A therapeutic or prophylactic agent for autoimmune diseases in humans with HLA-DRB1 genotype 09:01.

Abstract

To provide a therapeutic or prophylactic agent for autoimmune diseases in humans with the HLA-DRB1 genotype 09:01. [Solution] A therapeutic or prophylactic agent for human autoimmune diseases, comprising one or more polypeptides having an amino acid sequence with 90% or more sequence identity with the amino acid sequence described in Sequence ID No. 1, and having a total length of 7 to 17 residues. The HLA-DRB1 genotype is 09:01.

Description

[Technical Field]

[0001] This disclosure relates to a therapeutic or prophylactic agent for autoimmune diseases in humans with the HLA-DRB1 genotype 09:01. [Background technology]

[0002] In autoimmune diseases, the immune system, which is normally a mechanism for removing foreign substances originating from outside the body, mistakenly recognizes substances originating from within the body as antigens, leading to abnormal activation of the immune system. This abnormal activation of the immune system is known to cause various abnormalities in the bodily functions of patients suffering from autoimmune diseases.

[0003] Early-onset myasthenia gravis is a type of myasthenia gravis that develops in individuals between the ages of 10 and 49. Myasthenia gravis is designated as a rare disease in Japan and is an organ-specific autoimmune disease against molecules on the postsynaptic membrane at the neuromuscular junction, with muscle weakness being its main symptom.

[0004] In myasthenia gravis, autoantibodies are produced in the patient's body against acetylcholine receptors (AChR) and muscle-specific receptor tyrosine kinases (MuSK), molecules present on the postsynaptic membrane at the neuromuscular junction. These antibodies inhibit neurotransmission, leading to various symptoms. It is known that approximately 80% of myasthenia gravis cases are caused by autoantibodies against AChR.

[0005] In general humoral immunity, antigen-presenting cells use naive CD4 to detect antigens. + By presenting to T cells, naive CD4 + T cells effector CD4 + Antibody production occurs when T cells are differentiated and then activate B cells. In recent years, a new type of antigen-presenting cell has emerged: naive CD4 cells that present antigens. +By presenting to T cells, naive CD4 + T cells, effector CD4 + The existence of antigen-presenting cells that differentiate into regulatory T cells rather than T cells has been reported (Non-patent Literature 1, 2). Such antigen-presenting cells activate regulatory T cells by presenting the antigen, thereby affecting effector CD4 cells that have presented the antigen. + Because it inhibits the activation of B cells by T cells, it has the opposite effect of previously known antigen-presenting cells, suppressing antibody production. In fact, clinical trials using antibody production inhibitors that target this new type of antigen-presenting cell are being conducted for autoimmune diseases (Non-Patent Literature 3).

[0006] In autoimmune diseases, human leukocyte antigens (HLAs), which are protein complexes responsible for presenting antigens on the surface of antigen-presenting cells, recognize a portion of the antigen and form a binding link with it. In particular, when the antigen is a protein, the site on the antigen protein recognized by HLA is called an epitope. Such epitopes hold potential as targets for the treatment and diagnosis of autoimmune diseases, and attempts are being made to identify them in various autoimmune diseases. For example, epitopes in early-onset myasthenia gravis have also been reported (Non-Patent Literature 4). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Laura Passerini and Silvia Gregori.,"Induction of Antigen-Specific Tolerance in T Cell MediatedDiseases", Frontiers in Immunology 11(2194) (2020) [Non-Patent Document 2] Matthias G. von Herrath and Leonard C.Harrison.,"ANTIGEN-INDUCED REGULATORY T CELLS IN AUTOIMMUNITY", NATURE REVIEWIMMUNOLOGY 3:223-232 (2003)) [Non-Patent Document 3] Ciaran P Kelly et al., "TAK-101 Nanoparticles InduceGluten-Specific Tolerance in Celiac Disease: A Randomized, Double-Blind, Placebo-Controlled Study", Gastroenterology 161(1):66-80.e8 (2021). [Non-Patent Document 4] Takayuki Kanai et al, "Immuno-suppressive peptides for a human T cell clone autoreactive to a unique acetylcholinereceptor alpha subunit peptide presented by the disease-susceptible HLA-DQ6 ininfant-onset myasthenia gravis", Hum Immunol56(1-2):28-38 (1997). [Overview of the project] [Problems that the invention aims to solve]

[0008] This disclosure aims to provide a therapeutic or prophylactic agent for autoimmune diseases in humans with the HLA-DRB1 genotype 09:01. [Means for solving the problem]

[0009] The inventors have identified a peptide containing an amino acid sequence corresponding to the epitope of AChR, and have confirmed that such peptide binds to HLA-DRB1*09:01 among human HLA. They have found that an agent containing a polypeptide comprising this amino acid sequence may be applicable to the treatment or diagnosis of humans with HLA-DRB1 genotype 09:01, and have completed this disclosure.

[0010] This disclosure relates, for example, to the following: [1] An activator or activating composition for human regulatory T cells having HLA-DRB1 genotype 09:01, comprising one or more polypeptides having an amino acid sequence with 90% or more sequence identity with the amino acid sequence described in Sequence ID No. 1, and having a total length of 7 to 17 residues. [2] A composition for inhibiting or suppressing the activity of human effector T cells having HLA-DRB1 genotype 09:01, comprising one or more polypeptides having an amino acid sequence with 90% or more sequence identity with the amino acid sequence described in Sequence ID No. 1, and having a total length of 7 to 17 residues. [3] A composition for inhibiting or suppressing the activation of human B cells, comprising one or more polypeptides having an amino acid sequence with 90% or more sequence identity with the amino acid sequence described in Sequence ID No. 1, and having a total length of 7 to 17 residues. [4] An inhibitor or composition for suppressing humoral immunity in humans, comprising one or more polypeptides having an amino acid sequence with 90% or more sequence identity with the amino acid sequence described in Sequence ID No. 1, and having a total length of 7 to 17 residues. [5] A therapeutic agent, prophylactic agent, therapeutic composition, or prophylactic composition for human autoimmune diseases, comprising one or more polypeptides having an amino acid sequence with 90% or more sequence identity with the amino acid sequence described in Sequence ID No. 1, and having a total length of 7 to 17 residues. [6]A method for treating or preventing an autoimmune disease, comprising administering to a human subject with HLA-DRB1 genotype 09:01 one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and having a full length of 7 to 17 residues. [7]One or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and having a full length of 7 to 17 residues, for use in the treatment or prevention of an autoimmune disease in a human subject with HLA-DRB1 genotype 09:01. [8]Use of one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and having a full length of 7 to 17 residues in the manufacture of a therapeutic agent, prophylactic agent, therapeutic composition or prophylactic composition for an autoimmune disease in a human subject with HLA-DRB1 genotype 09:01. [9]One or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and having a full length of 7 to 17 residues, for use in the manufacture of a therapeutic agent, prophylactic agent, therapeutic composition or prophylactic composition for an autoimmune disease in a human subject with HLA-DRB1 genotype 09:01.

[10] A kit for diagnosing an autoimmune disease in a human subject with HLA-DRB1 genotype 09:01, comprising one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and having a full length of 7 to 17 residues, and a human leukocyte antigen that binds to at least one of the polypeptides.

[11] The kit according to

[10] , further comprising a labeling dye that binds to the human leukocyte antigen.

[12] A diagnostic agent for an autoimmune disease in a human subject with HLA-DRB1 genotype 09:01, comprising a complex comprising a polypeptide, a human leukocyte antigen and a labeling dye, wherein the polypeptide is one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and having a full length of 7 to 17 residues.

[13] A method for assisting in the diagnosis of an autoimmune disease in a human subject with an HLA-DRB1 genotype of 09:01, which includes detecting T cells having antigen specificity for one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and having a full length of 7 to 17 residues.

[14] A method for obtaining data for the diagnosis of an autoimmune disease in a human subject with an HLA-DRB1 genotype of 09:01, which includes detecting T cells having antigen specificity for one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and having a full length of 7 to 17 residues.

[15] The activator, composition, activity inhibitor, activation inhibitor, inhibitor, therapeutic agent, prophylactic agent, method, at least one polypeptide, use, kit or diagnostic agent according to any one of [1] to

[14] , wherein the one or more polypeptides are one or more polypeptides having 90% or more sequence identity to an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 5 to 15.

[16] The therapeutic agent, prophylactic agent, composition, method, at least one polypeptide, use, kit or diagnostic agent according to any one of [5] to

[15] , wherein the autoimmune disease is myasthenia gravis.

[17] The therapeutic agent, prophylactic agent, composition, method, at least one polypeptide, use, kit or diagnostic agent according to any one of [5] to

[15] , wherein the autoimmune disease is early-onset myasthenia gravis.

[18] A T cell receptor that binds to a complex of HLA-DRB1*09:01 and a polypeptide having a total length of 7 to 17 residues and containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO: 1, wherein the T cell receptor comprises an α chain and a β chain, the variable region of the α chain comprises a complementarity-determining region (CDR) 1α consisting of the amino acid sequence described in SEQ ID NO: 27, CDR2α consisting of the amino acid sequence described in SEQ ID NO: 28, and CDR3α consisting of the amino acid sequence described in SEQ ID NO: 29, and the variable region of the β chain comprises CDR1β consisting of the amino acid sequence described in SEQ ID NO: 30, CDR2β consisting of the amino acid sequence described in SEQ ID NO: 31, and CDR3β consisting of the amino acid sequence described in SEQ ID NO: 32.

[19] The T cell receptor according to

[18] , wherein the variable region of the α chain contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 33, and the variable region of the β chain contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 34.

[20] A T cell receptor that binds to a complex of HLA-DRB1*09:01 and a polypeptide having an amino acid sequence with 90% or more sequence identity with the amino acid sequence described in SEQ ID NO: 1 and having a total length of 7 to 17 residues, wherein the T cell receptor comprises an α chain and a β chain, the variable region of the α chain comprises CDR1α consisting of the amino acid sequence described in SEQ ID NO: 39, CDR2α consisting of the amino acid sequence described in SEQ ID NO: 40, and CDR3α consisting of the amino acid sequence described in SEQ ID NO: 41, and the variable region of the β chain comprises CDR1β consisting of the amino acid sequence described in SEQ ID NO: 42, CDR2β consisting of the amino acid sequence described in SEQ ID NO: 43, and CDR3β consisting of the amino acid sequence described in SEQ ID NO: 44.

