Design method and manufacturing method of 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. According to the present invention, provided is a nucleic acid construct that induces the production of an antibody against a target protein, said nucleic acid construct comprising: a polynucleotide that encodes two or more types of T-cell epitopes; and a polynucleotide encoding one or more B cell epitopes of the target protein. According to the present invention, it is possible to strongly induce an antibody against a target protein in a subject, and therefore it is advantageous in that the production of an antibody not only can be induced against an exogenous antigen such as a bacterium or a virus, but also the production of an antibody can be induced against a protein produced in itself.

Description

Technical Field

[0001] This application enjoys the priority benefit of Japanese Patent Application No. 2022-162824 (filing date: October 7, 2022), which is a prior Japanese application, and all the disclosed contents thereof are incorporated herein by reference as a part of this specification.

[0002] The present invention relates to nucleic acid constructs and methods for designing and manufacturing the same. Background Art

[0003] Many antibody drugs show excellent therapeutic effects in the treatment of chronic diseases and difficult diseases, but their target molecules are mostly self-proteins (for example, inflammatory cytokines as etiological substances in autoimmune diseases, etc.). On the other hand, vaccines induce effective antibodies against exogenous antigens such as viruses, and the main indications are infectious diseases. However, the existing vaccine technology has the following problems: due to immune tolerance, it is impossible to induce the production of antibodies against self-proteins, and it cannot be applied to the treatment of chronic diseases and difficult diseases (Non-Patent Document 1, Non-Patent Document 2, Non-Patent Document 3 and Non-Patent Document 4).

[0004] Prior art literature

[0005] Non-patent literature

[0006] Non-patent document 1: CA Janeway Jr., Immunol Today; 13(1):11-6 (1992).

[0007] Non-patent document 2: Ada G., N Engl J Med; 345(14):1042-53 (2001).

[0008] Non-patent document 3: Ada GL., Lancet; 335(8688):523-6 (1990).

[0009] Non-patent document 4: Anderson RM et al., Lancet; 350(9089):1466-70 (1997). Summary of the invention

[0010] Problems to be solved by the invention

[0011] The object of the present invention is to provide a novel nucleic acid construct that induces antibody production, and a design method and a production method thereof.

[0012] Means for solving problems

[0013] The inventors of the present application have found that the production of antibodies can be induced by using 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 aforementioned target protein. The inventors of the present application have also found that the nucleic acid construct is effective as a vaccine by administering it to animals. The present invention is an invention based on the above insights.

[0014] According to the present invention, the following solutions are provided.

[0015] [1] A nucleic acid construct that induces the production of antibodies against a target protein, the nucleic acid construct comprising: a polynucleotide encoding two or more T cell epitopes; and a polynucleotide encoding one or two or more B cell epitopes of the aforementioned target protein.

[0016] [2] The nucleic acid construct according to [1] above, wherein the two or more T cell epitopes comprise an amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 82, or an amino acid sequence substantially identical to the amino acid sequence.

[0017] [3] The nucleic acid construct according to [1] or [2] above, wherein the target protein is a protein produced in the body itself.

[0018] [4] The nucleic acid construct according to any one of [1] to [3] above, wherein the target protein is IL-17A and / or IL-23.

[0019] [5] The nucleic acid construct according to any one of [1] to [4] above, wherein the target protein is a protein that causes a disease or a protein produced by a disease, and the disease is an autoimmune disease or an allergic disease.

[0020] [6] The nucleic acid construct as described in [5] above, wherein the autoimmune disease is one or more autoimmune diseases selected from the group consisting of psoriasis, rheumatoid arthritis, systemic lupus erythematosus, Basedow's disease, chronic thyroiditis, type 1 diabetes, vasculitis, Addison's disease, polymyositis, Sjögren's syndrome, systemic sclerosis and glomerulonephritis.

[0021] [7] The nucleic acid construct as described in [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.

[0022] [8] The nucleic acid construct according to any one of [1] to [7] above, which is DNA or RNA.

[0023] [9] A preventive or therapeutic agent for a disease, comprising the nucleic acid construct according to any one of [1] to [8] above as an active ingredient.

[0024]

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

[0025]

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

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

[0026]

[12] A method for producing a preventive or therapeutic agent for a disease, comprising a step of using a nucleic acid construct that induces the production of antibodies 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.

[0027]

[13] The production method according to

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

[0028]

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

[0029]

[15] A method for producing a preventive or therapeutic agent for a disease, comprising the following steps:

[0030] (P) a step of implementing the design method according to claim 14 to design a nucleic acid construct or a protein complex that induces the production of antibodies against a target protein; and

[0031] (Q) A step of preparing the nucleic acid construct or protein complex designed in step (P).

[0032]

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

[0033]

[102] The design method as described in

[101] above, wherein the T cell epitope has a high binding ability to MHC class II molecules.

[0034]

[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 by a disease.

[0035]

[104] The design method according to any one of

[101] to

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

[0036]

[105] A design method as described in

[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.

[0037]

[106] The design method as described in

[105] above, wherein the autoimmune disease is one or more autoimmune diseases selected from the group consisting of psoriasis, rheumatoid arthritis, systemic lupus erythematosus, Basedow's disease, chronic thyroiditis, type 1 diabetes, vasculitis, Addison's disease, polymyositis, Sjögren's syndrome, systemic sclerosis and glomerulonephritis.

[0038]

[107] The design method as described in

[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.

[0039]

[108] The design method as described in

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

[0040]

[109] The design method as described in

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

[0041]

[110] The design method as described in

[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.

[0042]

[111] A design method as described in

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

[0043]

[112] The design method as described in

[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.

[0044]

[113] A design method as described in 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, β-amyloid protein, RS virus antigen protein, Zika virus antigen protein, SARS-CoV-2 virus antigen protein and influenza virus antigen protein.

[0045]

[114] The design method according to any one of

[101] to

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

[0046]

[115] A design method as described in any one of

[101] to

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

[0047]

[116] A method for producing a preventive or therapeutic agent for a disease, comprising the following steps:

[0048] (P) a step of implementing the design method described in any one of

[101] to

[115] above to design a nucleic acid construct or protein complex that induces the production of antibodies against the target protein; and

[0049] (Q) A step of preparing the nucleic acid construct or protein complex designed in step (P).

