tert-octadecyl group, a tert-nonadecyl group, a tert-icosyl group, a neopentadecyl group, a neohexadecyl group, a neoheptadecyl group, a neooctadecyl group, a neononadecyl group, and a neoicosyl group. １５ ~C ２０ Specific examples of the alkenyl group include a 1-pentadecenyl group, a 3-pentadecenyl group, a 5-pentadecenyl group, a 7-pentadecenyl group, a 13-pentadecenyl group, a 1-hexadecenyl group, a 2-hexadecenyl group, a 15-hexadecenyl group, a 1-heptadecenyl group, a 2-heptadecenyl group, a 15-heptadecenyl group, a 16-heptadecenyl group, a 1-octadecenyl group, a 2-octadecenyl group, a 9-octadecenyl group, a 16-octadecenyl group, a 17-octadecenyl group, a 1-nonadecenyl group, a 2-nonadecenyl group, a 16-nonadecenyl group, a 1-icosenyl group, a 2-icosenyl group, and an 18-icosenyl group.

[0042] One embodiment of the structure of the "phosphate group-containing linker" is exemplified by the chemical structure represented by the following general formula (I).

[0043]

[0044] (Wherein, L is absent; C ２ ~C ２０ Alkylene group; C ２ ~C ６ A group consisting of 1 to 6 repeating units of an alkyleneoxy group and C １ ~C６ a linking group to an alkylene group; or a C linked to an amino acid residue or an oligopeptide consisting of 2 to 5 amino acid residues via an amide bond １ ~C ６ is a linking group to an alkylene group, D is an oxygen atom or a sulfur atom, # indicates a linking portion to the oligonucleotide side, and Lipid indicates a lipid portion.

[0045] In general formula (I), when L is absent, Lipid is directly bonded to the phosphate group. When L is present, Lipid may be linked to the end of L via a desired bond (e.g., an amide bond, an ester bond, a phosphodiester bond, a phosphorothioate bond, etc.).

[0046] In the general formula (I), when L is an alkylene group, it is preferably C ２ ~C １０ It is an alkylene group. ２ ~C １０ Specific examples of the alkylene group include linear alkylene groups such as methylene, ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, heptamethylene, octamethylene, nonamethylene, and decamethylene; and branched alkylene groups such as 2-methyltrimethylene, 1,1-dimethylethylene, 1-methyltetramethylene, 2-methyltetramethylene, 1,1-dimethyltrimethylene, 1,2-dimethyltrimethylene, 2,2-dimethyltrimethylene, and 1-ethyltrimethylene.

[0047] In the general formula (I), when L is an alkylene group, it is more preferably C ２ ~C ６ alkylene group, more preferably C ２ ~C ４ alkylene groups, and even more preferably C ３ It is an alkylene group. ３ The alkylene group includes a trimethylene group (—CH ２ CH ２ CH ２ -) is preferred.

[0048] In the general formula (I), when L is a linking group between a group consisting of a repeating unit of an alkyleneoxy group and an alkylene group, it is preferably C ２ ~C ４ is a linking group between a group consisting of repeating units of an alkyleneoxy group and an alkylene group, and more preferably C ２ ~C ３ is a linking group between a group consisting of repeating units of an alkyleneoxy group and an alkylene group, and more preferably C ２ It is a linking group between a group consisting of repeating units of an alkyleneoxy group and an alkylene group.

[0049] In the general formula (I), when L is a linking group between a group consisting of repeating units of an alkyleneoxy group and an alkylene group, it is preferable that the linking group between the group consisting of repeating units of an alkyleneoxy group and C １ ~C ４ It is a linking group with an alkylene group, and more preferably a group consisting of a repeating unit of an alkyleneoxy group and C １ ~C ３ It is a linking group with an alkylene group, and more preferably a group consisting of repeating units of an alkyleneoxy group and C ２ It is a linking group to an alkylene group.

[0050] In general formula (I), when L is a linking group between a group consisting of repeating units of an alkyleneoxy group and an alkylene group, the number of repeating units is preferably 1 to 4, and more preferably 1 to 3, in any case.

[0051] In general formula (I), when L is a linking group between an oligopeptide consisting of amino acid residues and an alkylene group linked via an amide bond, it is preferably a linking group between an oligopeptide consisting of 2 to 4 amino acid residues and an alkylene group linked via an amide bond, and more preferably a linking group between an oligopeptide consisting of 2 or 3 amino acid residues and an alkylene group linked via an amide bond.

[0052] In the general formula (I), when L is a linking group of an alkylene group linked to an amino acid residue or an oligopeptide consisting of amino acid residues via an amide bond, it is preferably a C １ ~C ４ C is a linking group to an alkylene group, and more preferably is linked to an amino acid residue or an oligopeptide consisting of amino acid residues via an amide bond. １ ~C ３ It is a linking group to an alkylene group.

[0053] In general formula (I), when L is a linking group between an amino acid residue or an oligopeptide consisting of an amino acid residue and an alkylene group linked via an amide bond, the amino acid residue or oligopeptide preferably consists of at least one amino acid residue selected from the group consisting of glycine residue, alanine residue, β-alanine residue, valine residue, leucine residue, isoleucine residue, cysteine ​​residue, methionine residue, serine residue, threonine residue, aspartic acid residue, glutamic acid residue, asparagine residue, glutamine residue, arginine residue, lysine residue, histidine residue, phenylalanine residue, tyrosine residue, tryptophan residue, and proline residue, more preferably consists of a glycine residue, alanine residue, and / or β-alanine residue, and even more preferably consists of a glycine residue.

[0054] The lipid-modified 5'-end of a type B CpG ODN can be synthesized by directly introducing a lipid after completion of elongation of an oligonucleotide chain having a target sequence by subsequently coupling an amidite unit corresponding to the desired lipid moiety, or by synthesizing an oligonucleotide having a phosphate group-containing linker attached to the 5'-end, which has a functional group that can be used to introduce a lipid moiety, using 5'-Amino-Modifier C6 (Glen Research), 5'-TFA-Amino-Modifier C6-CE Phosphoramidite, 5'-TFA-Amino-Modifier-C5-CE Phosphoramidite (Link Technologies), or the like, and then using that functional group to introduce the lipid moiety.

[0055] Furthermore, a lipid-modified 3'-terminus of a B-type CpG ODN can be synthesized by using 3'-amino-Modifier C3 CPG and 3'-amino-Modifier C7 CPG (Glen Research) or the like to synthesize an oligonucleotide having a linker containing a phosphate group having a functional group that can be used to introduce a lipid moiety at the 3'-terminus, and then using that functional group to introduce the lipid moiety.

[0056] Furthermore, an —O—(CH ２ Those having a linker such as -O-P(=X)(OH)- (n is 3 to 18, X is an oxygen atom or a sulfur atom) can be synthesized using an amidite reagent corresponding to the number of n. For example, a phosphoramidite reagent such as DMT-propane-Diol phosphoramidite (ChemGenes, catalog number: CLP-9908) or DMT-hexane-Diol phosphoramidite (ChemGenes, catalog number: CLP-1120) can be used. ２ CH ２ Oligonucleotides having a glycol-containing linker such as (O)n-P(=X)(OH)- (n is 1 to 6, X is an oxygen atom or a sulfur atom) can be synthesized using an amidite reagent corresponding to the number of n. For example, DMT-tetraethyloxy-glycol CED phosphoramideite (Chemgenes, catalog number: CLP-1368) can be used. Furthermore, oligonucleotides having natural ribonucleotide units as linkers can be synthesized using the corresponding appropriately protected ribonucleoside phosphoramidite.

[0057] Lipid-modified B-type CpG ODNs of the present invention having a lipid moiety derived from a fatty acid at the 5' or 3' end can be synthesized by reacting an oligonucleotide having an aminoalkyl phosphate group (e.g., an alkyl group having 3 to 9 carbon atoms) attached to the 5' or 3' end with an activated ester such as a pentafluorophenyl ester of a fatty acid such as myristic acid, palmitic acid, stearic acid, arachidic acid, or behenic acid (Nucleic Acids Res. (2020) 47, 6029-6044). Alternatively, the oligonucleotide can be synthesized by solid-phase synthesis of an oligonucleotide having a target sequence on a solid support having an aminoalkyl phosphate group (e.g., an alkyl group having 3 to 9 carbon atoms) attached to the 5' end, condensing the oligonucleotide with a fatty acid such as myristic acid, palmitic acid, stearic acid, arachidic acid, or behenic acid using a condensing agent such as HATU, followed by deprotection and purification (WO 2017 / 192679).

[0058] A lipid-modified B-type CpG ODN of the present invention having a lipid moiety at the 5'-end or 3'-end can be synthesized by coupling an amidite unit corresponding to the desired lipid moiety. After chain elongation of the oligonucleotide having the target sequence is completed, 2-cyanoethyl (6-palmitamidohexyl) diisopropylphosphoramidite (Nucleic Acids Res. (2020) 47, 6029-6044, Link Technologies), 2-cyanoethyl (6-stearamidohexyl) diisopropylphosphoramidite (compound described in Reference Example 1 of WO 2021 / 010301), or C-18 Phosphoramidite (WO 98 / 18480) It can be synthesized by coupling an amidite unit corresponding to a fatty acid such as phosphoramidite.

[0059] The modified product in which a type B CpG ODN is linked to polydeoxyadenylic acid is, for example, a modified product in which a polydeoxyadenylic acid of 20 to 60 nucleotides in length (specifically, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides in length) is linked to the 3'-side of an oligodeoxynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1, more preferably an oligodeoxynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1. Modified oligodeoxynucleotides include those in which a 30-50 nucleotide long (specifically, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotide long) polydeoxyadenylic acid is linked to the 3' end of the oligodeoxynucleotide, and most preferably, those in which a 30-45 nucleotide long (specifically, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 nucleotide long) polydeoxyadenylic acid is linked to the 3' end of an oligodeoxynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 1. The modified type B CpG ODN can be administered in combination with an mRNA vaccine without particular limitation, and can enhance CTL induction. For example, the modified B-type CpG ODN (K3-dA) shown in Table 1 ４０ It was confirmed in the Examples that ) enhances CTL induction.

[0060] These modified forms of B-type CpG ODN bound to polydeoxyadenylic acid are, as described below, soluble in β-1,3-glucan (e.g., lentinan (LNT), schizophyllan (SPG)) and complexes (K3-dA). ３０ ~dA ４５ -LNT, K3-dA ３０ ~dA ４５ Specifically, K3-dA, in which a 40-nucleotide polydeoxyadenylic acid is bound to the 3'-terminus of K3, can be formed. ４０ (SEQ ID NO: 13) plus a 35 nucleotide-long linked K3-dA ３５, 30 nucleotides long bound K3-dA ３０ , 25 nucleotides long bound K3-dA ２５ and 20 nucleotide-long bound K3-dA ２０ Alternatively, the K3 CpG ODN and polydeoxyadenylic acid may be linked via a spacer.