[21] The T cell receptor according to

[20] , wherein the variable region of the α chain contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 45, and the variable region of the β chain contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 46. A nucleic acid encoding a T cell receptor, as described in any one of

[22]

[18] ~

[21] . Regulatory T cells expressing any one of the T cell receptors described in

[23]

[18] ~

[21] . A therapeutic or prophylactic agent for human autoimmune diseases, including regulatory T cells as described in

[24]

[23] , wherein the HLA-DRB1 genotype is 09:01.

[25] The polypeptide is a polypeptide having 90% or more sequence identity with an amino acid sequence selected from the group consisting of amino acid sequences described in SEQ ID NOs. 5 to 15, the T cell receptor, nucleic acid, regulatory T cell, therapeutic agent or prophylactic agent according to any one of

[18] to

[24] .

[26] The therapeutic or prophylactic agent according to

[24] or

[25] , wherein the autoimmune disease is myasthenia gravis.

[27] The therapeutic or prophylactic agent according to

[24] or

[25] , wherein the autoimmune disease is early-onset myasthenia gravis. [Effects of the Invention]

[0011] The present disclosure provides an agent that may be applied to the treatment or diagnosis of an autoimmune disease in humans having an HLA-DRB1 genotype of 09:01, comprising a polypeptide containing an amino acid sequence corresponding to an AChR epitope. [Brief explanation of the drawing]

[0012] [Figure 1] This figure shows the results of ICS (left) and ELISA (right) in evaluating the peptide-specific Th1 cell induction ability of SEQ ID NO: 2 using peripheral blood mononuclear cells (PBMCs) derived from healthy individuals that possess HLA-DRB1*09:01, which has been reported to be associated with myasthenia gravis (MG) in Asians, but lack DRB1*03:01 and DRB3*01:01. [Figure 2] This figure shows the results of an inductive cytological study (ICS) using DRB1*09:01-positive PBMCs derived from healthy individuals. For the triplet measurement (N=3) samples, the standard deviation is shown as an error bar, and the p-value was calculated using a t-test (***: p<0.005, Ψ: p<0.0005, NS: no significant difference). [Figure 3]This figure shows the results of an ELISA (ELISA) evaluation test for specific T cell induction ability using DRB1*09:01-positive PBMCs derived from healthy individuals. Error bars in the ELISA measurement values ​​indicate the standard deviation, and the p-value was calculated using a t-test (***: p<0.005, Ψ: p<0.0005, NS: no significant difference). [Figure 4] This figure shows the results of the peptide reactivity evaluation test (ELISA) of the acquired Th1 clones. [Figure 5] This figure shows the results of the DRB1*09:01 restriction evaluation test (ELISA) for the acquired Th1 clone (5B12). [Figure 6] This figure shows the results of the DRB1*09:01 restriction evaluation test (ELISA) for the acquired Th1 clone (6F5). [Modes for carrying out the invention]

[0013] The following describes the forms for implementing this disclosure, but this disclosure is not limited to the following embodiments.

[0014] <Sequence Identity> In this disclosure, “sequence identity” means the percentage (%) of identical residues in the overlapping amino acid sequences in the optimal alignment when two amino acid sequences are aligned. In this disclosure, mutations in an amino acid sequence that does not have 100% sequence identity with respect to a given amino acid sequence may be, for example, substitutions, deletions, or insertions of amino acid residues, and more specifically, may be, for example, a single-residue substitution, a single-residue deletion, or a single-residue insertion, and these deletions or insertions may occur at the terminal or elsewhere in the amino acid sequence.

[0015] <Human leukocyte antigen> Human leukocyte antigens (HLAs) are major histocompatibility complexes (MHCs) in humans. In humoral immunity, HLAs are exposed to the surface of antigen-presenting cells (APCs) while bound to antigens, thereby driving the antibody production mechanism against those antigens. HLAs are classified into HLA class I, which includes HLA-A, HLA-C, and HLA-B and is responsible for cellular immunity, and HLA class II, which includes HLA-DR, HLA-DQ, and HLA-DP and is responsible for humoral immunity.

[0016] Human white blood cells have tens of thousands of possible genotypes depending on the HLA genotype. HLA genotype is one of the risk factors in autoimmune diseases, and it is known that people with HLA-specific genotypes have a higher likelihood of developing autoimmune diseases. In particular, in early-onset myasthenia gravis, an HLA-DRB3 genotype of 01:01 or an HLA-DRB1 genotype of 03:01 are known risk factors.

[0017] According to the notation established by the WHO Nomenclature Committee for Factors of the HLA System, the HLA genotype (allyle) is expressed in the format "HLA-(gene symbol)*(region 1):(region 2):(region 3):(region 4)". Here, regions 1 through 4 are each represented by a two-digit number. Region 1 represents HLA specificity (antigen type), region 2 represents non-synonymous substitutions (variations in the amino acid sequence within the same antigen type), region 3 represents synonymous substitutions (variations in the exon region that do not change the coding amino acid sequence), and region 4 represents extracoding base substitutions (variations in the intron region sequence). In these notations, the gene symbol, region 1, and region 2 are clinically important information that directly affects the amino acid sequence of HLA.

[0018] In this disclosure, when the genotype of an HLA gene with the gene symbol XXX has a first region of YY and a second region of ZZ (i.e., the first two regions in the above notation are represented as HLA-XXX*YY:ZZ), it is also stated as "the genotype of HLA-XXX is YY:ZZ." That is, for example, "the genotype of HLA-DRB1 is 09:01" means that the genotype of an HLA gene with the gene symbol DRB1 (HLA-DRB1 gene) has a first region of 09 and a second region of 01, i.e., the genotype is HLA-DRB1*09:01.

[0019] <Acetylcholine receptor> Acetylcholine receptors (AChRs) are essential receptors for neurotransmission, playing a crucial role in the transmission of information between nerve cells through the binding of extracellularly secreted acetylcholine. In autoimmune diseases where antibodies against acetylcholine receptors are overproduced, a portion of neurotransmission mediated by acetylcholine receptors is inhibited, leading to various symptoms. Furthermore, the α1 subunit of the human acetylcholine receptor (human AChRα1) is known to be recognized as an autoantigen in approximately 80% of patients with early-onset myasthenia gravis. Human AChRα1 has the amino acid sequence (SEQ ID NO: 16) with accession number NP_000070.1 from the National Center for Biotechnology Information (NCBI).

[0020] <Activators or compositions for activating regulatory T cells> The first aspect of this disclosure relates to an activator for human regulatory T cells with HLA-DRB1 genotype 09:01, comprising one or more polypeptides having a total length of 7 to 17 residues and containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO: 1. The first aspect of this disclosure may also, in one embodiment, be an activating composition for human regulatory T cells with HLA-DRB1 genotype 09:01, comprising at least one of the above polypeptides.

[0021] If one or more of the above-mentioned polypeptides contain or consist of an amino acid sequence that has 90% or more sequence identity with a predetermined sequence (for example, the sequence represented by SEQ ID NOs: 1 to 15, or a part of the sequence represented by SEQ ID NO: 17), the amino acid sequence may have more than 90% sequence identity with the predetermined sequence, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity. Furthermore, if one or more of the above-mentioned polypeptides contain or consist of an amino acid sequence that has 90% or more sequence identity with a predetermined sequence (for example, the sequence represented by SEQ ID NOs: 1 to 15, or a part of the sequence represented by SEQ ID NO: 17), the amino acid sequence may be, for example, the predetermined sequence or an amino acid sequence that contains one, two, or three residue mutations in the predetermined sequence, and in one preferred embodiment, it may be the predetermined sequence or an amino acid sequence that contains one residue mutation in the predetermined sequence.

[0022] The amino acid sequence described in Sequence ID No. 1 corresponds to amino acids 334-343 of the amino acid sequence (Sequence ID No. 16) of the α1 subunit of the human acetylcholine receptor, whose Ref seq accession number is NP_000070.1 according to the National Center for Biotechnology Information (NCBI). Furthermore, the amino acid sequence described in Sequence ID No. 1 corresponds to amino acids 314-323 of the amino acid sequence (Sequence ID No. 17) obtained by removing amino acids 1-20, which correspond to the signal peptide, from the amino acid sequence shown in Sequence ID No. 16. Sequence ID 1: KVFIDTIPNI

[0023] In one embodiment of the present disclosure, the total length of the above one or more polypeptides may be, for example, 7 residues or more, 8 residues or more, 9 residues or more, 10 residues or more, 11 residues or more, 12 residues or more, 13 residues or more, or 14 residues or more, and may be 17 residues or less, 16 residues or more, 15 residues or less, 14 residues or less, 13 residues or less, 12 residues or less, or 11 residues or less. Also, in one embodiment of the present disclosure, the total length of the above one or more polypeptides may be, for example, 7 residues or more and 17 residues or less, 8 residues or more and 17 residues or less, 9 residues or more and 17 residues or less, 10 residues or more and 17 residues or less, 11 residues or more and 16 residues or less, 11 residues or more and 14 residues or less, 11 residues or more and 13 residues or less, 11 residues or more and 12 residues or less, or 11 residues. In a preferred embodiment, the total length of the above one or more polypeptides may be 11 residues or more and 16 residues or less.

[0024] In one embodiment of the present disclosure, the one or more polypeptides described above include an amino acid sequence having 90% or more, 95% or more, 98% or more, or 100% sequence identity with the amino acid sequence described in SEQ ID NO: 1. In one embodiment of the present disclosure, the one or more polypeptides described above include an amino acid sequence described in SEQ ID NO: 1 or an amino acid sequence having one, two, or three residue mutations in said amino acid sequence.

