Composition and use thereof

Abstract

Provided is a new composition capable of inducing an immune response against a target antigen. A composition according to the present disclosure comprises: a target antigen or a polynucleotide encoding the same; and an expression inducer of major histocompatibility gene complex class II (MHC class II molecule) and / or an expression inhibitor of an invariant chain thereof.

Description

Composition and its uses

[0001] This disclosure relates to compositions and their uses.

[0002] It is known that cancer patients have tumor-associated antigens, which are autoantigens strongly expressed on cancer cells. Therefore, attempts have been made to treat cancer by administering the tumor-associated antigens and peptides derived from them to cancer patients, thereby activating cancer-specific T cells that recognize the peptides in the cancer patients (Non-Patent Documents 1 and 2).

[0003] Kimberly R. J. et al. , (2010). “Peptide vaccines prevent tumor growth by activating T cells that respond to native tumor antigens.”, Proceedings of The National Academy of Sciences of The United States of America, 107, 10, p. 4652-4657 Luis A R. et al. , (2023). “Personalized RNA neoantigen vaccines stimulate T cells in pancreatic cancer.” Nature, 618, p. 144-150

[0004] However, there are cases in which an immune response to the tumor-associated antigen and the peptide derived from the tumor-associated antigen is not induced.

[0005] Therefore, this disclosure aims to provide a novel composition capable of inducing an immune response to a target antigen.

[0006] To achieve the aforementioned objectives, the compositions of this disclosure comprise a target antigen or a polynucleotide encoding it, and an expression inducer and / or an invariant chain inhibitor of major histocompatibility complex class II (MHC class II molecule).

[0007] The pharmaceutical compositions of this disclosure include the compositions of this disclosure.

[0008] According to this disclosure, an immune response to a target antigen can be induced. Therefore, it is expected that the compositions of this disclosure can induce an immune response to the tumor-associated antigen.

[0009] Figure 1 is a schematic diagram showing the mechanism of induction of immune tolerance in living organisms and the mechanism by which an immune response to neoself antigens is induced. Figure 2 is a graph showing the tumor volume in Example 1. Figure 3 is a graph showing the tumor volume in Example 2. Figure 4 is a graph showing the tumor volume in Example 3. Figure 5 is a graph showing the tumor volume in Example 4. Figure 6 is a graph showing the tumor volume in Example 5. Figure 7 is a graph showing the tumor volume in Example 6. Figure 8 is a graph showing the tumor volume in Example 7. Figure 9 is a graph showing the tumor volume in Example 8. Figure 10 is a graph showing the tumor volume in Example 9. Figure 11 is a graph showing the tumor volume in Example 10. Figure 12 is a graph showing the tumor volume in Example 11. Figure 13 is a graph showing the expression level of the invariant chain in Reference Example 1. Figure 14 is a graph showing the expression level of the invariant chain in Example 12. Figure 15 is a graph showing the tumor volume in Example 12. Figure 16 is a graph showing the tumor volume in Example 13. Figure 17 is a graph showing the protocol and anti-hIgG antibody titer in Example 14. Figure 18 is a graph showing the protocol and anti-hgp100 antibody titer in Example 15. Figure 19 is a graph showing the expression of NY-ESO-1 on the cell surface in Example 16. Figure 20 is a graph showing the protocol and anti-NY-ESO-1 antibody titer in Example 16. Figure 21 is a graph showing the protocol and the percentage of activated cells in Example 17. Figure 22 is a graph showing the protocol and the percentage of activated cells in Example 18. Figure 23 is a graph showing the expression of invariant chains in Example 19. Figure 24 is a graph showing the expression level of NY-ESO-1 in Example 19. Figure 25 is a graph comparing the expression level of invariant chains and NY-ESO-1 in Example 19. Figure 26 is a graph showing the protocol and anti-hIgG antibody titer in Example 20. Figure 27 is a graph showing the protocol and anti-HEL antibody titer in Example 21. Figure 28 is a graph showing the protocol and anti-ERVW-1 antibody titer in Example 22.

[0010] <Definitions> In this specification, “nucleic acid molecule,” “polynucleotide,” or “oligonucleotide” means a chain of polymers of deoxyribonucleotides (DNA), ribonucleotides (RNA), and / or modified nucleotides. The nucleic acid molecule may be a single-stranded nucleic acid molecule or a double-stranded nucleic acid molecule. The polynucleotide may consist of natural nucleotides, modified or artificial nucleotides, or both. The nucleic acid includes, for example, DNA, RNA, and / or DNA / RNA composed of natural and / or non-natural nucleic acid residues.

[0011] In this specification, “RNA” means natural or unnatural ribonucleic acid. The RNA is a chain of polymerized nucleotides. The RNA may be modified (modified nucleic acid) or unmodified. Examples of such modifications include methylation, pseudouridine, and thiolation. In this disclosure, it is preferable that the uridine in the RNA is pseudouridine. The pseudouridine may be partial or complete.

[0012] In this specification, "polyA" refers to adenosine added to mRNA by polyadenylation. PolyA is known to contribute to protein synthesis and mRNA stabilization. PolyA can be added, for example, by treating an RNA molecule with polyA polymerase.

[0013] In this specification, “gene expression” means the translation of mRNA into a polypeptide or protein, or post-translational modification of a polypeptide or protein.

[0014] In this disclosure, "gene expression induction" means that the expression of the target gene is induced, and may also mean a change from a state in which the target gene is not expressed to a state in which it is expressed.

[0015] In this specification, "hybridize" can be detected, for example, by various hybridization assays under stringent conditions. The hybridization assay is not particularly limited, and methods such as those described in Sambrook et al., "Molecular Cloning: A Laboratory Manual 2nd Ed." [Cold Spring Harbor Laboratory Press (1989)] may be employed.

[0016] In this specification, "stringent conditions" may be, for example, low-stringent conditions, medium-stringent conditions, or high-stringent conditions. "Low-stringent conditions" are, for example, 5×SSC, 5×Denhardt solution, 0.5% SDS, 50% formamide, and 32°C. "Medium-stringent conditions" are, for example, 5×SSC, 5×Denhardt solution, 0.5% SDS, 50% formamide, and 42°C. "High-stringent conditions" are, for example, 5×SSC, 5×Denhardt solution, 0.5% SDS, 50% formamide, and 50°C. The degree of stringency can be set by those skilled in the art by appropriately selecting conditions such as temperature, salt concentration, probe concentration and length, ionic strength, and time. The "stringent conditions" can also be those described in Sambrook et al.'s "Molecular Cloning: A Laboratory Manual 2nd Ed." [Cold Spring Harbor Laboratory Press (1989)], for example.

[0017] In this specification, “complementarity” means that one polynucleotide and another polynucleotide can form a nucleotide pair, i.e., a base pair.

[0018] In this specification, “corresponding” amino acid or nucleic acid means an amino acid or nucleotide in a polypeptide or polynucleotide that has, or is expected to have, a similar function to a given amino acid or nucleotide or portion in a standard polypeptide or polynucleotide.

[0019] In this specification, "identity" refers to the degree of identity when, for example, two sequences being compared (e.g., amino acid sequences or nucleotide sequences) are properly aligned, and means the percentage of exact amino acid matches between the sequences. This identity can be calculated using, for example, analysis software such as BLAST or FASTA with default parameters (the same applies hereinafter).

[0020] In this specification, “protein,” “peptide,” or “polypeptide” means a polymer of unmodified amino acids (natural amino acids), modified amino acids, and / or artificial amino acids. The polypeptide is, for example, a peptide having a length of 10 amino acids, 15 amino acids, or 20 amino acids or more.

[0021] In this specification, "antigen" means a molecule or part of a molecule that can be bound by a selective binding factor such as an antibody, an antigen-binding protein such as a B cell receptor or a T cell receptor.

[0022] In this specification, “tumor-associated antigen” (TAA) refers to an antigen relatively specific to tumor cells. Such tumor-associated antigens may be useful in diagnosing various tumors and are known to be helpful in determining response to treatment or recurrence.

[0023] In this specification, the term MHC class II (Major histocompatibility complex Class II, MHCII) molecule refers to a major histocompatibility complex (MHC) molecule expressed in B cells and antigen-presenting cells, etc., that is classified as a class II antigen. The MHCII molecule is a protein complex composed of an α chain and a β chain. The human MHCII molecule is composed of an HLA complex encoded at the HLA locus. The mouse MHCII molecule is composed of an H-2 complex encoded at the H-2 locus.

[0024] In this specification, "Class II transactivator" (CIITA) refers to a protein that activates the transcription of the MHCII gene. It is known that when CIITA is expressed, the expression of the MHCII molecule is induced.

[0025] In this specification, "interferon-gamma" (IFN-γ) is a cytokine secreted primarily by T cells, NK cells, dendritic cells, etc., in response to antigen stimulation or microbial infection. IFN-γ is known to contribute to the establishment of acquired immunity by inducing the expression of antigen-presenting genes, including the MHCII molecule, in antigen-presenting cells.

[0026] In this specification, "granulocyte-macrophage colony-stimulating factor" (GM-CSF) is a hematopoietic factor known to promote the proliferation of granulocyte and macrophage progenitor cells and induce colony formation in vitro. GM-CSF is known, for example, to induce the expression of MHCII molecules in antigen-presenting cells such as macrophages.

[0027] In this specification, "EBV immediate-early protein" (BZLF1) refers to the immediate early protein expressed in EBV (Epstein-Barr virus). BZLF1 is known to be involved in virus production in EBV-infected cells and to suppress the expression of invariant chains in order to evade the host's immune system.

[0028] In this specification, the invariant chain (Ii, CD74) refers to a protein known to form a complex with the MHCII molecule within the endoplasmic reticulum and prevent the binding of peptides to the MHCII molecule.

[0029] In this specification, “vector” (expression vector) means a nucleic acid molecule, a complex of the nucleic acid molecule with another molecule, or a recombinant plasmid or virus containing the nucleic acid molecule, which is delivered to a cell in vitro or in vivo.

[0030] In this specification, "RNA replicase" means an enzyme that uses RNA as a template and transcribes RNA. The RNA replicase can also be called an RNA-dependent RNA polymerase. As an example, in the case of alphavirus RNA replicase, it is known that RNA is replicated by the sequential synthesis of the (-) strand complementary strand and the (+) strand genomic RNA of the genomic RNA.

[0031] In this specification, "lipid" means a molecule having polar groups (hydrophilic groups) and nonpolar groups (hydrophobic groups) (amphiphilic molecules).

[0032] In this specification, "lipid nanoparticles" (LNPs) refer to nanoparticles with a diameter of approximately 10 nm to 1000 nm, mainly composed of lipids. These lipid nanoparticles can deliver nucleic acids through mechanisms such as cellular uptake, intracellular transport, endosomal release, and endosomal escape, and are used as nonviral drug delivery systems (DDS).

[0033] In this specification, “label” means a label used to distinguish a target molecule or substance from other molecules or substances. Such labels include, for example, fluorescent labels such as fluorescent dyes or fluorescent substances (e.g., fluorescein, fluorescein isothiocyanate, rhodamine); chemiluminescent labels such as luciferin and aequorin; luminescent substances such as luminol and acridinium derivatives; electroluminescent substances such as ruthenium complexes; and enzymatic labels such as horseradish peroxidase, alkaline phosphatase, β-galactosidase (β-gal), glucose oxidase, and luciferase. ３ H,１４ C, ３２ P, ３５ S, １２５ radioisotope (RI) labels such as I; ２ H, １３ C, １５ N, １７ O, １８ O, ３３ S, ３４ S, ３６ stable isotope labels such as S, etc. can be mentioned.

[0034] In this specification, "isolation" means identifying, separating, recovering from components in the natural state, and being in an identified and separated state, and / or being in a state recovered from components in the natural state.

[0035] In this specification, "immune checkpoint inhibitor" means a drug that inhibits the immunosuppressive effect by immune cells such as cancer cells or antigen-presenting cells. Examples of the immune checkpoint inhibitor include antibodies against immunosuppressive receptors.

[0036] In this specification, the form of the "antibody" may be a full-length immunoglobulin (antibody having an Fc region and an Fab region, full-length antibody), or F(ab')2, Fab', Fab, Fv antibody (variable fragment of antibody), disulfide bond Fv (dsFv), single-chain antibody (scFv), and polymers thereof (e.g., diabody), etc. The antibody may be a monoclonal antibody or a polyclonal antibody. The antibody may be, for example, a chimeric antibody, a humanized antibody, or a fully humanized antibody. The antibody may be, for example, a bispecific or oligospecific antibody.

[0037] In this specification, "pharmaceutical composition" means a chemical compound, protein, nucleic acid, composition, drug or medicine that can induce a desired therapeutic effect when appropriately administered to a subject. The pharmaceutical composition may contain one component or may contain multiple components.

[0038] In this specification, “treatment” means therapeutic treatment and / or preventive treatment. In this specification, “treatment” means the treatment, cure, prevention, suppression, remission (remission), or improvement of a disease, condition, or disorder, or the cessation, suppression, reduction, or delay of the progression of a disease, condition, or disorder. In this specification, “prevention” means a reduction in the likelihood of developing a disease or condition, or a delay in the development of a disease or condition. The “treatment” may be, for example, treatment of a patient who develops the disease in question, or treatment of a model animal of the disease in question.

[0039] In this specification, “subject” or “target of administration” means an animal or cells, tissues, or organs derived from an animal. The term “subject” is used in particular to include humans. The term “animal” means both humans and non-human animals. Examples of non-human animals include mammals such as mice, rats, rabbits, dogs, cats, cattle, horses, pigs, monkeys, dolphins, and sea lions. In this specification, “patient” means a subject receiving prophylactic or therapeutic treatment. The term “subject” includes, for example, a patient in addition to a healthy person.

[0040] In this disclosure, “therapeutic dose” refers to the amount of active ingredient determined to produce a therapeutic response in the subject of administration. The therapeutic dose can be readily determined by those skilled in the art.

[0041] In this specification, “kit” typically means a unit in which the components to be provided (e.g., compositions, pharmaceutical compositions, instructions, etc.) are provided in two or more compartments. The kit is suitably used to provide compositions that, for example, are not provided mixed, but are preferably mixed immediately before use for stability or other reasons. The kit preferably includes, for example, instructions or instructions on how to use the provided components (e.g., compositions, pharmaceutical compositions, etc.), or instructions or instructions describing how to process the components. In this specification, when the kit is used as a pharmaceutical kit, the kit may include instructions describing how to use the pharmaceutical composition, etc.

[0042] In this specification, “Instructions” or “Instructions” are instructions for a physician or other user on how to use the compositions, pharmaceutical compositions, or kits of the Disclosure. Such instructions may, for example, describe how to use the compositions, pharmaceutical compositions, or kits of the Disclosure. Such instructions may be prepared in accordance with the format prescribed by the supervisory authority of the country where the Disclosure is implemented (e.g., the Ministry of Health, Labour and Welfare in Japan, the Food and Drug Administration (FDA) in the United States, the European Medicines Agency (EMA) in Europe, etc.) and may be explicitly stated to have been approved by that supervisory authority. Such instructions may be so-called package inserts and are usually provided in paper format, but are not limited to that, and may also be provided in electronic format (e.g., a homepage provided on the Internet, email).

[0043] Sequence information for proteins or nucleic acids (e.g., DNA or RNA) encoding them as described herein can be obtained from Protein Data Bank, UniProt, or GenBank, etc. RNA nucleic acid sequences can also be obtained from the corresponding DNA base sequence or protein amino acid sequence using appropriate sequence conversion software.

[0044] The following explanation of this disclosure will be based on examples, but this disclosure is not limited to the following examples and can be modified and implemented as desired. Furthermore, the descriptions in this disclosure and each embodiment are mutually interchangeable unless otherwise specified. In this specification, when the expression "~" is used, it is used to mean including the numerical or physical values ​​before and after it. Also, in this specification, the expression "A and / or B" includes "A only," "B only," and "both A and B."

[0045] <Composition> In one embodiment, the present disclosure provides a composition capable of inducing an immune response to a target antigen. The composition of the present disclosure comprises a target antigen or a polynucleotide encoding it, and an expression inducer and / or an invariant chain inhibitor of major histocompatibility complex class II (MHC class II molecule).

[0046] The inventors have found that in patients with autoimmune diseases, a complex of the MHCII molecule and self-proteins (neo-self antigen) is recognized by autoantibodies. In a steady state, the MHCII molecule presents peptides derived from self-antigens, inducing immune tolerance in self-responsive T cells and self-responsive B cells (Figure 1(A)). On the other hand, as a result of further research, the inventors have found that in patients with autoimmune diseases, an abnormality in the antigen presentation mechanism of the MHCII molecule leads to the presentation of neo-self antigens, resulting in the activation of self-responsive T cells and self-responsive B cells, and consequently, autoimmune diseases (Figure 1(B)). The inventors conceived the idea of ​​whether this mechanism of activation of self-responsive T cells in autoimmune diseases could be used to induce immune responses to other antigens, such as tumor-associated antigens (TAAs). The present inventors have found that by using TAA as a target antigen and expressing the target antigen and MHCII, an immune response to the target antigen, particularly an immune response by T cells, can be induced, and that the immune response can be further enhanced by suppressing the expression of the invariant chain or by using it in combination with the immune checkpoint inhibitor, thus establishing this disclosure. Therefore, according to this disclosure, by expressing the target antigen together with the MHCII molecule, an immune response to the target antigen, particularly an immune response by T cells, can be induced.

[0047] Furthermore, the inventors conceived the idea that a similar activation mechanism could induce an immune response by B cells against a target antigen using the neoself antigen. The inventors then discovered that an immune response by B cells could be induced by using the target antigen in combination with the invariant chain expression inhibitor, leading to the establishment of this disclosure. Therefore, according to this disclosure, an immune response against the target antigen, particularly an immune response by B cells, can be induced by using the target antigen in combination with the invariant chain expression inhibitor.

[0048] The compositions of this disclosure include an expression inducer for a neoself antigen containing a target antigen, i.e., an inducer for a complex of the target antigen with an MHC II molecule. The neoself antigen expression inducer includes, for example, the target antigen or a polynucleotide encoding it, and an expression inducer for major histocompatibility complex class II (MHC class II molecule). In the compositions of this disclosure, it is presumed that the expression of the neoself antigen can be improved, for example, by overexpression of the MHC II molecule that the target antigen complexes. Alternatively, the neoself antigen expression inducer may also include, for example, the target antigen or a polynucleotide encoding it, and an expression inhibitor for the invariant chain. In this case, it is presumed that the expression of the neoself antigen can be improved, for example, by suppressing the expression of the invariant chain that blocks the peptide binding groove of the MHC II molecule, thereby improving the binding ability of the target antigen. As will be described later, the composition of this disclosure may include both the expression inducer of the MHC class II molecule and the expression inhibitor of the invariant chain.

[0049] The target antigen is not particularly limited and can be, for example, any antigen that induces an immune response. The target antigen is, for example, an antigen expressed in a subject to whom the composition of the disclosure is administered, or an antigen whose expression can be induced in a subject to whom the composition of the disclosure is administered. Examples of the type of target antigen include proteins, peptides, glycans, nucleic acid molecules, or mixtures thereof, and is preferably a protein or peptide because it can suitably induce an immune response. Examples of the target antigen include tumor-associated antigens (TAAs), tumor-specific antigens (TSAs), viral antigens, bacterial antigens, and parasitic antigens. The tumor-associated antigens include, for example, gp100, Tyrp1, Tyrp2, gp70, Melan A, 5T4, α5β1-integrin, activated integrin β7, 707-AP, α-fetoprotein (AFP), lectin-reactive AFP, placental alkaline phosphatase (Alppl2), ANXA2 (Annexin A2), ART-4, AURKA (AURORA A), B7H3 (CD276), B7H4, BAGE, β-catenin, BCMA, Bcr-abl, BTAA, MN / CA IX antigen, CA125, CA19-9, CA72-4, CAMEL, CAP-1, CASP-8, CD4, CD19, CD20, CD22, CD25, CD27, CD30, CD33, CD 47, CD52, CD56, CD70, CD80, CD96, CD123, CDK4, carcinoembryonic antigen (CEA), Claudin6, Claudin18.2, CLL1, CT, Cyclin A1, Cyp-B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, ENO1 (α-enolase), EpCam, ETV6-AML1, G250, GAGE ​​(GAGE-1, GAGE-2, etc.), GD2 (Ganglioside) G2), GnT-V, gp100, GPC3 (glypican-3), GUCY2C, HAGE, β-human chorionic gonadotropin (HCG), HER2 / neu, HLA-A*0201-R170I, HPV-E7, HSP70-2M, HST -2, iCE, insulin growth factor (IGF)-1, IGF-2, IGF-1R, IL-2R, IL-5, IL13Rα2, KIAA0205, K-Ras, LAGE, LDLR / FUT, MAGE (MAGE-3, MAGE-4, MAGE-5,MAGE-6, MART-1 / melan-A, MART-2 / Ski, MC1R, mesothelin (MSLN), myosin, MUC1, MUC4, MUM-1, MUM-2, MUM-3, NA88-A, Nectin-4, prostatic acid phosphatase (PAP), proteinase-3, PRAME (Melanoma antibody preferredly expressed in Examples include tumors, p53, p190 minor bcr-abl, Pml / RARα, prostate tumor antigen-1 (PCTA-1), prostate-specific antigen (PSA), PSM, PSMA, RAGE, RAS, RHAMM (CD168), RU1, RU2, SAGE, SART-1, SART-3, thyroglobulin, survivorbin, telomerase reverse transcriptase (TERT or TRT), TEL / AML1, TGFβ, TIM3, Tmem176b, TPI / m, TROP2, TRP-1, TRP-2, TRP-2 / INT2, VEGF, WT1, NY-ESO-1, NY-ESO-B, etc. In humans, endogenous retroviruses are known to contribute to the promotion of cancer growth and metastasis upon reactivation. Therefore, the tumor-associated antigen may be, for example, an antigen derived from human endogenous retroviruses (ERV), specifically including ERVW-1, ERV3-1, ERVE-1, ERVFRD-1, ERVFRD-3, ERVH-1, RVH48-1, ERVK-28, ERVK13-1, ERVK3-1, ERVK9-11, etc.

[0050] The aforementioned viral antigens include, for example, adenoviruses (adenovirus, etc.), coronaviruses (coronavirus, etc.), filoviridae (Ebolavirus, etc.), hepatitis C virus (HCV), dengue virus, Japanese encephalitis virus, West Nile virus, and yellow fever virus. Flaviviridae (e.g., *Hepatitis B* virus), Hepadnaviridae (e.g., *Hepatitis B* virus), herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), varicella-zoster virus (VZV), human cytomegalovirus (HCMV), Epstein-Barr virus (EBV) Herpesviridae (family of viruses), including Kaposi's sarcoma-associated herpesvirus (KSHV); Orthomyxoviridae (family of viruses), including influenza A virus (Influenza virus A), influenza B virus (Influenza virus B), and influenza C virus (Influenza virus C); measles virus, human parainfluenza virus types 1-4, mumps virus (Mumps virus) Paramyxoviridae, including viruses such as RSV (Respiratory Syncytial virus);Parvoviridae (Parvovirus B19, etc.); Picornaviridae (Enterovirus, Poliovirus, Human Rhinovirus A-B, Hepatitis A virus, Coxsackievirus, Echovirus, etc.); Poxviridae (Smallpox virus, Vaccinia virus, etc.); Human Immunodeficiency Virus (HIV) Antigens derived from viruses such as: Retroviridae (e.g., immunodeficiency virus-1, 2, human T-lymphophotropic virus-1, II); Rhabdoviridae (e.g., rabies virus, vesicular stomatitis virus); and Togaviridae (e.g., rubella virus, chikungunya virus).

[0051] The aforementioned bacterial antigens include, for example, Clostridium species such as Clostridium tetanus; Escherichia species such as Escherichia coli; Helicobacter species such as Helicobacter pylori; Legionella species such as Legionella pneumophila; Listeria species such as Listeria monocytogenes; and Mycobacterium tuberculosis. Mycobacterium species such as tuberculosis, Mycobacterium leprae, Mycobacterium avium, Mycobacterium intracellularae, Mycobacterium kansasii, Mycobacterium gordonae, etc.; Neisseria gonorrhoeae, Neisseria meningitidis Neisseria species (e.g., Neisseria meningitidis); Pseudomonas species (e.g., Pseudomonas aeruginosa); Salmonella species (e.g., Salmonella enterica serova typhi, Salmonella enterica serova paratyphi A); Staphylococcus species (e.g., Staphylococcus aureus); Streptococcus pneumoniae Antigens derived from bacteria such as Streptococcus species (including Pneumoniae and Streptococcus pyogenes) are examples of such antigens.

[0052] Examples of the aforementioned parasitic antigens include antigens derived from parasites such as liver fluke (Clonorchis sinensis), Japanese schistosoma (Schistosoma japonica), human roundworm (Ascaris lumbricoides), human pinworm (Enterobius vermicularis), cysticercus (Cysticercus cellulosae), broad tapeworm (Diphyllobothrium latum), echinococcus, amoeba histolytica, and malaria parasite (Plasmodium).

[0053] If the target antigen is a protein, the target antigen may consist of the full-length amino acid sequence of the target protein (target protein), or it may consist of a partial sequence. If the target antigen consists of a partial sequence of the target protein, the target antigen may be, for example, a polypeptide consisting of an amino acid sequence that does not include the intracellular domain and / or transmembrane domain of the target protein, or a polypeptide consisting of the extracellular domain of the target protein. Each domain in the target protein can be estimated, for example, using the amino acid sequence of the target protein and an algorithm for estimating domains such as transmembrane regions. Examples of algorithms capable of estimating the transmembrane domain include SOSUI (https: / / harrier.nagahama-i-bio.ac.jp / sosui / ), TMHMM (Transmembrane Hidden Markov Mode, https: / / services.healthtech.dtu.dk / services / TMHMM-2.0 / l), etc. When the target antigen is a membrane protein, the target antigen is, for example, a peptide composed of the extracellular domain of the membrane protein.

[0054] If the target antigen is a protein, the protein may be an intracellular protein, an extracellular protein (i.e., a secreted protein), or a membrane protein.

[0055] The target antigen may be, for example, a target antigen of the same species as the target of administration of the composition of this disclosure, or a target antigen of a different species. The target antigen may be, for example, one type or multiple types in combination.

