Formulations comprising a polypeptide comprising an amino acid sequence corresponding to an HLA-DR binding epitope of AChR

Abstract

Disclosed is an activator of regulatory T cells, an inhibitor of activity of effector T cells, an inhibitor of activation of B cells, an inhibitor of humoral immunity, or a therapeutic or prophylactic agent for an autoimmune disease, containing a polypeptide including an amino acid sequence equivalent to an HLA-DR binding epitope of an AChR.

Description

Technical Field

[0001] This disclosure relates to a formulation containing a polypeptide comprising an amino acid sequence including an HLA-DR binding epitope equivalent to AChR. Background Technology

[0002] In autoimmune diseases, the immune mechanisms normally used to clear foreign substances from outside the body recognize substances from within the body as antigens, resulting in abnormal activation of the immune system. Such abnormal immune activation is known to cause various functional abnormalities in patients with autoimmune diseases.

[0003] Early-onset myasthenia gravis is a type of myasthenia gravis that occurs in individuals aged 10 to 49 years. Myasthenia gravis is designated as a rare disease in Japan; it is an organ-specific autoimmune disease affecting molecules on the postsynaptic membrane of the neuromuscular junction, with muscle weakness as the primary symptom.

[0004] In myasthenia gravis, autoantibodies are produced in patients targeting acetylcholine receptors (AChR) and muscle-specific receptor tyrosine kinases (MuSK), molecules located on the postsynaptic membrane at the neuromuscular junction. These antibodies inhibit neurotransmission, leading to a variety of 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 present antigens to naïve CD4 cells. + T cells, primate CD4 + T cells differentiate into effector CD4 cells. + When T cells activate B cells to produce antibodies, in recent years, as novel antigen-presenting cells, antigen-presenting cells with the following functions have been reported: presenting antigens to naïve CD4 cells. + T cells, primate CD4 + T cells differentiate into regulatory T cells rather than effector CD4 cells. + T cells (Non-Patent Literature 1, 2). These antigen-presenting cells activate regulatory T cells through antigen presentation, thereby inhibiting effector CD4 cells that have been presented with the antigen. + T cell activation of B cells, therefore, has the opposite effect of antigen-presenting cells as previously known, suppressing antibody production. In fact, clinical trials are underway for autoimmune diseases using antibody production inhibitors targeting this novel type of antigen-presenting cell (Non-Patent Literature 3).

[0006] In autoimmune diseases, human leukocyte antigens (HLA), which are protein complexes that function as antigen-presenting proteins on the surface of antigen-presenting cells, recognize a portion of the antigen and form a binding with it. Especially when the antigen is a protein, the site within the antigen protein that is recognized by HLA is called an epitope. Because such epitopes hold the potential to be therapeutic and diagnostic targets for autoimmune diseases, attempts have been made to identify them in various autoimmune diseases; for example, epitopes in early-onset myasthenia gravis have been reported (Non-Patent Literature 4).

[0007] Existing technical documents Non-patent literature Non-patent document 1: Laura Passerini and Silvia Gregori., "Induction ofAntigen-Specific Tolerance in T Cell Mediated Diseases", Frontiers in Immunology 11(2194) (2020) Non-patent literature 2: Matthias G. von Herrath and Leonard C. Harrison., "Anti-Induced Registrar T Cells in Autoimmunity", Nature Review Immunology 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 ahuman T cell clone autoreactive to a unique acetylcholine receptor alphasubunit peptide presented by the disease-susceptible HLA-DQ6 in infant-onsetmyasthenia gravis", Hum Immunol56(1-2):28-38 (1997). Summary of the Invention

[0008] The problem the invention aims to solve The purpose of this disclosure is to provide a formulation containing a polypeptide comprising an amino acid sequence equivalent to an AChR epitope, which has the potential to be used for the treatment or diagnosis of autoimmune diseases.

[0009] Technical means to solve the problem The inventors discovered peptide fragments of AChR, secreted as a complex with human leukocyte antigen (HLA), from human dendritic cells that have taken up AChR, using the NAPA (Natural Antigen Processing Assay) method prepared from classical human monocytes. Furthermore, the inventors found that these peptide fragments contain amino acid sequences corresponding to AChR epitopes, and that formulations containing polypeptides comprising these amino acid sequences have the potential for application in the treatment or diagnosis of autoimmune diseases, thus completing this disclosure.

[0010] This disclosure relates to, for example, the following.

[0011] [1] An activator or composition for activating regulatory T cells, comprising at least one polypeptide selected from the group consisting of: (A) One or more polypeptides having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence described in Serial No. 1 and a total length of 8 or more residues and less than 20 residues. (B) One or more polypeptides having a total length of 12 residues or more and 21 residues or less, which contain an amino acid sequence having more than 90% sequence identity with the amino acid sequence recorded in sequence number 2; (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in Serial No. 4, and having a full length of 7 or more residues and less than 17 residues; and (E) One or more polypeptides having a full length of 10 or more residues and less than 19 amino acids, which contain an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence described in sequence number 5.

[0012] [2] An inhibitor of effector T cell activity or a composition for inhibiting activity, comprising at least one polypeptide selected from the group consisting of: (A) One or more polypeptides having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence described in Serial No. 1 and a total length of 8 or more residues and less than 20 residues. (B) One or more polypeptides having a total length of 12 residues or more and 21 residues or less, which contain an amino acid sequence having more than 90% sequence identity with the amino acid sequence recorded in sequence number 2; (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in Serial No. 4, and having a full length of 7 or more residues and less than 17 residues; and (E) One or more polypeptides having a full length of 10 or more residues and less than 19 amino acids, which contain an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence described in sequence number 5.

[0013] [3] An inhibitor of B cell activation or a composition for inhibiting activation, comprising at least one polypeptide selected from the group consisting of: (A) One or more polypeptides having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence described in Serial No. 1 and a total length of 8 or more residues and less than 20 residues. (B) One or more polypeptides having a total length of 12 residues or more and 21 residues or less, which contain an amino acid sequence having more than 90% sequence identity with the amino acid sequence recorded in sequence number 2; (D) One or more polypeptides comprising an amino acid sequence having more than 90% sequence identity with the amino acid sequence described in Serial No. 4, and having a full length of 7 or more residues but less than 17 residues; and (E) One or more polypeptides having a full length of 10 or more residues and less than 19 amino acids, which contain an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence described in sequence number 5.

[0014] [4] An inhibitor or composition for inhibiting humoral immunity, comprising at least one polypeptide selected from the group consisting of: (A) One or more polypeptides having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence described in Serial No. 1 and a total length of 8 or more residues and less than 20 residues. (B) One or more polypeptides having a total length of 12 residues or more and 21 residues or less, which contain an amino acid sequence having more than 90% sequence identity with the amino acid sequence recorded in sequence number 2; (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in Serial No. 4, and having a full length of 7 or more residues and less than 17 residues; and (E) One or more polypeptides having a full length of 10 or more residues and less than 19 amino acids, which contain an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence described in sequence number 5.

[0015] [5] A therapeutic agent, preventive agent, therapeutic composition, or preventive composition for an autoimmune disease, comprising at least one polypeptide selected from the group consisting of: (A) One or more polypeptides having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence described in Serial No. 1 and a total length of 8 or more residues and less than 20 residues. (B) One or more polypeptides having a total length of 12 residues or more and 21 residues or less, which contain an amino acid sequence having more than 90% sequence identity with the amino acid sequence recorded in sequence number 2; (D) One or more polypeptides having a full length of 7 residues or more and 17 residues or less, comprising an amino acid sequence with a sequence identity of 90% or more that is identical to the amino acid sequence described in sequence number 4; and (E) One or more polypeptides having a full length of 10 or more residues and less than 19 amino acids, which contain an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence described in sequence number 5.

[0016] [6] A treatment or prevention method for an autoimmune disease, comprising administering at least one polypeptide selected from the group consisting of: (A) One or more polypeptides having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence described in Serial No. 1 and a total length of 8 or more residues and less than 20 residues. (B) One or more polypeptides having a total length of 12 residues or more and 21 residues or less, which contain an amino acid sequence having more than 90% sequence identity with the amino acid sequence recorded in sequence number 2; (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in Serial No. 4, and having a full length of 7 or more residues and less than 17 residues; and (E) One or more polypeptides having a full length of 10 or more residues and less than 19 amino acids, which contain an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence described in sequence number 5.

[0017] [7] A polypeptide selected from the group consisting of at least one polypeptide for use in the treatment or prevention of autoimmune diseases: (A) One or more polypeptides having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence described in Serial No. 1 and a total length of 8 or more residues and less than 20 residues. (B) One or more polypeptides having a total length of 12 residues or more and 21 residues or less, which contain an amino acid sequence having more than 90% sequence identity with the amino acid sequence recorded in sequence number 2; (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in Serial No. 4, and having a full length of 7 or more residues and less than 17 residues; and (E) One or more polypeptides having a full length of 10 or more residues and less than 19 amino acids, which contain an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence described in sequence number 5.

[0018] [8] Use of at least one polypeptide selected from the group consisting of in the manufacture of a therapeutic agent, preventive agent, therapeutic composition or preventive composition for an autoimmune disease: (A) One or more polypeptides having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence described in Serial No. 1 and a total length of 8 or more residues and less than 20 residues. (B) One or more polypeptides having a total length of 12 residues or more and 21 residues or less, which contain an amino acid sequence having more than 90% sequence identity with the amino acid sequence recorded in sequence number 2; (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in Serial No. 4, and having a full length of 7 or more residues and less than 17 residues; and (E) One or more polypeptides having a full length of 10 or more residues and less than 19 amino acids, which contain an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence described in sequence number 5.

[0019] [9] A polypeptide selected from the group consisting of at least one polypeptide for use in the manufacture of a therapeutic agent, preventive agent, therapeutic composition or preventive composition for autoimmune diseases: (A) One or more polypeptides having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence described in Serial No. 1 and a total length of 8 or more residues and less than 20 residues. (B) One or more polypeptides having a total length of 12 residues or more and 21 residues or less, which contain an amino acid sequence having more than 90% sequence identity with the amino acid sequence recorded in sequence number 2; (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in Serial No. 4, and having a full length of 7 or more residues and less than 17 residues; and (E) One or more polypeptides having a full length of 10 or more residues and less than 19 amino acids, which contain an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence described in sequence number 5.

[0020]

[10] A diagnostic kit for an autoimmune disease, comprising: At least one polypeptide selected from the group consisting of: (A) Containing an amino acid sequence with more than 90% sequence identity to the amino acid sequence described in Serial No. 1, or one or more polypeptides with a full length of 8 residues or more and less than 20 residues. (B) Containing an amino acid sequence with more than 90% sequence identity to the amino acid sequence described in Serial No. 2, or one or more polypeptides with a total length of 12 residues or more and less than 21 residues. (D) An amino acid sequence containing more than 90% sequence identity with the amino acid sequence described in Serial No. 4, one or more polypeptides having a full length of 7 residues or more and less than 17 residues, and (E) One or more polypeptides containing an amino acid sequence that has more than 90% sequence identity with the amino acid sequence described in sequence number 5 and has a full length of more than 10 residues and less than 19 bases; And human leukocyte antigens that bind to at least one of the aforementioned polypeptides.

[0021]

[11] The kit described in

[10] further comprises a marker pigment that binds to the aforementioned human leukocyte antigen.

[0022]

[12] A diagnostic agent comprising a complex of a polypeptide, a human leukocyte antigen, and a labeling pigment for an autoimmune disease, wherein the polypeptide is at least one polypeptide selected from the group consisting of: (A) One or more polypeptides having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence described in Serial No. 1 and a total length of 8 or more residues and less than 20 residues. (B) One or more polypeptides having a total length of 12 residues or more and 21 residues or less, which contain an amino acid sequence having more than 90% sequence identity with the amino acid sequence recorded in sequence number 2; (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in Serial No. 4, and having a full length of 7 or more residues and less than 17 residues; and (E) One or more polypeptides having a full length of 10 or more residues and less than 19 amino acids, which contain an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence described in sequence number 5.

[0023]

[13] A method for assisting in the diagnosis of an autoimmune disease, comprising detecting T cells that are antigen-specific to at least one polypeptide selected from the group consisting of: (A) One or more polypeptides having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence described in Serial No. 1 and a total length of 8 or more residues and less than 20 residues. (B) One or more polypeptides having a total length of 12 residues or more and 21 residues or less, which contain an amino acid sequence having more than 90% sequence identity with the amino acid sequence recorded in sequence number 2; (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in Serial No. 4, and having a full length of 7 or more residues and less than 17 residues; and (E) One or more polypeptides having a full length of 10 or more residues and less than 19 amino acids, which contain an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence described in sequence number 5.