[0025] In one embodiment of the present disclosure, the one or more polypeptides described above may contain an amino acid sequence having 90% or more sequence identity with at least one selected from the group consisting of amino acid positions 311-327, 312-327, 313-327, 314-327, 311-326, 312-326, 313-326, 314-326, 311-325, 312-325, 313-325, 314-325, 311-324, 312-324, 313-324 and 314-324 of the amino acid sequence shown in SEQ ID NO: 17, or consist of an amino acid sequence having 90% or more sequence identity with the at least one, or consist of the at least one amino acid sequence. For example, amino acid sequences 311 to 327 in SEQ ID NO: 17 are the same as the amino acid sequence shown in SEQ ID NO: 2. In one embodiment of the present disclosure, the above one or more polypeptides may include an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, or consist of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2, or consist of such an amino acid sequence. Sequence ID 2: WVRKVFIDTIPNIMFFS

[0026] In one embodiment of this disclosure, the one or more polypeptides described above may be one or more polypeptides having a sequence identity of 90% or more with an amino acid sequence selected from the group consisting of amino acid sequences described in SEQ ID NOs. 5 to 15, and may be one or more polypeptides consisting of an amino acid sequence selected from the group consisting of amino acid sequences described in SEQ ID NOs. 5 corresponds to amino acid positions 311 to 323 of the amino acid sequence shown in SEQ ID NO. 17. 6 corresponds to amino acid positions 311 to 324 of the amino acid sequence shown in SEQ ID NO. 17. 7 corresponds to amino acid positions 312 to 324 of the amino acid sequence shown in SEQ ID NO. 17. 8 corresponds to amino acid positions 312 to 325 of the amino acid sequence shown in SEQ ID NO. 17. 9 corresponds to amino acid positions 312 to 327 of the amino acid sequence shown in SEQ ID NO. 17. 10 corresponds to amino acid positions 313 to 324 of the amino acid sequence shown in SEQ ID NO. 17. The amino acid sequence shown in SEQ ID NO: 11 corresponds to amino acids 313-325 in the amino acid sequence shown in SEQ ID NO: 17. The amino acid sequence shown in SEQ ID NO: 12 corresponds to amino acids 313-326 in the amino acid sequence shown in SEQ ID NO: 17. The amino acid sequence shown in SEQ ID NO: 13 corresponds to amino acids 313-327 in the amino acid sequence shown in SEQ ID NO: 17. The amino acid sequence shown in SEQ ID NO: 14 corresponds to amino acids 314-324 in the amino acid sequence shown in SEQ ID NO: 17. The amino acid sequence shown in SEQ ID NO: 15 corresponds to amino acids 314-326 in the amino acid sequence shown in SEQ ID NO: 17. Sequence ID 5: WVRKVFIDTIPNI Sequence ID 6: WVRKVFIDTIPNIM Sequence ID 7: VRKVFIDTIPNIM Sequence ID 8: VRKVFIDTIPNIMF Sequence ID 9: VRKVFIDTIPNIMFFS Sequence ID 10: RKVFIDTIPNIM Sequence ID 11: RKVFIDTIPNIMF Sequence ID 12: RKVFIDTIPNIMFF Sequence ID 13: RKVFIDTIPNIMFFS Sequence ID 14: KVFIDTIPNIM Sequence ID 15: KVFIDTIPNIMFF

[0027] The above polypeptide binds to HLA-DR. Therefore, although we do not wish to be bound by any theory, by contacting the above polypeptide with antigen-presenting cells that have the effect of promoting the activation (differentiation) of regulatory T cells as described in Non-Patent Document 1 and allowing phagocytosis, the polypeptide will be bound to HLA-DR and naive CD4 + It is thought that this can be presented to T cells, thereby triggering the activation (differentiation) of regulatory T cells.

[0028] The agent according to one embodiment may further contain, in addition to the polypeptide described above, an additive commonly used in the pharmaceutical technology, which may be at least one selected from the group consisting of, for example, excipients, buffers, stabilizers, antioxidants, binders, disintegrants, fillers, emulsifiers, and flow additive modifiers.

[0029] The components contained in the agent according to one embodiment may be encapsulated in a drug carrier intended for intracellular delivery. Examples of such drug carriers include metal nanoparticles (e.g., gold nanoparticles), liposomes, and capsids.

[0030] The dosage form of the agent according to one embodiment is not particularly limited and may be, for example, a liquid or a tablet. However, if the components contained in the agent according to one embodiment are encapsulated in a drug carrier intended for intracellular delivery, the dosage form is preferably a liquid.

[0031] The method of administration of the agent according to one embodiment is not particularly limited and can include, for example, intramedullary administration, intravenous injection, subcutaneous injection, intramuscular injection, oral administration, or spinal injection. As an example of a specific dosage, for example, when administered to an adult male (weighing 60 kg), the daily dose of the agent may be 0.001 μg to 1000 mg / day / person in terms of the amount of active ingredient.

[0032] The content of the polypeptide in the agent according to one embodiment is not particularly limited, and for example, the total content may be 0.001 to 100% by mass based on the total amount of the agent.

[0033] The target population for administration of the agent according to one embodiment is a human subject with an HLA-DRB1 genotype of 09:01, and may be a human subject who has, may have had, or has a genetic predisposition to an autoimmune disease. The autoimmune disease may be, for example, an autoimmune disease caused by the overproduction of antibodies against human AchRα1 or its peptide fragment, and is preferably myasthenia gravis, and more preferably early-onset myasthenia gravis. Myasthenia gravis is broadly classified into early-onset myasthenia gravis, which develops in subjects aged 10 to 49 years, and late-onset myasthenia gravis, which develops in subjects aged 50 years or older, and there are some differences in the HLA genotypes that can be risk factors between the two.

[0034] The above polypeptide was discovered using the NAPA method with dendritic cells prepared from human classical monocytes, and is highly likely to actually exist in the bodies of patients suffering from autoimmune diseases. Therefore, it has the potential to be more suitably applied to the treatment or diagnosis of autoimmune diseases than polypeptides with sequences corresponding to epitopes that have been discovered to date. The suitability of the above polypeptide for the treatment or diagnosis of autoimmune diseases compared to polypeptides with sequences corresponding to epitopes that have been discovered to date (other polypeptides) can be evaluated, for example, by the fact that the above polypeptide exhibits a higher T cell activation effect when it comes into contact with T cells than the other polypeptides. Such T cell activation effect can be evaluated, for example, by a T cell activation test. A T cell activation test is a test that confirms whether T cells present in PBMCs are activated by exposure to the polypeptide using an ELISA such as IFN-γ. When a polypeptide with a sequence corresponding to the discovered epitope is added to human PBMCs, it is presented as an epitope by antigen-presenting cells within the PBMC. T cells that have a T cell receptor (TCR) capable of capturing this HLA-binding polypeptide receive a signal and release activating cytokines such as IFN-γ. Activation is determined by measuring the released cytokines. Such assays can be performed using commercially available kits, for example, the BD OptEIA® Human IFN-γ ELISA Set (BD Biosciences, Cat. No. 555142).

[0035] <Inhibitors or compositions for inhibiting the activity of effector T cells> The second aspect of the present disclosure relates to an inhibitor of the activity of effector T cells in a human subject with the genotype of HLA-DRB1 being 09:01, which contains the above polypeptide. In one aspect, the second aspect of the present disclosure may also be a composition for inhibiting the activity of effector T cells in a human subject with the genotype of HLA-DRB1 being 09:01, which contains the above polypeptide. As the inhibitor of the activity of effector T cells according to the second aspect of the present disclosure, those similar to those described in the activator of regulatory T cells according to the first aspect of the present disclosure can be used, and the explanations and definitions described in the section of the activator of regulatory T cells according to the first aspect of the present disclosure are also incorporated into the inhibitor of the activity of effector T cells according to the second aspect of the present disclosure.

[0036] Although not wishing to be bound by any theory, the inhibitor of the activity of effector T cells containing the above polypeptide is considered to be able to inhibit the activity of effector T cells by promoting the activation (differentiation) of regulatory T cells through the function of antigen-presenting cells as described in Non-Patent Document 1, as described in the first aspect of the present disclosure.

[0037] <Inhibitor or composition for inhibiting activation of B cells>[ The third aspect of the present disclosure relates to an inhibitor of the activation of B cells in a human subject with the genotype of HLA-DRB1 being 09:01, which contains the above polypeptide. In one aspect, the third aspect of the present disclosure may also be a composition for inhibiting the activation of B cells in a human subject with the genotype of HLA-DRB1 being 09:01, which contains the above polypeptide. As the inhibitor of the activation of B cells according to the third aspect of the present disclosure, those similar to those described in the activator of regulatory T cells according to the first aspect of the present disclosure can be used, and the explanations and definitions described in the section of the activator of regulatory T cells according to the first aspect of the present disclosure are also incorporated into the inhibitor of the activation of B cells according to the third aspect of the present disclosure.

[0038] While we do not wish to be bound by any theory, it is thought that B cell activation inhibitors, including the polypeptides described above, can inhibit the activity of effector T cells through the action of antigen-presenting cells as described in Non-Patent Document 1, as explained in Aspect II of this disclosure, and thereby inhibit B cell activation.

[0039] <Suppressants or compositions for suppressing humoral immunity> The fourth aspect of this disclosure relates to a humoral immunity inhibitor (e.g., an antibody production inhibitor) for human subjects with HLA-DRB1 genotype 09:01, comprising the polypeptide described above. In one embodiment, the fourth aspect of this disclosure may also be a composition for suppressing humoral immunity for human subjects with HLA-DRB1 genotype 09:01, comprising the polypeptide described above. The humoral immunity inhibitor relating to the fourth aspect of this disclosure may be the same as those described in the regulatory T cell activators relating to the first aspect of this disclosure, and the descriptions and definitions described in the section on regulatory T cell activators relating to the first aspect of this disclosure shall also apply to the humoral immunity inhibitor relating to the fourth aspect of this disclosure.

[0040] While we do not wish to be bound by any theory, it is thought that humoral immunity suppressors, including the polypeptides described above, can suppress humoral immunity by inhibiting B cell activation through the function of antigen-presenting cells as described in Non-Patent Document 1, as explained in the third aspect of this disclosure.