[0061] (3) When a Type-B CpG ODN is Included in an Adjuvant in the Form of a Complex: The present invention further provides a complex of a Type-B CpG ODN or a modified version thereof with a β-1,3-glucan (hereinafter also referred to as the "complex of the present invention"). The complex of the present invention can also enhance CTL induction. Examples of β-1,3-glucans include lentinan, schizophyllan, scleroglucan, curdlan, perchyman, grifolan, and laminaran. The β-1,3-glucan is preferably a β-1,3-glucan containing a large amount of 1,6-glucopyranoside branches (side chain ratio 33-40%), such as lentinan, schizophyllan, or scleroglucan, more preferably lentinan or schizophyllan, and most preferably lentinan. Here, the Type-B CpG ODN may be a modified portion of a Type-B CpG ODN.

[0062] Lentinan (LNT) is a known β-1,3-1,6-glucan derived from shiitake mushrooms, with the molecular formula (C ６ H １０ O ５ )n, and the molecular weight of LNT, which has been put to practical use as an antitumor drug as described below, is controlled to approximately 300 kD to 700 kD. The molecular weight of LNT that forms a complex with B-type CpG is not particularly limited as long as it is a molecular weight that allows the formation of a complex, but is, for example, 5 kD to 2000 kD, preferably 15 kD to 1500 kD. This LNT is almost insoluble in water, methanol, ethanol (95), or acetone, but is soluble in polar organic solvents such as DMSO and aqueous sodium hydroxide solution.

[0063] Lentinan enhances the activity of activated macrophages, killer T cells, natural killer cells, and antibody-dependent macrophage-mediated cytotoxicity (ADMC) (Hamuro, J., et al.: Immunology, 39, 551-559, 1980; Hamuro, J., et al.: Int. J. Immunopharmacol., 2, 171, 1980; Herlyn, D., et al.: Gann, 76, 37-42, 1985). In animal experiments, combined administration of lentinan with chemotherapeutic agents has been shown to inhibit tumor growth and prolong life in syngeneic and autologous tumors. Furthermore, administration of lentinan alone has also been shown to inhibit tumor growth and prolong life. In clinical trials, the combination of oral tegafur administration with LNT was shown to extend survival times in patients with inoperable or recurrent gastric cancer (Drug Interview Form "Lentinan 1 mg for Intravenous Injection "Ajinomoto""). LNT has been approved and sold in Japan. The effectiveness of LNT administered alone has not yet been confirmed.

[0064] Schizophyllan (SPG) is a well-known soluble β-glucan derived from Schizophyllum commune. SPG consists of a β-(1→3)-D-glucan main chain and one β-(1→6)-D-glucosyl side chain for every three glucose units (Tabata, K., Ito, W., Kojima, T., Kawabata, S. and Misaki, A., "Carbohydr. Res.", 1981, 89, 1, pp. 121-135). SPG has been used for over 20 years as an intramuscular injection clinical drug for immunopotentiation against gynecological cancers (Shimizu, Chin, Nami, Masubuchi, Biotherapy, 1990, 4, p. 1390; Hasegawa, Oncology and Chemotherapy, 1992, 8, p. 225), and its safety in vivo has been confirmed (Theresa, M. McIntire and David, A. Brant, J. Am. Chem. Soc., 1998, 120, p. 6909).

[0065] In the present invention, the term "complex" refers to a product obtained by the association of multiple molecules via non-covalent or covalent bonds such as electrostatic bonds, van der Waals bonds, hydrogen bonds, and hydrophobic interactions.

[0066] The complex of the present invention preferably has a triple helix structure. In a preferred embodiment, of the three strands forming the triple helix structure, two are β-1,3-glucan strands and one is a polydeoxyadenylic acid (dA) strand bound to the 3' end of the B-type CpG ODN. The complex may also contain a portion that does not form a triple helix structure.

[0067] The composition ratio of type B CpG ODN to β-1,3-glucan in the complex of the present invention can vary depending on the chain length of the polydeoxyadenylic acid bound to the 3' end of the type B CpG ODN, the length of the β-1,3-glucan, etc. For example, when the lengths of the β-1,3-glucan chain and the polydeoxyadenylic acid chain are equivalent, two β-1,3-glucan chains and one polydeoxyadenylic acid can associate to form a triple helix structure. Generally, since the chain length of the polydeoxyadenylic acid is shorter than that of the β-1,3-glucan chain, multiple type B CpG ODN of the present invention can associate with two β-1,3-glucan chains via polydeoxyadenylic acid to form a triple helix structure.

[0068] The complex of the present invention is a complex containing a humanized type-B CpG ODN and a β-1,3-glucan (e.g., lentinan, schizophyllan, scleroglucan, curdlan, perchyman, grifolan, laminaran), and preferably a complex in which the β-1,3-glucan is selected from lentinan or schizophyllan. For example, a complex (e.g., K3-dA) comprising an oligodeoxynucleotide in which a 20- to 60-nucleotide-long polydeoxyadenylic acid is bound to the 3'-terminus of the type-B CpG ODN consisting of the nucleotide sequence represented by SEQ ID NO: 1 and in which all of the phosphodiester bonds have been replaced with phosphorothioate bonds, and a β-1,3-glucan (e.g., LNT, SPG) is also suitable. ２０ ~dA ６０ -LNT, K3-dA ２０ ~dA ６０-SPG), and more preferably, a complex (e.g., K3-dA) consisting of an oligodeoxynucleotide in which a 30-50 nucleotide long polydeoxyadenylic acid is bound to the 3'-terminus of a B-type CpG ODN consisting of the nucleotide sequence represented by SEQ ID NO: 1 and all of the phosphodiester bonds have been replaced with phosphorothioate bonds, and β-1,3-glucan (e.g., LNT, SPG). ３０ ~dA ５０ -LNT, K3-dA ３０ ~dA ５０ -SPG), and most preferably a complex (K3-dA) consisting of an oligodeoxynucleotide in which a 30-45 nucleotide long polydeoxyadenylic acid is bound to the 3'-terminus of a B-type CpG ODN consisting of the nucleotide sequence represented by SEQ ID NO: 1 and all of the phosphodiester bonds have been replaced with phosphorothioate bonds, and β-1,3-glucan (e.g., LNT, SPG). ３０ ~dA ４５ -LNT, K3-dA ３０ ~dA ４５ -SPG).

[0069] The conjugate of the present invention can be prepared by known methods, such as the conditions described in JP 2008-100919 A. Specifically, β-1,3-glucan, which naturally exists as a triple helix structure, is dissolved in an aprotic organic polar solvent (dimethyl sulfoxide (DMSO), acetonitrile, acetone, etc.) or an alkaline aqueous solution (sodium hydroxide, potassium hydroxide, ammonia, calcium hydroxide, etc.) and unwound into a single strand. The resulting single-stranded β-1,3-glucan solution is mixed with a solution of an oligodeoxynucleotide of the present invention in which polydeoxyadenylic acid is bound to the 3' end of the B-type CpG ODN (aqueous solution, aqueous buffer solution with a pH close to neutral, or acidic aqueous buffer solution, preferably aqueous solution or aqueous buffer solution with a pH close to neutral). The pH is adjusted to near neutral again as necessary, and the mixture is then maintained for a suitable period of time, for example, overnight at 5°C. As a result, two β-1,3-glucan chains and the poly dA chain in the oligodeoxynucleotide form a triple helix structure, thereby forming the complex of the present invention. The resulting complex can be purified by size exclusion chromatography, ultrafiltration, dialysis, or the like to remove any oligodeoxynucleotide that has not formed a complex. Furthermore, the resulting complex can be purified by anion exchange chromatography to remove any β-1,3-glucan that has not formed a complex. The complex can be appropriately purified by the above methods.

[0070] The formation of the complex of the present invention can be confirmed by, for example, but not limited to, measuring conformational changes by CD (circular dichroism) spectroscopy, UV absorption shifts by size exclusion chromatography, gel electrophoresis, microchip electrophoresis, or capillary electrophoresis.

[0071] The mixing ratio of the oligodeoxynucleotide having polydeoxyadenylic acid bound to the 3'-terminus of the B-type CpG ODN of the present invention to the β-1,3-glucan can be appropriately set taking into consideration the length of the poly dA chain, etc., but is usually 0.02 to 2.0, preferably 0.1 to 0.5, in terms of molar ratio (SPG / ODN). In a further embodiment, the molar ratio (β-1,3-glucan (e.g., LNT) / ODN) is, for example, 0.005 to 1.0, preferably 0.020 to 0.25.

[0072] More specifically, the method for preparing the complex of the present invention will be described using a CpG ODN-LNT complex as an example. LNT is dissolved in a 0.05-2N, preferably 0.1-1.5N, alkaline aqueous solution (e.g., 0.25N sodium hydroxide aqueous solution) and left at 1°C to 40°C for 10 hours to 4 days (e.g., left at room temperature overnight) to prepare a single-stranded LNT aqueous solution (e.g., a 50 mg / mL LNT aqueous solution). The LNT aqueous solution is mixed with a separately prepared CpG aqueous solution (e.g., a 100 μM CpG aqueous solution) at a molar ratio (LNT / ODN) of 0.005 to 1.0, and then the LNT aqueous solution is diluted with an acidic buffer solution (e.g., NaHCO3). ２ P.O. ４ ) is added to neutralize the mixture, and the mixture is maintained at 1 to 40°C for 6 hours to 4 days (for example, overnight at 4°C) to complete the complexation. Alternatively, the LNT aqueous solution may be added last for the complexation and mixed. Complex formation can be confirmed, for example, by using size exclusion chromatography to monitor the shift of the CpG ODN to higher molecular weights as absorbance at 240 to 280 nm (for example, 260 nm).

[0073] In one embodiment, the complex of the present invention is a rod-shaped particle or a rod-shaped particle bent into a highly ordered random coil. The particle size is reported as follows in Non-Patent Document 23: β-1,3-glucan is schizophyllan of a predetermined molecular weight, and CpG ODN is a modified form thereof, K3-dA. ４０When K3-SPG is used, the particle size of K3-SPG is equivalent to the particle size formed by naturally occurring schizophyllan exhibiting a triple helix structure, with an average particle size of approximately 30 nm. Furthermore, Patent Document 4 clarifies the relationship between the molecular weight of each β-1,3-glucan and the average particle size of the complex in a complex with a CpG ODN in which the β-1,3-glucan is lentinan or schizophyllan, and reveals that the average particle size can be controlled within the range of 20 to 180 nm. The particle size can be measured by dissolving the complex in an aqueous medium and using a device such as a Malvern Instruments Zeta Sizer under specified conditions using dynamic light scattering.

[0074] The complex of the present invention is preferably isolated. The purity of the "isolated complex" (the percentage of the weight of the target complex relative to the total weight of the object to be evaluated) is usually 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 99% or more.

[0075] The present invention has discovered that the above-mentioned type B CpG ODN, its modifications, and complexes thereof exhibit exceptional immune response enhancing activity as adjuvants that enhance CTL induction of mRNA vaccines.

[0076] 4. mRNA Vaccine: The present invention relates to an adjuvant that enhances CTL induction in mRNA vaccines. Conventional vaccines induce antibody responses by injecting humans with antigens (proteins or peptides), attenuated viruses, or viral vectors containing recombinantly encoded antigens. These antigenic components are prepared outside the human body. In contrast, mRNA vaccines involve chemically synthesizing mRNA sequences encoding the antigens, and then transfecting the full-length or fragments of the mRNA into human cells. The antigens are then translated within the cells, thereby inducing humoral and cellular immunity. In the present invention, we have found that the use of type B CpG ODN, its modifications, or its complexes as adjuvants enhances CTL induction and significantly enhances cellular immunity, as shown in the examples. Therefore, mRNA vaccines using the adjuvants of the present invention can induce enhanced immune responses not only as vaccines against infectious diseases but also as vaccines against cancer.

