Antigen peptide, nucleic acid, vector, hla / antigen peptide complex, detection reagent, and pharmaceutical composition

Abstract

To provide a novel peptide that can be used for detecting the sub-groove of HTLV-1 Tax.SOLUTION: An antigen peptide derived from the Tax protein encoded by HTLV-1 in the present disclosure includes a polypeptide of (P1), (P2), or (P3) defined below: (P1) a polypeptide consisting of at least one amino acid sequence of SEQ ID NO: 1 (SFHSLHLLF) and SEQ ID NO: 2 (SFHNLHLLF); (P2) a polypeptide consisting of an amino acid sequence in which the fourth amino acid from the N-terminus in (P1) is maintained and 1 to 3 amino acids are deleted, inserted, substituted, or added in the amino acid sequence of (P1); (P3) a polypeptide consisting of an amino acid sequence in which the fourth amino acid from the N-terminus in (P1) is maintained and which exhibits 80% or greater sequence identity to the amino acid sequence of (P1).SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present disclosure relates to antigen peptides, nucleic acids, vectors, HLA / antigen peptide complexes, detection reagents, pharmaceutical compositions, and the like. [Background technology]

[0002] The retrovirus human T-cell leukemia virus type-I (HTLV-1) is the causative virus that causes the onset of HTLV-1-associated diseases such as adult T-cell leukemia-lymphoma (ATL). It is said that there are approximately 800,000 HTLV-1-infected people (carriers) in Japan. However, the mechanism that causes the HTLV-1-associated diseases has not been fully elucidated.

[0003] The Tax protein of HTLV-1 is known as an antigen recognized by HTLV-1-specific CD8+ T cells. The Tax protein of HTLV-1 has two subgroups (Tax-A and Tax-B), and it has been reported that the subgroup of the Tax protein of HTLV-1 is associated with the risk of developing HTLV-1-associated diseases (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Naito, Tadasuke et al. “Distinct gene expression signatures induced by viral transactivators of different HTLV-1 subgroups that confer a different risk of HAM / TSP.” Retrovirology vol. 15,1 72. 6 Nov. 2018, doi:10.1186 / s12977-018-0454-x Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, an object of the present disclosure is to provide antigen peptides and the like that can be used for detecting the HTLV-1 Tax subgroup. [Means for solving the problem]

[0006] In order to achieve the above object, the disclosed antigenic peptide derived from the Tax protein encoded by human T-cell leukemia virus 1 (HTLV-1) comprises the following polypeptide (P1), (P2), or (P3): (P1) SEQ ID NO: 1 (SFHSLHLLF) and SEQ ID NO: 2 (SFH N A polypeptide consisting of at least one of the amino acid sequences: (P2) a polypeptide having an amino acid sequence in which the fourth amino acid from the N-terminus of (P1) is conserved and 1 to 3 amino acids have been deleted, inserted, substituted or added in the amino acid sequence of (P1); (P3) A polypeptide having an amino acid sequence which conserves the fourth amino acid from the N-terminus of (P1) and has an identity of 80% or more to the amino acid sequence of (P1).

[0007] The nucleic acids of the disclosure encode the antigenic peptides of the disclosure.

[0008] A vector of the present disclosure comprises a nucleic acid of the present disclosure.

[0009] The HLA / antigenic peptide complexes of the present disclosure include complexes of human leukocyte antigens (HLA) and antigenic peptides, The antigenic peptide includes an antigenic peptide derived from the Tax protein of the present disclosure.

[0010] A detection reagent for the HTLV-1 encoded Tax protein of the present disclosure comprises an HLA / antigenic peptide complex of the present disclosure.

[0011] The pharmaceutical compositions of the present disclosure comprise an antigenic peptide of the present disclosure and a pharma- ceutically acceptable carrier.

[0012] The method for detecting T cells disclosed herein includes a detection step of contacting a biological sample with an HLA / antigen peptide complex, thereby detecting T cells in the biological sample that bind to the HLA / antigen peptide complex, The HLA / antigenic peptide complex is an HLA / antigenic peptide complex of the present disclosure.

[0013] The method for evaluating a subtype of HTLV-1 according to the present disclosure includes a detection step of contacting a biological sample isolated from a subject with an HLA / antigenic peptide complex, thereby detecting T cells in the biological sample that bind to the HLA / antigenic peptide complex, an evaluation step of evaluating a subtype of the Tax protein encoded by HTLV-1 in the biological sample of the subject based on the detection result of the T cell in the detection step, The HLA / antigenic peptide complex is an HLA / antigenic peptide complex of the present disclosure. Effect of the Invention

[0014] According to the present disclosure, the Tax subgroup of HTLV-1 can be detected. [Brief description of the drawings]

[0015]

Figure 1

Figure 2

[0016] The present disclosure will be described below with reference to examples. In the following description, the descriptions of the disclosures can be mutually incorporated unless otherwise specified.

[0017] <Definition> As used herein, "peptide" refers to a compound composed of unmodified amino acids (natural amino acids), modified amino acids, and / or artificial amino acids, formed by condensing two or more amino acids through a peptide bond.

[0018] In this specification, "retrovirus" refers to a type of RNA virus that has reverse transcriptase. It is known that the retrovirus infects and grows in a host cell as follows. The retrovirus infects a host cell that is a target for infection, synthesizes double-stranded DNA by reverse transcription of viral RNA, and incorporates 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 the retroviral protease cleaves the immature viral proteins, and the mature viral proteins are assembled to form viral particles. The viral particles bud from the surface of the host cell.

[0019] In this specification, "HTLV-1" refers to a type of retrovirus, human T-cell leukemia virus type 1. The HTLV-1 is known as a virus that causes diseases such as 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 uveitis). The HTLV-1 belongs to the Oncoviridae 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 No. AB513134.1.

[0020] In this specification, "Tax protein" (hereinafter also referred to as "Tax") means a transcription activator that activates both the LTR (Long terminal repeat) and the intracellular promoter of a retrovirus. Tax is known to play an important role in the infectivity of the virus and the oncogenesis of infected cells. The HTLV-1 Tax is known to have two subtypes, Tax-A and Tax-B. An example of the HTLV-1 Tax-A is a protein (SEQ ID NO: 3) consisting of an amino acid sequence registered in Genbank under the accession number AB979451. An example of the HTLV-1 Tax-B is a protein (SEQ ID NO: 5) consisting of an amino acid sequence registered in Genbank under the accession number AB513134.