[0024]

[14] A method for obtaining data for diagnosing an autoimmune disease, comprising detecting T cells that are antigen-specific to at least one polypeptide selected from the group consisting of: (A) One or more polypeptides having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence described in Serial No. 1 and a total length of 8 or more residues and less than 20 residues. (B) One or more polypeptides having a total length of 12 residues or more and 21 residues or less, which contain an amino acid sequence having more than 90% sequence identity with the amino acid sequence recorded in sequence number 2; (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in Serial No. 4, and having a full length of 7 or more residues and less than 17 residues; and (E) One or more polypeptides having a full length of 10 or more residues and less than 19 amino acids, which contain an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence described in sequence number 5.

[0025]

[15] According to the activator or composition described in [1], the activity inhibitor or composition described in [2], the activation inhibitor or composition described in [3], the inhibitor or composition described in [4], the therapeutic agent, preventive agent or composition described in [5], the method described in [6], the at least one polypeptide described in [7], the use described in [8], the at least one polypeptide described in [9], the kit described in

[10] or

[11] , the diagnostic agent described in

[12] , or the method described in

[13] or

[14] , the at least one polypeptide described above contains (A) one or more polypeptides having a full length of 8 residues or more and 20 residues or less, which contain an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence described in Serial Number 1.

[0026]

[16] The at least one polypeptide described in [1], [2], [3], [4], [5], [6], [7], [8], [9],

[10] or

[11] ,

[12] ,

[13] or

[14] , comprises (B) one or more polypeptides having a total length of 12 or more residues but less than 21 residues, comprising an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence described in Serial No. 2.

[0027]

[17] According to the activator or composition described in [1], the activity inhibitor or composition described in [2], the activation inhibitor or composition described in [3], the inhibitor or composition described in [4], the therapeutic agent, preventive agent or composition described in [5], the method described in [6], the at least one polypeptide described in [7], the use described in [8], the at least one polypeptide described in [9], the kit described in

[10] or

[11] , the diagnostic agent described in

[12] , or the method described in

[13] or

[14] , the at least one polypeptide described above contains (D) one or more polypeptides having a total length of 7 or more residues but less than 17 residues, comprising an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence described in sequence number 4.

[0028]

[18] According to the activator or composition described in [1], the activity inhibitor or composition described in [2], the activation inhibitor or composition described in [3], the inhibitor or composition described in [4], the therapeutic agent, preventive agent or composition described in [5], the method described in [6], the at least one polypeptide described in [7], the use described in [8], the at least one polypeptide described in [9], the kit described in

[10] or

[11] , the diagnostic agent described in

[12] , or the method described in

[13] or

[14] , the at least one polypeptide described above contains (E) an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence described in sequence number 5, and having a full length of 10 or more residues and 19 or fewer polypeptides.

[0029]

[19] According to the activator or composition described in [1], the activity inhibitor or composition described in [2], the activation inhibitor or composition described in [3], the inhibitor or composition described in [4], the therapeutic agent, preventive agent or composition described in [5], the method described in [6], the at least one polypeptide described in [7], the use described in [8], the at least one polypeptide described in [9], the kit described in

[10] or

[11] , the diagnostic agent described in

[12] , or the method described in

[13] or

[14] , the at least one polypeptide described above contains: (A) One or more polypeptides having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence described in Serial No. 1 and a total length of 8 or more residues and less than 20 residues. (B) One or more polypeptides having a total length of 12 or more residues and less than 21 residues, which contain an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence described in sequence number 2.

[0030]

[20] According to the activator or composition described in [1], the activity inhibitor or composition described in [2], the activation inhibitor or composition described in [3], the inhibitor or composition described in [4], the therapeutic agent, preventive agent or composition described in [5], the method described in [6], the at least one polypeptide described in [7], the use described in [8], the at least one polypeptide described in [9], the kit described in

[10] or

[11] , the diagnostic agent described in

[12] , or the method described in

[13] or

[14] , the at least one polypeptide described above contains: (A) One or more polypeptides having a total length of 8 to 20 residues and containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in Serial No. 1; and (D) One or more polypeptides having a total length of 7 to 17 residues and containing an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence described in sequence number 4.

[0031]

[21] According to the activator or composition described in [1], the activity inhibitor or composition described in [2], the activation inhibitor or composition described in [3], the inhibitor or composition described in [4], the therapeutic agent, preventive agent or composition described in [5], the method described in [6], the at least one polypeptide described in [7], the use described in [8], the at least one polypeptide described in [9], the kit described in

[10] or

[11] , the diagnostic agent described in

[12] , or the method described in

[13] or

[14] , the at least one polypeptide described above contains: (A) One or more polypeptides having a total length of 8 to 20 residues and containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in Serial No. 1; and (E) One or more polypeptides having a total length of 10 or more residues and less than 19 amino acids, comprising an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence described in sequence number 5.

[0032]

[22] According to the activator or composition described in [1], the activity inhibitor or composition described in [2], the activation inhibitor or composition described in [3], the inhibitor or composition described in [4], the therapeutic agent, preventive agent or composition described in [5], the method described in [6], the at least one polypeptide described in [7], the use described in [8], the at least one polypeptide described in [9], the kit described in

[10] or

[11] , the diagnostic agent described in

[12] , or the method described in

[13] or

[14] , the at least one polypeptide described above contains: (B) One or more polypeptides comprising an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence described in Serial No. 2, and having a total length of 12 to 21 residues or more; and (D) One or more polypeptides having a total length of 7 to 17 residues and containing an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence described in sequence number 4.

[0033]

[23] According to the activator or composition described in [1], the activity inhibitor or composition described in [2], the activation inhibitor or composition described in [3], the inhibitor or composition described in [4], the therapeutic agent, preventive agent or composition described in [5], the method described in [6], the at least one polypeptide described in [7], the use described in [8], the at least one polypeptide described in [9], the kit described in

[10] or

[11] , the diagnostic agent described in

[12] , or the method described in

[13] or

[14] , the at least one polypeptide described above contains: (B) One or more polypeptides comprising an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence described in Serial No. 2, and having a total length of 12 to 21 residues or more; and (E) One or more polypeptides having a total length of 10 or more residues and less than 19 amino acids, comprising an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence described in sequence number 5.

[0034]

[24] According to the activator or composition described in [1], the activity inhibitor or composition described in [2], the activation inhibitor or composition described in [3], the inhibitor or composition described in [4], the therapeutic agent, preventive agent or composition described in [5], the method described in [6], the at least one polypeptide described in [7], the use described in [8], the at least one polypeptide described in [9], the kit described in

[10] or

[11] , the diagnostic agent described in

[12] , or the method described in

[13] or

[14] , the at least one polypeptide described above contains: (D) One or more polypeptides having a total length of 7 to 17 residues and containing an amino acid sequence having more than 90% sequence identity with the amino acid sequence described in Serial No. 4; and (E) One or more polypeptides having a total length of 10 or more residues and less than 19 amino acids, comprising an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence described in sequence number 5.

[0035]

[25] According to the activator or composition described in [1], the activity inhibitor or composition described in [2], the activation inhibitor or composition described in [3], the inhibitor or composition described in [4], the therapeutic agent, preventive agent or composition described in [5], the method described in [6], the at least one polypeptide described in [7], the use described in [8], the at least one polypeptide described in [9], the kit described in

[10] or

[11] , the diagnostic agent described in

[12] , or the method described in

[13] or

[14] , the at least one polypeptide described above contains: (A) One or more polypeptides having a total length of 8 residues or more but less than 20 residues, comprising an amino acid sequence having more than 90% sequence identity with the amino acid sequence described in sequence number 1; (B) One or more polypeptides comprising an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence described in Serial No. 2, and having a total length of 12 to 21 residues or more; and (D) One or more polypeptides having a total length of 7 to 17 residues and containing an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence described in sequence number 4.

[0036]

[26] According to the activator or composition described in [1], the activity inhibitor or composition described in [2], the activation inhibitor or composition described in [3], the inhibitor or composition described in [4], the therapeutic agent, preventive agent or composition described in [5], the method described in [6], the at least one polypeptide described in [7], the use described in [8], the at least one polypeptide described in [9], the kit described in

[10] or

[11] , the diagnostic agent described in

[12] , or the method described in

[13] or

[14] , the at least one polypeptide described above contains: (A) One or more polypeptides having a total length of 8 residues or more but less than 20 residues, comprising an amino acid sequence having more than 90% sequence identity with the amino acid sequence described in sequence number 1; (B) One or more polypeptides comprising an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence described in Serial No. 2, and having a total length of 12 to 21 residues or more; and (E) One or more polypeptides having a total length of 10 or more residues and less than 19 amino acids, comprising an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence described in sequence number 5.

[0037]

[27] According to the activator or composition described in [1], the activity inhibitor or composition described in [2], the activation inhibitor or composition described in [3], the inhibitor or composition described in [4], the therapeutic agent, preventive agent or composition described in [5], the method described in [6], the at least one polypeptide described in [7], the use described in [8], the at least one polypeptide described in [9], the kit described in

[10] or

[11] , the diagnostic agent described in

[12] , or the method described in

[13] or

[14] , the at least one polypeptide described above contains: (A) One or more polypeptides having a total length of 8 residues or more but less than 20 residues, comprising an amino acid sequence having more than 90% sequence identity with the amino acid sequence described in sequence number 1; (D) One or more polypeptides having a total length of 7 to 17 residues and containing an amino acid sequence having more than 90% sequence identity with the amino acid sequence described in Serial No. 4; and (E) One or more polypeptides having a total length of 10 or more residues and less than 19 amino acids, comprising an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence described in sequence number 5.

[0038]

[28] According to the activator or composition described in [1], the activity inhibitor or composition described in [2], the activation inhibitor or composition described in [3], the inhibitor or composition described in [4], the therapeutic agent, preventive agent or composition described in [5], the method described in [6], the at least one polypeptide described in [7], the use described in [8], the at least one polypeptide described in [9], the kit described in

[10] or

[11] , the diagnostic agent described in

[12] , or the method described in

[13] or

[14] , the at least one polypeptide described above contains: (B) One or more polypeptides having a total length of 12 to 21 residues and containing an amino acid sequence having more than 90% sequence identity with the amino acid sequence described in sequence number 2; (D) One or more polypeptides having a total length of 7 to 17 residues and containing an amino acid sequence having more than 90% sequence identity with the amino acid sequence described in Serial No. 4; and (E) One or more polypeptides having a total length of 10 or more residues and less than 19 amino acids, comprising an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence described in sequence number 5.

[0039]

[29] According to the activator or composition described in [1], the activity inhibitor or composition described in [2], the activation inhibitor or composition described in [3], the inhibitor or composition described in [4], the therapeutic agent, preventive agent or composition described in [5], the method described in [6], the at least one polypeptide described in [7], the use described in [8], the at least one polypeptide described in [9], the kit described in

[10] or

[11] , the diagnostic agent described in

[12] , or the method described in

[13] or

[14] , the at least one polypeptide described above contains: (A) One or more polypeptides having a total length of 8 residues or more but less than 20 residues, comprising an amino acid sequence having more than 90% sequence identity with the amino acid sequence described in sequence number 1; (B) One or more polypeptides having a total length of 12 to 21 residues and containing an amino acid sequence having more than 90% sequence identity with the amino acid sequence described in sequence number 2; (D) One or more polypeptides having a total length of 7 to 17 residues and containing an amino acid sequence having more than 90% sequence identity with the amino acid sequence described in Serial No. 4; and (E) One or more polypeptides having a total length of 10 or more residues and less than 19 amino acids, comprising an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence described in sequence number 5.

[0040]

[30] An activator, composition, activity inhibitor, activation inhibitor, inhibitor, therapeutic agent, preventive agent, method, at least one polypeptide, use, kit or diagnostic agent described in any one of [1] to

[29] , for use in subjects with HLA-DRB3 genotype 01:01 and / or HLA-DRB1 genotype 03:01, wherein the at least one polypeptide contains: (A) one or more polypeptides having an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence described in sequence number 1 and a total length of more than 8 residues and less than 20 residues.

[0041]

[31] An activator, composition, activity inhibitor, activation inhibitor, inhibitor, therapeutic agent, preventive agent, method, at least one polypeptide, use, kit or diagnostic agent described in any one of [1] to

[30] , for use in subjects with HLA-DRB3 genotype 01:01, wherein the at least one polypeptide comprises: (B) one or more polypeptides having an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence described in sequence number 2, and having a total length of more than 12 residues and less than 21 residues.