[0077] The mRNA in the mRNA vaccine may contain at least one modified nucleotide. For example, the modified nucleotide may be selected from the group consisting of 5-methylcytidine (5MeC), 5-methoxyuridine, 5-methyluridine (5MeU), pseudouridine (pU), and 1-alkylpseudouridine. The modification site may be one or more.

[0078] Infectious diseases targeted by mRNA vaccines include, but are not limited to, respiratory syncytial virus, 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. Preferred examples include papillomaviruses (HPV16, 18, etc.), respiratory syncytial virus, and influenza virus. When an mRNA vaccine is used as an infectious disease vaccine, mRNA encoding an infectious disease antigen protein derived from the pathogen (virus, bacteria, protozoan, etc.) of the target infectious disease is used.

[0079] Cancer antigens encoded by the mRNA of mRNA vaccines are not particularly limited, but include viral antigens (EBV [Epstein-Barr virus], HPV [human papillomavirus], KSHV [Kaposi's sarcoma-associated herpesvirus], etc.), tumor-associated antigens (tumor-associated antigens) such as differentiation antigens (gp100, MART-1, PSA, etc.), embryonic proteins (CEA, AFP, etc.), overexpressed proteins (HER-2, etc.), cancer testis antigens (MAGE, XAGE, NY-ESO-A, etc.), and tumor-specific antigens (tumor-specific antigens). Examples of antigens include driver mutation-derived antigens (such as KRAS, BRAF, and EGFR), passenger mutation-derived antigens (patient-specific mutant antigens), lncRNA-derived antigens, and non-exon region-derived antigens. Tumor-specific antigens are preferred. Further examples include peptides of partial sequences of these antigens, or antigens in which partial amino acids of peptides have been substituted to enhance antigenicity. Furthermore, it is envisioned that multiple mRNAs encoding these antigens or peptides may be linked together.

[0080] The mRNA vaccine can be produced by a known production method, for example, the known production method described in Patent Document 1 or Patent Document 2, or a method similar thereto.

[0081] The method of administering an mRNA vaccine is not particularly limited as long as it does not interfere with the translation of mRNA into protein in the body of the recipient, and examples include intramuscular administration, subcutaneous administration, intradermal administration, intratumoral administration, intralymph node administration, mucosal administration (application, spray into the nasal cavity, inhalation into the lungs), transdermal administration (application), intravenous administration, and oral administration.

[0082] The number of times an mRNA vaccine is administered may be one or more times, and can be determined appropriately depending on the immune response to the protein encoded by the mRNA.

[0083] 5. LNP-mRNA Vaccine An adjuvant containing the type B CpG ODN of the present invention can enhance CTL induction by an mRNA vaccine in the form of a particle in which mRNA is encapsulated. Examples of particles include CART (Non-Patent Document 8), PBAE (Non-Patent Document 9), and lipid nanoparticles (LNPs). Below, we will describe an LNP-mRNA vaccine in which mRNA is encapsulated in lipid nanoparticles (LNPs).

[0084] LNPs can be produced by the method described in Patent Document 1, and typically contain amphipathic lipids, sterols, ionizable lipids, and PEG lipids as lipid components.

[0085] The amphipathic lipid is a lipid that has affinity for both polar and nonpolar solvents, and specific examples thereof include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, and combinations thereof. The amphipathic lipid used in the particles of the present invention is preferably distearoylphosphatidylcholine and / or dioleoylphosphatidylethanolamine, and more preferably distearoylphosphatidylcholine.

[0086] Sterols are sterols having a hydroxy group, and specific examples thereof include cholesterol.

[0087] Examples of ionizable lipids include ALC-0315, SM-102, (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-diene-7,29-diyl diacetate, 3-dimethylaminopropyl(9Z,12Z)-octacosa-19,22-dien-11-yl carbonate, (7R,9Z)-18-({[3-(dimethylamino)propyloxy]carbonyl}oxy)octacosa-9-en-7-yl acetate, and Dlin-MC3-DMA.

[0088] The PEG lipid is a lipid modified with PEG, and specific examples thereof include 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol and / or N-[methoxypoly(ethylene glycol) 2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine, and combinations thereof, with 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol being preferred.

[0089] The combination of amphipathic lipids, sterols, ionizable lipids, and PEG lipids that constitute the LNPs can be selected from, for example, the following lipids. - Amphiphilic substance: DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) (NOF, #MC-8080) - Sterols: Cholesterol (Sigma, #C8667) - PEG lipid: DMG-PEG2000 (1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol) (NOF, #GM-020) - Ionized lipid: SM-102 (BROADPHARM, #BP-25499) - Ionized lipid: ALC-0315 (MEDCHEMEX, #HY-138170) - Ionized lipid: (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-diene-7,29-diyl diacetate (manufactured by Daiichi Sankyo Co., Ltd.)

[0090] The LNPs are prepared by mixing the above-mentioned amphipathic lipid, sterols, ionizable lipid, and PEG lipid with, for example, 20 mM citrate buffer, pH 4.0 (Nacalai Tesque, Inc., 11938-55) and 300 mM sucrose-containing 10 mM histidine buffer, pH 7.0 (Nacalai Tesque, Inc., 19755-05).

[0091] The lipid composition of the amphipathic lipid, sterols, ionized lipid, and PEG lipid constituting the LNP is preferably, for example, 10-22.5% amphipathic lipid, 15-55% sterols, 40-65% cationic lipid, and 1-5% PEG lipid in molar amounts. More preferably, the lipid composition of the amphipathic lipid, sterols, cationic lipid, and PEG lipid is 10-22.5% amphipathic lipid, 15-55% sterols, 40-65% cationic lipid, and 1-5% PEG lipid in molar amounts.

[0092] 6. Adjuvant Formulations and Dosages Adjuvants containing the type B CpG ODN, its modifications, or complexes thereof of the present invention can be formulated in any known form as a formulation for type B CpG ODN.

[0093] These preparations contain excipients (e.g., sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, alpha starch, and dextrin; cellulose derivatives such as crystalline cellulose; gum arabic; dextran; organic excipients such as pullulan; and silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and magnesium aluminometasilicate; phosphates such as calcium hydrogen phosphate; carbonates such as calcium carbonate; and calcium sulfate). Examples of suitable excipients include inorganic excipients such as sulfates, lubricants (e.g., metal stearates such as stearic acid, calcium stearate, and magnesium stearate; talc; colloidal silica; waxes such as beeswax and Gay's wax; boric acid; adipic acid; sulfates such as sodium sulfate; glycol; fumaric acid; sodium benzoate; DL-leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as anhydrous silicic acid and hydrated silicic acid; and the above-mentioned starch derivatives. Examples of the additives include: binders (for example, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, macrogol, and compounds similar to the excipients described above); disintegrants (for example, cellulose derivatives such as low-substituted hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium, and internally cross-linked sodium carboxymethyl cellulose; and chemically modified starches and celluloses such as carboxymethyl starch, carboxymethyl starch sodium, and cross-linked polyvinylpyrrolidone); emulsifiers (for example, colloidal clays such as bentonite and Veegum; metal hydroxides such as magnesium hydroxide and aluminum hydroxide; anionic surfactants such as sodium lauryl sulfate and calcium stearate; cationic surfactants such as benzalkonium chloride; and nonionic surfactants such as polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, and sucrose fatty acid esters.The composition is produced by a well-known method using additives such as stabilizers (including paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; phenols such as phenol and cresol; thimerosal; dehydroacetic acid; and sorbic acid), flavoring agents (including, for example, commonly used sweeteners, acidulants, fragrances, etc.), and diluents.

[0094] In one embodiment of the present invention, the adjuvant of the present invention may be in the form of an additive to a liquid for administering an mRNA vaccine. When the mRNA vaccine is a solution formulation, the adjuvant may be pre-formulated in a solution, or may be in the form of an additive that is added to the vaccine solution immediately before administration. When the mRNA vaccine is a lyophilized formulation, the administration liquid of the vaccine may be prepared using a solution that already contains the adjuvant of the present invention, or the adjuvant of the present invention may be added simultaneously with or after preparation of the administration liquid. The adjuvant of the present invention may be provided in the form of a formulation kit combined with an mRNA vaccine formulation.

[0095] The dosage of the adjuvant of the present invention varies depending on the type of mRNA vaccine to be combined, the disease, symptoms, age, etc. of the subject to be administered, but in the case of intramuscular administration, intradermal administration, subcutaneous administration, or intratumor tissue administration, it is desirable to administer to an adult a dose with a lower limit of 0.001 mg (preferably 0.01 mg, more preferably 0.03 mg, and more preferably 0.1 mg) and an upper limit of 1000 mg (preferably 100 mg, more preferably 10 mg, and even more preferably 3 mg) per dose, depending on the administration of the mRNA vaccine.

[0096] The adjuvants containing the type-B CpG ODN, its modifications, or complexes thereof of the present invention can enhance CTL induction by mRNA vaccines in the form of particles containing antigen-encoding mRNA, and can therefore be used to treat various diseases targeted by such mRNA vaccines. As used herein, the term "treatment" does not necessarily mean complete treatment, but rather encompasses various degrees of treatment or prevention. The adjuvants of the present invention enhance CTL induction by mRNA vaccines and can be used to treat the symptoms of various diseases thereby. "Treatment" includes alleviating (alleviating) symptoms characteristic of the target disease or accompanying symptoms, preventing or delaying the worsening of symptoms, etc. "Prevention" refers to preventing or delaying the onset / onset of a disease (disorder) or its symptoms, or reducing the risk of onset / onset. Meanwhile, "improvement" refers to alleviating (alleviating), improving, remission, or curing (including partial cure) a disease (disorder) or its symptoms. As such, treatment, prevention, and improvement are partially overlapping concepts, and it is difficult to clearly distinguish between them, and there is little practical benefit in doing so. In this specification, treatment for the purpose of prevention or improvement is also included in the concept of the term "treatment."

[0097] The present invention provides a pharmaceutical composition comprising an mRNA vaccine in which mRNA encoding a therapeutic antigen is encapsulated in particles, and an adjuvant containing an effective amount of type B CpG ODN, a modified form thereof, or a complex thereof, wherein the adjuvant is present in the composition independently of the particles encapsulating the mRNA; a method for treating a disease comprising administering the pharmaceutical composition to a subject treatable (including for preventive purposes) with the therapeutic antigen; use of the adjuvant for producing the pharmaceutical composition; and a kit comprising the adjuvant for preparing the pharmaceutical composition. When the therapeutic antigen encoded by the mRNA is an infectious disease antigen, the pharmaceutical composition is a therapeutic drug for infectious diseases. When the therapeutic antigen is a cancer vaccine, the pharmaceutical composition is a therapeutic drug for cancer treatable by the cancer antigen. The effective amount of the adjuvant of the present invention is not particularly limited as long as it is an amount that can enhance antigen-specific CTLs mediated by the mRNA vaccine, and the above-mentioned doses can be appropriately adopted.

