Design method and production method for nucleic acid construct and protein complex

Abstract

The purpose of the present invention is to provide a novel nucleic acid construct that induces antibody production. The present invention provides a nucleic acid construct that induces antibody production against a target protein, the nucleic acid construct including a polynucleotide that codes for at least two types of T cell epitopes and a polynucleotide that codes for one or more types of B cell epitopes for the target protein. The present invention makes it possible to powerfully induce antibodies against a target protein within the body of a subject, making it possible not only to induce antibody production against exogenous antigens such as bacteria and viruses but also to induce antibody production against proteins produced within the body of the self.

Description

Methods for designing and producing nucleic acid constructs and protein complexes REFERENCE TO RELATED APPLICATIONS

[0001] This application benefits from the priority of an earlier Japanese application, Patent Application No. 2022-162824 (filing date: October 7, 2022), the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a nucleic acid construct and a method for designing and producing the same.

[0003] Many antibody drugs have demonstrated excellent therapeutic effects in the treatment of chronic and intractable diseases, but many of their target molecules are self-proteins (e.g., inflammatory cytokines, which are causative agents in autoimmune diseases). Vaccines, on the other hand, induce effective antibodies against foreign antigens such as viruses, and are primarily used to treat infectious diseases. However, existing vaccine technologies have the problem of being unable to induce antibody production against self-proteins due to immune tolerance, making them inapplicable to the treatment of chronic and intractable diseases (Non-Patent Documents 1, 2, 3, and 4).

[0004] CA Janeway Jr., Immunol Today; 13(1): 11-6 (1992).Ada G., N Engl J Med; 345(14): 1042-53 (2001).Ada GL., Lancet; 335(8688): 523-6 (1990).Anderson RM, et al., Lancet; 350(9089): 1466-70 (1997).

[0005] An object of the present invention is to provide a novel nucleic acid construct that induces antibody production, as well as methods for designing and producing the same.

[0006] The present inventors have found that antibody production can be induced by a nucleic acid construct comprising a polynucleotide encoding two or more T cell epitopes and a polynucleotide encoding one or more B cell epitopes of the target protein. The present inventors have also found that the nucleic acid construct is effective as a vaccine through administration tests to animals. The present invention is based on these findings.

[0007] The present invention provides the following: [1] A nucleic acid construct for inducing antibody production against a target protein, comprising a polynucleotide encoding two or more T cell epitopes and a polynucleotide encoding one or more B cell epitopes of the target protein. [2] The nucleic acid construct according to [1] above, wherein the two or more T cell epitopes consist of an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 1 to 82, or an amino acid sequence substantially identical to that amino acid sequence. [3] The nucleic acid construct according to [1] or [2] above, wherein the target protein is a protein produced in the body. [4] The nucleic acid construct according to any of [1] to [3] above, wherein the target protein is IL-17A and / or IL-23. [5] The nucleic acid construct according to any of [1] to [4] above, wherein the target protein is a protein that causes a disease or a protein produced due to a disease, and the disease is an autoimmune disease or an allergic disease. [6] The nucleic acid construct according to [5] above, wherein the autoimmune disease is one or more autoimmune diseases selected from the group consisting of psoriasis, rheumatoid arthritis, systemic lupus erythematosus, Graves' disease, chronic thyroiditis, type I diabetes, vasculitis, Addison's disease, polymyositis, Sjögren's syndrome, systemic sclerosis, and glomerulonephritis. [7] The nucleic acid construct according to [5] above, wherein the allergic disease is one or more allergic diseases selected from the group consisting of atopic dermatitis, bronchial asthma, allergic rhinitis, hay fever, food allergy, and allergic conjunctivitis. [8] The nucleic acid construct according to any of [1] to [7] above, which is DNA or RNA. [9] A preventive or therapeutic agent for a disease, comprising as an active ingredient the nucleic acid construct according to any of [1] to [8] above.

[10] The preventive or therapeutic agent according to [9] above, wherein the disease is an autoimmune disease or an allergic disease.

[11] The preventive agent according to [9] or

[10] above, which is a pan-HLA compatible vaccine.

[12] A method for producing a preventive or therapeutic agent for a disease, comprising the step of using as an active ingredient a nucleic acid construct that induces antibody production against a target protein, wherein the nucleic acid construct comprises a polynucleotide encoding two or more T cell epitopes and a polynucleotide encoding one or more B cell epitopes of the target protein.

[13] The method according to

[12] above, wherein the disease is an autoimmune disease or an allergic disease.

[14] A method for designing a nucleic acid construct or a protein complex that induces antibody production against a target protein, comprising the step of combining two or more T cell epitopes with one or more B cell epitopes of the target protein.

[15] A method for producing a preventive or therapeutic agent for a disease, comprising the steps of: (P) carrying out the design method according to claim 14 to design a nucleic acid construct or a protein complex that induces antibody production against a target protein, and (Q) preparing the nucleic acid construct or protein complex designed in step (P).

[101] A method for designing a nucleic acid construct or protein complex that induces antibody production against a target protein, comprising a step of combining two or more T cell epitopes with one or more B cell epitopes of the target protein.

[102] The design method according to

[101] above, wherein the T cell epitopes have high binding affinity to MHC class II molecules.

[103] The design method according to

[101] or

[102] above, wherein the target protein is a protein that causes a disease or a protein produced due to a disease.

[104] The design method according to any of

[101] to

[103] above, wherein the target protein is a protein produced in the body itself.

[105] The design method according to

[103] or

[104] above, wherein the disease is one or more diseases selected from the group consisting of autoimmune diseases, allergic diseases, cancer, neurodegenerative diseases, infectious diseases, and autoinflammatory diseases.

[106] The design method according to

[105] above, wherein the autoimmune disease is one or more autoimmune diseases selected from the group consisting of psoriasis, rheumatoid arthritis, systemic lupus erythematosus, Graves' disease, chronic thyroiditis, type I diabetes, vasculitis, Addison's disease, polymyositis, Sjögren's syndrome, systemic sclerosis, and glomerulonephritis.

[107] The design method according to

[105] above, wherein the allergic disease is one or more allergic diseases selected from the group consisting of atopic dermatitis, bronchial asthma, allergic rhinitis, hay fever, food allergy, and allergic conjunctivitis.

[108] The design method according to

[105] above, wherein the cancer is one or more cancers selected from the group consisting of skin cancer, colon cancer, breast cancer, gastric cancer, leukemia, malignant lymphoma, and multiple myeloma.

[109] The design method according to

[105] above, wherein the neurodegenerative disease is Alzheimer's disease.

[110] The design method according to

[105] above, wherein the infectious disease is one or more infectious diseases selected from the group consisting of Zika fever, SARS-CoV-2 infection, and influenza infection.

[111] The design method according to

[105] above, wherein the autoinflammatory disease is one or more autoinflammatory diseases selected from the group consisting of familial Mediterranean fever, cryopyrin-associated periodic fever syndrome, TNF receptor-associated periodic syndrome, mevalonate kinase deficiency, Blau syndrome, periodic fever, aphthous stomatitis, pharyngitis, and cervical lymphadenitis syndrome.