[0041] <Therapeutic agents, preventive agents, therapeutic compositions, or preventive compositions for autoimmune diseases> The fifth aspect of this disclosure relates to a therapeutic or prophylactic agent for autoimmune diseases in humans with HLA-DRB1 genotype 09:01, comprising the polypeptide described above. The fifth aspect of this disclosure may, in one embodiment, also be a therapeutic or prophylactic composition for autoimmune diseases in humans with HLA-DRB1 genotype 09:01, comprising the polypeptide described above. The fifth aspect of this disclosure may, in one embodiment, also be a method for treating or prophylacticing an autoimmune disease, comprising administering the polypeptide described above to a human subject with HLA-DRB1 genotype 09:01. The fifth aspect of this disclosure may, in one embodiment, also be the polypeptide described above for use in the treatment or prophylactic use of autoimmune diseases in humans with HLA-DRB1 genotype 09:01. The fifth aspect of this disclosure may, in one embodiment, also be the use of the polypeptide in the manufacture of a therapeutic, prophylactic agent, therapeutic composition, or prophylactic composition for autoimmune diseases in humans with HLA-DRB1 genotype 09:01. Aspect five of this disclosure may, in one embodiment, also be the polypeptide described above for use in the manufacture of therapeutic agents, prophylactic agents, therapeutic compositions, or prophylactic compositions for autoimmune diseases in humans with the HLA-DRB1 genotype 09:01. The therapeutic or prophylactic agents for autoimmune diseases relating to Aspect five of this disclosure may be the same as those described in the section on regulatory T cell activators relating to Aspect one of this disclosure, and the descriptions and definitions described in the section on regulatory T cell activators relating to Aspect one of this disclosure shall also apply to the therapeutic or prophylactic agents for autoimmune diseases relating to Aspect five of this disclosure.

[0042] The autoimmune disease relating to the fifth aspect of this disclosure may be, for example, an autoimmune disease caused by the overproduction of antibodies against human AchRα1 or its peptide fragment, preferably myasthenia gravis, and more preferably early-onset myasthenia gravis. Since the polypeptide is a peptide fragment containing an amino acid sequence corresponding to the epitope of human AchRα1, which is known to be recognized as an autoantigen in myasthenia gravis and early-onset myasthenia gravis, the agent according to one embodiment of the fifth aspect of this disclosure can suitably treat and prevent myasthenia gravis and early-onset myasthenia gravis.

[0043] A therapeutic or prophylactic agent according to one embodiment of the fifth aspect of this disclosure is for administration to human subjects having the HLA-DRB1 genotype 09:01, and may also be for administration to human subjects who have, may have, or have a genetic predisposition to an autoimmune disease, and the autoimmune disease may be, for example, an autoimmune disease caused by the overproduction of antibodies against human AchRα1 or its peptide fragment, preferably myasthenia gravis, and more preferably early-onset myasthenia gravis. Furthermore, if the agent according to one embodiment of the fifth aspect of this disclosure is a therapeutic agent, the target of administration may be, for example, a human subject who has or may have an autoimmune disease. Furthermore, if the agent according to one embodiment of the fifth aspect of this disclosure is a prophylactic agent, the target of administration may be a human subject who has or may have a genetic predisposition to an autoimmune disease.

[0044] While we do not wish to be bound by any theory, the above polypeptide-containing therapeutic or prophylactic agents for autoimmune diseases can suppress humoral immunity through the action of antigen-presenting cells as described in Non-Patent Literature 1, as explained in the fourth aspect of this disclosure. Therefore, it is thought that these agents can treat and prevent autoimmune diseases, for example, by suppressing the production of anti-AchRα1 antibodies, thereby suppressing the degradation of AchRα1 and thus treating and preventing myasthenia gravis, such as early-onset myasthenia gravis.

[0045] <Methods to assist in the diagnosis of autoimmune diseases, methods for obtaining data for diagnosis, and diagnostic methods> A sixth aspect of this disclosure is a method to aid in the diagnosis of an autoimmune disease in a human subject with the HLA-DRB1 genotype 09:01, comprising detecting T cells having antigen specificity to the polypeptide. A sixth aspect of this disclosure may, in one embodiment, also be a method for obtaining data for the diagnosis of an autoimmune disease in a human subject with the HLA-DRB1 genotype 09:01, comprising detecting T cells having antigen specificity to the polypeptide. A sixth aspect of this disclosure may, in one embodiment, also be a diagnostic method for an autoimmune disease in a human subject with the HLA-DRB1 genotype 09:01, comprising detecting T cells having antigen specificity to the polypeptide. In these cases, the autoimmune disease may be, for example, an autoimmune disease resulting from the overproduction of antibodies against human AchRα1 or its peptide fragments, preferably myasthenia gravis, and more preferably early-onset myasthenia gravis. In these cases, the polypeptide may be the same as that described in the section on regulatory T cell activators relating to Aspect I of this Disclosure, and the description and definition of the at least one polypeptide described in the section on regulatory T cell activators relating to Aspect I of this Disclosure shall also apply to the at least one polypeptide relating to the methods relating to Aspect Six of this Disclosure.

[0046] The subjects to whom the method according to one embodiment of the sixth aspect of this disclosure is used are human subjects with an HLA-DRB1 genotype of 09:01, and may be humans who have a genetic predisposition to or are prone to an autoimmune disease, and the autoimmune disease may be, for example, an autoimmune disease caused by the overproduction of antibodies against human AchRα1 or its peptide fragment, preferably myasthenia gravis, and more preferably early-onset myasthenia gravis. Furthermore, the subjects to whom the method according to one embodiment of the sixth aspect of this disclosure is used may also be humans who do not have a genetic predisposition to or are prone to such an autoimmune disease, and in that case, the method according to one embodiment may be used for comprehensive exploration of the possibility of contracting the disease (so-called screening test).

[0047] In one embodiment of the sixth aspect of this disclosure, if T cells having antigen specificity to the polypeptide are detected in a sample taken from a subject, it may be used to assist in the diagnosis, obtain diagnostic data, or make a diagnosis that the subject is likely to have an autoimmune disease (e.g., an autoimmune disease caused by overproduction of antibodies against human AchRα1 or its peptide fragments, preferably myasthenia gravis, more preferably early-onset myasthenia gravis). Also in one embodiment of the sixth aspect of this disclosure, if the amount of T cells having antigen specificity to the polypeptide is greater in a sample taken from a subject than in a sample taken from a control (e.g., a healthy person) that is not suffering from an autoimmune disease (e.g., an autoimmune disease caused by overproduction of antibodies against human AchRα1 or its peptide fragments, preferably myasthenia gravis, more preferably early-onset myasthenia gravis), it may be used to assist in the diagnosis, obtain diagnostic data, or make a diagnosis that the subject is likely to have the autoimmune disease.

[0048] The sample used to measure the amount of T cells having antigen specificity to the polypeptide is not limited to any sample that can be obtained from the subject and can be used to assist in the diagnosis of autoimmune disease, to obtain diagnostic data, or to make a diagnosis based on the amount of T cells having antigen specificity to the polypeptide. For example, it may be bone marrow fluid, cerebrospinal fluid, lymph fluid, blood, plasma, serum, or saliva. In a preferred embodiment, it may be bone marrow fluid, which may be collected from, for example, the ilium or sternum.

[0049] In the sixth aspect of this disclosure, the "amount" of T cells having antigen specificity for the polypeptide is not limited to the concentration of the T cells, but may also be the intensity of a signal that can be used as an indicator of the amount of the T cells. For example, if the T cells are labeled with a polypeptide, the amount may be, for example, the intensity of a signal that can evaluate the amount of the labeled polypeptide, or, if the polypeptide is fluorescently labeled, the fluorescence intensity detected from the T cells. Also, if the T cells are labeled with a polypeptide-human leukocyte antigen complex, the amount may be, for example, the intensity of a signal that can evaluate the amount of the labeled complex, or, if the human leukocyte antigen complex is fluorescently labeled, the fluorescence intensity detected from the T cells. In these cases, the fluorescence intensity may be, for example, the fluorescence intensity measured by a microwell plate reader or a flow cytometer.

[0050] The detection method for detecting T cells having antigen specificity to the above polypeptide is not particularly limited as long as it is a method capable of detecting the T cells. For example, it can be carried out by labeling the T cells with a fluorescently labeled polypeptide or a complex of the polypeptide and a fluorescently labeled human leukocyte antigen, and then measuring the fluorescence intensity of the fluorescent dye in the cells contained in the sample.

[0051] In a preferred embodiment, T cells having antigen specificity to the polypeptide may be detected by labeling the T cells with a fluorescently labeled polypeptide-human leukocyte antigen complex (polypeptide-human leukocyte antigen complex) multimer (e.g., tetramer), and then measuring the fluorescence intensity of the fluorescent dye in the cells contained in the sample. When the T cells are labeled with a polypeptide-human leukocyte antigen complex multimer, the complex and the T cells can form a stable bond, allowing for more accurate detection by removing the nonspecifically bound complex through washing or other means. Such polypeptide-human leukocyte antigen complexes can be prepared, for example, using the polypeptide and a commercially available kit that can be used to create polypeptide-human leukocyte antigen complex multimers as described above (e.g., Quick Switch® series, MBL Life Sciences).

[0052] <Diagnostic kit for autoimmune diseases> The seventh aspect of this disclosure is a diagnostic kit for autoimmune diseases in humans with HLA-DRB1 genotype 09:01, comprising the polypeptide and a human leukocyte antigen bound to the polypeptide. In one embodiment, the kit according to the seventh aspect of this disclosure may further comprise a labeling dye bound to the human leukocyte antigen. In one embodiment, the kit according to the seventh aspect of this disclosure may comprise the human leukocyte antigen and the labeling dye as a complex, or the human leukocyte antigen and the labeling dye as a complex, or the polypeptide, the human leukocyte antigen and the labeling dye as a complex, or a polymer of the human leukocyte antigen and the labeling dye as a complex, or a polymer of the polypeptide-human leukocyte antigen complex and the labeling dye as a complex. That is, in one embodiment, the seventh aspect of this disclosure may also be a diagnostic agent for autoimmune diseases comprising a complex comprising a polypeptide, a human leukocyte antigen and a labeling dye. In these cases, the labeling dye may be a fluorescent dye. In these cases, the autoimmune disease may be, for example, an autoimmune disease caused by the overproduction of antibodies against human AchRα1 or its peptide fragments, and is preferably myasthenia gravis, and more preferably early-onset myasthenia gravis.

