Pharmaceutical composition

Abstract

[Problem] To provide a novel pharmaceutical composition that can be used for the induction of an immune response to an HTLV-1. [Solution] A pharmaceutical composition according to the present disclosure comprises: a human T-cell leukemia virus type-1 (HTLV-1) antigenic Gag protein p15 (Gag p15) or an immunogenic fragment thereof, a Gag protein p19 (Gag p19) or an immunogenic fragment thereof, and / or a Gag protein p24 (Gag p24) or an immunogenic fragment thereof; and a pharmaceutically acceptable carrier.

Description

Pharmaceutical Composition

[0001] The present disclosure relates to pharmaceutical compositions.

[0002] The retrovirus human T-cell leukemia virus type-I (HTLV-1) is known to cause diseases such as adult T-cell leukemia-lymphoma (ATL) and HTLV-1-associated inflammatory diseases, such as HTLV-1-associated myelopathy (HAM) and HTLV-1-associated uveitis (HU). These HTLV-1-associated diseases develop in HTLV-1-infected individuals (carriers). It is estimated that there are approximately 800,000 HTLV-1-infected individuals (carriers) in Japan. Furthermore, HTLV-1 infection is often asymptomatic.

[0003] Because ATL is resistant to treatment and has a poor prognosis, prevention of HTLV-1 infection is considered important for preventing the onset of ATL. Furthermore, ATL patients who have undergone hematopoietic stem cell transplantation may experience a relapse of ATL after transplantation. For these reasons, induction of an immune response against HTLV-1 is also important for improving the prognosis of ATL treatment. However, no pharmaceutical composition effective against HTLV-1 has been established.

[0004] Cook, Lucy B et al. “Revised Adult T-Cell Leukemia-Lymphoma International Consensus Meeting Report.” Journal of clinical oncology: official journal of the American Society of Clinical Oncology vol. 37,8 (2019): 677-687. doi:10.1200 / JCO.18.00501

[0005] Therefore, an object of the present disclosure is to provide a pharmaceutical composition that can be used to induce an immune response against HTLV-1.

[0006] To achieve the above object, the pharmaceutical composition of the present disclosure comprises human T-cell leukemia virus 1 (HTLV-1) antigenic Gag protein p15 (Gag p15) or an immunogenic fragment thereof, Gag protein p19 (Gag p19) or an immunogenic fragment thereof, and / or Gag protein p24 (Gag p24) or an immunogenic fragment thereof, and a pharmaceutically acceptable carrier.

[0007] The pharmaceutical compositions of the present disclosure comprise a nucleic acid encoding human T-cell leukemia virus 1 (HTLV-1) antigenic Gag protein p15 (Gag p15) or an immunogenic fragment thereof, Gag protein p19 (Gag p19) or an immunogenic fragment thereof, and / or Gag protein p24 (Gag p24) or an immunogenic fragment thereof, and a pharmaceutically acceptable carrier.

[0008] The pharmaceutical compositions of the present disclosure comprise a vector comprising a nucleic acid encoding human T-cell leukemia virus 1 (HTLV-1) antigenic Gag protein p15 (Gag p15) or an immunogenic fragment thereof, Gag protein p19 (Gag p19) or an immunogenic fragment thereof, and / or Gag protein p24 (Gag p24) or an immunogenic fragment thereof, and a pharmaceutically acceptable carrier.

[0009] The pharmaceutical composition of the present disclosure can induce an immune response against HTLV-1.

[0010] FIG. 1 is a graph showing the results of the LIPS assay in Example 1. FIG. 2 is a graph showing the results of the RT-qPCR in Example 1. FIG. 3 is a photograph showing the results of the proximity ligation assay in Example 1. FIG. 4 is a photograph showing the results of the ELISPOT assay in Example 1. FIG. 5 is a photograph showing the results of the ELISPOT assay in Example 1. FIG. 6 is a graph showing the results of the ELISPOT assay in Example 2. FIG. 7 is a photograph showing the results of the ELISPOT assay in Example 2. The sample numbers in FIG. 7A correspond to the numbers (1 to 12) shown in FIG. 7B, and indicate that samples 1 to 4 of the No peptide, Gag peptide, Positive Ctrl group in FIG. 7B were administered with saline, samples 5 to 8 with 0.2 mg / kg of lipid-mRNA particles, and samples 9 to 12 with 1 mg / kg of lipid-mRNA particles. The results in Figure 7C correspond to the sample positions shown in Figure 7B, with the first and second columns from the left showing the results for samples 1 to 12 with no peptide, the third and fourth columns from the left showing the results for samples 1 to 12 with Gag peptide, and the fifth and sixth columns from the left showing the results for samples 1 to 12 with Positive Ctrl.

[0011] The present disclosure will be described below with reference to examples. In the following description, the descriptions of the inventions can be mutually incorporated unless otherwise specified.

[0012] <Definition> As used herein, "protein" refers to a peptide polymer composed of unmodified amino acids (natural amino acids), modified amino acids, and / or artificial amino acids. The polymer may be, for example, linear, branched, or cyclic. The protein may also be referred to as a peptide or polypeptide.

[0013] As used herein, "retrovirus" refers to a type of RNA virus that possesses reverse transcriptase. Retroviruses are known to infect and replicate in host cells as follows: They infect target host cells, synthesize double-stranded DNA by reverse transcription of viral RNA, and integrate the viral DNA as a provirus into the genomic DNA of the host cell. The host cell then produces viral RNA and viral proteins, and retroviral protease cleaves the immature viral proteins, allowing the mature viral proteins to assemble into viral particles. The viral particles then bud from the surface of the host cell.

[0014] As used herein, "HTLV-1" refers to a type of retrovirus, human T-cell leukemia virus type-I. HTLV-1 is a virus that causes adult T-cell leukemia / lymphoma (ATL (or ATLL): Adult T-cell leukemia / lymphoma), HTLV-1 associated myelopathy (HAM: HTLV-1 associated myelopathy, TSP: Tropical spastic paraparesis), and HTLV-1 uveitis (HU: HTLV-1 HTLV-1 is known as a virus that causes diseases such as uveitis. HTLV-1 is a virus belonging to the Oncovirinae subfamily of the Retroviridae family. HTLV-1 has viral structural genes such as Gag, Pol, and Env. HTLV-1 also has regulatory genes such as Tax and Rex. HTLV-1 also has accessory genes such as p12, p13, p30, and HBZ. An example of the HTLV-1 provirus is a protein consisting of the amino acid sequence registered in Genbank under accession number AB513134.1.

[0015] As used herein, "HTLV-1 associated disease" is a general term for diseases caused by HTLV-1 infection. Examples of "HTLV-1 associated disease" include 1) ATL, 2) HAM, and 3) HU.

[0016] As used herein, "ATL" or "ATLL" refers to adult T-cell leukemia / lymphoma. It is known that in HTLV-1-infected individuals (carriers), HTLV-1 infects CD4-positive (+) T cells and the like, causing the infected cells to become cancerous, resulting in the development of ATL.

[0017] As used herein, "HAM" refers to HTLV-1 associated myelopathy (TSP: Tropical spastic paraparesis).

[0018] As used herein, "HU" refers to HTLV-1 uveitis or HTLV-1 associated uveitis.

[0019] As used herein, "processing" refers to a reaction in which a polypeptide translated as a precursor is cleaved or modified by a protease or the like to be processed into a more mature protein.

[0020] As used herein, "Gag protein (hereinafter also referred to as Gag)" refers to a retroviral capsid precursor protein (Gag protein before processing). The Gag is known to play a role in the assembly and budding of virus particles. The Gag is processed by a retroviral protease. The HTLV-1 Gag is processed, for example, by a protease into p15 (nucleocapsid protein), p19 (matrix protein), p24 (capsid protein), and the like. An example of the HTLV-1 Gag is a protein consisting of the amino acid sequence registered in Genbank under accession number AAA85841.1 (SEQ ID NO: 1).

[0021] Amino acid sequence of HTLV-1 Gag (SEQ ID NO: 1): MGQIFSRSASPIPRPPRGLAAHHWLNFLQAAYRLEPGPSSYDFHQLKKFLKIALETPVWICPINYSLLASLLPKGYPGRVNEILHILIQTQAQIPSRPAPPPPSSPTHDPPDSDPQIPPPYVEPTAPQVLPVMHPHGAPPNHRPWQMKDLQAIKQEVSQAAPGSPQFMQTIRLAVQQFDPTAKDLQDLLQYLCSSLVASLHHQQLDSLISEAET RGITGYNPLAGPLRVQANNPQQQGLRREYQQLWLAAFAALPGSAKDPSWASILQGLEEPYHAFVERLNIALDNGLPEGTPKDPILRSLAYSNANKECQKLLQARGHT NSPLGDMLRACQTWTPKDKTKVLVVQPKKPPPNQPCFRCGKAGHWSRDCTQPRPPPGPCPLCQDPTHWKRDCPRLKPTIPEPEPEEDALLLDLPADIPHPKNSIGGEV

[0022]

[0023] As used herein, "Tax protein (hereinafter also referred to as Tax)" refers to a transcriptional activator that activates both the long terminal repeat (LTR) and intracellular promoter of a retrovirus. Tax is known to play an important role in viral infectivity and canceration of infected cells. An example of the HTLV-1 Tax is a protein consisting of the amino acid sequence registered in Genbank under accession number P03409.2 (SEQ ID NO: 3).

[0024] Amino acid sequence of HTLV-1 Tax (SEQ ID NO: 3) MAHFPGFGQSLLFGYPVYVFGDCVQGDWCPISGGLCSARLHRHALLATCPEHQITWDPIDGRVIGSALQFLIPRLPSFPTQRTSKTLKVLTPPITHTTPNIPPSFLQAMRKYSPFRNGYMEPTLGQHLPTLSFPDPGLRPQNLYTLWGGSVVCMYLYQLSPPITWPLLPHVIFCHPGQLGAFLTNVPYKRIEELLYKISLTTGALIILPEDCLPTTLFQPARAPVTLTAWQNGLLPFHSTLTTPGLIWTFTDGTPMISGPCPKDGQPSLVLQSSSFIFHKFQTKAYHPSFLLSHGLIQYSSFHSLHLLFEEYTNIPISLLFNEKEADDNDHEPQISPGGLEPPSEKHFRETEV

[0025]

[0026] As used herein, "HBZ protein (hereinafter also referred to as HBZ)" refers to a retroviral bZIP factor. HBZ is known to contribute to the transcriptional regulation of viral RNA. The HTLV-1 HBZ can be, for example, a protein consisting of the amino acid sequence registered in Genbank under accession number BAE06226.1 (SEQ ID NO: 5). The HTLV-1 HBZ can be, for example, a polynucleotide encoding HBZ consisting of the nucleotide sequence (including a stop codon) of SEQ ID NO: 6 below. The HTLV-1 HBZ can also be, for example, a protein consisting of the amino acid sequence represented by SEQ ID NO: 7.

[0027] Amino acid sequence of HTLV-1 HBZ (SEQ ID NO: 5) MAASGLFRCLPVSCPEDLLVEELVDGLLSLEEELKDKEEEEAVLDGLLSLEEESRGRLRRGPPGEKAPPRGETHRDRQRRAEEKRKRKKEREKEEEKQTAEYLKRKEEEKARRRRRAEKKAADVARRKQEEQERRERKWRQGAEKAKQHSARKEKMQELGIDGYTRQLEGEVESLEAERRKLLQEKEDLMGEVNYWQGRLEAMWLQ

[0028] HTLV-1 HBZ base sequence (SEQ ID NO: 6) 5'--3'

[0029] Amino acid sequence of HTLV-1 HBZ (SEQ ID NO: 7) MAASGLFRCLPVSCPEDLLVEELVDGLLSLEEELKDKEEEEAVLDGLLSLEEESRGRLRRGPPGEKAPPRGETHRDRQRRAEEKRKRKKEREKEEEKQIAEYLKRKEEEKARRRRRAEKKAADVARRKQEEQERRERKWRQGAEKAKQHSARKEKMQELGIDGYTRQLEGEVESLEAERRKLLQEKEDLMGEVNYWQGRLEAMWLQ

[0030] HTLV-1 HBZ base sequence (SEQ ID NO: 8) 5'--3'

[0031] As used herein, "T cells" refer to a type of white blood cell, classified as lymphocytes, and expressing a T cell receptor (TCR). Known examples of T cells include CD4+ helper T cells and CD8+ cytotoxic T cells.

[0032] As used herein, "antigenicity" refers to the property of an antigen to induce immune responses such as antibody production and cellular immunity.

[0033] As used herein, "immunogenicity" refers to the property of an antigen to induce an immune response such as antibody production or cellular immunity. An "immunogenic fragment," also known as an immunogenic portion, refers to a fragment or truncation of a protein or polypeptide that induces an immune response.

[0034] As used herein, "vaccine" refers to a composition that generates an immune response for the prevention and / or treatment of a disease or condition. A vaccine is thus a pharmaceutical product containing an immunogenic agent, intended for use in humans or animals to generate specific protection and protection by vaccination.

[0035] As used herein, the term "pharmaceutically acceptable carrier" refers to a solvent and / or additive commonly used in the formulation of pharmaceutical compositions. Pharmaceutically acceptable carriers are well known in the art. Examples of such pharmaceutically acceptable carriers include aqueous solutions or other solvents, such as water or buffered saline, or solvents such as glycols, glycerol, oils (e.g., olive oil), or injectable organic esters. The pharmaceutically acceptable carrier may contain a physiologically acceptable compound that acts to stabilize or enhance drug absorption. Examples of such physiologically acceptable compounds include carbohydrates such as glucose, sucrose, or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low-molecular-weight proteins, or other stabilizers or excipients. Other ingredients known to those skilled in the art, such as diluents, excipients, additives, and / or adjuvants, may also be used in combination. Preferably, the pharmaceutically acceptable carrier is substantially or completely non-toxic to living organisms.