[112] The design method according to

[103] above, wherein the disease is one or more diseases selected from the group consisting of multiple sclerosis, neuromyelitis optica, myasthenia gravis, pemphigus, and autoimmune hemolytic anemia.

[113] The design method according to any one of

[101] to

[112] above, wherein the target protein is one or more proteins selected from the group consisting of IL-17A, IL-23, IL-1β, IL-13, IL-13R, IL-4Rα, TNF-α, IL-6, IL-6R, IgE, IL-5, IL-5Rα, FcRn, PD-1, PD-L1, CTLA-4, HER2, EGFR, CD22, CD20, RANKL, C5, IFNγ, amyloid β, respiratory syncytial virus antigen protein, Zika virus antigen protein, SARS-CoV-2 virus antigen protein, and influenza virus antigen protein.

[114] The design method according to any one of

[101] to

[112] above, wherein the target protein is IL-17A and / or IL-23.

[115] The design method according to any one of

[101] to

[114] above, wherein the nucleic acid construct is DNA or mRNA.

[116] A method for producing a preventive or therapeutic agent for a disease, comprising the steps of: (P) carrying out the design method according to any one of

[101] to

[115] above to design a nucleic acid construct or a protein complex that induces antibody production against the target protein, and (Q) preparing the nucleic acid construct or protein complex designed in step (P).

[0008] According to the present invention, it is possible to potently induce antibodies against a target protein in the body of a subject, which is advantageous in that it is possible to induce not only antibody production against foreign antigens such as bacteria and viruses, but also antibody production against proteins produced in the body itself (autoproteins) (for example, inflammatory cytokines produced in the body of a subject who has developed an autoimmune disease, etc.).

[0009] Figure 1-1 shows specific examples of amino acid sequences of human T cell epitopes. Figure 1-2 shows specific examples of amino acid sequences of human T cell epitopes. Figure 1-3 shows specific examples of amino acid sequences of human T cell epitopes. Figure 2A shows an image of the structure of the nucleic acid construct (mRNA vaccine) of the present invention. Figure 2B shows a schematic diagram of the nucleic acid construct prepared in Example 3. T-86 to T88 correspond to the nucleotide sequences encoding the T cell epitopes shown in SEQ ID NOs: 86 to 88, respectively, and B89 to B94 correspond to the nucleotide sequences encoding the B cell epitopes shown in SEQ ID NOs: 89 to 94, respectively. Figure 3 shows the antibody titers (absorbance at a wavelength of 450 nm) of antibodies against IL-17A and IL-23 induced by the nucleic acid constructs encoding T cell epitopes. Error bars indicate standard deviation. Figure 4 shows the antibody titers (absorbance at 450 nm) of subclass antibodies against IL-17A and IL-23 induced by a nucleic acid construct encoding a T cell epitope. IgG1, IgG2a, IgG2b, and IgG3 all represent antibody titers in the administration group. Figure 5 shows the antibody titers (absorbance at 450 nm) of antibodies against IL-17A and IL-23 induced by a nucleic acid construct encoding a T cell epitope. Error bars indicate standard deviation. Figure 6A shows the change in ear thickness in the control and normal mice after IMQ application. Figure 6B shows the inhibition rate of ear thickness increase 5 days after IMQ application in the control and treatment groups. Figure 6C shows representative photographs of the ears of psoriasis model mice (control and treatment groups) and normal mice 6 days after IMQ application. Figure 7A shows the change in ear thickness in the control and normal mice after IMQ application. Figure 7B shows the inhibition rate of ear thickness increase 6 days after IMQ application in the control and treatment groups. Figure 7C shows representative photographs of the ears of psoriasis model mice (control and treatment groups) and normal mice 6 days after IMQ application. Specific Description of the Invention

[0010] <<Nucleic Acid Construct>> The present invention provides a nucleic acid construct that induces antibody production against a target protein, and is characterized in that it comprises polynucleotides encoding two or more T cell epitopes and polynucleotides encoding one or more B cell epitopes of the target protein.

[0011] In the present invention, the term "T cell epitope" refers to a peptide that binds to an MHC class II molecule of the major histocompatibility complex (MHC) to form a complex, which is displayed on antigen-presenting cells (e.g., B cells, dendritic cells, macrophages) and is specifically recognized by the T cell receptor (TCR) of CD4-positive T cells.

[0012] In the present invention, any type of T cell epitope can be selected as the two or more types of T cell epitopes as long as it binds to the target MHC class II molecule. However, from the viewpoint of strongly inducing antibody production against the target protein (i.e., strongly activating CD4-positive T cells), it is preferable to select a T cell epitope that is taken up by antigen-presenting cells in the subject's living body and strongly binds to the MHC class II molecule (i.e., has a high binding ability to the MHC class II molecule).

[0013] In the present invention, T cell epitopes with high binding ability to MHC class II molecules can be selected using a known MHC binding prediction tool and a predicted binding score to MHC class II as an index. Examples of MHC binding prediction tools include Immune Epitope Database Analysis Resource (IEDB Analysis Resource, http: / / tools.iedb.org / main / ), MHCBN (http: / / crdd.osdd.net / raghava / mhcbn / ), NetMHCII (https: / / services.healthtech.dtu.dk / service.php?NetMHCII-2.3), SYFPEITHI (http: / / www.syfpeithi.de / 0-Home.htm), ANTIJEN (http: / / www.ddg-pharmfac.net / antijen / AntiJen / antijenhomepage.htm), IMGT / 3Dstructure-DB (https: / / www.imgt.org / IMGTindex / IMGT3Dstructure-db.php), and SEDB (http: / / sedb.bicpu.edu.in / ).

[0014] In humans, MHC refers to human leukocyte antigens (HLA), and examples of HLA class II molecules include HLA-DR, HLA-DQ, and HLA-DP. HLA-DR, HLA-DQ, and HLA-DP are all composed of an α chain and a β chain, and examples of the α chain include HLA-DRA, HLA-DQA, and HLA-DPA, and examples of the β chain include HLA-DRB, HLA-DQB, and HLA-DPB.

[0015] 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, and HLA-DR53.

[0016] Examples of HLA-DQ include HLA-DQ1, HLA-DQ2, HLA-DQ3, HLA-DQ4, HLA-DQ5, HLA-DQ6, HLA-DQ7, and HLA-DQ8.

[0017] Examples of HLA-DP include HLA-DP1, HLA-DP2, HLA-DP3, HLA-DP4, and HLA-DP5.

[0018] In the present invention, examples of HLA to which T cell epitopes bind include HLA-DR, which, when the subject is Japanese, has an α chain of HLA-DRA1. ＊ 01 and other alleles, and DRB1 as the β chain ＊ 01:01, DRB1 ＊ 04:03, DRB1 ＊ 04:05, DRB1 ＊ 04:06, DRB1 ＊ 08:02, DRB1 ＊ 08:03, DRB1 ＊ 09:01, DRB1 ＊ 12:01, DRB1 ＊ 13:02, DRB1 ＊ 14:54, DRB1 ＊ 15:01, DRB1 ＊ 15:02 and DRB1 ＊ In the case of Westerners, the α chain is HLA-DRA1. ＊ 01 and other alleles, and DRB1 as the β chain ＊ 07:01, DRB1 ＊ 03:01, DRB1 ＊ 04:01 and DRB1 ＊ 15:01 and other allyls.