[0056] The target antigen may be determined, for example, by comparing the expression of the target antigen (gene or protein) in the target cancer and the normal cells corresponding to the cancer, and selecting the target antigen whose expression level in the target cancer is significantly higher than that in the normal cells corresponding to the cancer. The expression level can be measured, for example, using semi-quantitative PCR, ELISA, etc., depending on the target gene or protein.

[0057] The target antigen is preferably an antigen that, when expressed in cells together with the MHCII molecule described later, can be presented on the cell surface by the MHCII molecule, i.e., an antigen capable of forming a complex with the MHCII molecule. Antigen presentation on the cell surface can be evaluated, for example, by flow cytometry or immunoprecipitation. Each evaluation method can be carried out, for example, by the following procedure. (Evaluation method using flow cytometry) (1) Transiently transfect HEK293 cells or B16F10 cells with a polynucleotide encoding the target antigen (e.g., plasmid or mRNA), or a polynucleotide encoding the target antigen and the MHCII molecule (e.g., plasmid or mRNA). (2) After transfection, culture for 36 hours (in the case of plasmid) or 16 to 24 hours (in the case of mRNA), and measure antigen expression on the cell surface by flow cytometry. (3) If the transfection group of polynucleotides encoding the target antigen and the MHCII molecule shows a higher expression level of the target antigen compared to the transfection group of the target antigen alone, the target antigen can be evaluated as an antigen capable of forming a complex with the MHCII molecule. (Method of evaluation by immunoprecipitation) (1) Transiently transfect HEK293 cells or B16F10 cells with a polynucleotide (e.g., plasmid or mRNA) encoding the target antigen (with a tag such as a Flag or His tag attached), or a polynucleotide (e.g., plasmid or mRNA) encoding the target antigen (with a tag such as a Flag or His tag attached) and the MHCII molecule. (2) After transfection, culture for 36 hours (in the case of plasmids) or 16 to 24 hours (in the case of mRNA), collect the transfected cells, lyse them, and obtain cell lysates. (3) Immunoprecipitation is performed on the cell lysate, and the complex of the target antigen and MHCII molecule is measured on the obtained precipitate by Western blotting or using beads. If coprecipitation of the target antigen and the MHCII molecule is confirmed, the target antigen can be evaluated as an antigen capable of forming a complex with the MHCII molecule.

[0058] The binding affinity of the target antigen to MHC class II molecules can be improved, for example, by adding an endoplasmic reticulum localization signal sequence (hereinafter also referred to as "endoplasmic reticulum localization signal" or "signal sequence") to the target antigen, removing the transmembrane region and optionally the intracellular region, and / or removing regions with high hydrophobicity and basic amino acid content (e.g., hydrophobic regions and / or hydrophilic regions). For this reason, the target antigen may be, for example, a protein or polypeptide that has undergone the addition of the endoplasmic reticulum localization signal sequence, the removal of the transmembrane region in the target antigen, and / or the removal of hydrophobic and / or basic amino acids in the target antigen. The hydrophobic region is, for example, a region mainly composed of hydrophobic amino acids such as valine, leucine, and isoleucine. The basic region is, for example, a region mainly composed of basic amino acids such as lysine, arginine, and histidine. If the target antigen is an intracellular protein, the intracellular protein can be presented on the cell surface by the MHC class II molecule by, for example, the addition of the endoplasmic reticulum localization signal sequence and / or the removal of the hydrophobic region, thereby enhancing the induction of an immune response. Furthermore, if the target antigen is a cell membrane protein, the cell membrane protein can be presented on the cell surface by the MHC class II molecule by, for example, the removal of the transmembrane region and optionally the intracellular region, thereby enhancing the induction of an immune response.

[0059] The polynucleotide encoding the target antigen may be, for example, a DNA nucleic acid molecule, an RNA nucleic acid molecule, or a DNA / RNA nucleic acid molecule. If the target antigen is a protein or peptide, the base sequence of the nucleic acid molecule can be set, for example, based on the amino acid sequence of the target antigen. The base sequence of the nucleic acid molecule may be, for example, codon-optimized. The codon-optimized base sequence encoding the target antigen can be designed, for example, using algorithms such as GeneArt (Life Technologies) or DNA2.0 (MenloParkCA), depending on the target to whom the composition of this disclosure is administered. The polynucleotide encoding the target antigen is preferably an RNA nucleic acid molecule. The RNA nucleic acid molecule preferably has a structure similar to mRNA. The structure of mRNA will be described later.

[0060] The polynucleotide may be, for example, a linear polynucleotide or a cyclic polynucleotide. When the polynucleotide is cyclic, it is preferable that the polynucleotide encoding the target antigen be located downstream of a cap-independent translation initiation site, such as an IRES (internal ribosome entry site). The IRES may be, for example, a natural IRES or a non-natural IRES. Examples of natural IRES include IRES present in RNA molecules of organisms (including entities that can reproduce using multicellular organisms, such as viruses). The IRES may be, for example, a known IRES that can be appropriately selected and used.

[0061] Examples of MHCII molecule expression inducers include drugs that can induce the expression of MHCII molecules in cells, particularly immune cells. The composition of this disclosure, for example, by including the MHCII molecule expression inducer, can create an environment in which the target antigen can easily form a complex with the MHCII molecule by causing the MHCII molecule to be expressed in the cells in a greater-than-normal amount. Examples of the MHCII molecule expression inducer include the MHCII molecule or a polynucleotide encoding it, a transcription inducer of the MHCII molecule or a polynucleotide encoding it. The MHCII molecule expression inducer is preferably a transcription inducer of the MHCII molecule or a polynucleotide encoding it, for example, because it can induce the expression of MHCII molecules inherent in the target to whom the composition of this disclosure is administered, and can induce an immune response regardless of the haplotype of the target MHCII molecule. The MHCII molecule expression inducer may be used alone or in combination of multiple types.

[0062] As described above, the MHCII molecule is composed of an α chain and a β chain. The origin of the α chain and β chain of the MHCII molecule is not particularly limited and may be, for example, human and non-human animals. The origin of the α chain and β chain of the MHCII molecule may be, for example, the same type of MHCII molecule as the target of administration of the composition of this disclosure, or a different type of MHCII molecule, but preferably the same type of MHCII molecule. The haplotype of the genes encoding the α chain and β chain is not particularly limited. The haplotype of the genes encoding the α chain and β chain may be, for example, the same haplotype as the target of administration of the composition of this disclosure, or a different haplotype, but preferably the same haplotype.

[0063] When the MHCII molecule is of human origin, the α-chain of the human MHCII molecule may be, for example, the α-chain of an MHCII molecule encoded at the HLA-DPA locus, HLA-DQA locus, or HLA-DRA locus. The β-chain of the human MHCII molecule may be, for example, the β-chain of an MHCII molecule encoded at the HLA-DPB locus, HLA-DQB locus, or HLA-DRB locus. The haplotypes of the α-chain and β-chain of the MHCII molecule at each of the aforementioned loci are not particularly limited. The MHCII molecule may be, for example, a molecule containing either an α-chain or a β-chain, and preferably a molecule containing both an α-chain and a β-chain. The MHCII molecule may be, for example, HLA-DR, HLA-DP, and HLA-DQ. The MHCII molecule may be, for example, one type or a combination of multiple types.

[0064] The MHCII molecule is preferably, for example, HLA-DR, HLA-DP, and HLA-DQ, and in particular, cancer-related MHCII molecules can be exemplified. The allele of the MHCII molecule may be the same as or different from the allele of the target to which the composition of this disclosure is administered, but it is preferably the same.

[0065] Examples of HLA-DR include HLA-DR1, HLA-DR2, HLA-DR3, HLA-DR4, HLA-DR5, HLA-DR6, HLA-DR7, HLA-DR8, HLA-DR9, HLA-DR10, HLA-DR11, HLA-DR12, HLA-DR13, HLA-DR14, HLA-DR15, HLA-DR52, HLA-DR53, and the like. Examples of HLA-DR include molecules containing an HLA-DRA such as HLA-DRA1 as the α-chain and an HLA-DRB such as HLA-DRB1, HLA-DRB3, HLA-DRB4, or HLA-DRB5 as the β-chain. Specifically, the HLA-DR includes, for example, an allele such as HLA-DRA1*01 as the α-chain, and for example, an allele such as HLA-DRB1*01, HLA-DRB1*03, HLA-DRB1*04, HLA-DRB1*07, HLA-DRB1*08, HLA-DRB1*09, HLA-DRB1*10, HLA-DRB1*11, HLA-DRB1*12, HLA-DRB1*13, HLA-DRB1*14, HLA-DRB1*15, HLA-DRB1*16, HLA-DRB3*01, HLA-DRB4*01, and HLA-DRB5*01 as the β-chain.

[0066] Examples of HLA-DQ include HLA-DQ1, HLA-DQ2, HLA-DQ3, HLA-DQ4, HLA-DQ5, HLA-DQ6, HLA-DQ7, HLA-DQ8, and so on. Examples of HLA-DQ include molecules containing an HLA-DQA such as HLA-DQA1 as the α-chain and an HLA-DQB such as HLA-DQB1 as the β-chain. Specifically, the HLA-DQ may include alleles such as HLA-DQA1*01, HLA-DQA1*02, HLA-DQA1*03, HLA-DQA1*04, HLA-DQA1*05, and HLA-DQA1*06 as the α-chain, and HLA-DQB1*02, HLA-DQB1*03, HLA-DQB1*04, HLA-DQB1*05, and HLA-DQB1*06 as the β-chain.

[0067] Examples of HLA-DP include HLA-DP1, HLA-DP2, HLA-DP3, HLA-DP4, HLA-DP5, etc. Examples of HLA-DP include molecules containing HLA-DPA such as HLA-DPA1 as the α-chain and HLA-DPB such as HLA-DPB1 as the β-chain. Specifically, examples of HLA-DP include alleles such as HLA-DPA1*01, HLA-DPA1*02, HLA-DPA1*03, HLA-DPA1*04, etc. as the α-chain and alleles such as HLA-DPB1*02, HLA-DPB1*04, HLA-DPB1*05, HLA-DPB1*09, etc. as the β-chain.

[0068] The MHCII molecule or the polynucleotide encoding it is preferably, for example, an MHCII molecule or polynucleotide encoding it that can bind to the target antigen.

[0069] The transcription inducer of the MHC class II molecule is, for example, a factor that can induce the expression of the MHC class II molecule in cells, particularly immune cells, when they come into contact with or are introduced into such cells. Examples of the transcription inducer of the MHC class II molecule include transcription factors such as major histocompatibility complex class II transactivator (CIITA); and cytokines such as interferon-γ (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The transcription inducer of the MHC class II molecule may be one type or multiple types may be used in combination.

[0070] The CIITA may be, for example, CIITA of the same species as the target organism to which the composition of this disclosure is administered, or CIITA of a different species. Information on CIITA from various animals can be referenced, for example, from a database. Furthermore, if CIITA from various animals has multiple isoforms, any of the isoforms of the CIITA may be used. The inventors have confirmed that any of the isoforms 1 to 5 of mouse CIITA can induce the expression of the MHCII molecule. As a specific example, isoform 2 of human CIITA may be, for example, a protein consisting of the following amino acid sequence (SEQ ID NO: 1) and a polynucleotide consisting of the following base sequence (SEQ ID NO: 2), which are registered under GenBank accession number: NM_000246.4. Furthermore, isoform 2 of mouse CIITA is, for example, a polynucleotide consisting of the following amino acid sequence (SEQ ID NO: 3) and base sequence (SEQ ID NO: 4), which are registered under GenBank accession number: NP_031601.1. The base sequence encoding CIITA below is a base sequence that includes a stop codon.

[0071]

[0072]

[0073]

[0074]

[0075] The CIITA may be, for example, a functional equivalent (mutant) within the range having transcriptional activation (transcriptional induction) ability of the MHCII molecule. Examples of the functional equivalent of CIITA include polypeptides having transcriptional activation ability of the MHCII molecule, consisting of an amino acid sequence that has 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the reference amino acid sequence of CIITA (SEQ ID NOs: 1 and 3). Examples of the functional equivalent include polypeptides having transcriptional activation ability of the MHCII molecule, consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added to the reference amino acid sequence of CIITA (SEQ ID NOs: 1 and 3). The aforementioned one or several are, for example, 1 to 226, 1 to 214, 1 to 169, 1 to 161, 1 to 113, 1 to 107, 1 to 56, 1 to 53, 1 to 45, 1 to 43, 1 to 33, 1 to 32, 1 to 22, 1 to 21, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 12, 1 to 11, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1. The substitution is preferably a conservative substitution, for example.

[0076] The aforementioned conservative substitution means the substitution of an amino acid residue with an amino acid residue having a similar side chain. Examples of such conservative substitutions include substitutions between amino acid residues having basic side chains such as lysine, arginine, and histidine; substitutions between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; substitutions between amino acid residues having non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; substitutions between amino acid residues having non-polar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; substitutions between amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and substitutions between amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0077] The IFN-γ may be, for example, IFN-γ derived from the same species as the target organism to which the composition of this disclosure is administered, or IFN-γ derived from a different species. Information on IFN-γ derived from various animals can be found, for example, in databases. As a specific example, human IFN-γ is the protein consisting of the following amino acid sequence (SEQ ID NO: 5) registered in GenBank accession number NP_000610.2. Mouse IFN-γ is the protein consisting of the following amino acid sequence (SEQ ID NO: 7) registered in NP_032363.1. The nucleotide sequence encoding the following IFN-γ is a nucleotide sequence including a stop codon.

[0078] Human IFN-γ (SEQ ID NO: 5) MKYTSYILAFQLCIVLGSLGCYCQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRGRRASQ

[0079] Polynucleotide encoding human IFN-γ (SEQ ID NO: 6) 5'-ATGAAATATACAAGTTATATCTTGGCTTTTCAGCTCTGCATCGTTTTGGGTTCTCTTGGCTGTTACTGCCAGGACCCATATGTAAAAGAAGCAGAAAACCTTAAGAAATATTTTAATGCAGGT CATTCAGATGTAGCGGATAATGGAACTCTTTTCTTAGGCATTTTGAAGAATTGGAAAGAGGAGAGTGACAGAAAAATAATGCAGAGCCAAATTGTCTCCTTTTACTTCAAACTTTTTAAAAACTTTA AAGATGACCAGAGCATCCAAAAGAGTGTGGAGACCATCAAGGAAGACATGAATGTCAAGTTTTTCAATAGCAACAAAAAGAAACGAGATGACTTCGAAAAGCTGACTAATTATTCGGTAACTGACTT GAATGTCCAACGCAAAGCAATACATGAACTCATCCAAGTGATGGCTGAACTGTCGCCAGCAGCTAAAACAGGGAAGCGAAAAAAGGAGTCAGATGCTGTTTCGAGGTCGAAGAGCATCCCAGTAA-3'

[0080] Mouse IFN-γ (SEQ ID NO: 7) MNATHCILALQLFLMAVSGCYCHGTVIESLESLNNYFNSSGIDVEEKSLFLDIWRNWQKDGDMKILQSQIISFYLRLFEVLKDNQAISNNISVIESHLITTFFSNSKAKKDAFMSIAKFEVNNPQVQRQAFNELIRVVHQLLPESSLRKRKRSRC

[0081] The aforementioned GM-CSF may be, for example, a GM-CSF derived from the same species as the target organism to which the composition of this disclosure is administered, or a GM-CSF derived from a different species. Information on GM-CSF derived from various animals can be referenced, for example, from databases. As a specific example, human GM-CSF is a protein consisting of the following amino acid sequence (SEQ ID NO: 8) registered in GenBank accession number NP_000749.2. Mouse GM-CSF is a protein consisting of the following amino acid sequence (SEQ ID NO: 10) registered in NP_034099.2. The base sequence encoding the following GM-CSF is a base sequence including a stop codon.

[0082] Human GM-CSF (SEQ ID NO: 8) MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE

[0083] Polynucleotide encoding human GM-CSF (SEQ ID NO: 9) 5'-ATGTGGCTGCAGAGCCTGCTGCTCTTGGGCACTGTGGCCTGCAGCATCTCTGCACCCGCCCGCTCGCCCAGCCCCAGCACGCAGCCCTGGGAGCATGTGAATGCCATCCAGGAGGCCCGGCGTCTCCTGAACCTGAGTAGAGACACTGCTGCTGAGATGAATGAAACAGTAGAAGTCATCTCAGAAATGTTTGACCTCCAGGAGCCGACCTGCCTAC AGACCCGCCTGGAGCTGTACAAGCAGGGCCTGCGGGGCAGCCTCACCAAGCTCAAGGGCCCCTTGACCATGATGGCCAGCCACTACAAGCAGCACTGCCCTCCAACCCCGGAAACTTCCTGTGCAACCCAGATTATCACCTTTGAAAGTTTCAAAGAGAACCTGAAGGACTTTCTGCTTGTCATCCCCTTTGACTGCTGGGAGCCAGTCCAGGAGTGA-3'

[0084] Mouse GM-CSF (Sequence ID 10) MWLQNLLFLGIVVYSLSAPTRSPITVTRPWKHVEAIKEALNLLDDMPVTLNEEVEVVSNEFSFKKLTCVQTRLKIFEQGLRGNFTKLKGALNMTASYYQTYCPPTPETDCETQVTTYADFIDSLKTFLTDIPFECKKPGQK

[0085] Polynucleotide encoding mouse GM-CSF (SEQ ID NO: 11) 5'-ATGTGGCTGCAGAATTTACTTTTCCTGGGCATTGTGGTCTACAGCCTCTCAGCACCCACCCGCTCACCCATCACTGTCACCCGGCCTTGGAAGCATGTAGAGGCCATCAAAGAAGCCCTGAACCTCCTGGATGACATGCCTGTCACGTTGAATGAAGAGGTAGAAGTCGTCTCTAACGAGTTCTCCTTCAAGAAGCTAACATGTGTGCAGACC CGCCTGAAGATATTCGAGCAGGGTCTACGGGGCAATTTCACCAAAACTCAAGGGCGCCTTGAACATGACAGCCAGCTACTACCAGACATACTGCCCCCCAACTCCGGAAACGGACTGTGAAACACAAGTTACCACCTATGCGGATTTCATAGACAGCCTTAAAACCTTTCTGACTGATATCCCCTTTGAATGCAAAAAACCAGGCCAAAAATGA-3'

[0086] The IFN-γ and GM-CSF mentioned above may be, for example, functional equivalents (mutants) within the range having transcriptional activation (transcriptional induction) ability of the MHCII molecule. Examples of functional equivalents of IFN-γ and GM-CSF include polypeptides having 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the reference amino acid sequence of IFN-γ or GM-CSF (SEQ ID NOs. 5, 7-8 and 10), respectively, and having transcriptional activation ability of the MHCII molecule. Functional equivalents of IFN-γ and GM-CSF include, for example, polypeptides having transcriptional activation ability for the MHCII molecule, which consist of amino acid sequences in which one or more amino acids are deleted, substituted, inserted, and / or added to the reference amino acid sequences of IFN-γ or GM-CSF (SEQ ID NOs. 5, 7-8, and 10). The one or more amino acids are, for example, 1-18, 1-17, 1-16, 1-15, 1-12, 1-11, 1-10, 1-8, 1-6, 1-5, 1-4, 1-3, 1 or 2, or 1. The substitution is preferably, for example, a conservative substitution as described above.

[0087] The polynucleotide encoding the MHC class II molecule transcription inducer may be, for example, a DNA nucleic acid molecule, an RNA nucleic acid molecule, or a DNA / RNA nucleic acid molecule. If the MHC class II molecule transcription inducer is a protein or peptide, the base sequence of the nucleic acid molecule can be set, for example, based on the amino acid sequence of the MHC class II molecule transcription inducer. The base sequence of the nucleic acid molecule may be, for example, codon-optimized. The codon-optimized base sequence encoding the MHC class II molecule transcription inducer can be designed, for example, using algorithms such as GeneArt (Life Technologies) or DNA2.0 (MenloParkCA), depending on the target to whom the composition of this disclosure is administered. The polynucleotide encoding the target antigen is preferably an RNA nucleic acid molecule. The RNA nucleic acid molecule preferably has a structure similar to mRNA. The structure of mRNA will be described later.

[0088] The compositions of this disclosure may, for example, include, in addition to, or instead of, the MHCII molecule expression inducer, an invariant chain expression inhibitor. When the expression of the invariant chain is suppressed, the relative number of MHCII molecules not bound to the invariant chain increases within the endoplasmic reticulum, thereby promoting the formation of a complex between the target antigen and the MHCII molecule. As a result, the compositions of this disclosure can, for example, induce a stronger immune response.

[0089] The amino acid sequence and nucleotide sequence of the invariant chain can, for example, refer to the amino acid sequence and nucleotide sequence of the target to which the composition of this disclosure will be administered. For invariant chains derived from various animals, information registered in a database can, for example, be referenced. As specific examples, human invariant chain isoforms 1 to 5 are proteins consisting of the following amino acid sequences (sequence numbers 12 to 16) registered in UniPro accession numbers P04233-1, P04233-3, P04233-4, and P04233-5, respectively. Similarly, mouse invariant chains isoforms 1 to 2 are proteins consisting of the following amino acid sequences (sequence numbers 17 to 18) registered in P04441-1 and P04441-2, respectively.

[0090] Human invariant strand isoform 1 (SEQ ID NO: 12) MHRRRSRSCREDQKPVMDDQRDLISNNEQLPMLGRRPGAPESKCSRGALYTGFSILVTLLLAGQATTAYFLYQQQGRLDKLTVTSQNLQLENLRMKLPKPPKPVSKMRMATPLLMQALPMGALPQGPMQNATKYGNMTEDHVMHLLQNADPLKVYPPLKGSFPENLRHLKNTMETIDWKVFESWMHHWLLFEMSRHSLEQKPTDAPPKVLTKCQEEVSHIPAVHPGSFRPKCDENGNYLPLQCYGSIGYCWCVFPNGTEVPNTRSRGHHNCSESLELEDPSSGLGVTKQDLGPVPM

[0091] Human invariant chain isoform 2 (SEQ ID NO: 13) MHRRRSRSCREDQKPVMDDQRDLISNNEQLPMLGRRPGAPESKCSRGALYTGFSILVTLLLAGQATTAYFLYQQQGRLDKLTVTSQNLQLENLRMKLPKPPKPVSKMRMATPLLMQALPMGALPQGPMQNATKYGNMTEDHVMHLLQNADPLKVYPPLKGSFPENLRHLKNTMETIDWKVFESWMHHWLLFEMSRHSLEQKPTDAPPKESLELEDPSSGLGVTKQDLGPVPM

[0092] Human invariant chain isoform 3 (SEQ ID NO: 14) MHRRRSRSCREDQKPVMDDQRDLISNNEQLPMLGRRPGAPESKCSRGALYTGFSILVTLLLAGQATTAYFLYQQQGRLDKLTVTSQNLQLENLRMKLPKPPKPVSKMRMATPLLMQALPMGALPQGPMQNATKYGNMTEDHVMHLLQSHWNWRTRLLGWV

[0093] Human invariant chain isoform 4 (SEQ ID NO: 15) MDDQRDLISNNEQLPMLGRRPGAPESKCSRGALYTGFSILVTLLLAGQATTAYFLYQQQGRLDKLTVTSQNLQLENLRMKLPKPPKPVSKMRMATPLLMQALPMGALPQGPMQNATKYGNMTEDHVMHLLQNADPLKVYPPLKGSFPENLRHLKNTMETIDWKVFESWMHHWLLFEMSRHSLEQKPTDAPPKVLTKCQEEVSHIPAVHPGSFRPKCDENGNYLPLQCYGSIGYCWCVFPNGTEVPNTRSRGHHNCSESLELEDPSSGLGVTKQDLGPVPM

[0094] Human invariant chain isoform 5 (SEQ ID NO: 16) MDDQRDLISNNEQLPMLGRRPGAPESKCSRGALYTGFSILVTLLLAGQATTAYFLYQQQGRLDKLTVTSQNLQLENLRMKLPKPPKPVSKMRMATPLLMQALPMGALPQGPMQNATKYGNMTEDHVMHLLQNADPLKVYPPLKGSFPENLRHLKNTMETIDWKVFESWMHHWLLFEMSRHSLEQKPTDAPPKESLELEDPSSGLGVTKQDLGPVPM

[0095] Mouse invariant strand isoform 1 (SEQ ID NO: 17) MDDQRDLISNHEQLPILGNRPREPERCSRGALYTGVSVLVALLLAGQATTAYFLYQQQGRLDKLTITSQNLQLESLRMKLPKSAKPVSQMRMATPLLMRPMSMDNMLLGPVKNVTKYGNMTQDHVMHLLTRSGPLEYPQLKGTFPENLKHLKNSMDGVNWKIFESWMKQWLLFEMSKNSLEEKKPTEAPPKVLTKCQEEVSHIPAVYPGAFRPKCDENGNYLPLQCHGSTGYCWCVFPNGTEVPHTKSRGRHNCSEPLDMEDLSSGLGVTRQELGQVTL

[0096] Mouse invariant strand isoform 2 (SEQ ID NO: 18) MDDQRDLISNHEQLPILGNRPREPERCSRGALYTGVSVLVALLLAGQATTAYFLYQQQGRLDKLTITSQNLQLESLRMKLPKSAKPVSQMRMATPLLMRPMSMDNMLLGPVKNVTKYGNMTQDHVMHLLTRSGPLEYPQLKGTFPENLKHLKNSMDGVNWKIFESWMKQWLLFEMSKNSLEEKKPTEAPPKEPLDMEDLSSGLGVTRQELGQVTL

[0097] The invariant chain expression inhibitor is, for example, one that can suppress the expression of the invariant chain. Examples of the invariant chain expression inhibitor include EBV immediate-early protein (BZLF1) or the polynucleotide encoding it, nucleic acid molecules that suppress the expression of the invariant chain, genome editing factors such as ZFN and TALEN.

[0098] The aforementioned BZLF1 is a protein expressed in EBV. The BZLF1 can, for example, refer to information registered in a database. Specifically, BZLF1 is a protein consisting of the following amino acid sequences (SEQ ID NOs. 19, 21-22) registered in GenBank accession number AIA57587.1, UniProt accession number Q3KSS8, and Q1HVG1.