[0098] The present invention will be specifically described below with reference to examples. Note that these examples are for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

[0099] In the examples, the following abbreviations may be used in relation to HPV16 E6_E7. - HPV16 E6_E7: a protein in which HPV16 E6 and E7 proteins are linked via a Furin sequence (SEQ ID NO: 7). - mRNA HPV16 E6_E7: mRNA encoding HPV16 E6_E7 (SEQ ID NO: 8). - mRNA HPV16 E6_E7(pU): mRNA of HPV16 E6_E7 having pseudouridine as a modified nucleotide. - mRNA HPV16 E6_E7(5MeC, 5MeU): mRNA of HPV16 E6_E7 having 5-methylcytidine and 5-methyluridine as modified nucleotides.

[0100] In the examples, the following abbreviations may be used in reference to gp70. - gp70: An antigen derived from a murine leukemia virus identified from tumor cells of the mouse colon cancer cell line CT26. - AH1: A CTL epitope sequence (SPSYVYHQF) (SEQ ID NO: 14) contained in gp70. - AH1-A5: A sequence in which the fifth amino acid of the AH1 sequence is substituted with A (SEQ ID NO: 9). - gp70 AH1-A5: A full-length gp70 sequence in which the AH1 sequence contained in gp70 is substituted with the AH1-A5 sequence (SEQ ID NO: 10). - mRNA gp70: An mRNA encoding gp70. - mRNA AH1: An mRNA encoding AH1. - mRNA AH1-A5: An mRNA encoding AH1-A5 (SEQ ID NO: 11). - mRNA gp70 AH1-A5: mRNA encoding gp70 AH1-A5 (SEQ ID NO: 12).

[0101]

[0102]

[0103] In the examples, the following abbreviations may be used for LNPs. - LNP (1): LNP having a lipid composition of ionizable lipid:DSPC:cholesterol:DMG-PEG200 in a molar ratio (%) of 45:12.5:41:1.5, with the ionizable lipid being (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-diene-7,29-diyl diacetate. - LNP (1, SM-102): LNP having a lipid composition of ionizable lipid:DSPC:cholesterol:DMG-PEG200 in a molar ratio (%) of 45:12.5:41:1.5, with the ionizable lipid being SM-102. - LNP (1, ALC-0315): LNP having a lipid composition of ionizable lipid:DSPC:cholesterol:DMG-PEG200 in a molar ratio (%) of 45:12.5:41:1.5, with the ionizable lipid being ALC-0315. - LNP (2): LNP having a lipid composition of ionizable lipid:DSPC:cholesterol:DMG-PEG200 in a molar ratio (%) of 55:8.5:35:1.5, with the ionizable lipid being (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-diene-7,29-diyl diacetate.

[0104] In the examples, the adjuvants added were as follows: The lyophilized product was dissolved in Otsuka distilled water. - K3 CpG ODN (Gene Design Co., Ltd., Cat. CN-65003, Lot. 20114610-001) (B-type CpG ODN) - K3 CpG ODN (GC) (Gene Design Co., Ltd., Cat. CN-65004) - K3 CpG ODN analog (Gene Design Co., Ltd.) (B-type CpG ODN) - D35 (Gene Design Co., Ltd., Cat. CN-65001, Lot. 20328606-001) (A-type CpG ODN) - Poly(I:C) (InvivoGen, Cat. vac-pic, Lot. 5819-44-02) (TLR3 agonist) - AS01B (Shingrix intramuscular injection AS01B formulation, Lot. AS016) (MPLA [TLR4 agonist] and QS-21 [saponin]) R848 (InvivoGen, Cat. vac-r848, Lot. 5820-44-02) (TLR7 / 8 agonist) - Alhydrogel (InvivoGen, Cat. vac-alu-250, Batch: 0001841976) (Alum adjuvant) - Adju-Phos (InvivoGen, Cat. vac-phos-250, Batch: 0002023203) (Alum adjuvant) - Addavax (InvivoGen, Cat. vac-adx-10, Lot: 5805-44-02) (Squalene) ADU-S100 (ChemieTek, Cat. CT-ADUS100, Lot: 02) (STING ligand) - ODN 2216 (InvivoGen, Cat. tlrl-2216-1, Lot. 6077-43-05) (A-type CpG ODN) - ODN 2006 (InvivoGen, Cat. tlrl-2006-1, Lot. 6057-44-03) (B-type CpG ODN) - ODN 1018 (MedChem, Cat. HY-150724, Lot. 230026) (B-type CpG ODN) - ODN 2395 (InvivoGen, Cat. tlrl-2395-1, Lot. 6094-44-01) (C-type CpG ODN)

[0105] The adjuvants added in the examples include K3-LNT (a complex of B-type CpG ODN) and K3-SPG (a complex of B-type CpG ODN). K3-LNT contains lentinan as the β(1→3) glucan and K3-dA as the nucleic acid. ４０ (complex formation rate: 100%, average particle size: about 60 nm), and K3-SPG contains β(1→3) glucan as schizophyllan and nucleic acid as K3-dA. ４０ (complex formation rate: 100%, average particle size: about 20 nm) Refer to Patent Document 4, and both were manufactured by Daiichi Sankyo and Daiichi Sankyo RD Novare Co., Ltd.

[0106] IMO-2125 (Tilsotolimod, Sodium, Statement on a Nonproprietary Name Adopted by the Usan Council, American Medical Association), a next-generation CpG, was synthesized at Daiichi Sankyo RD Novare Co., Ltd. according to Non-Patent Document 24.

[0107] The following materials were used for preparing LNP-mRNA. - DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) (NOF, #MC-8080) - Cholesterol (Sigma, #C8667) - DMG-PEG2000 (1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol) (NOF, #GM-020) - SM-102 (BROADPHARM, #BP-25499) - ALC-0315 (MEDCHEMEX, #HY-138170) - 20 mM citrate buffer, pH 4.0 (Nacalai Tesque, Inc., 11938-55) - 300 mM Sucrose-containing 10 mM histidine buffer, pH 7.0 (Nacalai Tesque, Inc., 19755-05)

[0108] LNP-K3, in which K3 is encapsulated in LNP, used in Example 5 was prepared as follows. First, K3-type CpG was dissolved in buffer (20 mM citric acid, pH 4.0) to prepare an aqueous solution with a weight concentration of 102.3 μg / mL. A lipid solution (vehicle: ethanol) was prepared with a molar ratio of DSPC, cholesterol, (7R,9Z)-18-({[3-(dimethylamino)propyloxy]carbonyl}oxy)octacos-9-en-7-yl acetate, and DMG-PEG2000 of 17.5:21:60:1.5, and a total lipid molar concentration of 5 mM (weight concentration: 3.07 mg / mL). Using a NanoAssemblr Benchtop (Precision Nanosystems), 4.9 mL of the above CpG solution was mixed with 1.6 mL of lipid solution to generate LNP-K3 nanoparticles in which K3-type CpG was encapsulated in the LNP, and the resulting nanoparticle dispersion was dialyzed and subjected to medium exchange. The resulting nanoparticles were then concentrated by ultrafiltration to obtain 300 μL of purified LNP-K3 dispersion (medium: buffer solution consisting of 10 mM histidine + 300 mM sucrose). The K3 concentration in the LNP-K3 dispersion was measured by fluorometry in the same manner as in WO 2015-005253 using a Quant-iT Oligreen ssDNA Reagent Kit (Thermo Fischer Scientific) for DNA detection. As a result, it was confirmed that the K3 concentration in the K3-LNP dispersion was 1.164 mg / mL and the K3 recovery rate was 70%. The particle size of LNP-K3 measured in the same manner as in other examples was 143.5 nm in terms of the average scattered light intensity.

[0109] In the examples, the mice used were C57BL / 6JJcl (supplied by CLEA Japan, age at time of arrival: 7 or 14 weeks, sex: female, age at start of administration: 8 or 16 weeks), BALB / cAnNCrlCrlj (supplied by Jackson Laboratory Japan, age at time of arrival: 6 weeks, sex: female, age at start of administration: 6 weeks), and BALB / cAjcl (supplied by CLEA Japan, age at time of arrival: 6 weeks, sex: female, age at start of administration: 7 weeks).

[0110] The mice were kept on FR-2 feed and chlorinated tap water ad libitum, at a temperature of 23±2° C., humidity of 55±20%, and with lights on for 12 hours.

[0111] Approximately 75 μL of blood was collected from the tail vein of the mice (hematocrit capillary tube, Heparin 501 Red, Sansho, Cat. No. 83-0546), added to 0.5 mL of PBS, and then allowed to stand on ice to prepare blood samples.

[0112] Animal Ethics This study was conducted with the approval of the Animal Experimentation Committee of Daiichi Sankyo Co., Ltd. All animal care, handling, and experimental procedures were carried out in accordance with the relevant internal regulations of Daiichi Sankyo Co., Ltd.

[0113] Antigen-specific CTLs in Examples 1 to 9 and 11 were measured as follows. Blood samples were centrifuged (800 g, 3 minutes), and 0.45 mL of the supernatant was removed. 1 mL of BD Pharm Lyse (Cat. No. 555899) diluted 1 / 10 with distilled water for injection (Otsuka) was added, and after centrifugation, the supernatant was removed. 1 mL of 1 / 10 BD Pharm Lyse was added again, and after centrifugation, the supernatant was removed. 1 mL of FACS Buffer (PBS, 1 mM EDTA, 0.1% BSA) was added, and after centrifugation, the supernatant was removed. 1 mL of PBS (without BSA) was added, and after centrifugation, the supernatant was removed. After adding 50 μL of PBS containing 0.05 μL of LIVE / DEAD Fixable Near-IR Dead Cell Stain Kit (Invitrogen, Cat. No. L34975), the cells were transferred to a 96-well V-Bottom Plate and left on ice for 10 minutes in the dark. After adding 150 μL of PBS and centrifuging (400 g, 5 minutes), the supernatant was removed. After adding 200 μL of FACS Buffer, the cells were centrifuged and the supernatant was removed. 25 μL of FACS buffer containing 0.25 μL of Mouse Fc Block (BD, Cat. No. 553142) was added, and the mixture was incubated at 4°C for 5 minutes in the dark. 25 μL of FACS buffer containing tetramer was added, and the mixture was incubated at 4°C for 30 minutes in the dark. 50 μL of FACS buffer containing anti-mouse CD45, anti-mouse CD3, and anti-mouse CD8 was added, and the mixture was incubated at 4°C for 20 minutes in the dark. After centrifugation, the supernatant was removed, and 200 μL of FACS buffer was added. For the analysis, an LSRFortessa X-20 or FACSVerse (BD Biosciences) was used.

[0114] The antibodies and tetramers used in the CTL assay are shown below.

[0115] Example 1: Effect of various adjuvants on CTL induction by LNP-mRNA vaccine LNP(1)-mRNA HPV16 E6_E7(pU) was prepared according to Patent Document 1. pU indicates that the mRNA contains pseudouridine as a modified nucleotide. (1) Preparation of mRNA-encapsulated nucleic acid lipid particles Distearoylphosphatidylcholine (1,2-distearoyl-sn-glycero-3-phosphocholine: hereinafter referred to as DSPC, NOF Cholesterol (Sigma-Aldrich, Inc.), (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-diene-7,29-diyl diacetate (LP), and 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol (PEG-DMG, NOF), each having a molecular weight of approximately 2,000. CHO:LP:PEG-DMG (DIMETHICONE CORPORATION) was dissolved in ethanol at a molar ratio of DSPC:Chol:LP:PEG-DMG = 12.5:41:45:1.5 to a total lipid concentration of 5 mM. HPV16 E6_E7 mRNA was prepared by the method described in Patent Document 1.