[0019] In the present invention, specific examples of T cell epitopes include, but are not limited to, those shown in Figure 1-1 (SEQ ID NOS: 1 to 31), Figure 1-2 (SEQ ID NOS: 32 to 64), and Figure 1-3 (SEQ ID NOS: 65 to 82). That is, in the present invention, two or more T cell epitopes can consist of amino acid sequences selected from the group consisting of the amino acid sequences shown in SEQ ID NOS: 1 to 82. In the present invention, the T cell epitope can also consist of an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOS: 1 to 64 or an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOS: 65 to 82.

[0020] In the present invention, a T cell epitope can consist of an amino acid sequence substantially identical to an amino acid sequence selected from the amino acid sequences shown in SEQ ID NOs: 1 to 82 (or the amino acid sequences shown in SEQ ID NOs: 1 to 64 or 65 to 82).

[0021] Here, in the present invention, "T cell epitopes consisting of substantially the same amino acid sequence" refers to T cell epitopes consisting of a specific amino acid sequence that have one or more alterations and have the ability to bind to an MHC class II molecule (HLA class II molecule).

[0022] In the present invention, examples of T cell epitopes consisting of substantially identical amino acid sequences include those having one or more alterations selected from the group consisting of deletions, substitutions, insertions, and additions in an amino acid sequence selected from the amino acid sequences set forth in SEQ ID NOS: 1 to 82 (or the amino acid sequences set forth in SEQ ID NOS: 1 to 64 or 65 to 82), and having the ability to bind to an MHC class II molecule (HLA class II molecule). The number of altered amino acids is, for example, 1 to 4 or 1 to 3, and particularly preferably 2 or 1. The number of altered amino acids can also be the number of mutations occurring by known methods such as site-directed mutagenesis, or the number of naturally occurring mutations. In the amino acid sequence, the alterations can be continuous or discontinuous. The alterations can also be multiple, homogeneous alterations (e.g., multiple substitutions) or multiple, heterogeneous alterations (e.g., a combination of one or more deletions and one or more substitutions).

[0023] The amino acid modification may be a conservative modification. "Conservative modification" refers to modifying one or more amino acids so as not to substantially alter the function of the protein. The amino acid substitution may also be a conservative substitution. "Conservative substitution" refers to substituting one or more amino acids with other amino acids and / or amino acid derivatives so as not to substantially alter the function of the protein. In conservative substitution, the substituted amino acid and the substituted amino acid preferably have similar properties and / or functions, for example. Specifically, chemical properties such as hydrophobicity and hydrophilicity index, polarity, and charge, or physical properties such as secondary structure, are preferably similar. Such amino acids or amino acid derivatives with similar properties and / or functions are known in the art. For example, nonpolar amino acids (hydrophobic amino acids) include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Polar amino acids (neutral amino acids) include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of positively charged amino acids (basic amino acids) include arginine, histidine, and lysine, and examples of negatively charged amino acids (acidic amino acids) include aspartic acid and glutamic acid.

[0024] In the present invention, T cell epitopes consisting of substantially the same amino acid sequence also include those that have 80% or more (preferably 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, and even more preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) sequence identity with an amino acid sequence selected from the amino acid sequences shown in SEQ ID NOs: 1 to 82 (or the amino acid sequences shown in SEQ ID NOs: 1 to 64 or SEQ ID NOs: 65 to 82) and have the ability to bind to an MHC class II molecule (HLA class II molecule). Here, "identity" refers to the degree of identity when the sequences to be compared are appropriately aligned, and means the occurrence (%) of exact amino acid matches between the sequences. When calculating identity, for example, the presence of gaps in the sequence and the properties of amino acids are taken into consideration (Wilbur, Natl. Acad. Sci. USA 80:726-730 (1983)). The alignment can be performed using, for example, any algorithm, and specifically, publicly available homology search software such as BLAST (Basic local alignment search tool) (Altschul et al., J. Mol. Biol. 215:403-410 (1990)), FASTA (Peasron et al., Methods in Enzymology 183:63-69 (1990)), and Smith-Waterman (Meth. Enzym., 164, 765 (1988)) can be used. Furthermore, identity can be calculated using, for example, a publicly available homology search program such as those described above, for example, by using the default parameters of the homology algorithm BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) of the National Center for Biotechnology Information (NCBI).

[0025] The T cell epitopes to be combined in the present invention can be selected from, for example, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, and 20 or more T cell epitopes. For example, the more types of T cell epitopes to be combined, the higher the probability that the combination will include T cell epitopes with high binding affinity to the target MHC class II molecule. Therefore, when the nucleic acid construct and protein complex of the present invention are used as active ingredients in a vaccine, the versatility of subjects to which the vaccine can be administered is expanded, thereby contributing to the universalization of vaccines. Here, in the present invention, the term "protein complex" refers to a fusion protein (typically a protein that induces antibody production against a target protein) containing a T cell epitope and a B cell epitope as components, which is produced by translating the nucleic acid construct of the present invention.

[0026] In the present invention, specific examples of combinations of amino acid sequences of T cell epitopes include combinations of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, or twenty or more amino acid sequences selected from the group consisting of the amino acid sequences set forth in SEQ ID NOS: 1 to 64, or combinations of amino acids substantially identical to such amino acid sequences. Furthermore, the above combinations are not limited to, but can be up to 64, 60 or less, 55 or less, 50 or less, 45 or less, 40 or less, 35 or less, or 30 or less. These lower and upper limits can be combined arbitrarily.

[0027] In the present invention, specific examples of combinations of amino acid sequences of T cell epitopes include combinations of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, or twenty or more amino acid sequences selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 1, 2, 7-9, 13-20, 24-27, 30, 31, 34-37, 42-44, 47-54, 56, 57, and 60-62, or combinations of amino acids substantially identical to such amino acid sequences. Furthermore, the above combinations are not limited to, but can be up to 39, 35 or less, 30 or less, or 25 or less. These lower and upper limits can be combined arbitrarily.

[0028] In the present invention, the T cell epitope may be a combination of amino acid sequences of T cell epitopes that bind to different MHC class II molecules (HLA class II molecules). Specific examples of such combinations include at least one, two, three, four, or five amino acid sequences selected from the group consisting of amino acid sequences shown in SEQ ID NOs: 1 to 7, at least one, two, three, four, or five amino acid sequences selected from the group consisting of amino acid sequences shown in SEQ ID NOs: 8 to 14, at least one, two, or three amino acid sequences selected from the group consisting of amino acid sequences shown in SEQ ID NOs: 15 to 18, at least one, two, three, four, or five amino acid sequences selected from the group consisting of amino acid sequences shown in SEQ ID NOs: 19 to 25, at least one amino acid sequence selected from the group consisting of amino acid sequences shown in SEQ ID NOs: 26 or 27, at least one, two, or three amino acid sequences selected from the group consisting of amino acid sequences shown in SEQ ID NOs: 28 to 31, and at least one amino acid sequence selected from the group consisting of amino acid sequences shown in SEQ ID NOs: 32 to 35. at least one, two, or three selected from the group consisting of amino acid sequences shown in SEQ ID NOs: 36 to 39; at least one, two, or three selected from the group consisting of amino acid sequences shown in SEQ ID NOs: 40 to 44; at least one, two, or three selected from the group consisting of amino acid sequences shown in SEQ ID NOs: 45 to 48; at least one, two, three, four, or five selected from the group consisting of amino acid sequences shown in SEQ ID NOs: 49 to 55; at least one, two, or three selected from the group consisting of amino acid sequences shown in SEQ ID NOs: 56 to 60, and at least one or two selected from the group consisting of amino acid sequences shown in SEQ ID NOs: 61 to 64.

