Agent containing polypeptide comprising amino acid sequence corresponding to HLA-dr-binding epitope of achr

Abstract

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

Description

Agent containing polypeptide containing amino acid sequence corresponding to HLA-DR binding epitope of AChR

[0001] The present disclosure relates to agents containing a polypeptide comprising an amino acid sequence corresponding to an HLA-DR binding epitope of an AChR.

[0002] In autoimmune diseases, the immune system, which is normally responsible for eliminating foreign substances from the outside of the body, recognizes substances from the inside of the body as antigens, resulting in abnormal activation of the immune system. Such abnormal activation of the immune system is known to cause various abnormalities in the body's biological functions in patients with autoimmune diseases.

[0003] Early-onset myasthenia gravis is a type of myasthenia gravis that develops in subjects aged 10 to 49. Myasthenia gravis is designated as an intractable disease in Japan and is an organ-specific autoimmune disease directed against molecules on the postsynaptic membrane of the neuromuscular junction, with muscle weakness as its main symptom.

[0004] In myasthenia gravis, autoantibodies are produced in the patient's body against acetylcholine receptors (AChRs) and muscle-specific receptor tyrosine kinases (MuSKs), which are molecules present in the postsynaptic membrane of the neuromuscular junction, and these antibodies inhibit neurotransmission, resulting in various symptoms. It is known that autoantibodies against AChRs account for approximately 80% of cases of myasthenia gravis.

[0005] In general humoral immunity, antigen-presenting cells transfer antigens to naive CD4 ＋ By presenting it to T cells, naive CD4 ＋ T cells into effector CD4 ＋ In recent years, a new type of antigen-presenting cell, naive CD4 ＋ By presenting it to T cells, naive CD4 ＋ T cells are expressed as effector CD4 ＋The existence of antigen-presenting cells that have the function of differentiating regulatory T cells, rather than T cells, has been reported (Non-Patent Documents 1 and 2). Such antigen-presenting cells activate regulatory T cells by presenting antigens, and then induce differentiation of effector CD4 ＋ Because they inhibit the activation of B cells by T cells, they have the opposite effect of the previously known action of antigen-presenting cells: they suppress antibody production. In fact, clinical trials are being conducted on autoimmune diseases using antibody production inhibitors that target this new type of antigen-presenting cell (Non-Patent Document 3).

[0006] In autoimmune diseases, human leukocyte antigens (HLA), a protein complex that plays a role in presenting antigens on the surface of antigen-presenting cells, recognize a portion of the antigen and form a bond with it. In particular, when the antigen is a protein, the portion of the antigen protein recognized by HLA is called an epitope. Such epitopes have the potential to serve as targets for the treatment and diagnosis of autoimmune diseases, and attempts have been made to identify them in various autoimmune diseases. For example, epitopes in early-onset myasthenia gravis have been reported (Non-Patent Document 4).

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

[0008] An object of the present disclosure is to provide an agent that contains a polypeptide comprising an amino acid sequence corresponding to an epitope of AChR and that may be applicable to the treatment or diagnosis of autoimmune diseases.

[0009] The present inventors have discovered peptide fragments of AChR that are secreted as complexes with human leukocyte antigens (HLA) from human dendritic cells that have incorporated AChR by the Natural Antigen Processing Assay (NAPA) method using dendritic cells prepared from human-derived classical monocytes. Furthermore, the present inventors have discovered that these peptide fragments contain amino acid sequences corresponding to epitopes of AChR, and that agents containing polypeptides containing these amino acid sequences may be applicable to the treatment or diagnosis of autoimmune diseases, thereby completing the present disclosure.

[0010] The present disclosure relates to, for example, the following: [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 comprising an amino acid sequence that has 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and a total length of 8 to 20 residues; (B) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 and a total length of 12 to 21 residues; (D) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 and a total length of 7 to 17 residues; and (E) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 and a total length of 10 to 19 residues. [2] An inhibitor of effector T cell activity or a composition for inhibiting the activity, comprising at least one polypeptide selected from the group consisting of: (A) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 and a total length of 8 to 20 residues; (B) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2 and a total length of 12 to 21 residues; (D) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 4 and a total length of 7 to 17 residues; and (E) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 5 and a total length of 10 to 19 residues.[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 comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 and a total length of 8 to 20 residues; (B) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2 and a total length of 12 to 21 residues; (D) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 4 and a total length of 7 to 17 residues; and (E) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 5 and a total length of 10 to 19 residues. [4] A suppressant or composition for suppressing humoral immunity, comprising at least one polypeptide selected from the group consisting of: (A) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 and a total length of 8 to 20 residues; (B) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2 and a total length of 12 to 21 residues; (D) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 4 and a total length of 7 to 17 residues; and (E) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 5 and a total length of 10 to 19 residues.[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 comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 and a total length of 8 to 20 residues; (B) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2 and a total length of 12 to 21 residues; (D) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 4 and a total length of 7 to 17 residues; and (E) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 5 and a total length of 10 to 19 residues. [6] A method for treating or preventing an autoimmune disease, comprising administering to a subject in need thereof at least one polypeptide selected from the group consisting of: (A) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 and a total length of 8 to 20 residues; (B) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2 and a total length of 12 to 21 residues; (D) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 4 and a total length of 7 to 17 residues; and (E) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 5 and a total length of 10 to 19 residues.[7] At least one polypeptide selected from the group consisting of: (A) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 and having a total length of 8 to 20 residues; (B) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2 and having a total length of 12 to 21 residues; (D) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 4 and having a total length of 7 to 17 residues; and (E) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 5 and having a total length of 10 to 19 residues. [8] In the production of a therapeutic agent, preventive agent, therapeutic composition, or preventive composition for an autoimmune disease, use of at least one polypeptide selected from the group consisting of: (A) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 and having a total length of 8 to 20 residues; (B) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2 and having a total length of 12 to 21 residues; (D) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 4 and having a total length of 7 to 17 residues; and (E) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 5 and having a total length of 10 to 19 residues.[9] At least one polypeptide selected from the group consisting of: (A) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 and having a total length of 8 to 20 residues; (B) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2 and having a total length of 12 to 21 residues; (D) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 4 and having a total length of 7 to 17 residues; and (E) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 5 and having a total length of 10 to 19 residues.

[10] A kit for diagnosing an autoimmune disease, comprising at least one polypeptide selected from the group consisting of: (A) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 and a total length of 8 to 20 residues; (B) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2 and a total length of 12 to 21 residues; (D) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 4 and a total length of 7 to 17 residues; and (E) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 5 and a total length of 10 to 19 residues; and a human leukocyte antigen that binds to the at least one polypeptide.

[11] The kit according to

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

[12] A diagnostic agent for an autoimmune disease, comprising a complex comprising a polypeptide, a human leukocyte antigen, and a labeling dye, wherein the polypeptide is at least one polypeptide selected from the group consisting of: (A) one or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 and having a total length of 8 to 20 residues; (B) one or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2 and having a total length of 12 to 21 residues; (D) one or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 4 and having a total length of 7 to 17 residues; and (E) one or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 5 and having a total length of 10 to 19 residues.

[13] A method for assisting in the diagnosis of an autoimmune disease, comprising detecting T cells having antigen specificity for at least one polypeptide selected from the group consisting of: (A) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 and a total length of 8 to 20 residues; (B) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2 and a total length of 12 to 21 residues; (D) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 4 and a total length of 7 to 17 residues; and (E) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 5 and a total length of 10 to 19 residues.

[14] A method for obtaining data for diagnosing an autoimmune disease, comprising detecting T cells having antigen specificity for at least one polypeptide selected from the group consisting of: (A) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 and a total length of 8 to 20 residues; (B) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2 and a total length of 12 to 21 residues; (D) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 4 and a total length of 7 to 17 residues; and (E) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 5 and a total length of 10 to 19 residues.

[15] The activator or composition according to [1], the at least one polypeptide comprising: (A) one or more polypeptides having an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 and a total length of 8 to 20 residues; the activity inhibitor or composition according to [2], the activation inhibitor or composition according to [3], the suppressor or composition according to [4], the therapeutic agent, preventive agent, or composition according to [5], the method according to [6], at least one polypeptide according to [7], the use according to [8], at least one polypeptide according to [9], the kit according to

[10] or

[11] , the diagnostic agent according to

[12] , or the method according to

[13] or

[14] .

[16] The activator or composition according to [1], the activity inhibitor or composition according to [2], the activation inhibitor or composition according to [3], the suppressor or composition according to [4], the therapeutic agent, preventive agent or composition according to [5], the method according to [6], the at least one polypeptide according to [7], the use according to [8], the at least one polypeptide according to [9], the kit according to

[10] or

[11] , the diagnostic agent according to

[12] , or the method according to

[13] or

[14] , wherein the at least one polypeptide comprises: (B) one or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2 and having a total length of 12 to 21 residues;

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

[10] or

[11] , the diagnostic agent according to

[12] , or the method according to

[13] or

[14] , wherein the at least one polypeptide comprises: (D) one or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 4 and having a total length of 7 to 17 residues;

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

[10] or

[11] , the diagnostic agent according to

[12] , or the method according to

[13] or

[14] , wherein the at least one polypeptide comprises: (E) one or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 5 and having a total length of 10 to 19 residues;

[19] The activator or composition according to [1], the at least one polypeptide comprising: (A) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and a total length of 8 to 20 residues; and (B) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 and a total length of 12 to 21 residues; the activator or composition according to [1], the activity inhibitor or composition according to [2], the activation inhibitor or composition according to [3], the suppressor or composition according to [4], the therapeutic agent, preventive agent, or composition according to [5], the method according to [6], the at least one polypeptide according to [7], the use according to [8], the at least one polypeptide according to [9], the kit according to

[10] or

[11] , the diagnostic agent according to

[12] , or the method according to

[13] or

[14] .

[20] The at least one polypeptide comprises: (A) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and a total length of 8 to 20 residues; and (D) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 and a total length of 7 to 17 residues; the activator or composition according to [1], the activity inhibitor or composition according to [2], the activation inhibitor or composition according to [3], the suppressor or composition according to [4], the therapeutic agent, preventive agent, or composition according to [5], the method according to [6], the at least one polypeptide according to [7], the use according to [8], the at least one polypeptide according to [9], the kit according to

[10] or

[11] , the diagnostic agent according to

[12] , or the method according to

[13] or

[14] .

[21] The at least one polypeptide comprises: (A) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and a total length of 8 to 20 residues; and (E) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 and a total length of 10 to 19 residues; the activator or composition according to [1], the activity inhibitor or composition according to [2], the activation inhibitor or composition according to [3], the suppressor or composition according to [4], the therapeutic agent, preventive agent, or composition according to [5], the method according to [6], the at least one polypeptide according to [7], the use according to [8], the at least one polypeptide according to [9], the kit according to

[10] or

[11] , the diagnostic agent according to

[12] , or the method according to

[13] or

[14] .