[0050] According to the present invention, antibodies against target proteins can be strongly induced in the body of a subject, and therefore it is advantageous in that antibody production can be induced not only against exogenous antigens such as bacteria and viruses, but also against proteins produced in the body itself (self-proteins) (for example, inflammatory cytokines produced in the body of a subject suffering from an autoimmune disease, etc.). BRIEF DESCRIPTION OF THE DRAWINGS

[0051] [ Figure 1-1 ] Figure 1-1 Specific examples of amino acid sequences of human T cell epitopes are shown.

[0052] [ Figure 1-2 ] Figure 1-2 Specific examples of amino acid sequences of human T cell epitopes are shown.

[0053] [ Figure 1-3 ] Figure 1-3 Specific examples of amino acid sequences of human T cell epitopes are shown.

[0054] [ Figure 2 ] Figure 2 A is a schematic diagram showing the structure of the nucleic acid construct (mRNA vaccine) of the present invention. Figure 2 B is a schematic diagram of the nucleic acid construct prepared in Example (Example 3). T-86 to T-88 correspond to the base sequences encoding T cell epitopes shown in sequence numbers 86 to 88, respectively, and B89 to 94 correspond to the base sequences encoding B cell epitopes shown in sequence numbers 89 to 94, respectively.

[0055] [ Figure 3 ] Figure 3 The antibody titers (absorbance at a wavelength of 450 nm) of antibodies against IL-17A and IL-23 induced by nucleic acid constructs encoding T cell epitopes are shown. The error bars indicate standard deviations.

[0056] [ Figure 4 ] Figure 4 The antibody titers (absorbance at a wavelength of 450 nm) of subclass antibodies against IL-17A and IL-23 induced by nucleic acid constructs encoding T cell epitopes are shown. IgG1, IgG2a, IgG2b, and IgG3 all represent the antibody titers of the administration groups.

[0057] [ Figure 5 ] Figure 5 The antibody titers (absorbance at a wavelength of 450 nm) of antibodies against IL-17A and IL-23 induced by nucleic acid constructs encoding T cell epitopes are shown. The error bars indicate standard deviations.

[0058] [ Figure 6 ] Figure 6 A shows the changes in auricle thickness of the control group and normal mice after IMQ coating. Figure 6 B shows the inhibition rate of the increase in auricle thickness in the control group and the administration group on the 5th day after IMQ application. Figure 6 C shows representative photographs of the auricles of psoriasis model mice (control group and administration group) and normal mice on day 6 after IMQ application.

[0059] [ Figure 7 ] Figure 7 A shows the changes in auricle thickness of the control group and normal mice after IMQ coating. Figure 7 B shows the inhibition rate of the increase in auricle thickness in the control group and the administration group on the 6th day after IMQ application. Figure 7 C shows representative photographs of the auricles of psoriasis model mice (control group and administration group) and normal mice on day 6 after IMQ application. DETAILED DESCRIPTION

[0060] <<Nucleic Acid Constructs>>

[0061] According to the present invention, a nucleic acid construct is provided. The nucleic acid construct of the present invention is a nucleic acid construct that induces the production of antibodies against a target protein, and is characterized by comprising: a polynucleotide encoding two or more T cell epitopes; and a polynucleotide encoding one or two or more B cell epitopes of the aforementioned target protein.

[0062] In the present invention, "T cell epitope" refers to the following peptide: the peptide binds to the MHC class II molecule in the major histocompatibility complex (MHC) to form a complex, which is presented on antigen presenting cells (e.g., B cells, dendritic cells, macrophages) and specifically recognized by the T cell receptor (TCR) of CD4-positive T cells.

[0063] In the present invention, two or more T cell epitopes may be selected as long as they bind to the MHC class II molecule of the target. From the viewpoint of strongly inducing the production of antibodies against the target protein (i.e., strongly activating CD4-positive T cells), it is preferred to select T cell epitopes that strongly bind to the MHC class II molecule (i.e., have a high binding ability to the MHC class II molecule) after being taken up by antigen-presenting cells in the subject's body.

[0064] In the present invention, T cell epitopes with high binding ability to MHC class II molecules can be selected using known MHC binding prediction tools, with the predicted binding score to MHC class II as an index. As MHC binding prediction tools, for example, there are 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 / ).

[0065] Here, in the case of humans, MHC refers to human leukocyte antigen (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 α chains include HLA-DRA, HLA-DQA, and HLA-DPA, and examples of β chains include HLA-DRB, HLA-DQB, and HLA-DPB.

[0066] Examples of HLA-DR include HLA-DR1, HLA-DR2, HLA-DR3, HLA-DR4, HLA-DR5, HLA-DR6, HLA-DR7, HLA-DR8, H LA-DR9, HLA-DR10, HLA-DR11, HLA-DR12, HLA-DR13, HLA-DR14, HLA-DR15, HLA-DR52, HLA-DR53, etc.

[0067] Examples of HLA-DQ include HLA-DQ1, HLA-DQ2, HLA-DQ3, HLA-DQ4, HLA-DQ5, HLA-DQ6, HLA-DQ7, and HLA-DQ8.

[0068] Examples of HLA-DP include HLA-DP1, HLA-DP2, HLA-DP3, HLA-DP4, and HLA-DP5.

[0069] In the present invention, as HLA to which the T cell epitope binds, for example, in the case of HLA-DR, when the subject is Japanese, the α chain may be HLA-DRA1. ＊ 01 and other alleles, as β chains, DRB1 ＊ 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 ＊ 11:01 etc. alleles, in the case of Europeans and Americans, as the α chain, HLA-DRA1 can be cited. ＊ 01 and other alleles, as β chain, DRB1 ＊ 07:01, DRB1 ＊ 03:01, DRB1 ＊ 04:01 and DRB1 ＊ 15:01 and so on.

[0070] In the present invention, T cell epitopes are not limited, and examples thereof include Figure 1-1 (Serial number 1 to 31), Figure 1-2 (Serial No. 32~64) and Figure 1-3 (SEQ ID NOs: 65 to 82) as a specific example. That is, in the present invention, two or more T cell epitopes may include an amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 82. In the present invention, the T cell epitope may also include an amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 1 to 64 or an amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 65 to 82.

[0071] In the present invention, the T cell epitope may include 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 (or the amino acid sequences represented by SEQ ID NOs: 1 to 64 or SEQ ID NOs: 65 to 82).