[0029] In the present invention, T cell epitopes can also be a combination of amino acid sequences of T cell epitopes that bind to different MHC class II molecules (HLA class II molecules). Specific examples of such combinations include a combination of two or more amino acid sequences selected from the group consisting of at least one amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NO: 65 or 66, at least one, two, or three amino acid sequences selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 67 to 70, at least one, two, or three amino acid sequences selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 71 to 75, and at least one, two, three, four, or five amino acid sequences selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 76 to 82.

[0030] As described above, since it is possible to combine amino acid sequences of T cell epitopes that bind to different MHC class II molecules (HLA class II molecules), the nucleic acid construct or protein complex of the present invention can be used as an active ingredient of a pan-HLA vaccine. Here, in the present invention, the term "pan-HLA vaccine" refers to a vaccine that can be applied to a wide range of subjects, and specifically refers to a vaccine that is configured to recognize two or more different HLA class II molecules present in humans.

[0031] The nucleic acid construct or protein complex of the present invention may contain multiple sets of the same T cell epitope, from the viewpoint of enhancing the induction of antibodies against the target protein in a subject. In the case of a nucleic acid construct, as shown in the Examples (Example 3) below, the nucleic acid construct may be configured to contain multiple repeats of a nucleotide sequence encoding the same T cell epitope, or multiple repeats randomly (for example, in the Examples (Example 3) below, the nucleic acid construct is configured to contain three repeats of each of the nucleotide sequences of SEQ ID NOs: 86 to 88).

[0032] In the present invention, the term "B cell epitope" refers to a portion (peptide) of a target protein (antigen) that is recognized by antigen-specific B cells.

[0033] In the present invention, any type of B cell epitope can be selected as the B cell epitope to be combined, as long as it is recognized by antigen-specific B cells.

[0034] In the present invention, target proteins include, but are not limited to, disease-causing proteins and proteins produced due to disease, including both foreign antigens (bacteria, viruses, etc.) and proteins produced within the body (autoproteins) (such as inflammatory cytokines produced within the body of a subject suffering from an autoimmune disease, etc.). Here, in the present invention, "disease-causing proteins" refer to proteins that cause disease, including proteins that cause disease or disease onset. Furthermore, "proteins produced due to disease" refer to proteins produced in response to the onset of disease, and particularly refer to proteins that have the effect of exacerbating or worsening the symptoms of the disease (e.g., inflammatory proteins).

[0035] In the present invention, examples of diseases associated with target proteins include autoimmune diseases, allergic diseases, cancer, neurodegenerative diseases, infectious diseases, and autoinflammatory diseases.

[0036] In the present invention, examples of autoimmune diseases include psoriasis, rheumatoid arthritis, systemic lupus erythematosus, Graves' disease, chronic thyroiditis (Hashimoto's disease), type I diabetes, vasculitis (e.g., antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis), Addison's disease, polymyositis, Sjogren's syndrome, systemic sclerosis, and glomerulonephritis (e.g., IgA nephropathy).

[0037] In the present invention, cancer includes solid cancers (carcinomas, sarcomas) and blood tumors, such as skin cancer (e.g., malignant melanoma, Merkel cell carcinoma, squamous cell carcinoma), colon cancer (colorectal cancer), breast cancer (including metastatic breast cancer), gastric cancer, leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia), malignant lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma), and multiple myeloma.

[0038] In the present invention, allergic diseases include, for example, atopic dermatitis, bronchial asthma, allergic rhinitis, hay fever, food allergy, and allergic conjunctivitis.

[0039] In the present invention, examples of neurodegenerative diseases include Alzheimer's disease.

[0040] In the present invention, infectious diseases include Zika fever (Zika virus infection), SARS-CoV-2 infection (so-called new coronavirus infection), and influenza infection.

[0041] In the present invention, examples of autoinflammatory diseases include familial Mediterranean fever, cryopyrin-associated periodic fever syndrome, TNF receptor-associated periodic syndrome, mevalonate kinase deficiency, Blau syndrome, periodic fever, aphthous stomatitis, pharyngitis, and cervical lymphadenitis syndrome.

[0042] In the present invention, diseases other than those mentioned above include rare diseases and intractable diseases, such as multiple sclerosis, neuromyelitis optica, myasthenia gravis, pemphigus, and autoimmune hemolytic anemia.

[0043] In the present invention, specific examples of target proteins include the proteins shown in Table 1.

[0044]

[0045] In the present invention, the B cell epitopes to be combined can be arbitrarily selected from B cell epitopes of one or more target proteins depending on the purpose, and when two or more types are selected, for example, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or nine or more B cell epitopes can be selected. When the target protein is a protein that causes a disease and / or a protein produced due to a disease, the B cell epitope may be selected from the perspective of preventing or treating the disease.

[0046] In the present invention, the combination of a T cell epitope and a B cell epitope can be selected from any type of T cell epitope and B cell epitope, respectively, and can be selected from, for example, the T cell epitopes and B cell epitopes exemplified above.

[0047] The nucleic acid construct or protein complex of the present invention can be designed and produced by known molecular biology methods based on a combination of selected T cell epitopes and B cell epitopes. Specifically, the protein complex can be designed and produced as a fusion protein containing the T cell epitope and the B cell epitope as components.

[0048] The nucleic acid construct of the present invention can be designed and produced as a single polynucleotide encoding a protein complex, i.e., a fusion protein containing a T cell epitope and a B cell epitope as components. Therefore, in this sense, the nucleic acid construct of the present invention can be rephrased as a "nucleic acid construct encoding a protein complex that induces antibody production against a target protein." In order to effectively induce antibody production in the subject's body, the order of the polynucleotide sequences encoding the T cell epitope and the B cell epitope can be adjusted as appropriate, and expression-regulating sequences or linker sequences can be inserted between the epitopes. Furthermore, the B cell epitope combined with the T cell epitope may consist of the entire target protein recognized by antigen-specific B cells, or it may consist of a portion of the target protein (e.g., a domain) containing the antigenic portion.

[0049] The nucleic acid construct of the present invention is composed of a polynucleotide, and can be a polynucleotide encoding two or more T cell epitopes and one or more B cell epitopes of a target protein. Polynucleotides include DNA and RNA, as well as modified forms thereof and artificial nucleic acids. However, from the viewpoint of using the nucleic acid construct as an active ingredient of a vaccine, RNA is preferred.

[0050] The protein complex of the present invention is configured as a fusion protein containing, as components, two or more T cell epitopes and one or more B cell epitopes of the target protein. However, the protein complex may be modified as long as the T cell epitopes and the B cell epitopes each function as epitopes.