[22] The activator or composition according to [1], the at least one polypeptide comprising: (B) one or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2 and having a total length of 12 to 21 residues; and (D) one or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 4 and having a total length of 7 to 17 residues; the activator or composition according to [1], the activity inhibitor or composition according to [2], the activation inhibitor or composition according to [3], the suppressor or composition according to [4], the therapeutic agent, preventive agent, or composition according to [5], the method according to [6], the at least one polypeptide according to [7], the use according to [8], the at least one polypeptide according to [9], the kit according to

[10] or

[11] , the diagnostic agent according to

[12] , or the method according to

[13] or

[14] .

[23] The activator or composition according to [1], the at least one polypeptide comprising: (B) one or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2 and having a total length of 12 to 21 residues; and (E) one or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 5 and having a total length of 10 to 19 residues; the activator or composition according to [1], the activity inhibitor or composition according to [2], the activation inhibitor or composition according to [3], the suppressor or composition according to [4], the therapeutic agent, preventive agent, or composition according to [5], the method according to [6], the at least one polypeptide according to [7], the use according to [8], the at least one polypeptide according to [9], the kit according to

[10] or

[11] , the diagnostic agent according to

[12] , or the method according to

[13] or

[14] .

[24] The activator or composition according to [1], the at least one polypeptide comprising: (D) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 and a total length of 7 to 17 residues; and (E) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 and a total length of 10 to 19 residues; the activator or composition according to [1], the activity inhibitor or composition according to [2], the activation inhibitor or composition according to [3], the suppressor or composition according to [4], the therapeutic agent, preventive agent, or composition according to [5], the method according to [6], the at least one polypeptide according to [7], the use according to [8], the at least one polypeptide according to [9], the kit according to

[10] or

[11] , the diagnostic agent according to

[12] , or the method according to

[13] or

[14] .

[25] The at least one polypeptide comprises: (A) one or more polypeptides having an overall length of 8 to 20 residues, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1; (B) one or more polypeptides having an overall length of 12 to 21 residues, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2; and (D) one or more polypeptides having an overall length of 7 to 17 residues, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 4; the activator or composition according to [1], the activity inhibitor or composition according to [2], the activation inhibitor or composition according to [3], the suppressor or composition according to [4], the therapeutic agent, preventive agent, or composition according to [5], the method according to [6], the at least one polypeptide according to [7], the use according to [8], the at least one polypeptide according to [9], the kit according to

[10] or

[11] , the diagnostic agent according to

[12] , or the method according to

[13] or

[14] .

[26] The at least one polypeptide comprises: (A) one or more polypeptides having an overall length of 8 to 20 residues, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1; (B) one or more polypeptides having an overall length of 12 to 21 residues, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2; and (E) one or more polypeptides having an overall length of 10 to 19 residues, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 5; the activator or composition according to [1], the activity inhibitor or composition according to [2], the activation inhibitor or composition according to [3], the suppressor or composition according to [4], the therapeutic agent, preventive agent, or composition according to [5], the method according to [6], the at least one polypeptide according to [7], the use according to [8], the at least one polypeptide according to [9], the kit according to

[10] or

[11] , the diagnostic agent according to

[12] , or the method according to

[13] or

[14] .

[27] The at least one polypeptide comprises: (A) one or more polypeptides having an overall length of 8 to 20 residues, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1; (D) one or more polypeptides having an overall length of 7 to 17 residues, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 4; and (E) one or more polypeptides having an overall length of 10 to 19 residues, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 5; the activator or composition according to [1], the activity inhibitor or composition according to [2], the activation inhibitor or composition according to [3], the suppressor or composition according to [4], the therapeutic agent, preventive agent, or composition according to [5], the method according to [6], the at least one polypeptide according to [7], the use according to [8], the at least one polypeptide according to [9], the kit according to

[10] or

[11] , the diagnostic agent according to

[12] , or the method according to

[13] or

[14] .

[28] The at least one polypeptide comprises: (B) one or more polypeptides having a total length of 12 to 21 residues, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2; (D) one or more polypeptides having a total length of 7 to 17 residues, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 4; and (E) one or more polypeptides having a total length of 10 to 19 residues, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 5; The activator or composition according to [1], the activity inhibitor or composition according to [2], the activation inhibitor or composition according to [3], the suppressor or composition according to [4], the therapeutic agent, preventive agent, or composition according to [5], the method according to [6], the at least one polypeptide according to [7], the use according to [8], the at least one polypeptide according to [9], the kit according to

[10] or

[11] , the diagnostic agent according to

[12] , or the method according to

[13] or

[14] .

[29] The at least one polypeptide is: (A) one or more polypeptides having a total length of 8 to 20 residues, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1; (B) one or more polypeptides having a total length of 12 to 21 residues, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2; (D) one or more polypeptides having a total length of 7 to 17 residues, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 4; and (E) one or more polypeptides having a total length of 10 to 19 residues, comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 5;

[30] The activator or composition according to any one of [1] to

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

[10] or

[11] , the diagnostic agent according to

[12] , or the method according to

[13] or

[14] .

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

[29] , which is for use in a subject whose HLA-DRB3 genotype is 01:01 and / or whose HLA-DRB1 genotype is 03:01, and which comprises: (A) one or more polypeptides having a total length of 8 to 20 residues and comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1;

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

[30] , which is intended for use in a subject whose HLA-DRB3 genotype is 01:01, and the at least one polypeptide comprises: (B) one or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2 and having a total length of 12 to 21 residues;

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

[31] , which is intended for use in a subject whose HLA-DRB3 genotype is 01:01 and / or whose HLA-DRB1 genotype is 09:01, and the at least one polypeptide comprises: (D) one or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO:4 and having a total length of 7 to 17 residues;

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

[32] , which is intended for use in a subject whose HLA-DRB3 genotype is 01:01 and / or whose HLA-DRB1 genotype is 03:01, and the at least one polypeptide comprises: (E) one or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 5 and having a total length of 10 to 19 residues;

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

[33] , wherein (A) the one or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 and having a total length of 8 to 20 residues are (A') one or more polypeptides having 90% or more sequence identity with an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 6 to 15.

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

[34] , wherein (B) the one or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2 and having a total length of 12 to 21 residues are (B') one or more polypeptides having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 16.

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

[35] , wherein (D) the one or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 4 and having a total length of 7 to 17 residues are (D') one or more polypeptides having 90% or more sequence identity with an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 22 to 32.

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

[36] , wherein (E) the one or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 5 and having a total length of 10 to 19 residues are (E') one or more polypeptides having 90% or more sequence identity with an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 33 to 35.

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

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

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

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

[0011] According to the present disclosure, there is provided an agent which contains a polypeptide comprising an amino acid sequence corresponding to an epitope of AChR and which may be applicable to the treatment or diagnosis of autoimmune diseases.

[0012] The polypeptide contained in the agent of the present disclosure, which comprises an amino acid sequence corresponding to an epitope of AChR, was discovered by the NAPA method using dendritic cells prepared from classical monocytes derived from humans, and is highly likely to actually be present in the bodies of patients suffering from autoimmune diseases. Therefore, it may be more suitable for application in the treatment or diagnosis of autoimmune diseases than polypeptides with sequences corresponding to epitopes that have been discovered so far.

[0013] 1 shows the coordinates of identified HLA-binding peptides on human AChRα1 in Example 1. In Example 2, an independent specific T cell induction assay was performed six times for each peptide using PBMCs derived from healthy individuals. This shows the results of ICS (top row) and ELISA (bottom row). In Example 3, five PBMC samples derived from MG patients carrying DRB3*01:01, a predicted restrictive allele of SEQ ID NO: 17, were used to evaluate the presence or absence of SEQ ID NO: 17-specific Th1 cells. This shows the results of ICS (left row) and ELISA (right row) in Example 2, where the specific Th1 cell induction ability of SEQ ID NO: 17 was evaluated using PBMCs derived from healthy individuals carrying HLA-DRB1*09:01, which has been reported to be associated with MG in Asians, but not DRB1*03:01 or DRB3*01:01.

[0014] Hereinafter, embodiments for carrying out the present disclosure will be described, but the present disclosure is not limited to the following embodiments.

[0015] <Sequence Identity> In the present disclosure, "sequence identity" refers to the percentage (%) of identical residues in overlapping amino acid sequences in optimal alignment of two amino acid sequences. In the present disclosure, a mutation in an amino acid sequence that does not have 100% sequence identity with a certain amino acid sequence may be, for example, a substitution, deletion, or insertion of an amino acid residue, more specifically, a single-residue substitution, deletion, or insertion, and the deletion or insertion may occur at or outside the terminal of the amino acid sequence.

[0016] <Human leukocyte antigens> Human leukocyte antigens (HLA) are major histocompatibility complexes (MHC) in humans. In humoral immunity, HLA is exposed to the cell surface of antigen-presenting cells (APCs) in an antigen-bound state, thereby activating the antibody production mechanism against the antigen. HLA is classified into HLA class I, which includes HLA-A, HLA-C, HLA-B, etc. and is responsible for cellular immunity, and HLA class II, which includes HLA-DR, HLA-DQ, HLA-DP, etc. and is responsible for humoral immunity.

[0017] Depending on the HLA genotype, human white blood cells have tens of thousands of different genotypes. The HLA genotype is one of the risk factors for autoimmune diseases, and it is known that people with specific HLA genotypes are more likely to develop autoimmune diseases. In particular, in early-onset myasthenia gravis, the HLA-DRB3 genotype being 01:01 or the HLA-DRB1 genotype being 03:01 are known as risk factors.

[0018] According to the notation method established by the WHO Nomenclature Committee for Factors of the HLA System, HLA genotypes (alleles) are represented in the following format: "HLA-(gene symbol) * (region 1): (region 2): (region 3): (region 4)." Regions 1 to 4 are each represented by a two-digit number, with region 1 representing HLA specificity (antigen type), region 2 representing non-synonymous substitutions (variations in amino acid sequences within the same antigen type), region 3 representing synonymous substitutions (variations in exon regions that do not result in changes to the encoded amino acid sequence), and region 4 representing base substitutions outside the coding region (variations in the sequence of intron regions). In these notations, the gene symbol, region 1, and region 2 represent information that directly affects the amino acid sequence of HLA and are clinically important.

[0019] In the present disclosure, when the genotype of an HLA gene having the gene symbol XXX has a first region YY and a second region ZZ (i.e., the first region and the second region in the above notation are represented as HLA-XXX*YY:ZZ), this is also referred to as "the genotype of HLA-XXX is YY:ZZ." For example, "the genotype of HLA-DRB3 is 01:01" means that the genotype of an HLA gene having the gene symbol DRB3 (the HLA-DRB3 gene) has a first region 01 and a second region 01, i.e., the genotype is HLA-DRB3*01:01. For example, "the genotype of HLA-DRB1 is 03:01" means that the genotype of the HLA gene (HLA-DRB1 gene) whose gene symbol is DRB1 has a first region of 03 and a second region of 01, i.e., the genotype is HLA-DRB1*03:01.