[0072] Here, in the present invention, "T cell epitopes comprising substantially the same amino acid sequence" refers to epitopes having one or more modifications in a T cell epitope comprising a specific amino acid sequence and having the ability to bind to MHC class II molecules (HLA class II molecules).

[0073] In the present invention, as a T cell epitope comprising a substantially identical amino acid sequence, for example, the following epitopes can be cited: in an amino acid sequence selected from the amino acid sequences represented by sequence numbers 1 to 82 (or the amino acid sequences represented by sequence numbers 1 to 64 or sequence numbers 65 to 82), one or more modifications selected from the group consisting of deletion, substitution, insertion and addition, and having the ability to bind to MHC class II molecules (HLA class II molecules). The number of modified amino acids is, for example, 1 to 4 or 1 to 3, and is particularly preferably 2 or 1. The number of modified amino acids can also be set to the number of mutations of the degree produced by known methods such as site-derected mutagenesis, or the number of mutations of the degree produced naturally. In the aforementioned amino acid sequence, the aforementioned modification can be produced continuously or discontinuously. The aforementioned modification can also be a plurality of modifications of the same kind (for example, a plurality of substitutions), or a plurality of modifications of different kinds (for example, a combination of more than one deletion and more than one substitution).

[0074] The modification of the above-mentioned amino acid can be a conservative modification. "Conservative modification" refers to modifying one or more amino acids in a way that does not substantially change the function of the protein. The substitution of the above-mentioned amino acids can also be a conservative substitution. "Conservative substitution" refers to replacing one or more amino acids with other amino acids and / or amino acid derivatives in a way that does not substantially change the function of the protein. In conservative substitution, the substituted amino acid is preferably similar to the substituted amino acid in properties and / or functions, for example. Specifically, it is preferred that the indexes of hydrophobicity and hydrophilicity, chemical properties such as polarity, charge, or physical properties such as secondary structure are similar. In this way, amino acids or amino acid derivatives with similar properties and / or functions are known in the art. For example, as non-polar amino acids (hydrophobic amino acids), for example, alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, etc. can be mentioned. Polar amino acids (neutral amino acids) can include glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine, etc. Examples of amino acids having a positive charge (basic amino acids) include arginine, histidine, and lysine, and examples of amino acids having a negative charge (acidic amino acids) include aspartic acid and glutamic acid.

[0075] In the present invention, examples of T cell epitopes comprising substantially identical amino acid sequences include epitopes having a sequence identity of 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) to an amino acid sequence selected from the amino acid sequences represented by SEQ ID NOs: 1 to 82 (or the amino acid sequences represented by SEQ ID NOs: 1 to 64 or 65 to 82), and having the ability to bind to MHC class II molecules (HLA class II molecules). Here, "identity" refers to, for example, the level of identity when the sequences to be compared are appropriately aligned (aligned), and refers to the occurrence rate (%) of exact matches of amino acids between the aforementioned sequences. When calculating the identity, for example, the presence of gaps in the sequence and the properties of the amino acids are taken into account (Wilbur, Natl. Acad. Sci. USA 80: 726-730 (1983)). The aforementioned comparison can be performed, for example, by utilizing any algorithm, 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. In addition, the calculation of identity can be performed, for example, using the publicly available homology search program described above, for example, it can be calculated in the homology algorithm BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) of the National Center for Biotechnology Information (NCBI) using default parameters.

[0076] The T cell epitopes 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 the types of T cell epitopes combined are increased, the more the probability that the combination contains T cell epitopes with high binding ability to the MHC class II molecules of the object can be increased. Therefore, when the nucleic acid construct and protein complex of the present invention are used as the active ingredient of the vaccine, the versatility of the object to which the vaccine is administered is expanded, thereby contributing to the generalization of the vaccine. Here, in the present invention, "protein complex" refers to a fusion protein (typically a protein that induces the production of antibodies against the target protein) produced by translating the nucleic acid construct of the present invention and containing T cell epitopes and B cell epitopes as constituent elements.

[0077] In the present invention, as a specific example of the combination of amino acid sequences of T cell epitopes, there can be cited more than 2, more than 3, more than 4, more than 5, more than 6, more than 7, more than 8, more than 9, more than 10, more than 11, more than 12, more than 13, more than 14, more than 15, more than 16, more than 17, more than 18, more than 19 or more than 20 kinds of amino acid sequences or a combination of amino acids substantially identical to the amino acid sequence selected from the group consisting of amino acid sequences represented by sequence numbers 1 to 64. In addition, the above combination is not limited and can be set to less than 64, less than 60, less than 55, less than 50, less than 45, less than 40, less than 35 or less than 30. These lower limits and upper limits can be combined arbitrarily.

[0078] In the present invention, as a specific example of the combination of amino acid sequences of T cell epitopes, there can be cited 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, 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 or 20 or more amino acid sequences or a combination of amino acids substantially identical to the amino acid sequences selected from the group consisting of amino acid sequences represented by sequence numbers 1, 2, 7 to 9, 13 to 20, 24 to 27, 30, 31, 34 to 37, 42 to 44, 47 to 54, 56, 57 and 60 to 62. In addition, the above combination is not limited and can be set to less than 39, less than 35, less than 30 or less than 25. These lower limits and upper limits can be arbitrarily combined.

[0079] In the present invention, the T cell epitope can also combine the amino acid sequences of T cell epitopes that bind to different MHC class II molecules (HLA class II molecules). As a specific example of such a combination, a combination of two or more amino acid sequences selected from the following groups can be cited: at least one, two, three, four or five selected from the group consisting of amino acid sequences represented by sequence numbers 1 to 7; at least one, two, three, four or five selected from the group consisting of amino acid sequences represented by sequence numbers 8 to 14; at least one, two or three selected from the group consisting of amino acid sequences represented by sequence numbers 15 to 18; at least one, two, three, four or five selected from the group consisting of amino acid sequences represented by sequence numbers 19 to 25; at least one selected from the group consisting of amino acid sequences represented by sequence numbers 26 or 27; at least one, two, three, four or five selected from the group consisting of amino acid sequences represented by sequence numbers 28 to 31. or 3; at least one, two or three selected from the group consisting of the amino acid sequences represented by sequence numbers 32 to 35; at least one, two or three selected from the group consisting of the amino acid sequences represented by sequence numbers 36 to 39; at least one, two or three selected from the group consisting of the amino acid sequences represented by sequence numbers 40 to 44; at least one, two or three selected from the group consisting of the amino acid sequences represented by sequence numbers 45 to 48; at least one, two, three, four or five selected from the group consisting of the amino acid sequences represented by sequence numbers 49 to 55; at least one, two or three selected from the group consisting of the amino acid sequences represented by sequence numbers 56 to 60; and at least one or two selected from the group consisting of the amino acid sequences represented by sequence numbers 61 to 64.