[0116] The lipid solution and mRNA solution were mixed in a microchannel using a NanoAssembler BenchTop (Precision Nanosystems Inc.) at a volume ratio of 1:3 to obtain a crude dispersion of nucleic acid-lipid particles. The nucleic acid-lipid particle dispersion was dialyzed (Float-A-Lyzer G2, MWCO: 1,000 kD, Spectra / Por) against approximately 25-50 times the volume of phosphate buffer (pH 7.4) for 12-18 hours to remove ethanol, yielding a dispersion of purified LNP(1)-mRNA HPV16 E6_E7(pU).

[0117] (2) Characterization of LNP-mRNA The dispersion containing LNP(1)-mRNA HPV16 E6_E7(pU) prepared in (1) was characterized. The methods for each characterization are described below.

[0118] (2-1) mRNA Encapsulation Rate The mRNA encapsulation rate was measured using the Quant-iT RiboGreen RNA Assay kit (Invitrogen) according to the package insert. Specifically, the amount of mRNA in the dispersion of LNP(1)-mRNA HPV16 E6_E7(pU) was quantified in the presence and absence of 0.015% Triton X-100 surfactant, and the encapsulation rate was calculated using the following formula: The mRNA encapsulation rate was 97%. {[Amount of mRNA in the presence of surfactant] - [Amount of mRNA in the absence of surfactant]} / [Amount of mRNA in the presence of surfactant]} x 100(%)

[0119] (2-2) Ratio of mRNA to Lipid The amount of mRNA in the nucleic acid-lipid particle dispersion was measured by reverse phase chromatography (System: Agilent 1100 series, Column: Bioshell A400 Protein C4 (10 cm × 4.6 mm, 3.4 μm) (SUPELCO), Buffer A: 0.1 M triethylamine acetate (pH 7.0), Buffer B: acetonitrile, (B%): 5-50% (0-15 min), Flow Rate: 1 mL / min, Temperature: 70°C, Detection: 260 nm). The amount of each lipid in the nucleic acid-lipid particle dispersion was measured by reverse phase chromatography (System: DIONEX UltiMate 3000, Column: XSelect CSH (50 mm × 3 mm, 5 μm) (Thermo Fisher Scientific), Buffer A: 0.2% formic acid, Buffer B: 0.2% formic acid, methanol, (B%): 75-95% (0-15 min), 95% (15-17 min), Flow Rate: 0.4 mL / min, Temperature: 50 ° C, Detection: Corona CAD (Charged Aerosol Detector)). The ratio of the total lipid amount to mRNA was calculated using the following formula. [Total lipid concentration] / [mRNA concentration] (wt / wt)

[0120] (2-3) Average particle size The particle size of the nucleic acid-lipid particles was measured using a Zeta Potential / Particle Sizer NICOMP™ 380ZLS (PARTICLE SIZING SYSTEMS). The average particle size represents the scattered light intensity average particle size, and was found to be 119.5 nm.

[0121] Various vaccine-adjuvant mixtures were prepared by mixing LNP(1)-mRNA HPV16 E6_E7(pU) (dose: 1 μg / mouse) and various adjuvants (doses / mouse shown in Figure 1 ) in 10 mM histidine buffer containing 300 mM sucrose so that a predetermined dose would be achieved with administration of 20 μL per mouse, and adjusting the pH to 7.0.

[0122] (3) Results Figure 1 shows the percentage of HPV16 E7 MHC Class I epitope-specific CTLs (%) among CD8-positive T cells in mouse blood. Mice were anesthetized with isoflurane by inhalation and 20 μL of either the vehicle or the various vaccine-adjuvant mixtures prepared as described above were administered intramuscularly into the gastrocnemius muscle three times at weekly intervals: week 0 (the day of administration), week 1 (7±1 days after the first administration), and week 2 (7±1 days after the week 1 administration). Data for the percentage of HPV16 E7 MHC Class I epitope-specific CTLs (%) among CD8-positive T cells in mouse blood at week 3 (1 week after the final administration) are shown. Open circles represent data from individual mice, and open bars represent the mean. Each group consisted of four mice.

[0123] As shown in Figure 1, the group administered LNP(1)-mRNA HPV16 E6_E7(pU) alone (second from the left) showed induction of E7 MHC Class I epitope-specific CTLs. The group administered K3-LNT intramuscularly together with LNP(1)-mRNA HPV16 E6_E7(pU) (third from the left) showed a tendency toward enhanced induction of E7 MHC Class I epitope-specific CTLs compared to the group administered LNP(1)-mRNA HPV16 E6_E7(pU) alone. On the other hand, in the group administered intramuscularly with LNP(1)-mRNA HPV16 E6_E7(pU), IMO-2125 (TLR9 ​​agonist), Poly(I:C) (TLR3 agonist), AS01B (MPLA [TLR4 agonist] and QS-21 [saponin]), R848 (TLR7 / 8 agonist), Alum (Alhydrogel or Adjuphos), Addavax (Squalene), and ADU-S100 (STING ligand), which are widely known adjuvants for recombinant proteins, no tendency toward enhanced induction of E7 MHC Class I epitope-specific CTL was observed compared to the group administered LNP(1)-mRNA HPV16 E6_E7(pU) alone (Figure 1).

[0124] These results demonstrate that adjuvants used in conventional vaccines using recombinant protein antigens cannot be used as adjuvants for mRNA vaccines, and that they induce CTLs at levels equivalent to or lower than those induced when no adjuvant is used (second from the left). Therefore, it was found that adjuvants used in conventional vaccines using recombinant protein antigens do not fully exert their immunostimulatory effects as adjuvants for mRNA vaccines. On the other hand, when the adjuvant of the present invention, K3-LNT, was used (third from the left), the induction of antigen-specific CTLs by LNP-mRNA was significantly enhanced.

[0125] Example 2 As in Example 1, various vaccine-adjuvant mixtures were prepared by mixing LNP(1)-mRNA HPV16 E6_E7(pU) (dose: 1 μg / mouse) and various adjuvants (K3, K3 dA40, K3 analog, ODN 1018, ODN 2006, ODN 2216, D35, ODN 2395, or IMO-2125; dose: 0.1 or 1 μg / mouse) in 10 mM histidine buffer containing 300 mM sucrose so that a predetermined dose would be achieved with an administration of 20 μL per mouse, and adjusting the pH to 7.0. 20 μL of either the vehicle or each of the vaccine-adjuvant mixtures prepared as described above was administered into the gastrocnemius muscle of mice anesthetized with isoflurane by inhalation three times in weeks 0, 1, and 2. Figure 2 shows data on HPV16 E7 MHC Class I epitope-specific CTLs (%) among CD8-positive T cells in mouse blood at week 3 (one week after the final administration). Open circles represent data from individual mice, and open bars represent the average. Each group consisted of four mice.

[0126] As shown in Figure 2, intramuscular administration of LNP(1)-mRNA HPV16 E6_E7(pU) induced E7 MHC Class I epitope-specific CTL. ４０In the group receiving intramuscular administration of LNP(1)-mRNA HPV16 E6_E7(pU) with K3 analog (modified type-B CpG ODN), ODN 1018 (type-B CpG ODN), or ODN 2006 (type-B CpG ODN), a tendency toward enhanced induction of E7 MHC Class I epitope-specific CTL was observed compared to the group receiving LNP(1)-mRNA HPV16 E6_E7(pU) alone. On the other hand, in the group receiving intramuscular administration of ODN 2216 (type A CpG ODN), D35 (type A CpG ODN), ODN 2395 (type C CpG ODN), or IMO-2125 (next-generation CpG ODN) simultaneously with LNP(1)-mRNA HPV16 E6_E7(pU), the tendency for enhancement of E7 MHC Class I epitope-specific CTL induction was weaker than in the group receiving LNP(1)-mRNA HPV16 E6_E7(pU) alone (Figure 2). These results indicate that the B-type CpG ODNs K3 CpG ODN, K3 analog, ODN 1018, and ODN 2006, as well as the modified B-type CpG ODN K3-dA, are suitable adjuvants for mRNA vaccines. ４０ It was shown that, compared with type A, type C, and next-generation CpG ODN, it more significantly enhanced the induction of antigen-specific CTLs by mRNA vaccines.

[0127] Example 3 As in Example 1, LNP(1)-mRNA HPV16 E6_E7(pU) (dose: 1 μg / mouse) and various adjuvants (K3, K3-LNT, K3 GpC (a negative control in which the cytosine and guanine of K3 are swapped), ODN 1018, ODN 2006, ODN 2216, D35, ODN 2395, or IMO-2125; dose: 1, 3, or 10 μg / mouse) were mixed in 10 mM histidine buffer containing 300 mM sucrose so that a predetermined dose would be achieved with an administration of 20 μL per mouse, and the mixture was adjusted to pH 7.0 to prepare various vaccine-adjuvant mixtures. 20 μL of either the vehicle or the vaccine-adjuvant mixture prepared as described above was administered into the gastrocnemius muscle of mice anesthetized with isoflurane by inhalation three times in weeks 0, 1, and 2. Figure 3 shows data on HPV16 E7 MHC Class I epitope-specific CTLs (%) among CD8-positive T cells in mouse blood at week 3 (one week after the final administration). Open circles represent data from individual mice, and open bars represent the average. Each group consisted of four mice.

[0128] As shown in Figure 3, intramuscular administration of LNP(1)-mRNA HPV16 E6_E7(pU) induced E7 MHC Class I epitope-specific CTLs. In the group receiving intramuscular administration of K3 (type B CpG ODN) or K3-LNT (type B CpG ODN complex) simultaneously with LNP(1)-mRNA HPV16 E6_E7(pU), a trend toward enhanced E7 MHC Class I epitope-specific CTL induction was observed compared with the group receiving LNP(1)-mRNA HPV16 E6_E7(pU) alone. For K3, an inverse correlation was observed between the dose and the induction of E7 MHC Class I epitope-specific CTLs. With K3, E7 MHC Class I epitope-specific CTLs were induced at doses lower than those used in Example 3 (Example 2), suggesting that the suppressive effect of K3 may be due to excessive administration within the dose range of Example 3. Furthermore, in the group administered ODN 1018 (type B CpG ODN) or ODN 2006 (type B CpG ODN) intramuscularly simultaneously with LNP(1)-mRNA HPV16 E6_E7(pU), a weak tendency toward enhanced induction of E7 MHC Class I epitope-specific CTLs was observed compared to the group administered LNP(1)-mRNA HPV16 E6_E7(pU) alone. It is possible that the enhancement of CTL induction is maximized with ODN 1018 and ODN 2006 at lower doses (less than 1 μg). On the other hand, in the group in which K3 GpC (negative control for K3 CpG ODN), ODN 2216 (A-type CpG ODN), D35 (A-type CpG ODN), ODN 2395 (C-type CpG ODN), or IMO-2125 (next-generation CpG ODN) was administered intramuscularly simultaneously with LNP(1)-mRNA HPV16 E6_E7(pU), no trend toward enhanced induction of E7 MHC Class I epitope-specific CTL was observed compared to the group administered LNP(1)-mRNA HPV16 E6_E7(pU) alone (Figure 3).