[0020] <Acetylcholine receptor> Acetylcholine receptors (AChRs) are receptors that play an essential role in neurotransmission by binding extracellularly secreted acetylcholine to transmit information between neurons. In autoimmune diseases in which antibodies against acetylcholine receptors are overproduced, a portion of neurotransmission mediated by acetylcholine receptors is inhibited, resulting in the development of various symptoms. Furthermore, among acetylcholine receptors, the α1 subunit of human acetylcholine receptors (human AChRα1) is known to be recognized as an autoantigen in approximately 80% of patients with early-onset myasthenia gravis. Human AChRα1 has the amino acid sequence (SEQ ID NO: 36) with the Ref seq accession number NP_000070.1 from the National Center for Biotechnology Information (NCBI).

[0021] <Activator or Composition for Activating Regulatory T Cells> A first aspect of the present disclosure relates to an activator of regulatory T cells, comprising at least one polypeptide selected from the group consisting of the following (A), (B), (D), and (E): (A) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and a total length of 8 to 20 residues; (B) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 and a total length of 12 to 21 residues; (D) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 and a total length of 7 to 17 residues; and (E) one or more polypeptides comprising an amino acid sequence that has 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 and a total length of 10 to 19 residues. In one embodiment, the first aspect of the present disclosure may also be a composition for activating regulatory T cells, comprising at least one of the above polypeptides.

[0022] An agent according to an embodiment may comprise (A) above. An agent according to an embodiment may comprise (B) above. An agent according to an embodiment may comprise (D) above. An agent according to an embodiment may comprise (E) above. An agent according to an embodiment may comprise (A) and (B) above. An agent according to an embodiment may comprise (A) and (D). An agent according to an embodiment may comprise (A) and (E). An agent according to an embodiment may comprise (B) and (D). An agent according to an embodiment may comprise (B) and (E). An agent according to an embodiment may comprise (D) and (E). An agent according to an embodiment may comprise (A), (B), and (D). An agent according to an embodiment may comprise (A), (B), and (E). An agent according to an embodiment may comprise (A), (D), and (E). An agent according to an embodiment may comprise (B), (D), and (E). An agent according to an embodiment may comprise (B), (D), and (E). An agent according to an embodiment may comprise (A), (B), (D), and (E).

[0023] When the one or more polypeptides represented by (A), (B), (D), and (E) above comprise or consist of an amino acid sequence that has 90% or more sequence identity to a predetermined sequence (e.g., a sequence represented by SEQ ID NOs: 1 to 17 and 22 to 35, or a part of the sequence represented by SEQ ID NO: 37), the amino acid sequence may have a sequence identity of more than 90% with the predetermined sequence, and 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, when the one or more polypeptides represented by (A), (B), (D), and (C) above contain an amino acid sequence that has 90% or more sequence identity with a predetermined sequence (e.g., a sequence represented by SEQ ID NOs: 1 to 17 and 22 to 35, or a part of the sequence represented by SEQ ID NO: 37) or consist of the amino acid sequence, the amino acid sequence may be, for example, the predetermined sequence or an amino acid sequence in which one, two, or three residues are mutated in the predetermined sequence, and in a preferred embodiment, the amino acid sequence may be the predetermined sequence or an amino acid sequence in which one residue is mutated in the predetermined sequence.

[0024] The amino acid sequence of SEQ ID NO: 1 corresponds to the 121st to 131st amino acid sequences of the α1 subunit of the human acetylcholine receptor (SEQ ID NO: 36), whose Ref seq accession number according to the National Center for Biotechnology Information (NCBI) is NP_000070.1. The amino acid sequence of SEQ ID NO: 1 also corresponds to the 101st to 111th amino acid sequences (SEQ ID NO: 37), obtained by excluding the 1st to 20th amino acid sequences corresponding to the signal peptide from the amino acid sequence of SEQ ID NO: 36. SEQ ID NO: 1: AIVKFTKVLLQ

[0025] In one embodiment of the present disclosure, the total length of the one or more types of polypeptides in (A) above may 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, or 20 or less, 19 or less, 18 or less, 17 or less, 16 or more residues, 15 or less, 14 or less, 13 or less, 12 or less, or 11 or less residues. Furthermore, in one embodiment of the present disclosure, the total length of the one or more types of polypeptides in (A) above may be, for example, 8 to 20 residues, 9 to 19 residues, 10 to 18 residues, 11 to 17 residues, 12 to 17 residues, 12 to 15 residues, 12 to 13 residues, or 12 residues. In a preferred embodiment, the total length of the one or more types of polypeptides in (A) above may be 12 to 17 residues.

[0026] In one embodiment of the present disclosure, the one or more types of polypeptides in (A) above comprise an amino acid sequence that has 90% or more, 92% or more, 95% or more, 98% or more, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In one embodiment of the present disclosure, the one or more types of polypeptides in (A) above comprise the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having one, two, or three residue mutations in the amino acid sequence.

[0027] In one embodiment of the present disclosure, the one or more polypeptides of (A) are selected from the group consisting of the 98th to 117th, 99th to 117th, 100th to 117th, 101st to 117th, 98th to 116th, 99th to 116th, 100th to 116th, 101st to 116th, 98th to 115th, 99th to 115th, 100th to 115th, 101st to 115th, 98th to 114th, 99th to 114th, 100th to 114th, 101st to 114th, 98th to 115 ...4th, 101st to 114th, 98th to 115th, 99th to 114th, 101st to 114th, 98th to 115th, 99th to 114th, 101st to 114th, 98th to 115th, 99th The amino acid sequence may comprise, consist of, or consist of an amino acid sequence having 90% or more sequence identity to at least one amino acid selected from the group consisting of amino acids 98 to 113, 99 to 113, 100 to 113, 101 to 113, 98 to 112, 99 to 112, 100 to 112, 101 to 112, 98 to 111, 99 to 111, 100 to 111, and 101 to 111. For example, the amino acid sequence of amino acids 98 to 117 of SEQ ID NO: 37 is the amino acid sequence of SEQ ID NO: 3. In one embodiment of the present disclosure, the one or more polypeptides of (A) above may comprise an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 3, or may consist of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 3. SEQ ID NO: 3: GDFAIVKFTKVLLQYTGHIT

[0028] In one embodiment of the present disclosure, the one or more polypeptides in (A) may be (A') one or more polypeptides having 90% or more sequence identity to an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 6 to 15, or may be one or more polypeptides consisting of an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 6 to 15. The amino acid sequence set forth in SEQ ID NO: 6 corresponds to positions 98 to 111 of the amino acid sequence set forth in SEQ ID NO: 37. The amino acid sequence set forth in SEQ ID NO: 7 corresponds to positions 98 to 113 of the amino acid sequence set forth in SEQ ID NO: 37. The amino acid sequence set forth in SEQ ID NO: 8 corresponds to positions 98 to 114 of the amino acid sequence set forth in SEQ ID NO: 37. The amino acid sequence set forth in SEQ ID NO: 9 corresponds to positions 99 to 111 of the amino acid sequence set forth in SEQ ID NO: 37. The amino acid sequence set forth in SEQ ID NO: 10 corresponds to positions 99 to 113 of the amino acid sequence set forth in SEQ ID NO: 37. The amino acid sequence set forth in SEQ ID NO: 11 corresponds to positions 99 to 114 of the amino acid sequence set forth in SEQ ID NO: 37. The amino acid sequence shown in SEQ ID NO: 12 corresponds to positions 100 to 111 of the amino acid sequence shown in SEQ ID NO: 37. The amino acid sequence shown in SEQ ID NO: 13 corresponds to positions 100 to 114 of the amino acid sequence shown in SEQ ID NO: 37. The amino acid sequence shown in SEQ ID NO: 14 corresponds to positions 101 to 114 of the amino acid sequence shown in SEQ ID NO: 37. The amino acid sequence shown in SEQ ID NO: 15 corresponds to positions 101 to 117 of the amino acid sequence shown in SEQ ID NO: 37. SEQ ID NO: 6: GDFAIVKFTKVLLQ SEQ ID NO: 7: GDFAIVKFTKVLLQYT SEQ ID NO: 8: GDFAIVKFTKVLLQYTG SEQ ID NO: 9: DFAIVKFTKVLLQ SEQ ID NO: 10: DFAIVKFTKVLLQYT SEQ ID NO: 11: DFAIVKFTKVLLQYTG SEQ ID NO: 12: FAIVKFTKVLLQ SEQ ID NO: 13: FAIVKFTKVLLQYTG SEQ ID NO: 14: AIVKFTKVLLQYTG SEQ ID NO: 15: AIVKFTKVLLQYTGHIT

[0029] The present inventors have found that one or more polypeptides of (A) above bind to HLA-DRB3*01:01 and HLA-DRB1*03:01 among human HLA. Thus, an agent according to one embodiment of the present disclosure may be an agent for use in a subject whose HLA-DRB3 genotype is 01:01 and / or whose HLA-DRB1 genotype is 03:01, and wherein the at least one polypeptide comprises one or more polypeptides of (A) above.

[0030] The amino acid sequence of SEQ ID NO: 2 corresponds to amino acids 110 to 124 of the amino acid sequence of the α1 subunit of the human acetylcholine receptor (SEQ ID NO: 36), whose Ref seq accession number according to the National Center for Biotechnology Information (NCBI) is NP_000070.1. The amino acid sequence of SEQ ID NO: 2 also corresponds to amino acids 90 to 104 of the amino acid sequence (SEQ ID NO: 37) obtained by excluding amino acids 1 to 20, which are the amino acid sequence corresponding to the signal peptide, from the amino acid sequence of SEQ ID NO: 36. SEQ ID NO: 2: LVLYNNNADGDFAIVK

[0031] In one embodiment of the present disclosure, the total length of the one or more types of polypeptides in (B) above may 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, or 21 or fewer residues, 20 or more residues, 19 or fewer residues, 18 or fewer residues, 17 or fewer residues, 16 or fewer residues, or 15 or fewer residues. Furthermore, in one embodiment of the present disclosure, the total length of the one or more types of polypeptides in (B) above may be, for example, 12 or more to 21 residues, 13 or more to 21 residues, 14 or more to 21 residues, 15 or more to 21 residues, 15 or more to 19 residues, 15 or more to 17 residues, 15 or more to 16 residues, or 15 residues. In a preferred embodiment, the total length of the one or more types of polypeptides in (B) above may be 15 or more to 16 residues.

[0032] In one embodiment of the present disclosure, the one or more polypeptides of (B) above comprise an amino acid sequence that has 90% or more, 93% or more, 95% or more, 98% or more, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2. In one embodiment of the present disclosure, the one or more polypeptides of (B) above comprise the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having one, two, or three residues mutated in the amino acid sequence. In a preferred embodiment, the one or more polypeptides of (B) above comprise an amino acid sequence that has 93% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2.