[0021] Amino acid sequence of HTLV-1 Tax-A (SEQ ID NO:3) MAHFPGGQSLLFGYPVYVFGDCVQGDWCPISGGLCSARLHRHALLATCPEHQITWDPIDGRVIGSALQFLIPRLPSFPTQRTSKTLKVLTPPITHTTPNIPPSFLQAMRKYSPFRNGYMEPTLGQHLPTLSFPDPGLRPQNLYTLWGGS VVCMYLYQLSPPITWPLLPHVIFCHPGQLGAFLTNVPYKRIEELLYKISLTTGALIILPEDCLPTTLFQPVRAPVTLTAWQNGLLPFHSTLTTPGLIWTFTDSTPMISGPCPKDGQPSLVLQSSSFIFHKFQTKAYHPSFLLSHGLIQYS SFHNLHLLF EEYTNIPISLLFNEKEADDNDHEPQISPGGLEPPSEKHFRETEV

[0022] Nucleotide sequence encoding HTLV-1 Tax-A (SEQ ID NO: 4)

[0023] Amino acid sequence of HTLV-1 Tax-B (SEQ ID NO:5) MAHFPGGQSLLFGYPVYVFGDCVQGDWCPISGGLCSARLHRHALLATCPEHQITWDPIDGRVIGSALQFLIPRLPSFPTQRTSKTLKVLTPPITHTTPNIPPSFLQAMRKYSPFRNGYMEPTLGQHLPTLSFPDPGLRPQNLYTLWGGS VVCMYLYQLSPPITWPLLPHVIFCHPGQLGAFLTNVPYKRIEELLYKISLTTGALIILPEDCLPTTLFQPARAPVTLTAWQNGLLPFHSTLTTPGLIWTFTDGTPMISGPCPKDGQPSLVLQSSSFIFHKFQTKAYHPSFLLSHGLIQYS SFHSLHLLF EEYTNIPISLLFNEKEADDNDHEPQISPGGLEPPSEKHFRETEV

[0024] Nucleotide sequence encoding Tax-B of HTLV-1 (SEQ ID NO:6)

[0025] As used herein, "T cells" refers to a type of white blood cell, classified as lymphocytes, and expressing a T cell receptor (TCR). Known examples of the T cells include CD4+ helper T cells and CD8+ cytotoxic T cells.

[0026] As used herein, "positive (+)" means that a higher signal is detected by an analysis method such as flow cytometry that utilizes an antigen-antibody reaction, compared to a negative control reaction using negative control cells that do not express the antigen or an antibody that does not react with the antigen. Also, as used herein, "negative (-)" means that a signal equal to or lower than a negative control reaction using negative control cells that do not express the antigen or an antibody that does not react with the antigen is detected.

[0027] In this specification, "human leukocyte antigen (HLA)" means human MHC (major histocompatibility complex). HLA has the function of presenting antigens to T cells. HLA is roughly divided into two groups, class I antigens and class II antigens. As the class I antigens, subtypes such as HLA-A, B, C, D, E, F, and G are known. The class I antigens form complexes with human β2 microglobulin protein and present peptides such as antigen peptides. As the class II antigens, subtypes such as HLA-DR, DQ, and DP are known. Alleles are known for each subtype of HLA. In addition, it is known that there are more than thousands of combinations of the alleles.

[0028] As used herein, the term "antigenicity" refers to the property of an antigen to induce immune responses such as antibody production and cellular immunity.

[0029] As used herein, "nucleic acid" refers to a polymer of deoxyribonucleotides (DNA), ribonucleotides (RNA), and / or modified nucleotides. When "nucleic acid" is used in combination with a specific protein, the "nucleic acid" refers to a polymer of nucleotides that encodes 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 "polynucleotide" or "oligonucleotide."

[0030] As used herein, a "vector" (expression vector) is in vitro or in vivo By "transfected vector" is meant a recombinant plasmid or virus that contains the nucleic acid to be delivered to a host cell.

[0031] As used herein, the term "pharmaceutical acceptable carrier" refers to a solvent and / or additive that can be commonly used in the formulation technology of pharmaceutical compositions. The pharmaceutical acceptable carrier is preferably one that is almost or completely non-toxic to living organisms.

[0032] As used herein, "adjuvant" refers to a compound that, when used in combination with a particular 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 the reduction, decrease, or suppression of a particular antigen-specific immune response.

[0033] As used herein, "vaccine" refers to a preparation or composition of, for example, an attenuated or inactivated (killed) infectious agent or an antigenic determinant (epitope, for example, peptide, glycan, lipid, etc.) from the infectious agent, which is used to induce humoral immunity (antibody production) or cellular immunity against the pathogen. As used herein, the vaccine may refer to, for example, a preparation or composition administered to provide immunity against HTLV-1 or protection against HTLV-1-associated disease caused by HTLV-1. The "vaccine" may also refer to, for example, a suspension or solution of an immunogen that, when administered to a subject, results in an immune response, i.e., immunity to prevent disease caused by infection or to reduce the severity of disease. The vaccine may also be referred to, for example, as a "vaccine composition" or a "vaccine formulation."

[0034] As used herein, the term "subject" refers to an animal or a cell, tissue, or organ derived from an animal. The subject is used to mean, in particular, a human. The animal refers to a human or a non-human animal. The non-human animal may be, for example, a mammal such as a mouse, a rat, a rabbit, a dog, a cat, a cow, a horse, a pig, a monkey, a dolphin, or a sea lion.

[0035] As used herein, "prevention" refers to reducing the possibility of onset of a disease or pathology, suppressing, delaying, or stopping the onset of a disease or pathology, suppressing, alleviating, delaying, or stopping the progression of a pathology, suppressing, reducing, delaying, or stopping the worsening of a disease or pathology, or suppressing, delaying, or stopping the recurrence of a disease or pathology. The "prevention" may be, for example, treatment of a subject (patient) who develops a target disease, or treatment of a model animal of the target disease.

[0036] 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 obtaining at least one purification step.

[0037] Hereinafter, the present disclosure will be described with examples, but the present disclosure is not limited to the following examples and the like, and can be arbitrarily changed and implemented. In addition, each description in the present disclosure can be mutually applied unless otherwise specified. In this specification, when the expression "~" is used, it is used in the sense of including the numerical values or physical values before and after it. In this specification, the expression "A and / or B" includes "only A", "only B", and "both A and B".

[0038] <Antigen peptide derived from Tax protein> In one aspect, the present disclosure discloses an antigen peptide derived from the Tax protein encoded by human T-cell leukemia virus type 1 (HTLV-1). The antigen peptide of the present disclosure includes the following polypeptide (P1), (P2), or (P3): (P1) A polypeptide consisting of at least one amino acid sequence of SEQ ID NO: 1 (SFH S LHLLF) and SEQ ID NO: 2 (SFH N LHLLF); (P2) A polypeptide consisting of an amino acid sequence in which the fourth amino acid from the N-terminus of the above (P1) is conserved and one or several amino acids are deleted, inserted, substituted, or added in the amino acid sequence of the above (P1); (P3) A polypeptide consisting of an amino acid sequence having 80% or more identity to the amino acid sequence of the above (P1), in which the fourth amino acid from the N-terminus of the above (P1) is conserved.

[0039] As a result of intensive research, the present inventors came up with the idea that since the Tax protein encoded by HTLV-1 has two subtypes and T cells with different reactivity to the subtypes may exist, the Tax subtype may be detected by using an antigen peptide derived from the Tax protein. The present inventors then found a polypeptide in a human HTLV-1 patient in which the amino acid sequence differs between the Tax subtypes and T cells capable of specifically recognizing antigen peptides derived from each subtype exist, and established the present disclosure. For this reason, the antigen peptide of the present disclosure can detect, for example, T cells specific to the Tax protein subtype, and therefore the subtype of the Tax protein encoded by the HTLV-1 can be evaluated.