[0042]

[32] An activator, composition, activity inhibitor, activation inhibitor, inhibitor, therapeutic agent, preventive agent, method, at least one polypeptide, use, kit or diagnostic agent described in any one of [1] to

[31] , for use in subjects with HLA-DRB3 genotype 01:01 and / or HLA-DRB1 genotype 09:01, wherein the at least one polypeptide comprises: (D) one or more polypeptides having an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence described in sequence number 4, and having a total length of 7 or more residues but less than 17 residues.

[0043]

[33] An activator, composition, activity inhibitor, activation inhibitor, inhibitor, therapeutic agent, preventive agent, method, at least one polypeptide, use, kit or diagnostic agent described in any one of [1] to

[32] , for use in subjects with HLA-DRB3 genotype 01:01 and / or HLA-DRB1 genotype 03:01, wherein the at least one polypeptide comprises: (E) one or more polypeptides having an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence described in sequence number 5, and having a full length of 10 or more residues and 19 or fewer.

[0044]

[34] The activator, composition, activity inhibitor, activation inhibitor, inhibitor, therapeutic agent, preventive agent, method, at least one polypeptide, use, kit or diagnostic agent described in any one of [1] to

[33] , wherein the above (A) comprises an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence described in Serial No. 1, and having a total length of 8 or more residues but less than 20 residues, is: (A') a polypeptide having a sequence identity of 90% or more with the amino acid sequence selected from the group consisting of the amino acid sequences described in Serial Nos. 6 to 15.

[0045]

[35] The activator, composition, activity inhibitor, activation inhibitor, inhibitor, therapeutic agent, preventive agent, method, at least one polypeptide, use, kit or diagnostic agent described in any one of [1] to

[34] , wherein (B) above comprises an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence described in Serial No. 2, and having a total length of 12 or more residues or less, and is one or more polypeptides: (B') one or more polypeptides having a sequence identity of 90% or more with the amino acid sequence described in Serial No. 16.

[0046]

[36] The activator, composition, activity inhibitor, activation inhibitor, inhibitor, therapeutic agent, preventive agent, method, at least one polypeptide, use, kit or diagnostic agent described in any one of [1] to

[35] , wherein the above (D) comprises an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence described in Serial No. 4, and having a total length of 7 or more residues but less than 17 residues, is: (D') a polypeptide having a sequence identity of 90% or more with the amino acid sequence selected from the group consisting of the amino acid sequences described in Serial Nos. 22 to 32.

[0047]

[37] The activator, composition, activity inhibitor, activation inhibitor, inhibitor, therapeutic agent, preventive agent, method, at least one polypeptide, use, kit or diagnostic agent described in any one of [1] to

[36] , wherein the above (E) comprises an amino acid sequence having a sequence identity of 90% or more with the amino acid sequence described in Serial No. 5, and having a total length of 10 residues or more and 19 bases or less: (E') is one or more polypeptides having a sequence identity of 90% or more with the amino acid sequence selected from the group consisting of the amino acid sequences described in Serial Nos. 33 to 35.

[0048]

[38] The above-mentioned autoimmune disease is myasthenia gravis, as described in any one of [5] to

[37] , including a therapeutic agent, preventive agent, composition, method, at least one polypeptide, use, kit or diagnostic agent.

[0049]

[39] The above-mentioned autoimmune disease is early-onset myasthenia gravis, as described in any one of [5] to

[38] , including a therapeutic agent, preventive agent, composition, method, at least one polypeptide, use, kit or diagnostic agent.

[0050] The effects of the invention According to this disclosure, a formulation containing a polypeptide comprising an amino acid sequence equivalent to an AChR epitope is provided, which has the potential to be applied to the treatment or diagnosis of autoimmune diseases.

[0051] The polypeptide contained in the formulation disclosed herein, which contains an amino acid sequence equivalent to an AChR epitope, was discovered using the NAPA method with dendritic cells prepared from classical human mononuclear cells. Because it is more likely to actually exist in patients with autoimmune diseases, it is more suitable for the treatment or diagnosis of autoimmune diseases than polypeptides with equivalent epitope sequences discovered to date. Attached Figure Description

[0052]

【 Figure 1 In Example 1, the identified HLA-binding peptide is shown as a graph of coordinates on human AChRα1.

[0053]

【 Figure 2 The figure shows the results of ICS (upper segment) and ELISA (lower segment) assays performed on each peptide using normal human PBMCs in Example 2, evaluating their specific T-cell induction capacity in six independent assays.

[0054]

【 Figure 3 [This refers to the use of 5 copies of the predicted restriction allele DRB3 with sequence number 17 in Example 3.] A figure showing the results of ELISA analysis evaluating the presence or absence of sequence number 17 specific Th1 cells in PBMC samples from MG patients at 01:01.

[0055]

【 Figure 4 [To use HLA-DRB1, which has been reported to be associated with MG in Asians] 09:01 and does not have DRB1 03:01, DRB3 A graph showing the results of ICS (left) and ELISA (right) on PBMCs derived from normal human at 01:01, evaluating the specific Th1 cell induction capacity of sequence number 17. Detailed Implementation

[0056] The following describes a method for implementing this disclosure, but this disclosure is not limited to the following implementation methods.

[0057] <Sequence Identity> In this disclosure, "sequence identity" refers to the percentage (%) of identical residues relative to residues in overlapping amino acid sequences during optimal alignment analysis of two amino acid sequences. In this disclosure, mutations in an amino acid sequence that do not represent 100% sequence identity with respect to a particular amino acid sequence can be, for example, substitutions, deletions, or insertions of amino acid residues; more specifically, they can be single-residue substitutions, single-residue deletions, or single-residue insertions, which can occur at the ends of the amino acid sequence or outside the ends.

[0058] Human leukocyte antigen (HLA) Human leukocyte antigens (HLA) are the major histocompatibility complex (MHC) in humans. In humoral immunity, HLA is exposed on the cell surface of antigen-presenting cells (APCs) in an antigen-bound state, thereby driving the antibody production mechanism against that antigen. HLA is divided into two classes: HLA class I (including HLA-A, HLA-C, and HLA-B) which are responsible for cellular immunity, and HLA class II (including HLA-DR, HLA-DQ, and HLA-DP) which are responsible for humoral immunity.

[0059] Based on HLA genotype, human white blood cells have tens of thousands of genotypes. HLA genotype is one of the risk factors for autoimmune diseases; it is known that individuals with specific HLA genotypes have a higher likelihood of developing autoimmune diseases. Particularly in early-onset myasthenia gravis, an HLA-DRB3 genotype of 01:01 or an HLA-DRB1 genotype of 03:01 is known to be a risk factor.

[0060] According to the notation system defined by the WHO's Nomenclature Committee for Factors of the HLA System, HLA genotypes (alleles) are indicated by "HLA- (gene symbol)". The regions are labeled in the format "(Region 1):(Region 2):(Region 3):(Region 4)". Regions 1 through 4 are represented by two-digit values: Region 1 indicates HLA specificity (antigen type), Region 2 indicates non-synonymous substitutions (variations in the amino acid sequence within the same antigen type), Region 3 indicates synonymous substitutions (variations in the encoded amino acid sequence within exons that do not change), and Region 4 indicates base substitutions outside the coding region (variations in the sequence of intron regions). Among these labels, the gene symbol, Region 1, and Region 2 provide information directly affecting the amino acid sequence of HLA and are clinically important.

[0061] In this disclosure, the first region of the genotype of the HLA gene with gene symbol XXX is YY, and the second region is ZZ (i.e., according to the above-described marking method, up to the second region is represented as HLA-XXX). YY:ZZ) is also recorded as "HLA-XXX genotype is YY:ZZ". For example, "HLA-DRB3 genotype is 01:01" means that the genotype of the HLA gene with the gene symbol DRB3 (HLA-DRB3 gene) has 01 in the first region and 01 in the second region, i.e., the genotype is HLA-DRB3. 01:01. For example, "HLA-DRB1 genotype is 03:01" means that the genotype of the HLA gene with the gene symbol DRB1 (HLA-DRB1 gene) has 03 in the first region and 01 in the second region, i.e., the genotype is HLA-DRB1. 03:01.

[0062] <Acetylcholine receptor> Acetylcholine receptors (AChRs) are receptors essential for neurotransmission, binding to acetylcholine secreted extracellularly and facilitating information transmission between nerve cells. In autoimmune diseases where antibodies against acetylcholine receptors are overproduced, a portion of neurotransmission mediated by acetylcholine receptors is inhibited, resulting in various symptoms. Furthermore, the α1 subunit of the acetylcholine receptor, particularly 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 (Sequence No. 36) of Ref seq accession number NP_000070.1 from the National Center for Biotechnology Information (NCBI).

[0063] <Activators or compositions for activating regulatory T cells> The first aspect of this disclosure relates to an activator of regulatory T cells, comprising a subset selected from (A), (B), (D), and (E) below: (A) One or more polypeptides having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence described in Serial No. 1 and a total length of 8 or more residues and less than 20 residues. (B) One or more polypeptides having a total length of 12 residues or more and 21 residues or less, which contain an amino acid sequence having more than 90% sequence identity with the amino acid sequence recorded in sequence number 2; (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in Serial No. 4, and having a full length of 7 or more residues and less than 17 residues; and (E) One or more polypeptides having a full length of 10 or more residues and less than 19 amino acids, which contain an amino acid sequence that has 90% or more sequence identity with the amino acid sequence described in sequence number 5. At least one polypeptide constitutes the group. In one embodiment, the first aspect of this disclosure may also be a composition for activating regulatory T cells comprising at least one of the above-mentioned polypeptides.

[0064] An embodiment of the formulation may include the above-described (A). An embodiment of the formulation may include the above-described (B). An embodiment of the formulation may include the above-described (D). An embodiment of the formulation may include the above-described (E). An embodiment of the formulation may include both (A) and (B). An embodiment of the formulation may include both (A) and (D). An embodiment of the formulation may include both (A) and (E). An embodiment of the formulation may include both (B) and (D). An embodiment of the formulation may include both (B) and (E). An embodiment of the formulation may include both (D) and (E). An embodiment of the formulation may include (A), (B), and (D). An embodiment of the formulation may include both (A), (B), and (E). An embodiment of the formulation may include both (A), (D), and (E). One embodiment of the formulation may include the above-described (B), (D), and (E). Another embodiment of the formulation may include the above-described (A), (B), (D), and (E).

[0065] In cases where one or more polypeptides shown in (A), (B), (D), and (E) above contain or are composed of an amino acid sequence that has a sequence identity of 90% or more with a given sequence (e.g., the sequences shown in sequence numbers 1-17 and 22-35, or a portion of the sequence shown in sequence number 37), the sequence identity of the amino acid sequence with the given sequence may exceed 90%, for example, the sequence identity may be 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%. Furthermore, in cases where the above-described (A), (B), (D) and the one or more polypeptides shown contain an amino acid sequence that has more than 90% sequence identity with a given sequence (e.g., the sequences shown in sequence numbers 1-17 and 22-35, or a portion of the sequence shown in sequence number 37), or is composed of such an amino acid sequence, the amino acid sequence may be, for example, the given sequence or an amino acid sequence containing a mutation of 1 residue, 2 residues, or 3 residues. In a preferred embodiment, it may be the given sequence or an amino acid sequence containing a mutation of 1 residue.

[0066] The amino acid sequence described in Serial No. 1 corresponds to positions 121–131 of the amino acid sequence of the α1 subunit of the human acetylcholine receptor (Serial No. 36) in Refseq accession number NP_000070.1 of the National Center for Biotechnology Information (NCBI). Furthermore, the amino acid sequence described in Serial No. 1 corresponds to positions 101–111 of the amino acid sequence (Serial No. 37) obtained by removing the amino acid sequences corresponding to the signal peptide, i.e., positions 1–20, from the amino acid sequence shown in Serial No. 36.

[0067] Serial Number 1: AIVKFTKVLLQ In one embodiment of this disclosure, the total length of one or more polypeptides in (A) above can be, for example, 8 or more residues, 9 or more residues, 10 or more residues, 11 or more residues, 12 or more residues, 13 or more residues, or 14 or more residues, and can be less than 20 residues, less than 19 residues, less than 18 residues, less than 17 residues, more than 16 residues, less than 15 residues, less than 14 residues, less than 13 residues, less than 12 residues, or less than 11 residues. Furthermore, in one embodiment of this disclosure, the total length of one or more polypeptides in (A) above can be, for example, 8 or more residues and less than 20 residues, 9 or more residues and less than 19 residues, 10 or more residues and less than 18 residues, 11 or more residues and less than 17 residues, 12 or more residues and less than 17 residues, 12 or more residues and less than 15 residues, 12 or more residues and less than 13 residues, or 12 residues. In a preferred embodiment, the full length of one or more polypeptides in (A) above can be more than 12 residues and less than 17 residues.