[0051] The nucleic acid construct or protein complex of the present invention can induce antibody production against a target protein. That is, by administering the nucleic acid construct or protein complex of the present invention to a subject, antibody production can be induced in the body of the subject.

[0052] Without being bound by the following theory, the mechanism of action from the preparation of a nucleic acid construct or protein complex to the administration of the prepared nucleic acid construct or protein complex to a subject, and the induction of antibody production in the subject's body will be explained.

[0053] [I. Nucleic Acid Constructs] (i) A nucleic acid construct to be administered to a subject is prepared using known molecular biology methods based on a nucleic acid construct designed by the design method of the present invention. (ii) The prepared nucleic acid construct is loaded onto a carrier (a lipid membrane such as a liposome) and administered to a subject by injection or other methods. (iii) The administered nucleic acid construct is translated in vivo to produce a protein (a fusion protein containing a T cell epitope and a B cell epitope as components). (iv) The produced fusion protein is recognized by antigen-specific B cells in vivo, taken up into the cells, and digested. (v) In B cells, the T cell epitope forms a complex with an MHC class II molecule, and the complex is presented on the B cell surface. (vi) CD4-positive T cells recognize and bind to the complex of the T cell epitope and an MHC class II molecule via the T cell receptor (TCR), becoming activated. (vii) The activated CD4-positive T cells undergo clonality and promote the proliferation and differentiation of B cells. (viii) B cells are activated, undergo clonally proliferation, and produce large amounts of the desired antibody.

[0054] [II. Protein Complexes] (i) Based on the protein complex designed by the design method of the present invention, a protein complex (a fusion protein containing a T cell epitope and a B cell epitope as components) to be administered to a subject is prepared by known molecular biology methods. (ii) The prepared protein complex is administered to a subject by injection or other methods. (iii) The administered protein complex is recognized by antigen-specific B cells in the subject's body, taken up into the cells, and digested. (iv) In B cells, the T cell epitope forms a complex with an MHC class II molecule, and the complex is presented on the B cell surface. (v) CD4-positive T cells recognize and bind to the complex of the T cell epitope and an MHC class II molecule via the T cell receptor (TCR), and are activated. (vi) The activated CD4-positive T cells undergo clonality and promote the proliferation and differentiation of B cells. (vii) B cells are activated and undergo clonality, resulting in the mass production of the desired antibody.

[0055] The nucleic acid construct of the present invention can induce antibody production against a target protein in a subject, and therefore can be used to prevent or treat a disease associated with the target protein, and can reduce the risk of contracting a disease associated with the target protein. That is, the present invention provides a preventive or therapeutic agent for a disease, which comprises the nucleic acid construct of the present invention as an active ingredient. The present invention also provides an agent for reducing the risk of contracting a disease, which comprises the nucleic acid construct of the present invention as an active ingredient. The preventive and therapeutic agents of the present invention can be administered to subjects who have a disease or who may have a disease. The risk-reducing agent of the present invention can be administered to subjects who may have a disease.

[0056] When the nucleic acid construct of the present invention is administered to a subject, the route of administration is not particularly limited as long as it provides a therapeutic or preventive effect for the target disease or a risk reduction effect for the target disease, but parenteral administration, for example, can be selected. Non-limiting examples of parenteral administration include intravenous administration, intramuscular administration, subcutaneous administration, topical administration, intraperitoneal administration, and intranasal administration.

[0057] The parenteral formulation can be selected in an appropriate dosage form depending on the specific administration route, and examples thereof include injections. Parenteral formulations can be in the form of aqueous or non-aqueous isotonic sterile solutions or suspensions. Both formulations can be formulated using pharmaceutically acceptable carriers by techniques commonly used in the art (e.g., known methods described in the General Provisions for Preparations of the Japanese Pharmacopoeia, 18th Edition). That is, the present invention provides pharmaceutical compositions comprising the nucleic acid constructs of the present invention and pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers include excipients, binders, diluents, additives, flavorings, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, preservatives, etc. The agent of the present invention can be formulated, for example, by using the nucleic acid construct of the present invention in a lipid nanoparticle (LNP), and examples of lipids constituting the LNP include pH-responsive lipids (DLin-MC3-DMA, DLin-DMA, DLin-KC2-DMA, etc.), PEGylated lipids (PEG-DMG, PEG-DSG, PEG-DPG, etc.), cholesterol, and neutral phospholipids.

[0058] The dosage of the nucleic acid construct of the present invention can be determined depending on the subject's sex, age, and weight, symptoms, dosage form, and route of administration, etc. When the nucleic acid construct of the present invention is administered for the purpose of treating or preventing a disease, the dosage per adult can be determined to be, for example, in the range of 0.01 μg to 100 mg, but is not limited to this. In the present invention, the active ingredient at the above dosage may be administered once a day or in two to four divided doses. In the present invention, the active ingredient at the above dosage may also be administered multiple times, initially once every one, two, three, or four weeks, and then once every one, two, three, or four weeks, or once every one, two, three, or four weeks, or once every month, two, three, three, or four weeks, or once every month, two, three, three, or four weeks, or once every month, two, three, three, or four weeks, or once every month, two, three, three, or four weeks, or once every month, two, three, three, or four weeks, or once every month, two, three, three, three, or four weeks, or once every month, two, three, three, three, or four weeks, or once every month, two, three, three, three, or four weeks, or once every month, two, three, three, three, four ... three

[0059] In the present invention, subjects include humans and non-human animals, and non-human animals are preferably non-human mammals (e.g., mice, rats, cows, pigs, horses, and monkeys).

[0060] <<Design Method>> Another aspect of the present invention provides a method for designing a nucleic acid construct or a protein complex. The design method of the present invention makes it possible to design a nucleic acid construct or a protein complex that induces antibody production against a target protein.

[0061] The design method of the present invention comprises the step of combining two or more T cell epitopes with one or more B cell epitopes of the target protein.

[0062] The design method of the present invention may include the steps of (X1) evaluating the T cell epitope's ability to bind to an MHC class II molecule using an MHC binding prediction tool, and (X2) selecting a T cell epitope with high MHC class II molecule-binding ability based on the evaluation results of step (X1). In the design method of the present invention, steps (X1) and (X2) may be performed prior to the step of combining two or more T cell epitopes with one or more B cell epitopes of target proteins. The design method of the present invention may also include a step of preliminarily screening T cell epitopes prior to steps (X1) and (X2). This preliminarily screening may be performed using AI (artificial intelligence) or a prediction algorithm.

[0063] In addition to the above, the design method of the present invention can be carried out according to the description of the nucleic acid construct of the present invention.

[0064] <<Preventive or Therapeutic Agent and Production Method Thereof>> Another aspect of the present invention provides a preventive or therapeutic agent for a disease. The preventive or therapeutic agent of the present invention is characterized by comprising the nucleic acid construct or protein complex of the present invention as an active ingredient.

[0065] In the preventive or therapeutic agent of the present invention, the target protein can be a protein that causes a disease or a protein produced due to a disease. In the production method of the present invention, the target protein can also be a protein produced in the body.