[0053] A kit relating to Aspect VII of this Disclosure can be used for the diagnosis of autoimmune diseases according to a method according to one embodiment of Aspect VII of this Disclosure. In one embodiment, a kit relating to Aspect VII of this Disclosure may further include a document, which may be electronic, and which may contain a protocol corresponding to a method according to one embodiment of Aspect VII of this Disclosure.

[0054] <Variation> The eighth aspect of this disclosure relates to agents, methods, and kits described above, wherein the agents, methods, and kits contain, instead of the polypeptide, a molecule containing the polypeptide as a substructure (hereinafter also referred to as the "molecule relating to the eighth aspect"). Such agents and kits can be implemented in the same manner as described above for the agents relating to the first to fifth aspects and the kit relating to the seventh aspect, except that they contain the molecule relating to the eighth aspect instead of the polypeptide. The molecule relating to the eighth aspect may be a non-natural molecule, for example, an artificial molecule.

[0055] The molecule relating to the eighth aspect may be, for example, a molecule (tandem molecule) in which at least two of the above polypeptides are directly or indirectly linked by peptide bonds. The tandem molecule may be linear or cyclic. The tandem molecule may be a molecule (tandem molecule) in which, for example, two, three, four, or five or more of the above polypeptides are directly or indirectly linked by peptide bonds. Examples of a tandem molecule in which at least two polypeptides are directly linked by peptide bonds include a polypeptide consisting of two consecutive amino acid sequences as shown in SEQ ID NO: 2, as shown in SEQ ID NO: 3, or a cyclic polypeptide in which peptide bonds are formed at both ends. A tandem molecule in which at least two polypeptides are indirectly linked by peptide bonds may be, for example, a molecule in which at least two polypeptides are linked by a peptide linker. Examples of peptide linkers include those disclosed in, for example, International Application No. 2019 / 233842, and specifically, GGGS (SEQ ID NO: 18), GGGGS (SEQ ID NO: 19), and the autodigestible peptides T2A (EGRGSLLTCGDVEENPGP (SEQ ID NO: 20) and GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 21)). Tandem molecules in which at least two polypeptides are indirectly linked by a peptide bond include, for example, polypeptides consisting of an amino acid sequence in which GGGS is inserted between the two amino acid sequences shown in SEQ ID NO: 2, as shown in SEQ ID NO: 4. Sequence ID 3: WVRKVFIDTIPNIMFFSWVRKVFIDTIPNIMFFS Accession number 4: WVRKVFIDTIPNIMFFSGGGSWVRKVFIDTIPNIMFFS

[0056] The molecule related to the eighth side surface may be, for example, a molecule containing the above polypeptide and a structure that is not a peptide as partial structures. Such a molecule may be, for example, a molecule in which at least two of the above polypeptides are bound via a bond other than a peptide bond, and such a molecule may further have a structure that is not a peptide at the peptide terminus not bound to a linker. Such a bond other than a peptide bond may be a covalent bond (e.g., carbon-carbon bond), or may be a bond by a non-covalent interaction typified by the interaction between biotin and streptavidin.

[0057] <T cell receptor> The ninth aspect of the present disclosure is a T cell receptor that binds to a complex of HLA-DRB1*09:01 and a polypeptide having an amino acid sequence with 90% or more sequence identity to the amino acid sequence described in SEQ ID NO: 1 and having a full length of 7 to 17 residues.

[0058] The T cell receptor according to the ninth aspect of the present disclosure, in one embodiment, includes an α chain and a β chain. The variable region of the α chain includes CDR1α consisting of the amino acid sequence described in SEQ ID NO: 27, CDR2α consisting of the amino acid sequence described in SEQ ID NO: 28, and CDR3α consisting of the amino acid sequence described in SEQ ID NO: 29. The variable region of the β chain includes CDR1β consisting of the amino acid sequence described in SEQ ID NO: 30, CDR2β consisting of the amino acid sequence described in SEQ ID NO: 31, and CDR3β consisting of the amino acid sequence described in SEQ ID NO: 32. The T cell receptor is composed of a dimer of an α chain and a β chain. The α chain and the β chain are each composed of a variable region and a constant region. The constant region penetrates the cell membrane and has a short cytoplasmic portion. There are three regions called complementary determining regions in the variable regions of the α chain and the β chain, which are greatly involved in the binding to antigens and MHC. In the present disclosure, the complementary determining regions of the α chain are denoted as CDR1α, CDR2α, and CDR3α, and the complementary determining regions of the β chain are denoted as CDR1β, CDR2β, and CDR3β.

[0059] In one embodiment of the present disclosure, the T cell receptor includes a variable region of the α chain that contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 33, and comprises CDR1α consisting of the amino acid sequence described in SEQ ID NO: 27, CDR2α consisting of the amino acid sequence described in SEQ ID NO: 28, and CDR3α consisting of the amino acid sequence described in SEQ ID NO: 29; and a variable region of the β chain that contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 34, and comprises CDR1β consisting of the amino acid sequence described in SEQ ID NO: 30, CDR2β consisting of the amino acid sequence described in SEQ ID NO: 31, and CDR3β consisting of the amino acid sequence described in SEQ ID NO: 32. The sequence identity of the variable region of the α chain with the amino acid sequence of SEQ ID NO: 33 may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%. The variable region of the β chain may have sequence identity with the amino acid sequence of SEQ ID NO: 34 of, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0060] In one embodiment of the present disclosure, the T cell receptor comprises an α chain containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 37, and including CDR1α consisting of the amino acid sequence described in SEQ ID NO: 27, CDR2α consisting of the amino acid sequence described in SEQ ID NO: 28, and CDR3α consisting of the amino acid sequence described in SEQ ID NO: 29; and a β chain containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 38, and including CDR1β consisting of the amino acid sequence described in SEQ ID NO: 30, CDR2β consisting of the amino acid sequence described in SEQ ID NO: 31, and CDR3β consisting of the amino acid sequence described in SEQ ID NO: 32. The sequence identity of the α chain with the amino acid sequence of SEQ ID NO: 37 may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%. The β-chain may have sequence identity with the amino acid sequence of SEQ ID NO: 38 of, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0061] In one embodiment of the present disclosure, the T cell receptor is composed of an α chain consisting of a variable region comprising the amino acid sequence of SEQ ID NO: 33 and a constant region containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 35, and a β chain consisting of a variable region comprising the amino acid sequence of SEQ ID NO: 34 and a constant region containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 36. The sequence identity of the constant region of the α chain with the amino acid sequence of SEQ ID NO: 35 may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%. The sequence identity of the constant region of the β chain with the amino acid sequence of SEQ ID NO: 36 may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0062] In one embodiment, the T cell receptor relating to the ninth aspect of this disclosure comprises an α chain and a β chain, wherein the variable region of the α chain comprises CDR1α consisting of the amino acid sequence described in SEQ ID NO: 39, CDR2α consisting of the amino acid sequence described in SEQ ID NO: 40, and CDR3α consisting of the amino acid sequence described in SEQ ID NO: 41, and the variable region of the β chain comprises CDR1β consisting of the amino acid sequence described in SEQ ID NO: 42, CDR2β consisting of the amino acid sequence described in SEQ ID NO: 43, and CDR3β consisting of the amino acid sequence described in SEQ ID NO: 44.

[0063] In one embodiment of the present disclosure, the T cell receptor includes a variable region of the α chain that contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 45, and comprises CDR1α consisting of the amino acid sequence described in SEQ ID NO: 39, CDR2α consisting of the amino acid sequence described in SEQ ID NO: 40, and CDR3α consisting of the amino acid sequence described in SEQ ID NO: 41; and a variable region of the β chain that contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 46, and comprises CDR1β consisting of the amino acid sequence described in SEQ ID NO: 42, CDR2β consisting of the amino acid sequence described in SEQ ID NO: 43, and CDR3β consisting of the amino acid sequence described in SEQ ID NO: 44. The sequence identity of the variable region of the α chain with the amino acid sequence of SEQ ID NO: 45 may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%. The variable region of the β chain may have sequence identity with the amino acid sequence of SEQ ID NO: 46 of, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0064] In one embodiment of the present disclosure, the T cell receptor comprises an α chain containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 49, and including CDR1α consisting of the amino acid sequence described in SEQ ID NO: 39, CDR2α consisting of the amino acid sequence described in SEQ ID NO: 40, and CDR3α consisting of the amino acid sequence described in SEQ ID NO: 41; and a β chain containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 50, and including CDR1β consisting of the amino acid sequence described in SEQ ID NO: 42, CDR2β consisting of the amino acid sequence described in SEQ ID NO: 43, and CDR3β consisting of the amino acid sequence described in SEQ ID NO: 44. The sequence identity of the α chain with the amino acid sequence of SEQ ID NO: 49 may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%. The β-chain may have sequence identity with the amino acid sequence of SEQ ID NO: 50 of, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0065] In one embodiment of the present disclosure, the T cell receptor is composed of an α chain comprising a variable region consisting of the amino acid sequence of SEQ ID NO: 45 and a constant region containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 47, and a β chain comprising a variable region consisting of the amino acid sequence of SEQ ID NO: 46 and a constant region containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 48. The sequence identity of the constant region of the α chain with the amino acid sequence of SEQ ID NO: 47 may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%. The sequence identity of the constant region of the β chain with the amino acid sequence of SEQ ID NO: 48 may be, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.