[0068] In one embodiment of this disclosure, the one or more polypeptides of (A) above comprise an amino acid sequence having a sequence identity of 90% or more, 92% or more, 95% or more, 98% or more, or 100% with the amino acid sequence described in Serial Number 1. In one embodiment of this disclosure, the one or more polypeptides of (A) above comprise the amino acid sequence described in Serial Number 1 or an amino acid sequence having a mutation of 1 residue, 2 residues, or 3 residues in the amino acid sequence.

[0069] In one embodiment of this disclosure, one or more polypeptides of (A) above may include positions 98-117, 99-117, 100-117, 101-117, 98-116, 99-116, 100-116, 101-116, 98-115, 99-115, 100-115, 101-115, 98-114, 99-114, 100-114, 101-115, 98-114, 99-114, 100-114, 101-115, 98-114, 99-114, 100-114, 101-115, 98-114, 99-114, 100-114, 101-115, 98-114, 99-114, 100-114, 101-114, 99-115, 100-114, 101-115, 99-114, 100-114, 101-114 ...100-114, 101-114, 100-114, 101-114, The amino acid sequence having at least one amino acid sequence with more than 90% sequence identity in the group consisting of positions 114, 98-113, 99-113, 100-113, 101-113, 98-112, 99-112, 100-112, 101-112, 98-111, 99-111, 100-111, and 101-111 can also be composed of the amino acid sequence having more than 90% sequence identity with the at least one sequence. For example, positions 98-117 of the amino acid sequence shown in sequence number 37 are the same as the amino acid sequence shown in sequence number 3. In one embodiment of this disclosure, one or more polypeptides of (A) above may comprise an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence shown in Serial No. 3, or may be composed of an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence shown in Serial No. 3, or may be composed of the amino acid sequence.

[0070] Serial Number 3: GDFAIVKFTKVLLQYTGHIT In one embodiment of this disclosure, the one or more polypeptides described in (A) can be one or more polypeptides whose sequence identity with an amino acid sequence selected from the group consisting of amino acid sequences described in serial numbers 6 to 15 is 90% or more, or can be one or more polypeptides consisting of amino acid sequences selected from the group consisting of amino acid sequences described in serial numbers 6 to 15. The amino acid sequence shown in serial number 6 corresponds to positions 98 to 111 of the amino acid sequence shown in serial number 37. The amino acid sequence shown in serial number 7 corresponds to positions 98 to 113 of the amino acid sequence shown in serial number 37. The amino acid sequence shown in serial number 8 corresponds to positions 98 to 114 of the amino acid sequence shown in serial number 37. The amino acid sequence shown in serial number 9 corresponds to positions 99 to 111 of the amino acid sequence shown in serial number 37. The amino acid sequence shown in serial number 10 corresponds to positions 99 to 113 of the amino acid sequence shown in serial number 37. The amino acid sequence shown in serial number 11 corresponds to positions 99 to 114 of the amino acid sequence shown in serial number 37. The amino acid sequence shown in sequence number 12 corresponds to positions 100-111 of the amino acid sequence shown in sequence number 37. The amino acid sequence shown in sequence number 13 corresponds to positions 100-114 of the amino acid sequence shown in sequence number 37. The amino acid sequence shown in sequence number 14 corresponds to positions 101-114 of the amino acid sequence shown in sequence number 37. The amino acid sequence shown in sequence number 15 corresponds to positions 101-117 of the amino acid sequence shown in sequence number 37.

[0071] Serial Number 6: GDFAIVKFTKVLLQ Serial Number 7: GDFAIVKFTKVLLQYT Serial Number 8: GDFAIVKFTKVLLQYTG Serial Number 9: DFAIVKFTKVLLQ Serial Number 10: DFAIVKFTKVLLQYT Serial Number 11: DFAIVKFTKVLLQYTG Serial Number 12: FAIVKFTKVLLQ Serial Number 13: FAIVKFTKVLLQYTG Serial Number 14: AIVKFTKVLLQYTG Serial Number 15: AIVKFTKVLLQYTGHIT The inventors have discovered that one or more polypeptides in (A) above are related to HLA-DRB3 in human HLA. 01:01 and HLA-DRB1 03:01 combination. Therefore, an embodiment of this disclosure may be a formulation for subjects with HLA-DRB3 genotype 01:01 and / or HLA-DRB1 genotype 03:01, and the above-mentioned at least one polypeptide comprises one or more polypeptides of (A) above.

[0072] The amino acid sequence described in sequence number 2 corresponds to positions 110–124 of the amino acid sequence of the α1 subunit of the human acetylcholine receptor (Sequence No. 36) in Refseq accession number NP_000070.1 of the National Center for Biotechnology Information (NCBI). Furthermore, the amino acid sequence described in sequence number 2 corresponds to positions 90–104 of the amino acid sequence (Sequence No. 37) obtained by removing the amino acid sequences corresponding to the signal peptide, i.e., positions 1–20.

[0073] Serial Number 2: LVLYNNADGDFAIVK In one embodiment of this disclosure, the total length of one or more polypeptides in (B) above can be, for example, 12 or more residues, 13 or more residues, 14 or more residues, 15 or more residues, 16 or more residues, 17 or more residues, or 18 or more residues, and can be less than 21 residues, 20 or more residues, 19 or less residues, 18 or less residues, 17 or less residues, 16 or less residues, or 15 or less residues. Furthermore, in one embodiment of this disclosure, the total length of one or more polypeptides in (B) above can be, for example, 12 or more residues and less than 21 residues, 13 or more residues and less than 21 residues, 14 or more residues and less than 21 residues, 15 or more residues and less than 21 residues, 15 or more residues and less than 19 residues, 15 or more residues and less than 17 residues, 15 or more residues and less than 16 residues, or 15 residues. In a preferred embodiment, the full length of one or more polypeptides in (B) above can be 15 residues or more and 16 residues or less.

[0074] In one embodiment of this disclosure, the one or more polypeptides in (B) above comprise an amino acid sequence having a sequence identity of 90% or more, 93% or more, 95% or more, 98% or more, or 100% with the amino acid sequence described in Serial No. 2. In one embodiment of this disclosure, the one or more polypeptides in (B) above comprise the amino acid sequence described in Serial No. 2 or an amino acid sequence having a mutation of 1 residue, 2 residues, or 3 residues in that amino acid sequence. In a preferred embodiment, the one or more polypeptides in (B) above comprise an amino acid sequence having a sequence identity of 93% or more with the amino acid sequence described in Serial No. 2.

[0075] In one embodiment of this disclosure, the one or more polypeptides described in (B) above may comprise an amino acid sequence having a sequence identity of 90% or more with at least one of the groups formed by positions 87-107, 88-107, 89-107, 90-107, 87-106, 88-106, 89-106, 90-106, 87-105, 88-105, 89-105, 90-105, 87-104, 88-104, 89-104, and 90-104 of the amino acid sequence shown in Serial No. 37. Alternatively, the polypeptide may be composed of the amino acid sequence having a sequence identity of 90% or more with the at least one sequence, or may be composed of the at least one amino acid sequence.

[0076] In one embodiment of this disclosure, the one or more polypeptides described in (B) may be one or more polypeptides whose sequence identity with the amino acid sequence selected from the group consisting of the amino acid sequence described in Serial No. 16 is 90% or more, or may be a polypeptide consisting of the amino acid sequence described in Serial No. 16. The amino acid sequence shown in Serial No. 16 corresponds to positions 90 to 104 of the amino acid sequence shown in Serial No. 37.

[0077] Serial Number 16: LVLYNNADGDFAIVK The inventors have discovered that one or more polypeptides in (B) above are related to HLA-DRB3 in human HLA. 01:01 combination. Therefore, an embodiment of this disclosure may involve a formulation for an HLA-DRB3 genotype of 01:01, and the above-mentioned at least one polypeptide may contain one or more polypeptides of (B) above.

[0078] The amino acid sequence described in sequence number 4 corresponds to positions 334–343 of the amino acid sequence of the α1 subunit of the human acetylcholine receptor (Sequence No. 36) in Refseq accession number NP_000070.1 of the National Center for Biotechnology Information (NCBI). Furthermore, the amino acid sequence described in sequence number 4 corresponds to positions 314–323 of the amino acid sequence obtained by removing the amino acid sequences corresponding to the signal peptide (positions 1–20) from the amino acid sequence shown in sequence number 36 (Sequence No. 37).

[0079] Serial Number 4: KVFIDTIPNI In one embodiment of this disclosure, the total length of one or more polypeptides in (D) can be, for example, 7 or more residues, 8 or more residues, 9 or more residues, 10 or more residues, 11 or more residues, 12 or more residues, 13 or more residues, or 14 or more residues, and can be 17 or less residues, 16 or more residues, 15 or less residues, 14 or less residues, 13 or less residues, 12 or less residues, or 11 or less residues. Furthermore, in one embodiment of this disclosure, the total length of one or more polypeptides in (D) can be, for example, 7 or more residues and 17 or less residues, 8 or more residues and 17 or less residues, 9 or more residues and 17 or less residues, 10 or more residues and 17 or less residues, 11 or more residues and 16 or less residues, 11 or more residues and 14 or less residues, 11 or more residues and 13 or less residues, 11 or more residues and 12 or less residues, or 11 residues. In a preferred embodiment, the full length of one or more polypeptides in (D) above can be 11 residues or more and 16 residues or less.

[0080] In one embodiment of this disclosure, the one or more polypeptides of (D) above comprise an amino acid sequence having a sequence identity of 90% or more, 95% or more, 98% or more, or 100% with the amino acid sequence described in Serial No. 4. In one embodiment of this disclosure, the one or more polypeptides of (D) above comprise the amino acid sequence described in Serial No. 4 or an amino acid sequence having a mutation of 1 residue, 2 residues, or 3 residues in that amino acid sequence.

[0081] In one embodiment of this disclosure, one or more polypeptides of (D) above may include positions 311-327, 312-327, 313-327, 314-327, 311-326, 312-326, 313-326, 314-326, 311-325, and 312 of the amino acid sequence shown in sequence number 37. The amino acid sequence having at least 90% or more sequence identity with the amino acid sequence formed by positions ~325, 313~325, 314~325, 311~324, 312~324, 313~324, and 314~324 can also be composed of the amino acid sequence having at least 90% or more sequence identity with the at least one sequence. For example, positions 311~327 of the amino acid sequence shown in sequence number 37 are the amino acid sequence shown in sequence number 17. In one embodiment of this disclosure, one or more polypeptides described above (D) can include an amino acid sequence having at least 90% or more sequence identity with the amino acid sequence shown in sequence number 17, or can be composed of the amino acid sequence having at least 90% or more sequence identity with the amino acid sequence shown in sequence number 17.

[0082] Serial Number 17: WVRKVFIDTIPNIMFFS In one embodiment of this disclosure, the one or more polypeptides described in (D) can be one or more polypeptides whose sequence identity with amino acid sequences selected from the group consisting of amino acid sequences described in serial numbers 22-32 is 90% or more, or can be one or more polypeptides consisting of amino acid sequences selected from the group consisting of amino acid sequences described in serial numbers 22-32. The amino acid sequence shown in serial number 22 corresponds to positions 311-323 of the amino acid sequence shown in serial number 37. The amino acid sequence shown in serial number 23 corresponds to positions 311-324 of the amino acid sequence shown in serial number 37. The amino acid sequence shown in serial number 24 corresponds to positions 312-324 of the amino acid sequence shown in serial number 37. The amino acid sequence shown in serial number 25 corresponds to positions 312-325 of the amino acid sequence shown in serial number 37. The amino acid sequence shown in serial number 26 corresponds to positions 312-327 of the amino acid sequence shown in serial number 37. The amino acid sequence shown in serial number 27 corresponds to positions 313-324 of the amino acid sequence shown in serial number 37. The amino acid sequence shown in sequence number 28 corresponds to positions 313-325 of the amino acid sequence shown in sequence number 37. The amino acid sequence shown in sequence number 29 corresponds to positions 313-326 of the amino acid sequence shown in sequence number 37. The amino acid sequence shown in sequence number 30 corresponds to positions 313-327 of the amino acid sequence shown in sequence number 37. The amino acid sequence shown in sequence number 31 corresponds to positions 314-324 of the amino acid sequence shown in sequence number 37. The amino acid sequence shown in sequence number 32 corresponds to positions 314-326 of the amino acid sequence shown in sequence number 37.