[0040] In the (P1) polypeptide, the amino acid sequence of SEQ ID NO: 1 corresponds, for example, to the 301st to 309th amino acids in the amino acid sequence of Tax-B (SEQ ID NO: 3). The amino acid sequence of SEQ ID NO: 2 corresponds, for example, to the 301st to 309th amino acids in the amino acid sequence of Tax-A (SEQ ID NO: 5).

[0041] The antigenic peptides of the present disclosure may be, for example, isolated antigenic peptides or may be antigenic peptides in mixture with other substances (eg, peptides or proteins).

[0042] In the antigen peptide of (P2), "one or several" may mean, for example, that (P2) is a polypeptide that functions as an antigen peptide having immune response-inducing activity and / or a polypeptide that can bind to HLA-A*24:02. "One or several" in (P2) means, for example, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2 in the amino acid sequence of (P1). In the present disclosure, a numerical range of the number discloses, for example, all positive integers within that range.

[0043] In the antigen peptide (P2), the substitution is preferably a conservative substitution. The conservative substitution means a substitution of an amino acid residue with an amino acid residue having a similar side chain. Examples of the conservative substitution include substitution between amino acid residues having a basic side chain such as lysine, arginine, histidine, etc.; substitution between amino acid residues having an acidic side chain such as aspartic acid, glutamic acid, etc.; substitution between amino acid residues having a non-charged polar side chain such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, etc.; substitution between amino acid residues having a non-polar side chain such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, etc.; substitution between amino acid residues having a β-branched side chain such as threonine, valine, isoleucine, etc.; substitution between amino acid residues having an aromatic side chain such as tyrosine, phenylalanine, tryptophan, histidine, etc.; and the like.

[0044] In the antigen peptide of (P3), the "identity" may be within a range in which (P3) is, for example, a polypeptide that functions as an antigen peptide having immune response-inducing activity and / or a peptide that functions as a polypeptide that can bind to HLA-A*24:02. The "identity" of (P3) is, for example, 70%, 75%, 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 (P1). 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) (hereinafter the same).

[0045] The antigenic peptide of the present disclosure may be, for example, a polypeptide consisting of an amino acid sequence in which "one or several" amino acids have been added to at least one of the C-terminus and N-terminus of the amino acid sequence of SEQ ID NO: 1. The "one or several" refers to the range in which the antigenic peptide of the present disclosure is, for example, a polypeptide that functions as an antigenic peptide having immune response-inducing activity and / or a peptide that functions as a polypeptide capable of binding to HLA-A*24:02. The one or several is, for example, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1.

[0046] The Tax may be, for example, Tax-A or Tax-B, and is preferably Tax-A.

[0047] The antigen peptide of the present disclosure preferably has binding affinity to, for example, human leukocyte antigen (HLA). The α chain of the HLA is, for example, HLA-A*24, preferably HLA-A*24:02. In this case, it can also be said that the antigen peptide of the present disclosure has binding affinity to a complex of the HLA-A*24 and human β2 microglobulin protein. The binding affinity to the HLA can be evaluated, for example, by determining whether the antigen peptide can be recovered from HLA / β2 microglobulin after coexistence of a cell expressing the HLA and human β2 microglobulin protein with the antigen peptide of the present disclosure. The HLA-A*24:02 is, for example, a protein consisting of the amino acid sequence of SEQ ID NO: 7 below. The human β microglobulin protein is, for example, a protein consisting of the amino acid sequence of SEQ ID NO: 8 below. The HLA may be, for example, a solubilized complex. In this case, the α chain and β2 microglobulin constituting the HLA may be, for example, composed of their respective extracellular domains. An example of the extracellular domain of HLA-A*24:02 is, for example, the 1st to 308th amino acids in the amino acid sequence of SEQ ID NO: 7 below (SEQ ID NO: 9).

[0048] Amino acid sequence of HLA-A*24:02 (SEQ ID NO:7) MAVMAPRTLVLLLSGALALTQTWAGSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIALRYYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRA YLEGTCVDGLRRYLENGKETLQRTDPPKTHMTHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEPSSQPTVPIVGIIAGLVLLGAVITGAVVAAVMWRRNSSDRKGGSYSQAASSDSAQGSDVSLTACKV

[0049] Amino acid sequence of human β2 microglobulin protein (SEQ ID NO:8) MSRSVALAVLALLSLSGLEAMIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVVTNHLSQPKIVKWDRDM

[0050] Amino acid sequence of HLA-A*24:02 extracellular domain (SEQ ID NO:9) MAVMAPRTLVLLLSGALALTQTWAGSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIALRYYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDL RSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEPSSQPTVPI

[0051] Nucleotide sequence encoding HLA-A*24:02 (SEQ ID NO: 10)

[0052] Nucleotide sequence encoding human β2 microglobulin protein (SEQ ID NO: 11, including stop codon) 5'-ATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTTTTTCTGGCCTGGAGGCTATGATTCAAAGAACTCCAAAAATTCAGGTTTACTCACGTCATCCAGCAGAGAATGGAAAGTCAAATTTCCTGAATTGCTATGTGTCTGGGTTTCATCCATCCGACATTGAAGTTGACTTAC TGAAGAATGGAGAGAGAATTGAAAAAGTGGAGCATTCAGACTTGTCTTTCAGCAAGGACTGGTCTTTCTATCTCTTGTACTACACTGAATTCACCCCCACTGAAAAAGATGAGTATGCCTGCCGTGTGAACCATGTGACTTTGTCACAGCCCAAGATAGTTAAGTGGGATCGAGACATGTAA-3'

[0053] The HLA and the human β2 microglobulin protein may be functionally equivalent to each other as long as the binding ability between the HLA and the human β2 microglobulin protein is maintained.

[0054] The functional equivalent of the HLA can be, for example, a polypeptide consisting of an amino acid sequence having 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of the HLA, and having the binding ability to the human β2 microglobulin protein. The functional equivalent of the HLA can be, for example, a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of the HAL, and having the binding ability to the human β2 microglobulin protein. The one or several can be, for example, 1 to 44, 1 to 33, 1 to 22, 1 to 11, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1 or 2, or 1. The substitution is preferably, for example, a conservative substitution.

[0055] The functional equivalent of the human β2 microglobulin protein may be, for example, a polypeptide having an amino acid sequence with 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of the human β2 microglobulin protein, and having the binding ability to the HLA. The functional equivalent of the human β2 microglobulin protein may be, for example, a polypeptide having an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of the human β2 microglobulin protein, and having the binding ability to the HLA. The one or several may be, for example, 1 to 44, 1 to 33, 1 to 22, 1 to 11, 1 to 8, 1 to 6, 1 to 4, 1 to 3, 1 or 2, or 1. The substitution is preferably, for example, a conservative substitution.

[0056] <Nucleic acid> In another aspect, the present disclosure provides a nucleic acid encoding the antigenic peptide. The nucleic acid of the present disclosure encodes the antigenic peptide of the present disclosure.

[0057] The nucleic acid may be composed of deoxynucleotide residues, ribonucleotide residues, or both. The nucleic acid may be composed of natural nucleic acid residues, non-natural nucleic acid residues, or both. Specific examples of the nucleic acid include DNA, RNA, and / or DNA / RNA composed of natural and / or non-natural nucleic acid residues. Examples of the non-natural nucleic acid residues 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 non-natural nucleic acid residue include cEt (constrained ethyl bicyclic nucleic acid, Ionis Pharmaceuticals), LNA (trademark, Locked Nucleic Acid), and ENA (registered trademark, 2'-O,4'-C-Ethylene bridged Nucleic Acid). The nucleic acid may have a 5' cap at the 5' end, for example.