[0066] The prophylactic agent of the present invention can typically be used as a vaccine. That is, the prophylactic agent of the present invention can be administered to a subject to induce antibody production against a target protein in the body of the subject, thereby preventing the onset or development of a disease, or to reduce the severity of the disease if the disease is onset or developed. Examples of the prophylactic agent of the present invention include mRNA vaccines, recombinant protein vaccines, and peptide vaccines, with mRNA vaccines being preferred from the viewpoint of production costs.

[0067] The therapeutic agent of the present invention can typically be used as an alternative to antibody drugs, i.e., by administering the therapeutic agent of the present invention to a subject, it can be used to induce antibody production against a target protein in the body of the subject, thereby treating a disease that the subject is suffering from or has developed.

[0068] Another aspect of the present invention provides a method for producing the prophylactic or therapeutic agent of the present invention, which comprises the steps of: (P) designing a nucleic acid construct or a protein complex that induces antibody production against a target protein; and (Q) preparing the nucleic acid construct or protein complex designed in step (P).

[0069] The design of the nucleic acid construct or protein complex in step (P) can be carried out according to the description of the design method of the present invention.

[0070] The preparation of the nucleic acid construct or protein complex in step (Q) can be carried out by known methods, including, but not limited to, for example, a nucleic acid construct can be prepared by chemical synthesis or in vitro transcription synthesis of oligonucleotides, and a protein complex can be prepared using an expression vector incorporating the nucleic acid construct.

[0071] The prophylactic or therapeutic agent and its production method of the present invention can be carried out in accordance with the above, as well as the description of the nucleic acid construct of the present invention and the design method of the present invention.

[0072] The present invention provides a method for preventing or treating a disease, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a nucleic acid construct or a composition comprising the same. The present invention also provides a method for reducing the risk of contracting a disease, comprising administering to a subject in need thereof an effective amount of a nucleic acid construct or a composition comprising the same. The method of the present invention can be carried out according to the description of the nucleic acid construct of the present invention and the preventive or therapeutic agent of the present invention.

[0073] The present invention also provides use of the nucleic acid construct of the present invention for the manufacture of an agent for preventing or treating a disease, or as an agent for preventing or treating a disease. The present invention also provides use of the nucleic acid construct of the present invention for the manufacture of an agent for reducing the risk of contracting a disease, or as an agent for reducing the risk of contracting a disease. The methods of the present invention can be carried out according to the descriptions related to the nucleic acid construct of the present invention and the preventive or therapeutic agent of the present invention.

[0074] The present invention will be described in more detail based on the following examples, but the present invention is not limited to these examples.

[0075] Example 1: Design of amino acid sequences of T cell epitopes (1) In Example 1, the amino acid sequences of mouse T cell epitopes shown in Table 2 were designed. In addition, the MHC class II binding ability of the amino acid sequences of the T cell epitopes shown in Table 2 was analyzed using the IEDB analysis resource.

[0076]

[0077] The results are shown in Tables 3 to 5. For the T cell epitopes shown here, a lower numerical value for MHC class II binding ability indicates a higher binding ability, suggesting that a lower numerical value for MHC class II binding ability indicates a higher antibody production-inducing activity. The results in Tables 3 to 5 suggest that these T cell epitopes have high MHC class II binding ability and high antibody production-inducing activity in Balb / c mice.

[0078]

[0079]

[0080]

[0081] Example 2: Design of amino acid sequences of T cell epitopes (2) In Example 2, amino acid sequences of human T cell epitopes were designed, and the MHC class II binding ability of the T cell epitopes was analyzed using the IEDB analysis resources.

[0082] The results are shown in Tables 6 and 7. For the T cell epitopes shown here, a lower numerical value for MHC class II binding ability indicates a higher binding ability, suggesting that a lower numerical value for MHC class II binding ability indicates a higher antibody production-inducing activity. The results in Tables 6 and 7 suggest that the MHC class II binding ability and antibody production-inducing activity are high in the majority of both Japanese and Caucasian populations. This indicates that the nucleic acid construct is pan-MHC compatible, and is applicable to the majority of human beings, including the Japanese population.

[0083]

[0084]

[0085] Example 3: Examination of antibody induction ability of nucleic acid constructs encoding T cell epitopes (1) In Example 3, the ability of nucleic acid constructs containing polynucleotides encoding T cell epitopes designed in Example 1 to induce antibody production in mice was examined.

[0086] (1) Method A. Preparation of Nucleic Acid Constructs (mRNA Vaccines) A ​​nucleic acid construct (IL-17A nucleic acid construct) containing a base sequence in which each of the base sequences encoding the three T cell epitopes shown in Table 8 (with T replaced by Ψ) is repeated three times and a base sequence in which each of the three mouse IL-17A B cell epitopes shown in Table 9 (with T replaced by Ψ) is repeated three times, and a nucleic acid construct (IL-23 nucleic acid construct) containing a base sequence in which each of the three T cell epitopes shown in Table 8 (with T replaced by Ψ) is repeated three times and a base sequence in which each of the three mouse IL-23 B cell epitopes shown in Table 10 (with T replaced by Ψ) was prepared according to standard methods (see also Figures 2A and B for the structure of the nucleic acid constructs). Ψ represents 1-methyl-3-pseudouridylyl.

[0087]

[0088]

[0089]

[0090] Balb / c mice were administered 10 μg of each of the IL-17A nucleic acid construct and IL-23 nucleic acid construct prepared in A above at 2-week intervals. Sera (200-fold diluted) collected before the start of administration (week 0) and at weeks 2, 4, 6, 8, and 10 after the start of administration were measured for antibody titers specific to mouse IL-17A and mouse IL-23 by ELISA using recombinant native mouse IL-17A or mouse IL-23 proteins, respectively. The antibody titer measurement procedure is shown in Table 11.

[0091]

[0092] (2) Results The results are shown in Figure 3. In mice administered with the nucleic acid construct (mRNA vaccine) (administration group), an increase in antibody titers against IL-17A and IL-23 was observed after the start of administration. In contrast, in mice not administered with the mRNA vaccine (control group), no increase in antibody titers against IL-17A and IL-23 was observed even after the start of administration. These results demonstrate that the nucleic acid construct (mRNA vaccine) containing the polynucleotide encoding the T cell epitope designed in Example 1 actually induces the production of antibodies against IL-17A and IL-23 in mice.

[0093] Example 4: Examination of antibody induction ability by nucleic acid constructs encoding T cell epitopes (2) In Example 4, the subclasses of antibodies induced to be produced in mice were examined using nucleic acid constructs containing polynucleotides encoding T cell epitopes designed in Example 1.

[0094] (1) Methods A. Nucleic Acid Constructs (mRNA Vaccines) In the same manner as in Example 3(1)A, IL-17A nucleic acid constructs and IL-23 nucleic acid constructs were prepared as mRNA vaccines.

[0095] (b) Antibody Titer Measurement Balb / c mice were administered 10 μg of each of the IL-17A nucleic acid construct and IL-23 nucleic acid construct prepared in (a) above at 2-week intervals. Six weeks after the start of administration, serum samples (200-fold dilution, 800-fold dilution, 3200-fold dilution, and 12800-fold dilution) were used to measure the antibody titers of each subclass antibody (IgG1, IgG2a, IgG2b, IgG3) against mouse IL-17A and mouse IL-23, respectively, by ELISA. The antibody titer measurement procedure is shown in Table 12.