[0036] As used herein, "adjuvant" refers to a compound that, when used in combination with a specific immunogen (antigen) in a formulation or composition, enhances, modifies, or modifies the resulting immune response. Enhancement, modification, or modification of the immune response refers, for example, to strengthening, increasing, or enhancing the specificity of at least one of an antibody response and a cellular immune response. Enhancement, modification, or modification of the immune response may also refer to lowering, reducing, or suppressing a specific antigen-specific immune response.

[0037] As used herein, "nucleic acid" refers to a polymer of deoxyribonucleotides (DNA), ribonucleotides (RNA), and / or modified nucleotides. When "nucleic acid" is used in conjunction with a specific protein, the "nucleic acid" refers to a polymer of nucleotides encoding the amino acid sequence of the protein. Examples of the nucleic acid include genomic DNA, cDNA, and mRNA. The nucleic acid may be, for example, single-stranded or double-stranded. The nucleic acid can be interchangeably referred to as a "polynucleotide" or an "oligonucleotide." In the present disclosure, when the nucleic acid is mRNA, the base sequence of the mRNA can be, for example, the thymine (t) in the examples of the polynucleotide base sequences shown in the SEQ ID NOs. hereinafter, the same explanation can be applied by replacing uracil (u) with thymine (u). Furthermore, the nucleic acid may be modified (modified nucleic acid) or unmodified. Examples of the modification include methylation, pseudouridylation, and thiolation. In the present disclosure, when the nucleic acid is mRNA, for example, it is preferable that the uridine of the nucleic acid is pseudouridylated, i.e., the uridine is substituted with pseudouridine. The pseudouridylation may be performed on some or all of the uridine.

[0038] As used herein, the term "vector" (expression vector) refers to a recombinant plasmid or virus containing a nucleic acid that can be delivered to a host cell in vitro or in vivo.

[0039] As used herein, the term "subject" refers to an animal or a cell, tissue, or organ derived from an animal. The term "subject" particularly includes humans. The term "animal" refers to humans and non-human animals. Examples of non-human animals include mammals such as mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys, dolphins, and sea lions.

[0040] As used herein, "treating" means ameliorating, alleviating, delaying the onset of, or inhibiting the progression of the diseases described herein.

[0041] As used herein, "prevention" means reducing the possibility of onset of a disease or pathological condition, suppressing, delaying, or stopping the onset of a disease or pathological condition, suppressing, alleviating, delaying, or stopping the progression of a pathological condition, suppressing, reducing, delaying, or stopping the worsening of the condition, or suppressing, delaying, or stopping the recurrence of a disease or pathological condition. The "prevention" may be, for example, treatment of a subject (patient) who develops the target disease, or treatment of an animal model of the target disease.

[0042] As used herein, "aggravation" means that the degree (severity) of a disease or condition worsens.

[0043] As used herein, "isolated" means identified and separated and / or recovered from components of its natural state. The "isolation" can be achieved, for example, by at least one purification step.

[0044] The present disclosure will be described below using examples, but the present disclosure is not limited to the following examples and can be implemented with any modifications. Furthermore, the descriptions in this disclosure can be mutually incorporated unless otherwise specified. In this specification, when the expression "~" is used, it is used to mean including the numerical or physical values ​​before and after it. In addition, in this specification, the expression "A and / or B" includes "A only," "B only," and "both A and B."

[0045] Pharmaceutical Composition Comprising HTLV-1 Gag Antigen In one aspect, the present disclosure provides a pharmaceutical composition capable of inducing an immune response against HTLV-1, particularly HTLV-1 Gag. The pharmaceutical composition of the present disclosure comprises human T-cell leukemia virus 1 (HTLV-1) antigenic Gag protein p15 (Gag p15) or an immunogenic fragment thereof, Gag protein p19 (Gag p19) or an immunogenic fragment thereof, and / or Gag protein p24 (Gag p24) or an immunogenic fragment thereof, and a pharmaceutically acceptable carrier.

[0046] As a result of extensive research, the present inventors discovered that Gag, a structural protein of HTLV-1, is expressed in the cells of ATL patients, and that cytotoxic T cells against Gag are present in patients who do not experience ATL relapse. This led to the idea that an effective immune response against HTLV-1 might be induced by using HTLV-1 Gag as an antigen. The present inventors then discovered that a cellular immune response of T cells can be induced by using a pharmaceutical composition containing HTLV-1 Gag p15, p19, and p24 as active ingredients, and have established the present disclosure. Therefore, the pharmaceutical composition of the present disclosure is capable of inducing an immune response against HTLV-1, for example.

[0047] The pharmaceutical composition of the present disclosure is characterized by comprising, as an active ingredient, an HTLV-1 antigen Gag protein p15 (Gag p15) or an immunogenic fragment thereof, Gag protein p19 (Gag p19) or an immunogenic fragment thereof, or Gag protein p24 (Gag p24) or an immunogenic fragment thereof (hereinafter collectively referred to as "immunogens"), and other configurations and conditions are not particularly limited. Because the pharmaceutical composition of the present disclosure comprises, as an active ingredient, an HTLV-1 antigen Gag protein p15 (Gag p15) or an immunogenic fragment thereof, Gag protein p19 (Gag p19) or an immunogenic fragment thereof, or Gag protein p24 (Gag p24) or an immunogenic fragment thereof, when used as a pharmaceutical composition, it can induce an immune response against HTLV-1.

[0048] Examples of the Gag p15 include the following proteins (a1), (a2), and (a3):

[0049] (a1) A protein consisting of the amino acid sequence of SEQ ID NO: 9 or 10. (a2) A protein consisting of the amino acid sequence of SEQ ID NO: 9 or 10 in which one or more amino acids have been deleted, inserted, substituted, or added. (a3) ​​A protein consisting of an amino acid sequence that is 80% or more identical to the amino acid sequence of SEQ ID NO: 9 or 10.

[0050] Amino acid sequence of Gag p15 (SEQ ID NO: 9) VVQPKKPPPNQPCFRCGKAGHWSRDCTQPRPPPGPCPLCQDPTHWKRDCPRLKPTIPEPEPEEDALLLDLPADIPHPKNLHRGGGLTSPPTLQQVLPNQDPASIL

[0051] Amino acid sequence of Gag p15 (SEQ ID NO: 10) VVQPKKPPPNQPCFRCGKAGHWSRDCTQPRPPPGPCPLCQDPTHWKRDCPRLKPTIPEPEPEEDALLLDLPADIPHPKNSIGGEV

[0052] In (a1), the amino acid sequence shown in SEQ ID NO: 9 or 10 is an amino acid sequence derived from HTLV-1. Furthermore, in (a1), the amino acid sequence shown in SEQ ID NO: 9 is, for example, the sequence of endogenous mature Gag p15 that can be expressed in infected cells, which is generated when ribosomal frameshifting occurs. The amino acid sequence shown in SEQ ID NO: 10 is, for example, the sequence of Gag p15 that is generated when ribosomal frameshifting does not occur. The amino acid sequence of (a1) is preferably, for example, the amino acid sequence of SEQ ID NO: 10, which is predicted to be expressed in large amounts in vivo when made into a vaccine composition.

[0053] In the protein (a2), "one or several" may be within a range in which (a2) is a protein immunogenic against HTLV-1, for example. "One or several" in (a2) means, for example, 1 to 31, 1 to 26, 1 to 25, 1 to 21, 1 to 15, 1 to 12, 1 to 10, 1 to 8, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1 in the amino acid sequence of (a1). In the present disclosure, a numerical range of the number discloses, for example, all positive integers falling within that range. That is, for example, the description "1 to 5" means the disclosure of all of "1, 2, 3, 4, and 5" (the same applies hereinafter).

[0054] In the protein (a2), the substitution is preferably a conservative substitution. The conservative substitution refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. Examples of the conservative substitution include substitutions between amino acid residues having basic side chains such as lysine, arginine, and histidine; substitutions between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; substitutions between amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; substitutions between amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; substitutions between amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and substitutions between amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0055] With respect to the protein (a3), the "identity" may be within a range such that the protein (a3) ​​is a peptide immunogenic to HTLV-1. The "identity" of the protein (a3) ​​is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more relative to the amino acid sequence of the protein (a1). The "identity" can be calculated, for example, by using default parameters in the homology algorithm BLAST (http: / / www.ncbi.nlm.nih.gov / BLAST / ) of the National Center for Biotechnology Information (NCBI) (the same applies hereinafter).

[0056] Examples of the Gag p19 include the following proteins (b1), (b2), and (b3):

[0057] (b1) A protein consisting of the amino acid sequence of SEQ ID NO: 11. (b2) A protein consisting of the amino acid sequence of SEQ ID NO: 11 in which one or more amino acids have been deleted, inserted, substituted, or added. (b3) A protein consisting of an amino acid sequence that is 80% or more identical to the amino acid sequence of SEQ ID NO: 11.

[0058] Amino acid sequence of Gag p19 (SEQ ID NO: 11) MGQIFSRSASPIPRPPRGLAAHHWLNFLQAAYRLEPGPSSYDFHQLKKFLKIALETPVWICPINYSLLASLLPKGYPGRVNEILHILIQTQAQIPSRPAPPPPSSPTHDPPDSDPQIPPPYVEPTAPQVL

[0059] In the protein (b2), "one or several" may be within a range in which the protein (b2) is immunogenic against HTLV-1. The "one or several" in (b2) may be, for example, 1 to 38, 1 to 32, 1 to 25, 1 to 19, 1 to 12, 1 to 6, 1 to 5, 1 to 3, 1 or 2, or 1 in the amino acid sequence of (b1).

[0060] In the protein (b3), the "identity" may be within a range such that the protein (b3) is a peptide having immunogenicity against HTLV-1. The "identity" of the protein (b3) to the amino acid sequence of the protein (b1) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0061] Examples of the Gag p24 include the following proteins (c1), (c2), and (c3):

[0062] (c1) A protein consisting of the amino acid sequence of SEQ ID NO: 12. (c2) A protein consisting of the amino acid sequence of SEQ ID NO: 12 in which one or more amino acids have been deleted, inserted, substituted, or added. (c3) A protein consisting of an amino acid sequence that is 80% or more identical to the amino acid sequence of SEQ ID NO: 12.

[0063] Amino acid sequence of Gag p24 (SEQ ID NO: 12) PVMHPHGAPPNHRPWQMKDLQAIKQEVSQAAPGSPQFMQTIRLAVQQFDPTAKDLQDLLQYLCSSLVASLHHQQLDSLISEAETRGITGYNPLAGPLRVQANNPQQQGLRREYQQLWLAAFAALPGSAKDPSWASILQGLEEPYHAFVERLNIALDNGLPEGTPKDPILRSLAYSNANKECQKLLQARGHTNSPLGDMLRACQTWTPKDKTKVL

[0064] In the protein of (c2), "one or several" may be within a range in which the protein of (c2) is immunogenic against HTLV-1. The "one or several" in (c2) may be, for example, 1 to 64, 1 to 53, 1 to 42, 1 to 32, 1 to 21, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 or 2, or 1 in the amino acid sequence of (c1).

[0065] In the protein (c3), the "identity" may be within a range such that the protein (c3) is a peptide having immunogenicity against HTLV-1. The "identity" of the protein (c3) to the amino acid sequence of the protein (c1) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0066] In the present disclosure, the immunogenic fragment of p15, p19, or p24 may be, for example, any protein fragment that is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 amino acids in length.

[0067] Examples of the immunogenic fragment of p19 include the amino acid sequence of positions 2 to 59 of SEQ ID NO: 1 (p19-1 in the Examples described below), the amino acid sequence of positions 52 to 109 of SEQ ID NO: 1 (p19-2 in the Examples described below), and the amino acid sequence of positions 102 to 130 of SEQ ID NO: 1 (p19-3 in the Examples described below), and preferably the amino acid sequence of positions 2 to 59 of SEQ ID NO: 1 (p19-1 in the Examples described below) or the amino acid sequence of positions 52 to 109 of SEQ ID NO: 1 (p19-2 in the Examples described below).

[0068] The immunogenic fragment of p19 may be, for example, a peptide encoding an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to any of the amino acid sequences of positions 2 to 59, 52 to 109, and 102 to 130 of SEQ ID NO: 1.

[0069] The pharmaceutical composition of the present disclosure may contain, for example, any one, more than one, or all of the Gag p15 or an immunogenic fragment thereof, the Gag p19 or an immunogenic fragment thereof, and the Gag p24 or an immunogenic fragment thereof. The pharmaceutical composition of the present disclosure preferably contains, for example, the Gag p15 or an immunogenic fragment thereof, the Gag p19 or an immunogenic fragment thereof, and the Gag p24 or an immunogenic fragment thereof. By adopting such a configuration, the pharmaceutical composition of the present disclosure can, for example, efficiently induce an immune response against HTLV-1.

[0070] The Gag p15, Gag p19, and Gag p24 are expressed by processing of unprocessed Gag proteins. Thus, the pharmaceutical composition of the present disclosure may contain, for example, unprocessed Gag proteins or immunogenic fragments thereof as the Gag p15, Gag p19, and / or Gag p24. Therefore, the unprocessed Gag proteins or immunogenic fragments thereof can also be referred to as precursors or precursor proteins of, for example, Gag p15 or immunogenic fragments thereof, Gag p19 or immunogenic fragments thereof, and / or Gag p24 or immunogenic fragments thereof.

[0071] Examples of the Gag before processing include the following proteins (d1), (d2), and (d3):

[0072] (d1) A protein consisting of the amino acid sequence of SEQ ID NO: 1. (d2) A protein consisting of the amino acid sequence of SEQ ID NO: 1 in which one or more amino acids have been deleted, inserted, substituted, or added. (d3) A protein consisting of an amino acid sequence that is 80% or more identical to the amino acid sequence of SEQ ID NO: 1.