[0058] The nucleic acid may be a single-stranded or double-stranded nucleic acid molecule.

[0059] The nucleic acid of the present disclosure can be designed by substituting corresponding codons based on the amino acid sequence of the antigen peptide of the present disclosure. The base sequence of the nucleic acid of the present disclosure may be, for example, codon-optimized.

[0060] In the present disclosure, various nucleic acids and antigen peptides 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.

[0061] The nucleic acid of the present disclosure may further comprise a nucleic acid encoding said HLA and / or said human β2 microglobulin protein.

[0062] Since the nucleic acid of the present disclosure encodes the antigen peptide, it can be suitably used for the synthesis of the antigen peptide. Furthermore, since the nucleic acid of the present disclosure encodes the antigen peptide, it can be used to induce cellular immunity against the antigen peptide when used as an active ingredient of a vaccine. Furthermore, the nucleic acid of the present disclosure can be suitably used, for example, for the genetic engineering synthesis of the complex described below.

[0063] <Expression vector> In another aspect, the present disclosure provides an expression vector that can be used to synthesize or express an antigen peptide. The expression vector of the present disclosure includes the nucleic acid of the present disclosure. According to the expression vector of the present disclosure, the antigen peptide of the present disclosure can be suitably produced by genetic engineering techniques. In addition, according to the expression vector of the present disclosure, for example, in vivo The peptides of the present disclosure can be expressed in

[0064] The expression vector of the present disclosure is, for example, an expression vector in which the nucleic acid of the present disclosure is inserted. The expression vector can also be said to be, for example, a vector in which the nucleic acid is functionally linked. The expression vector means, for example, a nucleic acid molecule that can transport an inserted gene into a target such as a cell.

[0065] The expression vector may contain a polynucleotide encoding an antigen peptide so as to express the antigen peptide encoded by the polynucleotide of the nucleic acid of the present disclosure, and the structure is not particularly limited. The expression vector may contain a nucleic acid encoding the HLA and / or the human β2 microglobulin protein in addition to the nucleic acid encoding the antigen peptide. In this case, a part or the whole of each polypeptide may be contained in the same expression vector or in different expression vectors.

[0066] The expression vector can be prepared, for example, by inserting a polynucleotide encoding an antigen peptide, i.e., the nucleic acid of the present disclosure, into a backbone vector (hereinafter also referred to as a "basic vector"). The type of the expression vector is not particularly limited and can be appropriately determined, for example, depending on the type of the host. Specifically, when the expression vector is synthesized by a genetic engineering technique, the expression vector is first synthesized by, for example, designing and synthesizing a nucleic acid encoding the antigen peptide. The design and synthesis can be performed, for example, by a PCR method using a vector containing a nucleic acid encoding the antigen peptide as a template and a primer designed to synthesize a desired nucleic acid region. Then, a recombinant vector for protein expression (expression vector) can be obtained by linking the obtained nucleic acid to an appropriate vector, and the recombinant vector can be 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)).

[0067] The host used for transformation is not particularly limited as long as it can express the target gene, 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. The prokaryotic organism is, for example, Escherichia coli ( Escherichia coli ) and other Escherichia species, Pseudomonas putida ( Pseudomonas putida Examples of the eukaryotic organism include bacteria of the genus Pseudomonas such as Saccharomyces cerevisiae ( Saccharomyces cerevisiae Examples of the animal cells include yeasts such as COS cells and CHO cells, and examples of the insect cells include Sf9 and Sf21 cells.

[0068] The expression vector may be a viral vector or a non-viral vector. When the introduction method is a method for transforming a host by a heat shock method, the expression vector may be, for example, a binary vector. The expression vector may be, for example, pETDuet-1, pQE-80L, pUCP26Km, etc. When transforming bacteria such as Escherichia coli, the expression vector may be, for example, a pETDuet-1 vector (Novagen), pQE-80L (QIAGEN), pBR322, pB325, pAT153, pUC8, etc. When transforming yeast, the expression vector may be, for example, pYepSec1, pMFa, pYES2, etc. When transforming insect cells, the expression vector may be, for example, pAc, pVL, etc. When transforming mammalian cells, examples of the expression vector include pCDM8 and pMT2PC.

[0069] The expression vector preferably has a regulatory sequence that regulates, for example, the expression of the polynucleotide encoding the antigen peptide and the expression of the antigen peptide of the present disclosure encoded by the polynucleotide of the antigen peptide. Examples of the regulatory sequence include a promoter, a terminator, an enhancer, a polyadenylation signal sequence, and an origin of replication (ori). In the expression vector, the arrangement of the regulatory sequence is not particularly limited. In the expression vector, the regulatory sequence may be arranged, for example, so long as it can functionally regulate the expression of the polynucleotide encoding the antigen peptide and the expression of the antigen peptide encoded thereby, and can be arranged based on a known method. For example, the regulatory sequence may utilize a sequence that is already included in the expression vector, or the regulatory sequence may be further inserted into the expression vector, or the regulatory sequence included in the basic vector may be replaced with another regulatory sequence.

[0070] The method of introducing the expression vector into the host is not particularly limited and can be carried out by a known method. The introduction method can be appropriately set depending on, for example, the type of the host. Examples of the introduction method include an introduction method using a gene gun such as a particle gun, a calcium phosphate method, a polyethylene glycol method, a lipofection method using liposomes, an electroporation method, an ultrasonic nucleic acid introduction method, a DEAE-dextran method, a direct injection method using a micro glass tube or the like, a hydrodynamic method, a cationic liposome method, a method using an introduction aid, a method mediated by Agrobacterium, a protoplast method, and the like. Examples of the liposome include lipofectamine and cationic liposome, and examples of the introduction aid include atelocollagen, nanoparticles, and polymers. When the host is a microorganism, for example, among others, E . coli or Ps. putida etc. The polynucleotide of the antigenic peptide of the present disclosure may be introduced into the host, for example, via an expression vector of the present disclosure.

[0071] In the production method of the present disclosure, for example, the transformant is cultured and the antigen peptide is collected from the culture. The culture may be a culture supernatant, a transformant such as cultured cells or cultured bacterial cells, or a treated or disrupted product thereof.

[0072] After the cultivation, when the antigen peptide is produced in the host, the production method of the present disclosure extracts the antigen peptide, for example, by disrupting the host. When the antigen peptide is produced or secreted outside the host, the production method of the present disclosure uses, for example, the culture solution as it is or removes the host by centrifugation or the like. Thereafter, the production method of the present disclosure can isolate or purify the antigen peptide by using, for example, general biochemical methods used for protein isolation and purification, specifically, concentration by an ultrafiltration membrane; salting out such as ammonium sulfate precipitation; chromatography using various columns such as gel filtration, ion exchange chromatography, and affinity chromatography; etc. alone or in appropriate combination.