[0033] In one embodiment of the present disclosure, the one or more polypeptides of (B) above are selected from the group consisting of positions 87 to 107, 88 to 107, 89 to 107, 90 to 107, 87 to 106, 88 to 106, 89 to 106, 90 to 106, 87 to 105, 88 to 105, 89 to 105, 90 to 105, 87 to 104, 88 to 104, 89 to 104, and 90 to 104 of the amino acid sequence shown in SEQ ID NO: 37. May contain an amino acid sequence having a sequence identity of 90% or more to at least one selected from the group consisting of, or may consist of, an amino acid sequence having a sequence identity of 90% or more to the at least one selected from the group consisting of,

[0034] In one embodiment of the present disclosure, the one or more polypeptides in (B) above may be (B') one or more polypeptides having 90% or more sequence identity to an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NO: 16, or may be a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 16. The amino acid sequence set forth in SEQ ID NO: 16 corresponds to the 90th to 104th amino acids of the amino acid sequence set forth in SEQ ID NO: 37. SEQ ID NO: 16: LVLYNNNADGDFAIVK

[0035] The present inventors have found that one or more polypeptides of (B) above bind to HLA-DRB3*01:01 among human HLA. Thus, an agent according to one embodiment of the present disclosure may be an agent for use in a subject whose HLA-DRB3 genotype is 01:01, and wherein the at least one polypeptide comprises one or more polypeptides of (B) above.

[0036] The amino acid sequence of SEQ ID NO: 4 corresponds to the 334th to 343rd amino acids of the amino acid sequence of the α1 subunit of the human acetylcholine receptor (SEQ ID NO: 36), whose Ref seq accession number according to the National Center for Biotechnology Information (NCBI) is NP_000070.1. The amino acid sequence of SEQ ID NO: 4 also corresponds to the 314th to 323rd amino acid sequences (SEQ ID NO: 37) obtained by excluding the 1st to 20th amino acids, which are the amino acid sequence corresponding to the signal peptide, from the amino acid sequence of SEQ ID NO: 36. SEQ ID NO: 4: KVFIDTIPNI

[0037] In one embodiment of the present disclosure, the total length of one or more types of polypeptides in (D) above may 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, or 17 or fewer residues, 16 or more residues, 15 or fewer residues, 14 or fewer residues, 13 or fewer residues, 12 or fewer residues, or 11 or fewer residues. Furthermore, in one embodiment of the present disclosure, the total length of one or more types of polypeptides in (D) above may be, for example, 7 to 17 residues, 8 to 17 residues, 9 to 17 residues, 10 to 17 residues, 11 to 16 residues, 11 to 14 residues, 11 to 13 residues, 11 to 12 residues, or 11 residues. In a preferred embodiment, the total length of one or more types of polypeptides in (D) above may be 11 to 16 residues.

[0038] In one embodiment of the present disclosure, the one or more types of polypeptides in (D) above comprise an amino acid sequence that has 90% or more, 95% or more, 98% or more, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 4. In one embodiment of the present disclosure, the one or more types of polypeptides in (D) above comprise the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence in which one, two, or three residues have been mutated in the amino acid sequence.

[0039] In one embodiment of the present disclosure, the one or more polypeptides of (D) above are selected from the group consisting of amino acid sequences 311 to 327, 312 to 327, 313 to 327, 314 to 327, 311 to 326, 312 to 326, 313 to 326, 314 to 326, 311 to 325, 312 to 325, 313 to 325, 314 to 325, 311 to 324, 312 to 324, 313 to 324, and 314 to 324 of the amino acid sequence shown in SEQ ID NO: 37. It may contain an amino acid sequence having a sequence identity of 90% or more with at least one selected from the group consisting of, or may consist of, an amino acid sequence having a sequence identity of 90% or more with the at least one selected from the group consisting of, or may consist of, an amino acid sequence having a sequence identity of 90% or more with the at least one selected from the group consisting of, For example, the 311th to 327th positions of the amino acid sequence shown in SEQ ID NO: 37 are the amino acid sequence shown in SEQ ID NO: 17. In one embodiment of the present disclosure, the one or more types of polypeptides in (D) above may comprise an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17, or may consist of an amino acid sequence that has 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 17. SEQ ID NO: 17: WVRKVFIDTIPNIMFFS

[0040] In one embodiment of the present disclosure, the one or more polypeptides in (D) above may be (D') one or more polypeptides having 90% or more sequence identity to an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 22 to 32, or may be one or more polypeptides consisting of an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 22 to 32. The amino acid sequence set forth in SEQ ID NO: 22 corresponds to positions 311 to 323 of the amino acid sequence set forth in SEQ ID NO: 37. The amino acid sequence set forth in SEQ ID NO: 23 corresponds to positions 311 to 324 of the amino acid sequence set forth in SEQ ID NO: 37. The amino acid sequence set forth in SEQ ID NO: 24 corresponds to positions 312 to 324 of the amino acid sequence set forth in SEQ ID NO: 37. The amino acid sequence set forth in SEQ ID NO: 25 corresponds to positions 312 to 325 of the amino acid sequence set forth in SEQ ID NO: 37. The amino acid sequence set forth in SEQ ID NO: 26 corresponds to positions 312 to 327 of the amino acid sequence set forth in SEQ ID NO: 37. The amino acid sequence shown in SEQ ID NO:27 corresponds to positions 313 to 324 of the amino acid sequence shown in SEQ ID NO:37. The amino acid sequence shown in SEQ ID NO:28 corresponds to positions 313 to 325 of the amino acid sequence shown in SEQ ID NO:37. The amino acid sequence shown in SEQ ID NO:29 corresponds to positions 313 to 326 of the amino acid sequence shown in SEQ ID NO:37. The amino acid sequence shown in SEQ ID NO:30 corresponds to positions 313 to 327 of the amino acid sequence shown in SEQ ID NO:37. The amino acid sequence shown in SEQ ID NO:31 corresponds to positions 314 to 324 of the amino acid sequence shown in SEQ ID NO:37. The amino acid sequence shown in SEQ ID NO:32 corresponds to positions 314 to 326 of the amino acid sequence shown in SEQ ID NO:37.SEQ ID NO:22: WVRKVFIDTIPNI SEQ ID NO:23: WVRKVFIDTIPNIM SEQ ID NO:24: VRKVFIDTIPNIM SEQ ID NO:25: VRKVFIDTIPNIMF SEQ ID NO:26: VRKVFIDTIPNIMFFS SEQ ID NO:27: RKVFIDTIPNIM SEQ ID NO:28: RKVFIDTIPNIMF SEQ ID NO:29: RKVFIDTIPNIMFF SEQ ID NO:30: RKVFIDTIPNIMFFS SEQ ID NO:31: KVFIDTIPNIM SEQ ID NO:32: KVFIDTIPNIMFF.

[0041] The present inventors have found that one or more polypeptides of (D) above bind to HLA-DRB3*01:01 and HLA-DRB1*09:01 among human HLA. Thus, an agent according to one embodiment of the present disclosure may be an agent for use in a subject whose HLA-DRB3 genotype is 01:01 and / or whose HLA-DRB1 genotype is 09:01, and wherein the at least one polypeptide comprises one or more polypeptides of (D) above.

[0042] The amino acid sequence of SEQ ID NO: 5 corresponds to the 165th to 177th amino acid sequence of the α1 subunit of the human acetylcholine receptor (SEQ ID NO: 36), whose Ref seq accession number according to the National Center for Biotechnology Information (NCBI) is NP_000070.1. The amino acid sequence of SEQ ID NO: 5 also corresponds to the 145th to 157th amino acid sequence (SEQ ID NO: 37), obtained by excluding the 1st to 20th amino acid sequences corresponding to the signal peptide from the amino acid sequence of SEQ ID NO: 36. SEQ ID NO: 5: KLGTWTYDGSVVA

[0043] In one embodiment of the present disclosure, the total length of one or more types of polypeptides in (E) above may 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, or 19 or fewer residues, 18 or more residues, 17 or fewer residues, 16 or fewer residues, 15 or fewer residues, 14 or fewer residues, or 13 or fewer residues. Furthermore, in one embodiment of the present disclosure, the total length of one or more types of polypeptides in (E) above may be, for example, 10 or more to 19 residues, 11 or more to 19 residues, 12 or more to 19 residues, 13 or more to 19 residues, 13 or more to 17 residues, 13 or more to 15 residues, 13 or more to 14 residues, or 13 residues. In a preferred embodiment, the total length of one or more types of polypeptides in (E) above may be 13 or more to 17 residues.

[0044] In one embodiment of the present disclosure, the one or more polypeptides of (E) above comprise an amino acid sequence that has 90% or more, 92%, 95% or more, 98% or more, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5. In one embodiment of the present disclosure, the one or more polypeptides of (E) above comprise the amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence having one, two, or three residues mutated in the amino acid sequence. In a preferred embodiment, the one or more polypeptides of (E) above comprise an amino acid sequence that has 92% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 5.

[0045] In one embodiment of the present disclosure, the one or more polypeptides of (E) are selected from the group consisting of the 142nd to 161st, 143rd to 161st, 144th to 161st, 145th to 161st, 142nd to 160th, 143rd to 160th, 144th to 160th, 145th to 160th, 142nd to 159th, 143rd to 159th, 144th to 159th, 145th to 159th, 146th to 159th, 147th to 159th, 148th to 159th, 149th to 160th, 150th to 150th, 151st to 151st, 152nd to 152nd, 153rd to 153rd, 154th to 154th, 155th to 155th, 156th to 157th, 158th to 159th, 160th to 161st, 161st to 162nd, 162nd to 163rd, 163rd to 164th, 164th to 165th, 165th to 166th, 166th to 167th, 167th to 168th, 168th to 169th, 169th to 170th, 171st to 172nd, 172nd to 173rd, 173rd to 174th, 175th to 176th, 176th to 177th, 177th to 178th, 178th The amino acid sequence may comprise an amino acid sequence having 90% or more sequence identity to at least one selected from the group consisting of amino acids 2 to 158, 143 to 158, 144 to 158, 145 to 158, 142 to 157, 143 to 157, 144 to 157, and 145 to 157, or may consist of an amino acid sequence having 90% or more sequence identity to said at least one amino acid sequence.