[0073] In the protein (d2), "one or several" may be within a range in which the protein (d2) is immunogenic against HTLV-1. The "one or several" in the (d2) may be, for example, 1 to 128, 1 to 107, 1 to 85, 1 to 64, 1 to 42, 1 to 21, 1 to 14, 1 to 12, 1 to 8, 1 to 4, 1 or 2, or 1 in the amino acid sequence of the (d1).

[0074] The "identity" of the protein (d3) may be within a range such that the protein (d3) is a peptide having immunogenicity against HTLV-1. The "identity" of the protein (d3) to the amino acid sequence of the protein (d1) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0075] The Gag before processing preferably comprises at least one of the amino acid sequence of amino acids 2 to 59 of SEQ ID NO: 1 (p19-1 in the Examples described below), the amino acid sequence of amino acids 52 to 109 of SEQ ID NO: 1 (p19-2 in the Examples described below), and the amino acid sequence of amino acids 102 to 130 of SEQ ID NO: 1 (p19-3 in the Examples described below), and the region other than the amino acid sequence is a protein consisting of an amino acid sequence other than the amino acid sequence in SEQ ID NO: 1 or an amino acid sequence having at least 80%, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence other than the amino acid sequence in SEQ ID NO: 1. More preferably, the unprocessed Gag comprises at least one of the amino acid sequence from positions 2 to 59 of SEQ ID NO: 1 (p19-1 in the Examples described below) and the amino acid sequence from positions 52 to 109 of SEQ ID NO: 1 (p19-2 in the Examples described below), and the region other than the amino acid sequence is a protein consisting of an amino acid sequence other than the amino acid sequence in SEQ ID NO: 1, or an amino acid sequence having at least 80%, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence other than the amino acid sequence in SEQ ID NO: 1.

[0076] In the present disclosure, the immunogenic fragment of pre-processed Gag may be, for example, any protein fragment that is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 amino acids in length from the protein, as long as the protein is immunogenic to HTLV-1.

[0077] The pharmaceutical composition of the present disclosure may further comprise, for example, the Tax protein or an immunogenic fragment thereof, and / or the HBZ protein or an immunogenic fragment thereof of HTLV-1. By further comprising, for example, the Tax or an immunogenic fragment thereof, and / or the HBZ protein or an immunogenic fragment thereof, the pharmaceutical composition of the present disclosure can more efficiently induce an immune response against HTLV-1.

[0078] Examples of the Tax include the following proteins (e1), (e2), and (e3):

[0079] (e1) A protein consisting of the amino acid sequence of SEQ ID NO: 3. (e2) A protein consisting of the amino acid sequence of SEQ ID NO: 3 in which one or more amino acids have been deleted, inserted, substituted, or added. (e3) A protein consisting of an amino acid sequence that is 80% or more identical to the amino acid sequence of SEQ ID NO: 3.

[0080] In the protein (e2), "one or several" may be within a range in which (e2) is a protein having immunogenicity against HTLV-1. The "one or several" in (e2) means, for example, 1 to 105, 1 to 88, 1 to 70, 1 to 52, 1 to 35, 1 to 17, 1 to 14, 1 to 10, 1 to 7, 1 to 3, 1 or 2, or 1 in the amino acid sequence of (e1).

[0081] In the protein (e3), the "identity" may be within a range such that the protein (e3) is a peptide having immunogenicity against HTLV-1. The "identity" of the protein (e3) to the amino acid sequence of the protein (e1) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0082] The HBZ may be, for example, the following protein (f1), (f2), or (f3):

[0083] (f1) A protein consisting of the amino acid sequence of SEQ ID NO: 5 or 7. (f2) A protein consisting of the amino acid sequence of SEQ ID NO: 5 or 7 in which one or more amino acids have been deleted, inserted, substituted, or added. (f3) A protein consisting of an amino acid sequence that is 80% or more identical to the amino acid sequence of SEQ ID NO: 5 or 7.

[0084] In the protein (f2), "one or several" may be within a range in which the protein (f2) is immunogenic against HTLV-1. The "one or several" in the (f2) may be, for example, 1 to 62, 1 to 52, 1 to 41, 1 to 31, 1 to 20, 1 to 10, 1 to 8, 1 to 6, 1 to 4, or 1 or 2 amino acids in the amino acid sequence of the (f1).

[0085] In the protein (f3), the "identity" may be within a range such that the protein (f3) is a peptide having immunogenicity against HTLV-1. The "identity" of the protein (f3) to the amino acid sequence of the protein (f1) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0086] In the present disclosure, the immunogenic fragment of Tax or HBZ may be, for example, any protein fragment of the protein that is immunogenic to HTLV-1. The immunogenic fragment of Tax or HBZ may be, for example, any protein fragment of the protein that is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 amino acids in length.

[0087] For example, when administered to a subject, the pharmaceutical composition of the present disclosure can induce cellular immunity against HTLV-1, particularly cellular immunity mediated by cytotoxic T cells. Therefore, the pharmaceutical composition of the present disclosure can also be referred to as a vaccine, vaccine composition, or vaccine preparation.

[0088] The pharmaceutical composition of the present disclosure is, for example, a vaccine for use in preventing the onset of ATL, the aggravation of ATL, or the recurrence of ATL.

[0089] The pharmaceutical composition of the present disclosure includes a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier may be a suspending agent, solubilizing agent, stabilizer, isotonicity agent, preservative, anti-adsorption agent, surfactant, diluent, vehicle, pH adjuster, soothing agent, buffer, sulfur-containing reducing agent, antioxidant, etc. for administering the active ingredient, and is appropriately added within a range that does not interfere with the effects of the present disclosure.

[0090] The suspending agent is not particularly limited, and examples thereof include methyl cellulose, polysorbate 80, hydroxyethyl cellulose, gum arabic, powdered tragacanth, sodium carboxymethyl cellulose, polyoxyethylene sorbitan monolaurate, and the like.

[0091] The solution adjuvant is not particularly limited, and examples thereof include polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, macrogol, and castor oil fatty acid ethyl ester.

[0092] The stabilizer is not particularly limited, and examples thereof include dextran 40, methylcellulose, gelatin, sodium sulfite, and sodium metasulfate.

[0093] The isotonic agent is not particularly limited, and examples thereof include D-mannitol, sorbitol, and the like.

[0094] The preservative is not particularly limited, and examples thereof include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, sorbic acid, phenol, cresol, and chlorocresol.

[0095] The adsorption inhibitor is not particularly limited, and examples thereof include human serum albumin, lecithin, dextran, ethylene oxide propylene oxide copolymer, hydroxypropyl cellulose, methyl cellulose, hydrogenated castor oil, and polyethylene glycol.

[0096] The sulfur-containing reducing agent is not particularly limited, and examples thereof include those having a sulfhydryl group, such as N-acetylcysteine, N-acetylhomocysteine, thiochitic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, sodium thiosulfate, glutathione, and thioalkanoic acids having 1 to 7 carbon atoms.

[0097] The antioxidant is not particularly limited, and examples thereof include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and salts thereof, L-ascorbyl palmitate, L-ascorbyl stearate, sodium hydrogen sulfite, sodium sulfite, triamyl gallate, propyl gallate, and chelating agents such as sodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, and sodium metaphosphate.

[0098] The pharmaceutical composition of the present disclosure may further contain, as appropriate, commonly added ingredients such as inorganic salts such as sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, and sodium bicarbonate; organic salts such as sodium citrate, potassium citrate, and sodium acetate; and sugars such as glucose.

[0099] The pharmaceutical composition of the present disclosure may further contain an adjuvant (immunostimulator), etc. Examples of the adjuvant include toll-like receptor stimulators such as CpG oligonucleotide (CpG ODN) and lipopolysaccharide (LPS); aluminum salts such as aluminum hydroxide, aluminum phosphate, and aluminum chloride; interferons such as interferon (IFN-γ); inflammatory cytokines such as TNF-α; interleukins such as IL-1, IL-2, IL-3, IL-4, IL-12, and IL-13; growth factors such as granulocyte-macrophage (GM-CSF) and granulocyte-colony-stimulating factor (G-CSF); cytokines such as Flt3 ligand, B7-1, and B7-2; chemokines; squalene-containing adjuvants such as squalane or squalene; complete or incomplete Freund's adjuvant; or mixtures thereof. An example of the LPS is MPL (Monophosphoryl lipid A). An example of the squalene-containing adjuvant is AddaVax (trademark) or MF-59 (registered trademark), which are oil-in-water emulsions of squalene.

[0100] The pharmaceutical composition of the present disclosure may be used, for example, in vitro or in vivo. The pharmaceutical composition of the present disclosure may be used, for example, as a research reagent or as a pharmaceutical. In the former case, the pharmaceutical composition of the present disclosure may also be referred to as a test reagent or test kit.

[0101] The subject to which the pharmaceutical composition of the present disclosure is administered is not particularly limited. When the pharmaceutical composition of the present disclosure is used in vivo, the subject (subject to administration) can be, for example, the above-mentioned examples. When the pharmaceutical composition of the present disclosure is used in vitro, the subject to administration can be, for example, a cell, tissue, organ, etc., and examples of the cell include cells collected from a living body, cultured cells, etc., and examples of the tissue or organ include tissue (biological tissue) or organ, etc. collected from a living body. Examples of the cell include immune cells such as T cells, B cells, NK cells, dendritic cells, etc.

[0102] When the pharmaceutical composition of the present disclosure is used in vivo, the subject may be a healthy individual not infected with HTLV-1, a person who may be infected with HTLV-1, or a patient infected with HTLV-1. The subject may also be a patient infected with HTLV-1 who has not yet developed ATL, a patient who has developed ATL, or a patient in remission from ATL. The subject is preferably a subject infected with HTLV-1 in whom prevention of the onset or recurrence of ATL is desired. The subject may also be a patient who has undergone bone marrow transplantation or hematopoietic stem cell transplantation after the onset of ATL.

[0103] The conditions for use (administration conditions) of the pharmaceutical composition of the present disclosure are not particularly limited, and the administration form, administration timing, dosage, etc. can be appropriately set depending on, for example, the type of the active ingredient in the pharmaceutical composition, the type of subject to administration, etc.

[0104] Examples of methods for administering the pharmaceutical composition of the present disclosure include intracerebral administration, intrathecal administration, intramuscular administration, subcutaneous administration, and intravenous administration. However, intramuscular administration or subcutaneous administration is preferred because it allows safe and stable administration regardless of the skill of the administerer.

[0105] The dosage of the pharmaceutical composition of the present disclosure is an amount capable of inducing an immune response in a subject, i.e., an effective dose. The dosage can be determined appropriately depending on, for example, the age, weight, symptoms, etc. of the subject.

[0106] The pharmaceutical composition of the present disclosure may be administered once or multiple times. The multiple times may be, for example, two, three, four, five, or more times. The number of administrations may be appropriately determined while confirming the preventive effect on the subject. When administering multiple times, the administration interval may be appropriately determined while confirming the preventive effect on the subject, and may be, for example, once a day, once a week, once every two weeks, once a month, once every three months, or once every six months.

[0107] The pharmaceutical composition of the present disclosure can prevent or alleviate at least one symptom caused by HTLV-1 infection in a subject to which it is administered. Therefore, the pharmaceutical composition of the present disclosure can induce an immune response against HTLV-1 and can therefore be used for the prevention and / or treatment of HTLV-1-associated diseases. An example of a symptom of HTLV-1 infection is the onset of ATL. Prevention of the symptom can be evaluated subjectively or objectively, and specific examples include self-assessment by the subject to which the composition is administered; evaluation by a physician; quality of life (QOL) evaluation; evaluation of the onset of ATL or delay in the progression of ATL symptoms, or reduction in the severity of ATL symptoms. The objective evaluation may be evaluation using animals or humans.

[0108] Each protein in the pharmaceutical composition of the present disclosure can be synthesized by genetic engineering techniques, specifically, by using the transformant described below. The protein may also be produced by in vitro synthesis, for example.

[0109] <Nucleic Acid-Containing Pharmaceutical Composition> In another aspect, the present disclosure provides a pharmaceutical composition capable of inducing an immune response against HTLV-1, particularly against HTLV-1 Gag. The pharmaceutical composition (nucleic acid-containing pharmaceutical composition) of the present disclosure comprises a nucleic acid encoding human T-cell leukemia virus 1 (HTLV-1) antigenic Gag protein p15 (Gag p15) or an immunogenic fragment thereof, Gag protein p19 (Gag p19) or an immunogenic fragment thereof, and / or Gag protein p24 (Gag p24) or an immunogenic fragment thereof, and a pharmaceutically acceptable carrier.

[0110] The nucleic acid-containing pharmaceutical composition of the present disclosure is characterized by comprising, as an active ingredient, a nucleic acid encoding the HTLV-1 antigen Gag protein p15 (Gag p15) or an immunogenic fragment thereof, Gag protein p19 (Gag p19) or an immunogenic fragment thereof, and / or Gag protein p24 (Gag p24) or an immunogenic fragment thereof, and other configurations and conditions are not particularly limited. Because the nucleic acid-containing pharmaceutical composition of the present disclosure comprises, as an active ingredient, a nucleic acid encoding the HTLV-1 antigen Gag protein p15 (Gag p15) or an immunogenic fragment thereof, Gag protein p19 (Gag p19) or an immunogenic fragment thereof, and / or Gag protein p24 (Gag p24) or an immunogenic fragment thereof, it can induce an immune response against HTLV-1 when used as a pharmaceutical composition. Unless otherwise specified, the nucleic acid-containing pharmaceutical composition of the present disclosure can be applied to the explanation of the pharmaceutical composition containing the Gag antigen of HTLV-1.