[0129] Example 4 As in Example 1, LNP(1)-mRNA HPV16 E6_E7(pU) (dose: 1 μg / mouse) and various adjuvants (K3-LNT or K3-SPG, dose: 10 μg / mouse) were mixed in 10 mM histidine buffer containing 300 mM sucrose to give a dose of 20 μL per mouse, and the pH was adjusted to 7.0 to prepare various vaccine-adjuvant mixtures. 20 μL each of the vehicle or the various vaccine-adjuvant mixtures prepared as described above was administered into the gastrocnemius muscle of mice anesthetized with isoflurane by inhalation three times at weeks 0, 1, and 2. Figure 4 shows data on HPV16 E7 MHC Class I epitope-specific CTL (%) among CD8-positive T cells in mouse blood at week 3 (one week after the final administration). Open circles represent data from individual mice, and open bars represent the average. Each group consisted of four mice.

[0130] As shown in Figure 4, intramuscular administration of LNP(1)-mRNA HPV16 E6_E7(pU) resulted in the induction of E7 MHC Class I epitope-specific CTLs. In the group receiving intramuscular administration of K3-LNT (a complex of type B CpG ODN) or K3-SPG (a complex of type B CpG ODN) simultaneously with LNP(1)-mRNA HPV16 E6_E7(pU), a trend toward enhanced E7 MHC Class I epitope-specific CTL induction was observed compared to the group receiving LNP(1)-mRNA HPV16 E6_E7(pU) alone (Figure 4). These results demonstrate that, like K3-LNT, K3-SPG enhances the induction of antigen-specific CTLs induced by LNP-mRNA.

[0131] Example 5 As in Example 1, LNP(1)-mRNA HPV16 E6_E7(pU) (dose: 1 μg / mouse) and various adjuvants (K3 or LNP-K3 prepared as described above, dose as K3: 10 μg / mouse) were mixed in 10 mM histidine buffer containing 300 mM sucrose to give a predetermined dose of 20 μL per mouse, and the pH was adjusted to 7.0 to prepare various vaccine-adjuvant mixtures. 20 μL each of vehicle or the various vaccine-adjuvant mixtures prepared as described above was administered into the gastrocnemius muscle of mice anesthetized with isoflurane by inhalation three times at weeks 0, 1, and 2. Figure 5 shows data on HPV16 E7 MHC Class I epitope-specific CTL (%) among CD8-positive T cells in mouse blood at week 3 (one week after the final administration). Open circles represent data from individual mice, and open bars represent the average. Each group consisted of four mice.

[0132] As shown in Figure 5, compared to the group administered with LNP(1)-mRNA HPV16 E6_E7(pU) alone, the group administered with K3 intramuscularly simultaneously with LNP(1)-mRNA HPV16 E6_E7(pU) showed a tendency toward enhanced induction of E7 MHC Class I epitope-specific CTL. On the other hand, compared to the group administered with LNP(1)-mRNA HPV16 E6_E7(pU) alone, the group administered with LNP-K3 (K3 encapsulated in LNP) simultaneously with LNP(1)-mRNA HPV16 E6_E7(pU) showed no tendency toward enhanced induction of E7 MHC Class I epitope-specific CTL (Figure 5). These results demonstrate that not including K3 in LNPs is important for enhancing the induction of antigen-specific CTLs by LNP-mRNA.

[0133] Example 6 As in Example 1, LNP(1)-mRNA HPV16 E6_E7(pU) (dose: 1 μg / mouse) and various adjuvants (K3-LNT (complex of type B CpG ODN), dose: 10 μg / mouse) were mixed in 10 mM histidine buffer containing 300 mM sucrose so that a predetermined dose was achieved with an administration of 20 μL per mouse, and the pH was adjusted to 7.0 to prepare a vaccine-adjuvant mixture. 20 μL each of the vehicle or vaccine-adjuvant mixture was administered intramuscularly (i.m.) or intradermally (i.d.) into the thigh muscle of mice anesthetized with isoflurane by inhalation three times at weeks 0, 1, and 2. Figure 6 shows data on HPV16 E7 MHC Class I epitope-specific CTL (%) among CD8-positive T cells in mouse blood at week 3 (one week after the final administration). Open circles represent data from individual mice, and open bars represent the average. Each group consisted of four mice.

[0134] As shown in Figure 6, intradermal administration of LNP(1)-mRNA HPV16 E6_E7(pU) induced higher levels of E7 MHC Class I epitope-specific CTL induction compared to intramuscular administration. In the group in which K3-LNT was administered intradermally simultaneously with LNP(1)-mRNA HPV16 E6_E7(pU), even higher levels of E7 MHC Class I epitope-specific CTL induction were observed compared to the group in which LNP(1)-mRNA HPV16 E6_E7(pU) was administered intradermally alone (Figure 6). These results demonstrate that intradermal administration of K3-LNT enhances the antigen-specific CTL induction induced by LNP-mRNA to a higher level than intramuscular administration.

[0135] Example 7 LNP(1)-mRNA HPV16 E6_E7(pU) similar to that in Example 1-6, and mRNA HPV16 E6_E7(5MeC, 5MeU) different from that in Example 1-6 were prepared by in vitro transcription using 5-methylcytidine and 5-methyluridine as substrates. An mRNA solution containing this was mixed with a lipid solution having the lipid composition of LNP(1), to prepare LNP(1)-mRNA HPV16 E6_E7(5MeC, 5MeU).

[0136] As in Example 1, LNP-mRNA (LNP(1)-mRNA HPV16 E6_E7(pU) or LNP(1)-mRNA HPV16 E6_E7(5MeC, 5MeU)) (dose: 1 μg / mouse) and various adjuvants (K3-LNT (complex of B-type CpG ODN), dose: 10 μg / mouse) were mixed in 10 mM histidine buffer containing 300 mM sucrose so that a predetermined dose was achieved with an administration of 20 μL per mouse, and the pH was adjusted to 7.0 to prepare various vaccine-adjuvant mixtures. 20 μL each of the vehicle or the various vaccine-adjuvant mixtures prepared as described above was administered into the gastrocnemius muscle of mice anesthetized with isoflurane by inhalation three times at weeks 0, 1, and 2. 7 shows data on HPV16 E7 MHC Class I epitope-specific CTL (%) among CD8-positive T cells in mouse blood at week 3 (one week after the final administration). Open circles represent data from individual mice, and open bars represent the average. Each group consisted of four mice.

[0137] As shown in Figure 7, intramuscular administration of LNP(1)-mRNA HPV16 E6_E7(pU) resulted in the induction of E7 MHC Class I epitope-specific CTLs. Furthermore, in the group receiving intramuscular administration of K3-LNT and LNP(1)-mRNA HPV16 E6_E7(pU) simultaneously, a trend toward enhanced induction of E7 MHC Class I epitope-specific CTLs was observed compared to the group receiving LNP(1)-mRNA HPV16 E6_E7(pU) alone. Next, intramuscular administration of LNP(1)-mRNA HPV16 E6_E7(5MeC, 5MeU) resulted in the induction of E7 MHC Class I epitope-specific CTLs. Furthermore, in the group where K3-LNT was administered intramuscularly simultaneously with LNP(1)-mRNA HPV16 E6_E7 (5MeC, 5MeU), a tendency toward enhanced induction of E7 MHC Class I epitope-specific CTLs was observed compared to the group administered LNP(1)-mRNA HPV16 E6_E7 alone (Figure 7). These results demonstrate that K3-LNT enhances antigen-specific CTL induction by mRNA vaccines, regardless of whether the modified nucleotides constituting the mRNA are pseudouridine or 5-methylcytidine and 5-methyluridine.

[0138] Example 8 An mRNA solution containing HPV16 E6_E7 (5MeC, 5MeU) was mixed with lipid solutions having three different lipid compositions containing different ionized lipids to prepare three types of LNP-mRNA. Specifically, the lipid composition was ionizable lipid:DSPC:cholesterol:DMG-PEG200 in a molar ratio (%) of 45:12.5:41:1.5, and the ionizable lipid was (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-diene-7,29-diyl diacetate, LNP(1)-mRNA HPV16 E6_E7(5MeC, 5MeU), the ionizable lipid was SM-102, and LNP(1,ALC-0315)-mRNA HPV16 E6_E7(5MeC, 5MeU), the ionizable lipid was ALC-0315. E6_E7 (5MeC, 5MeU) were prepared by the method described in Example 1.

[0139] Similarly to Example 1, LNP-mRNA (LNP(1)-mRNA HPV16 E6_E7(5MeC, 5MeU), LNP(1, SM-102)-mRNA HPV16 E6_E7(5MeC, 5MeU), or LNP(1, ALC-0315)-mRNA HPV16 E6_E7(5MeC, 5MeU), dose: 1 μg / mouse) and various adjuvants (K3, dose: 1 μg / mouse) were mixed in 10 mM histidine buffer containing 300 mM sucrose to give a dose of 20 μL per mouse, and the pH was adjusted to 7.0 to prepare a vaccine-adjuvant mixture. 20 μL of either the vehicle or each of the vaccine-adjuvant mixtures prepared as described above was administered three times in weeks 0, 1, and 2 into the gastrocnemius muscle of mice anesthetized with isoflurane by inhalation. Figure 8 shows data on HPV16 E7 MHC Class I epitope-specific CTL (%) among CD8-positive T cells in mouse blood at week 3 (one week after the final administration). Open circles represent data from individual mice, and open bars represent the average. Each group consisted of four mice.

[0140] As shown in Figure 8, intramuscular administration of LNP(1)-mRNA HPV16 E6_E7 (5MeC, 5MeU) resulted in the induction of E7 MHC Class I epitope-specific CTLs. Furthermore, in the group in which K3 was administered intramuscularly simultaneously with LNP(1)-mRNA HPV16 E6_E7 (5MeC, 5MeU), a tendency toward enhanced induction of E7 MHC Class I epitope-specific CTLs was observed compared to the group administered LNP(1)-mRNA HPV16 E6_E7 (5MeC, 5MeU) alone. Here, when LNP(1, SM-102)-mRNA HPV16 E6_E7(5MeC, 5MeU) or LNP(1, ALC-0315)-mRNA HPV16 E6_E7(5MeC, 5MeU), in which the ionized lipid constituting the LNP was SM-102 or ALC-0315, was administered intramuscularly, both LNP-mRNAs were found to induce E7 MHC Class I epitope-specific CTLs. Furthermore, in the group where K3 was administered intramuscularly simultaneously with LNP(1, SM-102)-mRNA HPV16 E6_E7(5MeC, 5MeU) or LNP(1, ALC-0315)-mRNA HPV16 E6_E7(5MeC, 5MeU), a tendency toward enhanced induction of E7 MHC Class I epitope-specific CTL was observed compared to the group administered with either LNP-mRNA alone (Figure 8). These results demonstrate that K3 CpG ODN enhances the induction of antigen-specific CTL by LNP-mRNA, regardless of the type of ionized lipid constituting the LNP, suggesting that the immune response-enhancing effect of K3 CpG ODN as an adjuvant is exerted regardless of the lipid constituting the LNP.