[0073] <HLA / antigen peptide complex> In another aspect, the present disclosure discloses an HLA / antigen peptide complex that can be used for detecting T cells responsive to Tax encoded by HTLV-1. The HLA / antigen peptide complex of the present disclosure includes a complex of a human leukocyte antigen (HLA) and an antigen peptide, and the antigen peptide includes the antigen peptide derived from the nucleocapsid of the present disclosure. According to the HLA / antigen peptide complex of the present disclosure, T cells responsive to Tax encoded by HTLV-1 can be detected.

[0074] The α-chain of the HLA is, for example, preferably HLA-A*24, and preferably HLA-A*24:02. In the HLA / antigen peptide complex, the HLA is a complex of the α-chain (H-chain) of HLA and β2-microglobulin.

[0075] The HLA / antigen peptide complex may be a monomer, or may form a multimer in which two or more of the HLA / antigen peptide complexes are aggregated. Examples of the multimer include a dimer, a trimer, a tetramer, etc. The multimer can be prepared by, for example, a general method, for example, using an α-chain with a biotinylated C-terminus, β2-microglobulin, and avidin.

[0076] <Detection Reagents> In another embodiment, the present disclosure discloses a detection reagent capable of detecting antigen-specific T cells derived from HTLV-1, particularly Tax of HTLV-1. The detection reagent of the present disclosure is characterized in that it comprises a detection reagent for the Tax protein encoded by HTLV-1, which contains the HLA / antigen peptide complex of the present disclosure. The detection reagent of the present disclosure allows detection of T cells capable of responding to HTLV-1.

[0077] The Tax may be, for example, Tax-A or Tax-B, and is preferably Tax-A.

[0078] <Pharmaceutical Composition> In another aspect, the present disclosure provides a pharmaceutical composition that can be used as a vaccine. The pharmaceutical composition is characterized by comprising the antigen peptide of the present disclosure and a pharma- ceutical acceptable carrier. The pharmaceutical composition of the present disclosure is characterized by comprising an antigen peptide as an active ingredient, and other configurations and conditions are not particularly limited. Since the pharmaceutical composition of the present disclosure comprises an antigen peptide as an active ingredient, it can induce an immune response against HTLV-1 when used as an active ingredient of a vaccine.

[0079] The pharmaceutical composition of the present disclosure can induce an immune response against an antigen peptide, for example, by administering it to a subject. Therefore, the pharmaceutical composition of the present disclosure can also be called a vaccine, a vaccine composition, or a vaccine preparation.

[0080] The pharmaceutical composition of the present disclosure is, for example, a vaccine for use in the prevention and / or treatment of HTLV-1-associated diseases, such as adult T-cell leukemia-lymphoma, HTLV-1-associated myelopathy, and HTLV-1 uveitis.

[0081] The pharmaceutical composition of the present disclosure may further include a pharma- ceutically acceptable carrier, which may include a suspending agent, a solubilizing agent, a stabilizing agent, an isotonic agent, a preservative, an adsorption inhibitor, a surfactant, a diluent, a medium, a pH adjuster, a soothing agent, a buffer, a sulfur-containing reducing agent, an antioxidant, etc., for administering the active ingredient, and may be appropriately added within a range that does not impair the effects of the present disclosure.

[0082] The suspending agent is not particularly limited, and examples thereof include methylcellulose, polysorbate 80, hydroxyethylcellulose, gum arabic, powdered tragacanth, sodium carboxymethylcellulose, polyoxyethylene sorbitan monolaurate, and the like.

[0083] The solution adjuvant is not particularly limited, and examples thereof include polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, macrogol, castor oil fatty acid ethyl ester, and the like.

[0084] The stabilizer is not particularly limited, and examples thereof include dextran 40, methylcellulose, gelatin, sodium sulfite, and sodium metasulfate.

[0085] The isotonicity agent is not particularly limited, and examples thereof include D-mannitol, sorbitol, and the like.

[0086] The preservative is not particularly limited, and examples thereof include methyl paraoxybenzoate, ethyl paraoxybenzoate, sorbic acid, phenol, cresol, and chlorocresol.

[0087] 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, polyethylene glycol, and the like.

[0088] The sulfur-containing reducing agent is not particularly limited, and examples thereof include those having a sulfohydryl group, such as N-acetylcysteine, N-acetylhomocysteine, thioxanthine, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, sodium thiosulfate, glutathione, and thioalkanoic acids having 1 to 7 carbon atoms.

[0089] The antioxidant is not particularly limited, and examples thereof include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, 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.

[0090] The pharmaceutical composition of the present invention may further contain, as appropriate, commonly added components, such as inorganic salts such as sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, sodium bicarbonate, etc.; organic salts such as sodium citrate, potassium citrate, sodium acetate, etc.; and sugars such as glucose, etc.

[0091] The pharmaceutical composition of the present disclosure may further include an adjuvant (immunostimulator), etc. Examples of the adjuvant include Toll-like receptor stimulants such as CpG oligonucleotides (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) colony-stimulating factor (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 is an oil-in-water emulsion of squalene.

[0092] The pharmaceutical compositions of the present disclosure include, for example: in vitro You can also use 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 be called a test reagent or test kit.

[0093] There are no particular limitations on the subjects to which the pharmaceutical composition of the present disclosure is administered. in vivo When used in the above, the subject (subject to be administered) can be, for example, the above-mentioned examples. in vitroWhen used in the above, the subject of administration can be, for example, a cell, a tissue, an organ, etc., the cell can be, for example, a cell collected from a living body, a cultured cell, etc., the tissue or organ can be, for example, a tissue (living tissue) or an organ collected from a living body, etc., the cell can be, for example, an immune cell such as a T cell, a B cell, a NK cell, a dendritic cell, etc.

[0094] The pharmaceutical composition of the present disclosure in vivo When used in the above, the subject of administration may be a healthy person not infected with HTLV-1, a person who may be infected with HTLV-1, or a patient infected with HTLV-1, but is preferably a subject for whom prevention or treatment of the onset of HTLV-1-related diseases is desired. In addition, since the active ingredient of the present disclosure has binding affinity to HLA, particularly binding affinity to HLA-A*24:02, the subject of administration preferably has the HLA-A*24:02 antigen gene as an MHC class I molecule in homozygous form.

[0095] The use conditions (administration conditions) of the pharmaceutical composition of the present disclosure are not particularly limited, and for example, the administration form, administration timing, administration amount, etc. can be appropriately set depending on the type of the active ingredient in the pharmaceutical composition, the type of subject to administration, etc.

[0096] Methods for administering the pharmaceutical composition of the present disclosure include, for example, intracerebral administration, intrathecal administration, intramuscular administration, subcutaneous administration, intravenous administration, and the like. However, intramuscular or subcutaneous administration by injection or infusion is preferable, for example, because it can be administered safely and stably regardless of the skill of the administerer.

[0097] 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 appropriately determined depending on, for example, the age, weight, symptoms, etc. of the subject.

[0098] The number of administrations of the pharmaceutical composition of the present disclosure is one or more times. The plurality of times is, for example, 2 times, 3 times, 4 times, 5 times or more. The number of administrations may be appropriately determined while confirming the preventive effect on the administration subject. When administering multiple times, the administration interval can be appropriately determined while confirming the preventive effect on the administration subject, and examples include once a day, once a week, once every two weeks, once a month, once every three months, once every six months, etc.