[0066] A polypeptide having an amino acid sequence with 90% or more sequence identity to the amino acid sequence described in SEQ ID NO:1 and having a full length of 7 to 17 residues incorporates the descriptions and definitions set forth in the section of the activator of regulatory T cells according to the first aspect of the present disclosure.

[0067] The above T cell receptor can be produced by recombinant methods known to those skilled in the art. For example, reference can be made to Walchli et al., "A Practical Approach to T-Cell Receptor Cloning and Expression", PLOS One, 6(11):e27930. The gene sequences that can be used in constructing the above T cell receptor are known to those skilled in the art and can be found, for example, in immunogenetic and immunoinformatics databases such as THE INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM® (https: / / www.imgt.org / ). Such genes can be used as a framework for inserting the sequences provided herein for the above T cell receptor. Since the T cell receptor is expressed on the surface of T cells, usually a signal peptide sequence is included (e.g., at the N-terminus). When a polypeptide containing a signal peptide is expressed on the cell surface, the signal peptide is usually proteolytically removed, for example, during processing of the polypeptide in the endoplasmic reticulum and translocation to the cell surface. Thus, usually the T cell receptor is expressed on the cell surface as a mature protein lacking the signal peptide, although the precursor form of the T cell receptor contains the signal peptide.

[0068] <Nucleic acid encoding a T cell receptor> The tenth aspect of this disclosure is the nucleic acid encoding the T cell receptor. In one embodiment, the nucleic acid is in the form of a vector. The nucleic acid and vector enable the expression of the T cell receptor in regulatory T cells. The vector is typically an expression vector in which the nucleic acid encoding the T cell receptor is operably ligated to a promoter. A typical cloning and / or expression vector includes a transcription and translation terminator, an initiation sequence, and a promoter useful for regulating the expression of the desired nucleic acid sequence. In one embodiment, the expression vector is in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., "Molecular cloning: a laboratory manual," Cold Spring Harbor Laboratory Press, 2001, 3rd ed., and other molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, a preferred vector includes at least one origin of replication, a promoter sequence, a restriction endonuclease site, and a selectable marker.

[0069] Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into a vector and packaged into retroviral particles using techniques known in the art. Recombinant viruses can then be isolated and delivered to target cells, either in vivo or ex vivo.

[0070] One example of a suitable promoter is the cytomegalovirus (CMV) promoter sequence. This promoter sequence is a potent constitutive promoter sequence that can drive high levels of expression of any polynucleotide sequence operatively ligated to it. Another example of a suitable promoter is the EF-1α promoter. Other constitutive promoter sequences are also available, such as the SV40 promoter.

[0071] To evaluate T cell receptor expression, expression vectors may also include selectable marker genes to facilitate the identification and selection of expressing cells from a cell population intended for transfection via a viral vector. Useful selectable markers include, for example, antibiotic resistance genes.

[0072] Methods for introducing and expressing genes in cells are known in this field. For example, expression vectors can be introduced into host cells by physical, chemical, or biological means.

[0073] Physical methods for introducing target nucleic acids into host cells include calcium phosphate precipitation, lipofection, particle impact, microinjection, and electroporation. Methods for producing cells containing vectors and / or foreign nucleic acids are well known in the art. For example, see Sambrook et al. mentioned above.

[0074] Biological methods for introducing target nucleic acids into host cells include the use of DNA vectors and RNA vectors. Viral vectors, particularly retroviral vectors, are the most widely used method for inserting genes into mammalian cells, such as human cells.

[0075] Chemical means for introducing target nucleic acids into host cells include colloidal dispersion systems such as polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is the liposome.

[0076] <Regulatory T cells> The eleventh aspect of this disclosure is regulatory T cells expressing the T cell receptor described above. Expressing the T cell receptor means that the T cell receptor is present on the surface of the T cell. The descriptions and definitions of the T cell receptor described in the ninth aspect of this disclosure also apply to regulatory T cells described in the eleventh aspect of this disclosure.

[0077] <Therapeutic agents, preventive agents, therapeutic compositions, or preventive compositions for autoimmune diseases> Aspect 12 of this disclosure relates to a therapeutic or prophylactic agent for autoimmune diseases in human subjects having the HLA-DRB1 genotype 09:01, comprising the regulatory T cells described above. Aspect 12 of this disclosure may, in one embodiment, also be a therapeutic or prophylactic composition for autoimmune diseases in human subjects having the HLA-DRB1 genotype 09:01, comprising the regulatory T cells described above. Aspect 12 of this disclosure may, in one embodiment, also be a method for treating or prophylacticizing an autoimmune disease, comprising administering the regulatory T cells described above to a human subject having the HLA-DRB1 genotype 09:01. Aspect 12 of this disclosure may, in one embodiment, also be the regulatory T cells described above for use in the treatment or prophylactic use of autoimmune diseases in human subjects having the HLA-DRB1 genotype 09:01. A twelfth aspect of this disclosure may, in one embodiment, be the use of the regulatory T cells in the manufacture of a therapeutic agent, prophylactic agent, therapeutic composition or prophylactic composition for a human autoimmune disease in which the HLA-DRB1 genotype is 09:01. A twelfth aspect of this disclosure may, in one embodiment, be the use of the regulatory T cells for use in the manufacture of a therapeutic agent, prophylactic agent, therapeutic composition or prophylactic composition for a human autoimmune disease in which the HLA-DRB1 genotype is 09:01.

[0078] The autoimmune disease relating to the twelfth aspect of this disclosure may be, for example, an autoimmune disease caused by the overproduction of antibodies against human AchRα1 or its peptide fragment, preferably myasthenia gravis, and more preferably early-onset myasthenia gravis. Since the polypeptide is a peptide fragment containing an amino acid sequence corresponding to the epitope of human AchRα1, which is known to be recognized as an autoantigen in myasthenia gravis and early-onset myasthenia gravis, the agent according to one embodiment of the fifth aspect of this disclosure can suitably treat and prevent myasthenia gravis and early-onset myasthenia gravis.

[0079] A therapeutic or prophylactic agent according to one embodiment of the twelfth aspect of this disclosure is for administration to human subjects having the HLA-DRB1 genotype 09:01, and may be for administration to human subjects who have, may have, or have a genetic predisposition to an autoimmune disease, and the autoimmune disease may be, for example, an autoimmune disease caused by the overproduction of antibodies against human AchRα1 or its peptide fragment, preferably myasthenia gravis, and more preferably early-onset myasthenia gravis. Furthermore, if the agent according to one embodiment of the twelfth aspect of this disclosure is a therapeutic agent, the target of administration may be, for example, a human subject who has or may have an autoimmune disease. Furthermore, if the agent according to one embodiment of the fifth aspect of this disclosure is a prophylactic agent, the target of administration may be a human subject who has or may have a genetic predisposition to an autoimmune disease.

[0080] While we do not wish to be bound by any theory, it is thought that therapeutic or prophylactic agents for autoimmune diseases, including the regulatory T cells mentioned above, can suppress humoral immunity, thereby treating and preventing autoimmune diseases. For example, by suppressing the production of anti-AchRα1 antibodies, the degradation of AchRα1 can be suppressed, thereby treating and preventing myasthenia gravis, such as early-onset myasthenia gravis. [Examples]

[0081] The present disclosure will be described in more detail below with reference to examples, but the present disclosure is not limited to the following examples.

[0082] [Example 1: Examination of the possibility of constraint on HLA-DRB1*09:01] Using PBMCs derived from healthy individuals that possess HLA-DRB1*09:01, which has been reported to be associated with MG in Asians, and lack DRB1*03:01 and DRB3*01:01, we evaluated the ability of a peptide consisting of the amino sequence shown in SEQ ID NO: 2 (hereinafter simply referred to as the peptide of SEQ ID NO: 2 or the peptide itself) to induce specific Th1 cells.

[0083] <Peptide preparation> The peptide of Sequence ID No. 2 was synthesized by GeneScript or Eurofins genomics and dissolved in DMSO to a concentration of 20 mg / mL. <Preparation of culture media> Human type AB serum (GeminiBio, Cat:100-512) and fetal bovine serum (Nichirei, Cat:175012) were inactivated at 56°C for 30 minutes and then filtered through a 0.2 μm filter before use. AIM-V (Gibco, Cat:12055-091) containing 5% human type AB serum was used as the culture medium for PBMCs. Cell culture was performed under 37°C, 5% CO2, and water vapor saturated conditions. D-PBS(-) containing 2% fetal bovine serum was used as the staining buffer.

[0084] <List of PBMCs derived from healthy individuals> The HLA alleles of the healthy human-derived PBMCs used in the study of Example 1 are shown in Table 1.

[0085] [Table 1]

[0086] <Induction of peptide-specific T cells using PBMCs derived from healthy individuals> PBMCs that had been stored in liquid nitrogen vapor phase were taken out and rapidly thawed in a 37°C water bath. They were washed once with AIM-V and resuspended in AIM-V containing 5% human AB serum (hereinafter referred to as complete medium), and seeded in a 24-well plate at 1.5×10 6 cells / well. Peptide at a final concentration of 20 μg / mL and IL-7 at a final concentration of 10 ng / mL (PeproTech, Cat: AF-200-07-50ug) were added to initiate the culture (Day0, total medium volume: 2 mL / well). One week after the start of the culture, PBMCs from the same donor as the PBMCs during the culture were thawed in the same manner as at Day0, washed once with AIM-V, and adjusted to 3×10 6 cells / mL or less. The peptide used for induction at Day0 was added at a concentration of 20 μg / mL and cultured for 2 hours. Subsequently, a mitomycin C solution at a final concentration of 50 μg / mL was added and cultured for an additional 45 minutes. Then, it was washed three times with AIM-V to obtain antigen-presenting cells. The cells cultured from Day0 for one week were collected, seeded in a new 24-well plate within the range of 1 - 1.5×10 6 / well, and the prepared antigen-presenting cells were seeded in the same number. After that, IL-7 at a final concentration of 10 ng / mL was added to resume the culture (Day7, total medium volume: 2 mL / well). Two days later, half of the culture medium was replaced with complete medium containing 40 U / L IL-2, and the culture was continued for one week while replacing half of the culture medium with complete medium containing 20 U / mL IL-2 more surely. During this period, when the cells reached confluence, the 2 mL / well culture medium was well suspended, 1 mL of which was seeded in a new well, and complete medium containing 20 U / mL IL-2 was added to each well to 1 mL / well (hereinafter referred to as subculture operation). The cultured cells were collected on Day14 and used for <intracellular cytokine staining (ICS)> and <evaluation of IFN-γ production ability by ELISA> described below. <S