[0096]

[0097] (2) Results The results are shown in Figure 4. In mice (administered group) administered with the nucleic acid construct (mRNA vaccine), antibody titers of IgG2a and IgG1 subclasses against IL-17A and IL-23 were high 6 weeks after the start of administration. In contrast, in mice (control group) not administered with the mRNA vaccine, antibody titers of both subclasses were close to 0. These results demonstrate that the antibodies induced by the nucleic acid construct (mRNA vaccine) containing the polynucleotide encoding the T cell epitope designed in Example 1 are both IgG2a, which is induced by a Th1-type immune response, and IgG1, which is induced by a Th2-type immune response.

[0098] Example 5: Examination of antibody induction ability by nucleic acid construct encoding T cell epitope (3) In Example 5, the nucleic acid construct containing the polynucleotide encoding the T cell epitope designed in Example 1 was used to induce antibodies in the Balb / c mice (H2-IE) used in Example 3. ｄ , H2-IA ｄ C57BL / 6 mice (H2-IA) with a different MHC class II ｂ ) We examined the ability of the antibody production induction in mice.

[0099] (1) Methods A. Preparation of nucleic acid constructs (mRNA vaccines) IL-17A nucleic acid constructs and IL-23 nucleic acid constructs were prepared as mRNA vaccines in the same manner as in Example 3(1)A. B. Antibody titer measurement Antibody titers were measured in the same manner as in Example 3(1)B, except that C57BL / 6 mice were used instead of Balb / c mice.

[0100] (2) Results The results are shown in Figure 5. In mice (administered group) administered with the nucleic acid construct (mRNA vaccine), an increase in antibody titers against IL-17A and IL-23 was observed after the start of administration. In contrast, in mice (control group) not administered with the mRNA vaccine, no increase in antibody titers against IL-17A and IL-23 was observed even after the start of administration. These results demonstrate that the nucleic acid construct (mRNA vaccine) containing the polynucleotide encoding the T cell epitope designed in Example 1 can induce the production of antibodies against IL-17A and IL-23 even in mice with different MHC class IIs, and also demonstrate that the nucleic acid construct of the present invention can be used as a pan-HLA-compatible vaccine.

[0101] Example 6: Examination of the efficacy of antibodies induced by nucleic acid constructs encoding T cell epitopes In Example 6, the efficacy of antibodies induced and produced by nucleic acid constructs (mRNA vaccines) containing polynucleotides encoding the T cell epitopes designed in Example 1 was examined.

[0102] (1) Methods A. Preparation of Nucleic Acid Constructs (mRNA Vaccines) In the same manner as in Example 3(1)A, an IL-17A nucleic acid construct and an IL-23 nucleic acid construct were prepared as mRNA vaccines.

[0103] (b) Antibody induction by mRNA vaccine Antibody production was induced by administering 10 μg each of the IL-17A nucleic acid construct and IL-23 nucleic acid construct prepared in (a) above to Balb / c mice at 2-week intervals, and plasma was collected 20 weeks after the start of administration.

[0104] (c) Antibody administration test: Balb / c mice (not administered antibodies) were given 10 mg of imiquimod (IMQ) 5% cream (Versena Cream 5%, Mochida Pharmaceutical) for 5 consecutive days to induce psoriasis (control group), and 0.15 mL of the plasma prepared in (a) above was intraperitoneally administered to Balb / c mice, and immediately after administration, 10 mg of IMQ 5% cream was applied to the front and back of the ears for 5 consecutive days to induce psoriasis (treatment group). The thickness of both ears was measured daily using a digital thickness gauge (Ozaki Seisakusho). The inhibition rate (%) was calculated based on the thickness of both ears 5 days after IMQ application (inhibition rate (%) in the treatment group = (1 - (treatment group mice - mean value of normal mice) / (mean value of IMQ mice - mean value of normal mice)) x 100; inhibition rate (%) in the control group = (1 - (control group mice - mean value of normal mice) / (mean value of control group mice - mean value of normal mice)) x 100), and photographs of the mouse ears were taken after repeated 6-day IMQ application. In addition, changes in the thickness of the ears of normal mice were measured, and photographs of the mouse ears were taken.

[0105] (2) Results The results are shown in Figure 6. In the control group, the thickness of the ear increased with each passing day (Figure 6A). Furthermore, the inhibition rate calculated based on the thickness of both ears 5 days after IMQ application was higher in the treatment group compared to the control group (Figure 6B). The photograph in Figure 6C also confirmed that the psoriasis-like symptoms (thickness and inflammation) of the ears in the treatment group were suppressed compared to the control group. These results demonstrated the effectiveness of the antibodies produced and induced by the nucleic acid construct (mRNA vaccine) containing the polynucleotide encoding the T cell epitope designed in Example 1.

[0106] Example 7: Examination of the efficacy of a nucleic acid construct (mRNA vaccine) encoding a T cell epitope In Example 7, the efficacy of a nucleic acid construct (mRNA vaccine) containing a polynucleotide encoding a T cell epitope designed in Example 1 was examined.

[0107] (1) Methods A. Preparation of Nucleic Acid Constructs (mRNA Vaccines) In the same manner as in Example 3(1)A, an IL-17A nucleic acid construct and an IL-23 nucleic acid construct were prepared as mRNA vaccines.

[0108] (b) mRNA vaccine administration test: Balb / c mice were administered 10 μg of each of the IL-17A nucleic acid construct and IL-23 nucleic acid construct prepared in (a) above twice, at a 2-week interval. Four weeks after the start of administration, 5 mg of IMQ 5% cream was applied to the anterior surface of the ear for six consecutive days to induce psoriasis in the mice (treatment group), and Balb / c mice (without mRNA vaccine administration) in which psoriasis was similarly induced (control group) to compare changes in ear thickness. The thickness of both ears was measured daily using a digital thickness gauge (Ozaki Seisakusho). Then, based on the thickness of both ears 6 days after IMQ application, the inhibition rate (%) (inhibition rate (%) in the treatment group = (1 - (treatment group mice - mean value of normal mice) / (mean value of IMQ mice - mean value of normal mice)) x 100; inhibition rate (%) in the control group = (1 - (control group mice - mean value of normal mice) / (mean value of control group mice - mean value of normal mice)) x 100) was calculated, and photographs of the mouse ears were taken after repeated 6-day IMQ application. In addition, changes in the thickness of the ears of normal mice were measured, and the mouse ears were photographed.

[0109] (2) Results The results are shown in Figure 7. In the control group, the thickness of the ear increased with each passing day (Figure 7A). In the treatment group, the inhibition rate calculated based on the thickness of both ears 6 days after IMQ application was higher than in the control group (Figure 7B). The photograph in Figure 7C also confirmed that the psoriasis-like symptoms (thickness and inflammation) of the ears in the treatment group were suppressed compared to the control group. These results demonstrated the effectiveness of the nucleic acid construct (mRNA vaccine) containing the polynucleotide encoding the T cell epitope designed in Example 1.