[0046] In one embodiment of the present disclosure, the one or more polypeptides in (E) above may be (E') one or more polypeptides having 90% or more sequence identity to an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 33 to 35, or may be one or more polypeptides consisting of an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 33 to 35. The amino acid sequence set forth in SEQ ID NO: 33 corresponds to positions 145 to 157 of the amino acid sequence set forth in SEQ ID NO: 37. The amino acid sequence set forth in SEQ ID NO: 34 corresponds to positions 145 to 160 of the amino acid sequence set forth in SEQ ID NO: 37. The amino acid sequence set forth in SEQ ID NO: 35 corresponds to positions 145 to 161 of the amino acid sequence set forth in SEQ ID NO: 37. SEQ ID NO: 33: KLGTWTYDGSVVA SEQ ID NO: 34: KLGTWTYDGSVVAINP SEQ ID NO: 35: KLGTWTYDGSVVAINPE

[0047] The present inventors have found that one or more polypeptides of (E) above bind to HLA-DRB3*01:01 and HLA-DRB1*03:01 among human HLA. Thus, an agent according to one embodiment of the present disclosure may be an agent for use in a subject whose HLA-DRB3 genotype is 01:01 and / or whose HLA-DRB1 genotype is 03:01, and wherein the at least one polypeptide comprises one or more polypeptides of (E) above.

[0048] At least one polypeptide selected from the group consisting of (A), (B), (D), and (E) binds to HLA-DR. Therefore, without wishing to be bound by any theory, at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) is brought into contact with antigen-presenting cells having the effect of promoting the activation (differentiation) of regulatory T cells as described in Non-Patent Document 1, and then phagocytosed, thereby naive CD4 T cells are phagocytosed in a state where the at least one polypeptide is bound to HLA-DR. ＋ It is thought that they are presented to T cells, thereby inducing the activation (differentiation) of regulatory T cells.

[0049] An agent according to one embodiment may further contain one or more types of HLA-DR in addition to at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above, and the HLA-DR is preferably one to which at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above can bind, and the genotype of the HLA-DR preferably matches the HLA-DR genotype of the subject to be administered. An agent according to one embodiment may further contain, in addition to at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above, an additive commonly used in the formulation technical field, and the additive may be, for example, at least one selected from the group consisting of an excipient, a buffer, a stabilizer, an antioxidant, a binder, a disintegrant, a filler, an emulsifier, and a flow additive adjuster.

[0050] The component contained in the agent according to one embodiment may be encapsulated in a drug carrier for intracellular delivery, such as metal nanoparticles (e.g., gold nanoparticles), liposomes, and capsids.

[0051] The dosage form of the agent according to one embodiment is not particularly limited and may be, for example, a liquid or a tablet. However, when the component contained in the agent according to one embodiment is encapsulated in a drug carrier intended for delivery into cells, the dosage form is preferably a liquid.

[0052] The administration form of the agent according to one embodiment is not particularly limited, and examples thereof include intramedullary administration, intravenous injection, subcutaneous injection, intramuscular injection, oral administration, spinal injection, etc. As an example of a specific dosage, when administered to a human adult male (body weight 60 kg), the daily dosage of the agent may be 0.001 μg to 1000 mg / day / person in terms of the amount of active ingredient.

[0053] The content of at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) in the agent according to one embodiment is not particularly limited, and for example, the total content thereof may be 0.001 to 100% by mass based on the total amount of the agent.

[0054] The subject to which the agent of one embodiment is administered may be, for example, a human, or may be a human with genetic factors that are afflicted with, may be afflicted with, or are susceptible to an autoimmune disease. The autoimmune disease may be, for example, an autoimmune disease caused by the overproduction of antibodies against human AchRα1 or a peptide fragment thereof, and is preferably myasthenia gravis, more preferably early-onset myasthenia gravis. Note that myasthenia gravis is broadly divided into early-onset myasthenia gravis, which occurs in subjects aged 10 to 49, and late-onset myasthenia gravis, which occurs in subjects aged 50 or older, and the HLA genotypes, which may be risk factors, differ partially between the two.

[0055] At least one polypeptide selected from the group consisting of (A), (B), (D), and (E) was discovered by the NAPA method using dendritic cells prepared from human classical monocytes. It is highly likely that the polypeptide is actually present in the body of a patient suffering from an autoimmune disease. Therefore, it may be more suitable for use in the treatment or diagnosis of autoimmune diseases than polypeptides having sequences corresponding to previously discovered epitopes. The suitability of at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) for the treatment or diagnosis of autoimmune diseases compared to polypeptides (other polypeptides) having sequences corresponding to previously discovered epitopes can be assessed, for example, by determining whether the at least one polypeptide has a higher T cell activation activity than the other polypeptides upon contact with T cells. Such T cell activation activity can be assessed, for example, by a T cell activation test. A T cell activation test is a test in which exposure to a polypeptide is confirmed by ELISA, such as IFN-γ, to determine whether T cells present in PBMCs are activated. When a polypeptide with a sequence corresponding to the identified epitope is added to human PBMC, it is presented as an epitope by antigen-presenting cells within the PBMC. T cells with a TCR capable of capturing this HLA-binding polypeptide receive a signal and release activating cytokines such as IFN-γ. Activation can be determined by measuring the released cytokines. Such an assay can be performed using a commercially available kit, for example, the BD OptEIA® Human IFN-γ ELISA Set (BD Biosciences, Cat. No. 555142).

[0056] <Effector T cell activity inhibitor or composition for inhibiting activity> A second aspect of the present disclosure relates to an effector T cell activity inhibitor comprising at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above. In one embodiment, the second aspect of the present disclosure can also be a composition for inhibiting effector T cell activity comprising at least one polypeptide. As the effector T cell activity inhibitor according to the second aspect of the present disclosure, the same agents as those described in the section regarding the regulatory T cell activator according to the first aspect of the present disclosure can be used, and the explanations and definitions described in the section regarding the regulatory T cell activator according to the first aspect of the present disclosure also apply to the effector T cell activity inhibitor according to the second aspect of the present disclosure.

[0057] Without wishing to be bound by any theory, it is believed that an inhibitor of effector T cell activity comprising at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above promotes the activation (differentiation) of regulatory T cells via the action of antigen-presenting cells as described in Non-Patent Document 1, as explained in the first aspect of the present disclosure, and thereby can inhibit the activity of effector T cells.

[0058] A third aspect of the present disclosure relates to an inhibitor of B cell activation, comprising at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above. In one embodiment, the third aspect of the present disclosure can also be a composition for inhibiting B cell activation, comprising at least one polypeptide. As the B cell activation inhibitor according to the third aspect of the present disclosure, the same inhibitors as those described in the section on the activator of regulatory T cells according to the first aspect of the present disclosure can be used, and the explanations and definitions described in the section on the activator of regulatory T cells according to the first aspect of the present disclosure also apply to the B cell activation inhibitor according to the third aspect of the present disclosure.

[0059] Without wishing to be bound by any theory, it is believed that a B cell activation inhibitor comprising at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above can inhibit the activity of effector T cells via the action of antigen-presenting cells as described in Non-Patent Document 1, as explained in the second aspect of the present disclosure, and thereby can inhibit B cell activation.

[0060] <Suppressant or composition for suppressing humoral immunity> The fourth aspect of the present disclosure relates to a suppressant of humoral immunity (e.g., an antibody production suppressant) comprising at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above. In one embodiment, the fourth aspect of the present disclosure can also be a composition for suppressing humoral immunity comprising at least one polypeptide. As the suppressant of humoral immunity according to the fourth aspect of the present disclosure, the same one as described in the section on the activator of regulatory T cells according to the first aspect of the present disclosure can be used, and the explanations and definitions described in the section on the activator of regulatory T cells according to the first aspect of the present disclosure also apply to the suppressant of humoral immunity according to the fourth aspect of the present disclosure.

[0061] Without wishing to be bound by any theory, it is believed that a humoral immunity suppressant comprising at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above can inhibit the activation of B cells via the action of antigen-presenting cells as described in Non-Patent Document 1, as explained in the third aspect of the present disclosure, and thereby can suppress humoral immunity.

[0062] <Therapeutic Agent, Preventive Agent, Therapeutic Composition, or Preventive Composition for Autoimmune Disease> The fifth aspect of the present disclosure relates to a therapeutic agent or preventive agent for autoimmune disease, comprising at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above. In one aspect, the fifth aspect of the present disclosure can also be a therapeutic composition or a preventive composition for autoimmune disease, comprising the at least one polypeptide. In one aspect, the fifth aspect of the present disclosure can also be a method for treating or preventing autoimmune disease, comprising administering the at least one polypeptide to a subject in need thereof. In one aspect, the fifth aspect of the present disclosure can also be the at least one polypeptide for use in the treatment or prevention of autoimmune disease. In one aspect, the fifth aspect of the present disclosure can also be use of the at least one polypeptide in the manufacture of a therapeutic agent, preventive agent, therapeutic composition, or preventive composition for autoimmune disease. In one aspect, the fifth aspect of the present disclosure can also be the at least one polypeptide for use in the manufacture of a therapeutic agent, preventive agent, therapeutic composition, or preventive composition for autoimmune disease. As the therapeutic or preventive agent for an autoimmune disease according to the fifth aspect of the present disclosure, the same agents as those explained in the section on the activator of regulatory T cells according to the first aspect of the present disclosure can be used, and the explanations and definitions given in the section on the activator of regulatory T cells according to the first aspect of the present disclosure are also applied to the therapeutic or preventive agent for an autoimmune disease according to the fifth aspect of the present disclosure.

[0063] The autoimmune disease according to the fifth aspect of the present disclosure may be, for example, an autoimmune disease caused by the overproduction of antibodies against human AchRα1 or a peptide fragment thereof, and is preferably myasthenia gravis, and 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 an epitope of human AchRα1, which is known to be recognized as an autoantigen in myasthenia gravis and early-onset myasthenia gravis, the agent according to an embodiment of the fifth aspect of the present disclosure can suitably treat and prevent myasthenia gravis and early-onset myasthenia gravis.

[0064] A therapeutic or prophylactic agent according to an embodiment of the fifth aspect of the present disclosure may be intended for administration to, for example, a human, or may be intended for administration to a human suffering from, having a genetic predisposition to, or susceptible to, an autoimmune disease. The autoimmune disease may be, for example, an autoimmune disease caused by overproduction of antibodies against human AchRα1 or a peptide fragment thereof, preferably myasthenia gravis, and more preferably early-onset myasthenia gravis. Furthermore, when the agent according to an embodiment of the fifth aspect of the present disclosure is a therapeutic agent, the subject of administration may be, for example, a human suffering from or having a genetic predisposition to an autoimmune disease. Furthermore, when the agent according to an embodiment of the fifth aspect of the present disclosure is a prophylactic agent, the subject of administration may be a human suffering from or having a genetic predisposition to an autoimmune disease.

[0065] Without wishing to be bound by any theory, 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, as explained in the fourth aspect of the present disclosure, can suppress humoral immunity through the action of antigen-presenting cells as described in Non-Patent Document 1, thereby treating and preventing autoimmune diseases. For example, it is believed that by suppressing the production of anti-AchRα1 antibodies, the degradation of AchRα1 can be suppressed, thereby treating and preventing myasthenia gravis, such as early-onset myasthenia gravis.