[0087] <S <Intracellular Cytokine Staining (ICS)> The collected cultured cells were seeded in a 96-well round-bottom plate at 2.0×10 6Seeded 2 to 6 wells with each one, added peptide at a concentration of 20 μg / mL to half of them and DMSO as a solvent at a concentration of 0.1% to the other half, and cultured for 2 hours. Added Brefeldin A (BioLegend, Cat: 420601) and Monensin (BioLegen, Cat: 420701) at a final concentration of 1X, and further cultured for 4 hours. Collected the cells, added FITC-labeled human CD3 antibody (BioLegend, Cat: 300440), PerCP / Cy5.5-labeled CD8a antibody (BioLegend, Cat: 100733), APC-labeled CD4 antibody (BioLegend, Cat: 100526), and 500-fold diluted LIVE / DEAD Fixable Far Red Dead reagent (Invitrogen, Cat: L34973) containing Fc Block (Miltenyi Biotec, Cat: 130 - 059 - 901), and reacted at 4°C for 15 minutes. Added Cytofix Cytoperm Fix / Perm Solution (Beckton Dickinson, Cat: 554714) and treated at 4°C for 20 minutes. Washed with Perm. / Wash Buffer (10X product included with Cat: 554714 diluted 10-fold with distilled water), added PE-labeled anti-human IFN-γ antibody (BioLegend, Cat: 502509), reacted at 37°C for 30 minutes, continued to wash with Perm. / Wash Buffer, then washed with Staining Buffer, and analyzed by FACS.

[0088] <Evaluation of IFN-γ production ability by ELISA> The collected cultured cells were placed in a 96-well round-bottom plate at 1.0×10 5Each cell was seeded in 6 wells. Half of the wells were treated with a peptide at a concentration of 20 μg / mL, and the other half with a solvent, DMSO, at a concentration of 0.1%. The cells were incubated for at least 16 hours. The culture supernatant was collected, diluted appropriately with Assay Diluent, and then subjected to ELISA analysis. IFN-γ absorption was measured using the BD OptEIA ELISA Set (human IFN-γ, Becton Dickinson, Cat: 555142) according to the instructions provided. If the absorbance was lower than that of the blank well, the IFN-γ concentration was treated as 0.

[0089] <Result> Figure 1 shows the results of ICS (left) and ELISA (right) in evaluating the ability of SEQ ID NO: 2 to induce specific Th1 cells using healthy human-derived PBMCs that possess HLA-DRB1*09:01, which has been reported to be associated with MG in Asians, but lack DRB1*03:01 and DRB3*01:01. As shown in Figure 1, SEQ ID NO: 2-specific Th1 cells were induced in 7 out of 9 samples subjected to ICS analysis (left, solid line). Of the 9 samples subjected to ICS analysis, 5 samples (4 with induction of SEQ ID NO: 2-specific Th1 cells, 1 without induction) were subjected to ELISA analysis. In the 4 samples in which induction was confirmed by ICS, SEQ ID NO: 2-specific Th1 cell-derived IFN-γ production was similarly detected (right, solid line). These results indicate that SEQ ID NO: 2 is also constrained by HLA-DRB1*09:01.

[0090] Examples 2-5 were conducted according to the following procedures. <Peptide preparation> The procedure was the same as in Example 1.

[0091] <Preparation of culture media> The procedure was the same as in Example 1.

[0092] <List of PBMCs derived from healthy individuals> The HLA allele information of PBMCs derived from healthy individuals used in the examination of Example 2 is as shown in the following table. Each PBMC was purchased from Precision for Medicine or STEMCELLS. The bold characters indicate the HLA alleles for which allelic restriction is to be demonstrated in this test.

Table 2

[0093] <List of feeder PBMCs used> The allogeneic PBMCs used during cloning and Th1 clone amplification are as shown in the following table and were purchased from Precision for Medicine or STEMCELLS.

Table 3

[0094] <List of feeder B-LCLs used> The allogeneic B-LCLs used during cloning and Th1 clone amplification are as shown in the following table and were all obtained from the RIKEN Cell Bank. The HLA allele information in the table was transcribed from the results of HLA typing by next-generation sequencing (NGS) performed in advance.

Table 4

[0095] <B-LCL cells for DRB1*09:01 allelic restriction evaluation> The B-LCLs used during the allelic restriction test are as shown in the following table and were all obtained from the RIKEN Cell Bank. The HLA allele information in the table was transcribed from the results of HLA typing by NGS performed in advance.

Table 5

[0096] <Induction of peptide-specific T cells using PBMCs from healthy individuals> The same procedure as in Example 1 was followed.

[0097] <Intracellular cytokine staining> The same procedure as in Example 1 was performed.

[0098] <Evaluation of IFN-γ production ability by ELISA> The same procedure as in Example 1 was performed.

[0099] <Lysis, seeding, and passage of B-LCL cells> About 3 - 4×10 5 cells / mL were used to initiate the culture. Subculture was performed at sub-confluent stage, and the B-LCL cells were used as feeder cells upon phytohemagglutinin (PHA) stimulation.

[0100] <Isolation of CD4 + cells by MACS> According to the manufacturer's recommended protocol, positive selection was performed using MACS Microbeads (Miltenyi Biotec, Cat: 130 - 045 - 101) from cultured cells to isolate CD4 + cells.

[0101] <Cloning and amplification of peptide-specific Th1 cells by limiting dilution method> Feeder B-LCL cells and any allogeneic PBMCs (cell concentration 3×10 6 / mL or less) were treated with mitomycin C solution (final concentration = 50 μg / mL) in a CO2 incubator for 45 minutes, washed with AIM-V, and then four types of cells including two types of PBMCs and two types of B-LCL cells were mixed (final concentration of each PBMC 2.5×10 5 cells / mL, final concentration of each B-LCL cell 2.5×10 4 cells / mL). According to the number of samples to be cloned, the required number of sets was prepared, and PHA-P (FUJIFILM Wako Pure Chemical Corporation, Cat: 161 - 15251) was added at a final concentration of 200 ng / mL to obtain a PHA-containing feeder cell mixture. Next, among the fractionated CD4 + cells, CD4 + IFN-γ +Select a specimen with a high cell ratio and prepare a PHA-containing feeder cell mixture to adjust the CD4 + cell concentration to 10 cells / mL, and seed 100 μL / well (total PBMC amount: 5.0×10 4 cells / well, B-LCL cells: 5.0×10 3 cells / well, PHA: 200 ng / mL, CD4 + cells: 1 cell / well) into a 96-well plate and start culturing. Five days later, add a medium containing 80 U / mL IL-2 equal to the volume of the culture medium, and then replace half of the culture medium with a medium containing 80 U / mL IL-2 every other day. During this period, wells with obvious cell amplification were scaled up to a 48-well plate and culturing was continued. From 10 to 14 days after the start of cloning, an amplification stimulus by PHA stimulation was applied in the same manner as above. At the time of seeding, transfer the culture dish to a 24-well plate, set the total amount of PBMC to 1.0×10 6 cells / well, the total amount of B-LCL cells to 1.0×10 5 cells / well, the final concentration of PHA to 50 ng / mL, and the seeding number of the Th1 clone to be amplified to 2.0×10 5 cells / well or less. Also, change the timing of IL-2 addition to 3 days after the start of culturing and the IL-2 content of the medium to be added to 200 U / mL.

[0102] <Peptide Stimulation of Th1 Clone> To confirm the antigen reactivity of the Th1 clone cells amplified after cloning, the recovered Th1 clone cells were seeded in a 96-well round-bottom plate at 1 well or 3 wells. If a sufficient amount of cells could be recovered, they were counted and the viable cells were seeded at 5.0×10 4 cells / well. Add 0.1% DMSO or 20 μg / mL of peptide to this and start culturing (total culture medium volume: 200 μL / well). After culturing for 16 hours or more, collect the culture supernatant and measure the IFN-γ concentration in the supernatant.

[0103] <Determination Test of Restricted Allergen of Established Specific Th1 Clone> In specific Th1 clones established by peptide addition, it was examined whether the peptide could activate T cells with specific HLA restriction, and its restricting allele was confirmed. Specifically, B-LCL cells having various HLA alleles were cultured for 2 hours in the presence of the peptide to associate the peptide on the HLA molecules on the B-LCL surface. Thereafter, excess peptide was removed by washing, and it was used as an antigen-presenting cell (peptide-pulsed B-LCL). Th1 clone cells with confirmed antigen reactivity were co-cultured with peptide-pulsed or non-pulsed B-LCL, and after culturing for 16 hours or more, the culture supernatant was collected, and the IFN-γ concentration in the supernatant was measured. The restricting allele was determined from the HLA allele pattern of B-LCL in which IFN-γ production was specifically observed in the peptide pulse.

[0104] <Total RNA extraction from Th1 clones> Th1 clone cells or cell pellets stored in a cell bank (Takara Bio, Cat: CB021) stored at -80°C were used. When using the cells stored in the cell bank, the storage vial was warmed in a 37°C water bath and rapidly thawed. The thawed cell suspension was transferred to a conical tube containing pre-warmed AIM-V and inverted and mixed. The cell suspension was centrifuged (400 x g, 5 minutes, room temperature) to recover the pellet, and the pellet was lysed with Buffer RLT (included in the RNeasy Mini Kit. QIAGEN, Cat: 169025782). The solubilized solution was treated with QIAshredder (QIAGEN, Cat: 79656) to cleave genomic DNA. Subsequent total RNA purification was performed according to the manufacturer's recommended protocol of the RNeasy Mini Kit. The obtained total RNA concentration was determined using NanoDrop8000 (Thermo Fisher Scientific).