[0111] The nucleic acid encoding Gag p15 is, for example, a polynucleotide selected from the group consisting of (A1) to (A6) and (A7) below. (A1) a polynucleotide consisting of the base sequence of SEQ ID NO: 13 or 14; (A2) a polynucleotide consisting of the base sequence of (A1) in which one or several bases have been deleted, inserted, substituted, and / or added; (A3) a polynucleotide consisting of a base sequence having 80% or more identity to any of the base sequences of (A1); (A4) a polynucleotide consisting of a base sequence complementary to a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of any of the base sequences of (A1); (A5) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 9 or 10; (A6) a polynucleotide encoding a protein consisting of the amino acid sequence of (A5) in which one or several amino acids have been deleted, inserted, substituted, and / or added; (A7) a polynucleotide encoding a protein consisting of an amino acid sequence having 80% or more identity to the amino acid sequence of (A5);

[0112] In the above (A1), the nucleotide sequence of SEQ ID NO: 13 or 14 is as follows: The nucleotide sequence of SEQ ID NO: 13 or 14 is a coding sequence for Gag p15 consisting of the amino acid sequence of SEQ ID NO: 9 or 10. The polynucleotide (A1) of SEQ ID NO: 13 or 14 can be obtained from, for example, HTLV-1.

[0113] Polynucleotide of (A1) (SEQ ID NO: 13) 5'-gttgtccagcctaaaaaaccccccccaaatcagccgtgcttccggtgcgggaaagcaggccactggagtcgggactgcactcagcctcgccccccccccgggccatgccccctatgtcaagacccaactcactggaagcgagactgccccgcctaa agccccactatcccagaaccagagccagaggaagatgccctcctattagacctccccgctgacatcccacacccaaaaaacctccatagggggggaggtttaacctccccccccacattacagcaagtccttcctaaccaagacccagcatctattctg-3'

[0114] Polynucleotide of (A1) (SEQ ID NO: 14) 5'-gttgtccagcctaaaaaaccccccccaaatcagccgtgcttccggtgcgggaaagcaggccactggagtcgggactgcactcagcctcgccccccccccgggccatgccccctatgtcaagacccaa ctcactggaagcgagactgcccccgcctaaagcccactatcccagaaccagagccagaggaagatgccctcctattagacctccccgctgacatcccacacccaaaaaactccatagggggggaggtt-3'

[0115] In (A2), the "one or several" may be within a range in which the protein encoded by the polynucleotide of (A2) has immunogenicity against HTLV-1. The "one or several" in (A2) may be, for example, 1 to 94, 1 to 78, 1 to 76, 1 to 63, 1 to 51, 1 to 47, 1 to 31, 1 to 25, 1 to 15, 1 to 12, 1 to 10, 1 to 9, 1 to 7, 1 to 6, 1 to 5, 1 or 2, or 1 in the base sequence of (A1).

[0116] In (A3), the "identity" may be within a range such that the protein encoded by the polynucleotide of (A3) has immunogenicity against HTLV-1. The "identity" in (A3) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more relative to the base sequence of (A1).

[0117] In (A4), the "hybridizing polynucleotide" is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide of (A1). The hybridization can be detected, for example, by various hybridization assays. The hybridization assay is not particularly limited, and for example, the method described in "Molecular Cloning: A Laboratory Manual 2nd Ed." edited by Sambrook et al. [Cold Spring Harbor Laboratory Press (1989)] can be used.

[0118] In (A4), "stringent conditions" may be, for example, low stringency conditions, moderate stringency conditions, or high stringency conditions. "Low stringency conditions" are, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 32°C. "Medium stringency conditions" are, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 42°C. "High stringency conditions" are, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 50°C. Those skilled in the art can set the degree of stringency by appropriately selecting conditions such as temperature, salt concentration, probe concentration and length, ionic strength, and time. The "stringent conditions" are, for example, those described in the aforementioned "Molecular Cloning: A Laboratory Manual 2nd Edition" edited by Sambrook et al. nd Ed.) (Cold Spring Harbor Laboratory Press (1989)) or the like can also be used.

[0119] The polynucleotide (A5) can be designed, for example, based on the amino acid sequence of SEQ ID NO: 9 or 10 by substituting the corresponding codons.

[0120] In (A6), the "one or several" may be within a range in which the protein encoded by the polynucleotide of (A6) has immunogenicity against HTLV-1. The "one or several" in (A6) may be, for example, 1 to 31, 1 to 26, 1 to 25, 1 to 21, 1 to 15, 1 to 12, 1 to 10, 1 to 8, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1 in the amino acid sequence of (A5).

[0121] In (A7), the "identity" may be within a range such that the protein encoded by the polynucleotide of (A7) has immunogenicity against HTLV-1. The "identity" in (A7) to the amino acid sequence of (A5) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0122] The nucleic acid encoding Gag p19 is, for example, a polynucleotide selected from the group consisting of (B1) to (B6) and (B7) below. (B1) A polynucleotide consisting of the base sequence of SEQ ID NO: 15; (B2) A polynucleotide consisting of a base sequence in which one or more bases have been deleted, inserted, substituted, and / or added in the base sequence of (B1); (B3) A polynucleotide consisting of a base sequence that is 80% or more identical to any of the base sequences of (B1); (B4) A polynucleotide consisting of a base sequence that is complementary to a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of any of the base sequences of (B1); (B5) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 11; (B6) A polynucleotide encoding a protein consisting of an amino acid sequence in which one or more amino acids have been deleted, inserted, substituted, and / or added in the amino acid sequence of (B5); (B7) A polynucleotide encoding a protein consisting of an amino acid sequence that is 80% or more identical to the amino acid sequence of (B5).

[0123] In (B1), the nucleotide sequence of SEQ ID NO: 15 is as follows: The nucleotide sequence of SEQ ID NO: 15 is a coding sequence for Gag p19 consisting of the amino acid sequence of SEQ ID NO: 11. The polynucleotide (B1) of SEQ ID NO: 15 can be obtained from, for example, HTLV-1.

[0124] Polynucleotide of (B1) (SEQ ID NO: 15) 5'-ggccaaatcttttcccgtagcgctagccctattccgcggccgccccgggggctggccgctcatcactggcttaacttcctccaggcggcatatcgcctagaacccggtccctccagttacgatttccaccagttaaaaaaatttcttaaaatagctttagaaacaccagtctggatctgtcccattaactact ccctcctagccagcctactcccaaaaggataccccggccgggtgaatgaaattttacacatactcatccaaacccaagcccagatcccgtcccgtcccgcgccaccgccgccgtcatccccccacccacgaccccccggattctgatccacaaatcccccctccctatgttgagcctacggccccccaagtcctt-3'

[0125] In (B2), the "one or several" may be within a range in which the protein encoded by the polynucleotide of (B2) has immunogenicity against HTLV-1. The "one or several" in (B2) may be, for example, 1 to 116, 1 to 96, 1 ​​to 77, 1 to 58, 1 to 38, 1 to 19, 1 to 15, 1 to 11, 1 to 7, 1 to 3, 1 or 2, or 1 in the base sequence of (B1).

[0126] In (B3), the "identity" may be within a range such that the protein encoded by the polynucleotide of (B3) has immunogenicity against HTLV-1. The "identity" in (B3) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more relative to the base sequence of (B1).

[0127] In (B4), the "hybridizing polynucleotide" is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide of (B1). The hybridization can be detected, for example, by various hybridization assays. The hybridization assay is not particularly limited, and for example, the method described in "Molecular Cloning: A Laboratory Manual 2nd Ed." edited by Sambrook et al. [Cold Spring Harbor Laboratory Press (1989)] can be used. In (B4), the stringent conditions can be the same as those described in (A4).

[0128] The polynucleotide (B5) can be designed, for example, based on the amino acid sequence of SEQ ID NO: 11, by substituting the corresponding codons.

[0129] In (B6), the "one or several" may be within a range in which the protein encoded by the polynucleotide of (B6) has immunogenicity against HTLV-1. The "one or several" in (B6) may be, for example, 1 to 38, 1 to 32, 1 to 25, 1 to 19, 1 to 12, 1 to 6, 1 to 5, 1 to 3, 1 or 2, or 1 in the amino acid sequence of (B5).

[0130] In (B7), the "identity" may be within a range such that the protein encoded by the polynucleotide of (B7) has immunogenicity against HTLV-1. The "identity" in (B7) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more relative to the amino acid sequence of (B5).

[0131] The nucleic acid encoding Gag p24 is, for example, a polynucleotide selected from the group consisting of (C1) to (C6) and (C7) below. (C1) A polynucleotide consisting of the base sequence of SEQ ID NO: 16; (C2) A polynucleotide consisting of a base sequence in which one or more bases have been deleted, inserted, substituted, and / or added in the base sequence of (C1); (C3) A polynucleotide consisting of a base sequence that is 80% or more identical to any of the base sequences of (C1); (C4) A polynucleotide consisting of a base sequence that is complementary to a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of any of the base sequences of (C1); (C5) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 12; (C6) A polynucleotide encoding a protein consisting of an amino acid sequence in which one or more amino acids have been deleted, inserted, substituted, and / or added in the amino acid sequence of (C5); (C7) A polynucleotide encoding a protein consisting of an amino acid sequence that is 80% or more identical to the amino acid sequence of (C5).

[0132] In the polynucleotide (C1), the nucleotide sequence of SEQ ID NO: 16 is as follows: The nucleotide sequence of SEQ ID NO: 16 is a coding sequence for Gag p24 consisting of the amino acid sequence of SEQ ID NO: 12. The polynucleotide (C1) of SEQ ID NO: 16 can be obtained from, for example, HTLV-1.

[0133] Polynucleotide of (C1) (SEQ ID NO: 16) 5'--3'

[0134] In (C2), the "one or several" may be within a range in which the protein encoded by the polynucleotide of (C2) has immunogenicity against HTLV-1. The "one or several" in (C2) may be, for example, 1 to 192, 1 to 160, 1 to 128, 1 to 96, 1 ​​to 64, 1 to 32, 1 to 25, 1 to 19, 1 to 12, 1 to 6, or 1 or 2 in the base sequence of (C1).

[0135] In (C3), the "identity" may be within a range such that the protein encoded by the polynucleotide of (C3) has immunogenicity against HTLV-1. The "identity" in (C3) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more relative to the base sequence of (C1).

[0136] In (C4), the "hybridizing polynucleotide" is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide of (C1). The hybridization can be detected, for example, by various hybridization assays. The hybridization assay is not particularly limited, and for example, the method described in "Molecular Cloning: A Laboratory Manual 2nd Ed." edited by Sambrook et al. [Cold Spring Harbor Laboratory Press (1989)] can be used. In (C4), the stringent conditions can be the same as those described in (A4).

[0137] The polynucleotide (C5) can be designed, for example, based on the amino acid sequence of SEQ ID NO: 12, by substituting the corresponding codons.

[0138] In (C6), the "one or several" may be within a range in which the protein encoded by the polynucleotide of (C6) has immunogenicity against HTLV-1. The "one or several" in (C6) may be, for example, 1 to 64, 1 to 53, 1 to 42, 1 to 32, 1 to 21, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 or 2, or 1 in the amino acid sequence of (C5).

[0139] In (C7), the "identity" may be within a range such that the protein encoded by the polynucleotide of (C7) has immunogenicity against HTLV-1. The "identity" in (C7) with respect to the amino acid sequence of (C5) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0140] In the present disclosure, the nucleic acid of the immunogenic fragment of p15, p19, or p24 may be, for example, within the scope of the protein encoded by the polynucleotide having immunogenicity against HTLV-1.

[0141] The nucleic acid-containing pharmaceutical composition of the present disclosure may contain, for example, a nucleic acid encoding any one, more than one, or all of the Gag p15 or an immunogenic fragment thereof, the Gag p19 or an immunogenic fragment thereof, and the Gag p24 or an immunogenic fragment thereof. The nucleic acid-containing pharmaceutical composition of the present disclosure preferably contains, for example, a nucleic acid encoding the Gag p15 or an immunogenic fragment thereof, a nucleic acid encoding the Gag p19 or an immunogenic fragment thereof, and a nucleic acid encoding the Gag p24 or an immunogenic fragment thereof. By adopting such a configuration, the nucleic acid-containing pharmaceutical composition of the present disclosure can, for example, efficiently induce an immune response against HTLV-1.

[0142] The nucleic acid-containing pharmaceutical composition of the present disclosure may contain a nucleic acid encoding the unprocessed Gag protein, or an immunogenic fragment thereof, as the Gag p15, Gag p19, and / or Gag p24.

[0143] The nucleic acid encoding the unprocessed Gag may be, for example, a polynucleotide selected from the group consisting of (D1) to (D6) and (D7) below. (D1) A polynucleotide consisting of the base sequence of SEQ ID NO: 2; (D2) A polynucleotide consisting of a base sequence in which one or more bases have been deleted, inserted, substituted, and / or added in the base sequence of (D1); (D3) A polynucleotide consisting of a base sequence that is 80% or more identical to any of the base sequences of (D1); (D4) A polynucleotide consisting of a base sequence that is complementary to a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of any of the base sequences of (D1); (D5) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1; (D6) A polynucleotide encoding a protein consisting of an amino acid sequence in which one or more amino acids have been deleted, inserted, substituted, and / or added in the amino acid sequence of (D5); (D7) A polynucleotide encoding a protein consisting of an amino acid sequence that is 80% or more identical to the amino acid sequence of (D5).

[0144] In the polynucleotide (D1), the nucleotide sequence of SEQ ID NO: 2 is as follows: The nucleotide sequence of SEQ ID NO: 2 is a coding sequence for unprocessed Gag consisting of the amino acid sequence of SEQ ID NO: 1. The polynucleotide (D1) of SEQ ID NO: 2 can be obtained from, for example, HTLV-1.