[0099] BZLF1 (BZLF1_EBVB9) (Sequence ID 19) MMDPNSTSEDVKFTPDPYQVPFVQAFDQATRVYQDLGGPSQAPLPCVLWPVLPEPLPQGQLTAYHVSTAPTGSWFSAPQPAPENAYQAYAAPQLFPVSDITQNQQTNQAGGEAPQPGDNSTVQTAAAVVFACPGANQGQQLADIGVPQPAPVAAPARRTRKPQQPESLEECDSELEIKRYKNRVASRKCRAKFKQLLQHYREVAAAKSSENDRLRLLLKQMCPSLDVDSIIPRTPDVLHEDLLNF

[0100] A polynucleotide (SEQ ID NO: 20) encoding BZLF1 (BZLF1_EBV B9) 5'-ATGATGGACCCAAACTCGACTTCTGAAGATGTAAAATTTACACCTGACCCATACCAGGTGCCTTTTGTACAAGCTTTTGACCAAGCTACCAGAGTCTATCAGGACCTGGGAGGGCCATCGCAAGCTCCTTTGCCTTGTGTGCTGTGGCCGGTGCTGCCAGAGCCTCTGCCACAAGGCCAGCTAACTGCCTATCATGTTTCAACCGCTCCGACTGGGTCGTGGTTTTCTGCCCCTCAGCCTGCTCCTGAGAATGCTTATCAAGCTTATGCAGCACCTCAGCTGTTCCCAGTCTCCGACATAACCCAGAATCAACAGACTAACCAAGCCGGGGGAGAAGCACCTCAACCTGGAGACAATTCTACTGTTCAAACAGCAGCAGCAGTGGTGTTTGCTTGCCCCGGGGCTAACCAAGGACAACAGCTAGCAGACATTGGTGTTCCACAGCCTGCACCAGTGGCTGCCCCGGCACGACGCACACGGAAACCACAACAGCCAGAATCGCTGGAGGAATGCGATTCTGAACTAGAAATAAAGCGATACAAGAATCGGGTGGCTTCCAGAAAATGCCGGGCCAAGTTTAAGCAACTGCTGCAGCACTACCGTGAGGTCGCTGCTGCCAAATCATCTGAAAATGACAGGCTGCGCCTCCTGTTGAAGCAGATGTGCCCAAGCCTGGATGTTGACTCCATTATCCCCCGGACACCAGATGTTTTACACGAGGATCTCTTAAATTTCTAA-3’

[0101] BZLF1 (BZLF1_EBVG) (Sequence ID 21) MMDPNSTSEDVKFTPDPYQVPFVQAFDQATRVYQDLGGPSQAPLPCVLWPVLPEPLPQGQLTAYHVSTAPTGSWFSAPQPAPENAYQAYAAPQLFPVSDITQNQQTNQAGGEAPQPGDNSTVQTAAAVVFACPGANQGQQLADIGVPQPAPVAAPARRTRKPQQPESLEECDSELEIKRYKNRVASRKCRAKFKQLLQHYREVAAAKSSENDRLRLLLKQMCPSLDVDSIIPRTPDVLHEDLLNF

[0102] BZLF1 (BZLF1_EBVA8) (Sequence ID 22) MMDPNSTSEDVKFTPDPYQVPFVQAFDQATRVYQDLGGPSQAPLPCVLWPVLPEPLPQGQLTAYHVSAAPTGSWFPAPQPAPENAYQAYAAPQLFPVSDITQNQQTNQAGGEAPQPGDNSTVQPAAAVVFACPGANQGQQLADIGAPQPAPAAAPARRTRKPLQPESLEECDSELDIKRYKNRVASRKCRAKFKHLLQHYREVASAKSSENDRLRLLLKQMCPSLDVDSIIPRTPDVLHEDLLNF

[0103] The BZLF1 may be, for example, a functional equivalent (mutant) within the range having the activity to suppress the expression of the invariant chain. The functional equivalent of BZLF1 may be a polypeptide having 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the reference amino acid sequence of BZLF1 (SEQ ID NOs. 19, 21-22), and having the activity to suppress the expression of the invariant chain. The functional equivalent of BZLF1 may be a polypeptide having the activity to suppress the expression of the invariant chain, for example, a polypeptide having the activity to suppress the expression of the invariant chain, in which one or more amino acids are deleted, substituted, inserted and / or added to the reference amino acid sequence of BZLF1 (SEQ ID NOs. 19, 21-22). The aforementioned one or several are, for example, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 12, 1 to 11, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1. The substitution is preferably, for example, the aforementioned conservative substitution.

[0104] Examples of nucleic acid molecules that suppress the expression of the invariant strand include siRNA, antisense, and genome editing factors that target the invariant strand. The sequence of the siRNA can be designed, for example, based on the sequence of the mRNA of the invariant strand expressed by the target of the composition of this disclosure. The siRNA can be designed, for example, using the Reynolds method. The siRNA may also be designed using a known algorithm such as siSPOTR (Reference 1). The antisense can be, for example, a nucleic acid molecule that can hybridize to the base sequence of the mRNA of the invariant strand. Examples of nucleic acid molecules that suppress the expression of the invariant strand include the nucleic acid molecules that suppress expression in the examples described later. Reference 1: Ryan L. Boudreau et al. (2013). “siSPOTR: a tool for designing highly specific and potent siRNAs for human and mouse”, Nucleic Acids Research, 41, 1, p. e9

[0105] The genome editing factor includes, for example, a protein having nuclease activity and a guide chain for localizing the protein to a target sequence. The protein is, for example, a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) enzyme, and specific examples include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, C Examples include sm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, etc. The guide strand may be, for example, crRNA and tracrRNA, or a single-stranded nucleic acid in which these are linked via a linker. In this case, the nucleic acid can be designed so that the base sequence that anneals with the target sequence in the crRNA is complementary to the base sequence that codes for the gene in the invariant strand.

[0106] If any of the components constituting the composition of the present disclosure is a polynucleotide, the composition of the present disclosure may include a replicase protein (replicase or replicon) or a polynucleotide encoding it. If the polynucleotide is DNA, the composition of the present disclosure includes, for example, a DNA replicase (DNA replicon) or a polynucleotide encoding it. Furthermore, if the polynucleotide is an RNA polynucleotide, the composition of the present disclosure includes, for example, an RNA replicase (RNA replicon) or a polynucleotide encoding it.

[0107] The RNA replicase described above is known to be present in most RNA viruses except retroviruses. For example, RNA replicases derived from positive-strand RNA viruses such as alpha viruses and flaviviruses can be used. The alpha virus is an enveloped single-stranded positive-sense RNA virus belonging to the Togaviridae family.

[0108] The aforementioned alpha viruses include, for example, Eastern Equine Encephalitis Virus (EEEV), Venezuelan Equine Encephalitis Virus (VEEV), Everglades Virus (EVEV), Mukambo Virus (MUCV), Semliki Forest Virus (Semliki Forest Virus, SFV), Pixna Virus (PIXV), Middleberg Virus (MIDV), Chikungunya Virus (CHIKV), Onyonnyon Virus (ONNV), Ross River Virus (RRV), Burma Forest Virus (BF), Geta Virus (GET), Sagiyama Virus (SAGV), Beval Virus (BEBV), Mayaro Virus (MAYV), Una Virus (UNAV), and Sindbis Worth Examples include Sindbisvirus (SINV), Auravirus (AURAV), Wataroavirus (WHAV), Babankivirus (BABV), Kyzylagachvirus (KYZV), Western equine encephalitis virus (WEEV), Highland J virus (HJV), Fort Morgan virus (FMV), Ndumu virus (NDUV), and Buggy Creek virus. Furthermore, the α-viral RNA replicase is, for example, the RNA replicase of SINV, SFV, RRV, VEEV, or EEEV, and is preferably the RNA replicase of VEEV.

[0109] The α-virus RNA replicase includes, for example, sequences encoding at least one, two, three, or four types of non-structural viral proteins (e.g., nsP1, nsP2, nsP3, nsP4).

[0110] The replicase is positioned, for example, at the 5' end of the polynucleotide of the component constituting the composition. This allows the replicase to efficiently replicate the polynucleotide of the component constituting the composition within the cell, thereby inducing a sustained immune response to the target antigen. Specifically, if the polynucleotide of the component constituting the composition is RNA, the composition of this disclosure includes, for example, RNA containing a base sequence encoding RNA replicase, optionally a subgenome promoter such as a 26S subgenome promoter, and the base sequences of the component constituting the composition, in this order. The RNA may also be a self-amplifying RNA vector. Preferably, the RNA includes a 5' cap and a polyA sequence.

[0111] Examples of polynucleotides constituting the composition to be combined with the replicase include a polynucleotide encoding the target antigen, a polynucleotide encoding the MHC class II molecule, a polynucleotide encoding the transcription inducer of the MHC class II molecule, and a polynucleotide encoding BZLF1. The polynucleotides constituting the composition to be combined with the replicase may be one type or two or more types.

[0112] The compositions of this disclosure may further include, for example, an immune checkpoint inhibitor. Examples of such immune checkpoint inhibitors include drugs that target PD-1; PDL1; PDL2; CTLA-4; TIGIT; LAG3; KIR; CD137; CCR4; LILRB1; LILRB2; NKG2A; BTLA; TIM-3; B7-H3; B7-H4; HVEM; GAL9; CD160; VISTA; BTNL2; PVR; BTN1A1; BTN2A2; BTN3A2; CSF1-R, etc., or drugs that inhibit the function of any of these molecules. As specific examples, the aforementioned immune checkpoint inhibitors include, for example, PD-1 antibodies (nivolumab, pembrolizumab, AMP-224, AMP-514 (MEDI0680), pidilizumab (CT-011), etc.); PD-L1 antibodies (Durvalumab (MEDI4736), MPDL3280A, BMS-936559, avelumab (MSB0010718C), etc.); PD-L2 antibodies; CTLA-4 antibodies (ipilimumab, tremelimumab, etc.); TIGIT antibodies; LAG3 antibodies (I Examples include MP-321, BMS-986016, etc.; KIR antibody (IPH2101, etc.); CD137 antibody; CCR4 antibody; LILRB1 antibody; LILRB2 antibody; NKG2A antibody; BTLA antibody; TIM-3 antibody; B7-H3 antibody (MGA-271, etc.); B7-H4 antibody; HVEM antibody; GAL9 antibody; CD160 antibody; VISTA antibody; BTNL2 antibody; PVR antibody; BTN1A1 antibody; BTN2A2 antibody; BTN3A2 antibody (References 2 and 3); CSF1-R antibody, etc. The aforementioned immune checkpoint inhibitors may include, for example, one type or multiple types. Reference 2: Yao Set et al., (2013). “Advances in targeting cell surface signaling molecules for immune modulation.” Nat Rev Drug Discov. 12, 2, p. 130-146 Reference 3: Arnett HA et. al. , (2014). “Immune modulation by butyrophilins.” Nat Rev Immunol. 14, 8, p. 559-569

[0113] In the compositions of this disclosure, the target antigen and the MHCII molecule expression inducer may each be, for example, a protein or a peptide, a nucleic acid molecule, or both. Specifically, the compositions of this disclosure may include, for example, (1) a protein or peptide as the target antigen and a nucleic acid molecule encoding the MHCII molecule expression inducer as the MHCII molecule expression inducer, (2) a nucleic acid molecule encoding the target antigen as the target antigen and a protein or peptide as the MHCII molecule expression inducer, or (3) a nucleic acid molecule encoding the target antigen as the target antigen and a nucleic acid molecule encoding the MHCII molecule expression inducer as the MHCII molecule expression inducer. The compositions of this disclosure are preferably, for example, able to efficiently induce neoself antigen expression within the same cell, and therefore preferably include a nucleic acid molecule encoding the target antigen as the target antigen and a nucleic acid molecule encoding the MHCII molecule expression inducer as the MHCII molecule expression inducer.

[0114] If the composition of the present disclosure includes a polynucleotide of a component constituting the composition, the polynucleotide may be, for example, a vector (expression vector) containing the polynucleotide. Examples of such vectors include viral vectors and non-viral vectors (e.g., plasmid vectors).

[0115] Examples of the aforementioned viral vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors, poxvirus vectors, herpes simplex virus vectors, mouse leukemia virus vectors, and hybrid viral vectors.

[0116] The plasmid vector may be, for example, a plasmid vector such as a binary vector. Examples of such vectors include pETDuet-1, pQE-80L, and pUCP26km. When introduced into mammalian cells, examples of such expression vectors include pCDM8 and pMT2PC.

[0117] The vector preferably has a regulatory sequence that, for example, regulates the expression of polynucleotides of components constituting the composition. Examples of the regulatory sequence include promoters, terminators, enhancers, polyadenylation signal sequences, origin of replication sequences (ORI), etc. The arrangement of the regulatory sequence in the vector is not particularly limited. In the vector, the regulatory sequence only needs to be arranged so as to functionally regulate, for example, the expression of polynucleotides of components constituting the composition and the expression of peptides encoded therein, and can be arranged according to known methods. The regulatory sequence may, for example, utilize a sequence already present in the vector, or the regulatory sequence may be further inserted into the vector, or the regulatory sequence present in the basic vector may be replaced with another regulatory sequence.

[0118] If the vector is a viral vector, it can be prepared, for example, by introducing all the nucleic acid molecules necessary for the production of the viral vector into the host. Specifically, the viral vector may be prepared by introducing the remaining nucleic acid molecules necessary for the production of the viral vector into a host that has already been introduced some of the nucleic acid molecules necessary for the production of the viral vector. Alternatively, the viral vector may be prepared, for example, by introducing the nucleic acid molecules to be delivered by the viral vector, and optionally the remaining nucleic acid molecules necessary for the production of viral particles, into a host cell (packaging cell) that has already been introduced some or all of the nucleic acid molecules necessary for the production of viral particles. Examples of packaging cells include cells into which retrovirus Gag and / or Pol and / or envelope proteins have been introduced, and cells into which the adenovirus E1A and / or E1B regions have been introduced. As the packaged cells, commercially available cells suitable for viral vector production may be used, for example, Plat-E cells (CosmoBio, #RV-101) and Lenti-X293T cells (Takara, #Z2180N) can be used.

[0119] Examples of polynucleotides constituting the composition contained in the vector include a polynucleotide encoding the target antigen, a polynucleotide encoding the MHC class II molecule, a polynucleotide encoding the transcription inducer of the MHC class II molecule, and a polynucleotide encoding BZLF1. The polynucleotide constituting the composition to be combined with the replicase may be, for example, one type or two or more types.

[0120] If the composition of the present disclosure includes an invariant chain expression inhibitor, the invariant chain expression inhibitor may be, for example, a protein or a peptide, a nucleic acid molecule, or composed of both. If the composition of the present disclosure includes an invariant chain expression inhibitor, it is preferable that the composition of the present disclosure includes, as the target antigen, a nucleic acid molecule encoding the target antigen, as the MHCII molecule expression inducer, and as the invariant chain expression inhibitor, a nucleic acid molecule encoding the invariant chain expression inhibitor.

[0121] In the compositions of this disclosure, if the target antigen, the MHCII molecule expression inducer, and / or the invariant chain are nucleic acid molecules, it is preferable that the nucleic acid molecules are RNA nucleic acid molecules. Furthermore, it is preferable that the RNA nucleic acid molecules have a structure similar to mRNA.

[0122] The RNA nucleic acid molecule is arranged such that, for example, from the 5' end to the 3' end, the base sequence encoding the target protein, such as the target antigen, the MHCII molecule expression inducer, and / or the invariant chain, and the polyA sequence are arranged in this order.

[0123] The RNA nucleic acid molecule may include, for example, a cap structure or a cap analogue at its 5' end. Examples of such cap structures include a 5' guanosine cap structure generated using RNA cap analogs such as 3'-O-Me-m7G(5')ppp(5')G[ARCA cap], G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, m7G(5')ppp(5')G, and m7G(5')ppp(5')G-2'-O-methyl. The generation of the 5' guanosine cap structure can be carried out using commercially available products sold by companies such as New England BioLabs.

[0124] The RNA nucleic acid molecule may have a structure similar to that of mRNA, for example. In this case, the RNA nucleic acid molecule may include one or more regions or portions that act or function as untranslated regions (UTRs) to enhance the stability of the RNA nucleic acid molecule. Specifically, the RNA nucleic acid molecule may include untranslated regions such as a 5'UTR and a 3'UTR. The 5'UTR is a region of mRNA located immediately upstream (5') of the start codon (the first codon of the mRNA transcript translated by the ribosome). The 3'UTR is a region of mRNA located immediately downstream (3') of the stop codon (the codon of the mRNA transcript that signals the end of translation).

[0125] The UTR sequence can be a known sequence. The 5'UTR may include, for example, a Kozak sequence. The 5'UTR may be, for example, a 5'UTR from a different cell species than the target cell, or a synthetic 5'UTR. The 5'UTR can be, for example, a 5'UTR derived from human hemoglobin, alpha-2 (HBA2). The RNA nucleic acid molecule may, for example, use an internal ribosome entry site (IRES) instead of a 5'UTR. The 5'UTR may be, for example, a sequence that does not include a start codon.

[0126] The length of the 5'UTR is, for example, 20 bases or more, and specific examples include a length of 30 to 200 bases, 40 to 150 bases, and 50 to 100 bases.

[0127] The 3'UTR may include, for example, AU-rich elements (AREs) such as class I AREs, class II AREs, and class III AREs. The 3'UTR may be, for example, a 3'UTR from a different cell species than the target cell, or a synthetic 3'UTR.

[0128] The UTR may include, for example, regulatory factors. Examples of regulatory factors include promoters, enhancers, IRESs, introns, poly-U sequences, etc. Examples of promoters include T7 promoters and SP6 promoters.

[0129] If the RNA nucleic acid molecule has a structure similar to mRNA, the RNA nucleic acid molecule may, for example, include the cap structure or a cap analogue, the 5'UTR, the base sequence encoding the target protein, the 3'UTR, and the polyA sequence in this order from the 5' end.

[0130] In the RNA nucleic acid molecule, the base sequence encoding the target protein and the polyA sequence, or the cap structure or cap analogue, the 5'UTR, the base sequence encoding the target protein, the 3'UTR, and the polyA sequence are linked directly or indirectly, respectively. The direct linkage is such that the 3' ribonucleotide residue of each sequence is covalently linked to the 5' ribonucleotide residue of the other sequence. The indirect linkage is such that the 3' ribonucleotide residue of each sequence is covalently linked to the 5' ribonucleotide residue of the other sequence via a linker sequence. The length of the linker sequence can be, for example, 1 to 10 bases. The linker sequence is not particularly limited as long as it is a linker used in the field.

[0131] The length of the RNA nucleic acid molecule is not particularly limited and can be determined, for example, according to the length of each sequence. Examples of RNA nucleic acid molecule lengths in this disclosure include 100 to 10,000 nucleotides, 300 to 7,000 nucleotides, 700 to 3,300 nucleotides, and so on.

[0132] The nucleic acid molecule may be an unmodified nucleic acid molecule or a modified nucleic acid molecule. Similarly, the RNA nucleic acid molecule may be an unmodified RNA nucleic acid molecule or a modified RNA nucleic acid molecule. If the RNA nucleic acid molecule is an unmodified RNA nucleic acid molecule, it is composed of unmodified nucleotide residues, for example, natural nucleotide residues. If the nucleic acid molecule or the RNA nucleic acid molecule is a modified nucleic acid molecule, it is composed of modified nucleotide residues, or a combination of modified and unmodified nucleotide residues. If the nucleic acid molecule or the RNA nucleic acid molecule is composed of modified and unmodified nucleotide residues, the number of modified nucleotide residues is not particularly limited and can be 1 to 10,000, 1 to 5,000, 1 to 3,000, 1 to 1,000, 1 to 500, 1 to 250, 1 to 100, 1 to 50, 1 to 25, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. Furthermore, the number of unmodified nucleotide residues is not particularly limited and can be 1 to 10,000, 1 to 5,000, 1 to 3,000, 1 to 1,000, 1 to 500, 1 to 250, 1 to 100, 1 to 50, 1 to 25, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. When the nucleic acid molecule or RNA nucleic acid molecule is composed of modified nucleotide residues and unmodified nucleotide residues, the proportion of the modified nucleotide residues is 1% or more, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, or 80% or more, relative to the total length of the RNA nucleic acid molecule of this disclosure. The proportion of the unmodified nucleotide residues is 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, and 99% or more, relative to the total length of the RNA nucleic acid molecule of this disclosure.

[0133] Modification of the nucleotide residues includes, for example, modification of the ribose-phosphate skeleton (hereinafter also referred to as the "ribophosphate skeleton"). Modification of the ribophosphate skeleton includes, for example, modification of ribose residues, substitution with a non-ribophosphate skeleton, and modification of phosphate groups.

[0134] Modifications to the ribose residue include, for example, modification of the 2' carbon and substitution with stereoisomers. Modifications to the 2' carbon include, for example, substitution of the hydroxyl group bonded to the 2' carbon with a hydrogen atom, a halogen atom such as a fluorohydrogen atom, an -O-alkyl group, an -O-acyl group, an amino group, etc. Substitution with stereoisomers includes, for example, substitution with an arabinose residue.

[0135] Substitution to the non-ribophosphate skeleton includes, for example, substitution to a non-ribose residue or a non-phosphate-containing non-ribophosphate skeleton. The non-ribophosphate skeleton includes, for example, the uncharged form of the ribophosphate skeleton. Substitutes for nucleotides substituted with a non-ribophosphate skeleton include, for example, morpholino, cyclobutyl, pyrrolidine, and artificial nucleic acid monomer residues. Examples of artificial nucleic acid monomer residues include PNA (peptide nucleic acid), LNA (Locked Nucleic Acid), and ENA (2'-O,4'-C-Etherene-bridged Nucleic Acid).

[0136] Examples of modifications to the phosphate group include modifications to make the α-phosphate group most adjacent to the sugar residue uncharged, modifications to make the charge distribution of the two oxygen atoms (unbonded oxygen atoms) that are not bound to the sugar residue in the α-phosphate group asymmetrical, and modifications to the two oxygen atoms (bonded oxygen atoms) that are bound to the sugar residue in the α-phosphate group.

[0137] In the phosphate group, for example, the unbonded oxygen atom may be substituted. Examples of substitution of the unbonded oxygen atom include substitution with sulfur (S), selenium (Se), boron (B), carbon (C), hydrogen (H), nitrogen (N), -OR (where R is an alkyl group or aryl group), etc. Examples of such modified phosphate groups include phosphorothioates, phosphorodithioates, phosphoroselenates, boranophosphates, boranophosphate esters, phosphonate hydrogens, phosphoramides, alkyl or arylphosphonates, phosphotryesters, etc. The substitution of the unbonded oxygen atom may, for example, be a substitution of both oxygen atoms or a substitution of either one. In the former case, the substitution of the unbonded oxygen atom may be by the same atom or by different atoms.

[0138] In the phosphate group, for example, the bound oxygen atom may be substituted. Substitution of the bound oxygen atom may include substitution with sulfur (S), carbon (C), nitrogen (N), etc. Examples of such modified phosphate groups include N-substituted cross-linked phosphoramide, S-substituted cross-linked phosphorothioate, and C-substituted cross-linked methylene phosphonate. The substitution of the bound oxygen may be, for example, substitution of both the 5' terminal nucleotide residue and the 3' terminal nucleotide residue of the nucleic acid molecule of the present invention, or substitution of either one. Substitution of the 5' terminal nucleotide residue is preferably with C, and substitution of the 3' terminal nucleotide residue is preferably with N.

[0139] The phosphate group may be substituted with, for example, a phosphorus-free linker. Examples of such linkers include siloxanes, carbonates, carboxymethyls, carbamates, amides, thioethers, ethylene oxide linkers, sulfonates, sulfonamides, thioformacetals, formacetals, oximes, methyleneiminos, methylenemethyliminos, methylenehydrazos, methylenedimethylhydrazos, methyleneoxymethyliminos, and the like.

[0140] The modification of the phosphate group may be the entire phosphate group or one or more atoms within the phosphate group. In the former case, the modification of the phosphate group may be, for example, substitution or deletion of the entire phosphate group.

[0141] The modifications of the terminal nucleotide residues include, for example, functional molecules such as labeling substances and protecting groups; dyes; intercalating agents such as acridine; crosslinking agents such as psoralen and mitomycin C; saffyrin porphyrins such as TPPC4 and texaphylline; polycyclic aromatic hydrocarbons such as phenazine and dihydrophenazine; artificial endonucleases such as EDTA; cholesterol, cholic acid, adamantane acetate, 1-pyrenebutyric acid, dihydrotestosterone, and 1,3-bis-O(hexadecyl)glyceride. Lipophilic carriers such as cerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholic acid, dimethoxytrityl, phenoxazine; peptide complexes such as Antennapedia peptide and Tat peptide; alkylating agents; phosphoric acid; amino acids; mercapto; PEG such as PEG-40K; MPEG; [MPEG] ２The additions include polyaminos, alkyls, substituted alkyls, radiolabeled markers, enzymes, haptens such as biotin, transport / absorption enhancers such as aspirin, vitamin E, and folic acid, and synthetic ribonucleases such as imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole complexes, and Eu3+ complexes of tetraazamacro rings. The protecting group may be, for example, S (sulfur), Si (silicon), B (boron), or an ester-containing group. Functional molecules such as the labeling substance can be used, for example, for the detection of RNA nucleic acid molecules of this disclosure. The addition may be an addition to the phosphate group of a nucleotide residue, or an addition to the phosphate group or sugar residue via a spacer. The terminal atom of the spacer may be added to or substituted with, for example, the bound oxygen of the phosphate group, or the oxygen (O), nitrogen (N), sulfur (S), or carbon (C) of a sugar residue. The binding site of the sugar residue is preferably, for example, the 3' position C, the 5' position C, or an atom bound to these. The spacer may be added to or substituted for, for example, the terminal atoms of a nucleotide substitute such as PNA. The spacer may be, for example, -(CH ２ ) ｎ -, - (CH ２ ) ｎ N-, -(CH ２ ) ｎ O-,-(CH ２ ) ｎ S-, O(CH ２ CH ２ O) ｎ CH ２ CH ２ Examples include OH, non-basic sugars, amides, carboxyl groups, amines, oxyamines, oxyimines, thioethers, disulfides, thioureas, sulfonamides, morpholino, biotin reagents, fluorescein reagents, etc. In the above formula, n is a positive integer, and n=3 or n=6 is preferred. In the RNA nucleic acid molecule of this disclosure, the 5' end may be modified with, for example, a phosphate group or a phosphate group analog. The phosphate group may be, for example, 5'-monophosphate ((HO)) ２ (O)P-O-5'), 5' diphosphate ((HO) ２ (O)P-O-P(HO)(O)-O-5'), 5'triphosphate((HO) ２(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'), 5'-Guanosine Cap (7-Methylated or Unmethylated, 7m-G-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'), 5'-Adenosine Cap (Appp), Any Modified or Unmodified nucleotide Cap Structure (N-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'), 5'-Thiophosphate (Phosphorothioate: (HO) ２ (S)P-O-5'), 5'-dithiophosphate (phosphorodithioate: (HO)(HS)(S)P-O-5'), 5'-phosphorothiolic acid ((HO) ２ (O)P-S-5'), sulfur-substituted monophosphates, diphosphates and triphosphates (e.g., 5'-α-thiotriphosphate, 5'-γ-thiotriphosphate, etc.), 5'-phosphoramides ((HO) ２ (O)P-NH-5', (HO)(NH ２ )(O)P-O-5'), 5'-alkylphosphonic acid (e.g., RP(OH)(O)-O-5', (OH) ２ (O) P-5'-CH ２ Examples include R being an alkyl group (e.g., methyl, ethyl, isopropyl, propyl, etc.)), 5'-alkyl ether phosphonic acid (e.g., RP(OH)(O)-O-5', where R is an alkyl ether (e.g., methoxymethyl, ethoxymethyl, etc.)), etc.