Claims

1. A nucleic acid construct that induces antibody production against a target protein, comprising a polynucleotide encoding two or more T cell epitopes and a polynucleotide encoding one or two or more B cell epitopes of the target protein, wherein each of the two or more T cell epitopes is specific for an allele of an MHC class II molecule (HLA class II molecule in the case of humans).

2. The nucleic acid construct according to claim 1, wherein each of the two or more T cell epitopes consists of an amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 82.

3. [[ID=⑤]]The nucleic acid construct according to claim 1 or 2, wherein the target protein is a protein produced in its own body.

4. The nucleic acid construct according to claim 1 or 2, wherein the target protein is IL-17A and / or IL-23.

5. The nucleic acid construct according to claim 1 or 2, wherein the target protein is a protein that causes a disease or a protein produced due to a disease, and the disease is an autoimmune disease or an allergic disease.

6. The nucleic acid construct according to claim 5, wherein the autoimmune disease is one or two or more autoimmune diseases selected from the group consisting of psoriasis, rheumatoid arthritis, systemic lupus erythematosus, Graves' disease, chronic thyroiditis, type I diabetes, vasculitis, Addison's disease, polymyositis, Sjögren's syndrome, systemic sclerosis, and glomerulonephritis.

7. The nucleic acid construct according to claim 5, wherein the allergic disease is one or two or more allergic diseases selected from the group consisting of atopic dermatitis, bronchial asthma, allergic rhinitis, hay fever, food allergy, and allergic conjunctivitis.

8. The nucleic acid construct according to claim 1 or 2, which is DNA or RNA.

9. A prophylactic or therapeutic agent for a disease, comprising the nucleic acid construct according to claim 1 or 2 as an active ingredient.

10. The prophylactic or therapeutic agent according to claim 9, wherein the disease is an autoimmune disease or an allergic disease.

11. The prophylactic agent according to claim 9, which is a pan-HLA compatible vaccine.

12. A method for producing a prophylactic or therapeutic agent for a disease, comprising the step of using a nucleic acid construct that induces antibody production against a target protein as an active ingredient, wherein the nucleic acid construct comprises a polynucleotide encoding two or more T cell epitopes and a polynucleotide encoding one or two or more B cell epitopes of the target protein, and each of the two or more T cell epitopes is specific for an allele of an MHC class II molecule (HLA class II molecule in the case of humans).

13. The production method according to claim 12, wherein the disease is an autoimmune disease or an allergic disease.

14. A method for designing a nucleic acid construct or a protein complex that induces antibody production against a target protein, comprising the step of combining two or more T cell epitopes and one or two or more B cell epitopes of the target protein, and each of the two or more T cell epitopes is specific for an allele of an MHC class II molecule (HLA class II molecule in the case of humans).

15. (Deleted)

16. A nucleic acid construct that induces antibody production against a target protein, comprising a polynucleotide encoding five or more T cell epitopes and a polynucleotide encoding one or two or more B cell epitopes of the target protein, and each of the five or more T cell epitopes is specific for an allele of an MHC class II molecule (HLA class II molecule in the case of humans).

17. The nucleic acid construct according to claim 16, wherein each of the five or more T cell epitopes consists of an amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 82.

18. The nucleic acid construct according to claim 1 or 2, wherein the allele of the HLA class II molecule is one or more selected from the group consisting of HLA-DR, HLA-DQ, and HLA-DP.

19. The allele of the HLA class II molecule is, as the β chain, DRB1 ＊ 01:01, DRB1 ＊ 08:03, DRB1 ＊ 13:02, DRB1 ＊ 04:03, DRB1 ＊ 11:01, DRB1 ＊ 04:06, DRB1 ＊ 15:02, DRB1 ＊ 09:01, DRB1 ＊ 14:54, DRB1 ＊ 07:01, DRB1 ＊ 03:01, DRB1 ＊ 15:01, DRB1 ＊ 04:05, DRB1 ＊ 04:01, DRB1 ＊ 12:01 and DRB1 ＊ The nucleic acid construct according to claim 1 or 2, which is one or more selected from the group consisting of 08:02

20. The nucleic acid construct according to claim 1 or 2, wherein the MHC class II molecule binding ability of the two or more T cell epitopes is less than 3.0, and the MHC class II binding ability is defined as a numerical value calculated using the IEDB analysis resource.

21. The nucleic acid construct according to claim 1 or 2, wherein the base sequence composition is connected in the order of the base sequence encoding the one or more B cell epitopes and the base sequence encoding the two or more T cell epitopes from the 5'-end side to the 3'-end side.

22. The production method according to claim 12, wherein each of the two or more T cell epitopes consists of an amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 82.

23. The design method according to claim 14, wherein each of the two or more T cell epitopes consists of an amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 82.

24. The following steps: (P) A step of designing a nucleic acid construct or a protein complex that induces antibody production against a target protein by carrying out the design method according to claim 14 or 23, and (Q) A step of preparing the nucleic acid construct or the protein complex designed in step (P) A method for producing a prophylactic or therapeutic agent for a disease, comprising the steps.

25. The nucleic acid construct according to claim 1, wherein each of the two or more T cell epitopes consists of an amino acid sequence substantially identical to an amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 82, and the substantially identical amino acid sequence has a sequence identity of 80% or more with the amino acid sequence selected from the amino acid sequences represented by SEQ ID NOs: 1 to 82 and has the ability to bind to MHC class II molecules.

26. The nucleic acid construct according to claim 1, wherein each of the two or more T cell epitopes consists of an amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 6, 8 to 12, 19 to 23, 28 to 35, 40, 41, 45, 46, 49 to 52, 56 to 59, 61 to 68, and 76 to 79.

27. The nucleic acid construct according to claim 1, wherein each of the two or more T cell epitopes consists of an amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 4, 8, 9, 19, 20, 28, 29, 40, and 41.

28. Each of the five or more T cell epitopes consists of an amino acid sequence substantially identical to an amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 82, and the substantially identical amino acid sequence has 80% or more sequence identity with an amino acid sequence selected from the amino acid sequences represented by SEQ ID NOs: 1 to 82 and has the ability to bind to MHC class II molecules. The nucleic acid construct according to claim 16.

29. Each of the five or more T cell epitopes consists of an amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 6, 8 to 12, 19 to 23, 28 to 35, 40, 41, 45, 46, 49 to 52, 56 to 59, 61 to 68, and 76 to 79. The nucleic acid construct according to claim 16.

30. Each of the five or more T cell epitopes consists of an amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 4, 8, 9, 19, 20, 28, 29, 40, and 41. The nucleic acid construct according to claim 16.

31. The target protein is IL-17A and / or IL-23. The nucleic acid construct according to claim 26 or 29.

32. Each of the two or more T cell epitopes consists of an amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 6, 8 to 12, 19 to 23, 28 to 35, 40, 41, 45, 46, 49 to 52, 56 to 59, 61 to 68, and 76 to 79. The production method according to claim 12.

33. Each of the two or more T cell epitopes consists of an amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 6, 8 to 12, 19 to 23, 28 to 35, 40, 41, 45, 46, 49 to 52, 56 to 59, 61 to 68, and 76 to 79. The design method according to claim 14.

34. The target protein is IL-17A and / or IL-23. The production method according to claim 32 or the design method according to claim 33.