[0141] Example 9 An mRNA solution containing HPV16 E6_E7 (5MeC, 5MeU) was mixed with two types of lipid solutions with different lipid compositions to prepare two types of LNP-mRNA. Specifically, LNP(1)-mRNA HPV16 E6_E7(5MeC, 5MeU) in which the ionized lipid was (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-diene-7,29-diyl diacetate and the lipid composition was ionized lipid:DSPC:cholesterol:DMG-PEG200 in a molar ratio (%) of 45:12.5:41:1.5, and LNP(2)-mRNA HPV16 E6_E7(5MeC, 5MeU) in which the ionized lipid was the same and the lipid composition was ionized lipid:DSPC:cholesterol:DMG-PEG200 in a molar ratio (%) of 55:8.5:35:1.5 were prepared by the method described in Example 1.

[0142] As in Example 1, LNP-mRNA (LNP(1)-mRNA HPV16 E6_E7 (5MeC, 5MeU) or LNP(2)-mRNA HPV16 E6_E7 (5MeC, 5MeU), dose: 1 μg / mouse) and various adjuvants (K3-LNT or K3, dose: 10 μg / mouse) were mixed in 10 mM histidine buffer containing 300 mM sucrose to give a dose of 20 μL per mouse, and the pH was adjusted to 7.0 to prepare various vaccine-adjuvant mixtures. 20 μL each of vehicle or the vaccine-adjuvant mixtures prepared as described above was administered into the gastrocnemius muscle of mice anesthetized with isoflurane by inhalation three times at weeks 0, 1, and 2. 9 shows data on HPV16 E7 MHC Class I epitope-specific CTL (%) among CD8-positive T cells in mouse blood at week 3 (one week after the final administration). Open circles represent data from individual mice, and open bars represent the average. Each group consisted of four mice.

[0143] As shown in Figure 9, intramuscular administration of LNP(1)-mRNA HPV16 E6_E7 (5MeC, 5MeU) resulted in the induction of E7 MHC Class I epitope-specific CTLs. Furthermore, in the group in which K3-LNT or K3 was administered intramuscularly simultaneously with LNP(1)-mRNA HPV16 E6_E7 (5MeC, 5MeU), a tendency toward enhanced induction of E7 MHC Class I epitope-specific CTLs was observed compared to the group administered LNP(1)-mRNA HPV16 E6_E7 (5MeC, 5MeU) alone. On the other hand, intramuscular administration of LNP(2)-mRNA HPV16 E6_E7 (5MeC, 5MeU) containing different lipid ratios in the LNP resulted in the induction of E7 MHC Class I epitope-specific CTLs. Furthermore, in the group in which K3-LNT or K3 was administered intramuscularly simultaneously with LNP(2)-mRNA HPV16 E6_E7 (5MeC, 5MeU), a tendency toward enhanced E7 MHC Class I epitope-specific CTL induction was observed compared to the group administered LNP(2)-mRNA HPV16 E6_E7 (5MeC, 5MeU) alone (Figure 9). These results demonstrate that K3-LNT and K3 enhance antigen-specific CTL induction by LNP-mRNA, regardless of the lipid ratio constituting the LNP.

[0144] Example 10 LNP(1)-mRNA gp70 AH1-A5(5MeC, 5MeU) was prepared by in vitro transcription using 5-methylcytidine and 5-methyluridine as substrates, and an mRNA solution containing mRNA gp70 AH1-A5(5MeC, 5MeU) encoding gp70 (gp70 AH1-A5) containing an IgM signal and the AH1-A5 sequence was mixed with a lipid solution having the lipid composition of LNP(1) to prepare LNP(1)-mRNA gp70 AH1-A5(5MeC, 5MeU).

[0145] As in Example 1, LNP-mRNA (LNP(1)-mRNA gp70 AH1-A5 (5MeC, 5MeU, dose: 1.5 μg / mouse)) and various adjuvants (K3-LNT, dose: 10 μg / mouse) were mixed in 10 mM histidine buffer containing 300 mM sucrose so that a predetermined dose would be achieved with an administration of 20 μL per mouse, and the mixture was adjusted to pH 7.0 to prepare various vaccine-adjuvant mixtures. 20 μL each of the vehicle or the vaccine-adjuvant mixtures prepared as described above was administered to the base of the tail of mice anesthetized with isoflurane by inhalation a total of three times at weeks 0, 1, and 2. 10 shows data on gp70 AH1 MHC Class I epitope-specific CTL (%) among CD8-positive T cells in mouse blood at week 4 (2 weeks after the final administration). Open circles represent data from individual mice, and open bars represent the average. Each group consisted of 8 mice.

[0146] As shown in Figure 10, when LNP(1)-mRNA gp70 AH1-A5 (5MeC, 5MeU) was administered to the base of the tail, AH1-specific CTL induction was observed. In the group in which K3-LNT was administered to the base of the tail simultaneously with LNP(1)-mRNA gp70 AH1-A5 (5MeC, 5MeU), a tendency toward enhanced AH1-specific CTL induction was observed compared to the group administered LNP(1)-mRNA gp70 AH1-A5 (5MeC, 5MeU) alone (Figure 10). These results demonstrate that K3-LNT enhances CTL induction by LNP-mRNA, regardless of the antigen encoded by the mRNA.

[0147] Example 11 After CT26 tumor cells were transplanted into mice, the tumor volume of each individual or the survival rate of each group was examined.

[0148] First, as in Example 10, LNP-mRNA (LNP(1)-mRNA gp70 AH1-A5 (5MeC, 5MeU, dose: 1.5 μg / mouse)) and various adjuvants (K3-LNT, dose: 10 μg / mouse) were mixed in 10 mM histidine buffer containing 300 mM sucrose so that a predetermined dose would be achieved with an administration of 20 μL per mouse, and the pH was adjusted to 7.0 to prepare an adjuvant solution, a vaccine solution, and a vaccine-adjuvant mixture. 20 μL each of the vehicle adjuvant solution, the vaccine solution, and the vaccine-adjuvant mixture was administered to the base of the tail of each mouse four times in total, at weeks 0, 1, 2, and 4.

[0149] Next, one day after the final administration, CT26 tumor cells were subcutaneously implanted. CT26.WT (ATCC, Cat. No. CRL-2638) cells were used as CT26 tumor cells and incubated at 37°C, 5% CO ２ The cells were cultured in RPMI-1640 (10% FBS, 100 Units / mL Penicillin, 0.1 mg / mL Streptomycin) in an incubator set at 4°C. To detach the cells, they were treated with 0.25% Trypsin-EDTA diluted 4-fold with PBS for approximately 2-3 minutes, and then collected and used in experiments or passaged. For transplantation of CT26 tumor cells, hair was shaved under isoflurane anesthesia the day before cell transplantation. CT26 tumor cells were used at a concentration of 5 x 10 ６ The cells were prepared in RPMI-1640 (without FBS or penicillin streptomycin) to a concentration of 5 × 10 cells / mL. ５ CT26 tumor cells were implanted subcutaneously into the left flank at a rate of 100 μL / cell.

[0150] For tumor volume measurement and humane endpoints, the major and minor diameters (mm) of tumors were measured using calipers three times a week from day 6 to day 50 after tumor cell inoculation. Tumor volume was calculated as "minor diameter × minor diameter × major diameter / 2 (mm)." ３ Mice with tumors exceeding 15 mm in average long and short diameter were given a humane endpoint.

[0151] Figures 11A-D show the change in tumor volume in each individual after CT26 tumor cell transplantation, indicated by broken lines. Figure 11E shows the survival rate for each group. Eight animals were used per group. As shown in Figures 11A and 11B, when CT26 tumor cells were transplanted into the vehicle-administered group or the K3-LNT-alone-administered group, tumor growth was observed in all animals (8 animals), and all animals reached the humane endpoint 15 days (vehicle-administered group) or 18 days (K3-LNT-alone-administered group) after CT26 tumor cell transplantation. On the other hand, as shown in Figure 11C, tumor growth was delayed in the LNP(1)-mRNA gp70 AH1-A5 (5MeC, 5MeU)-alone-administered group. While tumor growth was observed in five of the eight animals, tumor formation was not observed in three animals up to the 50-day observation period after CT26 tumor cell transplantation. The survival rate 50 days after CT26 tumor inoculation was 50% (4 of 8 mice survived). Furthermore, as shown in Figure 11D, the group administered K3-LNT simultaneously with LNP(1)-mRNA gp70 AH1-A5 (5MeC, 5MeU) showed a tendency toward prolonged delay in tumor growth, and tumor formation was not observed in 4 of 8 mice up to 50 days after CT26 tumor cell inoculation, the observation period. As shown in Figure 11E, the survival rate 50 days after CT26 tumor inoculation was 75% (6 of 8 mice survived). These results demonstrated that K3-LNT can be used as an adjuvant for LNP-mRNA vaccines that utilize cancer-associated antigen-specific CTLs as effectors.

[0152] Example 12 In addition to the LNP(1)-mRNA gp70 AH1-A5(5MeC, 5MeU) similar to that of Example 10, LNP(1)-mRNA AH1-A5(5MeC, 5MeU) was also prepared. The latter LNP(1)-mRNA AH1-A5(5MeC, 5MeU) was obtained by mixing an mRNA solution containing mRNA AH1-A5(5MeC, 5MeU) with a lipid solution having the lipid composition of LNP(1). Furthermore, the mRNA AH1-A5(5MeC, 5MeU) contained in the mRNA solution is mRNA encoding AH1-A5, and is mRNA prepared by in vitro transcription using 5-methylcytidine and 5-methyluridine as substrates. AH1-A5 is a region known as the CD8 epitope of gp70 AH1-A5. In terms of the base length of mRNA, gp70 AH1-A5 is 121 bases long, while AH1-A5 is 9 bases long.

[0153] As in Example 1, LNP-mRNA (LNP(1)-mRNA AH1-A5 (5MeC, 5MeU), or LNP(1)-mRNA gp70 AH1-A5 (5MeC, 5MeU), dose: 2 μg / mouse) and adjuvant (K3, dose: 3 μg / mouse) were mixed in 10 mM histidine buffer containing 300 mM sucrose so that a 20 μL administration per mouse would yield the prescribed dose, and the mixture was adjusted to pH 7.0 to prepare a vaccine-adjuvant mixture. 20 μL each of vehicle or each of the vaccine-adjuvant mixtures prepared as described above was administered to the base of the tail of mice anesthetized with isoflurane by inhalation three times at weeks 0, 1, and 2. Figure 12 shows data on gp70 AH1 MHC Class I epitope-specific CTL (%) among CD8-positive T cells in mouse blood at week 3 (one week after the final administration). Open circles represent data from individual mice, and open bars represent the average. Groups consisted of four mice (vehicle administration group) or six mice (LNP(1)-mRNA AH1-A5 (5MeC, 5MeU) administration group or LNP(1)-mRNA gp70 AH1-A5 (5MeC, 5MeU) administration group).