[0083] Serial Number 22: WVRKVFIDTIPNI Serial Number 23: WVRKVFIDTIPNIM Serial Number 24: VRKVFIDTIPNIM Serial Number 25: VRKVFIDTIPNIMF Serial Number 26: VRKVFIDTIPNIMFFS Serial Number 27: RKVFIDTIPNIM Serial Number 28: RKVFIDTIPNIMF Serial Number 29: RKVFIDTIPNIMFF Serial Number 30: RKVFIDTIPNIMFFS Serial Number 31: KVFIDTIPNIM Serial number 32: KVFIDTIPNIMFF The inventors have discovered that one or more polypeptides of (D) above interact with HLA-DRB3 in human HLA. 01:01 and HLA-DRB1 09:01 combination. Therefore, the formulation involved in one embodiment of this disclosure can be a formulation for subjects with HLA-DRB3 genotype 01:01 and / or HLA-DRB1 genotype 09:01, and the above-mentioned at least one polypeptide comprises one or more polypeptides of the above (D).

[0084] The amino acid sequence described in sequence number 5 corresponds to positions 165–177 of the amino acid sequence of the α1 subunit of the human acetylcholine receptor (Sequence No. 36) in Refseq accession number NP_000070.1 of the National Center for Biotechnology Information (NCBI). Furthermore, the amino acid sequence described in sequence number 5 corresponds to positions 145–157 of the amino acid sequence (Sequence No. 37) obtained by removing the amino acid sequences corresponding to the signal peptide, i.e., positions 1–20, from the amino acid sequence shown in sequence number 36.

[0085] Serial Number 5: KLGTWTYDGSVVA In one embodiment of this disclosure, the total length of one or more polypeptides in (E) can be, for example, 10 or more residues, 11 or more residues, 12 or more residues, 13 or more residues, 14 or more residues, 15 or more residues, or 16 or more residues, and can be 19 or less residues, 18 or more residues, 17 or less residues, 16 or less residues, 15 or less residues, 14 or less residues, or 13 or less residues. Furthermore, in one embodiment of this disclosure, the total length of one or more polypeptides in (E) can be, for example, 10 or more residues and 19 or less residues, 11 or more residues and 19 or less residues, 12 or more residues and 19 or less residues, 13 or more residues and 19 or less residues, 13 or more residues and 17 or less residues, 13 or more residues and 15 or less residues, 13 or more residues and 14 or less residues, or 13 residues. In a preferred embodiment, the full length of one or more polypeptides in (E) can be 13 or more residues and less than 17 residues.

[0086] In one embodiment of this disclosure, the one or more polypeptides of (E) above comprise an amino acid sequence having a sequence identity of 90% or more, 92% or more, 95% or more, 98% or more, or 100% with the amino acid sequence described in Serial No. 5. In one embodiment of this disclosure, the one or more polypeptides of (E) above comprise the amino acid sequence described in Serial No. 5 or an amino acid sequence having a mutation of 1 residue, 2 residues, or 3 residues in the amino acid sequence. In a preferred embodiment, the one or more polypeptides of (E) above comprise an amino acid sequence having a sequence identity of 92% or more with the amino acid sequence described in Serial No. 5.

[0087] In one embodiment of this disclosure, one or more polypeptides of (E) above may include positions 142-161, 143-161, 144-161, 145-161, 142-160, 143-160, 144-160, 145-160, 142-159, 143-159, 144-159, and 145 of the amino acid sequence shown in sequence number 37. The amino acid sequence having a sequence identity of 90% or more with at least one of the groups consisting of positions ~159, 142-158, 143-158, 144-158, 145-158, 142-157, 143-157, 144-157, and 145-157 can also be composed of the amino acid sequence having a sequence identity of 90% or more with the at least one sequence, or can also be composed of the at least one amino acid sequence.

[0088] In one embodiment of this disclosure, the one or more polypeptides described in (E) may be one or more polypeptides whose sequence identity with an amino acid sequence selected from the group consisting of amino acid sequences described in sequences 33-35 is 90% or more, or may be one or more polypeptides consisting of amino acid sequences selected from the group consisting of amino acid sequences described in sequences 33-35. The amino acid sequence shown in sequence 33 corresponds to positions 145-157 of the amino acid sequence shown in sequence 37. The amino acid sequence shown in sequence 34 corresponds to positions 145-160 of the amino acid sequence shown in sequence 37. The amino acid sequence shown in sequence 35 corresponds to positions 145-161 of the amino acid sequence shown in sequence 37.

[0089] Serial Number 33: KLGTWTYDGSVVA Serial number 34: KLGTWTYDGSVVAINP Serial number 35: KLGTWTYDGSVVAINPE The inventors have discovered that one or more polypeptides in (E) above are related to HLA-DRB3 in human HLA. 01:01 and HLA-DRB1 03:01 combination. Therefore, the formulation involved in one embodiment of this disclosure can be a formulation for subjects with HLA-DRB3 genotype 01:01 and / or HLA-DRB1 genotype 03:01, and the above-mentioned at least one polypeptide comprises one or more polypeptides of the above (E).

[0090] At least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above binds to HLA-DR. Therefore, although not expected to be bound by any theory, it is believed that by bringing at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above into contact with and being phagocytosed by an antigen-presenting cell that promotes the activation (differentiation) of regulatory T cells as described in Non-Patent Literature 1, the at least one polypeptide is presented to the naïve CD4 in an HLA-DR-bound state. + T cells, which in turn can induce the activation (differentiation) of regulatory T cells.

[0091] One embodiment of the formulation, in addition to at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above, may further contain one or more HLA-DRs, preferably HLA-DRs capable of binding to at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above, and preferably having a genotype consistent with the genotype of the HLA-DR in the target drug. Another embodiment of the formulation, in addition to at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above, may further contain additives commonly used in the field of formulation technology, such additives being at least one selected from the group consisting of excipients, buffers, stabilizers, antioxidants, binders, disintegrants, fillers, emulsifiers, and flow modifiers.

[0092] One embodiment of the formulation contains components that can 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.

[0093] The dosage form of the formulation involved in one embodiment is not particularly limited, for example, it can be a liquid drug or a tablet, but when the ingredients contained in the formulation involved in one embodiment are encapsulated in a drug carrier for intracellular delivery, the dosage form is preferably a liquid drug.

[0094] The administration method of the formulation involved in one embodiment is not particularly limited, and examples include intraosseous administration, intravenous injection, subcutaneous injection, intramuscular injection, oral administration, or spinal injection. As a specific dosage example, when administering the formulation to an adult male (weighing 60 kg), the dosage per day, based on the amount of active ingredient, can be 0.001 μg to 1000 mg / day / person.

[0095] The content of at least one polypeptide selected from the group consisting of (A), (B), (D) and (E) in the formulation involved in one embodiment is not particularly limited. For example, based on the total amount of the formulation, the total content can be 0.001 to 100 by mass.

[0096] As one embodiment, the target population for administering the formulation can be, for example, a human being, a person already suffering from an autoimmune disease, a person who may have an autoimmune disease, or a person with a genetic predisposition to the disease. The aforementioned autoimmune disease can be, for example, an autoimmune disease caused by the overproduction of antibodies against human AchRα1 or its peptide fragments, preferably myasthenia gravis, more preferably early-onset myasthenia gravis. Furthermore, myasthenia gravis is broadly classified into early-onset myasthenia gravis, which occurs in individuals aged 10 to 49 years, and late-onset myasthenia gravis, which occurs in individuals aged 50 years and older, with some differences in HLA genotypes that can be risk factors between the two.

[0097] Because at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above was discovered using the NAPA method with dendritic cells prepared from classical human monocytes, it is highly likely to be present in patients already suffering from autoimmune diseases. Therefore, compared to polypeptides with epitope sequences discovered to date, it is potentially more suitable for the treatment or diagnosis of autoimmune diseases. The fact that at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above is more suitable for the treatment or diagnosis of autoimmune diseases than polypeptides with epitope sequences discovered to date (other polypeptides) can be evaluated, for example, by the higher T cell activation activity of the at least one polypeptide upon contact with T cells compared to the other polypeptides mentioned above. Such T cell activation activity can be evaluated, for example, by a T cell activation assay. A T cell activation assay is an ELISA test using IFN-γ, etc., to confirm whether T cells present in PBMCs are activated by exposure to a polypeptide. When a polypeptide sequence corresponding to the discovered epitope is added to human PBMCs, it is presented as an epitope by antigen-presenting cells within the PBMCs. T cells with TCRs capable of capturing this HLA-binding polypeptide receive the signal and release activating cytokines such as IFN-γ. Activation is determined by measuring the released cytokines. Such a measurement can be performed using commercially available kits, such as the BD OptEIA (registered trademark) Human IFN-γ ELISA Set (BD Biosciences, Cat. No. 555142).

[0098] <Inhibitors or compositions for inhibiting the activity of effector T cells> The second aspect of the present disclosure relates to an effector T cell activity inhibitor, which contains at least one polypeptide selected from the group consisting of the above (A), (B), (D), and (E). In one embodiment, the second aspect of the present disclosure may also be a composition for inhibiting the activity of effector T cells containing the above at least one polypeptide. As the effector T cell activity inhibitor related to the second aspect of the present disclosure, the same substances as those described in the activator of regulatory T cells related to the first aspect of the present disclosure can be used, and the descriptions and definitions recorded in the requirements of the activator of regulatory T cells related to the first aspect of the present disclosure are also incorporated into the effector T cell activity inhibitor related to the second aspect of the present disclosure.

[0099] Although not wishing to be bound by any theory, it is considered that, as described in the first aspect of the present disclosure, the effector T cell activity inhibitor containing at least one polypeptide selected from the group consisting of the above (A), (B), (D), and (E) promotes the activation (differentiation) of regulatory T cells through the action of antigen-presenting cells as described in Non-Patent Document 1, and thereby can inhibit the activity of effector T cells.

[0100] <Inhibitor of B cell activation or composition for inhibiting B cell activation> The third aspect of the present disclosure relates to an inhibitor of B cell activation, which contains at least one polypeptide selected from the group consisting of the above (A), (B), (D), and (E). In one embodiment, the third aspect of the present disclosure may also be a composition for inhibiting B cell activation containing the above at least one polypeptide. As the inhibitor of B cell activation related to the third aspect of the present disclosure, the same substances as those described in the activator of regulatory T cells related to the first aspect of the present disclosure can be used, and the descriptions and definitions recorded in the requirements of the activator of regulatory T cells related to the first aspect of the present disclosure are also incorporated into the inhibitor of B cell activation related to the third aspect of the present disclosure.

[0101] Although not wishing to be bound by any theory, it is considered that, as described in the second aspect of the present disclosure, the inhibitor of B cell activation containing at least one polypeptide selected from the group consisting of the above (A), (B), (D), and (E) can inhibit the activity of effector T cells through the action of antigen-presenting cells as described in Non-Patent Document 1, and thereby can inhibit the activation of B cells.

[0102] <Inhibitor of humoral immunity or composition for inhibiting humoral immunity>[ The fourth aspect of this disclosure relates to an inhibitor of humoral immunity (e.g., an antibody production inhibitor) comprising at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above. In one embodiment, the fourth aspect of this disclosure may also be a composition for inhibiting humoral immunity comprising at least one of the aforementioned polypeptides. As the inhibitor of humoral immunity according to the fourth aspect of this disclosure, the same substances described in the regulatory T cell activators according to the first aspect of this disclosure may be used. The descriptions and definitions set forth in the essentials of the regulatory T cell activators according to the first aspect of this disclosure are also applied to the inhibitor of humoral immunity according to the fourth aspect of this disclosure.

[0103] While not intended to be bound by any theory, it is believed that, as described in the third aspect of this disclosure, an inhibitor of humoral immunity comprising at least one polypeptide selected from the group consisting of (A), (B), (D) and (E) above can inhibit the activation of B cells by the action of antigen-presenting cells as described in Non-Patent Document 1, thereby inhibiting humoral immunity.

[0104] <Therapeutic agents, preventive agents, therapeutic compositions, or preventive compositions for autoimmune diseases> The fifth aspect of this disclosure relates to a therapeutic or preventive agent for an autoimmune disease, comprising at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above. In one embodiment, the fifth aspect of this disclosure may also be a therapeutic or preventive composition for an autoimmune disease comprising the at least one polypeptide. In one embodiment, the fifth aspect of this disclosure may also be a method of treating or preventing an autoimmune disease including administering the at least one polypeptide to a subject in need. In one embodiment, the fifth aspect of this disclosure may also be the use of the at least one polypeptide in the treatment or prevention of an autoimmune disease. In one embodiment, the fifth aspect of this disclosure may also be the use of the at least one polypeptide in the manufacture of a therapeutic, preventive, therapeutic, or preventive composition for an autoimmune disease. In one embodiment, the fifth aspect of this disclosure may also be the use of the at least one polypeptide in the manufacture of a therapeutic, preventive, therapeutic, or preventive composition for an autoimmune disease. As a therapeutic or preventive agent for the autoimmune disease covered by the fifth aspect of this disclosure, the same substances described in the regulatory T cell activators covered by the first aspect of this disclosure may be used. The descriptions and definitions set forth in the essentials of the regulatory T cell activators covered by the first aspect of this disclosure are also referenced in the therapeutic or preventive agent for the autoimmune disease covered by the fifth aspect of this disclosure.