[0145]

[0146] In (D2), the "one or several" may be within a range in which the protein encoded by the polynucleotide of (D2) has immunogenicity against HTLV-1. The "one or several" in (D2) may be, for example, 1 to 387, 1 to 322, 1 to 258, 1 to 192, 1 to 129, 1 to 64, 1 to 51, 1 to 42, 1 to 38, 1 to 25, 1 to 12, 1 to 6, 1 to 3, 1 or 2, or 1 in the base sequence of (D1).

[0147] In (D3), the "identity" may be within a range such that the protein encoded by the polynucleotide of (D3) has immunogenicity against HTLV-1. The "identity" in (D3) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more relative to the base sequence of (D1).

[0148] In (D4), the "hybridizing polynucleotide" is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide of (D1). The hybridization can be detected, for example, by various hybridization assays. The hybridization assay is not particularly limited, and for example, the method described in "Molecular Cloning: A Laboratory Manual 2nd Ed." edited by Sambrook et al. [Cold Spring Harbor Laboratory Press (1989)] can be used. In (D4), the stringent conditions can be the same as those described in (A4).

[0149] The polynucleotide (D5) can be designed, for example, based on the amino acid sequence of SEQ ID NO: 1, by substituting the corresponding codons.

[0150] In (D6), the "one or several" may be within a range in which the protein encoded by the polynucleotide of (D6) has immunogenicity against HTLV-1. The "one or several" in (D6) may be, for example, 1 to 128, 1 to 107, 1 to 85, 1 to 64, 1 to 42, 1 to 21, 1 to 14, 1 to 12, 1 to 8, 1 to 4, 1 or 2, or 1 in the amino acid sequence of (D5).

[0151] In (D7), the "identity" may be within a range such that the protein encoded by the polynucleotide of (D7) has immunogenicity against HTLV-1. The "identity" of (D7) to the amino acid sequence of (D5) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0152] In the present disclosure, the nucleic acid of the immunogenic fragment of pre-processed Gag may be, for example, within the range in which the protein encoded by the polynucleotide has immunogenicity against HTLV-1.

[0153] The nucleic acid-containing pharmaceutical composition of the present disclosure may further comprise a nucleic acid encoding the Tax protein or an immunogenic fragment thereof and / or the HBZ protein or an immunogenic fragment thereof of HTLV-1. For example, by further comprising a nucleic acid encoding the Tax or an immunogenic fragment thereof and / or a nucleic acid encoding the HBZ protein or an immunogenic fragment thereof, the nucleic acid-containing pharmaceutical composition of the present disclosure can more efficiently induce an immune response against HTLV-1.

[0154] The nucleic acid encoding Tax may be, for example, a polynucleotide selected from the group consisting of (E1) to (E6) and (E7) below. (E1) A polynucleotide consisting of the base sequence of SEQ ID NO: 4; (E2) A polynucleotide consisting of a base sequence in which one or more bases have been deleted, inserted, substituted, and / or added in the base sequence of (E1); (E3) A polynucleotide consisting of a base sequence having 80% or more identity to any of the base sequences of (E1); (E4) A polynucleotide consisting of a base sequence complementary to a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of any of the base sequences of (E1); (E5) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 3; (E6) A polynucleotide encoding a protein consisting of an amino acid sequence in which one or more amino acids have been deleted, inserted, substituted, and / or added in the amino acid sequence of (E5); (E7) A polynucleotide encoding a protein consisting of an amino acid sequence having 80% or more identity to the amino acid sequence of (E5).

[0155] In the polynucleotide (E1), the nucleotide sequence of SEQ ID NO: 4 is as follows: The nucleotide sequence of SEQ ID NO: 4 is a coding sequence for Tax consisting of the amino acid sequence of SEQ ID NO: 3. The polynucleotide (E1) of SEQ ID NO: 4 can be obtained from, for example, HTLV-1.

[0156]

[0157] In (E2), the "one or several" may be within a range in which the protein encoded by the polynucleotide of (E2) has immunogenicity against HTLV-1. The "one or several" in (E2) may be, for example, 1 to 318, 1 to 265, 1 to 212, 1 to 159, 1 to 106, 1 to 53, 1 to 42, 1 to 31, 1 to 21, 1 to 10, 1 to 6, 1 or 2, or 1 in the base sequence of (E1).

[0158] In (E3), the "identity" may be within a range such that the protein encoded by the polynucleotide of (E3) has immunogenicity against HTLV-1. The "identity" in (E3) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more relative to the base sequence of (E1).

[0159] In (E4), the "hybridizing polynucleotide" is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide of (E1). The hybridization can be detected, for example, by various hybridization assays. The hybridization assay is not particularly limited, and for example, the method described in "Molecular Cloning: A Laboratory Manual 2nd Ed." edited by Sambrook et al. [Cold Spring Harbor Laboratory Press (1989)] can be used. In (E4), the stringent conditions can be the same as those described in (A4).

[0160] The polynucleotide (E5) can be designed, for example, based on the amino acid sequence of SEQ ID NO: 3, by substituting the corresponding codons.

[0161] In (E6), the "one or several" may be within a range in which the protein encoded by the polynucleotide of (E6) has immunogenicity against HTLV-1. The "one or several" in (E6) may be, for example, 1 to 105, 1 to 88, 1 to 70, 1 to 52, 1 to 35, 1 to 17, 1 to 14, 1 to 10, 1 to 7, 1 to 3, 1 or 2, or 1 in the amino acid sequence of (E5).

[0162] In (E7), the "identity" may be within a range such that the protein encoded by the polynucleotide of (E7) has immunogenicity against HTLV-1. The "identity" of (E7) to the amino acid sequence of (E5) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0163] The nucleic acid encoding HBZ is, for example, a polynucleotide selected from the group consisting of (F1) to (F6) and (F7) below. (F1) A polynucleotide consisting of the base sequence of SEQ ID NO: 6 or 8; (F2) A polynucleotide consisting of a base sequence in which one or more bases have been deleted, inserted, substituted, and / or added in the base sequence of (F1); (F3) A polynucleotide consisting of a base sequence that is 80% or more identical to any of the base sequences of (F1); (F4) A polynucleotide consisting of a base sequence that is complementary to a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of any of the base sequences of (F1); (F5) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 5 or 7; (F6) A polynucleotide encoding a protein consisting of an amino acid sequence in which one or more amino acids have been deleted, inserted, substituted, and / or added in the amino acid sequence of (F5); (F7) A polynucleotide encoding a protein consisting of an amino acid sequence that is 80% or more identical to the amino acid sequence of (F5).

[0164] In (F1), the nucleotide sequence of SEQ ID NO: 6 or 8 is as follows: The nucleotide sequence of SEQ ID NO: 6 or 8 is a coding sequence for HBZ consisting of the amino acid sequence of SEQ ID NO: 5 or 7. The polynucleotide (F1) of SEQ ID NO: 6 or 8 can be obtained from, for example, HTLV-1.

[0165] Polynucleotide of (F1) (SEQ ID NO: 6) 5'--3'

[0166] Polynucleotide of (F1) (SEQ ID NO: 8) 5'--3'

[0167] In (F2), the "one or several" may be within a range in which the protein encoded by the polynucleotide of (F2) has immunogenicity against HTLV-1. The "one or several" in (F2) may be, for example, 1 to 186, 1 to 156, 1 to 123, 1 to 93, 1 to 60, 1 to 30, 1 to 24, 1 to 18, 1 to 12, 1 to 6, 1 to 3, 1 or 2, or 1 in the base sequence of (F1).

[0168] In (F3), the "identity" may be within a range such that the protein encoded by the polynucleotide of (F3) has immunogenicity against HTLV-1. The "identity" in (F3) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more relative to the base sequence of (F1).

[0169] In (F4), the "hybridizing polynucleotide" is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide of (F1). The hybridization can be detected, for example, by various hybridization assays. The hybridization assay is not particularly limited, and for example, the method described in "Molecular Cloning: A Laboratory Manual 2nd Ed." edited by Sambrook et al. [Cold Spring Harbor Laboratory Press (1989)] can be used. In (F4), the stringent conditions can be the same as those described in (A4).

[0170] The polynucleotide (F5) can be designed, for example, based on the amino acid sequence of SEQ ID NO: 5 or 7 by substituting the corresponding codons.

[0171] In (F6), the "one or several" may be within a range in which the protein encoded by the polynucleotide of (F6) has immunogenicity against HTLV-1. The "one or several" in (F6) may be, for example, 1 to 62, 1 to 52, 1 to 41, 1 to 31, 1 to 20, 1 to 10, 1 to 8, 1 to 6, 1 to 4, or 1 or 2 amino acids in the amino acid sequence of (F5).

[0172] In (F7), the "identity" may be within a range such that the protein encoded by the polynucleotide of (F7) has immunogenicity against HTLV-1. The "identity" of (F7) with respect to the amino acid sequence of (F5) is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0173] In the present disclosure, the nucleic acid of the immunogenic fragment of Tax or HBZ may be, for example, within the scope of a protein encoded by the polynucleotide, which has immunogenicity against HTLV-1.

[0174] The nucleic acid may be composed of deoxynucleotide residues, ribonucleotide residues, or both. The nucleic acid may also be composed of natural nucleic acid residues, unnatural nucleic acid residues, or both. Specific examples of the nucleic acid include DNA, RNA, and / or DNA / RNA composed of natural and / or unnatural nucleic acid residues. Examples of the unnatural nucleic acid residue include modified nucleotide residues or modified ribonucleotide residues in which the base, sugar residue, or sugar phosphate backbone of the nucleotide residue is modified. When the sugar residue is modified, examples of the unnatural nucleic acid residue include cEt (constrained ethyl bicyclic nucleic acid, manufactured by Ionis Pharmaceuticals), LNA (trademark, Locked Nucleic Acid), and ENA (registered trademark, 2'-O,4'-C-Ethylenebridged Nucleic Acid). The nucleic acid may have, for example, a 5' cap at the 5' end.

[0175] The nucleic acid may be a single-stranded or double-stranded nucleic acid molecule.

[0176] In the nucleic acid-containing pharmaceutical composition of the present disclosure, the nucleic acid encoding each protein can be designed, for example, by substituting corresponding codons based on the amino acid sequence of each protein. In the pharmaceutical composition of the present disclosure, the base sequence of the nucleic acid may be, for example, codon-optimized.

[0177] In the present disclosure, various nucleic acids and proteins can be synthesized, for example, by genetic engineering techniques or organic synthesis techniques, and can also be referred to as synthetic DNA such as cDNA, or synthetic RNA.

[0178] In the present disclosure, the nucleic acid is preferably a modified nucleic acid. Specifically, when the nucleic acid is RNA, some or all of the uridines in the RNA are preferably pseudouridines.

[0179] In the present disclosure, when the nucleic acid is mRNA, the nucleic acid may include a 5' cap structure, a 5'-UTR, a 3'-UTR, a polyA sequence, etc. at its end. In the present disclosure, the nucleic acid may form a complex with, for example, a lipid complex (lipid nanoparticle).

[0180] <Vector-Containing Pharmaceutical Composition> In another aspect, the present disclosure provides a pharmaceutical composition capable of inducing an immune response against HTLV-1, particularly against HTLV-1 Gag. The pharmaceutical composition (vector-containing pharmaceutical composition) of the present disclosure comprises a vector containing a nucleic acid encoding human T-cell leukemia virus 1 (HTLV-1) antigenic Gag protein p15 (Gag p15) or an immunogenic fragment thereof, Gag protein p19 (Gag p19) or an immunogenic fragment thereof, and / or Gag protein p24 (Gag p24) or an immunogenic fragment thereof, and a pharmaceutically acceptable carrier.

[0181] The vector-containing pharmaceutical composition of the present disclosure is characterized by comprising, as an active ingredient, a vector comprising a nucleic acid encoding the HTLV-1 antigen Gag protein p15 (Gag p15) or an immunogenic fragment thereof, Gag protein p19 (Gag p19) or an immunogenic fragment thereof, and / or Gag protein p24 (Gag p24) or an immunogenic fragment thereof, and other configurations and conditions are not particularly limited. Because the vector-containing pharmaceutical composition of the present disclosure comprises, as an active ingredient, a vector comprising a nucleic acid encoding the HTLV-1 antigen Gag protein p15 (Gag p15) or an immunogenic fragment thereof, Gag protein p19 (Gag p19) or an immunogenic fragment thereof, and / or Gag protein p24 (Gag p24) or an immunogenic fragment thereof, when used as a pharmaceutical composition, it can induce an immune response against HTLV-1. Unless otherwise specified, the vector-containing pharmaceutical composition of the present disclosure can be applied to the explanations of the pharmaceutical composition containing the Gag antigen of HTLV-1 and the nucleic acid-containing pharmaceutical composition.

[0182] In the vector-containing pharmaceutical composition of the present disclosure, the vector has inserted therein, for example, a nucleic acid encoding Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and / or Gag protein p24 (Gag p24) or an immunogenic fragment thereof. The vector can also be referred to as, for example, a vector in which the nucleic acid is operably linked. The vector refers to, for example, a nucleic acid molecule capable of transporting an inserted gene into a target, such as a cell.

[0183] The vector is not particularly limited in its configuration, as long as it contains a polynucleotide encoding a protein so that the protein encoded by the polynucleotide of the nucleic acid can be expressed.

[0184] The vector can be prepared, for example, by inserting a polynucleotide encoding a protein that is an active ingredient in the pharmaceutical composition of the present disclosure, i.e., a nucleic acid encoding Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and / or Gag protein p24 (Gag p24) or an immunogenic fragment thereof, into a backbone vector (hereinafter also referred to as a "basic vector"). The type of the vector is not particularly limited and can be appropriately determined depending on, for example, the type of subject to be administered.