[0154] As shown in Figure 12, when LNP(1)-mRNA AH1-A5 (5MeC, 5MeU) encoding only AH1-A5 or LNP(1)-mRNA gp70 AH1-A5 (5MeC, 5MeU) encoding gp70 AH1-A5 were administered to the base of the tail, both LNP-mRNAs induced AH1-specific CTLs. In the group administered K3 simultaneously with LNP(1)-mRNA AH1-A5 (5MeC, 5MeU) or LNP(1)-mRNA gp70 AH1-A5 (5MeC, 5MeU) to the base of the tail, a tendency toward enhanced AH1-specific CTL induction was observed compared to the group administered each LNP-mRNA alone (Figure 12). These results suggest that K3 may enhance CTL induction by LNP-mRNA, even when the mRNA encodes only the CD8 epitope sequence. The use of mRNA encoding only the CD8 epitope sequence as a vaccine may reduce vaccine production costs, facilitate production, enhance efficacy per unit mRNA amount, and potentially produce a safer vaccine. Furthermore, the particle size may be reduced when encapsulated in LNP, allowing for a smaller vaccine administration volume.

[0155] The sample name, modified nucleotides, lipid ratio, mRNA encapsulation rate (%), and particle size of the LNP-mRNA used in the above examples are summarized below. The mRNA encapsulation rate (%) and "particle size" were measured by the methods described in "(2-1) mRNA encapsulation rate" and "(2-3) Average particle size" in Example 1.

[0156]

[0157]

[0158] Example 13: Effect of lipid-modified B-type CpG ODN on CTL induction by LNP-mRNA vaccine. １６The effect of alkyl-linked lipid-modified products (Lipid-K3(1) and Lipid-K3(2)) on the induction of CTLs specific to the antigen encoded by the mRNA vaccine was evaluated. (1) Preparation of Lipid-K3(1) and Lipid-K3(2) The structures of the oligonucleotides Lipid-K3(1) and Lipid-K3(2) are shown in Table 7. Each oligonucleotide was synthesized using the phosphoramidite method (Nucleic Acids Research, 12, 4539 (1984), Nature Communications 6, Article number: 6317 (2015)). Oligonucleotides containing an "X" or "Xo" moiety were synthesized using C-18 phosphoramidite as described in WO 98 / 18480. The "molecular weight" in the table indicates the value actually measured by negative ion ESI mass spectrometry.

[0159]

[0160] In the sequences in the table, a, g, c, and t represent 2'-deoxyribonucleotides. All nucleosides are linked by phosphorothioate bonds (-P(=S)(OH)-). ∧ In the table, "Xo" bonded to the hydroxyl group at the 5' end of the oligonucleotide via a phosphodiester bond (-P(=O)(OH)-) and "X" bonded to the hydroxyl group at the 5' end of the oligonucleotide via a phosphorothioate bond represent the structures described below.

[0161]

[0162] (2) Antigen-specific CTL induction test According to the method of Example 1, LNP-mRNA (LNP(1)-mRNA HPV16 E6_E7(pU), dose: 1 μg / mouse) and various adjuvants (K3, Lipid-K3(1) or Lipid-K3(2), dose: 0.1 or 1 μg / mouse) were mixed in 10 mM histidine buffer containing 300 mM sucrose so that a predetermined dose was achieved with an administration of 30 μL per mouse, and the mixture was adjusted to pH 7.0 to prepare a vaccine-adjuvant mixture. 30 μL of vehicle or the various vaccine-adjuvant mixtures prepared as described above were administered into the gastrocnemius muscle of mice anesthetized by inhalation with isoflurane, three times in total, at weeks 0, 1, and 2. 14 shows data on HPV16 E7 MHC Class I epitope-specific CTL (%) among CD8-positive T cells in mouse blood at week 3 (one week after the final administration). Open circles represent data for individual mice, and open bars represent the average. Each group consisted of four mice.

[0163] As shown in Figure 14, intramuscular administration of LNP(1)-mRNA HPV16 E6_E7(pU) resulted in the induction of E7 MHC Class I epitope-specific CTLs. In the group administered K3, Lipid-K3(1), or Lipid-K3(2) simultaneously with LNP(1)-mRNA HPV16 E6_E7(pU) intramuscularly, a tendency toward enhanced E7 MHC Class I epitope-specific CTL induction was observed compared to the group administered LNP(1)-mRNA HPV16 E6_E7(pU) alone. These results demonstrate that K3, Lipid-K3(1), or Lipid-K3(2) enhances antigen-specific CTL induction by LNP-mRNA.

[0164] According to the present invention, an adjuvant that enhances CTL induction by an mRNA vaccine can be provided. The adjuvant of the present invention was observed to enhance antigen-specific CTL induction even when mRNA encoding only a CD8 epitope sequence was used as the mRNA encoding the antigen protein. Therefore, shortening the length of the mRNA used in the vaccine is expected to reduce vaccine production costs, facilitate production, enhance efficacy per unit mRNA amount, enable the production of even safer vaccines, and reduce the administration volume of the vaccine by reducing the particle size when encapsulated in LNPs. This application is based on Patent Application No. 2024-134532 filed in Japan (filing date: August 9, 2024), the contents of which are incorporated in their entirety herein.

Claims

1. An adjuvant comprising a type B CpG oligodeoxynucleotide (CpG ODN), a modified form thereof, or a complex thereof, for administration in combination with an mRNA vaccine in which mRNA encoding an antigen is encapsulated in particles, wherein the adjuvant exists independently of the particles encapsulating the mRNA.

2. The adjuvant according to claim 1, wherein the B-type CpG ODN portion in the B-type CpG ODN, its modified form, or its conjugate has all of its phosphodiester bonds replaced with phosphorothioate bonds and contains one or more unmethylated CpG motifs.

3. The adjuvant according to claim 1 or 2, wherein the chain length of the type B CpG ODN portion in the type B CpG ODN, its modified form, or its complex is 100 nucleotides or less.

4. An adjuvant according to any one of claims 1 to 3, characterized in that the adjuvant enhances the ability to induce cellular immunity specific to the antigen encoded by the mRNA.

5. The adjuvant according to any one of claims 1 to 4, wherein the type B CpG ODN portion in the type B CpG ODN, its modified form, or its complex comprises a sequence consisting of TCG.

6. The adjuvant described in any one of claims 1 to 5, wherein the mRNA vaccine is an infectious disease vaccine or a cancer vaccine.

7. The adjuvant according to any one of claims 1 to 6, wherein the B-type CpG ODN, a modified version thereof, or a complex thereof comprises one selected from the group consisting of the nucleic acid sequences of SEQ ID NOs: 1 to 6 and SEQ ID NO:

13.

8. The adjuvant according to any one of claims 1 to 6, wherein the type B CpG ODN, a modified version thereof, or a conjugate thereof comprises K3 CpG ODN, K3-dA40, K3 CpG ODN analog, ODN 2006, ODN 1018, ODN 1826, or ODN 1668.

9. The adjuvant according to any one of claims 1 to 8, wherein the B-type CpG ODN complex is a complex of B-type CpG ODN or a modified form thereof with β(1→3) glucan.

10. The adjuvant according to claim 9, wherein the β(1→3) glucan is lentinan or schizophyllan.

11. The adjuvant according to any one of claims 1 to 8, wherein the modified B-type CpG ODN is lipid-modified.

12. The adjuvant of any one of claims 1 to 11, wherein in the mRNA vaccine, the particles encapsulating the mRNA comprise lipid nanoparticles (LNPs).

13. An adjuvant according to any one of claims 1 to 12, wherein the mRNA comprises at least one modified nucleotide.

14. The adjuvant of claim 13, wherein the modified nucleotides include at least one pyrimidine nucleotide substituted at the 5-position and / or a pseudouridine nucleotide optionally substituted at the 1-position.

15. The adjuvant of claim 13, wherein the modified nucleotide comprises at least one selected from the group consisting of 5-methylcytidine, 5-methoxyuridine, 5-methyluridine, pseudouridine, and 1-alkylpseudouridine.

16. The adjuvant of claim 13, wherein the modified nucleotide comprises at least one selected from the group consisting of 5-methylcytidine, 5-methyluridine, and 1-methylpseudouridine.

17. The adjuvant of claim 12, wherein the LNP comprises an amphipathic lipid, sterols, an ionizable lipid, and a PEG-lipid.

18. The adjuvant of claim 17, wherein the ionizable lipid is selected from ALC-0315, SM-102, and (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-diene-7,29-diyl diacetate.

19. The adjuvant described in claim 17 or 18, wherein the lipid composition of the amphipathic lipid, sterols, ionizable lipid, and PEG lipid constituting the LNP is, in molar amounts, 10 to 22.5% amphipathic lipid, 15 to 55% sterols, 40 to 65% ionizable lipid, and 1 to 5% PEG lipid.

20. A method for enhancing the induction of antigen-specific cytotoxic T cells by an mRNA vaccine, comprising administering an adjuvant containing a type B CpG oligodeoxynucleotide (CpG ODN), a modified form thereof, or a complex thereof to a subject who will receive or has received an mRNA vaccine in which mRNA encoding an antigen is encapsulated in particles, wherein the adjuvant exists independently of the particles encapsulating the mRNA.

21. The method of claim 20, wherein the adjuvant and the mRNA vaccine are administered in combination.

22. A method for treating a disease treatable by the antigen, comprising administering to a subject suffering from the disease an mRNA vaccine in which mRNA encoding the antigen is encapsulated in particles, and an adjuvant comprising a B-type CpG oligodeoxynucleotide (CpG ODN), a modified form thereof, or a complex thereof, wherein the adjuvant exists independently of the particles encapsulating the mRNA.

23. The method of claim 22, wherein the antigen is an infectious disease antigen or a cancer antigen, and the disease is an infectious disease or a cancer.

24. An adjuvant comprising a B-type CpG oligodeoxynucleotide (CpG ODN), a modified form thereof, or a complex thereof, for use in enhancing the induction of antigen-specific cytotoxic T cells by an mRNA vaccine in which antigen-encoding mRNA is encapsulated in particles, wherein the adjuvant exists independently of the particles encapsulating the mRNA.

25. An adjuvant comprising a type B CpG oligodeoxynucleotide (CpG ODN), a modified form thereof, or a complex thereof, for use in treating a disease treatable by an antigen using an mRNA vaccine in which the mRNA encoding the antigen is encapsulated in particles, wherein the adjuvant exists independently of the particles encapsulating the mRNA.

26. The adjuvant of claim 25, wherein the antigen is an infectious disease antigen or a cancer antigen, and the disease is an infectious disease or cancer.

27. Use of a type B CpG oligodeoxynucleotide (CpG ODN), a modified form thereof, or a complex thereof for producing an adjuvant comprising a type B CpG ODN, a modified form thereof, or a complex thereof for administration in combination with an mRNA vaccine in which mRNA encoding an antigen is encapsulated in particles, wherein the adjuvant exists independently of the particles encapsulating the mRNA.

28. The use according to claim 27, wherein the antigen is an infectious disease antigen or a cancer antigen, and the vaccine is used to treat an infectious disease and / or cancer.

29. A pharmaceutical composition comprising an mRNA vaccine in which mRNA encoding an antigen is encapsulated in particles, and an adjuvant comprising a type B CpG oligodeoxynucleotide (CpG ODN), a modified form thereof, or a complex thereof, wherein the adjuvant is present independently of the particles encapsulating the mRNA.

30. The pharmaceutical composition according to claim 29, wherein the antigen is an infectious disease antigen or a cancer antigen, for treating an infectious disease or cancer.

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