[0142] In the nucleotide residue, the base is not particularly limited and may be a natural base or a non-natural base. For example, a common base, a modified analog thereof, etc., can be used.

[0143] Examples of the aforementioned bases include purine bases such as adenine and guanine, and pyrimidine bases such as cytosine, uracil, and thymine. Other examples of the aforementioned bases include inosine, thymine, xanthine, hypoxanthine, nubularine, isoguanisine, and tubercidine. Examples of the aforementioned bases include alkyl derivatives such as 2-aminoadenine and 6-methylated purine; alkyl derivatives such as 2-propylated purine; 5-halouracil and 5-halocytosine; 5-propynyluracil and 5-propynylcytosine; 6-azouracil, 6-azocytosine, and 6-azothymine; 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, and 5-aminoallylu Racil; 8-halo-, amin-, thio-, thio-alkyl-, hydroxy-, and other 8-substituted purines; 5-trifluoromethyl- and other 5-substituted pyrimidines; 7-methylguanine; 5-substituted pyrimidines; 6-azapyrimidines; N-2, N-6, and O-6 substituted purines (including 2-aminopropyladenine); 5-propynyluracil and 5-propynylcytosine; dihydrouracil; 3-deaza-5-azacytosine; 2- Aminopurine; 5-alkyluracil; 7-alkylguanine; 5-alkylcytosine; 7-deazaadenine; N6,N6-dimethyladenine; 2,6-diaminopurine; 5-amino-allyl-uracil; N3-methyluracil; substituted 1,2,4-triazole; 2-pyridinone; 5-nitroindole; 3-nitropyrrole; 5-methoxyuracil; uracil-5-oxyacetic acid; 5-methoxycarbonylmethyluracil; 5-methyl- Examples include 2-thiouracil; 5-methoxycarbonylmethyl-2-thiouracil; 5-methylaminomethyl-2-thiouracil; 3-(3-amino-3-carboxypropyl)uracil; 3-methylcytosine; 5-methylcytosine; N4-acetylcytosine; 2-thiocytosine; N6-methyladenine; N6-isopentyladenine; 2-methylthio-N6-isopentenyladenine; N-methylguanine; and O-alkylated bases.The purines and pyrimidines are, for example, described in U.S. Patent No. 3,687,808, edited by Kroschwitz J. I., Concise Encyclopedia of Polymer Science and Engineering, pp. 858-859, John Wiley & Sons (1990), and Uwe English et al., (1991), "Chemically Modified Oligonucleotides as Probes and Inhibitors," Angela and Chemie, International Edition, 30, p. This includes what is disclosed in 613.

[0144] The aforementioned base may or may not be modified. Examples of modified nucleotides include methyl-5-uridine (m5U), 2-thio-uridine (s2U), 2'-O-methyl-5-uridine (Ome5U), pseudouridine (ψ), methyl-1-pseudridine (m1ψ), methyl-5-cytosine (m5C), 2'O-methyl-5-cytosine (Om5C), N6-methyl-adenosine (m6A), N1-methyl-adenosine (m1A), etc. When the base is U, pseudouridine is preferred as the modified base.

[0145] The modified nucleotide residues may also include, for example, residues that delete a base, i.e., a baseless ribophosphate skeleton. The modified nucleotide residues may be, for example, those described in U.S. Provisional Application No. 60 / 465,665 (filed April 25, 2003) and International Application No. PCT / US04 / 07070 (filed March 8, 2004).

[0146] The nucleic acid molecule or RNA nucleic acid molecule may include, for example, a label. Examples of such labels include fluorescent labels, enzyme labels, dye labels, radioisotopes, or isotope labels.

[0147] In the compositions of this disclosure, the components such as the target antigen, the MHCII molecule expression inducer, the invariant chain expression inhibitor, and the immune checkpoint inhibitor can be prepared, for example, by any method depending on their type.

[0148] If the aforementioned component is a protein or a partial peptide thereof, the protein may be prepared by any method. For example, if the target antigen is a tumor-associated antigen in cancer, the tumor-associated antigen may be, for example, a protein or a partial peptide isolated and purified from cancer cells of mammals (including model animals such as mice and humans) or tissues in which cancer cells are present. Furthermore, the protein may be a protein or a partial peptide synthesized chemically or biochemically by a cell-free translation system, or it may be a recombinant protein or a partial peptide produced from a transformant into which nucleic acid having a base sequence encoding the protein or partial peptide has been introduced.

[0149] If the constituent is a DNA nucleic acid molecule, the DNA nucleic acid molecule may be prepared by designing its base sequence based on the amino acid sequence of the protein or peptide of the constituent and then chemically synthesizing it. As a specific example, if the target antigen is a tumor-associated antigen, the DNA nucleic acid molecule encoding the tumor-associated antigen may be, for example, cDNA derived from cancer cells or any tissue containing cancer cells in mammals (including model animals such as mice and humans). The cDNA encoding the tumor-associated antigen can also be directly amplified by Polymerase Chain Reaction (hereinafter abbreviated as "PCR method") and Reverse Transcription-PCR (hereinafter abbreviated as "RT-PCR method") using total RNA or mRNA (mRNA encoding a protein antigen) prepared from the cancer cells or tissue containing cancer cells as templates. The cDNA encoding the tumor-associated antigen can also be cloned from a cDNA library prepared by inserting cDNA fragments prepared from total RNA or mRNA prepared from the cancer cells or tissue into a suitable vector, using methods such as colony or plaque hybridization or PCR. The vector used for the library may be a bacteriophage, plasmid, cosmid, or phagemid. The DNA encoding the tumor-associated antigen can be used as is, or optionally digested with restriction enzymes or linked with a linker, and then ligated downstream of a promoter in a suitable expression vector. The DNA may have an ATG translation start codon at its 5' end and a TAA, TGA, or TAG translation stop codon at its 3' end. These translation start and stop codons can be added using a suitable synthetic DNA adapter.

[0150] If the constituent is an RNA nucleic acid molecule, the RNA nucleic acid molecule may be prepared by designing its base sequence based on the amino acid sequence of the protein or peptide of the constituent and then performing chemical synthesis or the like. For example, the RNA nucleic acid molecule can be produced by chemical synthesis such as solid-phase synthesis or liquid-phase synthesis based on the base sequence constituting the RNA nucleic acid molecule. Alternatively, the RNA nucleic acid molecule may be produced by an in vitro transcription system. In this case, the RNA nucleic acid molecule can be synthesized, for example, using the above-mentioned DNA nucleic acid molecule encoding the RNA nucleic acid molecule as a template and using phage RNA polymerase derived from T7 phage, T3 phage, SP6 phage, etc. The in vitro transcription may be carried out by methods described in, for example, International Publication No. 2014 / 152027, International Publication No. 2018 / 053209, International Publication No. 2019 / 036682, etc.

[0151] In the compositions of the present disclosure, when the components such as the target antigen, the MHCII molecule expression inducer, the invariant chain expression inhibitor, and the immune checkpoint inhibitor are nucleic acid molecules, particularly RNA nucleic acid molecules, the compositions of the present disclosure preferably include a lipid nucleic acid complex, wherein the lipid nucleic acid complex includes a lipid and a nucleic acid molecule encoding the component. By including the component as the lipid nucleic acid complex, for example, when administered into a living organism, the compositions of the present disclosure can suitably induce the expression of the target antigen, the MHCII molecule expression inducer, the invariant chain expression inhibitor, and the immune checkpoint inhibitor from the nucleic acid molecule encoding the component.

[0152] Examples of the lipids include lipid nanoparticles, liposomes, extracellular vesicles (EVs), etc. When the lipids are lipid nanoparticles, the lipid-nucleic acid complex is a nucleic acid lipid nanoparticle. In the lipid-nucleic acid complex, it is preferable that the nucleic acid molecules are encapsulated within the lipids, for example.

[0153] The lipid nanoparticles may include, for example, cationic lipids, non-cationic lipids, sterols (cholesterol), PEG lipids, etc. Examples of non-cationic lipids include neutral lipids, anionic lipids, amphiphilic lipids, etc. Specific examples of each lipid constituting the lipid nanoparticles, their composition, and methods for producing them can be found, for example, in International Publication No. 2009 / 127060.

[0154] Examples of the cationic lipids include cationic lipids containing tertiary amines, such as heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), and N-(1-(2,3-dioleyl Oxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2-dimyristyloxypropane-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide ( DMRIE), 2,3-dioleyloxy-N-[2(spermine-carboxamide)ethyl]-N,N-dimethyl-1-propaneaminium trifluoroacetate (DOSPA), dioctadecylamideglycylspermine (DOGS), 3-dimethylamino-2-(cholesta-5-ene-3-β-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholesta-5-ene-3β-oxy)-3'-oxapentoxy]-3-dimethyl Examples include til-1-(cis,cis-9',1-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dirinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dirinoleylcarbamyl-3-dimethylaminopropane (DLinCDAP), and mixtures thereof.

[0155] The noncationic lipids are 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dipalmitoyl-sn-glycero Cello-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 dietherPC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero -3-Phosphocholine (C16LysoPC), 1,2-Dilinolenoyl-sn-Glycerol-3-Phosphocholine, 1,2-Diarachidonoyl-sn-Glycerol-3-Phosphocholine, 1,2-Didocosahexaenoyl-sn-Glycerol-3-Phosphocholine, 1,2-Difitanoyl-sn-Glycerol-3-Phosphoethanolamine (ME16.0PE), 1,2-Distearoyl-sn-Glycerol-3-Phosphoethanolamine, 1,2-Dilinoleoyl-sn Examples include glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof.

[0156] The PEG-modified lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, DMG-PEG, PEG-c-DOMG (also called PEG-DOMG), PEG-DSG, PEG-DPG, and mixtures thereof. Examples of DMG-PEG include 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000-DMG).

[0157] Examples of the sterols include cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof.

[0158] In the compositions of the present disclosure, the components such as the target antigen, the MHCII molecule expression inducer, the invariant chain expression inhibitor, and the immune checkpoint inhibitor may be, for example, mixed, or some or all of them may be unmixed. In the latter case, each component may be provided, for example, separated into two or more compartments. In this case, the compositions of the present disclosure may also be, for example, a composition kit of the present disclosure. The kit may include, for example, instructions or a manual describing how to use the compositions of the present disclosure.

[0159] The compositions of this disclosure may be used, for example, in vitro or in vivo. The compositions of this disclosure may also be used, for example, as research reagents or as pharmaceuticals. In the former case, the compositions of this disclosure may also be called test reagents or test kits.

[0160] The subjects to whom the compositions of this disclosure are administered are not particularly limited. When the compositions of this disclosure are used in vivo, the subjects (subjects to administration) include, for example, humans and non-human animals. Examples of non-human animals include mammals such as mice, rats, rabbits, dogs, cats, cattle, horses, pigs, monkeys, dolphins, and sea lions. When the compositions of this disclosure are used in vitro, the subjects to administration include, for example, cells, tissues, organs, etc., where examples of cells include cells collected from living organisms, cultured cells, etc., and examples of tissues or organs include tissues (living tissues) or organs collected from living organisms, etc. Examples of cells include immune cells such as T cells, B cells, NK cells, and dendritic cells; etc.

[0161] When the compositions of this disclosure are used in vivo, the recipients may be healthy individuals who do not suffer from a specific disease, individuals who may have a specific disease, or patients who have a specific disease, but it is preferable that the recipients are those for whom treatment of a specific disease is desired. Examples of such diseases include cancer. Examples of such cancers include cancers for which the tumor-associated antigen is known, and specific examples include skin cancer, colorectal cancer, kidney cancer, breast cancer, lung cancer, hematological cancer, brain tumor, bladder cancer, pancreatic cancer, liver cancer, uterine cancer, ovarian cancer, bone tumor, stomach cancer, esophageal cancer, tongue cancer, nasopharyngeal cancer, etc.

[0162] The conditions for use (administration conditions) of the compositions disclosed herein are not particularly limited, and the administration form, timing of administration, dosage, etc. can be appropriately set depending on the type of active ingredient in the composition, the type of target for administration, etc.

[0163] Methods of administering the composition of this disclosure include, for example, parenteral administration such as intravenous administration, intramuscular administration, and subcutaneous administration.

[0164] The dosage of the composition disclosed herein is an amount that can induce an immune response to the target antigen in the recipient, i.e., an effective dose or therapeutically effective dose. The dosage can be appropriately determined, for example, by the age, weight, symptoms, etc., of the recipient. When the composition disclosed herein is administered intramuscularly to a mouse, the single dose can be, for example, 0.01 to 1 mg / kg body weight. When the composition disclosed herein is administered intravenously to a human, the daily dose can be, for example, 0.001 to 100 g. Furthermore, when the composition disclosed herein is administered intravenously to a human, the daily dose can also be, for example, 0.01 to 1000 mg / kg body weight.

[0165] The composition of this disclosure is administered one or more times. The number of times is, for example, two, three, four, five or more times. The number of times may be determined as appropriate while confirming the preventive effect on the target of administration. When multiple doses are administered, the interval between doses can be determined as appropriate while confirming the therapeutic effect on the target of administration, for example, once a day, once a week, once every two weeks, once a month, once every three months, once every six months, etc.

[0166] The compositions of this disclosure can prevent or alleviate at least one symptom caused by a specific disease in the subject receiving the treatment. The prevention of such symptoms can be evaluated subjectively or objectively, and specific examples include self-assessment by the subject receiving the treatment; assessment by a physician; QOL (Quality of Life) assessment; and assessment of delay in the progression of symptoms of the specific disease or alleviation of symptoms of the specific disease. The objective assessment may be conducted by animals or by humans.

[0167] The compositions of this disclosure can induce an immune response to a target antigen contained in the neoself antigen by expressing the neoself antigen, for example, as described above.

[0168] The immune response induced by the compositions of this disclosure is, for example, an immune response by T cells and / or B cells, as shown in the examples described below. The immune response may also be an immune response by immune cells other than T cells, specifically myeloid cells such as dendritic cells and macrophages, and lymphocytes such as B cells and NK cells. The T cells are, for example, CD4 ＋ T cells and CD8 ＋ Examples include T cells. If the immune response is an immune response mediated by B cells, the immune response may also be, for example, the induction of antibodies against the target antigen.

[0169] Furthermore, the compositions of this disclosure can be used, for example, to activate immune cells responsive to a target antigen. The immune cells are, for example, CD4 ＋ T cells, CD8 ＋ Examples include T cells and B cells.

[0170] The compositions of this disclosure can, for example, activate the target antigen-responsive immune cells, and therefore, when used in combination with the immune checkpoint inhibitor, the target antigen-responsive immune cells can be further activated. For this reason, the compositions of this disclosure can be used, for example, in combination with the immune checkpoint inhibitor.

[0171] The compositions of this disclosure can be used, for example, to induce an immune response to the target antigen, and are expected to be suitably used in pharmaceutical compositions such as vaccine compositions.

[0172] <Pharmaceutical Compositions> In another embodiment, the Disclosure provides pharmaceutical compositions. The pharmaceutical compositions of the Disclosure comprise the compositions of the Disclosure. The pharmaceutical compositions of the Disclosure also comprise immune checkpoint inhibitors, which are used in combination with the compositions of the Disclosure. The pharmaceutical compositions of the Disclosure can induce an immune response to a target antigen and can therefore be used in the treatment of cancer or infectious diseases. The pharmaceutical compositions of the Disclosure are expected to enable suitable treatment of cancer or infectious diseases because they can induce an immune response to a target antigen.

[0173] The pharmaceutical compositions of this disclosure may further include pharmaceutically acceptable carriers. Examples of such carriers include suspensions for administering the active ingredient, solubilizers, stabilizers, isotonic agents, preservatives, anti-adsorption agents, surfactants, diluents, media, pH adjusters, analgesics, buffers, sulfur-containing reducing agents, antioxidants, etc., and can be appropriately added insofar as they do not interfere with the effects of this disclosure.

[0174] The suspending agent is not particularly limited and includes, for example, methylcellulose, polysorbate 80, hydroxyethylcellulose, gum arabic, tragacanth powder, sodium carboxymethylcellulose, polyoxyethylene sorbitan monolaurate, and the like.

[0175] The aforementioned solution additive is not particularly limited and includes, for example, polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, macrogol, castor oil fatty acid ethyl ester, and the like.

[0176] The stabilizer is not particularly limited and examples include dextran 40, methylcellulose, gelatin, sodium sulfite, sodium metasulfate, and the like.

[0177] The isotonic agent is not particularly limited, and examples include D-mannitol and sorbitol.

[0178] The preservative is not particularly limited and examples include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, sorbic acid, phenol, cresol, chlorocresol, and the like.

[0179] The adsorption inhibitor is not particularly limited and includes, for example, human serum albumin, lecithin, dextran, ethylene oxide propylene oxide copolymer, hydroxypropyl cellulose, methylcellulose, hydrogenated castor oil, polyethylene glycol, and the like.

[0180] The sulfur-containing reducing agent is not particularly limited and includes, for example, N-acetylcysteine, N-acetylhomocysteine, thioctic acid (α-lipoic acid), thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodium thiosulfate, glutathione, and thioalkanoates having 1 to 7 carbon atoms, which have a sulfohydryl group.

[0181] The antioxidant is not particularly limited, and examples include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbic acid palmitate, L-ascorbic acid stearate, sodium bisulfite, sodium sulfite, triamyl gallate, propyl gallate, or chelating agents such as ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, and sodium metaphosphate.

[0182] The pharmaceutical composition of the present invention may further contain, as appropriate, commonly added components such as inorganic salts like sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, and sodium bicarbonate; organic salts like sodium citrate, potassium citrate, and sodium acetate; and sugars like glucose.

[0183] The pharmaceutical compositions of this disclosure may be in the form of a kit, for example, similar to the composition kit of this disclosure.

[0184] The administration conditions for the pharmaceutical compositions of this disclosure can be found by referring to the description of the administration conditions for the compositions of this disclosure.

[0185] <Method for Inducing an Immune Response to a Target Antigen and Method for Activating Target Antigen-Responsive T Cells> In another embodiment, the present disclosure provides a method for inducing an immune response to a target antigen or a method for activating immune cells responsive to a target antigen. The method for inducing an immune response to a target antigen according to the present disclosure uses the composition and / or the pharmaceutical composition of the present disclosure. The method for activating target antigen-responsive T cells according to the present disclosure uses the composition and / or the pharmaceutical composition of the present disclosure. The method for activating target antigen-responsive B cells according to the present disclosure uses the composition and / or the pharmaceutical composition of the present disclosure. The method for inducing antibodies against a target antigen according to the present disclosure uses the composition and / or the pharmaceutical composition of the present disclosure. According to the induction method of the present disclosure, an immune response to a target antigen can be induced. According to the activation method of the present disclosure, immune cells responsive to the target antigen can be activated.

[0186] The induction and activation methods of this disclosure include, for example, administering the compositions and / or pharmaceutical compositions of this disclosure to a subject. The subject is preferably an animal, including a human. The induction and activation methods of this disclosure can be used, for example, in vitro or in vivo.

[0187] <Methods of Treatment> In another embodiment, the Disclosure provides methods for treating cancer or infectious diseases (hereinafter also referred to as "methods of treatment"). The method of treating cancer according to the Disclosure includes the step of administering (using) the composition and / or pharmaceutical composition of the Disclosure to a subject. The method of treating cancer according to the Disclosure is expected to be suitable for treating cancer because it can induce an immune response to the target antigen even in an environment in which cancer is present. The method of treating infectious diseases according to the Disclosure also includes the step of administering (using) the composition and / or pharmaceutical composition of the Disclosure to a subject. The method of treating infectious diseases according to the Disclosure is expected to be suitable for treating infectious diseases, particularly preventive measures, because it can induce an immune response even to antigens that cannot adequately induce an immune response on their own.

[0188] In the treatment method of the present disclosure, the target antigen or the polynucleotide encoding it and the MHC class II molecule expression inducer in the composition may be administered together or separately. In the latter case, the order in which the target antigen or the polynucleotide encoding it and the MHC II molecule expression inducer are administered is not particularly limited, for example, the MHC II molecule expression inducer may be administered to the subject before, during, and / or after the administration of the target antigen or the polynucleotide encoding it.

[0189] The treatment method of this disclosure may include, for example, administering an immune checkpoint inhibitor to the subject before, during, and / or after administration of the composition and / or pharmaceutical composition.

[0190] <Use> In another embodiment, the Disclosure is the use of the compositions and / or pharmaceutical compositions of the Disclosure for use in methods of treating cancer. The Disclosure is the use of the compositions and / or pharmaceutical compositions of the Disclosure for manufacturing pharmaceutical compositions for use in treating cancer. The Disclosure is the use of the compositions and / or pharmaceutical compositions of the Disclosure for use in methods of treating infectious diseases. The Disclosure is the use of the compositions and / or pharmaceutical compositions of the Disclosure for manufacturing pharmaceutical compositions for use in treating infectious diseases.

[0191] Next, examples of the present disclosure will be described. However, the present disclosure is not limited to the following examples. Commercial reagents were used according to their protocols unless otherwise specified. Note that "mol / l" may also be denoted as "M".

[0192] [Example 1] We confirmed that antitumor activity can be induced in skin cancer model mice by administering an LNP vaccine containing mRNA of gp100, TYRP2, and MHC class II (MHC-II) molecules.

[0193] (1) Preparation of a DNA template for mRNA transcription I-A is a polynucleotide that encodes the MHCII molecule and is registered in GenBank accession number NM_010378.3.ｂ The α chain (SEQ ID NO: 24) and I-A registered with GenBank accession number NM_207105.3 ｂ The β-chain (SEQ ID NO: 26) was used. As polynucleotides encoding tumor-associated antigens of skin cancer, mouse melanoma gp100 (SEQ ID NO: 28), registered in GenBank accession number MH882516.1, and mouse tyrosinase-related protein TYRP2 (SEQ ID NO: 32), registered in GenBank accession number NM_010024.3, were used. The above I-A ｂ The α chain and I-A ｂ The nucleotide sequence (SEQ ID NO: 37) obtained by linking the coding region (CDS) of the β chain of TYRP2 with P2A (SEQ ID NO: 36) was cloned into the pME18s vector. The CDS sequences of the extracellular region of gp100 and the extracellular region of TYRP2 were cloned into the pME18s vector, respectively. The T7 promoter and 5'-untranslated region were positioned upstream of the CDS sequence. Furthermore, the 3'-untranslated region and polyA tail (polyA sequence) were positioned downstream of the CDS sequence, and the restriction enzyme (NotI) recognition site was positioned downstream of the polyA tail. After cloning, Proteinase K (Takara Bio, 9034) and SDS were added to the pME18s vector to remove RNase. RNase was removed from a pME18s empty vector without an insert in the same manner. After the aforementioned removal, restriction enzyme NotI (NEB, R0189L) was added to the pME18s vector. This linearized the pME18s vector and prepared a DNA template for mRNA transcription.