[0185] The vector may, for example, further comprise a nucleic acid encoding the Tax protein of HTLV-1 or an immunogenic fragment thereof, and / or the HBZ protein or an immunogenic fragment thereof.

[0186] When the pharmaceutical composition of the present disclosure contains multiple proteins as active ingredients, the vector may contain nucleic acids encoding multiple proteins, or multiple vectors may each contain nucleic acids encoding one or multiple proteins.

[0187] Examples of the vector include viral vectors and non-viral vectors. Examples of the viral vector include baculovirus; poxviruses such as vaccinia virus, avipox virus, canarypox virus, fowlpox virus, raccoon pox virus, and swinepox virus; adenoviruses such as canine adenovirus; adeno-associated viruses; herpes viruses; and retroviruses. When the heat shock method is used to transform the host, examples of the vector include binary vectors. Examples of the vector include pETDuet-1, pQE-80L, and pUCP26Km. When transforming bacteria such as Escherichia coli, examples of the vector include pETDuet-1 vector (Novagen), pQE-80L (QIAGEN), pBR322, pB325, pAT153, and pUC8. When transforming yeast, examples of the vector include pYepSec1, pMFa, pYES2, etc. When transforming insect cells, examples of the vector include pAc, pVL, etc. When transforming mammalian cells, examples of the vector include pCDM8, pMT2PC, etc.

[0188] The vector preferably has a regulatory sequence that regulates, for example, expression of a polynucleotide encoding Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and / or Gag p24 or an immunogenic fragment thereof of the present disclosure, and expression of Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and / or Gag p24 or an immunogenic fragment thereof encoded by the polynucleotide encoding Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and / or Gag p24 or an immunogenic fragment thereof of the present disclosure. Examples of the regulatory sequence include a promoter, a terminator, an enhancer, a polyadenylation signal sequence, and an origin of replication (ori). The location of the regulatory sequence in the vector is not particularly limited. In the vector, the regulatory sequence may be arranged in a manner known in the art so long as it is capable of functionally regulating the expression of the polynucleotide encoding Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and / or Gag p24 or an immunogenic fragment thereof of the present disclosure, and the expression of Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and / or Gag p24 or an immunogenic fragment thereof encoded thereby. For example, the regulatory sequence may be a sequence already present in the vector, or an additional regulatory sequence may be inserted into the vector, or the regulatory sequence present in the vector may be replaced with another regulatory sequence.

[0189] The vector may be used, for example, to produce a protein in the pharmaceutical composition of the present disclosure. In this case, the protein can be produced by introducing the vector into a host and expressing the protein encoded by the vector.

[0190] The type of vector is not particularly limited and can be appropriately determined depending on, for example, the type of host. Specifically, when the vector is synthesized by genetic engineering techniques, the vector is first synthesized by, for example, designing and synthesizing a nucleic acid encoding Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and / or Gag p24 or an immunogenic fragment thereof of the present disclosure. The design and synthesis can be performed, for example, by PCR using a vector or the like containing a nucleic acid encoding Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and / or Gag p24 or an immunogenic fragment thereof of the present disclosure as a template and primers designed to synthesize a desired nucleic acid region. The resulting nucleic acid is then ligated to an appropriate vector to obtain a recombinant vector for protein expression, and this recombinant vector is then introduced into a host so that the target gene can be expressed to obtain a transformant (Sambrook J. et al., Molecular Cloning, A Laboratory Manual (4th edition) (Cold Spring Harbor Laboratory Press (2012))).

[0191] The host used for transformation is not particularly limited as long as it can express the gene of interest, and examples thereof include non-human hosts such as microorganisms, animal cells, insect cells, or cultured cells thereof, isolated human cells or cultured cells thereof, and mammalian cells. Examples of the prokaryotic organisms include bacteria such as Escherichia genus bacteria such as Escherichia coli and Pseudomonas genus bacteria such as Pseudomonas putida. Examples of the eukaryotic organisms include yeasts such as Saccharomyces cerevisiae. Examples of the animal cells include COS cells and CHO cells, and examples of the insect cells include Sf9 and Sf21.

[0192] The method for introducing the vector into the host is not particularly limited and can be performed by a known method. The introduction method can be appropriately selected depending on, for example, the type of the host. Examples of the introduction method include introduction using a gene gun such as a particle gun, the calcium phosphate method, the polyethylene glycol method, lipofection using liposomes, electroporation, ultrasonic nucleic acid transfer, DEAE-dextran, direct injection using glass microtubes, the hydrodynamic method, the cationic liposome method, methods using introduction adjuvants, methods mediated by Agrobacterium, and the protoplast method. Examples of the liposome include lipofectamine and cationic liposomes, and examples of the introduction adjuvants include atelocollagen, nanoparticles, and polymers. When the host is a microorganism, a method mediated by E. coli or Ps. putida is preferred. The Gag p15 or immunogenic fragment thereof, Gag p19 or immunogenic fragment thereof, and / or Gag p24 or immunogenic fragment thereof polynucleotides of the present disclosure may be introduced into the host, for example, via a vector of the present disclosure.

[0193] In the production method of the present disclosure, for example, the transformant is cultured, and Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and / or Gag p24 or an immunogenic fragment thereof of the present disclosure is collected from the culture. The culture may be a culture supernatant, or a transformant such as cultured cells or cultured bacterial cells, or a processed or disrupted product thereof.

[0194] After the culture, if the Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and / or Gag p24 or an immunogenic fragment thereof of the present disclosure is produced within the host, the production method of the present disclosure involves, for example, disrupting the host to extract Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and / or Gag p24 or an immunogenic fragment thereof.Furthermore, if the Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and / or Gag p24 or an immunogenic fragment thereof of the present disclosure is produced or secreted outside the host, the production method of the present disclosure involves, for example, using the culture medium as is or removing the host by centrifugation or the like. Thereafter, the production method of the present disclosure can isolate or purify proteins such as Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and / or Gag p24 or an immunogenic fragment thereof by using, for example, a general biochemical method used for isolating and purifying proteins, specifically, concentration using an ultrafiltration membrane; salting out such as ammonium sulfate precipitation; chromatography using various columns such as gel filtration, ion exchange chromatography, and affinity chromatography, either alone or in appropriate combination.

[0195] <Preventive Method> In another aspect, the present disclosure provides a method for treating and / or preventing an HTLV-1-associated disease, or for preventing the onset, aggravation, or recurrence of ATL. The presently disclosed method for treating and / or preventing an HTLV-1-associated disease, or for preventing the onset, aggravation, or recurrence of ATL, is characterized by administering to a subject a pharmaceutical composition, nucleic acid-containing pharmaceutical composition, and / or vector-containing pharmaceutical composition comprising an HTLV-1 Gag antigen of the present disclosure; other configurations and conditions are not particularly limited. The preventive method of the present disclosure comprises, as an active ingredient, Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and / or Gag p24 or an immunogenic fragment thereof, or a nucleic acid encoding the same, and therefore may be able to induce an immune response against HTLV-1 when administered to a subject. The prevention method of the present disclosure can be applied to, for example, the explanations of the pharmaceutical composition containing the HTLV-1 Gag antigen, the nucleic acid-containing pharmaceutical composition, and the vector-containing pharmaceutical composition of the present disclosure.

[0196] The preventive method of the present disclosure can induce, for example, an immune response against HTLV-1, and therefore can also be referred to as, for example, a vaccination method against HTLV-1, a method for inducing protective immunity, a method for stimulating an immune response, or a method for inducing an immune response. Furthermore, the preventive method of the present disclosure can also be referred to as, for example, the prevention and / or treatment of an HTLV-1-associated disease.

[0197] In the preventive method of the present disclosure, the active ingredient is administered to a subject to induce an immune response in the subject, thereby conferring immunity to HTLV-1 in the subject. Thus, the preventive method of the present disclosure includes, for example, a step (administration step) of administering to the subject a pharmaceutical composition, nucleic acid-containing pharmaceutical composition, and / or vector-containing pharmaceutical composition comprising an HTLV-1 Gag antigen of the present disclosure that includes the active ingredient. As a result, in the preventive method of the present disclosure, for example, an immune response is induced in the subject against Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and / or Gag p24 or an immunogenic fragment thereof, and a cellular immune response by T cells or the like is generated against Gag p15 or an immunogenic fragment thereof, Gag p19 or an immunogenic fragment thereof, and / or Gag p24 or an immunogenic fragment thereof.

[0198] In the prevention method of the present disclosure, the administration step is carried out once or multiple times. The administration conditions in the administration step can be referenced from the explanation of the pharmaceutical composition of the present disclosure.

[0199] <Use> In another aspect, the present disclosure relates to use of a pharmaceutical composition, nucleic acid-containing pharmaceutical composition, and / or vector-containing pharmaceutical composition comprising an HTLV-1 Gag antigen of the present disclosure for use in a method for preventing the onset of ATL, the aggravation of ATL, or the recurrence of ATL. The present disclosure relates to use of a pharmaceutical composition, nucleic acid-containing pharmaceutical composition, and / or vector-containing pharmaceutical composition comprising an HTLV-1 Gag antigen of the present disclosure for use in the manufacture of a vaccine for use in suppressing the onset of ATL, the aggravation of ATL, or the recurrence of ATL. The use of the pharmaceutical composition, nucleic acid-containing pharmaceutical composition, and / or vector-containing pharmaceutical composition comprising an HTLV-1 Gag antigen of the present disclosure can be explained by reference to, for example, the descriptions of the pharmaceutical composition, nucleic acid-containing pharmaceutical composition, vector-containing pharmaceutical composition, and method for prevention comprising an HTLV-1 Gag antigen of the present disclosure.

[0200] The present disclosure will be described in detail below using examples, but the present disclosure is not limited to the embodiments described in the examples. Unless otherwise specified, commercially available reagents, kits, etc. were used according to their protocols.

[0201] Example 1 It was confirmed that Gag p15, Gag p19 and / or Gag p24, which are active ingredients of the pharmaceutical composition of the present disclosure, can induce an immune response against HTLV-1.

[0202] (1) Study of HTLV-1 Antigens. Antibody responses to HTLV-1 antigens were examined using serum or plasma from HTLV-1-infected individuals, ATL patients, and other individuals. Specifically, luciferase immunoprecipitation (LIPS assay) was performed. Serum or plasma was obtained from HTLV-1-infected individuals, ATL patients, HAM / TSP patients, and HTLV-1-uninfected individuals. All clinical samples were collected after obtaining written informed consent in accordance with the Declaration of Helsinki. The experiments described in the Examples were approved by the Institutional Ethics Committee of Kumamoto University (Accession Nos. G489, G499, and E2214). The antigens used were Tax (SEQ ID NO: 17), Env (SEQ ID NO: 18), Gag p15 (SEQ ID NO: 19), Gag p19 (SEQ ID NO: 20), and Gag p24 (SEQ ID NO: 21). As the antigen, vectors expressing fusion proteins of various proteins and NanoLuc were prepared, and after the preparation, the vectors were introduced into the 293T cell line, and the cell lysate of the cells was used. ７A count of antigen, LIPS buffer, and 1 μl of the serum or plasma were mixed to a total volume of 100 μl. After the preparation, the plate was incubated at room temperature (hereinafter, approximately 24°C) for 30 minutes using a plate mixer. After the incubation, Protein A / G magnetic beads (Thermo Fisher Scientific) were washed 3 to 5 times with LIPS buffer using a magnetic plate. After the washing, the magnetic beads were resuspended in LIPS buffer. After the resuspension, 10 μl of the magnetic beads were added to each well of the plate. After the addition, the plate was incubated at room temperature for 30 minutes using a plate mixer. After the incubation, to concentrate immune complexes consisting of antibodies and NanoLuc-viral antigen fusion proteins in the patient samples, the plate was placed on a magnetic plate, and the pellets in each well were washed five times with 150 to 200 μl of LIPS buffer. After washing, the LIPS buffer used for washing was removed, and 100 μl of Nano-Glo luciferase assay substrate (Promega) was added to each well. After the addition, luminescence was measured using a luminometer (Promega). The results are shown in FIG. 1.