[0080] In the present invention, the T cell epitope can also combine the amino acid sequences of T cell epitopes that bind to different MHC class II molecules (HLA class II molecules). As a specific example of such a combination, a combination of two or more amino acid sequences selected from the following groups can be cited: at least one selected from the group consisting of amino acid sequences represented by sequence numbers 65 or 66; at least one, two or three selected from the group consisting of amino acid sequences represented by sequence numbers 67 to 70; at least one, two or three selected from the group consisting of amino acid sequences represented by sequence numbers 71 to 75; and at least one, two, three, four or five selected from the group consisting of amino acid sequences represented by sequence numbers 76 to 82.

[0081] As described above, the amino acid sequences of T cell epitopes that bind to different MHC class II molecules (HLA class II molecules) can be combined, so 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, "pan-HLA vaccine" refers to a vaccine that can be widely applied to a subject, specifically, a vaccine constructed in a manner that recognizes two or more different HLA class II molecules present in humans.

[0082] From the viewpoint of improving the induction of antibodies against the target protein in the subject, the nucleic acid construct or protein complex of the present invention may also contain multiple sets of the same T cell epitopes. In the case of a nucleic acid construct, as shown in the following example (Example 3), the base sequence encoding the same T cell epitope may be repeatedly included multiple times, or may be randomly included multiple times (for example, in the following example (Example 3), the base sequence of sequence numbers 86 to 88 is repeated 3 times in the nucleic acid construct).

[0083] In the present invention, "B cell epitope" refers to a portion (peptide) of a target protein (antigen) that is recognized by antigen-specific B cells.

[0084] In the present invention, the combined B cell epitopes may be any type of B cell epitopes as long as they are recognized by antigen-specific B cells.

[0085] In the present invention, the target protein is not limited, and examples include proteins that are the cause of the disease and proteins produced by the disease, and also include any of exogenous antigens (bacteria, viruses, etc.) and proteins produced in the body itself (self-proteins) (inflammatory cytokines produced in the body of a subject who has developed an autoimmune disease, etc.). Here, in the present invention, "proteins that are the cause of the disease" refers to proteins that are the root cause of the disease, including proteins that are the root cause of the disease or the onset of the disease. In addition, "proteins produced by the disease" refers to proteins that are produced as a trigger for the onset of the disease, and specifically refers to proteins that have the effect of exacerbating or worsening the symptoms of the disease (for example, inflammatory proteins).

[0086] In the present invention, examples of target protein-related diseases include autoimmune diseases, allergic diseases, cancer, neurodegenerative diseases, infectious diseases, and autoinflammatory diseases.

[0087] In the present invention, examples of autoimmune diseases include psoriasis, rheumatoid arthritis, systemic lupus erythematosus, Basedow's disease, chronic thyroiditis (Hashimoto's thyroid disease), type 1 diabetes, vasculitis (e.g., antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis), Addison's disease, polymyositis, Sjögren's syndrome, systemic sclerosis, and glomerulonephritis (e.g., IgA nephropathy).

[0088] In the present invention, examples of cancer include solid cancers (cancer, sarcoma) and blood tumors, for example, skin cancer (e.g., malignant melanoma, Merkel cell carcinoma, squamous cell carcinoma), colorectal cancer (colon and rectal cancer), breast cancer (including metastatic breast cancer), gastric cancer, leukemia (e.g., acute lymphocytic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia), malignant lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma) and multiple myeloma.

[0089] In the present invention, examples of allergic diseases include atopic dermatitis, bronchial asthma, allergic rhinitis, hay fever, food allergy, and allergic conjunctivitis.

[0090] In the present invention, examples of neurodegenerative diseases include Alzheimer's disease.

[0091] In the present invention, examples of infectious diseases include Zika fever (Zika virus infection), SARS-CoV-2 infection (so-called novel coronavirus infection), and influenza infection.

[0092] In the present invention, examples of autoinflammatory diseases include familial Mediterranean fever, cold pyrine-associated periodic syndrome, TNF receptor-associated periodic syndrome, mevalonate kinase deficiency, Blau syndrome, periodic fever-aphthous stomatitis-pharyngitis and cervical lymphadenitis syndrome.

[0093] In the present invention, diseases other than the above-mentioned diseases include rare diseases and intractable diseases, for example, multiple sclerosis, neuromyelitis optica, myasthenia gravis, pemphigus, autoimmune hemolytic anemia, and the like.

[0094] In the present invention, specific examples of target proteins include the proteins shown in Table 1.

[0095] [Table 1]

[0096] Table 1: Examples of target proteins

[0097]

[0098] Table 1: Examples of target proteins (continued 1)

[0099]

[0100] Table 1 : Examples of target proteins (continued 2)

[0101]

[0102] The B cell epitopes combined in the present invention can be arbitrarily selected from one or more B cell epitopes of the target protein according to the purpose. When two or more B cell epitopes 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, and 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 by a disease, the B cell epitope can be selected based on the viewpoint of preventing or treating the disease.

[0103] In the present invention, the combination of a T cell epitope and a B cell epitope can be selected from any kind of T cell epitopes and B cell epitopes, respectively, for example, can be selected from the T cell epitopes and B cell epitopes exemplified above.

[0104] The nucleic acid construct or protein complex of the present invention can be designed and manufactured based on the combination of the selected T cell epitope and B cell epitope using known molecular biological methods. Specifically, the protein complex can be designed and manufactured in the form of a fusion protein containing T cell epitopes and B cell epitopes as constituent elements.

[0105] The nucleic acid construct of the present invention can be designed and manufactured as a polynucleotide encoding a protein complex (i.e., a fusion protein containing T cell epitopes and B cell epitopes as constituent elements). 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 the production of antibodies against a target protein". With respect to the nucleic acid construct of the present invention, from the perspective of effectively inducing antibody production in the subject's organism, the order of the polynucleotide sequences encoding T cell epitopes and B cell epitopes, respectively, can be appropriately adjusted, and sequences and linker sequences that regulate expression can also be inserted between each epitope. In addition, the B cell epitope combined with the T cell epitope can be composed of the entire target protein recognized by antigen-specific B cells, or can be composed of a portion of the target protein (e.g., a domain, etc.) containing an antigen portion.