[0194] I-A ｂThe amino acid sequence of the α chain (SEQ ID NO: 23) is MPRSRALILGVLALTTMLSLCGGEDDIEADHVGTYGISVYQSPGDIGQYTFEFDGDELFYVDLDKKETVWMLPEFGQLASFDPQGGLQNIAVVKHNLGVLTKRSNSTPATNEAPQATVFPKSPVLLGQPNTLICFVDNIFPPVINITWLRNSKSVADGVYETSFFVNRDYSFHKLSYLTFIPSDDDIYDCKVEHWGLEEPVLKHWEPEIPAPMSELTETVVCALGLSVGLVGIVVGTIFIIQGLRSGGTSRHPGPL

[0195] I-A ｂPolynucleotide encoding the α chain (SEQ ID NO: 24) 5'-ATGCCGCGCAGCAGAGCTCTGATTCTGGGGGTCCTCGCCCTGACCACCATGCTCAGCCTCTGTGGAGGTGAAGACGACATTGAGGCCGACCACGTAGGCACCTATGGTATAAGTGTATATCAGTCTCCTGGAGACATTGGCCAGTACACATTTGAATTTGATGGTGATGAGTTGTTCTATGTGGACTTGGATAAGAAGGAGACTGTCTGGATGCTTCCTGAGTTTGGCCAATTGGCAAGCTTTGACCCCCAAGGTGGACTGCAAAACATAGCTGTAGTAAAACACAACTTGGGAGTCTTGACTAAGAGGTCAAATTCCACCCCAGCTACCAATGAGGCTCCTCAAGCGACTGTGTTCCCCAAGTCCCCTGTGCTGCTGGGTCAGCCCAACACCCTCATCTGCTTTGTGGACAACATCTTCCCTCCTGTGATCAACATCACATGGCTCAGAAATAGCAAGTCAGTCGCAGACGGTGTTTATGAGACCAGCTTCTTCGTCAACCGTGACTATTCCTTCCACAAGCTGTCTTATCTCACCTTCATCCCTTCTGACGATGACATTTATGACTGCAAGGTGGAACACTGGGGCCTGGAGGAGCCGGTTCTGAAACACTGGGAACCTGAGATTCCAGCCCCCATGTCAGAGCTGACAGAGACTGTGGTCTGTGCCCTGGGGTTGTCTGTGGGCCTTGTGGGCATCGTGGTGGGCACCATCTTCATCATTCAAGGCCTGCGATCAGGTGGCACCTCCAGACACCCAGGGCCTTTATGA-3'

[0196] I-A ｂβ-chain amino acid sequence (SEQ ID NO: 25) MALQIPSLLLSAAVVVLMVLSSPGTEGGDSERHFVYQFMGECYFTNGTQRIRYVTRYIYNREEYVRYDSDVGEHRAVTELGRPDAEYWNSQPEILERTRAELDTVCRHNYEGPETHTSLRRLEQPNVVISLSRTEALNHHNTLVCSVTDFYPAKIKVRWFRNGQEETVGVSSTQLIRNGDWTFQVLVMLEMTPRRGEVYTCHVEHPSLKSPITVEWRAQSESAWSKMLSGIGGCVLGVIFLGLGLFIRHRSQKGPRGPPPAGLLQ

[0197] I-A ｂPolynucleotide encoding the β chain (SEQ ID NO: 26) 5'-ATGGCTCTGCAGATCCCCAGCCTCCTCCTCTCGGCTGCTGTGGTGGTGCTGATGGTGCTGAGCAGCCCAGGGACTGAGGGCGGAGACTCCGAAAGGCATTTCGTGTACCAGTTCATGGGCGAGTGCTACTTCACCAACGGGACGCAGCGCATACGATATGTGACCAGATACATCTACAACCGGGAGGAGTACGTGCGCTACGACAGCGACGTGGGCGAGCACCGCGCGGTGACCGAGCTGGGGCGGCCAGACGCCGAGTACTGGAACAGCCAGCCGGAGATCCTGGAGCGAACGCGGGCCGAGCTGGACACGGTGTGCAGACACAACTACGAGGGGCCGGAGACCCACACCTCCCTGCGGCGGCTTGAACAGCCCAATGTCGTCATCTCCCTGTCCAGGACAGAGGCCCTCAACCACCACAACACTCTGGTCTGCTCAGTGACAGATTTCTACCCAGCCAAGATCAAAGTGCGCTGGTTCCGGAATGGCCAGGAGGAGACGGTGGGGGTCTCATCCACACAGCTTATTAGGAATGGGGACTGGACCTTCCAGGTCCTGGTCATGCTGGAGATGACCCCTCGGCGGGGAGAGGTCTACACCTGTCACGTGGAGCATCCCAGCCTGAAGAGCCCCATCACTGTGGAGTGGAGGGCACAGTCTGAGTCTGCCTGGAGCAAGATGTTGAGCGGCATCGGGGGCTGCGTGCTTGGGGTGATCTTCCTCGGGCTTGGCCTTTTCATCCGTCACAGGAGTCAGAAAGGACCTCGAGGCCCTCCTCCAGCAGGGCTCCTGCAGTGA-3'

[0198] Amino acid sequence of melanoma gp100 (SEQ ID NO: 27) (Underlined region is the extracellular region) MGVQRRSFLPVLVLSALLAVGALEGSRNQDWLGVPRQLVTKTWNRQLYPEWTEVQGSNCWRGGQVSLRVINDGPTLVGANASFSIALHFPGSQKVLPDGQVIWANNTIINGSQVWGGQPVYPQEPDDACVFPDGGPCPSGPKPPKRSFVYVWKTWGKYWQVLGGPVSRLSIATGHAKLGTHTMEVTVYHRRGSQSYVPLAHASSTFTITDQVPFSVSVSQLQALDGETKHFLRNHPLIFALQLHDPSGYLAEADLSYTWDFGDGTGTLISRALDVTHTYLESGSVTAQVVLQA AIPLVSCGSSPVPGTTDGYMPTAEAPGTTSRQGTTTKVVGTTPGQMPTTQPSGTTVVQMPTTEVTATTSEQMLTSAVIDTTLAEVSTTEGTGTTPTRPSGTTVAQATTTEGPDASPLLPTQSSTGSISPLLDDTDTIMLVKRQVPLDCVLYRYGSFSLALDIVQGI ESAEILQAVPFSEGDAFELTVSCQGGLPKEACMDISSPGCQPPAQRLCQSVPPSPDCQLVLHQVLKGGSGTYCLNVSLADANSLAVASTQLVVPGQDGGLGQAPLLVGILLVLVAVVLASLIHRHRLKKQGSVSQMPHGSTHWLRLPPVFRARGLGENSPLLSGQQV

[0199]

[0200] The amino acid sequence of TYRP1 (SEQ ID NO: 29) (the underlined region is the extracellular region): MKSYNVLPLAYISLFLMLFYQVWAQFPRECANIEALRRGVCCPDLLPSSGPGTDPCGSSSGRGRCVAVIADSRPHSRHYPHDGKDDREAWPLRFFNRTCQCNDNFSGHNCGTCRPGWRGAACNQKILTVRRNLLDLSPEEKSHFVRALDMAKRTTHPQFVIATRRLEDILGPDGNTPQFENISVYNYFVWTHYYSVKKTFLGTGQESFGDVDFSHEGPAFLTWHRYHLLQLERDMQEMLQEPSFSLPYWNF ATGKNVCDVCTDDLMGSRSNFDSTLISPNSVFSQWRVVCESLEEYDTLGTLCNSTEGGPIRRNPAGNVGRPAVQRLPEPQDVTQCLEVRVFDTPPFYSNSTDSFRNTVEGYSAPTGKYDPAVRSLHNLAHLFLNGTGGQTHLS PNDPIFVLLHTFTDAVFDEWLRRYNADISTFPLENAPIGHNRQYNMVPFWPPVTNTEMFVTAPDNLGYAYEVQWPGQEFTVSEIITIAVVAALLLVAAIFGVASCLIRSRSTKNEANQPLLTDHYQRYAEDYEELPNPNHSMV

[0201]

[0202] The amino acid sequence of TYRP2 (SEQ ID NO: 31) (the underlined region is the extracellular region): MGLVGWGLLLGCLGCGILLRARAQFPRVCMTLDGVLNKECCPPLGPEATNICGFLEGRGQCAEVQTDTRPWSGPYILRNQDDREQWPRKFFNRTCKCTGNFAGYNCGGCKFGWTGPDCNRKKPAILRRNIHSLTAQEREQFLGALDLAKKSIHPDYVITTQHWLGLLGPNGTQPQIANCSVYDFFVWLHYYSVRDTLLGPGRPYKAIDFSHQGPAFVTWHRYHLLWLERELQRLTGNESFA LPYWNFATGKNECDVCTDELLGAARQDDPTLISRNSRFSTWEIVCDSLDDYNRRVTLCNGTYEGLLRRNKVGRNNEKLPTLKNVQDCLSLQKFDSPPFFQNSTFSFRNALEGFDKADGTLDSQVMNLHNLAHSFLNGT NALPHSAANDPVFVVLHSFTDAIFDEWLKRNNPSTDAWPQELAPIGHNRMYNMVPFFPPVTNEELFLTAEQLGYNYAVDLSEEEAPVWSTTLSVVIGILGAFVLLLGLLAFLQYRRLRKGYAPLMETGLSSKRYTEEA

[0203]

[0204] - T7 promoter sequence (SEQ ID NO: 33) 5'-TAATACGACTCACTATA-3' - 5'-UTR sequence (SEQ ID NO: 34) 5'-TCTTCTGGTCCCCACAGACTCAGAGAGAACCC-3' - 3'-UTR sequence (SEQ ID NO: 35) 5'-GCTGGAGCCTCGGTGGCCTAGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC-3' - P2A sequence (SEQ ID NO: 36) 5'-GGAAGCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGATGTTGAAGAAAACCCCGGGCCT-3'

[0205] ・I-A ｂ The α chain and I-A ｂ The base sequence obtained by linking the CDS of the β chain with P2A (I-A ｂ α-P2A-I-A b

[0206] (2) In vitro transcription mRNA was produced using an in vitro transcription system (IVT reaction system). The reagents used were RiboMAX Large Scale RNA Production Systems (Promega, P1300), m1ΨTP (TriLink, N-1081), and CleanCap® Reagent AG (3'-OMe) (TriLink, N-7413). The reagents of the IVT reaction system and the DNA template were incubated at 37°C for 5-6 hours to obtain the composition of the IVT reaction solution described below. Subsequently, the DNA template in the reaction solution was removed with RQ1 RNase-Free DNAse(s). Subsequently, the mRNA in the reaction solution was purified using the RNeasy Mini Kit (QIAGEN, 74106). For C57BL / 6 mouse administration mRNA, an ethanol-containing buffer was added to the reaction solution after the IVT reaction system reaction to remove double-stranded RNA (dsRNA), a byproduct of the IVT reaction system. Subsequently, the mRNA in the reaction solution was purified using 3M sodium acetate (pH 5.2) (Promega, P135A) and isopropanol (Nacalai Tesque, 29113-95).

[0207]

[0208] (3) Preparation of lipid nanoparticle (LNP) vaccine The prepared mRNA was mixed in the ratio shown in Table 2 below and adjusted to a final concentration of 0.276 mg / mL with 50 mM sodium acetate buffer (pH 4.0) to obtain the mRNA mixture.

[0209]

[0210] The lipids used were heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102, BroadPharm, BP-25499), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, MERCK, 850365P), cholesterol (MERCK, C8667), and 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000-DMG, MERCK, 880151P). Each lipid was mixed in a predetermined mixing ratio (SM-102:DSPC:Cholesterol:PEG2000-DMG = 50:10:38.5:1.5), and the mixture was prepared so that the final total concentration of all lipids was 25 mM, thereby obtaining a lipid mixture. The mRNA mixture (M) and the lipid mixture (L) were mixed in a ratio of M:L = 3:1, and the stock solution for the LNP vaccine was prepared using NanoAssemblr® Ignite® or Ignite+® (manufactured by Neppageen). Subsequently, the stock solution was dialyzed with 8% sucrose buffer (20 mM Tris·HCl, pH 7.5) using Slide-A-Lyzer® Dialesis Cassettes, 10K MWCO (Thermo Fisher, 66810). The mixture was then diluted 2-fold with 8% sucrose buffer to prepare the LNP vaccine.

[0211] (4) For the antitumor effect on skin cancer model mice, C57BL / 6 mice (Nippon SLC Co., Ltd.) were used. The LNP vaccine was administered to each mouse at a dose of 100 μl (10.3 μg in mRNA equivalent, the same applies hereafter) once every two weeks for a total of three doses. The administration method was intramuscular injection. One week after the three doses of the LNP vaccine were administered, the B16F1 cells (melanoma) were placed subcutaneously in the mice at a dose of 7 × 10⁻⁶. ５ The mice were inoculated with cells / mice. Tumor volume was then measured twice a week at predetermined intervals (1, 2, 3, 4, or 5 weeks) after inoculation. The negative control group was administered the same procedure, except that they did not receive the LNP vaccine. These results are shown in Figure 2.

[0212] Figure 2 is a graph showing tumor volume. In Figure 2, the horizontal axis represents the number of days after melanoma inoculation, and the vertical axis represents tumor volume. In Figure 2, the upper panel shows the results for negative control (untreated mice) and control 1 (MHC-II alone) from left to right, while the lower panel shows the results for control 2 (TAA alone: ​​gp100 + Tyrp2 only) and the example (MHC-II + TAA: gp100 + Tyrp2 + MHCII). As shown in Figure 2, tumor volume increased over time in the negative control, control 1, and control 2. In contrast, the increase in tumor volume was significantly suppressed in the MHC-II + TAA administration group (gp100 + Tyrp2 + MHCII). Since tumor volume increased in control 1 and control 2, it was found that combining the MHCII molecule with tumor-associated antigens induces an immune response to tumor-associated antigens, thereby inducing antitumor activity against melanoma.

[0213] [Example 2] We confirmed that antitumor activity can be induced in colorectal cancer model mice by administering an LNP vaccine containing gp70 and MHC-II mRNA.

[0214] (1) Preparation of a DNA template for mRNA transcription As the polynucleotide encoding the tumor-associated antigen of colorectal cancer, we used the polynucleotide encoding gp70 (SEQ ID NO: 39), which is registered in GenBank with accession number KP087798.1. As the polynucleotide encoding the MHCII molecule, we used I-A, which is registered in GenBank with accession number AY452201.1. d I-A, registered with GenBank accession number BC010322.1, is part of the α chain (SEQ ID NO: 41). ｄ The β-chain (SEQ ID NO: 43) was used. gp100, TYRP2, I-A ｂ α chain and I-A ｂ Instead of the polynucleotide encoding the β chain, use gp70, I-A ｄ α chain and I-A ｄA DNA template for mRNA transcription was prepared in the same manner as in Example 1(1), except that a polynucleotide encoding the β chain was used.

[0215] The amino acid sequence of gp70 (SEQ ID NO: 38) (the underlined region is the extracellular region) MESTTLSKPFKNQVNPWGPLIVLLILGGVNPVALGNSPHQVFNLSWEVTNGDRETVWAITGNHPLWTWWPDLTPDLCMLALHGPSYWGLEYRAPFSPPPGPPCCSGSSDSTPGCSRDCEEPLTSYTPRCNTAWNRLKLSKVTHAHNEGFYVCPGPHRPRWARSCGGPESFYCASWGCETTGRASWKPSSSWDYITVSNNLTSDQATPVCKGNEWCNSLTIRFTSFGKQATSWVTGHWWGLRLYVSGHDPGLIFGIRLKITDSGPRVPIGPNPVLSDRRPPSRPRPTRSPPPSNSTPTETPLTLPEPPPAGVENRLLN LVKGAYQALNLTSPDKTQECWLCLVSGPPYYEGVAVLGTYSNHTSAPANCSVASQHKLTLSEVTGQGLCIGAVPKTHQVLCNTTQKTSDGSYYLAAPTGTTWACSTGLTPCISTTILDLTTDYCVLVELWPRVTYHSPSYVYHQFERRAKYKREPVSLTLALLLGGLTMGGIAAGV GTGTTALVATQQFQQLQAAMHDDLKEVEKSITNLEKSLTSLSEVVLQNRRGLDLLFLKEGGLCAALKEECCFYADHTGLVRDSMAKLRERLSQRQKLFESQQGWFEGLFNKSPWFTTLISTIMGPLIILLLILLFGPCILNRLVQFIKDRISVVQALVLTQQYHQLKTIGDCKSRE

[0216]

[0217] I-A ｄ α-chain amino acid sequence (SEQ ID NO: 40) (AAR19089.1) MPCSRALILGVLALNTMLSLCGGEDVIEADHVGFYGTTVYQSPGDIGQYTHEFDGDELFYVDLDKKKTVWRLPEFGQLILFEPQGGLQNIAAEKHNLGILTKRSNFTPATNEAPQATVFPKSPVLLGQPHTLICFVDNIFPPVINITWLRNSKSVTDGVYETSFLVNRDHSFHKLSYLTFIPSDDDIYDCKVEHWGLEEPVLKHWEPEIPAPMSGLTETVVCALGLSVGLVGIVVGTIFIIQGLRSGGTSRHPGPL

[0218] I-A ｄPolynucleotide encoding the α chain (SEQ ID NO: 41) (AY452201.1) 5'-ATGCCGTGCAGCAGAGCTCTGATTCTGGGGGTCCTCGCCCTGAACACCATGCTCAGCCTCTGCGGAGGTGAAGACGTCATTGAGGCCGACCACGTAGGCTTCTATGGTACAACTGTTTATCAGTCTCCTGGAGACATTGGCCAGTACACACATGAATTTGATGGTGATGAGTTGTTCTATGTGGACTTGGATAAGAAGAAAACTGTCTGGAGGCTTCCTGAGTTTGGCCAATTGATACTCTTTGAGCCCCAAGGTGGACTGCAAAACATAGCTGCAGAAAAACACAACTTGGGAATCTTGACTAAGAGGTCAAATTTCACCCCAGCTACCAATGAGGCTCCTCAAGCGACTGTGTTCCCCAAGTCCCCTGTGCTGCTGGGTCAGCCCCACACCCTTATCTGCTTTGTGGACAACATCTTCCCACCTGTGATCAACATCACATGGCTCAGAAATAGCAAGTCAGTCACAGACGGCGTTTATGAGACCAGCTTCCTCGTCAACCGTGACCATTCCTTCCACAAGCTGTCTTATCTCACCTTCATCCCTTCTGATGATGACATTTATGACTGCAAGGTGGAGCACTGGGGCCTGGAGGAGCCGGTTCTGAAACACTGGGAACCTGAGATTCCAGCCCCCATGTCAGGGCTGACAGAAACTGTGGTGTGTGCCCTGGGGTTGTCTGTGGGCCTTGTGGGCATCGTGGTGGGCACCATCTTCATCATTCAAGGCCTGCGATCAGGTGGCACCTCCAGACACCCAGGGCCTTTATGA-3'

[0219] I-A ｄβ-chain amino acid sequence (SEQ ID NO: 42) MALQIPSLLLSAAVVVLMVLSSPGTEGGDSERHFVVQFKGECYYTNGTQRIRLVTRYIYNREEYVRYDSDVGEYRAVTELGRPDAEYWNSQPEILERTRAEVDTACRHNYEETEVPTSLRRLEQPNVAISLSRTEALNHHNTLVCSVTDFYPAKIKVRWFRNGQEETVGVSSTQLIRNGDWTFQVLVMLEMTPHQGEVYTCHVEHPSLKSPITVEWRAQSESARSKMLSGIGGCVLGVIFLGLGLFIRHRSQKGPRGPPPAGLLQ

[0220] I-A ｄPolynucleotide encoding the β chain (SEQ ID NO: 43) (BC010322.1) 5'-ATGGCTCTGCAGATCCCCAGCCTCCTCCTCTCGGCTGCTGTGGTGGTGCTGATGGTGCTGAGCAGCCCAGGGACTGAGGGCGGAGACTCCGAAAGGCATTTCGTGGTCCAGTTCAAGGGCGAGTGCTACTACACCAACGGGACGCAGCGCATACGGCTCGTGACCAGATACATCTACAACCGGGAGGAGTACGTGCGCTACGACAGCGACGTGGGCGAGTACCGCGCGGTGACCGAGCTGGGGCGGCCAGACGCCGAGTACTGGAACAGCCAGCCGGAGATCCTGGAGCGAACGCGGGCCGAGGTGGACACGGCGTGCAGACACAACTACGAGGAGACGGAGGTCCCCACCTCCCTGCGGCGGCTTGAACAGCCCAATGTCGCCATCTCCCTGTCCAGGACAGAGGCCCTCAACCACCACAACACTCTGGTCTGTTCGGTGACAGATTTCTACCCAGCCAAGATCAAAGTGCGCTGGTTCAGGAATGGCCAGGAGGAGACAGTGGGGGTCTCATCCACACAGCTTATTAGGAATGGGGACTGGACCTTCCAGGTCCTGGTCATGCTGGAGATGACCCCTCATCAGGGAGAGGTCTACACCTGCCATGTGGAGCATCCCAGCCTGAAGAGCCCCATCACTGTGGAGTGGAGGGCACAGTCCGAGTCTGCCCGGAGCAAGATGTTGAGCGGCATCGGGGGCTGCGTGCTTGGGGTGATCTTCCTCGGGCTCGGCCTTTTCATCCGTCACAGGAGTCAGAAAGGACCTCGAGGCCCTCCTCCAGCAGGGCTCCTGCAGTGA-3'

[0221] ・I-A ｄ α chain of and I-A ｄ CDS of the β chain of ligated with P2A (I-A ｄ α-P2A-I-A ｄ

[0222] (2) In vitro transfer The gp70 and I-A prepared in Example 2(1) in the same manner as in Example 1(2) above. ｄ α-P2A-I-A ｄ mRNA was prepared from a β DNA template.

[0223] (3) Preparation of Lipid Nanoparticle (LNP) Vaccine In the preparation of the LNP vaccine, the LNP vaccine was prepared in the same manner as in Example 1 (3) above, except that either gp70 mRNA alone or gp70 mRNA and MHCII molecule mRNA were used as the mRNA. The mixing ratio of the mRNA mixture was prepared according to the ratios shown in Table 3 below.

[0224]

[0225] (4) Antitumor effect on colorectal cancer model mice The tumor volume was measured in the same manner as in Example 1 (4), except that BALB / c mice (Nippon SLC Co., Ltd.) were used instead of C57BL / 6 mice, and CT26 cells (colorectal cancer) were used instead of B16F1 cells (melanoma). The CT26 cells (colorectal cancer) were placed subcutaneously in the mice at a rate of 5 × 10⁻⁶ ５ The mice were inoculated to produce cells / mice. The negative control was administered the same way, except that the LNP vaccine was not administered, and the tumor volume was measured. These measurement results are shown in Figure 3.

[0226] Figure 3 is a graph showing tumor volume. In Figure 3, the horizontal axis represents the number of days after inoculation with colorectal cancer cells, and the vertical axis represents tumor volume. In Figure 3, the upper panel shows the results for negative control (untreated mice) and control 1 (MHC-II alone) from left to right, while the lower panel shows the results for control 2 (TAA alone: ​​gp70 only) and the example (MHC-II + TAA: gp70 + MHC-II). As shown in Figure 3, tumor volume increased over time in the negative control, control 1, and control 2. In contrast, the increase in tumor volume was completely suppressed in the MHC-II + TAA administration group (gp70 + MHC-II). Since tumor volume increased in control 1 and control 2, it was found that combining the MHC-II molecule with tumor-associated antigens induces an immune response to tumor-associated antigens, thereby inducing antitumor activity against colorectal cancer.

[0227] [Example 3] We confirmed that antitumor activity can be induced in kidney cancer model mice by administering an LNP vaccine containing gp70 and MHC-II mRNA.

[0228] (1) Preparation of a DNA template for mRNA transcription. As polynucleotides encoding tumor-associated antigens of kidney cancer, gp70 and I-A used in Example 2(1) above. ｄ α-P2A-I-A ｄ mRNA was prepared from a β DNA template.

[0229] (2) In vitro transcription mRNA was prepared from the DNA template prepared in Example 4(1) in the same manner as in Example 2(2).

[0230] (3) Preparation of lipid nanoparticle (LNP) vaccine Using the mRNA from Example 3 (3), an LNP vaccine was prepared in the same manner as in Example 2 (3).

[0231] (4) Antitumor effect on kidney cancer model mice The tumor volume was measured in the same manner as in Example 2(4) above, except that Renca cells (kidney cancer) were used instead of CT26 cells (colon cancer). The Renca cells (kidney cancer) were placed subcutaneously in mice at a rate of 5 × 10⁻⁶５ The cells were inoculated into mice. These results are shown in Figure 4.

[0232] Figure 4 is a graph showing tumor volume. In Figure 4, the horizontal axis represents the number of days after inoculation with kidney cancer cells, and the vertical axis represents tumor volume. In Figure 4, the upper panel shows the results for negative control (untreated mice) and control 1 (MHC-II alone) from left to right, while the lower panel shows the results for control 2 (TAA alone: ​​gp70 only) and Example 3 (MHC-II + TAA: gp70 + MHC-II). As shown in Figure 4, tumor volume increased over time in the negative control, control 1, and control 2. In contrast, the increase in tumor volume was completely suppressed in the MHC-II + TAA administration group (gp70 + MHC-II). Since tumor volume increased in control 1 and control 2, it was found that combining the MHC-II molecule with tumor-associated antigens induces an immune response to tumor-associated antigens, thereby inducing antitumor activity against kidney cancer.

[0233] [Example 4] We confirmed that antitumor activity could be induced in breast cancer model mice by administering an LNP vaccine containing gp70 and MHC-II mRNA.

[0234] (1) Preparation of a DNA template for mRNA transcription. As polynucleotides encoding tumor-associated antigens of breast cancer, gp70 and I-A used in Example 2(1) above. ｄ α-P2A-I-A ｄ mRNA was prepared from a β DNA template.

[0235] (2) In vitro transcription mRNA was prepared from the DNA template prepared in Example 3(1) in the same manner as in Example 2(2).

[0236] (3) Preparation of lipid nanoparticle (LNP) vaccine Using the mRNA from Example 4(2), an LNP vaccine was prepared in the same manner as in Example 2(3).

[0237] (4) Antitumor effect on breast cancer model mice The tumor volume was measured in the same manner as in Example 2(4) above, except that EMT6 cells (breast cancer) were used instead of CT26 cells (colon cancer). The EMT6 cells (breast cancer) were placed subcutaneously in mice at a rate of 2.5 × 10 ５ The cells were inoculated to form mice. These results are shown in Figure 5.

[0238] Figure 5 is a graph showing tumor volume. In Figure 5, the horizontal axis represents the number of days after inoculation with breast cancer cells, and the vertical axis represents tumor volume. In Figure 5, the upper panel shows the results for negative control (untreated mice) and control 1 (MHC-II alone) from left to right, while the lower panel shows the results for control 2 (TAA alone: ​​gp70 only) and the example (MHC-II + TAA: gp70 + MHC-II). As shown in Figure 5, tumor volume increased over time in the negative control, control 1, and control 2. In contrast, the increase in tumor volume was completely suppressed in the MHC-II + TAA administration group (gp70 + MHC-II). Since tumor volume increased in control 1 and control 2, it was found that combining the MHC-II molecule with tumor-associated antigens induces an immune response to tumor-associated antigens, thereby inducing antitumor activity against breast cancer.

[0239] [Example 5] Antitumor activity against tumor-associated antigens is CD4 ＋ T cells and CD8 ＋ It was confirmed that the cause was T cells.

[0240] In the colorectal cancer mouse model described in Example 2, the antitumor activity after T cell removal was investigated. Specifically, in the colorectal cancer mouse model described in Example 2, the LNP vaccine was administered to mice by intramuscular injection once every two weeks for a total of three doses. Then, one week after the third vaccine administration, 200 μg of anti-CD4 antibody (BioXcell, GK1.5, BE0003-1) and / or 200 μg of anti-CD8 antibody (BioXcell, 2.43, BE0061) were administered intraperitoneally once a week for a total of three doses. On the day following the first administration of anti-CD4 antibody and / or anti-CD8 antibody, tumor cells were administered, and tumor observation was also started. Except for these points, the tumor volume was measured in the same manner as in Example 2(4). The negative control was measured in the same manner except that the LNP vaccine was not administered, and the control was measured in the same manner except that the antibodies were not administered. These measurement results are shown in Figure 6.