[0066] <Method for assisting in the diagnosis of autoimmune disease, method for obtaining data for diagnosis, and diagnostic method> A sixth aspect of the present disclosure relates to a method for assisting in the diagnosis of autoimmune disease, comprising detecting T cells having antigen-specificity for at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above. In one embodiment, the sixth aspect of the present disclosure may also relate to a method for obtaining data for the diagnosis of autoimmune disease, comprising detecting T cells having antigen-specificity for at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above. In one embodiment, the sixth aspect of the present disclosure may also relate to a method for diagnosing autoimmune disease, comprising detecting T cells having antigen-specificity for 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 a peptide fragment thereof, preferably myasthenia gravis, and more preferably early-onset myasthenia gravis. In these cases, the at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above can be the same as that described in the section on the activator of regulatory T cells according to the first aspect of the present disclosure, and the explanations and definitions of the at least one polypeptide described in the section on the activator of regulatory T cells according to the first aspect of the present disclosure also apply to the at least one polypeptide in the method according to the sixth aspect of the present disclosure.

[0067] In subjects suffering from an autoimmune disease (e.g., an autoimmune disease caused by the overproduction of antibodies against human AchRα1 or a peptide fragment thereof, preferably myasthenia gravis, more preferably early-onset myasthenia gravis), T cells having antigen specificity for at least one polypeptide selected from the group consisting of (A), (B), (D), and (E), i.e., T cells expressing antibodies that recognize at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) on their cell surface, are likely to be produced. Therefore, by using the presence or concentration of such T cells having antigen specificity for 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 the presence or absence of an autoimmune disease, obtain diagnostic data, or perform a diagnosis.

[0068] The subject for use of the method according to an embodiment of the sixth aspect of the present disclosure may be, for example, a human, or may be a human with genetic factors that may be susceptible to or have a risk of suffering from an autoimmune disease, such as an autoimmune disease caused by overproduction of antibodies against human AchRα1 or a peptide fragment thereof, preferably myasthenia gravis, and more preferably early-onset myasthenia gravis. The subject for use of the method according to an embodiment of the sixth aspect of the present disclosure may also be a human without genetic factors that may be susceptible to or have a risk of suffering from such an autoimmune disease, in which case the method according to an embodiment may be used for comprehensive screening of the possibility of suffering from the disease (so-called screening test).

[0069] In one embodiment of the sixth aspect of the present disclosure, if T cells having antigen-specificity for at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above are detected in a sample collected from a subject, diagnostic aids, diagnostic data may be obtained, or a diagnosis may be made that the subject is likely to be suffering from an autoimmune disease (e.g., an autoimmune disease caused by the overproduction of antibodies against human AchRα1 or a peptide fragment thereof, preferably myasthenia gravis, more preferably early-onset myasthenia gravis). Furthermore, in one embodiment of the sixth aspect of the present disclosure, if 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 in a sample collected from a subject is higher than that in a sample collected from a control (e.g., a healthy individual) not suffering from an autoimmune disease (e.g., an autoimmune disease caused by the overproduction of antibodies against human AchRα1 or a peptide fragment thereof, preferably myasthenia gravis, more preferably early-onset myasthenia gravis), diagnostic aids, diagnostic data may be obtained, or a diagnosis may be made that the subject is likely to be suffering from the autoimmune disease.

[0070] The sample for measuring 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 as long as it can be obtained from a subject and can be used to assist in the diagnosis of autoimmune disease, obtain data for diagnosis, or make a diagnosis based on 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. For example, the sample may be bone marrow fluid, cerebrospinal fluid, lymphatic fluid, blood, plasma, serum, or saliva. In a preferred embodiment, the sample may be bone marrow fluid, which may be collected from, for example, the ilium or sternum.

[0071] In the sixth aspect of the present disclosure, the "quantity" of T cells having antigen-specificity for at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) is not limited to the concentration of the T cells, but may also be the intensity of a signal that can be used as an indicator of the quantity of the T cells. For example, when the T cells are labeled with a polypeptide, the quantity may be, for example, the intensity of a signal that allows the quantity of the labeled polypeptide to be evaluated. For example, if the polypeptide is fluorescently labeled, the quantity may be, for example, the intensity of a signal that allows the quantity of the labeled complex to be evaluated. For example, if the human leukocyte antigen complex is fluorescently labeled, the quantity may be, for example, the intensity of a signal that allows the quantity of the labeled complex to be evaluated. For example, if the human leukocyte antigen complex is fluorescently labeled, the quantity may be, for example, the intensity of fluorescence that can be detected from the T cells. In these cases, the fluorescence intensity may be measured, for example, by a microwell plate reader or a flow cytometer.

[0072] The method for detecting T cells having antigen specificity for at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above is not particularly limited as long as it is a method that can detect the T cells. For example, the method can be performed by labeling the T cells with a fluorescently labeled polypeptide or a complex of a polypeptide and a fluorescently labeled human leukocyte antigen, and then measuring the fluorescence intensity of the fluorescent dye in cells contained in a sample.

[0073] In a preferred embodiment, detection of T cells having antigen-specificity for at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above may be carried out by labeling the T cells with a fluorescently labeled multimer (e.g., a tetramer) of a complex of a polypeptide and a human leukocyte antigen (HLA) (polypeptide-HLA complex), followed by measuring the fluorescence intensity of the fluorescent dye in cells contained in a sample. When the T cells are labeled with a multimer of a polypeptide-HLA complex, the complex can form a stable bond with the T cells, allowing for more accurate detection by removing nonspecifically bound complexes by a washing procedure or the like. Such polypeptide-HLA complexes can be prepared, for example, using at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above and a commercially available kit (e.g., QuickSwitch® series, MBL Life Sciences) that can be used to prepare the above-described polypeptide-HLA complex multimers.

[0074] <Kit for Diagnosing Autoimmune Disease> A seventh aspect of the present disclosure is a kit for diagnosing an autoimmune disease, comprising at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above, and a human leukocyte antigen (HLA) that binds to the at least one polypeptide. In one embodiment, the kit according to the seventh aspect of the present disclosure may further comprise a labeling dye that binds to the HLA. In one embodiment, the kit according to the seventh aspect of the present disclosure may comprise the HLA and the labeling dye as a complex, the HLA and the labeling dye as a complex, the polypeptide, the HLA, and the labeling dye as a complex, a human leukocyte antigen multimer and the labeling dye, or a polypeptide-human leukocyte antigen complex multimer and the labeling dye. That is, in one embodiment, the seventh aspect of the present disclosure may also be a diagnostic agent for an autoimmune disease, comprising a complex comprising a polypeptide, a human leukocyte antigen, and a labeling dye. In these cases, the labeling dye may be a fluorescent dye. In these cases, the autoimmune disease may be, for example, an autoimmune disease caused by overproduction of an antibody against human AchRα1 or a peptide fragment thereof, and is preferably myasthenia gravis, and more preferably early-onset myasthenia gravis.

[0075] The kit according to the seventh aspect of the present disclosure can be used to diagnose an autoimmune disease according to a method according to an embodiment of the sixth aspect of the present disclosure. In one embodiment, the kit according to the seventh aspect of the present disclosure may further comprise an accompanying document, which may be electronic and which may include a protocol corresponding to the method according to an embodiment of the sixth aspect of the present disclosure.

[0076] <Modifications> The eighth aspect of the present disclosure relates to the agents, methods, and kits described above, which contain, instead of at least one polypeptide selected from the group consisting of (A), (B), (D), and (E), 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 a "molecule according to the eighth aspect"). Such agents and kits can be implemented in the same manner as described above for the agents according to the first to fifth aspects and the kit according to the seventh aspect, except that they contain a molecule according to the eighth aspect instead of the at least one polypeptide. The molecule according to the eighth aspect may be a non-natural molecule, for example, an artificial molecule.

[0077] The molecule according to the eighth aspect may be, for example, a molecule (tandem molecule) in which at least two polypeptides selected from the group consisting of (A), (B), (D), and (E) are directly or indirectly linked by peptide bonds. The tandem molecule may be linear or cyclic. The tandem molecule may be, for example, a molecule (tandem molecule) in which two, three, four, or five or more polypeptides selected from the group consisting of (A), (B), (D), and (E) are directly or indirectly linked by peptide bonds. Examples of tandem molecules in which at least two polypeptides are directly linked by peptide bonds include a polypeptide consisting of two consecutive amino acid sequences of SEQ ID NO: 17, such as that shown in SEQ ID NO: 18, and a polypeptide consisting of an amino acid sequence consisting of a consecutive amino acid sequence of SEQ ID NO: 17 and an amino acid sequence of SEQ ID NO: 3, such as that shown in SEQ ID NO: 19, or a cyclic polypeptide in which peptide bonds are formed at both ends thereof. A tandem molecule in which at least two polypeptides are indirectly linked by a peptide bond may be, for example, one in which at least two polypeptides are linked by a peptide linker. Examples of the peptide linker include those disclosed in International Application No. 2019 / 233842, etc., and specific examples include GGGS (SEQ ID NO: 38), GGGGS (SEQ ID NO: 39), and the autolytic peptide T2A (EGRGSLLTCGDVEENPGP (SEQ ID NO: 40) and GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 41)). Examples of tandem molecules in which at least two polypeptides are indirectly linked by peptide bonds include a polypeptide consisting of an amino acid sequence in which GGGS is inserted between two amino acid sequences shown in SEQ ID NO: 17, as shown in SEQ ID NO: 20, and a polypeptide consisting of an amino acid sequence in which GGGGS is inserted between the amino acid sequence shown in SEQ ID NO: 17 and the amino acid sequence shown in SEQ ID NO: 3, and between the amino acid sequence shown in SEQ ID NO: 3 and the amino acid sequence shown in SEQ ID NO: 17, as shown in SEQ ID NO: 21.SEQ ID NO: 18: WVRKVFIDTIPNIMFFSWVRKVFIDTIPNIMFFS SEQ ID NO: 19: WVRKVFIDTIPNIMFFSGDFAIVKFTKVLLQYTGHIT SEQ ID NO: 20: WVRKVFIDTIPNIMFFFSGGGSWVRKVFIDTIPNIMFFS SEQ ID NO: 21: WVRKVFIDTIPNIMFFFSGGGGGSGDFAIVKFTKVLLQYTGHITGGGGSWVRKVFIDTIPNIMFFS.

[0078] The molecule according to the eighth aspect may be, for example, a molecule 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 a molecule may be, for example, a molecule in which at least two polypeptides selected from the group consisting of (A), (B), (D), and (E) are bonded via a bond other than a peptide bond, and such a molecule may further have a non-peptide structure at the peptide terminus not bonded to the linker. Such a bond other than a peptide bond may be a covalent bond (e.g., a carbon-carbon bond), or may be a bond formed by a non-covalent interaction, such as the interaction between biotin and streptavidin.

[0079] The present disclosure will be described in more detail below using examples, but the present disclosure is not limited to the following examples.