[0106] The nucleic acid construct of the present invention is composed of a polynucleotide, which may 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, and also include their modified forms and artificial nucleic acids. From the perspective of using the nucleic acid construct as an active ingredient of a vaccine, RNA is preferred.

[0107] The protein complex of the present invention is constituted as a fusion protein comprising two or more T cell epitopes and one or two or more B cell epitopes of the aforementioned target protein as constituent elements, but the aforementioned protein complex may also be modified as long as the aforementioned T cell epitope and the aforementioned B cell epitope each function as an epitope.

[0108] The nucleic acid construct or protein complex of the present invention can induce the production of antibodies 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 subject's body.

[0109] Without being limited to the following theory, the mechanism of action from preparation of a nucleic acid construct or a protein complex to administration of the prepared nucleic acid construct or protein complex to a subject and induction of antibody production in the subject will be described.

[0110] [I. Case of Nucleic Acid Construct]

[0111] (i) Based on the nucleic acid construct designed by the design method of the present invention, a nucleic acid construct to be administered to a subject is prepared using known molecular biological methods.

[0112] (ii) The prepared nucleic acid construct is loaded onto a carrier (lipid membrane such as liposome) and administered to a subject by injection or the like.

[0113] (iii) The administered nucleic acid construct is translated in the subject organism to produce a protein (a fusion protein containing a T cell epitope and a B cell epitope as components).

[0114] (iv) The produced fusion protein is recognized by antigen-specific B cells in the subject organism and taken up into the cells and digested.

[0115] (v) Within B cells, the T cell epitope forms a complex with MHC class II molecules, and the complex is presented on the surface of the B cells.

[0116] (vi) CD4-positive T cells recognize and bind to the complex of T cell epitopes and MHC class II molecules via T cell receptors (TCR), thereby being activated.

[0117] (vii) Activated CD4-positive T cells undergo clonal proliferation, promoting the proliferation and differentiation of B cells.

[0118] (viii) B cells are activated and undergo clonal proliferation, producing large amounts of target antibodies.

[0119] [II. Case of protein complex]

[0120] (i) Based on the protein complex designed by the design method of the present invention, a protein complex (a fusion protein comprising a T cell epitope and a B cell epitope as components) to be administered to a subject is prepared using known molecular biological methods.

[0121] (ii) The prepared protein complex is administered to a subject by injection or the like.

[0122] (iii) The administered protein complex is recognized by antigen-specific B cells in the subject's body and taken up into the cells and digested.

[0123] (iv) Within B cells, the T cell epitope forms a complex with MHC class II molecules, and the complex is presented on the surface of the B cells.

[0124] (v) CD4-positive T cells recognize and bind to the complex of T cell epitopes and MHC class II molecules through T cell receptors (TCR), thereby being activated.

[0125] (vi) Activated CD4-positive T cells undergo clonal proliferation, promoting the proliferation and differentiation of B cells.

[0126] (vii) B cells are activated, undergo clonal proliferation, and produce large amounts of target antibodies.

[0127] The nucleic acid construct of the present invention can induce the production of antibodies against the target protein in the subject, and therefore can be used for the prevention or treatment of target protein-related diseases, and can reduce the risk of suffering from target protein-related diseases. That is, according to the present invention, a disease prevention agent or therapeutic agent is provided, which has the nucleic acid construct of the present invention as an effective ingredient. According to the present invention, a preparation for reducing the risk of suffering from a disease is also provided, which has the nucleic acid construct of the present invention as an effective ingredient. The preventive and therapeutic agents of the present invention can be administered to a subject suffering from the disease or a subject who may suffer from the disease. The risk reducing agent of the present invention can be administered to a subject who may suffer from the disease.

[0128] When the nucleic acid construct of the present invention is administered to a subject, the administration route is not particularly limited as long as the therapeutic or preventive effect of the subject disease or the effect of reducing the risk of the subject disease can be obtained, and for example, parenteral administration can be selected. Non-limiting examples of parenteral administration include intravenous administration, intramuscular administration, subcutaneous administration, local administration, intraperitoneal administration, and intranasal administration.

[0129] As a non-oral administration agent, an appropriate dosage form can be selected according to the specific administration form, for example, an injection can be cited. The non-oral administration agent can be made into an aqueous or non-aqueous isotonic sterile solution or suspension. Any preparation can be formulated using a method commonly implemented in the art (for example, a known method recorded in the eighteenth revision of the Japanese Pharmacopoeia General Rules for Preparations, etc.), using a pharmaceutically acceptable carrier. That is, according to the present invention, a pharmaceutical composition comprising a nucleic acid construct of the present invention and a pharmaceutically acceptable carrier is provided. As a pharmaceutically acceptable carrier, excipients, binders, diluents, additives, spices, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, preservatives, etc. can be cited. As for the preparation of the present invention, for example, the nucleic acid construct of the present invention can be formulated using lipid nanoparticles (LNP), and lipids constituting 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.

[0130] The dosage of the nucleic acid construct in the present invention can be determined depending on the sex, age and weight of the subject, symptoms, dosage form and administration route, etc. In the present invention, when the nucleic acid construct is administered for the purpose of treating or preventing a disease, the single dosage for adults can be determined, for example, within the range of 0.01 μg to 100 mg, but is not limited thereto. In the present invention, the above-mentioned dosage of the active ingredient can be administered once a day, or it can be administered in 2 to 4 times. In the present invention, the above-mentioned dosage of the active ingredient can also be initially administered multiple times at intervals of once a week, every 2 weeks, every 3 weeks, or every 4 weeks, and then administered multiple times at intervals of once a month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, or once a year.

[0131] In the present invention, the subject includes humans and non-human animals, and the non-human animal is preferably a non-human mammal (eg, mouse, rat, cow, pig, horse, monkey).

[0132] <<Design Method>>

[0133] According to another aspect of the present invention, a method for designing a nucleic acid construct or a protein complex is provided. According to the design method of the present invention, a nucleic acid construct or a protein complex that induces the production of antibodies against a target protein can be designed.