[0241] Figure 6 is a graph showing tumor volume. In Figure 6, the horizontal axis represents the number of days after colon cancer cell inoculation, and the vertical axis represents tumor volume. In Figure 6, the upper panel shows the results from left to right for the negative control (untreated mouse), control (no antibody administration), and anti-CD4 antibody administration group (CD4 removal), while the lower panel shows the results for the anti-CD8 antibody administration group (CD8 removal) and the anti-CD4 antibody and anti-CD8 antibody administration group (CD4 + CD8 removal). As shown in Figure 6, the increase in tumor volume was completely suppressed in the control group. In contrast, the anti-CD4 antibody administration group and the anti-CD8 antibody administration group showed a slight increase in tumor volume compared to the control, and the anti-CD4 antibody and anti-CD8 antibody administration groups showed an increase in tumor volume equivalent to that of the negative control. From these results, it was estimated that the antitumor activity by mRNA encoding tumor-associated antigens and MHC-II molecules is caused by the activation of T cells by tumor-associated antigens and MHC-II. Furthermore, the aforementioned antitumor activity is related to CD4 ＋ T cells and CD8 ＋ The involvement of T cells was suggested.

[0242] [Example 6] We confirmed that administering an LNP vaccine containing gp70 and CIITA, or gp70, CIITA, and BZLF1 mRNA, to a colorectal cancer model mouse could more effectively induce antitumor activity.

[0243] (1) Preparation of mRNA transcription DNA template Mouse CIITA (SEQ ID NO: 4) was used as the polynucleotide encoding the transcription inducer of the MHCII molecule. In addition, a polynucleotide encoding BZLF1 (SEQ ID NO: 20) was used as the polynucleotide encoding the invariant chain expression inhibitor. An mRNA transcription DNA template was prepared in the same manner as in Example 2(1), except that a polynucleotide encoding mouse CIITA and a polynucleotide encoding BZLF1 were used in addition to the polynucleotides of Example 2(1).

[0244] (2) In vitro transfer: In the same manner as in Example 1 (2), gp70 and I-A prepared in Example 2 (1) ｄ α-P2A-I-A ｄ mRNA was prepared from β, CIITA, and BZLF1 DNA templates.

[0245] (3) Preparation of lipid nanoparticle (LNP) vaccine An LNP vaccine was prepared in the same manner as in Example 2(3), except that the mRNA from Example 6(2) was used. The mixing ratios of the mRNA mixture containing CIITA and the mRNA mixture containing CIITA and BZLF1 were prepared in the ratios shown in Table 4 below.

[0246]

[0247] (4) Antitumor effect on colorectal cancer model mice Tumor volume was measured in the same manner as in Example 2(4), except that the LNP vaccine of Example 6(3) was used instead of the LNP vaccine of Example 2(3). The tumor volume of the negative control was measured in the same manner, except that the LNP vaccine was not administered. These measurement results are shown in Figure 7.

[0248] Figure 7 is a graph showing tumor volume. In Figure 7, the horizontal axis represents the number of days after colon cancer cell inoculation, and the vertical axis represents tumor volume. In Figure 7, the upper panel shows the results from left to right for negative control (untreated mouse), control 2 (TAA alone: ​​gp70 only), and example 6-1 (MHC-II + TAA: gp70 + MHC-II), while the lower panel shows the results for example 6-2 (MHC-II allele-independent gp70 + CIITA) and example 6-3 (MHC-II allele-independent gp70 + CIITA + BZLF1). As shown in Figure 7, tumor volume increased over time in the negative control and control groups. In contrast, the increase in tumor volume was completely suppressed in example 6-1 (gp70 + MHC-II). Furthermore, in Examples 6-2 (MHC-II allele-independent gp70 + CIITA) and 6-3 (MHC-II allele-independent gp70 + CIITA + BZLF1), the increase in tumor volume was suppressed compared to the negative control and the control. Moreover, in Example 6-3 (MHC-II allele-independent gp70 + CIITA + BZLF1), the increase in tumor volume was suppressed compared to Example 6-2 (MHC-II allele-independent gp70 + CIITA). These results indicate that even without directly inducing the expression of the MHCII molecule, indirectly inducing its expression using an MHCII molecule expression inducer such as CIITA can induce an immune response to tumor-associated antigens and thus induce antitumor activity. It was also found that antitumor activity can be enhanced by using an MHCII molecule expression inducer in combination with an invariant chain expression inhibitor. Furthermore, since the suppression of the invariant chain enhanced antitumor activity, it was hypothesized that in mice administered the LNP vaccine, a complex of the MHCII molecule and the target antigen, i.e., a neoself antigen, was expressed, and that the expression of this antigen induced antitumor activity.

[0249] [Example 7] We confirmed that antitumor activity can be more effectively induced in skin cancer model mice by using an LNP vaccine containing gp100, TYRP2, and MHC-II mRNA in combination with an anti-PD-1 antibody.

[0250] (1) Preparation of mRNA transcription DNA template Mouse melanoma gp100 (SEQ ID NO: 28) and mouse tyrosinase-related protein TYRP2 (SEQ ID NO: 32) were used as polynucleotides encoding tumor-associated antigens of skin cancer. The mRNA transcription DNA template was prepared in the same manner as in Example 1(1) above, except that TYRP2 was used instead of TYRP1.

[0251] (2) In vitro transfer In the same manner as in Example 1(2), gp100, TYRP2 and I-A prepared in Example 7(1) ｂ α-P2A-I-A ｂ mRNA was prepared from a β DNA template.

[0252] (3) Preparation of lipid nanoparticle (LNP) vaccine An LNP vaccine was prepared in the same manner as in Example 1(3), except that the mRNA from Example 7(2) was used. The mixing ratios of each mRNA mixture were prepared according to the ratios shown in Table 5 below.

[0253]

[0254] (4) Antitumor effect on skin cancer model mice B16F1 cells were inoculated subcutaneously into C57BL / 6 mice (7 × 10 ５ Cells / mouse). The group administered the LNP vaccine containing mRNA prepared in Example 7(3) above received 100 μl / mouse of the mRNA-containing LNP vaccine at weekly doses for a total of three times, starting one week after tumor inoculation. The group administered the anti-PD-1 antibody (Bio X Cell, RMP1-14, BE0146) received 200 μg / mouse of the anti-PD-1 antibody for a total of three times, on the day of the first LNP vaccination and every four days thereafter, and the tumor volume was measured. The negative control group did not receive the LNP vaccine, but the tumor volume was measured in the same manner. These measurement results are shown in Figure 8.

[0255] Figure 8 is a graph showing tumor volume. In Figure 8, the horizontal axis represents the number of days after melanoma inoculation, and the vertical axis represents tumor volume. In the upper section of Figure 8, each figure from left to right shows the results for negative control (untreated mouse), control 1 (TAA alone: ​​gp100 + Tyrp2 only), and example 7-1 (gp100 + Tyrp2 + MHC-II). In the lower section of Figure 8, each figure from left to right shows the results for anti-PD-1 antibody alone (anti-PD-1 antibody), gp100, Tyrp2 and PD-1 antibodies (TAA + anti-PD-1 antibody: gp100 + Tyrp2 + anti-PD-1 antibody), and example 7-2 (MHC-II + TAA + anti-PD-1 antibody: gp100 + Tyrp2 + MHC-II + anti-PD-1 antibody). As shown in Figure 8, the increase in tumor volume was suppressed in the MHC-II + TAA + anti-PD-1 antibody administration group, indicating that the LNP vaccine exhibits antitumor activity even when administered after tumor tissue formation. Furthermore, when the LNP vaccine of Example 7 (gp100 + Tyrp2 + MHC-II) was used in combination with the anti-PD-1 antibody, the increase in tumor volume was completely suppressed. From the above, it was found that the antitumor effect of the composition of this disclosure can be enhanced by using it in combination with an immune checkpoint inhibitor such as an anti-PD-1 antibody.

[0256] [Example 8] We confirmed that antitumor activity can be more effectively induced in colorectal cancer model mice by using an LNP vaccine containing gp70 and MHC-II mRNA in combination with an anti-PD-1 antibody.

[0257] (1) Preparation of a DNA template for mRNA transcription A DNA template for mRNA transcription was prepared in the same manner as in Example 2(1) above.

[0258] (2) In vitro transcription mRNA was prepared from the DNA template prepared in Example 8(1) in the same manner as in Example 2(2).

[0259] (3) Preparation of lipid nanoparticle (LNP) vaccine Using the mRNA from Example 8(2), an LNP vaccine was prepared in the same manner as in Example 2(3).

[0260] (4) Antitumor effect on colorectal cancer model mice CT26 cells were inoculated subcutaneously into BALB / c mice (5 × 10 ５ (Cells / mouse). The group administered the LNP vaccine containing mRNA prepared in Example 8(3) above received 100 μl / mouse of the mRNA-containing LNP vaccine at weekly doses for a total of three times, starting one week after tumor inoculation. The group administered anti-PD-1 antibody received 200 μg / mouse of the anti-PD-1 antibody for a total of three times, on the day of the initial LNP vaccination and every four days thereafter, and the tumor volume was measured. The negative control group was not administered the LNP vaccine, but the tumor volume was measured in the same manner. These measurement results are shown in Figure 9.

[0261] Figure 9 is a graph showing tumor volume. In Figure 9, the horizontal axis represents the number of days after colon cancer cell inoculation, and the vertical axis represents tumor volume. In the upper section of Figure 9, each figure from left to right shows the results for negative control (untreated mouse), control 1 (TAA alone: ​​gp70 only), and Example 8-1 (MHC-II + TAA: gp70 + MHC-II). In the lower section of Figure 9, each figure from left to right shows the results for anti-PD-1 antibody alone (anti-PD-1 antibody), gp70 and anti-PD-1 antibody (TAA + anti-PD-1 antibody), and Example 8-2 (MHC-II + TAA + anti-PD-1 antibody: gp70 + MHC-II + anti-PD-1 antibody). As shown in Figure 9, the increase in tumor volume was suppressed in the Example 8-1 treatment group (MHC-II + TAA), indicating that the LNP vaccine in the example can induce antitumor activity even after tumor tissue formation. Furthermore, when an anti-PD-1 antibody was used in combination with the LNP vaccine (MHC-II + TAA) of Example 8-1 (Example 8-2), the increase in tumor volume was suppressed even more effectively. From the above, it was found that the antitumor effect of the composition of this disclosure can be enhanced by using it in combination with an immune checkpoint inhibitor such as an anti-PD-1 antibody.

[0262] [Example 9] We confirmed that antitumor activity can be more effectively induced in kidney cancer model mice by using an LNP vaccine containing gp70 and MHC-II mRNA in combination with an anti-PD-1 antibody.

[0263] (1) Preparation of a DNA template for mRNA transcription A DNA template for mRNA transcription was prepared in the same manner as in Example 3(1) above.

[0264] (2) In vitro transcription mRNA was prepared from the DNA template prepared in Example 9(1) in the same manner as in Example 3(2).

[0265] (3) Preparation of lipid nanoparticle (LNP) vaccine Using the mRNA from Example 9(2), an LNP vaccine was prepared in the same manner as in Example 3(3).

[0266] (4) Antitumor effect on kidney cancer model mice Renca cells were inoculated subcutaneously into BALB / c mice (2 × 10⁻¹⁰ ５ Cells / mouse). The group administered the LNP vaccine containing mRNA prepared in Example 9(3) above received 100 μl / mouse of the mRNA-containing LNP vaccine at weekly intervals for a total of three times, starting two weeks later. The group administered anti-PD-1 antibody received 200 μg / mouse of anti-PD-1 antibody at a rate of three times: on the day of the initial LNP vaccination and every four days thereafter. Tumor volume was measured. The negative control group was not administered the LNP vaccine, but tumor volume was measured in the same manner. These measurement results are shown in Figure 10.

[0267] Figure 10 is a graph showing tumor volume. In Figure 10, the horizontal axis represents the number of days after inoculation with kidney cancer cells, and the vertical axis represents tumor volume. In the upper section of Figure 10, each figure from left to right shows the results for negative control (untreated mouse), control 1 (TAA alone: ​​gp70 only), and example 9-1 (MHC-II + TAA: gp70 + MHC-II). In the lower section of Figure 10, each figure from left to right shows the results for anti-PD-1 antibody alone (anti-PD-1 antibody), gp70 and PD-1 antibody (TAA + anti-PD-1 antibody: gp70 + anti-PD-1 antibody), and example 9-2 (MHC-II + TAA + anti-PD-1 antibody: gp70 + MHC-II + anti-PD-1 antibody). As shown in Figure 10, in the treatment group of Example 9-1 (MHC-II + TAA), the increase in tumor volume was suppressed, indicating that antitumor activity can be induced even when the LNP vaccine is administered after tumor tissue formation. Furthermore, when the LNP vaccine of Example 9-1 was used in combination with an anti-PD-1 antibody (Example 9-2), the increase in tumor volume was suppressed even more effectively. From the above, it was found that the composition of this disclosure can further enhance antitumor activity when used in combination with an immune checkpoint inhibitor such as an anti-PD-1 antibody.

[0268] [Example 10] We confirmed that antitumor activity could be more effectively induced in colorectal cancer model mice by using an LNP vaccine containing gp70 and CIITA mRNA in combination with siRNA against the invariant strand.

[0269] (1) Preparation of mRNA transcription DNA template and siRNA A mRNA transcription DNA template was prepared in the same manner as in Example 6(1) above. In addition, an invariant strand siRNA having the following base sequence was synthesized by Nippon Gene Co., Ltd. In the following sequence, "dT" means deoxythymidine.

[0270] Invariant strand (CD74) siRNA (targeting the untranslated region at the 3' end) Sense strand (SEQ ID NO: 45): 5'-ACCUAGGCUGGACACAUUUdTdT-3' Antisense strand (SEQ ID NO: 46): 3'-dTdTUGGAUCCGACCUGUGUAAA-5'

[0271] (2) In vitro transcription Similar to Example 2(2) above, gp70 and I-A prepared in Example 10(1) ｄ α-P2A-I-A ｄ β, and mRNA was prepared from the DNA templates of CIITA.

[0272] (3) Preparation of lipid nanoparticle (LNP) vaccine The annealed siRNA synthesized by Nippon Gene Co., Ltd. was dissolved in RNase-Free water to a final concentration of 50 μM (0.665 μg / μL). Next, gp70:CIITA:siRNA was mixed at a ratio of 10:9:1. Then, the mRNA and siRNA mixture was prepared to a final concentration of 0.276 mg / mL with 50 mM sodium acetate buffer (pH 4.0). Except for these points, an LNP vaccine was prepared in the same manner as in Example 6(3).

[0273] (4) Antitumor effect on colorectal cancer model mice CT26 cells were inoculated subcutaneously into BALB / c mice (5×10 ５ cells / mouse). The group administered the LNP vaccine containing the mRNA prepared in Example 10(3) was given the LNP vaccine containing mRNA or the LNP vaccine containing mRNA and siRNA at 100 μl / mouse once a week for a total of 3 times starting 1 week after cancer inoculation. Also, the group administered the anti-PD-1 antibody was given the anti-PD-1 antibody at 200 μg / mouse, a total of 3 times every 4 days from the first LNP vaccine inoculation day and the inoculation day, and the tumor volume was measured. These measurement results are shown in FIG. 11.

[0274] Figure 11 is a graph showing tumor volume. In Figure 11, the horizontal axis represents the number of days after colon cancer cell inoculation, and the vertical axis represents tumor volume. In the upper section of Figure 11, each figure from left to right shows the results for negative control (untreated mouse), control 1 (TAA alone: ​​gp70 only), and example 10-1 (gp70 + MHC-II). In the middle section of Figure 11, each figure from left to right shows the results for example 10-2 (gp70 + CIITA + CD74 siRNA) and control 2 (TAA + anti-PD-1 antibody: gp70 + anti-PD-1 antibody). In the lower section of Figure 11, each figure from left to right shows the results for example 10-3 (gp70 + CIITA + anti-PD-1 antibody) and example 10-4 (gp70 + CIITA + CD74 siRNA + anti-PD-1 antibody). As shown in Figure 11, in the group administered in Example 10-1, the increase in tumor volume was suppressed to the same extent as in Example 7, indicating that antitumor activity was induced even when the LNP vaccine of the example was administered after the formation of tumor tissue. Furthermore, in the group administered in Example 10-2, the increase in tumor volume was significantly suppressed compared to Example 10-1, indicating that the same antitumor activity enhancement effect as BZLF1 can be obtained by using siRNA as an invariant chain (CD74) expression inhibitor. In addition, in the groups administered in Examples 10-3 and 10-4, the increase in tumor volume was significantly suppressed compared to the groups administered in Examples 10-1 and 10-2, respectively, indicating that antitumor activity can be enhanced by using it in combination with immune checkpoint inhibitors such as anti-PD-1 antibodies. In particular, in the group administered in Example 10-4, the colorectal cancer tumor tissue disappeared when administered after the formation of tumor tissue. From the above, it has been found that the composition of this disclosure can further enhance antitumor activity when used in combination with an invariant chain expression inhibitor and an immune checkpoint inhibitor such as an anti-PD-1 antibody.

[0275] [Example 11] We confirmed that antitumor activity could be more effectively induced in skin cancer model mice by using an LNP vaccine containing gp100, TYRP2, and MHC-II mRNA in combination with siRNA or an anti-PD-1 antibody against the invariant chain.

[0276] (1) Preparation of mRNA transcription DNA template and siRNA A mRNA transcription DNA template was prepared in the same manner as in Example 7(1). The invariant strand siRNA was prepared in the same manner as in Example 10(1).

[0277] (2) In vitro transfer In the same manner as in Example 1(2), gp100, TYRP2, I-A prepared in Example 11(1) ｄ α-P2A-I-A ｄ mRNA was prepared from β and CIITA DNA templates.

[0278] (3) Preparation of lipid nanoparticle (LNP) vaccine An LNP vaccine was prepared in the same manner as in Example 7(3), except that the mRNA from Example 11(2) was used to mix the mRNA and siRNA in the ratio gp100:TYRP2:CIITA:siRNA = 5:5:9:1.

[0279] (4) Antitumor effect on skin cancer model mice B16F1 cells were inoculated subcutaneously into C57BL / 6 mice (7 × 10 ５ Cells / mouse). The group administered the LNP vaccine containing mRNA prepared in Example 11(3) above received 100 μl / mouse of the mRNA-containing LNP vaccine or the mRNA and siRNA-containing LNP vaccine at weekly intervals for a total of three times, starting two weeks after cancer vaccination. The group administered the anti-PD-1 antibody received 200 μl / mouse of the anti-PD-1 antibody at a rate of three times: on the day of the initial LNP vaccination and every four days thereafter. Tumor volume was measured. These measurement results are shown in Figure 12.

[0280] Figure 12 is a graph showing tumor volume. In Figure 12, the horizontal axis represents the number of days after melanoma inoculation, and the vertical axis represents tumor volume. In the upper section of Figure 12, each figure from left to right shows the results for negative control (untreated mouse) and control 1 (TAA alone: ​​gp100 + Tyrp2). In the middle section of Figure 12, each figure from left to right shows the results for Example 11-1 (CIITA + CD74 siRNA + TAA: gp100 + Tyrp2 + CIITA + CD74 siRNA) and control 2 (TAA + anti-PD-1 antibody: gp100 + Tyrp2 + anti-PD-1 antibody). In the lower section of Figure 12, the figures from left to right show the results for Example 11-2 (CIITA + TAA + anti-PD-1 antibody: gp100 + Tyrp2 + CIITA + PD-1 antibody) and Example 11-3 (CIITA + TAA + CD74 siRNA + anti-PD-1 antibody: gp100 + Tyrp2 + CIITA + CD74 siRNA + PD-1 antibody). As shown in Figure 12, the group administered in Example 11-1 showed a significantly suppressed increase in tumor volume compared to Control 1, indicating that using siRNA as an invariant chain expression inhibitor can provide an enhanced antitumor activity similar to that of BZLF1. Furthermore, the group administered in Example 11-3 showed a significantly suppressed increase in tumor volume compared to the group administered in Example 11-1, indicating that antitumor activity can be enhanced by using it in combination with immune checkpoint inhibitors such as anti-PD-1 antibodies. In particular, in the 11-3 administration group, inoculation after tumor tissue formation resulted in the disappearance of melanoma tumor tissue. From the above, it was found that the composition of this disclosure can further enhance antitumor activity when used in combination with an invariant chain expression inhibitor and an immune checkpoint inhibitor such as an anti-PD-1 antibody.

[0281] [Reference Example 1] We confirmed that invariant chain expression can be suppressed using the siRNA used in Example 10 and siRNA targeting an invariant chain at a different site than the siRNA used in Example 10.

[0282] (1) Preparation of mRNA transcription DNA template and siRNA A mRNA transcription DNA template encoding mouse CIITA was prepared in the same manner as in Example 6(1). In addition, an mRNA transcription DNA template encoding human CIITA (human CIITA isoform 2) was prepared in the same manner as in Example 6(1) instead of mouse CIITA.

[0283] The invariant strand siRNAs (CD74 siRNA #1 to #4) having the following base sequence as the sense strand were purchased from Dharmacon. The invariant strand siRNAs from Example 10(1) were also used. Human CD74 siRNA #1 (targets CDS) 5'-GACCUUAUCUCCAACAAUG-3' (SEQ ID NO: 47) Human CD74 siRNA #2 (targets CDS) 5'-AAGCAGGAGCUGUCGGGAA-3' (SEQ ID NO: 48) Human CD74 siRNA #3 (targets untranslated region at 3' end) 5'-AGACAAACCAAGUCGGAAC-3' (SEQ ID NO: 49) Human CD74 siRNA #4 (targets untranslated region at 3' end) 5'-GUGCCUGGCUCUUUCGUCA-3' (SEQ ID NO: 50) Mouse CD74 siRNA #1 (targets CDS) 5'-CCAGAAAGGUGCAGCCGUG-3' (SEQ ID NO: 51) Mouse CD74 siRNA #2 (targets CDS) 5'-GGGCCUGUGAAGAACGUUA-3' (SEQ ID NO: 52) Mouse CD74 siRNA #3 (targets CDS) 5'-UCGCAUGAAGCUUCCGAAA-3' (SEQ ID NO: 53) Mouse CD74 siRNA #4 (targets CDS) 5'-GGCGUGAACUGGAAGAUCU-3' (SEQ ID NO: 54)

[0284] (2) In vitro transcription mRNA was prepared from the mouse CIITA or human CIITA DNA template prepared in Reference Example 1(1) in the same manner as in Example 1(2).

[0285] (3) Preparation of lipid nanoparticle (LNP) vaccine LNPs were prepared in the same manner as in Example 2(3), except that the mRNA and siRNA mixture was mixed in a ratio of CIITA:siRNA = 1:1 using the mRNA from Reference Example 1(2). The siRNA used was the siRNA from Example 10.

[0286] (4) Measurement of invariant chain expression levels B16 cells were cultured for 16 hours after introducing LNPs containing CIITA and siRNA. After the culture, B16 cells were harvested and stained with Alexa Fluor 647-labeled anti-CD74 antibody. After staining, CD74 expression was measured by flow cytometry. The negative control was performed in the same manner except that no LNPs were introduced, and the positive control was performed in the same manner except that LNPs containing only CIITA were introduced. These results are shown in Figure 13.

[0287] Figure 13 is a graph showing the expression of the invariant chain. In Figure 13, the horizontal axis represents the sample type, and the vertical axis represents the expression level of the invariant chain. As shown in Figure 13, the siRNA against the invariant chain in Example 10 was able to suppress the expression level of the invariant chain. Therefore, it was estimated that the improvement in antitumor activity by the combined use of siRNA against the invariant chain in Example 10 was due to the decrease in the expression level of the invariant chain by the siRNA, and as a result, the expression level of the complex of tumor antigen and MHC-II increased.

[0288] (5) Measurement of invariant strand expression levels using other siRNAs The insert encoding mouse or human CIITA, introduced into the mRNA transcription DNA template prepared in Reference Example 1 (1) above, was cloned into a pME18S vector. The effect of the siRNAs was investigated by introducing the resulting CIITA plasmid vector and human or mouse CD74 siRNAs #1 to #4 into B16 cells or HEK293 cells.

[0289] Specifically, B16 cells were introduced using polyethyleneimine (PEI) as the plasmid vector for mouse CIITA and using a lipofection reagent (Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher Scientific, 13778150)) and cultured for 48 hours. After the culture, the expression level of the invariant chain in the B16 cells was measured in the same manner as in Reference Example 1(4). In addition, HEK293 cells were introduced using PEI as the plasmid vector for human CIITA and using the aforementioned lipofection reagent, and cultured for 48 hours. After culturing, the expression level of the invariant chain in the B16 cells was measured in the same manner as in Reference Example 1(4). These results are shown in Figure 14.

[0290] Figure 14 is a graph showing the expression of invariant chains. In Figure 14, (A) shows the results using mouse CIITA, and (B) shows the results using human CIITA. In Figure 14, the horizontal axis indicates the type of sample, and the vertical axis indicates the expression level of invariant chains. As shown in Figures 14(A) and (B), in both human and mouse CIITA, the expression of invariant chains could be suppressed by siRNA targeting the invariant chain.

[0291] [Example 12] We confirmed that administering an LNP vaccine containing gp70 and CIITA mRNA to kidney cancer model mice could more effectively induce antitumor activity. We also confirmed that combining this vaccine with siRNA against the invariant chain or an anti-PD-1 antibody could more effectively induce antitumor activity in kidney cancer model mice.

[0292] (1) Preparation of mRNA transcription DNA templates and siRNAs mRNA transcription DNA templates for gp70 and CIITA were prepared in the same manner as in Example 6(1). The invariant strand siRNA was prepared in the same manner as in Example 10(1).

[0293] (2) In vitro transcription mRNA was prepared from the gp70 and CIITA DNA templates prepared in Example 12(1) in the same manner as in Example 1(2).

[0294] (3) Preparation of lipid nanoparticle (LNP) vaccine An LNP vaccine was prepared in the same manner as in Example 6(3), except that the mRNA obtained in Example 12(2) was used and the mRNA and siRNA mixture was mixed in the ratio gp70:CIITA:siRNA = 9.5:9.5:1.