[0080] Example 1: Identification of antigen peptides derived from human AChRα1 that bind to HLA-DR Step 1: Collection of clinical samples The peripheral blood mononuclear cells (hereinafter referred to as PBMCs) used in this example were frozen samples from healthy individuals and purchased from VERITAS Co., Ltd. The HLA genotypes of the purchased PBMCs are shown in Table 1 below.

[0081] <Step 2: CD14 ＋ CD16 －Isolation of monocytes> Frozen PBMCs were quickly thawed in a 37°C water bath and washed twice with RPMI-1640 medium containing 10% FBS (RPMI-1640 / 10% FBS medium). CD14 was isolated using EasySep Human Monocyte Isolation Kit (VERITAS, Cat. No. ST-19359) according to the package insert of the kit. ＋ CD16 － Monocytes were isolated by negative selection, which resulted in the isolation of classical monocytes from PBMCs.

[0082] <Step 3: Preparation of monocyte-derived dendritic cells and addition of human AChRα1> Isolated CD14 ＋ CD16 － Monocytes were cultured at 1.5 × 10 per well of a 6-well dish in RPMI-1640 / 10% FBS medium containing 50 ng / mL GM-CSF and 50 ng / mL IL-4. ６ The culture was started at 37°C in 5% CO ２ Monocyte-derived dendritic cells were produced by culturing the cells in an incubator for 7 days. Human AChRα1 was then added at 100 μg / well, and the cells were cultured for an additional 24 hours. The human AChRα1 used in this process was prepared by gene synthesis based on the AChRα sequence information, inserting the synthetic gene into a transfer vector, cotransfecting the expression vector into SF9 insect cells, and then producing and purifying the gene.

[0083] <Step 4: Isolation of HLA-DR-bound peptides> The cultured samples were subjected to isolation according to the protocol for Dynabeads Protein G (Invitrogen, Cat. No. 10004D). 5 μg of anti-HLA-DR antibody was bound to the beads. 0.1% TFA was added to the solution containing the resulting peptide-bound HLA-DR, and the mixture was allowed to react at room temperature for 1 minute to release the peptide from HLA-DR. Microcon® Centrifugal Filter Devices (Merck, Cat. No. MRCPRT010), capable of separating particles of 10 kD or less, were attached to a dedicated 1.5 mL tube, and the solution containing the recovered peptide and HLA-DR was added thereto. The mixture was centrifuged at 14,000 × g for 30 minutes to separate HLA-DR and peptide. The peptide solution in the tube was subjected to a freeze-dryer to remove the solvent, and the solution was stored as a freeze-dried sample.

[0084] <Step 5: LC-MS / MS analysis> Purified water was added to the obtained freeze-dried sample and stirred to prepare a measurement sample, which was subjected to LC-MS / MS analysis under the conditions shown in Table 2 below.

[0085] <Step 6: LC-MS / MS Spectral Data Analysis> Mascot Server ver. 2.8 (Matrix Science) was used to analyze the spectral data obtained by LC-MS / MS analysis, and analysis was performed under the conditions shown in Table 3 below. When searching for NCBI prot and AChRα1 sequences, peptides satisfying a false discovery rate (FDR) of p<0.01 were searched for, but FDR was not calculated when searching for AChRα1 sequences alone. Peptides satisfying a false discovery rate (FDR) of p<0.01 were searched for. Skyline ver. 22.2 (MacCoss Lab.) was used for quantitative analysis. Spectral data obtained by LC-MS / MS analysis and the identification results obtained from Mascot Server were imported, and then the peak area values ​​in the extracted ion chromatograms of the peptides were calculated.

[0086] <Step 7: Identifying HLA alleles by sample comparison> The LC-MS / MS analysis data obtained in step 6 was compared for each donor to identify HLA alleles. Peptides commonly identified in samples bearing DRB1*03:01 were designated as DRB1*03:01-binding peptides, and peptides commonly identified in samples bearing DRB3*01:01 were designated as DRB3*01:01-binding peptides.

[0087] Table 4 shows the HLA-DRB1*03:01-binding peptides thus identified, along with their corresponding amino acid positions in the amino acid sequence set forth in SEQ ID NO: 37. Table 5 shows the HLA-DRB3*01:01-binding peptides thus identified, along with their corresponding amino acid positions in the amino acid sequence set forth in SEQ ID NO: 37. Figure 1 shows the coordinates of the HLA-binding peptides thus identified on human AChRα1. In Figure 1, clusters of identified DRB-binding peptides are shown as boxes, with the shaded clusters derived from DRB1*03:01, the dotted clusters derived from DRB3*01:01, and the solid black clusters derived from other HLAs. In Figure 1, the light gray regions in the row below the amino acid numbers indicate the AChRα1 sequence, and the dark gray regions indicate the transmembrane moieties.

[0088]

[0089]

[0090] These results revealed that peptides shown in SEQ ID NOs: 6 to 15 were commonly found in samples derived from subjects with the HLA-DRB3 genotype 01:01 and samples derived from subjects with the HLA-DRB1 genotype 03:01, and these peptides shared the amino acid sequence shown in SEQ ID NO: 1. Therefore, it was revealed that the peptide having the amino acid sequence shown in SEQ ID NO: 1 is a DRB3*01:01-binding peptide and a DRB1*03:01-binding peptide, and that the site in human AChRα1 having the amino acid sequence shown in SEQ ID NO: 1 is an epitope for DRB3*01:01 and DRB1*03:01.

[0091] Furthermore, these results demonstrated that the peptide shown in SEQ ID NO: 16 (SEQ ID NO: 2) was commonly found in samples derived from subjects with the HLA-DRB3 genotype 01:01. Therefore, it was revealed that the peptide having the amino acid sequence shown in SEQ ID NO: 2 is a DRB3*01:01-binding peptide, and that the site in human AChRα1 having the amino acid sequence shown in SEQ ID NO: 2 is an epitope for DRB3*01:01.

[0092] Furthermore, these results revealed that peptides shown in SEQ ID NOs: 22 to 32 were commonly found in samples derived from subjects with the HLA-DRB3 genotype 01:01, and these peptides shared the amino acid sequence shown in SEQ ID NO: 4. Therefore, it was revealed that the peptide having the amino acid sequence shown in SEQ ID NO: 4 is a DRB3*01:01-binding peptide, and that the site in human AChRα1 having the amino acid sequence shown in SEQ ID NO: 4 is an epitope for DRB3*01:01.

[0093] These results revealed that the peptides shown in SEQ ID NOs: 33 to 35 were commonly found in samples derived from subjects with the HLA-DRB3 genotype 01:01 and samples derived from subjects with the HLA-DRB1 genotype 03:01, and these peptides shared the amino acid sequence shown in SEQ ID NO: 5. Therefore, it was revealed that the peptide having the amino acid sequence shown in SEQ ID NO: 5 is a DRB3*01:01-binding peptide and a DRB1*03:01-binding peptide, and that the site in human AChRα1 having the amino acid sequence shown in SEQ ID NO: 5 is an epitope for DRB3*01:01 and DRB1*03:01.

[0094] [Example 2: Test to evaluate specific T cell induction ability using peripheral blood mononuclear cells (PBMCs) from healthy individuals] We investigated whether peptides containing the epitopes discovered in Example 1 have the ability to induce specific T cells from PBMCs derived from healthy individuals.

[0095] <Preparation of media> Human AB type serum (GeminiBio, Cat: 100-512) and fetal bovine serum (Nichirei, Cat: 175012) were inactivated at 56°C for 30 minutes and then filtered through a 0.2 μm filter before use. AIM-V (Gibco, Cat: 12055-091) containing 5% human AB type serum was used as the culture medium for PBMC. Cells were cultured at 37°C with 5% CO ２ - The staining was carried out under water vapor saturated conditions. D-PBS(-) containing 2% fetal bovine serum was used as the staining buffer.

[0096] <List of PBMCs derived from healthy individuals> The HLA alleles of PBMCs derived from healthy individuals and the evaluation peptides used in the study of this example are shown in Table 6. Six experiments to evaluate induction ability were performed for each peptide. Each PBMC was purchased from Precision for Medicine or STEMCELLS. Bold and italicized text indicates predicted restriction alleles for peptides consisting of the amino acid sequences set forth in SEQ ID NOs: 3, 16, 35, and 17, and underlined text indicates predicted restriction alleles for a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 17. Note that samples marked with * in Table 6 were evaluated in two independent experiments.

[0097]

[0098] <List of Evaluated Peptides> The peptides evaluated 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.

[0099]

[0100] <Induction of peptide-specific T cells using PBMCs derived from healthy individuals> PBMCs stored in the vapor phase of liquid nitrogen were removed and quickly thawed in a 37°C water bath. They were washed once with AIM-V and resuspended in AIM-V containing 5% human AB type serum (hereinafter referred to as complete medium). 1.5 × 10 ６The cells were seeded at 3 × 10 cells / well in a 24-well plate. The peptide was added at a final concentration of 20 μg / mL and IL-7 (PeproTech, Cat: AF-200-07-50 μg) at a final concentration of 10 ng / mL, and the culture was initiated (Day 0, total medium volume: 2 mL / well). One week after the initiation of culture, PBMCs derived from the same donor as the PBMCs in culture were thawed in the same manner as on Day 0, washed once with AIM-V, and then cultured at 3 × 10 cells / well. ６ The cells were adjusted to a concentration of 100 cells / mL or less, and the same peptide used for induction on Day 0 was added at a concentration of 20 μg / mL, followed by incubation for 2 hours. A mitomycin C solution was then added at a final concentration of 50 μg / mL, and the cells were incubated for another 45 minutes. The cells were then washed three times with AIM-V to obtain antigen-presenting cells. The cells were incubated for one week from Day 0, and collected at a concentration of 1-1.5 × 10 cells / mL. ６ The cells were seeded into a new 24-well plate within the range of 1 / well, and the same number of prepared antigen-presenting cells were seeded. IL-7 was then added to a final concentration of 10 ng / mL, and the culture was resumed (Day 7, total medium volume: 2 mL / well). Two days later, half of the culture medium was replaced with complete medium containing 40 U / L IL-2, and the culture was continued for one week, with further half-replacements being repeated with complete medium containing 20 U / mL IL-2. During this period, if the cells reached confluence, 2 mL / well of the culture medium was thoroughly suspended, 1 mL of which was seeded into a new well, and 20 U / mL IL-2-containing complete medium was added to each well to make the volume 1 mL / well (hereinafter referred to as the passaging process). The cultured cells were harvested on Day 14 and used for the <Intracellular Cytokine Staining (ICS)> and <Assessment of IFN-γ Production by ELISA> described below.