[0099] The pharmaceutical composition of the present disclosure can prevent or reduce at least one symptom caused by HTLV-1 infection in the administration subject. Symptoms of HTLV-1 infection include, for example, the onset of HTLV-1-related diseases. The prevention of the above symptoms can be evaluated subjectively or objectively. Specific examples include self-evaluation by the administration subject; evaluation by a doctor; QOL (Quality of Life) evaluation; evaluation of delaying the progression of symptoms of HTLV-1-related diseases or reducing the severity of symptoms of HTLV-1-related diseases, etc. The objective evaluation may be by animals or by humans.

[0100] <Method for detecting T cells> In another aspect, the present disclosure provides a method for detecting antigen-specific T cells derived from HTLV-1, particularly Tax of HTLV-1. The method for detecting T cells of the present disclosure includes a detection step of contacting a biological sample with an HLA / antigen peptide complex to detect T cells that bind to the HLA / antigen peptide complex in the biological sample. The HLA / antigen peptide complex is characterized by being the HLA / antigen peptide complex of the present disclosure, and other configurations and conditions are not limited. The method for detecting T cells of the present disclosure can incorporate the descriptions of the antigen peptide, nucleic acid, expression vector, HLA / antigen peptide complex, detection reagent, and pharmaceutical composition of the present disclosure.

[0101] The living body is, for example, a human or non-human animal (for example, cow, pig, sheep, mouse, rat, rabbit, horse, monkey, etc.). The biological sample may be, for example, a body fluid, a cell, a tissue, an organ, etc., and is preferably a biological sample containing T cells. The body fluid may be, for example, blood, etc. The cells may be peripheral blood mononuclear cells, etc. When the biological sample is a liquid sample, the biological sample may be diluted with a solvent such as water or a buffer solution. When the biological sample is a solid sample, the biological sample is preferably dispersed in the solvent.

[0102] In the method for detecting T cells of the present disclosure, the HLA / antigen peptide complex preferably includes a complex of HLA and an antigen peptide derived from a Tax protein. Examples of the Tax include Tax-A and Tax-B, and preferably Tax-A. When the antigen peptide derived from Tax-A is used as the antigen peptide of the present disclosure, T cells specific to the antigen peptide derived from Tax-A can be detected. On the other hand, when the antigen peptide derived from Tax-B is used as the antigen peptide of the present disclosure, T cells specific to the antigen peptide derived from Tax-B can be detected.

[0103] The contact is a step of contacting the biological sample with the HLA / antigen peptide complex. In the contact step, the method of contacting the biological sample with the HLA / antigen peptide complex is not particularly limited, and is preferably performed in a liquid, for example. The contact may be performed, for example, by mixing the biological sample with the HLA / antigen peptide complex. Examples of the liquid include water, physiological saline, and buffer solutions. The contact conditions in the contact step can be set, for example, as general conditions that cause binding between the TCR in the T cells and the HLA / antigen peptide complex. Specific examples of the temperature of the contact are, for example, 0 to 40°C, 4 to 40°C, and the contact time is, for example, 1 to 120 minutes, 5 to 60 minutes.

[0104] In the detection step, by detecting the presence or absence of T cells that bind to the HLA / antigen peptide complex, for example, the presence or absence of T cells specific to the HLA / antigen peptide complex in the biological sample can be analyzed (qualitatively). Also, by detecting the degree of binding (binding amount) between the two, for example, the amount of T cells specific to the HLA / antigen peptide complex in the biological sample can be analyzed (quantitatively). The binding can be measured, for example, by ELISA using an antigen-antibody reaction, flow cytometry, or the like.

[0105] When the binding between the HLA / antigen peptide complex and the T cell cannot be detected, it can be determined that there are no T cells specific to the HLA / antigen peptide complex in the biological sample. When the binding is detected, it can be determined that there are T cells specific to the HLA / antigen peptide complex in the biological sample. Also, the number of T cells specific to the HLA / antigen peptide complex in the biological sample can be analyzed in advance from the amount of binding of the HLA / antigen peptide complex to the T cell. Therefore, the detection method of the present disclosure can also be referred to as an analysis method, for example.

[0106] The method for detecting the binding between the HLA / antigen peptide complex and the T cell is not particularly limited. As the method, for example, a conventionally known method for detecting the binding between substances can be adopted.

[0107] <Method for evaluating subtypes of HTLV-1> In another aspect, the present disclosure provides a method for evaluating the subtype of HLA / antigen peptide. The evaluation method of the present disclosure includes a detection step of detecting T cells that bind to the HLA / antigen peptide complex in a biological sample isolated from a subject by contacting the biological sample with the HLA / antigen peptide complex, and an evaluation step of evaluating the subtype of the Tax protein encoded by HTLV-1 in the biological sample of the subject based on the detection result of the T cells in the detection step, characterized in that the HLA / antigen peptide complex is the HLA / antigen peptide complex of the present disclosure, and other configurations and conditions are not limited. The evaluation method of the present disclosure can be applied to the explanations of the antigen peptide, nucleic acid, expression vector, HLA / antigen peptide complex, detection reagent, pharmaceutical composition, and detection method of the present disclosure.

[0108] The living body is, for example, a human or non-human animal (for example, cow, pig, sheep, mouse, rat, rabbit, horse, monkey, etc.). The biological sample is, for example, a body fluid, a cell, a tissue, an organ, etc. The body fluid is, for example, blood, etc. The cell is, for example, a peripheral blood mononuclear cell, etc.

[0109] In the evaluation method of the present disclosure, the HLA / antigen peptide complex preferably includes a complex of HLA and an antigen peptide derived from a Tax protein. The Tax can be, for example, Tax-A or Tax-B, and is preferably Tax-A. When the antigen peptide derived from Tax-A is used as the antigen peptide of the present disclosure, HTLV-1 expressing the Tax-A can be detected. On the other hand, when the antigen peptide derived from Tax-B is used as the antigen peptide of the present disclosure, T cells specific to the antigen peptide derived from Tax-B can be detected.

[0110] In the evaluation step, if the detection rate of T cells is higher when the Tax-A-derived peptide is used than when the Tax-B-derived peptide is used, the subtype of the Tax protein encoded by HTLV-1 in the biological sample of the subject can be evaluated as Tax-A. Also, if the detection rate of T cells is higher when the Tax-B-derived peptide is used than when the Tax-A-derived peptide is used, the subtype of the Tax protein encoded by HTLV-1 in the biological sample of the subject can be evaluated as Tax-B.

[0111] The detection step can be carried out, for example, by flow cytometry, and the method described in Example 1 can be referred to.