[0105] The autoimmune disease involved in the fifth aspect of this disclosure can be, for example, an autoimmune disease caused by the overproduction of antibodies against human AchRα1 or its peptide fragments, preferably myasthenia gravis, more preferably early-onset myasthenia gravis. Since at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above is a peptide fragment containing an amino acid sequence corresponding to a human AchRα1 epitope known to be recognized as an autoantigen in myasthenia gravis and early-onset myasthenia gravis, the formulation according to one embodiment of the fifth aspect of this disclosure is suitable for treating and preventing myasthenia gravis and early-onset myasthenia gravis.

[0106] The therapeutic or preventative agent according to one embodiment of the fifth aspect of this disclosure can be used, for example, to administer to a person, a person already suffering from an autoimmune disease, a person who may have an autoimmune disease, or a person with a genetic predisposition to the disease. The autoimmune disease can be, for example, an autoimmune disease caused by the overproduction of antibodies against human AchRα1 or its peptide fragments, preferably myasthenia gravis, more preferably early-onset myasthenia gravis. Furthermore, when the formulation according to one embodiment of the fifth aspect of this disclosure is a therapeutic agent, it can be administered to, for example, a person who already suffers from or is likely to suffer from an autoimmune disease. Furthermore, when the formulation according to one embodiment of the fifth aspect of this disclosure is a preventative agent, it can be administered to a person who may have an autoimmune disease or a person with a genetic predisposition to the disease.

[0107] While not intended to be bound by any theory, it is believed that, as described in the fourth aspect of this disclosure, a therapeutic or preventive agent for autoimmune diseases comprising at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above can suppress humoral immunity through the action of antigen-presenting cells as described in Non-Patent Document 1, thereby enabling the treatment and prevention of autoimmune diseases, for example, by inhibiting the production of anti-AchRα1 antibodies and inhibiting the breakdown of AchRα1, thereby enabling the treatment and prevention of myasthenia gravis such as early-onset myasthenia gravis.

[0108] <Diagnostic aids for autoimmune diseases, methods for obtaining diagnostic data, diagnostic methods> A sixth aspect of this disclosure is a method for assisting in the diagnosis of autoimmune diseases, comprising detecting T cells that are antigen-specific to at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above. In one embodiment, the sixth aspect of this disclosure may also be a method for obtaining data for diagnosing autoimmune diseases, comprising detecting T cells that are antigen-specific to at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above. In one embodiment, the sixth aspect of this disclosure may also be a method for diagnosing autoimmune diseases, comprising detecting T cells that are antigen-specific to at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above. 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 fragment pairs, preferably myasthenia gravis, more preferably early-onset myasthenia gravis. In these cases, at least one of the polypeptides selected from the group consisting of (A), (B), (D), and (E) above may be used with the same substances described in the regulatory T cell activator according to the first aspect of this disclosure. The description and definition of the at least one polypeptide described in the section on the regulatory T cell activator according to the first aspect of this disclosure are also applied to the at least one polypeptide in the method according to the sixth aspect of this disclosure.

[0109] In individuals already suffering from autoimmune diseases (e.g., autoimmune diseases caused by the overproduction of antibodies against human AchRα1 or its peptide fragments, preferably myasthenia gravis, more preferably early-onset myasthenia gravis), T cells with antigen specificity against at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above are readily produced; that is, T cells expressing antibodies on their cell surface that recognize at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above. Therefore, by using the presence or concentration of such T cells with antigen specificity against at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) as an indicator, it is possible to assist in the diagnosis of whether an autoimmune disease is present, obtain data for diagnosis, or perform a diagnosis.

[0110] As an object of the method according to one embodiment of the sixth aspect of this disclosure, it can be, for example, a person, a person who may suffer from an autoimmune disease or a person with a genetic predisposition to an autoimmune disease, such as an autoimmune disease caused by the overproduction of antibodies against human AchRα1 or its peptide fragments, preferably myasthenia gravis, more preferably early-onset myasthenia gravis. Furthermore, as an object of the method according to one embodiment of the sixth aspect of this disclosure, it can also be a person who does not have the possibility of suffering from such an autoimmune disease or does not have a genetic predisposition to it; in this case, the method according to the embodiment can be a method for comprehensively exploring the likelihood of disease (so-called screening tests).

[0111] In one embodiment of the sixth aspect of this disclosure, if T cells with antigen specificity for at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) are detected in a sample collected from the subject, it may help to diagnose that the subject has an autoimmune disease (e.g., an autoimmune disease caused by the overproduction of antibodies corresponding to human AchRα1 or its peptide fragments, preferably myasthenia gravis, more preferably early-onset myasthenia gravis), obtain diagnostic data, or make a diagnosis. Furthermore, in one embodiment of the sixth aspect of this disclosure, if the amount of T cells with antigen specificity to at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above is greater in a sample collected from a subject than in a sample collected from a control (e.g., a normal person) who does not have an autoimmune disease (e.g., an autoimmune disease caused by an overproduction of antibodies against human AchRα1 or its peptide fragments, preferably myasthenia gravis, more preferably early-onset myasthenia gravis), it may help to diagnose that the subject has the possibility of having the autoimmune disease, obtain data for diagnosis, or make a diagnosis.

[0112] As a sample for measuring the amount of T cells having antigen specificity against at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above, any sample that can be obtained from the subject and can be used to assist in the diagnosis of whether an autoimmune disease is present, to obtain data for diagnosis, or to perform diagnosis based on the amount of T cells having antigen specificity against at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) is acceptable. There are no limitations on the sample. For example, it can be bone marrow fluid, cerebrospinal fluid, lymph, blood, plasma, serum, or saliva. In a preferred embodiment, it can be bone marrow fluid, which can be collected, for example, from the iliac bone or sternum.

[0113] In the sixth aspect of this disclosure, the "amount" of T cells having antigen specificity for at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above is not limited to the concentration of the T cells, but can also be a signal intensity that can be used as an indicator of the amount of the T cells. For example, when the T cells are labeled with a polypeptide, the amount can be, for example, a signal intensity that can evaluate the amount of the labeled polypeptide, such as the fluorescence intensity detected from the T cells when the polypeptide is fluorescently labeled. Furthermore, when the T cells are labeled with a polypeptide-human leukocyte antigen complex, the amount can be, for example, a signal intensity that can evaluate the amount of the labeled complex, such as the fluorescence intensity detected from the T cells when the human leukocyte antigen complex is fluorescently labeled. In these cases, the fluorescence intensity can be, for example, the fluorescence intensity measured by a microplate reader or flow cytometer.

[0114] In the detection method for at least one polypeptide selected from the group consisting of (A), (B), (D) and (E) above, there are no particular limitations as long as the T cell can be detected. For example, the T cell can be labeled with a fluorescently labeled polypeptide or a complex of a polypeptide and a fluorescently labeled human leukocyte antigen, and then the fluorescence intensity of the fluorescent pigment in the cells contained in the sample can be measured.

[0115] In a preferred embodiment, the detection of T cells with antigen specificity to at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) can be performed by labeling the T cells with a fluorescently labeled polymer (e.g., a tetramer) of a polypeptide-human leukocyte antigen complex (peptide-human leukocyte antigen complex), 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 polymer of the polypeptide-human leukocyte antigen complex, since the complex can form a stable binding with the T cells, non-specifically bound complexes can be removed by washing or other methods, allowing for more accurate detection. Such a polypeptide-human leukocyte antigen complex can be prepared, for example, using at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) and a commercially available kit (e.g., Quick Switch series, MBL LIFE SCIENCE) capable of preparing a polypeptide-human leukocyte antigen complex polymer as described above.

[0116] Diagnostic kits for autoimmune diseases A seventh aspect of this disclosure is a diagnostic kit for autoimmune diseases, comprising: at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above; and a human leukocyte antigen bound to the at least one 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 in the form of a complex, may comprise the human leukocyte antigen and the labeling dye in the form of a complex, may comprise a polypeptide, the human leukocyte antigen, and the labeling dye in the form of a complex, may comprise a polymer of the human leukocyte antigen and the labeling dye in the form of a complex, or may comprise a polymer of a polypeptide-human leukocyte antigen complex and the labeling dye in the form of 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 containing a polypeptide, 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, preferably myasthenia gravis, more preferably early-onset myasthenia gravis.

[0117] The kit according to the seventh aspect of this disclosure can be used for the diagnosis of autoimmune diseases according to the method according to an embodiment of the sixth aspect of this disclosure. In one embodiment, the kit according to the seventh aspect of this disclosure may further include an appendix, which may be electronic and may contain specifications corresponding to the method according to an embodiment of the sixth aspect of this disclosure.

[0118] <Variation Example> The eighth aspect of this disclosure relates to a formulation and kit, wherein the formulation, method, and kit described above, instead of at least one polypeptide selected from the group consisting of (A), (B), (D), and (E), contains a molecule comprising at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) as a partial structure (hereinafter also referred to as "the molecule involved in the eighth aspect"). Such a formulation and kit, except that it contains the molecule involved in the eighth aspect instead of the aforementioned at least one polypeptide, can be implemented in the same manner as described above in the formulations of the first to fifth aspects and the kit of the seventh aspect. The molecule involved in the eighth aspect can be a non-natural molecule, for example, an artificial molecule.

[0119] The molecules involved in the eighth aspect can be, for example, molecules (tandem molecules) formed by directly or indirectly linking at least two polypeptides selected from the group consisting of (A), (B), (D), and (E) via peptide bonds. Tandem molecules can be chain-like or cyclic. Tandem molecules can be molecules (tandem molecules) formed by directly or indirectly linking, for example, two, three, four, or five or more polypeptides selected from the group consisting of (A), (B), (D), and (E) via peptide bonds. For tandem molecules with at least two polypeptides directly linked by peptide bonds, examples include polypeptides consisting of amino acid sequences formed by linking two amino acid sequences shown in Serial No. 17, as shown in Serial No. 18; and polypeptides consisting of amino acid sequences formed by linking the amino acid sequences shown in Serial No. 17 and Serial No. 3, as shown in Serial No. 19; or cyclic polypeptides with peptide bonds formed at both ends. For tandem molecules with at least two polypeptides indirectly linked by peptide bonds, for example, at least two polypeptides can be linked by a peptide linker. As the peptide linker, examples include peptide linkers disclosed in International Application No. 2019 / 233842, such as GGGS (Serial No. 38), GGGGS (Serial No. 39), and the self-digested peptide T2A (EGRGSLLTCGDVEENPGP (Serial No. 40) and GSGEGRGGSLLTCGDVEENPGP (Serial No. 41)). For tandem molecules in which at least two polypeptides are indirectly linked by peptide bonds, examples include polypeptides such as those shown in Serial No. 20, which consist of an amino acid sequence of GGGS inserted between the two amino acid sequences shown in Serial No. 17; and polypeptides such as those shown in Serial No. 21, which consist of amino acid sequences of GGGGS inserted between the amino acid sequence shown in Serial No. 17 and the amino acid sequence shown in Serial No. 3, and between the amino acid sequence shown in Serial No. 3 and the amino acid sequence shown in Serial No. 17.

[0120] Serial Number 18: WVRKVFIDTIPNIMFFSWVRKVFIDTIPNIMFFS Serial Number 19: WVRKVFIDTIPNIMFFSGDFAIVKFTKVLLQYTGHIT Serial number 20: WVRKVFIDTIPNIMFFSGGGSWVRKVFIDTIPNIMFFS Serial number 21: WVRKVFIDTIPNIMFFSGGGGSGDFAIVKFTKVLLQYTGHITGGGGSWVRKVFIDTIPNIMFFS The molecules involved in the eighth aspect may, for example, be molecules comprising at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) and a non-peptide structure as a partial structure. Such molecules may, for example, be molecules in which at least two polypeptides selected from the group consisting of (A), (B), (D), and (E) are linked by a bond other than a peptide bond, and such molecules may further have a non-peptide structure at the peptide terminus that is not further linked to a linker. Such a bond other than a peptide bond may be a covalent bond (e.g., a carbon-carbon bond) or a bond formed by a non-covalent interaction, such as the interaction between biotin and streptavidin.

[0121]

Example

[0122] [Example 1: Identification of antigenic peptides derived from human AChRα1 that bind to HLA-DR] <Step 1: Clinical Sample Collection> The peripheral blood mononuclear cells (hereinafter referred to as PBMCs) used in this embodiment were frozen samples from normal individuals and purchased from VERITAS Corporation. The HLA genotypes of the purchased PMBCs are shown in Table 1 below.