[0295] (4) Antitumor effect on kidney cancer model mice Renca cells were inoculated subcutaneously into BALB / c mice (2.5 × 10⁻¹⁴). ５ Cells / mouse). The group administered the LNP vaccine containing mRNA prepared in Example 12(3) above received 100 μl each of either the mRNA-containing LNP vaccine or the mRNA and siRNA-containing LNP vaccine per mouse, once a week for a total of three doses, starting one week after tumor inoculation. The administration method was intramuscular injection into four locations on the hands and feet. The group administered anti-PD-1 antibody received 200 μg / mouse of anti-PD-1 antibody for a total of three doses, on the day of the initial LNP vaccination and every four days thereafter, and the tumor volume was measured. The negative control group did not receive the LNP vaccine, but the tumor volume was measured in the same manner. These results are shown in Figure 15.

[0296] Figure 15 is a graph showing tumor volume. In Figure 15, the horizontal axis represents the number of days after inoculation with kidney cancer cells, and the vertical axis represents tumor volume. In the upper section of Figure 15, each figure from left to right shows the results for negative control (untreated mouse), control 1 (TAA alone: ​​gp70 only), and example 12-1 (gp70 + CIITA). In the lower section of Figure 15, each figure from left to right shows the results for example 12-2 (gp70 + CIITA + CD74 siRNA), control 2 (anti-PD-1 antibody alone), and example 12-3 (gp70 + CIITA + CD74 siRNA + anti-PD-1 antibody). As shown in Figure 15, it was confirmed that the increase in tumor volume was suppressed in the example 12-1 administration group, and it was found that antitumor activity was induced even when the LNP vaccine of the example was administered after tumor tissue formation. Furthermore, the group administered in Example 12-2 showed a significantly suppressed increase in tumor volume compared to Example 12-1, indicating that using siRNA as an invariant chain (CD74) expression inhibitor enhances antitumor activity. Moreover, Example 12-3 showed a significantly suppressed increase in tumor volume compared to Example 12-2, indicating that combining it with an immune checkpoint inhibitor such as an anti-PD-1 antibody can enhance antitumor activity. From the above, it was found that combining MHC-II molecules and tumor-associated antigens induces an immune response to tumor-associated antigens, thereby inducing antitumor activity against renal cancer. The composition of this disclosure can further enhance antitumor activity when used in combination with an invariant chain expression inhibitor and an immune checkpoint inhibitor such as an anti-PD-1 antibody.

[0297] [Example 13] We confirmed that administering an LNP vaccine containing gp70 and CIITA mRNA to breast cancer model mice could more effectively induce antitumor activity. We also confirmed that combining this vaccine with siRNA against the invariant chain or an anti-PD-1 antibody could more effectively induce antitumor activity in breast cancer model mice.

[0298] (1) Preparation of mRNA transcription DNA templates and siRNAs mRNA transcription DNA templates for gp70 and CIITA were prepared in the same manner as in Example 6(1). The invariant strand siRNA was prepared in the same manner as in Example 10(1).

[0299] (2) In vitro transcription mRNA was prepared from the gp70 and CIITA DNA templates prepared in Example 13(1) in the same manner as in Example 1(2).

[0300] (3) Preparation of lipid nanoparticle (LNP) vaccine An LNP vaccine was prepared in the same manner as in Example 6(3), except that the mRNA obtained in Example 13(2) was used and the mRNA and siRNA mixture was mixed in the ratio gp70:CIITA:siRNA = 9.5:9.5:1.

[0301] (4) Antitumor effect on breast cancer model mice EMT6 cells were used instead of Renca cells, and tumor volume was measured in the same manner as in Example 12(4), except that an LNP vaccine containing mRNA or an LNP vaccine containing mRNA and siRNA was administered as the LNP vaccine starting 3 days after tumor inoculation. These results are shown in Figure 16.

[0302] Figure 16 is a graph showing tumor volume. In Figure 16, the horizontal axis represents the number of days after inoculation with breast cancer cells, and the vertical axis represents tumor volume. In the upper section of Figure 16, each figure from left to right shows the results for negative control (untreated mouse), control 1 (TAA alone: ​​gp70 only), and example 13-1 (gp70 + CIITA). In the lower section of Figure 16, each figure from left to right shows the results for example 13-2 (gp70 + CIITA + CD74 siRNA), control 2 (anti-PD-1 antibody), and example 13-3 (gp70 + CIITA + CD74 siRNA + anti-PD-1 antibody). As shown in Figure 16, it was confirmed that the increase in tumor volume was suppressed in the example 13-1 administration group, and it was found that antitumor activity was induced even when the LNP vaccine of the example was administered after tumor tissue formation. Furthermore, the group administered in Example 13-2 showed a significantly suppressed increase in tumor volume compared to Example 13-1, indicating that using siRNA as an invariant chain (CD74) expression inhibitor enhances antitumor activity. In addition, Example 13-3 showed a significantly suppressed increase in tumor volume compared to Example 13-2, indicating that antitumor activity can be enhanced by using it in combination with immune checkpoint inhibitors such as anti-PD-1 antibodies. From the above, it was found that combining MHC-II molecules and tumor-associated antigens induces an immune response to tumor-associated antigens, thereby inducing antitumor activity against breast cancer. The composition of this disclosure can further enhance antitumor activity when used in combination with an invariant chain expression inhibitor and immune checkpoint inhibitors such as anti-PD-1 antibodies.

[0303] [Example 14] We confirmed that antibody induction ability can be enhanced by an LNP vaccine containing siRNA against human IgG, CIITA, and invariant strands.

[0304] (1) Preparation of mRNA transcription DNA template and siRNA A mRNA transcription DNA template of the CH domain of human IgG (hIgG) (NCBI accession number: JQ917464.2) was prepared in the same manner as in Example 6(1), except that the CH domain of human IgG (hIgG) (NCBI accession number: JQ917464.2) was used instead of gp70. The CH domain of hIgG had an underlined endoplasmic reticulum localization signal (derived from SLAM protein) added to its N-terminus in the amino acid sequence of Sequence ID No. 55 and the nucleotide sequence of Sequence ID No. 56 below. The invariant strand siRNA was prepared in the same manner as in Example 10(1).

[0305] The amino acid sequence of the CH domain of IgG (SEQ ID NO: 55) is MDPKGSLSWRILLFLSLAFELSYGLEASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLHSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0306]

[0307] (2) In vitro transcription mRNA was prepared from the IgG CH domain and CIITA DNA template prepared in Example 14(1) in the same manner as in Example 1(2). In addition, I-A prepared in Example 2(1) ｄ α-P2A-I-A ｄ mRNA was prepared from a β DNA template.

[0308] (3) Preparation of lipid nanoparticle (LNP) vaccine An LNP vaccine was prepared in the same manner as in Example 6(3), except that the mRNA obtained in Example 14(2) was used and the mRNA and siRNA mixture was mixed in the ratio of IgG:CIITA:siRNA = 9.5:9.5:1.

[0309] (4) BALB / c mice were used as the antibody-inducing mice. As shown in Figure 17(A), the group administered the LNP vaccine containing mRNA prepared in Example 14(3) was administered either the mRNA-containing LNP vaccine or the mRNA and siRNA-containing LNP vaccine at a dose of 100 μl / mouse. The administration method was intramuscular injection at four sites on the hands and feet. Six days after administration, blood was collected from the mice and the antibody titer against IgG in the obtained serum was measured. mRNA encoding IgG (CH domain), MHC-II (I-Adα + I-Adβ), and GFP was introduced into HEK293T cells, and the serum diluted 100-fold was added and allowed to stand. After the aforementioned standing period, the HEK293T cells were stained with APC-labeled anti-mouse IgG Fc antibody (Jackson Immunoresearch, Cat No. 115-136-071), and the fluorescence intensity of the HEK293T cells was measured using a flow cytometer. These results are shown in Figure 17(B).

[0310] Figure 17 is a graph showing the protocol and anti-hIgG antibody titer. In Figure 17, (A) shows the administration protocol for the LNP vaccine, and (B) shows the anti-hIgG antibody titer. In Figure 17(B), the horizontal axis shows the sample type, and the vertical axis shows the anti-hIgG antibody titer. As shown in Figure 17(B), the group administered Example 14 (hIgG + CIITA + CD74 siRNA) showed a significant increase in anti-hIgG antibody titer compared to the negative control (untreated) and the control (hIgG alone). In other words, it was found that even for antigens that do not sufficiently induce an immune response when used alone, a strong immune response can be induced by combining them with an MHCII molecule expression inducer and siRNA against the invariant chain.

[0311] [Example 15] We confirmed that antibody induction ability can be enhanced by an LNP vaccine containing siRNA against human gp100, CIITA, and invariant strands.

[0312] (1) Preparation of mRNA transcription DNA template and siRNA As the polynucleotide encoding human gp100 (hgp100), the polynucleotide encoding the extracellular region (SEQ ID NO: 58) from among the hgp100 registered in GenBank accession number: NM_006928.5 was used. The mRNA transcription DNA template was prepared in the same manner as in Example 6(1), except that the polynucleotide encoding hgp100 was used instead of gp70. The invariant strand siRNA was prepared in the same manner as in Example 10(1).

[0313] The amino acid sequence of the extracellular region of hgp100 (SEQ ID NO: 57) is: MDLVLKRCLLHLAVIGALLAVGATKVPRNQDWLGVSRQLRTKAWNRQLYPEWTEAQRLDCWRGGQVSLKVSNDGPTLIGANASFSIALNFPGSQKVLPDGQVIWVNNTIINGSQVWGGQPVYPQETDDACIFPDGGPCPSGSWSQKRSFVYVWKTWGQYWQVLGGPVSGLSIGTGRAMLGTHTMEVTVYHRRGSRSYVPLAHSSSAFTITDQVPFSVSVSQLRALDGGNKHFLRNQPLTFALQLHDPSGYLAEADLSYTWDFGDSSGTLISRALVVTHTYLEPGPVTAQVVLQAAI PLTSCGSSPVPGTTDGHRPTAEAPNTTAGQVPTTEVVGTTPGQAPTAEPSGTTSVQVPTTEVISTAPVQMPTAESTGMTPEKVPVSEVMGTTLAEMSTPEATGMTPAEVSIVVLSGTTAAQVTTTEWVETTARELPIPEPEGPDASSI MSTESITGSLGPLLDGTATLRLVKRQVPLDCVLYRYGSFSVTLDIVQGIESAEILQAVPSGEGDAFELTVSCQGGLPKEACMEISSPGCQPPAQRLCQPVLPSPACQLVLHQILKGGSGTYCLNVSLADTNSLAVVSTQLIMPGQEAG

[0314]

[0315] (2) In vitro transcription mRNA was prepared from the hgp100 and CIITA DNA templates prepared in Example 15(1) in the same manner as in Example 1(2).

[0316] (3) Preparation of lipid nanoparticle (LNP) vaccine An LNP vaccine was prepared in the same manner as in Example 6(3), except that the mRNA obtained in Example 15(2) was used and the mRNA and siRNA mixture was mixed in the ratio hgp100:CIITA:siRNA = 9.5:9.5:1.

[0317] (4) Antibody induction effect As shown in Figure 18(A), the antibody induction effect was confirmed in the same manner as in Example 14(4) above, except that hgp100+CIITA+CD74siRNA was used instead of hIgG+CIITA+CD74siRNA. These results are shown in Figure 18(B).

[0318] Figure 18 is a graph showing the protocol and anti-hgp100 antibody titer. In Figure 18, (A) shows the administration protocol for the LNP vaccine, and (B) shows the anti-hgp100 antibody titer. In Figure 18(B), the horizontal axis shows the sample type, and the vertical axis shows the anti-hgp100 antibody titer. As shown in Figure 18(B), the group administered Example 15 (hgp100 + CIITA + CD74 siRNA) showed a significant increase in anti-hgp100 antibody titer compared to the negative control (untreated) and the control (hgp100 alone). In other words, it was found that even for antigens that do not sufficiently induce an immune response when administered alone, a strong immune response can be induced by combining them with an MHCII molecule expression inducer and siRNA against the invariant chain.

[0319] [Example 16] By adding a signal sequence, it was confirmed that the peptide can be expressed on the cell surface as a complex with MHC-II, and that an antibody production response and a T cell response to the peptide can be induced in vivo.

[0320] (1) DNA plasmid construction A polynucleotide consisting of the base sequence of Sequence ID No. 60 below was used as the polynucleotide encoding the tumor antigen (human NY-ESO-1). The base sequences of Sequence ID Nos. 60, 62, and 64 below are codon-optimized base sequences of the base sequence registered under NCBI accession number NM_001327.3. In this example, in order to investigate the effect of endoplasmic reticulum localization signals (derived from BM40 or SLAM protein), three types of NY-ESO-1 described below (i) to (iii) were prepared. Note that NY-ESO-1 was a protein from which the hydrophobic region at the C-terminus (Sequence ID No. 66) was removed. In addition, in Sequence ID Nos. 59 to 64 below, the underlined amino acid sequences or base sequences indicate the amino acid sequences or base sequences (including linker sequences) added to the N-terminus of NY-ESO-1. Furthermore, DNA templates for mRNA transcription of NY-ESO-1 (with signal sequence, without Flag) and CIITA were prepared in the same manner as in Example 6(1), except that NY-ESO-1 (with signal sequence, without Flag) was used instead of gp70. The invariant strand siRNA was prepared in the same manner as in Example 10(1). (i) NY-ESO-1 (without signal sequence, with Flag) (SEQ ID NOs. 59-60) A Flag tag (DYKDDDDK: SEQ ID NO. 65) was added to the N-terminus of NY-ESO-1 without a signal sequence. (ii) NY-ESO-1 (with signal sequence and Flag) (SEQ ID NOs. 61-62) NY-ESO-1 with a BM40 protein-derived endoplasmic reticulum localization signal sequence added to its N-terminus, and the Flag tag added between the signal sequence and NY-ESO-1. (iii) NY-ESO-1 (with signal sequence and without Flag) (SEQ ID NOs. 63-64) NY-ESO-1 with a SLAM protein-derived endoplasmic reticulum localization signal sequence added to its N-terminus, and the Flag tag not added between the signal sequence and NY-ESO-1.

[0321] The amino acid sequence of NY-ESO-1 (without signal sequence, with Flag) (SEQ ID NO: 59) is MDYKDDDDKQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRGAGAARASGPGGGAPRGPHGGAASGLNGCCRCGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPLPVPGVLLKEFTVSGNILTIRLTAADHRQLQLSISSCLQQLSLLMWITQCFLPVFLAQPPSGQRR

[0322] Polynucleotide (SEQ ID NO: 60) encoding NY-ESO-1 (no signal sequence, with flag) 5'--3'

[0323] The amino acid sequence of NY-ESO-1 (with signal sequence and Flag) (SEQ ID NO: 61) is MRAWIFFLLCLAGRALAASDYKDDDDKLEQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRGAGAARASGPGGGAPRGPHGGAASGLNGCCRCGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPLPVP

[0324] Polynucleotide (SEQ ID NO: 62) encoding NY-ESO-1 (with signal sequence and flag). 5'-ATGAGGGCCTGGATCTTCTTTCTCCTTTGCCTGGCCGGGAGGGCTCTGGCAGCTAGCGACTACAAAGACGATGACGACAAGCTCGAGCAAGCGGAGGGACGAGGCACTGGTGGTAGCACGGGTGACGCAGATGGGCCCGGTGGGCCAGGAATTCCAGACGGGCCGGGAGGTAATGCGGGTGGACCGGGAGAAGCTGGCGCTACTGGCGGGAGAGGCCCAC GGGGAGCAGGTGCTGCACGAGCATCAGGACCAGGCGGAGGTGCGCCGCGCGGGCCCCATGGAGGGGCCGCGAGTGGCCTCAATGGCTGCTGTAGGTGCGGTGCGAGGGGCCCTGAGAGCAGACTGTTGGAGTTTTATCTGGCTATGCCGTTCGCGACCCCCATGGAGGCTGAACTGGCTAGGAGATCTCTCGCACAGGATGCTCCCCCATTGCCTGTCCCG-3'

[0325] The amino acid sequence of NY-ESO-1 (with signal sequence, without Flag) (SEQ ID NO: 63) is MDPKGSLSWRILLFLSLAFELSYGLEQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRGAGAARASGPGGGAPRGPHGGAASGLNGCCRCGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPLPVP

[0326] Polynucleotide (SEQ ID NO: 64) encoding NY-ESO-1 (with signal sequence, without flag): 5'-ATGGACCCCAAAGGCTCCCTTTCCTGGAGAATACTTCTGTTTCTCTCTCTGGCTTTTGAGTTGAGCTACGGACTCGAGCAAGCGGAGGGACGAGGCACTGGTGGTAGCACGGGTGACGCAGATGGGCCCGGTGGGCCAGGAATTCCAGACGGGCCGGGAGGTAATGCGGGTGGACCGGGAGAAGCTGGCGCTACTGGCGGGAGAGGCCCACGGGGAGCAGGTGCTGCACGAGCATCAGGACCAGGCGGAGGTGCGCCGCGCGGGCCCCATGGAGGGGCCGCGAGTGGCCTCAATGGCTGCTGTAGGTGCGGTGCGAGGGGCCCTGAGAGCAGACTGTTGGAGTTTTATCTGGCTATGCCGTTCGCGACCCCCATGGAGGCTGAACTGGCTAGGAGATCTCTCGCACAGGATGCTCCCCCATTGCCTGTCCCG-3'

[0327] The amino acid sequence of the hydrophobic C-terminal portion of NY-ESO-1 (SEQ ID NO: 66) is GVLLKEFTVSGNILTIRLTAADHRQLQLSISSCLQQLSLLMWITQCFLPVFLAQPPSGQRR

[0328] The base sequence encoding the hydrophobic C-terminal portion of NY-ESO-1 (SEQ ID NO: 67) is 5'-GGTGTGCTTTTGAAGGAGTTCACCGTGTCCGGCAACATCCTCACTATACGACTCACAGCCGCGGATCATAGGCAACTCCAACTTAGCATAAGCAGCTGTCTCCAGCAGCTGTCTTTGCTTATGTGGATCACCCAGTGTTTCTTGCCAGTCTTTCTGGCGCAGCCGCCCTCTGGGCAACGCCGCTAG-3'

[0329] (2) In vitro transcription In the same manner as in Example 1 (2) above, mRNA was prepared from the DNA templates of NY-ESO-1 (with signal sequence, without Flag) and CIIITA prepared in Example 16 (1). Also, using the DNA template of I-A ｂ α-P2A-I-A ｂ β, mRNA of MHC-II (I-Abα + I-Abβ) was prepared.

[0330] (3) Measurement of the expression level of tumor antigen Each DNA plasmid prepared in Example 16 (1) (NY-ESO-1 (with signal sequence, with Flag) or NY-ESO-1 (without signal sequence, with Flag)), MHC-II (I-Abα + I-Abβ), and GFP were introduced into HEK293T cells using a transfection reagent (Polyethylenimine Max, manufactured by Polyscience). The mixing ratio of the DNA plasmids was set to NY-ESO-1 (with signal sequence, with Flag) or NY-ESO-1 (without signal sequence, with Flag): I-Abα: I-Abβ = 1:1:1. After the introduction, staining was performed using an APC-anti-Flag antibody (manufactured by BioLegend, clone name: L5). After the staining, the expression of NY-ESO-1 on the cell surface was measured by flow cytometry. These results are shown in FIG. 19.

[0331] Figure 19 is a graph showing the expression of NY-ESO-1 on the cell surface. In Figure 19, the horizontal axis represents the type of DNA plasmid introduced, and the vertical axis represents the expression level of NY-ESO-1 on the cell surface. In Figure 19, each figure from left to right shows the results with and without the NY-ESO-1 signal sequence. As shown in Figure 19, the expression level of NY-ESO-1 on the cell surface was very low in the case without the signal sequence, whereas expression on the cell surface was observed even when alone with the NY-ESO-1 signal sequence. Furthermore, as shown in Figure 19, combining the NY-ESO-1 with the signal sequence with MHC-II significantly increased the expression level of NY-ESO-1 on the cell surface. NY-ESO-1 is an intracellular protein and is normally hardly presented on MHC-II. Therefore, it was found that by adding an endoplasmic reticulum localization signal to the intracellular protein, a complex of the intracellular protein and MHC-II can be expressed on the cell surface.

[0332] (4) Preparation of lipid nanoparticle (LNP) vaccine Using the mRNA obtained in Example 16(2), an LNP vaccine was prepared in the same manner as in Example 6(3), except that the mRNA and siRNA mixture was mixed in the ratio of NY-ESO-1 (with signal sequence, without flag):CIITA:siRNA = 9.5:9.5:1.

[0333] (5) C57 / B6 mice were used as the antibody-inducing mice. As shown in Figure 20(A), the group receiving the LNP vaccine containing mRNA prepared in Example 16(4) received either the mRNA-containing LNP vaccine or the mRNA and siRNA-containing LNP vaccine, 100 μl each, per mouse, once every two weeks for a total of two doses. The administration method was intramuscular injection into four locations on the hands and feet. One week after the second administration of the LNP vaccine, blood was collected from the mice, and the antibody titer against NY-ESO-1 in the obtained serum was analyzed. The antibody titer was determined by introducing mRNA of NY-ESO-1 (with signal sequence, without flag), MHC-II (I-Abα + I-Abβ), and GFP into HEK293T cells, and then adding the serum diluted 100-fold and allowing it to stand. After the aforementioned standing period, the HEK293T cells were stained with the APC-IgG Fc antibody, and the fluorescence intensity of the HEK293T cells was measured using a flow cytometer. These results are shown in Figure 20(B).

[0334] Figure 20 is a graph showing the protocol and anti-NY-ESO-1 antibody titer. In Figure 20, (A) shows the administration protocol for the LNP vaccine, and (B) is a graph showing the anti-NY-ESO-1 antibody titer. In Figure 20(B), the horizontal axis shows the sample type, and the vertical axis shows the anti-NY-ESO-1 antibody titer. As shown in Figure 20(B), the group administered Example 16 (NY-ESO-1 + CIITA + CD74 siRNA) showed an increased anti-NY-ESO-1 antibody titer compared to the negative control (untreated) and the control (NY-ESO-1 alone). In other words, it was found that even for antigens that do not sufficiently induce an immune response when used alone, a strong immune response can be induced by using them in combination with an MHCII molecule expression inducer and siRNA against the invariant chain.

[0335] [Example 17] We confirmed that CD4 T cells that react with gp70 are induced by an LNP vaccine containing gp70, CIITA, and siRNA against the invariant chain, even in the absence of the invariant chain.

[0336] (1) Preparation of mRNA transcription DNA templates and siRNA mRNA transcription DNA templates for gp70 and CIITA were prepared in the same manner as in Example 12(1). The invariant strand siRNA was prepared in the same manner as in Example 10(1).

[0337] (2) In vitro transcription mRNA was prepared from the gp70 and CIITA DNA templates prepared in Example 17(1) in the same manner as in Example 2(2). In addition, I-A prepared in Example 2(1) ｄ α-P2A-I-A ｄ mRNA was prepared from a β DNA template.

[0338] (3) Preparation of lipid nanoparticle (LNP) vaccine An LNP vaccine was prepared in the same manner as in Example 6(3), except that the mRNA obtained in Example 17(2) was used and the mRNA and siRNA mixture was mixed in the ratio gp70:CIITA:siRNA = 9.5:9.5:1.

[0339] (4) BALB / c mice were used as the mice for CD4 T cell induction. As shown in Figure 21(A), the group receiving the LNP vaccine containing mRNA and siRNA prepared in Example 17(3) was administered 100 μl of either the mRNA-containing LNP vaccine or the mRNA and siRNA-containing LNP vaccine to each mouse, once every two weeks for a total of three doses. The administration method was intramuscular injection into four locations on the hands and feet. One week after the third administration of the LNP vaccine, blood was collected from the mice, and then the lymph nodes of the mice were collected. After preparing a cell suspension from the lymph nodes, it was stained with Brilliant Violet 421-labeled anti-CD45.2 antibody (Biolegend, clone name: 104) and APC-labeled anti-CD69 antibody (Biolegend, clone name: H1.2F3). The cell suspension after staining was sorted using a cell sorter to detect activated CD4 ＋ T cells, CD45.2 ＋ CD69 ＋ The cells were sorted. The resulting activated CD4 ＋High-throughput sequencing was performed on T cells to elutiary TCRs. Then, clonal TCRs (clonal numbers: A12, C10, G2, G8, H8) were selected from the lymph node-derived cell suspension, and the polynucleotides encoding these TCRs were cloned into NFAT-GFP reporter cells. Next, to detect TCRs that recognize gp70 and the MHC-II complex, gp70 reporter cells were prepared by introducing mRNA encoding gp70, MHC-II (I-Adα + I-Adβ), invariant chain (Ii), and H2-M into HEK293 T cells. The NFAT-GFP reporter cells and the gp70 reporter cells were co-cultured for one day. Subsequently, the expression of the activation marker (CD69) and GFP was detected in the NFAT-GFP reporter cells by flow cytometry. And the activated reporter cells (CD69) in the NFAT-GFP reporter cells ＋ GFP ＋ The proportion of ) was calculated. These results are shown in Figure 21(B).

[0340] Figure 21 is a graph showing the protocol and the percentage of activated cells. In Figure 21, (A) shows the LNP vaccine administration and subsequent analysis protocol, and (B) shows the percentage of activated cells. In Figure 21(B), the horizontal axis shows the mRNA components in the LNP, and the vertical axis shows the percentage of activated reporter cells. In Figure 21(B), each No. indicates the clone number, and from left to right, the results for A12, C10, G2, G8, and H8 are shown. As shown in Figure 21(B), none of the clones reacted much with cells expressing gp70 in the presence of the invariant chain. In contrast, none of the clones reacted strongly with cells expressing gp70 in the absence of the invariant chain. In the absence of the invariant chain, it is thought that gp70 forms a complex with MHC-II, i.e., forms a neoself antigen and is expressed on the cell surface. Therefore, it was found that the composition of this disclosure induces neoself antigen-specific CD4 T cells. Although not shown in the figure, among the NFAT-GFP reporter cells expressing clonally proliferated TCRs, there were also TCRs that responded to gp70 reporter cells expressing gp70 alone. Therefore, it was found that with the composition of this disclosure, even for antigens that do not sufficiently induce an immune response when used alone, a strong immune response can be induced when used in combination with an MHCII molecule expression inducer.