[0112] <Use> In another aspect, the present disclosure relates to the use of the antigen peptide, nucleic acid, vector, HLA / antigen peptide complex, detection reagent, and / or pharmaceutical composition of the present disclosure for use in a method for preventing the onset of HTLV-1-associated disease and the aggravation of HTLV-1-associated disease. The present disclosure relates to the use of the antigen peptide, nucleic acid, vector, HLA / antigen peptide complex, detection reagent, and / or pharmaceutical composition of the present disclosure for use in the manufacture of a vaccine for use in suppressing the onset of HTLV-1-associated disease and the aggravation of HTLV-1-associated disease. The use of the antigen peptide, nucleic acid, vector, HLA / antigen peptide complex, detection reagent, and / or pharmaceutical composition of the present disclosure can be described, for example, by referring to the explanation of the antigen peptide, nucleic acid, vector, HLA / antigen peptide complex, detection reagent, and / or pharmaceutical composition, detection method, and evaluation method for HTLV-1 subtype of the present invention. EXAMPLES

[0113] The present disclosure will be described in detail below using examples, but the present disclosure is not limited to the aspects described in the examples. Unless otherwise specified, commercially available reagents, kits, etc. were used according to their protocols.

[0114] [Example 1] It has been confirmed that the antigen peptide derived from the Tax protein encoded by HTLV-1 of the present disclosure induces the activity of T cells derived from HTLV-1-infected individuals.

[0115] Whole blood was collected from an HTLV-1-infected individual, and peripheral blood mononuclear cells (PBMCs) were obtained. The PBMCs were obtained by density gradient centrifugation from the whole blood using Ficoll-Paque (trademark) Plus (GE Healthcare Life Sciences, Cat No: 17-1440-03). After the centrifugation, 1 × 10 5 The PBMCs of the cells were washed with a washing buffer (1% BSA / PBS). HLA-A24 / Tax-A tetramer (outsourced to MBL, Inc., Tax-A peptide (SEQ ID NO: 2: SFH N LHLLF) with HLA-A*24:02 and β2 microglobulin) or HLA-A24 / Tax-B tetramer (MBL, Code No: TS-M018-1, Tax-B peptide (SEQ ID NO: 1: SFH SLHLLF) and HLA-A*24:02 and β2 microglobulin complex tetramer) were diluted with a washing buffer to a final concentration of 10 μg / ml and added to the PBMCs. After the addition, the cells were left to stand for 30 minutes at room temperature (about 24° C.). After that, the cells were washed with a washing buffer. After the washing, the surface was stained with an antibody. The antibody used was a CD8a antibody (A82791, manufactured by Beckman Coulter). After the staining, the cells were left to stand for 20 minutes on ice. After that, the cells were washed with a washing buffer. After the staining, LIVE / DEAD™ Fixable Near-IR Dead Cell Stain Kit (manufactured by Thermo Fisher Scientific) was added to stain dead cells. After the addition, the cells were left to stand for 30 minutes on ice. After that, the cells were washed with a washing buffer. After the washing, the PBMCs were fixed with 1% paraformaldehyde (Nacalai Tesque, Cat No: 09154-85). After the fixation, the expression levels of surface molecules were measured by flow cytometry using FACSVerse™ (BD). The data obtained by the flow cytometry was analyzed using FlowJo™ (BD). The negative control was performed in the same manner, except that no tetramer was added. These results are shown in Figures 1 and 2.

[0116] FIG. 1 is a dot plot showing the results of flow cytometry. In FIG. 1, the horizontal axis indicates the mean fluorescence intensity of CD8a, and the vertical axis indicates the mean fluorescence intensity of the tetramer. As shown in FIG. 1, in PBMCs derived from HTLV-1 / Tax-A patients, the mean fluorescence intensity of the tetramer was significantly higher when stained with HLA-A24 / Tax-A tetramer than when stained with HLA-A24 / Tax-B tetramer. + / CD8a + In PBMCs from HTLV-1 / Tax-B patients, the tetramer-specific expression level was increased when stained with HLA-A24 / Tax-B tetramers compared to when stained with HLA-A24 / Tax-A tetramers. + / CD8a+ These results indicate that accurate measurement results can be obtained by tetramer staining when the Tax subtype of the tetramer matches the Tax subtype of the HTLV-1 infecting the patient, but not when they do not match.

[0117] Figure 2 shows the tetramer + / CD8a + Graph showing percentage of T cells and CD8a + 2(A) is a graph showing the median fluorescence intensity of tetramers in T cells. + / CD8a + The percentage of T cells (%) is shown, and the horizontal axis indicates whether the subtype is matched or mismatched. In FIG. 2(B), the vertical axis indicates the median fluorescence intensity (MFI) of the tetramer, and the horizontal axis indicates whether the subtype is matched or mismatched. As shown in FIG. 2(A), in all of the CD8 T cells derived from HTLV-1-infected individuals, the number of cells positive for tetramer staining was reduced in the mismatched combinations compared to the matched combinations. Also, as shown in FIG. 2(B), in five of six cases of CD8 T cells derived from HTLV-1-infected individuals, the median fluorescence intensity was reduced in the mismatched combinations compared to the matched combinations. These results show that the use of both HLA-A24 / Tax-A and HLA-A24 / Tax-B tetramers allows accurate measurement of the HTLV-1 in which the patient is infected.

[0118] Although the present invention has been described above with reference to the embodiment, the present invention is not limited to the above embodiment. Various modifications that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

[0119] All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety as if the contents were specifically set forth herein.