[0203] Amino acid sequence of Tax (SEQ ID NO: 17) MAHFPGFGQSLLFGYPVYVFGDCVQGDWCPISGGLCSARLHRHALLATCPEHQITWDPIDGRVIGSALQFLIPRLPSFPTQRTSKTLKVLTPPITHTTPNIPPSFLQAMRKYSPFRNGYMEPTLGQHLPTLSFPDPGLRPQNLYTLWGGSVVCMYLYQLSPPITWPLLPHVIFCHPGQLGAFLTNVPYKRIEELLYKISLTTGALIILPEDCLPTTLFQPARAPVTLTAWQNGLLPFHSTLTTPGLIWTFTDGTPMISGPCPKDGQPSLVLQSSSFIFHKFQTKAYHPSFLLSHGLIQYSSFHSLHLLFEEYTNIPISLLFNEKEADDNDHEPQISPGGLEPPSEKHFRETEV

[0204] Amino acid sequence of Env (SEQ ID NO: 18) MGKFLATLILFFQFCPLIFGDYSPSCCTLTIGVSSYHSKPCNPAQPVCSWTLDLLALSADQALQPPCPNLVSYSSYHATYSLYLFPHWIKKPNRNGGGYYSASYSDPCSLKCPYLGCQSWTCPYTGAVSSPYWKFQHDVNFTQEVSRLNINLHFSKCGFPFSLLVDAPGYDPIWFLNTEPSQLPPTAPPLLPHSNLDHILEPSIPWKSKLLTLVQLTLQSTNYTCIVCIDRASLSTWHVLYSPN VSVPSSSSTPLLYPSLALPAPHLTLPFNWTHCFDPQIQAIVSSPCHNSLILPPFSLSPVPTLGSRSRRAVPVAVWLVSALAMGAGVAGGITGSMSLASGKSLLHEVDKDISQLTQAIVKNHK NLLKIAQYAAQNRRGLDLLFWEQGGLCKALQEQCCFLNITNSHVSILQERPPLENRVLTGWGLNWDLGLSQWAREALQTGITLVALLLLVILAGPCILRQLRHLPSRVRYPHYSLINPESL

[0205] Amino acid sequence of Gag p15 (SEQ ID NO: 19) VVQPKKPPPNQPCFRCGKAGHWSRDCTQPRPPPGPCPLCQDPTHWKRDCPRLKPTIPEPEPEEDALLLDLPADIPHPKNLHRGGGLTSPPTLQQVLPNQDPASIL

[0206] Amino acid sequence of Gag p19 (SEQ ID NO: 20) MGQIFSRSASPIPRPPRGLAAHHWLNFLQAAYRLEPGPSSYDFHQLKKFLKIALETPVWICPINYSLLASLLPKGYPGRVNEILHILIQTQAQIPSRPAPPPPSSPTHDPPDSDPQIPPPYVEPTAPQVL

[0207] Amino acid sequence of Gag p24 (SEQ ID NO: 21) PVMHPHGAPPNHRPWQMKDLQAIKQEVSQAAPGSPQFMQTIRLAVQQFDPTAKDLQDLLQYLCSSLVASLHHQQLDSLISEAETRGITGYNPLAGPLRVQANNPQQQGLRREYQQLWLAAFAALPGSAKDPSWASILQGLEEPYHAFVERLNIALDNGLPEGTPKDPILRSLAYSNANKECQKLLQARGHTNSPLGDMLRACQTWTPKDKTKVL

[0208] FIG. 1 is a graph showing the results of the LIPS assay. In FIG. 1, the horizontal axis indicates the type of subject, and the vertical axis indicates luminescence intensity. FIG. 1(A) shows the antibody response to Tax, FIG. 1(B) shows the antibody response to Env, FIG. 1(C) shows the antibody response to Gag p15, FIG. 1(D) shows the antibody response to Gag p19, and FIG. 1(E) shows the antibody response to Gag p24. As shown in FIG. 1, it was found that the antibody responses to Tax and Env were lower in ATL patients than in HTLV-1-infected patients. It was also found that the antibody responses to Gag p19 and Gag p24 were higher in ATL patients than in HTLV-1-infected patients.

[0209] (2) Study of Gag Expression In Example 1(1), the ATL patients showed low antibody responses to Tax and high antibody responses to Gag. Therefore, gene expression of Tax and Gag was studied. Specifically, quantitative reverse transcription PCR (RT-qPCR) was performed. For HTLV-1 cases, ATL cell lines, MT1 cells, MT4 cells, 55T(+) cells, ED cells, and TLOm1 cells, and an HTLV-1-negative T cell line, Jurkat cells, were used. For ATL cases, MT1 cells and serum collected from ATL patients (ATL-1, ATL-2, ATL-3, ATL-4, and ATL-5) were used. Total RNA was extracted from each cell line using TRIzol Reagent (Invitrogen). After extraction, cDNA was synthesized using SuperScript IV Reverse Transcriptase (Invitrogen). Gag cDNA was prepared using the following Gag cDNA synthesis primers. Tax cDNA and 18Sr RNA were synthesized using random primers. After synthesis, Gag expression was measured using Taqman real-time PCR. Tax expression was measured using SYBR-green real-time PCR. 18Sr RNA was quantified using Taqman Gene Expression Assays (Hs99999901_s1; Applied Biosystems). The amplification conditions were 50°C for 2 minutes, 95°C for 10 minutes, 95°C for 15 seconds, and 60°C for 1 minute, with each cycle consisting of 40 cycles. The results are shown in Figure 2.

[0210] Gag cDNA synthesis primer Primer 1 (SEQ ID NO: 22) 5'- TGCAGGATATGGGCC-3'. Gag primer set Primer 2 (SEQ ID NO: 23) 5'-AGTACCTTTGCTCCTCCCTC-3' Primer 3 (SEQ ID NO: 24) 5'-TAATACCTCGGGTTTCGGCC-3'. Gag probe Probe (SEQ ID NO: 25) 5'-TTCCCTCCATCACCAGCTAGATAGCCT-3'. Tax primer set Primer 4 (SEQ ID NO: 26) 5'-CCGCCGATCCCAAAGAA-3' Primer 5 (SEQ ID NO: 27) 5'-CCTGTCCAAACCCTGGGAA-3'

[0211] Figure 2 is a graph showing the results of RT-qPCR. In Figure 2, the horizontal axis indicates the type of sample, and the vertical axis indicates the relative mRNA expression level. Figure 2(A) shows the expression level of Gag mRNA in various cell lines, Figure 2(B) shows the expression level of Tax mRNA in various cell lines, Figure 2(C) shows the expression level of Gag mRNA in ATL case samples, and Figure 2(D) shows the expression level of Tax mRNA in ATL case samples. As shown in Figure 2, it was found that Gag was expressed in ATL cells and ATL case samples that did not express Tax. These results revealed the mechanism of Tax-independent Gag expression.

[0212] (3) Examination of Gag Expression in Tax-Nonexpressing Cells Next, we examined whether Gag expression was observed in cells that did not express Tax. Specifically, a proximity ligation assay (PLA) was performed. PLA was performed using the Duolink PLA kit (Sigma-Aldrich) according to the manufacturer's instructions. The cells used were ATL cell lines, MT4 cells, ATL-55T(+) cells, and ED cells, with Jurkat cells, a T cell line, used as a negative control. The cells were concentrated onto glass coverslips using a cytospin (Cytospin 2, Shandon). After concentration, the cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes. After fixation, the cells were permeabilized with 0.2% Triton® X-100 at room temperature for 15 minutes. After permeabilization, the cells were blocked with Duolink In Situ PLA Probe-Blocking Solution. After blocking, the cells were stained with antibodies. For staining, mouse anti-HTLV-1 p24 monoclonal antibody (clone 46 / 3.24.4, ZeptoMetrix), rabbit polyclonal antibody against HTLV-1 p24 Gag (Cat No. 5418, ABL), or mouse anti-HTLV-1 p19 Gag monoclonal antibody (Cat No. 0801003, ZeptoMetrix) were used. After staining, the glass coverslips were sealed with DAPI-containing In Situ Mounting Medium (Sigma-Aldrich). After sealing, the cells were observed using a Nikon C2 confocal microscope (Nikon). The results are shown in Figure 3.

[0213] Figure 3 is a photograph showing the results of a proximity ligation assay. Figure 3(A) shows the results for Gag p19 antibody. Figure 3(B) shows the results for Gag p24 antibody. In Figure 3, the upper row shows the results of DAPI staining, the middle row shows the results of Gag p19 or Gag p24 staining, and the lower row shows the results of DAPI and Gag p19 or Gag p24 staining (Merge). In Figure 3, the area surrounded by the dashed line indicated by the arrow indicates the expression of Gag p19 or Gag p24. As shown in Figure 3(A), it was found that Gag p19 was expressed not only in MT4 cells that express Tax, but also in ATL-55T(+) cells and ED cells that do not express Tax. Furthermore, as shown in Figure 3(B), Gag p24 was found to be expressed not only in MT4 cells that express Tax, but also in ATL-55T(+) cells and ED cells that do not express Tax. These results demonstrate that Gag p19 and p24 are expressed in ATL cells, regardless of the presence or absence of Tax expression.

[0214] (4) Study of cellular immune responses induced by Gag in ATL patients after hematopoietic stem cell transplantation Next, we investigated whether Gag induces cellular immune responses in ATL patients who have undergone hematopoietic stem cell transplantation and are maintaining long-term remission. Specifically, we used the ELISPOT assay. Based on the amino acid sequence of Gag (GenBank accession number AAA85841.1, SEQ ID NO: 1), overlapping peptides of 9 amino acids in length (offset: 1 amino acid) were designed. The Gag p19 peptide was designed starting from the second amino acid in SEQ ID NO: 1. The 97 peptides of Gag p15 were cloned into p15-1 (p15 １－５０ ), and p15-2 (p15 ５１－９７ ) and pooled as p19-1 (p19). １－５０ ), p19-2 (p19 ５１－１００ ), and p19-3 (p19 １０１－１２１) were pooled. Peripheral blood mononuclear cells (PBMCs) were collected from ATL patients who had undergone hematopoietic stem cell transplantation and maintained long-term remission. ELISPOT assays were performed using a human IFN-γ ELISPOT kit (MABTECH). The PBMCs were seeded onto ELISPOT plates. After seeding, 0.25 μmol / L of the pooled peptides and 1 μg / ml of purified NA / LE mouse anti-human CD28 antibody (Cat No. 555725, BD Biosciences) were added. After the addition, stimulation was carried out for 6 hours. Spots of IFN-γ-producing cells were developed using an AP Conjugate Kit (Bio-Rad) and counted using an ImmunoSpot S6 Analyzer (CTL). The results are shown in Figure 4.

[0215] Figure 4 is a photograph showing the results of the ELISPOT assay. In Figure 4, the left panel shows the results of stimulation with Gag p19 peptide. The upper and middle panels on the right panel show the results of stimulation with Gag p15 peptide, and the lower panel on the right panel shows the results of unstimulated stimulation. As shown in Figure 4, spots were formed when stimulated with Gag p19 or Gag p15 peptide. These results demonstrated that IFN-γ was produced by Gag p19 or Gag p15 in PBMCs from ATL patients who had undergone hematopoietic stem cell transplantation and maintained long-term remission. Furthermore, the production of IFN-γ suggested the induction of a cellular immune response.

[0216] (5) Study of Gag-induced cellular immune response in mice Next, we investigated whether a cellular immune response was induced in mice immunized with recombinant Gag protein. Specifically, we used an ELISPOT assay. Gag p15 peptides and Gag p19 peptides were designed and pooled in the same manner as in Example 1(4). Gag p24 peptides were designed in the same manner as in Example 1(4). 206 Gag p24 peptides were cloned into p24-1 (p24 １－５１ ), p24-2 (p24 ５２－１０３ ), p24-3 (p24 １０４－１５４), and p24-4 (p24 １５５－２０６ ) and pooled. Mice (C57BL / 6J, Jackson Laboratory Japan) were immunized with Gag protein, and 14 days later, splenocytes were collected from the mice. The ELISPOT assay was performed in the same manner as in Example 1(4), except that splenocytes collected from mice were used as samples.

[0217] Figure 5 is a photograph showing the results of the ELISPOT assay. In Figure 5, the upper row (A) shows the results for splenocytes from mouse 1, the middle row (B) shows the results for splenocytes from mouse 2, and the lower row (C) shows the results for splenocytes from mouse 3. In Figure 5, from left to right, two rows (Gag p15 1 and 2, respectively) and p15-1 (p15 １－５０ ) and p15-2 (p15 ５１－９７ )) shows stimulation with Gag p15, and 3 rows (Gag p19 3-5, respectively) show stimulation with Gag p19-1 (p19 １－５０ ), p19-2 (p19 ５１－１００ ), and p19-3 (p19 １０１－１２１ )) shows stimulation with Gag p19, and 4 (Gag p24 6-9, respectively, p24-1 (p24 １－５１ ), p24-2 (p24 ５２－１０３ ), p24-3 (p24 １０４－１５４ ), and p24-4 (p24 １５５－２０６ )) indicates stimulation with Gag p24, 1st row (10) indicates the positive control, and 1st row (11) indicates the negative control. As shown in Figure 5, spots were formed when stimulated with Gag p15, Gag p19, or Gag p24 peptides. In particular, Gag p19-1 (p19 １－５０ These results indicate that IFN-γ is produced by Gag p15, Gag p19, or Gag p24 in mice immunized with Gag proteins. Furthermore, the production of IFN-γ suggests that a cellular immune response is induced.

[0218] Example 2 (1) Preparation of Template DNA To prepare template plasmid DNA for use in in vitro transcription (IVT), a plasmid was synthesized having a DNA fragment in which the T7 promoter sequence (TAATACGACTCACTATA: SEQ ID NO: 28), 5'UTR sequence (AGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGA: SEQ ID NO: 29), KOZAK sequence (GCCACC: SEQ ID NO: 30), each antigen sequence (Gag antigen), and 3'UTR sequence (TAAGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGG: SEQ ID NO: 31) were linked in this order. 1 μg of the plasmid was dissolved in 210 μL of nuclease-free water, and then Q5 Hot Start High-Fidelity 2X Master Mix (250 μL, NEB# M0494L), 10 μM sense primer (20 μL), and 10 μM poly-T-containing antisense primer (20 μL) were added. The mixture was incubated at 95°C for 1 minute, followed by 35 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 3 minutes, followed by a further 5 minutes of incubation at 72°C to amplify the template DNA by PCR. The resulting mixture was purified using a NucleoSpin Gel and PCR Clean Up Kit (TaKaRa #U0609A) to obtain template DNA (SEQ ID NO: 32) bearing the antigen sequence of interest.