[0341] [Example 18] We confirmed the presence of CD4 T cells that react to neoself antigen in melanoma patients.

[0342] As shown in Figure 22(A), the presence of neoself antigen-reactive CD4 T cells was confirmed using T cell-derived TCRs from tumor tissue derived from melanoma patients. The target antigen was PMEL (Premelanosome protein, gp100).

[0343] Specifically, using sequencing data of single-cell TCRs of T cells infiltrating tumor tissue derived from melanoma patients, we can analyze clonally proliferated CD4 ＋TCRs (clonal numbers: B6, B11) from T cells were extracted. The polynucleotides encoding the extracted TCRs were cloned into NFAT-GFP reporter cells. Next, to detect TCRs that recognize gp100 and the MHC-II complex, gp100 reporter cells were prepared by introducing mRNA encoding gp100 and patient-derived HLA-II, invariant chain (Ii), and HLA-DM into HEK293 T cells. The NFAT-GFP reporter cells and the gp100 reporter cells were co-cultured for one day. Subsequently, the expression of the activation marker (CD69) and GFP was detected in the NFAT-GFP reporter cells by flow cytometry. Then, the expression of activated reporter cells (CD69) in the NFAT-GFP reporter cells was detected. ＋ GFP ＋ The proportion of ) was calculated. These results are shown in Figure 22(B).

[0344] Figure 22 is a graph showing the protocol and the percentage of activated cells. In Figure 22, (A) shows the protocol for TCR identification and subsequent analysis in melanoma-infiltrating T cells, and (B) is a graph showing the percentage of activated cells. In Figure 22(B), the horizontal axis shows the mRNA components in LNPs, and the vertical axis shows the percentage of activated reporter cells. In Figure 22(B), each No. indicates the clone number, and from left to right, the results for B6 and B11 are shown. As shown in Figure 22(B), all clones showed a weak reaction with cells expressing gp100 in the presence of the invariant chain. In contrast, all clones showed a strong reaction with cells expressing gp100 in the absence of the invariant chain. In the absence of the invariant chain, it is thought that gp100 forms a complex with MHC-II, i.e., forms a neoself antigen and is expressed on the cell surface. Therefore, it was found that neoself antigen-specific CD4 T cells exist among the CD4 T cells infiltrating the tumor tissue of the melanoma patient. Although not shown in the figure, among the NFAT-GFP reporter cells expressing clonally proliferated TCRs, there were also TCRs that responded to gp100 reporter cells expressing gp100 alone. Therefore, it was found that, according to the composition of this disclosure, even with antigens that do not sufficiently induce an immune response when used alone, a strong immune response can be induced when used in combination with an MHCII molecule expression inducer.

[0345] [Example 19] Using siRNAs of various invariant strands, it was confirmed that when the expression level of the invariant strand decreases, the expression level of the NY-ESO-1 and MHC-II complex on the cell surface increases.

[0346] (1) Comparison of invariant strand and NY-ESO-1 expression levels. 51 types of invariant strand siRNAs (human CD74 siRNAs) were purchased from Dharmacon and 7 types from Nippongene. The base sequences of the sense strands of each siRNA were as shown in Tables 6-7 below.

[0347]

[0348]

[0349] HEK293T cells were introduced with mRNA encoding human NY-ESO-1, the aforementioned human CIITA, and GFP to create reporter cells. The human NY-ESO-1 used was the aforementioned NY-ESO-1 (with signal sequence and flag). Next, each siRNA was introduced into the reporter cells. Two days after introduction, the reporter cells were stained with anti-human CD74 antibody and APC-anti-mouse IgG Fc antibody. After staining, the expression level of the invariant chain was measured using a flow cytometer. The expression level of NY-ESO-1 on the cell surface was measured in the same manner, except that APC-labeled anti-flag antibody was used instead of anti-human CD74 antibody and APC-anti-mouse IgG Fc antibody. The control was performed in the same manner except that no siRNA was introduced. These results are shown in Figures 23 and 24.

[0350] Figure 23 is a graph showing the expression of invariant chains. In Figure 23, the horizontal axis represents the type of sample, and the vertical axis represents the expression level of invariant chains. As shown in Figure 23, the expression level of invariant chains was suppressed by each siRNA.

[0351] Next, Figure 24 is a graph showing the expression level of NY-ESO-1. In Figure 24, the horizontal axis represents the sample type, and the vertical axis represents the expression level of NY-ESO-1 on the cell surface. As shown in Figure 24, the introduction of invariant chain siRNA increased the expression level of NY-ESO-1 on the cell surface. Furthermore, compared with the expression level of the invariant chain in Figure 23, a decrease in the expression level of the invariant chain was associated with an increase in the expression level of NY-ESO-1 on the cell surface, i.e., an increase in the expression level of the neoself antigen.

[0352] (2) Comparison of expression levels of tumor antigen and invariant chain As mentioned above, a certain trend was observed in the expression levels of the invariant chain and the neoself antigen. Therefore, the expression levels (average fluorescence intensity) of the invariant chain obtained in Example 20(1) and the expression levels (average fluorescence intensity) of NY-ESO-1 were compared. These results are shown in Figure 25.

[0353] Figure 25 is a graph comparing the expression levels of the invariant chain and NY-ESO-1. In Figure 25, the horizontal axis represents the expression level of the invariant chain, the vertical axis represents the expression level of NY-ESO-1, and R in the figure represents the correlation coefficient. As shown in Figure 25, as the expression level of the invariant chain increased, the expression level of NY-ESO-1 decreased, and the two showed a significantly negative correlation. In other words, suppressing the expression level of the invariant chain increases the amount of neoself antigen presented on the cell surface. From these results, it was estimated that suppressing the expression level of the invariant chain increases the amount of neoself antigen presented on the cell surface, and can further enhance the immune response to the target antigen.

[0354] [Example 20] We confirmed that antibody induction can be achieved by an LNP vaccine containing siRNA against IgG and the invariant strand.

[0355] (1) Preparation of mRNA transcription DNA template and siRNA A mRNA transcription DNA template for the CH domain of IgG was prepared in the same manner as in Example 14(1). The invariant strand siRNA was prepared in the same manner as in Example 10(1).

[0356] (2) In vitro transcription mRNA was prepared from the IgG CH domain DNA template prepared in Example 20(1) in the same manner as in Example 1(2).

[0357] (3) Preparation of lipid nanoparticle (LNP) vaccine An LNP vaccine was prepared in the same manner as in Example 6(3), except that the mRNA obtained in Example 20(2) was mixed with a siRNA mixture in a ratio of IgG:siRNA = 10:1.

[0358] (4) Antibody induction effect As shown in Figure 26(A), instead of using an LNP vaccine containing the CH domain of IgG and CIITA mRNA and CD74 siRNA, an LNP vaccine containing the CH domain of IgG and CD74 siRNA was used, and the anti-human IgG antibody titer was measured in the same manner as in Example 14(4), except that blood collection was performed on the 9th day after administration of the LNP vaccine. These results are shown in Figure 26(B).

[0359] Figure 26 is a graph showing the protocol and anti-hIgG antibody titer. In Figure 26, (A) shows the administration protocol for the LNP vaccine, and (B) shows the anti-hIgG antibody titer. In Figure 26(B), the horizontal axis shows the sample type, and the vertical axis shows the anti-hIgG antibody titer. As shown in Figure 26(B), the group administered with Example 20 (hIgG + CD74 siRNA) showed a significant increase in anti-hIgG antibody titer compared to the negative control (untreated) and the control (hIgG alone). In other words, it was found that even for antigens that do not sufficiently induce an immune response when administered alone, a strong immune response can be induced by combining them with siRNA against the invariant chain.

[0360] [Example 21] We confirmed that antibodies can be induced by an LNP vaccine containing siRNA against HEL and invariant strands.

[0361] (1) Preparation of mRNA transcription DNA template and siRNA A HEL mRNA transcription DNA template was prepared in the same manner as in Example 20(1), except that HEL (Hen Egg Lysozyme, SEQ ID NO: 125) was used instead of the CH domain of IgG. HEL is a modified protein that has been modified to accumulate in cells by introducing C30A and C64A mutations into the amino acid sequence (wild-type HEL) based on the nucleotide sequence registered under NCBI accession number NM_205281.2. The amino acid sequences or nucleotide sequences shown in SEQ ID NOs: 124-125 below correspond to the endoplasmic reticulum localization signal sequence, the C30A mutation, and the C64A mutation, respectively. The invariant strand siRNA was prepared in the same manner as in Example 10(1).

[0362] The amino acid sequence of HEL (SEQ ID NO: 124) is MRSLLILVLCFLPLAALGKVFGRCELAAAMKRHGLDNYRGYSLGNWVAAAKFESNFNTQATNRNTDGSTDYGILQINSRWWANDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDGNGMNAWVAWRNRCKGTDVQAWIRGCRL

[0363] Polynucleotide encoding HEL (SEQ ID NO: 125) 5'-ATGAGGTCTTTGCTAATCTTGGTGCTTTGCTTCCTGCCCCTGGCTGCTCTGGGGAAAGTCTTTGGACGATGTGAGCTGGCAGCGGCTATGAAGCGTCACGGACTTGATAACTATCGGGATACAGCCTGGGAAACTGGGTGGCTGCCGCAAAATTCGAGAGTAACTTCAACACCCAGGCTACAAACCGTAACACCGATGGGAGTACCGACTACGGAATCCTA CAGATCAACAGCCGCTGGTGGCCAACGATGGCAGGACCCCAGGCTCCAGGAACCTGTGCAACATCCCGTGCTCAGCCCTGCTGAGCTCAGACATAACAGCGAGCGTGAACTGCGCGAAGAAGATCGTCAGCGATGGAAACGGCATGAACGCGTGGGTCGCCTGGCGCAACCGCTGCAAGGGCACCGACGTCCAGGCGTGGATCAGAGGCTGCCGGCTGTGA-3'

[0364] (2) In vitro transcription mRNA was prepared from the HEL DNA template prepared in Example 21(1) in the same manner as in Example 1(2).

[0365] (3) Preparation of lipid nanoparticle (LNP) vaccine An LNP vaccine was prepared in the same manner as in Example 20(3), except that the mRNA of the CH domain of IgG was replaced with the mRNA of HEL.

[0366] (4) Antibody induction effect As shown in Figure 27(A), an LNP vaccine containing HEL mRNA and CD74 siRNA was used instead of an LNP vaccine containing the CH domain of IgG and CIITA mRNA and CD74 siRNA, and the anti-HEL antibody titer was measured in the same manner as in Example 14(4), except that blood collection was performed on the 9th day after administration of the LNP vaccine. These results are shown in Figure 27(B).

[0367] Figure 27 is a graph showing the protocol and anti-HEL antibody titer. In Figure 27, (A) shows the administration protocol for the LNP vaccine, and (B) shows the anti-HEL antibody titer. In Figure 27(B), the horizontal axis shows the sample type, and the vertical axis shows the anti-HEL antibody titer. As shown in Figure 27(B), the group administered with Example 21 (HEL + CD74 siRNA) showed a significant increase in anti-HEL antibody titer compared to the negative control (untreated) and the control (HEL alone). In other words, it was found that even for antigens that do not sufficiently induce an immune response when administered alone, a strong immune response can be induced by combining them with siRNA against the invariant chain.

[0368] [Example 22] We confirmed that antibody induction ability can be enhanced by an LNP vaccine containing siRNA against ERVW-1 and the invariant strand.

[0369] (1) Preparation of mRNA transcription DNA template and siRNA A mRNA transcription DNA template for ERVW-1 was prepared in the same manner as in Example 6(1), except that the extracellular region of the endogenous retrovirus-derived protein ERVW-1 (SEQ ID NO: 127) was used instead of gp70. The amino acid sequences or nucleotide sequences shown in SEQ ID NOs: 126-127 correspond to the endoplasmic reticulum localization signals (derived from SLAM protein), respectively. The invariant strand siRNA was prepared in the same manner as in Example 10(1).

[0370] The amino acid sequence of the extracellular region of ERVW-1 (SEQ ID NO: 126) is MDPKGSLSWRILLFLSLAFELSYGAPPPCRCMTSSSPYQEFLWRMQRPGNIDAPSYRSLSKGTPTFTAHTHMPRNCYHSATLCMHANTHYWTGKMINPSCPGGLGVTVCWTYFTQTGMSDGGGVQDQAREKHVKEVISQLTRVHGTSSPYKGLDLSKLHETLRTHTRLVSLFNTTLTGLHEVSAQNPTNCWICLPLNFRPYVSIPVPEQWNNFST EINTTSVLVGPLVSNLEITHTSNLTCVKFSNTTYTTNSQCIRWVTPPTQIVCLPSGIFFVCGTSAYRCLNGSSESMCFLSFLVPPMTIYTEQDLYSYVISKPRNKRVPILPFVIGAGVLGA LGTGIGGITTSTQFYYKLSQELNGDMERVADSLVTLQDQLNSLAAVVLQNRRALDLLTAERGGTCLFLGEECCYYVNQSGIVTEKVKEIRDRIQRRAEELRNTGPWGLLSQWMPWILPFLGP

[0371]

[0372] The amino acid sequence of the intracellular region of ERVW-1 (SEQ ID NO: 128) is: LAAIILLLLFGPCIFNLLVNFVSSRIEAVKLQMEPKMQSKTKIYRRPLDRPASPRSDVNDIKGTPPEEISAAQPLLRPNSAGSS

[0373] The polynucleotide encoding the intracellular region of ERVW-1 (SEQ ID NO: 129) is 5'-CTAGCAGCTATAATATTGCTACTCCTCTTTGGACCCTGTATCTTTAACCTCCTTGTTAACTTTGTCTCTTCCAGAATCGAAGCTGTAAAACTACAAATGGAGCCCAAGATGCAGTCCAAGACTAAGATCTACCGCAGACCCCTGGACCGGCCTGCTAGCCCACGATCTGATGTTAATGACATCAAAGGCACCCCTCCTGAGGAAATCTCAGCTGCACAACCTCTACTACGCCCCAATTCAGCAGGAAGCAGTTAG-3'

[0374] (2) In vitro transcription mRNA was prepared from the ERVW-1 DNA template prepared in Example 21(1) in the same manner as in Example 1(2).

[0375] (3) Preparation of lipid nanoparticle (LNP) vaccine An LNP vaccine was prepared in the same manner as in Example 20(3), except that the mRNA of ERVW-1 was used instead of the mRNA of the CH domain of IgG.

[0376] (4) C3H mice were used as the antibody-inducing mice. As shown in Figure 28(A), the group receiving the LNP vaccine containing mRNA prepared in Example 22(3) was administered either the LNP vaccine containing mRNA or the LNP vaccine containing mRNA and siRNA to each mouse at a dose of 100 μl each, once every two weeks, for a total of two doses. The administration method was intramuscular injection to four sites on the hands and feet. Next, seven days after the second administration, blood was collected from the mice and the antibody titer against ERVW-1 in the obtained serum was measured. The extracellular domain of ERVW-1, MHC-II (I-Ekα + I-Ekβ), and GFP mRNA were introduced into HEK293T cells, and the serum diluted 100-fold was added and allowed to stand. After the aforementioned standing period, the HEK293T cells were stained with the APC-labeled anti-mouse IgG Fc antibody, and the fluorescence intensity of the HEK293T cells was measured using a flow cytometer. These results are shown in Figure 28(B).

[0377] Figure 28 is a graph showing the protocol and anti-ERVW-1 antibody titer. In Figure 28, (A) shows the administration protocol for the LNP vaccine, and (B) shows the anti-ERVW-1 antibody titer. In Figure 28(B), the horizontal axis shows the sample type, and the vertical axis shows the anti-ERVW-1 antibody titer. As shown in Figure 28(B), the group administered Example 22 (ERVW-1 + CIITA + CD74 siRNA) showed a significant increase in anti-ERVW-1 antibody titer compared to the negative control (untreated) and control (ERVW-1 alone). Therefore, it was found that a strong immune response can be induced by combining an MHCII molecule expression inducer with siRNA against the invariant chain.

[0378] While the present disclosure has been described above with reference to embodiments and examples, the present disclosure is not limited to the above embodiments and examples. Various modifications to the structure and details of the present disclosure are possible, as can be understood by those skilled in the art within the scope of the present disclosure.

[0379] This application claims priority based on Japanese Patent Application No. 2025-024549, filed on 18 February 2025, and incorporates all of its disclosures herein.

[0380] The patents, patent applications, and documents cited herein are incorporated herein by reference in the same manner as their contents are specifically described herein.

[0381] <Notes> Some or all of the above embodiments and examples may be described as follows, but are not limited to the following. <Compositions> (Note 1) A composition comprising a target antigen or a polynucleotide encoding the same, and an expression inducer and / or an invariant chain inhibitor of major histocompatibility complex class II (MHC class II molecule). (Note 2) The composition according to Note 1, wherein the expression inducer of the MHC class II molecule is an MHC class II molecule or a polynucleotide encoding the same. (Note 3) The composition according to Note 1 or 2, wherein the expression inducer of the MHC class II molecule is a transcription inducer of the MHC class II molecule or a polynucleotide encoding the same. (Note 4) The composition according to Note 3, wherein the transcription inducer of the MHC class II molecule comprises at least one selected from the group consisting of major histocompatibility complex class II transactivator (CIITA), interferon-γ (IFN-γ), and granulocyte-macrophage colony-stimulating factor (GM-CSF). (Note 5) The composition according to any one of Notes 1 to 4, comprising the invariant chain expression inhibitor. (Note 6) The composition according to Note 5, wherein the invariant chain expression inhibitor is EBV immediate-early protein (BZLF1) or a polynucleotide encoding it. (Note 7) The composition according to Note 5 or 6, wherein the invariant chain expression inhibitor is an invariant chain expression inhibitory nucleic acid molecule. (Note 8) The composition according to any one of Notes 1 to 7, comprising a replicase protein or a polynucleotide encoding it. (Note 9) The composition according to any one of Notes 1 to 8, for use in combination with an immune checkpoint inhibitor. (Note 10) The composition according to any one of Notes 1 to 8, comprising an immune checkpoint inhibitor.(Note 11) The composition according to Note 9 or 10, wherein the immune checkpoint inhibitor is selected from the group consisting of PD-1 antibody, PD-L1 antibody, PD-L2 antibody, CTLA4 antibody, TIGIT antibody, LAG3 antibody, KIR antibody, CD137 antibody, CCR4 antibody, LILRB1 antibody, LILRB2 antibody, NKG2A antibody, BTLA antibody, TIM-3 antibody, B7-H3 antibody, B7-H4 antibody, HVEM antibody, GAL9 antibody, CD160 antibody, VISTA antibody, BTNL2 antibody, PVR antibody, BTN1A1 antibody, BTN2A2 antibody, BTN3A2 antibody, and CSF1-R antibody. (Note 12) The composition according to any one of Notes 1 to 11, comprising lipid nanoparticles (LNP). (Note 13) The composition according to any one of Notes 1 to 12, wherein the target antigen is selected from the group consisting of tumor-associated antigens, viral antigens, bacterial antigens, and parasitic antigens. (Note 14) The composition according to any one of Notes 1 to 13, wherein the target antigen is bindable to an MHC class II molecule. (Note 15) The composition according to any one of Notes 1 to 14 for use in inducing an immune response to a target antigen. (Note 16) The composition according to Note 15, wherein the immune response is an immune response by T cells or B cells. (Note 17) The composition according to Note 16, comprising an expression inducer for an MHC class II molecule, wherein the immune response is an immune response by T cells. (Note 18) The T cell is CD4. ＋ T cells and CD8 ＋ A composition according to Appendix 16 or 17, wherein at least one of the T cells. (Appendix 19) A composition according to any one of Appendix 16 to 18, comprising an invariant chain expression inhibitor, wherein the immune response is an immune response by B cells. (Appendix 20) A composition according to any one of Appendix 16 to 19, wherein the immune response is an antibody production response to the target antigen. (Appendix 21) A composition according to any one of Appendix 1 to 20 for use in activating target antigen-responsive T cells. (Appendix 22) The target antigen-responsive T cells are CD4 ＋ T cells and CD8 ＋A composition according to Appendix 21, wherein at least one of the T cells. (Appendix 23) A composition according to any one of Appendix 1 to 22 for use in activating target antigen-responsive B cells. <Pharmaceutical composition> (Appendix 24) A pharmaceutical composition comprising a composition according to any one of Appendix 1 to 23. (Appendix 25) A pharmaceutical composition comprising an immune checkpoint inhibitor, for use in combination with any one of Appendix 1 to 23. (Appendix 26) A pharmaceutical composition according to Appendix 24 or 25 for use in the treatment of cancer or infectious diseases. (Appendix 27) A pharmaceutical composition according to Appendix 26, wherein the cancer is one selected from the group consisting of skin cancer, colorectal cancer, kidney cancer, breast cancer, lung cancer, hematological cancer, brain tumor, bladder cancer, pancreatic cancer, liver cancer, uterine cancer, ovarian cancer, bone tumor, stomach cancer, esophageal cancer, tongue cancer, and nasopharyngeal cancer. (Note 28) The pharmaceutical composition according to Note 26 or 27, wherein the target antigen is a tumor-associated antigen (TAA) and an antigen that can be presented on the cell surface by an MHC class II molecule. (Note 29) The pharmaceutical composition according to Note 28, wherein the TAA is selected from the group consisting of gp100, Tyrp1, Tyrp2, gp70, and Melan A. (Note 30) The pharmaceutical composition according to any one of Notes 24 to 29 for use in combination with an immune checkpoint inhibitor. (Note 31) The pharmaceutical composition according to Note 25 or 30, wherein the immune checkpoint inhibitor is selected from the group consisting of PD-1 antibody, PD-L1 antibody, PD-L2 antibody, CTLA4 antibody, TIGIT antibody, LAG3 antibody, KIR antibody, CD137 antibody, CCR4 antibody, LILRB1 antibody, LILRB2 antibody, NKG2A antibody, BTLA antibody, TIM-3 antibody, B7-H3 antibody, B7-H4 antibody, HVEM antibody, GAL9 antibody, CD160 antibody, VISTA antibody, BTNL2 antibody, PVR antibody, BTN1A1 antibody, BTN2A2 antibody, BTN3A2 antibody, and CSF1-R antibody. (Note 32) The pharmaceutical composition according to Note 26, wherein the infectious disease is one selected from the group consisting of influenza virus, malaria, EB virus, herpes simplex virus, and cytomegalovirus. (Note 33) The pharmaceutical composition according to Note 32, wherein the target antigen is selected from the group consisting of viral antigens, bacterial antigens, and parasitic antigens.<Methods> (Note 34) A method for inducing an immune response to a target antigen using the composition described in any of Notes 1 to 23 and / or the pharmaceutical composition described in any of Notes 24 to 33. (Note 35) A method for activating target antigen-responsive T cells using the composition described in any of Notes 1 to 23 and / or the pharmaceutical composition described in any of Notes 24 to 33. (Note 36) A method for activating target antigen-responsive B cells using the composition described in any of Notes 1 to 23 and / or the pharmaceutical composition described in any of Notes 24 to 33. (Note 37) A method for inducing antibodies against a target antigen using the composition described in any of Notes 1 to 23 and / or the pharmaceutical composition described in any of Notes 24 to 33. (Note 38) The method according to any of Notes 34 to 37, comprising the step of administering the composition and / or the pharmaceutical composition to a subject. (Note 39) The method according to any of Notes 34 to 38, for use in vitro or in vivo. <Treatment Method> (Note 40) A method for treating cancer, comprising using the composition described in any of Notes 1 to 23 and / or the pharmaceutical composition described in any of Notes 24 to 31 on a subject. (Note 41) The treatment method according to Note 40, further comprising the step of administering an immune checkpoint inhibitor to the subject before, during, and / or after administration of the composition and / or the pharmaceutical composition. (Note 42) A method for treating an infectious disease, comprising using the composition described in any one of Notes 1 to 23 and / or the pharmaceutical composition described in any of Notes 24, 26, 32, and 33 on a subject. <Use> (Note 43) The composition described in any of Notes 1 to 23 for use in a method for treating cancer. (Note 44) The composition described in any of Notes 1 to 23 for use in a method for treating an infectious disease. (Note 45) Use of the composition described in any of Notes 1 to 23 for manufacturing a pharmaceutical composition for use in treating cancer. (Note 46) Use of any of the compositions described in Notes 1 to 23 for the manufacture of pharmaceutical compositions for use in the treatment of infectious diseases.

[0382] As explained above, this disclosure can induce an immune response to a target antigen. Therefore, this disclosure is extremely useful, for example, in the pharmaceutical field.

Claims

1. A composition comprising a target antigen or a polynucleotide encoding it, and an expression inducer and / or an invariant chain inhibitor of major histocompatibility complex class II (MHC class II molecule).

2. The composition according to claim 1, wherein the expression inducer for the MHC class II molecule is an MHC class II molecule or a polynucleotide encoding it.

3. The composition according to claim 1 or 2, wherein the expression inducer of the MHC class II molecule is a transcription inducer of the MHC class II molecule or a polynucleotide encoding it.

4. The composition according to claim 3, wherein the transcription inducer of the MHC class II molecule comprises at least one selected from the group consisting of major histocompatibility complex class II transactivator, interferon-γ, and granulocyte-macrophage colony-stimulating factor.

5. The composition according to any one of claims 1 to 4, comprising the invariant chain expression inhibitor.

6. The composition according to claim 5, wherein the invariant chain expression inhibitor is EBV immediate-early protein or a polynucleotide encoding it.

7. The composition according to claim 5 or 6, wherein the invariant chain expression inhibitor is an invariant chain expression inhibitory nucleic acid molecule.

8. The composition according to any one of claims 1 to 7, for use in combination with an immune checkpoint inhibitor.

9. The composition according to any one of claims 1 to 8, comprising an immune checkpoint inhibitor.

10. The composition according to any one of claims 1 to 9, comprising lipid nanoparticles.

11. The composition according to any one of claims 1 to 10, wherein the target antigen is a tumor-associated antigen.

12. The composition according to any one of claims 1 to 11, wherein the target antigen is capable of binding to an MHC class II molecule.

13. A composition according to any one of claims 1 to 12, for use in inducing an immune response to a target antigen.

14. A pharmaceutical composition comprising the composition described in any one of claims 1 to 13.

15. The pharmaceutical composition according to claim 14, for use in the treatment of cancer or infectious diseases.

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