[0123] Table 1 Process 2: CD14 + CD16 - Isolation of monocytes Frozen PBMCs were rapidly thawed in a 37°C water bath and washed twice with RPMI-1640 medium containing 10% FBS (RPMI-1640 / 10% FBS medium). CD14 cells were isolated by negative selection using the EasySep Human Monocyte Isolation Kit (VERITAS, Cat. No. ST-19359) according to the kit's accompanying documentation. + CD16 - Monocytes. Thus, classic monocytes were isolated from PBMCs.

[0124] <Step 3: Preparation of monocyte-derived dendritic cells and addition of human AChRα1> Separate CD14 + CD16 -Monocytes were cultured in RPMI-1640 / 10% FBS medium containing 50 ng / mL GM-CSF and 50 ng / mL IL-4 at 1.5 × 10⁻⁶ cells per well in 6-well dishes. 6 Dendritic cells derived from monocytes were cultured starting at cells / mL in a 37°C, 5% CO2 incubator for 7 days. Then, human AChRα1 was added to 100 μg / well, and the cells were cultured for another 24 hours. The human AChRα1 used in this procedure was synthesized based on the AChRα sequence information. The synthesized gene was inserted into a transfer vector, and the expression vector was co-transfected into SF9 cells (used as insect cells) for production and purification.

[0125] <Step 4: Isolation of HLA-DR binding peptide> For the cultured samples, the procedure was performed according to the specifications of Dynabeads Protein G (Invitrogen, Cat. No. 10004D). 5 μg of anti-HLA-DR antibody was bound to the beads. The peptide was released from HLA-DR by adding 0.1% TFA to the solution containing HLA-DR bound with the desired peptide and allowing it to react at room temperature for 1 minute. A Microcon (trademarked) centrifugal filter device (Merck, Cat. No. MRCPRT010) capable of separating samples below 10 kDa was attached to a dedicated 1.5 mL tube. The recovered solution containing the peptide and HLA-DR was added, and the tube was centrifuged at 14000 × g for 30 minutes to separate HLA-DR and the peptide. The peptide solution in the tube was then lyophilized to remove the solvent and stored as a lyophilized sample.

[0126] <Step 5: LC-MS / MS Analysis> Purified water was added to the obtained freeze-dried sample and stirred to prepare the sample for analysis. The sample was then analyzed by LC-MS / MS under the conditions shown in Table 2 below.

[0127] Table 2 <Step 6: LC-MS / MS Spectral Data Analysis> The spectral data obtained from LC-MS / MS analysis were analyzed using Mascot Server version 2.8 (MascotServer ver. 2.8, Matrix Science) under the conditions shown in Table 3 below. In the NCBI prot and AChRα1 sequence searches, peptides meeting the false discovery rate (FDR) p < 0.01 were selected as search targets; FDR was not calculated only for AChRα1 sequence searches. Furthermore, Skyline Ver. 22.2 (MacCoss Lab.) was used for quantitative analysis. After importing the spectral data obtained from LC-MS / MS analysis and the identification results obtained through Mascot Server, the peak area values ​​in the extracted ion chromatograms of the peptides were calculated.

[0128] Table 3 <Step 7: Identification of HLA alleles through sample comparison> The LC-MS / MS analysis data obtained in step 6 were compared for each donor to identify HLA alleles. Those with DRB1 The peptides commonly identified in the 03:01 sample were designated as DRB1. 03:01 binding peptide, will have DRB3 The peptides commonly identified in the 01:01 sample were designated as DRB3. 01:01 binding peptide.

[0129] HLA-DRB1 identified in this way The results of the 03:01 binding peptide, along with its corresponding amino acid sequence position shown in SEQ ID NO. 37, are presented in Table 4. The HLA-DRB3 thus identified... The results of the 01:01 binding peptide, along with its corresponding amino acid sequence position shown in sequence number 37, are presented in Table 5. The results of plotting these identified HLA-binding peptides as coordinates on human AChRα1 are shown below. Figure 1 .exist Figure 1 In the diagram, clusters of identified DRB-binding peptides are represented by boxes, while clusters indicated by slashes originate from DRB1. At 03:01, the clusters indicated by the dotted shading are from DRB3. 01:01, the clusters indicated by black fill are from other HLA types. Additionally, in Figure 1 In the diagram, the light gray area below the amino acid number represents the AChRα1 sequence, and the dark gray area represents the transmembrane portion.

[0130] Table 4 Table 5 Based on these results, peptides with sequence numbers 6–15 were found in samples from individuals with HLA-DRB3 genotype 01:01 and individuals with HLA-DRB1 genotype 03:01. These peptides share the amino acid sequence shown in sequence number 1. Therefore, the peptide with the amino acid sequence shown in sequence number 1 is clearly identified as DRB3. 01:01 binding peptide and is DRB1 03:01 binding peptide, and the location of the amino acid sequence shown in sequence number 1 in human AChRα1 is clearly relative to DRB3. 01:01 and DRB1 Table position 03:01.

[0131] Furthermore, based on these results, the peptide represented by sequence number 16 (sequence number 2) was co-founded in samples from individuals with the HLA-DRB3 genotype 01:01. Therefore, the peptide with the amino acid sequence represented by sequence number 2 is definitively identified as DRB3. 01:01 binding peptide, and the location of the amino acid sequence shown in sequence number 2 in human AChRα1 is clearly relative to DRB3. Table position 01:01.

[0132] Furthermore, based on these results, peptides with sequence numbers 22–32 were commonly found in samples from individuals with the HLA-DRB3 genotype 01:01, and these peptides share the amino acid sequence shown in sequence number 4. Therefore, the peptide with the amino acid sequence shown in sequence number 4 is definitively identified as DRB3. 01:01 binding peptide, and the location of the amino acid sequence shown in sequence number 4 in human AChRα1 is clearly relative to DRB3. Table position 01:01.

[0133] Based on these results, peptides with sequence numbers 33–35 were found in samples from individuals with HLA-DRB3 genotype 01:01 and individuals with HLA-DRB1 genotype 03:01. These peptides share the amino acid sequence shown in sequence number 5. Therefore, the peptide with the amino acid sequence shown in sequence number 5 is clearly identified as DRB3. 01:01 binding peptide and is DRB1 03:01 binding peptide, and the site in human AChRα1 with the amino acid sequence shown in sequence number 5 is clearly identified as corresponding to DRB3. 01:01 and DRB1 Table position 03:01.

[0134] [Example 2: Evaluation of T-cell induction capacity using peripheral blood mononuclear cells (PBMCs) from healthy individuals] This study investigates whether the epitope-containing peptides discovered in Example 1 have the effect of inducing specific T cells from normal human PBMCs.

[0135] <Preparation of Culture Media> For human AB serum (GeminiBio, Cat: 100-512) and fetal bovine serum (Nichirei, Cat: 175012), the serum was 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 AB serum was used as the culture medium for PBMCs. Cell culture was performed at 37°C under 5% CO2-water vapor saturation. D-PBS (-) containing 2% fetal bovine serum was used as the staining buffer.

[0136] A list of PBMCs from normal human sources The HLA alleles and evaluation peptides of normal human PBMCs used in this study are shown in Table 6. Six induction capacity evaluation experiments were performed for each peptide. Each PBMC was purchased from Precision for Medicine or STEMCELLS. Bold and italic text indicates the predicted limiting alleles of peptides consisting of the amino acid sequences described in sequence numbers 3, 16, 35, and 17; underlined text indicates the predicted limiting allele of peptides consisting of the amino acid sequence described in sequence number 17. Furthermore, for samples indicated by ※ in Table 6, evaluation was performed in two independent experiments.

[0137] Table 6 <Evaluation Peptide Overview> The evaluation peptides used in the study are shown in Table 7. These peptides were dissolved in DMSO (ATCC, Cat:4-X) to a concentration of 20 mg / mL.

[0138] Table 7 <Induction of peptide-specific T cells using normal human PBMCs> Remove the PBMCs stored in liquid nitrogen vapor phase and thaw them rapidly in a 37°C water bath. Wash once with AIM-V and then resuspend them in AIM-V containing 5% human AB serum (hereinafter referred to as complete culture medium) at a concentration of 1.5 × 10⁻⁶. 6 1 peptide / well was seeded into a 24-well plate. Add a final concentration of 20 μg / mL peptide and a final concentration of 10 ng / mL IL-7 (PeproTech, Cat: AF-200-07-50 ug) to start the culture (day 0, total culture volume: 2 mL / well).

[0139] One week after the start of culture, PBMCs from the same donor as those cultured on day 0 were thawed, washed once with AIM-V, and prepared in 3×10⁻⁶ doses. 6 Cells were cultured at concentrations below 20 μg / mL, with the same peptide used for induction on day 0 added to a concentration of 20 μg / mL, and incubated for 2 hours. Mitomycin C solution was then added to a final concentration of 50 μg / mL, and the cells were incubated for another 45 minutes. Afterward, the cells were washed three times with AIM-V and used as antigen-presenting cells. Cells cultured for one week from day 0 were harvested and cultured at a concentration of 1–1.5 × 10⁻⁶ cells / mL. 6 Cells were seeded at a rate of 2 mL / well in new 24-well plates, along with the same number of prepared antigen-presenting cells. IL-7 was then added to a final concentration of 10 ng / mL, and culture was restarted (day 7, total culture volume: 2 mL / well). Two days later, the culture medium was partially exchanged with complete medium containing 40 U / L IL-2, followed by another partial exchange with complete medium containing 20 U / mL IL-2, while continuing culture for one week. During this period, when cells reached confluence, 2 mL / well of culture medium was thoroughly resuspended, and 1 mL of this medium was seeded into new wells. Complete medium containing 20 U / mL IL-2 was added to each well to a final concentration of 1 mL / well (hereinafter referred to as passage). Cultured cells were harvested on day 14 for intracellular cytokine staining (ICS) and ELISA-based IFN-γ production assessment, as described later.

[0140] <Intracellular Cytokine Staining (ICS)> The recovered cultured cells were cultured at 2.0 × 10⁻⁶. 6Seeds were applied to 2–6 wells of a 96-well round-bottom plate. In one half of the wells, the peptide for induction was added to a concentration of 20 μg / mL, and DMSO was added as a solvent to a concentration of 0.1% in the other half. The plates were incubated for 2 hours. Then, 1X Brefeldin A (BioLegend, Cat: 420601) and Monensin (BioLegen, Cat: 420701) were added, and the plates were incubated for another 4 hours. Cells were recovered and treated with staining buffer containing Fc Block (Miltenyi Biotec, Cat: 130-059-901) reagent, 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). The mixture was incubated at 4°C for 15 minutes. Cytofix Cytoperm Fix / Perm Solution (Beckton Dickinson, Cat: 554714) was then added, and the mixture was treated at 4°C for 20 minutes. The sample was washed with Perm. / Wash buffer (diluted 10-fold with distilled water to a 10-fold concentration of the Cat: 554714 package), and then PE-labeled anti-human IFN-γ antibody (BioLegend, Cat: 502509) was added. The mixture was reacted at 37°C for 30 minutes, washed with Perm. / Wash buffer, and then washed with staining buffer before analysis by FACS.

[0141] <Evaluation of IFN-γ production capacity based on ELISA> The recovered cultured cells were cultured in a 96-well round-bottom plate at a concentration of 1.0 × 10⁻⁶. 5 Six peptides were seeded per well. In one half of the wells, the peptide for induction was added to a concentration of 20 μg / mL, and DMSO was added as a solvent to a concentration of 0.1% in the other half. The wells were incubated for at least 16 hours. The culture supernatant was collected, appropriately diluted with assay diluent, and used for ELISA analysis. For the determination of IFN-γ concentration, the BD OptEIA ELISA SET (human IFN-γ, Becton Dickinson, Cat: 555142) was used, following the instructions in the supplementary documentation. If the absorbance was lower than that of the blank well, the IFN-γ concentration was treated as 0.

[0142] <Results> Figure 2The graph shows the results of ICS (upper segment) and ELISA (lower segment) assays performed on six independent peptide-specific T cell induction assays using PBMCs from healthy individuals. As shown in the ICS and ELISA results, samples with significant peptide-specific T cell induction are represented by solid lines, while other samples are represented by dashed lines. Peptide-specific T cell induction was confirmed in 1 out of 6 samples (serial number 16), 3 and 35 in 3 out of 6 samples (serial numbers 3 and 35), and in all 6 samples (serial number 17). These results indicate that helper T cells with T cell receptors (TCRs) that recognize HLA complexes with each peptide do indeed exist in healthy individuals. In particular, sequence 17, based on the ICS results, showed significantly higher IFN-γ production of CD4+ upon peptide stimulation compared to the solvent control group (solvent = DMSO). + The high T cell rate indicates its high potential.