[0120] <Supplementary Note> Some or all of the above embodiments and examples can be described as follows, but are not limited thereto. <Antigenic peptide derived from Tax protein> (Supplementary Note 1) An antigenic peptide derived from the Tax protein encoded by human T-cell leukemia virus type 1 (HTLV-1), comprising a polypeptide of the following (P1), (P2), or (P3): (P1) A polypeptide consisting of the amino acid sequence of at least one of SEQ ID NO: 1 (SFH S LHLLF) and SEQ ID NO: 2 (SFH N LHLLF); (P2) A polypeptide consisting of an amino acid sequence in which the fourth amino acid from the N-terminus of the above (P1) is conserved, and 1 to 3 amino acids are deleted, inserted, substituted, or added in the amino acid sequence of the above (P1); (P3) A polypeptide consisting of an amino acid sequence having 80% or more identity to the amino acid sequence of the above (P1), in which the fourth amino acid from the N-terminus of the above (P1) is conserved. (Supplementary Note 2) The antigenic peptide according to claim 1, wherein the Tax is Tax-A. (Supplementary Note 3) The antigenic peptide according to Supplementary Note 1 or 2, wherein the (P2) is a polypeptide consisting of an amino acid sequence in which 1, 2, or 3 amino acids are added to at least one of the C-terminus and N-terminus of the amino acid sequence of the above (P1). (Supplementary Note 4) The antigenic peptide according to any one of Supplementary Notes 1 to 3, wherein the peptide has binding affinity to human leukocyte antigen (HLA). (Supplementary Note 5) The antigenic peptide according to Supplementary Note 4, wherein the HLA includes HLA-A*24. <Nucleic acid> (Supplementary Note 6) A nucleic acid encoding the antigenic peptide according to any one of Supplementary Notes 1 to 5. <Vector> (Supplementary Note 7) A vector containing the nucleic acid according to Supplementary Note 6. <HLA / antigen peptide complex> (Appendix 8) Comprising a complex of human leukocyte antigen (HLA) and an antigen peptide, The antigen peptide contains an antigen peptide derived from the Tax protein described in any of Appendices 1 to 5, and is an HLA / antigen peptide complex. (Appendix 9) The HLA contains HLA-A*24, and is the HLA / antigen peptide complex described in Appendix 8. (Appendix 10) The complex is a multimer of the complex, and is the HLA / antigen peptide complex described in Appendix 8 or 9. <Detection (diagnosis) reagent> (Appendix 11) A detection reagent for the Tax protein encoded by HTLV-1, comprising the HLA / antigen peptide complex described in any of Appendices 8 to 10. (Appendix 12) The Tax is Tax-A, and is the detection reagent described in Appendix 11. <Pharmaceutical composition> (Appendix 13) A pharmaceutical composition comprising an antigen peptide described in any of Appendices 1 to 5 and a pharmaceutically acceptable carrier. (Appendix 14) Furthermore, the pharmaceutical composition described in Appendix 13, which contains an adjuvant. (Appendix 15) A vaccine for use in the prevention and / or treatment of the onset of HTLV-1-related diseases, and is the pharmaceutical composition described in Appendix 13 or 14. <Use> (Appendix 16) An antigen peptide described in any of Appendices 1 to 5, a nucleic acid described in Appendix 6, a vector described in Appendix 7, an HLA / antigen peptide complex described in any of Appendices 8 to 10, a detection reagent described in Appendix 11 or 12, or a pharmaceutical composition described in any of Appendices 13 to 15, for use in the prevention and / or treatment method of the onset of HTLV-1-related diseases. <Method for detecting T cells> (Appendix 17) A detection step of detecting T cells that bind to the HLA / antigen peptide complex in the biological sample by contacting the biological sample with the HLA / antigen peptide complex is included. The method for detecting T cells, wherein the HLA / antigen peptide complex is the HLA / antigen peptide complex according to any one of Appendices 8 to 10. (Appendix 18) The detection method according to Appendix 17, wherein the HLA / antigen peptide complex includes a complex of HLA and a peptide derived from the Tax-A protein. (Appendix 19) The detection method according to Appendix 17 or 18, wherein the biological sample is selected from the group consisting of blood and peripheral blood mononuclear cells. <Method for evaluating subtypes of HTLV-1> (Appendix 20) A detection step of detecting T cells that bind to the HLA / antigen peptide complex in the biological sample by contacting the biological sample isolated from the subject with the HLA / antigen peptide complex is included. An evaluation step of evaluating the subtype of the Tax protein encoded by HTLV-1 in the biological sample of the subject is included based on the detection result of the T cells in the detection step. The method for evaluating subtypes of HTLV-1, wherein the HLA / antigen peptide complex is the HLA / antigen peptide complex according to any one of Appendices 8 to 10. (Appendix 21) The evaluation method according to Appendix 20, wherein the HLA / antigen peptide complex includes a complex of HLA and a peptide derived from the Tax-A protein. (Appendix 22) The evaluation method according to Appendix 20 or 21, wherein the biological sample is selected from the group consisting of blood and peripheral blood mononuclear cells.

Industrial Applicability

[0121] As described above, according to the antigen peptide of the present disclosure, for example, specific CD8a against Tax encoded by HTLV-1 +It is possible to measure T cells. Therefore, the antigen peptide of the present disclosure can detect, for example, a subgroup of HTLV-1 Tax. Therefore, the present disclosure is extremely useful, for example, in the medical field.

Claims

1. An antigenic peptide derived from the Tax protein encoded by human T-cell leukemia virus 1 (HTLV-1), comprising the following polypeptide (P1), (P2), or (P3): (P1) a polypeptide consisting of at least one of the amino acid sequences of SEQ ID NO: 2 (SFHNLHLLF) and SEQ ID NO: 1 (SFHSLHLLF); (P2) a polypeptide consisting of an amino acid sequence in which the fourth amino acid from the N-terminus of (P1) is conserved and 1 to 3 amino acids have been deleted, inserted, substituted or added in the amino acid sequence of (P1); (P3) A polypeptide having an amino acid sequence which conserves the fourth amino acid from the N-terminus of (P1) and has an identity of 80% or more to the amino acid sequence of (P1).

2. The antigen peptide of claim 1, wherein the Tax is Tax-A.

3. The antigen peptide according to claim 1 or 2, wherein the (P2) is a polypeptide consisting of an amino acid sequence in which 1, 2, or 3 amino acids have been added to at least one of the C-terminus and N-terminus of the amino acid sequence of the (P1).

4. The antigen peptide according to claim 1 , wherein the peptide has binding affinity to a human leukocyte antigen (HLA).

5. The antigen peptide of claim 4, wherein the HLA includes HLA-A*24.

6. A nucleic acid encoding an antigenic peptide according to any one of claims 1 to 5.

7. A vector comprising the nucleic acid of claim 6.

8. comprising a complex of a human leukocyte antigen (HLA) and an antigenic peptide, The antigenic peptide is an HLA / antigenic peptide complex comprising an antigenic peptide derived from a Tax protein according to any one of claims 1 to 5.

9. The HLA / antigen peptide complex of claim 8, wherein the HLA comprises HLA-A*24.

10. 10. The HLA / antigen peptide complex of claim 8 or 9, wherein the complex is a multimer of the complex.

11. A detection reagent for the Tax protein encoded by HTLV-1, comprising the HLA / antigen peptide complex according to any one of claims 8 to 10.

12. The detection reagent according to claim 11, wherein the Tax is Tax-A.

13. A pharmaceutical composition comprising an antigenic peptide according to any one of claims 1 to 5 and a pharma- ceutically acceptable carrier.

14. The pharmaceutical composition of claim 13, further comprising an adjuvant.

15. The pharmaceutical composition according to claim 13 or 14, which is a vaccine for use in preventing and / or treating the onset of an HTLV-1 associated disease.

16. An antigenic peptide according to any one of claims 1 to 5, a nucleic acid according to claim 6, a vector according to claim 7, an HLA / antigenic peptide complex according to any one of claims 8 to 10, a detection reagent according to claim 11 or 12, or a pharmaceutical composition according to any one of claims 13 to 15, for use in a method for preventing and / or treating the onset of an HTLV-1-associated disease.

17. a detection step of contacting a biological sample with an HLA / antigenic peptide complex and detecting T cells in the biological sample that bind to the HLA / antigenic peptide complex; A method for detecting T cells, wherein the HLA / antigen peptide complex is an HLA / antigen peptide complex according to any one of claims 8 to 10.

18. The method of claim 17, wherein the HLA / antigen peptide complex comprises a complex of HLA and a peptide derived from Tax-A protein.

19. The method of claim 17 or 18, wherein the biological sample is selected from the group consisting of blood and peripheral blood mononuclear cells.

20. a detection step of contacting a biological sample isolated from a subject with an HLA / antigenic peptide complex, thereby detecting T cells in the biological sample that bind to the HLA / antigenic peptide complex; an evaluation step of evaluating a subtype of the Tax protein encoded by HTLV-1 in the biological sample of the subject based on the detection result of the T cell in the detection step; A method for evaluating a subtype of HTLV-1, wherein the HLA / antigen peptide complex is an HLA / antigen peptide complex according to any one of claims 8 to 10.

21. The evaluation method according to claim 20 , wherein the HLA / antigen peptide complex comprises a complex of HLA and a peptide derived from Tax-A protein.

22. The evaluation method according to claim 20 or 21, wherein the biological sample is selected from the group consisting of blood and peripheral blood mononuclear cells.

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