[0219]

[0220] (2) Preparation of mRNA Using each of the obtained template DNAs, mRNA was prepared by in vitro transcription (IVT). 300μg / mL template DNA (80μL), 100mM CleanCap AG (32μL, TriLink catalog #N-7113), 100mM ATP (40μL, TriLink catalog #N-1510), 100mM CTP (40μL, TriLink catalog #N-1511), 100mM GTP (40μL, TriLink catalog #N-1512), 100mM N1-methyl-ψ-Uridine-5'-Triphosphate (40μL), UltraPure DNase / RNase-Free Distilled Water (405.6 μL, Thermo Fisher catalog #10977015), T7 Transcription 10x buffer (80 μL), RNase inhibitor (20 μL, NEB catalog #M0314L), yeast inorganic pyrophosphatase (16 μL, NEB catalog #M2403L), and T7 RNA polymerase (6.4 μL, Roche catalog #08140669103) were mixed and incubated at 37°C for 3 hours. RNase-Free DNase I (24 μL, TaKaRa catalog #2270A) was added and incubated at 37°C for 30 minutes. 10x phosphatase buffer (96 μL, NEB catalog #B0289S) and Antarctic phosphatase (48 μL, NEB catalog #M0289L) were then added and incubated at 37°C for 30 minutes. The mixture was mixed with 8 M LiCl solution (484 μL, Sigma-Aldrich catalog #L7026), allowed to stand at -20°C for at least 1 hour, centrifuged (4°C, 15,000 rpm, 30 minutes), and the supernatant was discarded. 75% ethanol was then added, followed by centrifugation (4°C, 15,000 rpm, 5 minutes), and the supernatant was discarded. This procedure was repeated three times.The resulting precipitate was dissolved in nuclease-free water and purified using an RNeasy Maxi kit (Qiagen catalog #75162) according to the accompanying manual. Alternatively, the resulting precipitate was treated with RNase III (NEB, catalog #M0245S) for 10 minutes at room temperature, followed by purification on an Oligo dT column (Sartorius, catalog #311.1218-2). From the above, mRNA (SEQ ID NO:33) expressing a protein having the amino acid sequence (SEQ ID NO:1) was obtained. A cap structure and poly(A) sequence were added to each mRNA. The cap structure was the structure shown below, CleanCap (Cap1) (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0221]

[0222] <SEQ ID NO: 1> Amino acid sequence of Gag MGQIFSRSASPIPRPPRGLAAHHWLNFLQAAYRLEPGPSSYDFHQLKKFLKIALETPVWICPINYSLLASLLPKGYPGRVNEILHILIQTQAQIPSRPAPPPPSSPTHDPPDSDPQIPPPYVEPTAPQVLPVMHPHGAPPNHRPWQMKDLQAIKQEVSQAAPGSPQFMQTIRLAVQQFDPTAKDLQDLLQYLCSSLVASLHHQQLDSLISEAET RGITGYNPLAGPLRVQANNPQQQGLRREYQQLWLAAFAALPGSAKDPSWASILQGLEEPYHAFVERLNIALDNGLPEGTPKDPILRSLAYSNANKECQKLLQARGHT NSPLGDMLRACQTWTPKDKTKVLVVQPKKPPPNQPCFRCGKAGHWSRDCTQPRPPPGPCPLCQDPTHWKRDCPRLKPTIPEPEPEEDALLLDLPADIPHPKNSIGGEV

[0223] (3) Intravenous Immunization of Mice and Splenocyte Preparation Mouse test conditions were based on a previously published study (Molecular Therapy Nucleic Acids 2016, 5, e326). Specifically, lipid-mRNA particles were prepared by mixing Lipofectamin RNAiMAX Transfection Reagent (Invitrogen #56532) and mRNA for the Gag antigen (SEQ ID NO:1) (SEQ ID NO:33) in an RNAiMAX:mRNA ratio of 2:1 (wt:wt) in Opti-MEM (Gibco #31985062). The resulting lipid-mRNA particles were administered intravenously to C57BL / 6 mice (female, 10 weeks old, n=4) at doses of 0.2 mg / kg and 1 mg / kg mRNA on days 0 and 4. As a negative control, Opti-MEM was administered in the same manner. On day 10, mice were euthanized and their spleens were removed. The spleens were crushed on a cell strainer using a sterile syringe plunger and washed with 10 ml of RPMI to collect the cell suspension. The cell suspension was centrifuged, the supernatant was removed, and the cells were hemolyzed with 1 ml of NH4Cl for 2 minutes. The reaction was then stopped with 9 ml of RPMI and centrifuged. The cell pellet was resuspended in 20 ml of RPMI with 10% FBS.

[0224] (4) Analysis of cellular immune response The ability to induce cellular immune responses was evaluated by ELISpot assay. Peptides used for cell stimulation were designed and pooled using the following method. Based on the amino acid sequence of Gag, overlapping peptides of 9 amino acids in length (offset: 1 amino acid) were designed. 97 peptides from Gag p15, 121 peptides from Gag p19, and 206 peptides from Gag p24 were pooled to form a peptide pool. ELISpot assay was performed using MABTECH ELISpot Flex: IFN-gamma, Mouse-ALP according to the following procedure. A Millipore assay plate MAIPS4510 was treated with a coating antibody, and 10 mouse splenocytes were added. 6After 24 hours of stimulation with 0.25 μM of the above peptide pool, IFN-gamma-producing cells were stained with the detection antibody. The stained spots were analyzed using an ImmunoSpot S6 ENTRY Analyzer (CTL), and the number of spots was counted.

[0225] The results are shown in Figures 6 and 7. Figure 7 is a photograph showing the results of the ELISPOT assay, in which No Peptide represents the results when 0 mg / kg, 0.2 mg / kg, and 1.0 mg / kg of lipid-mRNA particles were administered as a negative control group (no peptide stimulation), and Gag Peptide pool represents the results when 0 mg / kg, 0.2 mg / kg, and 1.0 mg / kg of lipid-mRNA particles were administered. The first and second columns from the left in Figure 7C show the results for Samples 1 to 12 (Samples 1 to 4: saline, Samples 5 to 8: 0.2 mg / kg, Samples 9 to 12: 1 mg / kg) treated with no peptide (negative control group; no peptide stimulation). The third and fourth columns show the results for Samples 1 to 12 (Samples 1 to 4: saline, Samples 5 to 8: 0.2 mg / kg, Samples 9 to 12: 1 mg / kg) treated with Gag peptide. The fifth and sixth columns show the results for Samples 1 to 12 (Samples 1 to 4: saline, Samples 5 to 8: 0.2 mg / kg, Samples 9 to 12: 1 mg / kg) treated with Positive Control. Figure 6 shows the counts of the spots in Figure 7C for the no peptide and Gag peptide groups.

[0226] As shown in Figures 6 and 7, spots were formed in the Gag peptide pools (samples 5-8 and 9-12). These results demonstrated that IFN-γ was produced in mice immunized with lipid-mRNA particles encapsulating mRNA encoding Gag protein. Furthermore, the amount of IFN-γ produced increased with increasing concentrations of lipid-mRNA particles administered. The production of IFN-γ suggested the induction of a cellular immune response.

[0227] Although the present disclosure has been described above with reference to the embodiments, the present disclosure is not limited to the above embodiments. Various modifications that can be understood by those skilled in the art can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

[0228] The patents, patent applications, and publications cited herein are incorporated by reference into this specification in their entirety as if the contents were specifically set forth herein.

[0229] <Supplementary Notes> Some or all of the above embodiments and examples can be described as in the following supplementary notes, but are not limited to the following. <Pharmaceutical Compositions Comprising HTLV-1 Gag Antigen> (Supplementary Note 1) A pharmaceutical composition comprising human T-cell leukemia virus 1 (HTLV-1) antigenic Gag protein p15 (Gag p15) or an immunogenic fragment thereof, Gag protein p19 (Gag p19) or an immunogenic fragment thereof, and / or Gag protein p24 (Gag p24) or an immunogenic fragment thereof, and a pharmaceutically acceptable carrier. (Supplementary Note 2) The pharmaceutical composition according to Supplementary Note 1, comprising unprocessed Gag protein as the Gag p15, Gag p19, and / or Gag p24. (Appendix 3) The pharmaceutical composition according to Appendices 1 or 2, further comprising an HTLV-1 Tax protein or an immunogenic fragment thereof, and / or an HBZ protein or an immunogenic fragment thereof. (Appendix 4) The pharmaceutical composition according to any of Appendices 1 to 3, further comprising an adjuvant. (Appendix 5) The pharmaceutical composition according to any of Appendices 1 to 4, for use in treating and / or preventing an HTLV-1-associated disease, or for use in preventing the onset of ATL, the aggravation of ATL, or the recurrence of ATL. <Nucleic Acid-Containing Pharmaceutical Compositions> (Appendix 6) A pharmaceutical composition comprising a nucleic acid encoding human T-cell leukemia virus 1 (HTLV-1) antigenic Gag protein p15 (Gag p15) or an immunogenic fragment thereof, Gag protein p19 (Gag p19) or an immunogenic fragment thereof, and / or Gag protein p24 (Gag p24) or an immunogenic fragment thereof, and a pharmaceutically acceptable carrier. (Appendix 7) The pharmaceutical composition according to Appendix 6, comprising a nucleic acid encoding a pre-processed Gag protein as the Gag p15, the Gag p19, and / or the Gag p24. (Appendix 8) The pharmaceutical composition according to Appendix 6 or 7, further comprising a nucleic acid encoding an HTLV-1 Tax protein or an immunogenic fragment thereof, and / or an HBZ protein or an immunogenic fragment thereof. (Appendix 9) The pharmaceutical composition according to any of Appendixes 6 to 8, further comprising an adjuvant.(Appendix 10) The pharmaceutical composition according to any one of Appendices 6 to 9, for use in treating and / or preventing an HTLV-1-associated disease, or for use in preventing the onset of ATL, the aggravation of ATL, or the recurrence of ATL. <Vector-Containing Pharmaceutical Compositions> (Appendix 11) A pharmaceutical composition comprising a vector comprising a nucleic acid encoding human T-cell leukemia virus 1 (HTLV-1) antigenic Gag protein p15 (Gag p15) or an immunogenic fragment thereof, Gag protein p19 (Gag p19) or an immunogenic fragment thereof, and / or Gag protein p24 (Gag p24) or an immunogenic fragment thereof, and a pharmaceutically acceptable carrier. (Appendix 12) The pharmaceutical composition according to Appendices 11, comprising a vector comprising a nucleic acid encoding unprocessed Gag protein as the Gag p15, the Gag p19, and / or the Gag p24. (Appendix 13) The pharmaceutical composition according to Appendix 11 or 12, further comprising a vector comprising a nucleic acid encoding HTLV-1 Tax protein or an immunogenic fragment thereof, and / or HBZ protein or an immunogenic fragment thereof. (Appendix 14) The pharmaceutical composition according to any of Appendixes 11 to 13, further comprising an adjuvant. (Appendix 15) The pharmaceutical composition according to any of Appendixes 11 to 14, for use in treating and / or preventing an HTLV-1-associated disease, or for use in preventing the onset of ATL, aggravation of ATL, or ATL recurrence. <Preventive Method> (Appendix 16) A method for treating and / or preventing an HTLV-1-associated disease, or preventing the onset of ATL, aggravation of ATL, or ATL recurrence, using the pharmaceutical composition according to any of Appendixes 1 to 15. (Appendix 17) The preventive method according to Appendix 16, comprising an administration step of administering the pharmaceutical composition to a subject. <Use> (Appendix 18) The pharmaceutical composition according to any one of Appendices 1 to 17, for use in the treatment and / or prevention of an HTLV-1-associated disease, or for use in the prevention of the onset of ATL, the aggravation of ATL, or the recurrence of ATL.

[0230] As described above, the pharmaceutical composition of the present disclosure is capable of inducing an immune response against HTLV-1, for example. Therefore, the pharmaceutical composition of the present disclosure is expected to be able to prevent, for example, the onset or recurrence of HTLV-1-associated diseases. For this reason, the present disclosure is extremely useful, for example, in the field of medicine.

Claims

1. A pharmaceutical composition comprising human T-cell leukemia virus 1 (HTLV-1) antigenic Gag protein p15 (Gag p15) or an immunogenic fragment thereof, Gag protein p19 (Gag p19) or an immunogenic fragment thereof, or Gag protein p24 (Gag p24) or an immunogenic fragment thereof, and a pharmaceutically acceptable carrier.

2. The pharmaceutical composition of claim 1 , wherein the Gag p15, the Gag p19, or the Gag p24 comprises an unprocessed Gag protein.

3. The pharmaceutical composition according to claim 1 or 2, further comprising HTLV-1 Tax protein or an immunogenic fragment thereof, or HBZ protein or an immunogenic fragment thereof.

4. The pharmaceutical composition according to claim 1 or 2, further comprising an adjuvant.

5. The pharmaceutical composition according to claim 1 or 2, for use in preventing the onset, aggravation, or recurrence of ATL.

6. A pharmaceutical composition comprising a nucleic acid encoding human T-cell leukemia virus 1 (HTLV-1) antigenic Gag protein p15 (Gag p15) or an immunogenic fragment thereof, Gag protein p19 (Gag p19) or an immunogenic fragment thereof, or Gag protein p24 (Gag p24) or an immunogenic fragment thereof, and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition of claim 6 , comprising a nucleic acid encoding an unprocessed Gag protein as the Gag p15, the Gag p19, or the Gag p24.

8. The pharmaceutical composition according to claim 6 or 7, further comprising a nucleic acid encoding the Tax protein or an immunogenic fragment thereof, or the HBZ protein or an immunogenic fragment thereof, of HTLV-1.

9. A pharmaceutical composition comprising a vector containing a nucleic acid encoding human T-cell leukemia virus 1 (HTLV-1) antigenic Gag protein p15 (Gag p15) or an immunogenic fragment thereof, Gag protein p19 (Gag p19) or an immunogenic fragment thereof, or Gag protein p24 (Gag p24) or an immunogenic fragment thereof, and a pharmaceutically acceptable carrier.

10. 10. The pharmaceutical composition of claim 9, comprising a vector comprising a nucleic acid encoding an unprocessed Gag protein as the Gag p15, the Gag p19, or the Gag p24.

11. The pharmaceutical composition according to claim 9 or 10, further comprising a vector comprising a nucleic acid encoding the HTLV-1 Tax protein or an immunogenic fragment thereof, or the HBZ protein or an immunogenic fragment thereof.