[0143] [Example 3: Evaluation of specific T cell induction capacity using peripheral blood mononuclear cells (PBMCs) from MG patients] In order to confirm the presence of peptide-specific T cell-induced Th1 cells in MG patients, the peptide identified in all examples in Example 2 was identified as 17, and MG-derived PBMCs were used for the study.

[0144] The HLA alleles of PBMCs derived from myasthenia gravis patients used in this study are shown in Table 8. Evaluation peptides consisted only of the amino acid sequence described in sequence number 17. All PBMCs were purchased from Eolas Bio. Bold and italic text indicates the predicted limiting allele of sequence number 17.

[0145] Table 8 <Induction of peptide-specific T cells using PBMCs derived from MG patients> Induction was performed using essentially the same method as in Example 2, "Induction of Peptide-Specific T Cells from Normal Human PBMCs," but the aim was to expand memory T cells or effector T cells in patient PBMCs. Therefore, peptides and IL-7 were added on day 0, and culture medium replacement and passage were performed from day 2 onwards using complete culture medium containing IL-2. The cells were ready for ELISA analysis on day 7. ELISA analysis was performed using the same method as in Example 2, "Evaluation of IFN-γ Production Capacity Based on ELISA."

[0146] <Results> Figure 3 To represent the use of 5 copies of the predicted restriction allele DRB3 with sequence number 17 The image shows the results of an ELISA analysis evaluating the presence or absence of sequence number 17-specific Th1 cells in PBMC samples from MG patients at 01:01. The results indicated the presence of sequence number 17-specific Th1 cells in 3 out of 4 samples. These results suggest that sequence number 17-specific Th1 cells are present in DRB3. In patients with positive MG at 01:01, etiology is suspected.

[0147] [Example 4: Exploring the possibility of further restrictions on other alleles] In the ICS results of Example 2, the percentage of Th1 cells specific to sequence number 17 (IFNγ-positive CD4+ upon peptide stimulation and DMSO stimulation) was [data missing]. + The T-cell differential percentage was as high as 0.70–7.47% (median: 2.3%). In normal individuals, activation (priming) by endogenous antigens is not considered. However, naive T cells typically differentiate into Th1 cells 2–3 days after antigen stimulation, followed by additional stimulation and clonal expansion in the presence of IL-2. The doubling time of activated T cells is approximately 6–8 hours, and within 5 days from day 9 of IL-2 addition to day 14 of cell recovery, one cell expands to approximately 1 million. On the other hand, TCR diversity is considered to be 10... 8 Up to 10 9 If there is only one Naive T cell that recognizes the HLA-peptide complex, the presence ratio is 10. -9 ~10 -8 After the above cloning and amplification, the percentage was considered to be around 0.1% to 1%. Compared to this theoretical value, the experimental value obtained in Example 2 was higher, therefore, the further restriction of sequence number 17 on other alleles is suspected. Therefore, HLA-DRB1, which has been reported to be associated with MG in Asians, was used. 09:01 and does not have DRB1 03:01, DRB3 01:01 normal human PBMCs were used to evaluate the specific Th1 cell induction ability of sequence number 17.

[0148] A list of PBMCs from normal human sources The HLA alleles of normal human PBMCs used in Example 4 are shown in Table 9. Evaluation peptides are only peptides consisting of the amino acid sequence described in sequence number 17. Bold and italicized alleles indicate those reported to be associated with the pathogenesis of myasthenia gravis in Asians.

[0149] Table 9 <Results> Figure 4 To indicate the use of HLA-DRB1 in Asians that has been reported to be associated with MG 09:01 and does not have DRB1 03:01, DRB3 A graph showing the results of ICS (left) and ELISA (right) assays evaluating the specific Th1 cell induction capacity of PBMCs derived from normal individuals at a 01:01 scale. Figure 4 As shown, in 7 out of 9 samples submitted for ICS analysis, sequence number 17-specific Th1 cells were induced (left side, solid line). Of the 9 samples submitted for ICS analysis, 5 samples (4 with sequence number 17-specific Th1 cell induction and 1 without induction) were submitted for ELISA analysis. In these 4 samples confirmed by ICS to have been induced, IFNγ production from sequence number 17-specific Th1 cells was also detected (right side, solid line). From these results, it can be concluded that, in addition to the initially hypothesized restriction allele (DRB1... 03:01, DRB3 In addition to 01:01), serial number 17 is also affected by HLA-DRB1. The restriction is 09:01.

Claims

1. A therapeutic or preventative agent for an autoimmune disease, comprising at least one polypeptide selected from the group consisting of: (D) One or more polypeptides having a total length of 7 residues or more and 17 residues or less, which contain an amino acid sequence having more than 90% sequence identity with the amino acid sequence recorded in sequence number 4. (A) One or more polypeptides having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence described in Serial No. 1 and a total length of 8 or more residues and less than 20 residues. (B) One or more polypeptides comprising an amino acid sequence having a sequence identity of 90% or more with respect to the amino acid sequence described in Serial No. 2, and having a full length of 12 or more residues and less than 21 residues; and (E) One or more polypeptides having a total length of 10 or more residues and less than 19 residues, comprising an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence described in sequence number 5.

2. An activator of regulatory T cells, comprising at least one polypeptide selected from the group consisting of: (D) One or more polypeptides having a total length of 7 residues or more and 17 residues or less, which contain an amino acid sequence having more than 90% sequence identity with the amino acid sequence recorded in sequence number 4. (A) One or more polypeptides having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence described in Serial No. 1 and a total length of 8 or more residues and less than 20 residues. (B) One or more polypeptides comprising an amino acid sequence having a sequence identity of 90% or more with respect to the amino acid sequence described in Serial No. 2, and having a full length of 12 or more residues and less than 21 residues; and (E) One or more polypeptides having a total length of 10 or more residues and less than 19 residues, comprising an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence described in sequence number 5.

3. An inhibitor of effector T cell activity, comprising at least one polypeptide selected from the group consisting of: (D) One or more polypeptides having a total length of 7 residues or more and 17 residues or less, which contain an amino acid sequence having more than 90% sequence identity with the amino acid sequence recorded in sequence number 4. (A) One or more polypeptides having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence described in Serial No. 1 and a total length of 8 or more residues and less than 20 residues. (B) One or more polypeptides comprising an amino acid sequence having a sequence identity of 90% or more with respect to the amino acid sequence described in Serial No. 2, and having a full length of 12 or more residues and less than 21 residues; and (E) One or more polypeptides having a total length of 10 or more residues and less than 19 residues, comprising an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence described in sequence number 5.

4. An inhibitor of B cell activation, comprising at least one polypeptide selected from the group consisting of: (D) One or more polypeptides having a total length of 7 residues or more and 17 residues or less, which contain an amino acid sequence having more than 90% sequence identity with the amino acid sequence recorded in sequence number 4. (A) One or more polypeptides having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence described in Serial No. 1 and a total length of 8 or more residues and less than 20 residues. (B) One or more polypeptides comprising an amino acid sequence having a sequence identity of 90% or more with respect to the amino acid sequence described in Serial No. 2, and having a full length of 12 or more residues and less than 21 residues; and (E) One or more polypeptides having a total length of 10 or more residues and less than 19 residues, comprising an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence described in sequence number 5.

5. An inhibitor of humoral immunity, comprising at least one polypeptide selected from the group consisting of: (D) One or more polypeptides having a total length of 7 residues or more and 17 residues or less, which contain an amino acid sequence having more than 90% sequence identity with the amino acid sequence recorded in sequence number 4. (A) One or more polypeptides having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence described in Serial No. 1 and a total length of 8 or more residues and less than 20 residues. (B) One or more polypeptides comprising an amino acid sequence having a sequence identity of 90% or more with respect to the amino acid sequence described in Serial No. 2, and having a full length of 12 or more residues and less than 21 residues; and (E) One or more polypeptides having a total length of 10 or more residues and less than 19 residues, comprising an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence described in sequence number 5.

6. A treatment or prevention method for an autoimmune disease, comprising administering at least one polypeptide selected from the group consisting of: (D) One or more polypeptides having a total length of 7 residues or more and 17 residues or less, which contain an amino acid sequence having more than 90% sequence identity with the amino acid sequence recorded in sequence number 4. (A) One or more polypeptides having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence described in Serial No. 1 and a total length of 8 or more residues and less than 20 residues. (B) One or more polypeptides comprising an amino acid sequence having a sequence identity of 90% or more with respect to the amino acid sequence described in Serial No. 2, and having a full length of 12 or more residues and less than 21 residues; and (E) One or more polypeptides having a total length of 10 or more residues and less than 19 residues, comprising an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence described in sequence number 5.

7. At least one polypeptide selected from the group consisting of, for use in the treatment or prevention of autoimmune diseases: (D) One or more polypeptides having a total length of 7 residues or more and 17 residues or less, which contain an amino acid sequence having more than 90% sequence identity with the amino acid sequence recorded in sequence number 4. (A) One or more polypeptides having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence described in Serial No. 1 and a total length of 8 or more residues and less than 20 residues. (B) One or more polypeptides comprising an amino acid sequence having a sequence identity of 90% or more with respect to the amino acid sequence described in Serial No. 2, and having a full length of 12 or more residues and less than 21 residues; and (E) One or more polypeptides having a total length of 10 or more residues and less than 19 residues, comprising an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence described in sequence number 5.

8. The use of at least one polypeptide selected from the group consisting of: in the manufacture of a therapeutic agent, preventive agent, therapeutic composition, or preventive composition for an autoimmune disease: (D) One or more polypeptides having a total length of 7 residues or more and 17 residues or less, which contain an amino acid sequence having more than 90% sequence identity with the amino acid sequence recorded in sequence number 4. (A) One or more polypeptides having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence described in Serial No. 1 and a total length of 8 or more residues and less than 20 residues. (B) One or more polypeptides comprising an amino acid sequence having a sequence identity of 90% or more with respect to the amino acid sequence described in Serial No. 2, and having a full length of 12 or more residues and less than 21 residues; and (E) One or more polypeptides having a total length of 10 or more residues and less than 19 residues, comprising an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence described in sequence number 5.

9. A diagnostic agent for an autoimmune disease, comprising a complex of a polypeptide, a human leukocyte antigen, and a labeling pigment, wherein the polypeptide is at least one polypeptide selected from the group consisting of: (D) One or more polypeptides having a total length of 7 residues or more and 17 residues or less, which contain an amino acid sequence having more than 90% sequence identity with the amino acid sequence recorded in sequence number 4. (A) One or more polypeptides having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence described in Serial No. 1 and a total length of 8 or more residues and less than 20 residues. (B) One or more polypeptides comprising an amino acid sequence having a sequence identity of 90% or more with respect to the amino acid sequence described in Serial No. 2, and having a full length of 12 or more residues and less than 21 residues; and (E) One or more polypeptides having a full length of 10 or more residues and less than 19 amino acids, which contain an amino acid sequence having a sequence identity of more than 90% with the amino acid sequence described in sequence number 5.

10. A diagnostic kit for an autoimmune disease, comprising at least one polypeptide selected from the group consisting of: and a human leukocyte antigen bound to said at least one polypeptide: (D) One or more polypeptides having a total length of 7 residues or more and 17 residues or less, which contain an amino acid sequence having more than 90% sequence identity with the amino acid sequence recorded in sequence number 4. (A) One or more polypeptides having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence described in Serial No. 1 and a total length of 8 or more residues and less than 20 residues. (B) One or more polypeptides comprising an amino acid sequence having a sequence identity of 90% or more with respect to the amino acid sequence described in Serial No. 2, and having a full length of 12 or more residues and less than 21 residues; and (E) One or more polypeptides having a full length of 10 or more residues and less than 19 amino acids, which contain an amino acid sequence that has a sequence identity of more than 90% with the amino acid sequence described in sequence number 4.

11. The kit according to claim 10, further comprising a labeling pigment bound to the human leukocyte antigen.

12. A method for assisting in the diagnosis of an autoimmune disease, comprising the step of detecting T cells that are antigen-specific to at least one polypeptide selected from the group consisting of: (D) One or more polypeptides having a total length of 7 residues or more and 17 residues or less, which contain an amino acid sequence having more than 90% sequence identity with the amino acid sequence recorded in sequence number 4. (A) One or more polypeptides having an amino acid sequence that has 90% or more sequence identity with the amino acid sequence described in Serial No. 1 and a total length of 8 or more residues and less than 20 residues. (B) One or more polypeptides comprising an amino acid sequence having a sequence identity of 90% or more with respect to the amino acid sequence described in Serial No. 2, and having a full length of 12 or more residues and less than 21 residues; and (E) One or more polypeptides having a full length of 10 or more residues and less than 19 amino acids, which contain an amino acid sequence that has a sequence identity of more than 90% with the amino acid sequence described in sequence number 4.

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