[0105] <Cloning of T cell receptor by 5'-RACE method> T cell receptor (TCR) cDNA cloning was performed from total RNA purified from each Th1 clone using the SMARTer® RACE 5' / 3' Kit (Clontech, Cat: 634858). The cloning process followed the manufacturer's recommended protocol. The input total RNA amount ranged from 8.2 to 500 TCRs. The 3' end primers used for specific sequence amplification are shown in the table below. PCR conditions were (94°C 30 sec, 72°C 3 min) x 5 cycles, (94°C 30 sec, 70°C 30 sec, 72°C 3 min) x 5 cycles, and (94°C 30 sec, 68°C 30 sec, 72°C 3 min) x 25 cycles, followed by 72°C 7 min. [Table 6]

[0106] <Colony Direct PCR and Amplicon Sequencing> Transformed colonies were collected, suspended in EmeraldAmp® PCR Master Mix (Takara Bio, Cat: RR300A), and subjected to PCR reaction with the primers listed in the table below. The PCR conditions were (98°C for 10 seconds, 60°C for 30 seconds, 72°C for 1 minute) × 30 cycles. The PCR products were analyzed by 1% agarose gel electrophoresis and SYBR® Gold Nucleic Acid Gel Stain (Thermo Fisher Scientic, Cat: S11494). For samples in which the target product was observed, the DNA concentration was determined using Quant-iT® dsDNA HR Assay Kits, Broad Range (Thermo Fisher Scientic, Cat: Q33130). 15 ng of PCR product was cleaned of residual primers using ExoSAP-IT® ExpressPCR Product Cleanup (Thermo Fisher Scientic, Cat: 75001) according to the recommended protocol. Using primer-removed PCR products as templates, sequencing analysis was performed from both sides using the primers shown in the table below. [Table 7]

[0107] <Analysis of TCR sequences> The obtained sequences were subjected to V(D)J analysis in the THE INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM (registered trademark) (https: / / www.imgt.org / ) database to determine VDJ segments and complementarity-determining regions (CDRs).

[0108] [Example 2: Evaluation test of specific T cell induction ability using PBMCs derived from healthy individuals] Results An evaluation test of specific T cell induction ability was carried out using 10 specimens including PBMCs from different lots derived from the same donor and PBMCs from the same lot evaluated in different tests. The results are shown in FIGS. 2 and 3. From the results of ICS in FIG. 2, in all specimens except No. 6, the rate of IFN-γ-producing CD4-positive T cells increased during peptide stimulation compared with DMSO stimulation in the Solvent group, so it was judged that peptide-specific T cells were induced. Among the 10 specimens, ELISA analysis (FIG. 3) was performed on 6 specimens, and peptide-specific IFN-γ production was observed in all specimens except No. 6. From the above results, peptide-specific T cells were induced in specimens having DRB1*09:01 with a probability of 90%, and this peptide was considered to have DRB1*09:01 restriction.

[0109] [Example 3: Establishment of peptide-specific type I helper T clone cells (Th1 clones)] Results Samples from Example 2 to 9 were selected, and after fractionation of CD4-positive T cells by MACS, they were subjected to limiting dilution. Allogeneic PBMCs and B-LCL cell lines were added as feeder cells, and peptide-specific Th1 cells were cloned by PHA stimulation. Cells were partially collected from wells where amplification was observed and treated with DMSO or peptide stimulation as a solvent group, and the amount of IFN-γ in the culture supernatant was measured. The results are shown in Figure 4. The graph shows the difference obtained by subtracting the amount of IFN-γ after DMSO stimulation from the amount of IFN-γ after peptide stimulation. As shown in Figure 4, significant IFN-γ production was observed in clones 1B5, 5B12, and 6F5 upon peptide stimulation. Of these, 5B12 and 6F5 were further amplified by PHA stimulation in the presence of feeder cells, and antigen reactivity was confirmed again (Table 8), clearly showing that peptide reactivity was retained. [Table 8]

[0110] [Example 4: Th1 clone-restricted allele determination test using B-LCL cells for DRB1*09:01 restriction evaluation] result Of the Th1 clones 5B12 and 6F5 established in Example 3, HLA typing was performed using the NGS method with 6F5, which had an abundance of cells. As shown in Table 9, compared with the allele information provided by the PBMC supplier, HLA-DRB4 was "01:03" instead of "01:01", and HLA-DQB1 was "02:02" instead of "02:01". Each clone cell was used as an effector cell, and B-LCL cells with DRB1*09:01-restricted peptide treatment were co-cultured as antigen-presenting cells, and the amount of IFN-γ released into the supernatant was evaluated. As shown in Figures 5 and 6, IFN-γ production was observed only when B-LCL cells with DRB1*09:01-restricted peptide treatment were co-cultured as antigen-presenting cells. From these results, clones 5B12 and 6F5 were shown to be Th1 clones that are DRB1*09:01-restricted and peptide-specific. [Table 9]

[0111] [Example 5: Identification of DRB1*09:01-restrictive peptide-specific TCR sequences] result The results of T cell receptor (TCR) analysis of DRB1*09:01-restrictive peptide-specific Th1 clones were as follows. [Table 10]

[0112] The sequencing results showed the following amino acid sequence of the TCR of clone 5B12. [Table 11]

[0113] The sequencing results showed the following amino acid sequence of the TCR of clone 6F5. [Table 12]

Claims

1. A therapeutic or prophylactic agent for human autoimmune diseases, comprising one or more polypeptides having a total length of 7 to 17 residues, and containing an amino acid sequence with 90% or more sequence identity with the amino acid sequence described in Sequence ID No.

1.

2. An activator for human regulatory T cells with HLA-DRB1 genotype 09:01, comprising one or more polypeptides having an amino acid sequence with 90% or more sequence identity with the amino acid sequence described in Sequence ID No. 1, and having a total length of 7 to 17 residues.

3. An effector T cell activation inhibitor for human subjects with HLA-DRB1 genotype 09:01, comprising one or more polypeptides with a total length of 7 to 17 residues, and containing an amino acid sequence that has 90% or more sequence identity with the amino acid sequence described in SEQ ID NO:

1.

4. A human B cell activation inhibitor for HLA-DRB1 with genotype 09:01, comprising one or more polypeptides with a total length of 7 to 17 residues, and containing an amino acid sequence that is 90% or more identical to the amino acid sequence described in Sequence ID No.

1.

5. A humoral immunosuppressant for humans with HLA-DRB1 genotype 09:01, comprising one or more polypeptides having an amino acid sequence with 90% or more sequence identity with the amino acid sequence described in Sequence ID No. 1, and having a total length of 7 to 17 residues.

6. A diagnostic agent for autoimmune diseases in humans with HLA-DRB1 genotype 09:01, comprising a complex comprising a polypeptide, a human leukocyte antigen, and a labeling dye, wherein the polypeptide comprises one or more polypeptides having an amino acid sequence with 90% or more sequence identity with the amino acid sequence described in SEQ ID NO: 1, and having a total length of 7 to 17 residues.

7. A diagnostic kit for autoimmune diseases in humans with HLA-DRB1 genotype 09:01, comprising one or more polypeptides having a total length of 7 to 17 residues and containing an amino acid sequence with 90% or more sequence identity with the amino acid sequence described in Sequence ID No. 1, and a human leukocyte antigen that binds to at least one of the polypeptides.

8. The kit according to claim 7, further comprising a labeling dye that binds to the aforementioned human leukocyte antigen.

9. A T cell receptor that binds to a complex of HLA-DRB1*09:01 and a polypeptide having a total length of 7 to 17 residues and containing an amino acid sequence that has 90% or more sequence identity with the amino acid sequence described in Sequence ID No. 1, The T cell receptor comprises an α chain and a β chain. The variable region of the α chain includes a complementarity-determining region (CDR) 1α consisting of the amino acid sequence described in SEQ ID NO: 27, a CDR 2α consisting of the amino acid sequence described in SEQ ID NO: 28, and a CDR 3α consisting of the amino acid sequence described in SEQ ID NO:

29. The variable region of the β chain comprises CDR1β consisting of the amino acid sequence described in SEQ ID NO: 30, CDR2β consisting of the amino acid sequence described in SEQ ID NO: 31, and CDR3β consisting of the amino acid sequence described in SEQ ID NO: 32, thereby providing a T cell receptor.

10. The variable region of the α chain contains an amino acid sequence that has 90% or more sequence identity with the amino acid sequence of SEQ ID NO:

33. The T cell receptor according to claim 9, wherein the variable region of the β chain includes an amino acid sequence that has 90% or more sequence identity with the amino acid sequence of SEQ ID NO:

34.

11. A T cell receptor that binds to a complex of HLA-DRB1*09:01 and a polypeptide having a total length of 7 to 17 residues and containing an amino acid sequence that has 90% or more sequence identity with the amino acid sequence described in Sequence ID No. 1, The T cell receptor comprises an α chain and a β chain. The variable region of the α chain includes a complementarity-determining region (CDR) 1α consisting of the amino acid sequence described in SEQ ID NO: 39, a CDR 2α consisting of the amino acid sequence described in SEQ ID NO: 40, and a CDR 3α consisting of the amino acid sequence described in SEQ ID NO:

41. The variable region of the β chain comprises CDR1β consisting of the amino acid sequence described in SEQ ID NO: 42, CDR2β consisting of the amino acid sequence described in SEQ ID NO: 43, and CDR3β consisting of the amino acid sequence described in SEQ ID NO: 44, thereby providing a T cell receptor.

12. The variable region of the α chain contains an amino acid sequence that has 90% or more sequence identity with the amino acid sequence of SEQ ID NO:

45. The T cell receptor according to claim 11, wherein the variable region of the β chain includes an amino acid sequence that has 90% or more sequence identity with the amino acid sequence of SEQ ID NO:

46.

13. Regulatory T cells expressing the T cell receptor according to any one of claims 9 to 12.

14. A therapeutic or prophylactic agent for human autoimmune diseases comprising regulatory T cells as described in claim 13, wherein the HLA-DRB1 genotype is 09:01.

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