[0134] The design method of the present invention includes the step of combining two or more T cell epitopes with one or two or more B cell epitopes of the target protein.

[0135] The design method of the present invention may include: step (X1), using an MHC binding prediction tool to evaluate the ability of T cell epitopes to bind to MHC class II molecules; and step (X2), based on the evaluation results of step (X1), selecting T cell epitopes with high binding ability to MHC class II molecules. In the design method of the present invention, the above steps (X1) and (X2) can be performed before 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 pre-screening T cell epitopes before the above steps (X1) and (X2). This pre-screening can be implemented using AI (Artificial Intelligence) or a prediction algorithm.

[0136] In addition to the above, the design method of the present invention can be implemented according to the description of the nucleic acid construct of the present invention.

[0137] <<Preventive or therapeutic agent and method for producing the same>>

[0138] According to another aspect of the present invention, a preventive or therapeutic agent for a disease is provided. The preventive or therapeutic agent of the present invention is characterized in that it contains the nucleic acid construct or protein complex of the present invention as an active ingredient.

[0139] In the preventive or therapeutic agent of the present invention, the target protein may be a protein that causes a disease or a protein produced by a disease. In the production method of the present invention, the target protein may be a protein produced in one's own body.

[0140] The preventive agent of the present invention can typically be used as a vaccine. That is, the preventive agent of the present invention can be used for the following purposes: by administering to a subject, thereby inducing the production of antibodies to the target protein in the subject's organism, preventing the onset or onset of a disease; or, even if the disease has already occurred or the disease has developed, reducing its degree. As the preventive agent of the present invention, for example, mRNA vaccines, genetically recombinant protein vaccines, and peptide vaccines can be cited, and from the perspective of manufacturing cost, mRNA vaccines are preferred.

[0141] The therapeutic agent of the present invention can typically be used in place of an antibody drug. That is, the therapeutic agent of the present invention can be used for the purpose of inducing the production of antibodies against the target protein in the subject's body by administering the agent to the subject, thereby treating a disease suffered by or developing in the subject.

[0142] According to another aspect of the present invention, a method for producing a preventive or therapeutic agent of the present invention is also provided. The method of the present invention comprises: (P) a step of designing a nucleic acid construct or protein complex that induces the production of antibodies against a target protein; and (Q) a step of preparing the nucleic acid construct or protein complex designed by step (P).

[0143] 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.

[0144] The preparation of the nucleic acid construct or protein complex in step (Q) can be implemented using known methods, but is not limited to them. For example, the nucleic acid construct can be prepared by chemical synthesis or in vitro transcription synthesis to prepare oligonucleotides, and the protein complex can be prepared using an expression vector that incorporates the nucleic acid construct.

[0145] In addition to the above, the preventive or therapeutic agent of the present invention and the method for producing the same can be implemented according to the description of the nucleic acid construct of the present invention and the design method of the present invention.

[0146] According to the present invention, a method for preventing or treating a disease is provided, comprising administering a therapeutically or preventively effective amount of a nucleic acid construct or a composition comprising the same to a subject in need thereof. According to the present invention, a method for reducing the risk of a disease is also provided, comprising administering an effective amount of a nucleic acid construct or a composition comprising the same to a subject in need thereof. The method of the present invention can be implemented according to the records related to the nucleic acid construct of the present invention and the preventive or therapeutic agent of the present invention.

[0147] According to the present invention, the use of the nucleic acid construct of the present invention for manufacturing a preventive or therapeutic agent for a disease, or as a preventive or therapeutic agent for a disease, is also provided. According to the present invention, the use of the nucleic acid construct of the present invention for manufacturing a preparation that reduces the risk of suffering from a disease, or as a preparation that reduces the risk of suffering from a disease, is also provided. The method of the present invention can be implemented according to the records related to the nucleic acid construct of the present invention and the preventive or therapeutic agent of the present invention.

[0148] Example

[0149] The present invention will be described in more detail based on the following examples, but the present invention is not limited to these examples.

[0150] Example 1: Design of amino acid sequence of T cell epitope (1)

[0151] 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 abilities of the amino acid sequences of T cell epitopes shown in Table 2 were analyzed using the IEDB analysis resource.

[0152] [Table 2]

[0153] Table 2: Amino acid sequences of T cell epitopes

[0154] Serial Number 83 FASVYAWNRKRIVTQQL Serial Number 84 YNYLYRLFRKSNLK Serial Number 85 QLIRAAEIRASANLAAGG

[0155] The results are shown in Tables 3 to 5. Among the T cell epitopes shown here, the lower the value of MHC class II binding ability, the higher the binding ability, suggesting that the lower the value of MHC class II binding ability, the higher the antibody production induction activity. According to the results of Tables 3 to 5, it is suggested that these T cell epitopes have high MHC class II binding ability in Balb / c mice and high antibody production induction activity.

[0156] [Table 3]

[0157] Table 3: MHC class II binding ability and antibody production induction activity

[0158]

[0159] [Table 4]

[0160] Table 4: MHC class II binding ability and antibody production induction activity

[0161]

[0162] [Table 5]

[0163] Table 5: MHC class II binding ability and antibody production induction activity

[0164]

[0165] Example 2: Design of amino acid sequence of T cell epitope (2)

[0166] In Example 2, the amino acid sequence of a human T cell epitope is designed, and the MHC class II binding ability of the T cell epitope is analyzed using the IEDB analysis resource.

[0167] The results are shown in Table 6 and Table 7. In the T cell epitope shown here, the lower the numerical value of MHC II class binding ability, the higher the binding ability, which implies that the lower the numerical value of MHC II class binding ability, the higher the antibody production induction activity. According to the results of Table 6 and Table 7, it is implied that in most of the Japanese population and the European and American population, the MHC II class binding ability is high, and the antibody production induction activity is high. That is, it is shown that this nucleic acid construct is a pan-MHC type, and it is shown that this nucleic acid construct can be applied to most of the humans represented by the Japanese population.

[0168] [Table 6]

[0169] Table 6: MHC class II binding capacity and antibody production induction activity in Japanese

[0170]

[0171] ※Frequency quoted from Ikeda N et al., Tissue Antigens; 85(4):252-9(2015).

[0172] [Table 7]

[0173] Table 7: MHC class II binding capacity and antibody production induction activity in European and American people

[0174]

[0175] ※Frequency quoted from Mack SJ et al., Tissue Antigens; 73(1): 17-32 (2009).