[0101] <Intracellular cytokine staining (ICS)> The collected cultured cells were placed in a 96-well round-bottom plate at 2.0 × 10 ６The cells were seeded in 2 to 6 wells each, and the peptide used for induction was added to half of the wells at a concentration of 20 μg / mL, and the solvent DMSO was added to the other half at a concentration of 0.1%. After incubation for 2 hours, Brefeldin A (BioLegend, Cat: 420601) and Monensin (BioLegen, Cat: 420701) were added to a final concentration of 1X, and the cells were further incubated for 4 hours. The cells were collected, and a 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) were added, followed by incubation at 4°C for 15 minutes. Cytofix Cytoperm Fix / Perm Solution (Beckton Dickinson, Cat. No. 554714) was added and treated at 4°C for 20 minutes. The cells were washed with Perm. / Wash Buffer (the 10X product included with Cat. No. 554714 was diluted 10-fold with distilled water), and PE-labeled anti-human IFN-γ antibody (BioLegend, Cat. No. 502509) was added and incubated at 37°C for 30 minutes. After washing with Perm. / Wash Buffer and then with Staining Buffer, the cells were analyzed by FACS.

[0102] <Evaluation of IFN-γ production ability by ELISA> The collected cultured cells were plated in a 96-well round-bottom plate at 1.0 × 10 ５The cells were seeded into six wells, and the peptide used for induction was added to half of the wells at a concentration of 20 μg / mL, and the solvent DMSO was added to the other half at a concentration of 0.1%. The wells were then cultured for at least 16 hours. The culture supernatant was collected, appropriately diluted with Assay Diluent, and subjected to ELISA analysis. IFN-γ concentration was measured using a BD OptEIA ELISA Set (human IFN-γ, Becton Dickinson, Cat: 555142) according to the package insert. If the absorbance was lower than that of the blank well, the IFN-γ concentration was treated as 0.

[0103] <Results> Figure 2 shows the results of ICS (top panel) and ELISA (bottom panel) in which an independent specific T cell induction test was performed six times for each peptide using PBMCs derived from healthy individuals. From the ICS and ELISA results, samples in which peptide-specific T cell induction was evident are shown with a solid line, and other samples are shown with a dotted line. Peptide-specific T cell induction was confirmed in one of six cases for SEQ ID NO: 16, in three of six cases for SEQ ID NOs: 3 and 35, and in all six cases for SEQ ID NO: 17. It was revealed that healthy individuals have helper T cells that have T cell receptors (TCRs) that recognize HLA and each peptide complex. In particular, the ICS results for SEQ ID NO: 17 showed a significant increase in IFN-γ-producing CD4 T cells upon peptide stimulation compared to the solvent control group (solvent = DMSO). ＋ The high T cell rate indicated that the cells had great potential.

[0104] Example 3: Test to evaluate the ability to induce specific T cells using peripheral blood mononuclear cells (PBMCs) from MG patients. In Example 2, the peptide shown in SEQ ID NO: 17 was confirmed to induce peptide-specific T cells in all cases. To confirm the presence of Th1 cells specific to this peptide in MG patients, we decided to conduct a study using MG-derived PBMCs.

[0105] <List of PBMCs derived from myasthenia gravis patients> The HLA alleles of the PBMCs derived from myasthenia gravis patients used in the study are as shown in Table 8. The only peptide evaluated was the peptide consisting of the amino acid sequence set forth in SEQ ID NO: 17. All PBMCs were purchased from Eolas Bio. The bold and italicized text indicates the predicted restriction allele of SEQ ID NO: 17.

[0106]

[0107] <Induction of peptide-specific T cells using PBMCs derived from MG patients> Peptide-specific T cells were induced using essentially the same method as in <Induction of peptide-specific T cells using PBMCs derived from healthy individuals> in Example 2, but since the purpose was to expand memory T cells or effector T cells in the patient's PBMCs, peptide and IL-7 were added on Day 0, and two days later, medium replacement with IL-2-containing complete medium and passage work were performed, and the cells were subjected to ELISA analysis on Day 7. ELISA analysis was performed using the same method as in <Evaluation of IFN-γ production ability by ELISA> in Example 2.

[0108] <Results> Figure 3 shows the results of ELISA analysis in which the presence or absence of SEQ ID NO: 17-specific Th1 cells was evaluated using five PBMC samples derived from MG patients carrying DRB3*01:01, a predicted restriction allele of SEQ ID NO: 17. As a result, the presence of SEQ ID NO: 17-specific Th1 cells was suggested in three of four samples. These results indicated that SEQ ID NO: 17-specific Th1 cells exist in DRB3*01:01-positive MG patients, and their pathogenic role was suspected.

[0109] [Example 4: Examination of the possibility of further restriction to other alleles] In the results of ICS in Example 2, the rate of SEQ ID NO: 17-specific Th1 cells (IFNγ-positive CD4 ＋The difference between T cells was high, ranging from 0.70% to 7.47% (median: 2.3%). Healthy individuals are not thought to be primed by endogenous antigens, but normally naive T cells receive antigen stimulation and take 2-3 days to differentiate into Th1 cells, after which they receive additional stimulation and undergo clonal amplification in the presence of IL-2. The doubling time of activated T cells is said to be approximately 6-8 hours, and one cell amplifies to approximately 1 million cells in the five days from Day 9, when IL-2 is added, to Day 14, when the cells are collected. On the other hand, TCR diversity is 10 ８ From 10 ９ If there is one naive T cell that recognizes the complex of HLA and peptide, the ratio is 10 －９ ~10 －８ %, which is thought to become approximately 0.1 to 1% after the above-mentioned clonal amplification. Since the experimental value obtained in Example 2 was higher than this theoretical value, further restriction of SEQ ID NO: 17 to other alleles was suspected. Therefore, the ability of SEQ ID NO: 17 to induce specific Th1 cells was evaluated using PBMCs derived from healthy individuals who had HLA-DRB1*09:01, which has been reported to be associated with MG in Asians, but did not have DRB1*03:01 or DRB3*01:01.

[0110] <List of PBMCs derived from healthy individuals> The HLA alleles of the PBMCs derived from healthy individuals used in the study of Example 4 are as shown in Table 9. The peptide evaluated was only the peptide consisting of the amino acid sequence set forth in SEQ ID NO: 17. Bold and italic letters indicate alleles that have been reported to be associated with the onset of myasthenia gravis in Asians.

[0111]

[0112] <Results> Figure 4 shows the results of ICS (left) and ELISA (right) in which the ability of SEQ ID NO: 17 to induce specific Th1 cells was evaluated using PBMCs derived from healthy individuals who had HLA-DRB1*09:01, which has been reported to be associated with MG in Asians, but did not have DRB1*03:01 or DRB3*01:01. As shown in Figure 4, SEQ ID NO: 17-specific Th1 cells were induced in seven of nine samples subjected to ICS analysis (left, solid line). Of the nine samples subjected to ICS analysis, five samples (four samples with induction of SEQ ID NO: 17-specific Th1 cells and one sample without induction) were subjected to ELISA analysis, and IFNγ production derived from SEQ ID NO: 17-specific Th1 cells was similarly detected in the four samples in which induction was confirmed by ICS (right, solid line). The above results demonstrated that SEQ ID NO: 17 is restricted by not only the initially assumed restrictive alleles (DRB1*03:01, DRB3*01:01) but also HLA-DRB1*09:01.

Claims

1. A therapeutic or prophylactic agent for autoimmune diseases, comprising at least one polypeptide selected from the group consisting of: (D) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 and having a full length of 7 to 17 residues; (A) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and having a full length of 8 to 20 residues; (B) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 and having a full length of 12 to 21 residues; and (E) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 and having a full length of 10 to 19 residues.

2. An activator of regulatory T cells, comprising at least one polypeptide selected from the group consisting of: (D) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 and having a full length of 7 to 17 residues; (A) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and having a full length of 8 to 20 residues; (B) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 and having a full length of 12 to 21 residues; and (E) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 and having a full length of 10 to 19 residues.

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 an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 and having a full length of 7 to 17 residues; (A) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and having a full length of 8 to 20 residues; (B) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 and having a full length of 12 to 21 residues; and (E) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 and having a full length of 10 to 19 residues.

4. An inhibitor of B cell activation, comprising at least one polypeptide selected from the group consisting of: (D) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 and having a full length of 7 to 17 residues; (A) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and having a full length of 8 to 20 residues; (B) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 and having a full length of 12 to 21 residues; and (E) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 and having a full length of 10 to 19 residues.

5. An inhibitor of humoral immunity, comprising at least one polypeptide selected from the group consisting of: (D) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 and having a full length of 7 to 17 residues; (A) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and having a full length of 8 to 20 residues; (B) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 and having a full length of 12 to 21 residues; and (E) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 and having a full length of 10 to 19 residues.

6. A method for treating or preventing an autoimmune disease, comprising administering to a subject in need thereof at least one polypeptide selected from the group consisting of: (D) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 and having a full length of 7 to 17 residues; (A) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and having a full length of 8 to 20 residues; (B) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 and having a full length of 12 to 21 residues; and (E) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 and having a full length of 10 to 19 residues.

7. At least one polypeptide selected from the group consisting of: (D) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 4, with a full length of 7 to 17 residues; (A) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, with a full length of 8 to 20 residues; (B) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, with a full length of 12 to 21 residues; and (E) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 5, with a full length of 10 to 19 residues, for use in the treatment or prevention of autoimmune diseases.

8. Use of at least one polypeptide selected from the group consisting of: (D) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 4, with a full length of 7 to 17 residues; (A) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, with a full length of 8 to 20 residues; (B) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, with a full length of 12 to 21 residues; and (E) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 5, with a full length of 10 to 19 residues, in the manufacture of a therapeutic agent, prophylactic agent, therapeutic composition or prophylactic composition for autoimmune diseases.

9. A diagnostic agent for autoimmune diseases, comprising a complex comprising a polypeptide, a human leukocyte antigen, and a labeled dye, wherein the polypeptide comprises: (D) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 and having a full length of 7 to 17 residues; (A) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and having a full length of 8 to 20 residues; (B) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 and having a full length of 12 to 21 residues; and (E) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 5 and having a full length of 10 to 19 residues; The diagnostic agent is at least one polypeptide selected from the group consisting of.

10. A kit for diagnosing autoimmune diseases, comprising: (D) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 and having a full length of 7 to 17 residues; (A) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and having a full length of 8 to 20 residues; (B) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 and having a full length of 12 to 21 residues; and (E) one or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 and having a full length of 10 to 19 residues; A kit comprising at least one polypeptide selected from the group consisting of and a human leukocyte antigen that binds to the at least one polypeptide.

11. The kit according to claim 10, further comprising a labeled dye that binds to the human leukocyte antigen.

12. (D) One or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 and having a full length of 7 to 17 residues; (A) One or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and having a full length of 8 to 20 residues; (B) One or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 and having a full length of 12 to 21 residues; and (E) One or more polypeptides having an amino acid sequence with 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 and having a full length of 10 to 19 residues; A method for assisting in the diagnosis of an autoimmune disease, comprising detecting T cells having antigen specificity for at least one polypeptide selected from the group consisting of

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