[0176] Example 3: Study on the antibody inducing ability of nucleic acid constructs encoding T cell epitopes (1)

[0177] In Example 3, the ability to induce antibody production in mice was studied using a nucleic acid construct comprising a polynucleotide encoding the T cell epitope designed in Example 1.

[0178] (1) Methods

[0179] I. Preparation of nucleic acid construct (mRNA vaccine)

[0180] Prepared according to conventional methods (see also the composition of nucleic acid constructs Figure 2 A and B): a nucleic acid construct (IL-17A nucleic acid construct) comprising a base sequence obtained by repeating three times the three base sequences encoding T cell epitopes shown in Table 8 (base sequences obtained by replacing T with Ψ) and three base sequences encoding mouse IL-17A B cell epitopes shown in Table 9 (base sequences obtained by replacing T with Ψ); and a nucleic acid construct (IL-23 nucleic acid construct) comprising a base sequence obtained by repeating three times the three base sequences encoding T cell epitopes shown in Table 8 (base sequences obtained by replacing T with Ψ) and three base sequences encoding mouse IL-23 B cell epitopes shown in Table 10 (base sequences obtained by replacing T with Ψ). It should be noted that Ψ represents 1-methyl-3-pseudouridylyl.

[0181] [Table 8]

[0182] Table 8: Base sequences encoding T cell epitopes ＊

[0183]

[0184] * The base sequences of SEQ ID NOs: 86 to 88 correspond to the amino acid sequences of the T cell epitopes of SEQ ID NOs: 83 to 5 shown in Table 2, respectively.

[0185] [Table 9]

[0186] Table 9: Base sequences encoding B cell epitopes of IL-17A

[0187]

[0188] [Table 10]

[0189] Table 10: Base sequences encoding B cell epitopes of IL-23

[0190]

[0191] II Antibody titer determination

[0192] 10 μg of each of the IL-17A nucleic acid construct and IL-23 nucleic acid construct prepared in the above-mentioned 1 was administered to Balb / c mice at intervals of 2 weeks. Then, the antibody titers specific to mouse IL-17A and mouse IL-23 were measured by ELISA using recombinant native proteins of mouse IL-17A or mouse IL-23 for serum (200-fold dilution) before administration (week 0) and 2, 4, 6, 8, and 10 weeks after administration, respectively. Table 11 shows the steps for measuring antibody titers.

[0193] [Table 11]

[0194] Table 11: Steps for antibody titer determination

[0195]

[0196] (2) Results

[0197] The results are as follows Figure 3 As shown. In mice (administered group) to which the nucleic acid construct (mRNA vaccine) was administered, an increase in antibody titers against IL-17A and IL-23 was observed after the start of administration. In contrast, in mice (control group) to which the mRNA vaccine was not administered, an increase in antibody titers against IL-17A and IL-23 was not observed after the start of administration. According to this result, the use of a nucleic acid construct (mRNA vaccine) containing a polynucleotide encoding a T cell epitope designed in Example 1 actually induced the production of antibodies against IL-17A and IL-23 in mice.

[0198] Example 4: Study on the antibody inducing ability of nucleic acid constructs encoding T cell epitopes (2)

[0199] In Example 4, the nucleic acid construct comprising the polynucleotide encoding the T cell epitope designed in Example 1 was used to study the subclasses of antibodies induced in mice.

[0200] (1) Methods

[0201] I Nucleic acid construct (mRNA vaccine)

[0202] In the same manner as in Example 3(1)I, IL-17A nucleic acid construct and IL-23 nucleic acid construct were prepared in the form of mRNA vaccines.

[0203] II Antibody titer determination

[0204] 10 μg of each of the IL-17A nucleic acid construct and IL-23 nucleic acid construct prepared in the above-mentioned 1 was administered to Balb / c mice at intervals of 2 weeks. Then, the antibody titers of each subclass antibody (IgG1, IgG2a, IgG2b, IgG3) against mouse IL-17A and mouse IL-23 were measured by ELISA for serum 6 weeks after the start of administration (diluted 200 times, diluted 800 times, diluted 3200 times, diluted 12800 times). Table 12 shows the steps of antibody titer measurement.

[0205] [Table 12]

[0206] Table 12: Steps for antibody titer determination

[0207]

[0208] (2) Results

[0209] The results are as follows Figure 4 As shown. In mice (administered group) administered with a nucleic acid construct (mRNA vaccine), the antibody titers of IgG2a and IgG1 were high among the subclasses of antibodies against IL-17A and IL-23 6 weeks after the start of administration. In contrast, in mice (control group) not administered with an mRNA vaccine, the antibody titers of all subclasses were close to 0. According to this result, the antibodies induced by the nucleic acid construct (mRNA vaccine) containing a polynucleotide encoding the T cell epitope designed in Example 1 are: IgG2a induced in a Th1 type immune response; and IgG1 induced in a Th2 type immune response.

[0210] Example 5: Study on the antibody inducing ability of nucleic acid constructs encoding T cell epitopes (3)

[0211] In Example 5, a nucleic acid construct comprising a polynucleotide encoding a T cell epitope designed in Example 1 was used to culture Balb / c mice (H2-IE d 、H2-IA d ) Different MHC class II C57BL / 6 mice (H2-IA b ) were used to study the ability to induce antibody production in mice.

[0212] (1) Methods

[0213] 1. Preparation of nucleic acid construct (mRNA vaccine)

[0214] In the same manner as in Example 3(1)I, IL-17A nucleic acid construct and IL-23 nucleic acid construct were prepared in the form of mRNA vaccines.

[0215] II Antibody titer determination

[0216] The antibody titer was measured in the same manner as in Example 3(1)II except that C57BL / 6 mice were used instead of Balb / c mice.

[0217] (2) Results

[0218] The results are as follows Figure 5 As shown. In mice (administered group) to which the nucleic acid construct (mRNA vaccine) was administered, an increase in the antibody titer against IL-17A and IL-23 was observed after the start of administration. In contrast, in mice (control group) to which the mRNA vaccine was not administered, an increase in the antibody titer against IL-17A and IL-23 was not observed after the start of administration. According to this result, the use of a nucleic acid construct (mRNA vaccine) comprising a polynucleotide encoding a T cell epitope designed in Example 1 also induced the production of antibodies against IL-17A and IL-23 in mice with different MHC class II, and indicated that the nucleic acid construct of the present invention can be used as a pan-HLA vaccine.

[0219] Example 6: Study on the effectiveness of antibodies induced by nucleic acid constructs encoding T cell epitopes

[0220] In Example 6, the effectiveness of antibodies induced by a nucleic acid construct (mRNA vaccine) comprising a polynucleotide encoding the T cell epitope designed in Example 1 was studied.

[0221] (1) Methods

[0222] I. Preparation of nucleic acid construct (mRNA vaccine)

[0223] The same procedure as in Example 3 (1) I was followed to prepare IL-17A nucleic acid construct and IL-23 nucleic acid construct in the form of mRNA vaccines.

[0224] II. Antibody induction based on mRNA vaccines

[0225] 10 μg each of the IL-17A nucleic acid construct and the IL-23 nucleic acid construct prepared in the above-mentioned I was administered to Balb / c mice at intervals of 2 weeks to induce antibody production, and plasma was collected 20 weeks after the start of the administration.

[0226] III Antibody administration test

[0227] A comparison study was conducted on the changes in auricle thickness between two groups of mice: a control group of mice (no antibody administration) in which psoriasis was induced by applying 10 mg of imiquimod (IMQ) 5% cream (BESELNA CREAM 5%, Mochida Pharmaceutical Co., Ltd.) to the front and back of the auricle for 5 consecutive days; and a group of mice (administered group) in which psoriasis was induced by intraperitoneal administration of 0.15 mL of the plasma prepared in II above and applying 10 mg of IMQ 5% cream to the front and back of the auricle for 5 consecutive days from immediately after the administration. The thickness of both auricles was measured every day using a digital thickness gauge (Ozaki Seisakusho). Based on the thickness of both auricles on the 5th day after IMQ application, the inhibition rate (%) was calculated (inhibition rate (%) of the administration group = (1-(average value of mice in the administration group - normal mice) / (average value of IMQ mice - average value of normal mice)) × 100; inhibition rate (%) of the control group = (1-(average value of mice in the control group - normal mice) / (average value of mice in the control group - average value of normal mice)) × 100), and photographs of the auricles of mice after repeated application of IMQ for 6 days were taken. In addition, the change in the thickness of the auricles of normal mice was also measured, and photographs of the auricles of mice were taken.

[0228] (2) Results

[0229] The results are as follows Figure 6 As shown in the control group, the thickness of the auricle increased day by day ( Figure 6 A). In addition, the inhibition rate calculated based on the thickness of both auricles 5 days after IMQ application was higher in the treated group than in the control group ( Figure 6 B) According to Figure 6 It was also observed in the photograph C that the psoriasis-like symptoms (thickness and inflammation) of the auricle of the administration group were suppressed compared with the control group. Based on these results, the effectiveness of the antibodies induced by the nucleic acid construct (mRNA vaccine) containing the polynucleotide encoding the T cell epitope designed in Example 1 was shown.

[0230] Example 7: Study on the effectiveness of nucleic acid constructs encoding T cell epitopes (mRNA vaccines)

[0231] In Example 7, the effectiveness of a nucleic acid construct (mRNA vaccine) comprising a polynucleotide encoding the T cell epitope designed in Example 1 was studied.

[0232] (1) Methods

[0233] I. Preparation of nucleic acid construct (mRNA vaccine)

[0234] In the same manner as in Example 3 (1) I, IL-17A nucleic acid construct and IL-23 nucleic acid construct were prepared as mRNA vaccines.

[0235] II mRNA vaccine administration trial

[0236] Balb / c mice were administered 10 μg of each of the IL-17A nucleic acid construct and IL-23 nucleic acid construct prepared in the above-mentioned I twice at intervals of 2 weeks. The changes in the thickness of the auricle were compared between the following two groups of mice: mice induced with psoriasis by applying 5 mg of IMQ 5% cream to the front of the auricle for 6 consecutive days 4 weeks after the start of administration (administration group); and mice induced with psoriasis in the same manner as Balb / c mice (no mRNA vaccine was administered) (control group). The thickness of both auricles was measured every day using a digital thickness gauge (Ozaki Seisakusho). Then, based on the thickness of both auricles 6 days after IMQ application, the inhibition rate (%) was calculated (inhibition rate (%) of the administration group = (1-(average value of mice in the administration group - normal mice) / (average value of IMQ mice - average value of normal mice)) × 100; inhibition rate (%) of the control group = (1-(average value of mice in the control group - normal mice) / (average value of mice in the control group - average value of normal mice)) × 100), and photographs of the mouse auricles after repeated application of IMQ for 6 days were taken. In addition, the change in the thickness of the auricles of normal mice was also measured, and photographs of the mouse auricles were taken.

[0237] (2) Results

[0238] The results are as follows Figure 7 As shown in the control group, the thickness of the auricle increased day by day ( Figure 7 A). In the administration group, the inhibition rate calculated based on the thickness of both auricles 6 days after IMQ application was higher than that of the control group ( Figure 7 B) According to Figure 7 It was also observed in the photograph of C that psoriasis-like symptoms (thickness and inflammation) of the auricle of the administration group were suppressed compared with the control group. Based on these results, the effectiveness of the nucleic acid construct (mRNA vaccine) containing the polynucleotide encoding the T cell epitope designed in Example 1 was shown.

Claims

1. A nucleic acid construct that induces the production of antibodies against a target protein, the 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.

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

3. The nucleic acid construct according to claim 1 or 2, wherein: The target protein is a protein produced in one's 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 is a cause of a disease or a protein produced by 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 more autoimmune diseases selected from the group consisting of psoriasis, rheumatoid arthritis, systemic lupus erythematosus, Basedow's disease, chronic thyroiditis, type 1 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 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 preventive or therapeutic agent for a disease, comprising the nucleic acid construct according to claim 1 or 2 as an active ingredient.

10. The preventive or therapeutic agent according to claim 9, wherein The disease is an autoimmune disease or an allergic disease. The preventive agent according to claim 9 , which is a pan-HLA vaccine.

12. A method for producing a preventive or therapeutic agent for a disease, comprising the step of using a nucleic acid construct that induces the production of antibodies 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.

13. The manufacturing 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 production of antibodies against a target protein, comprising the step of combining two or more T cell epitopes with one or two or more B cell epitopes of the target protein.

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

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