A preparation containing a polypeptide comprising an amino acid sequence corresponding to the HLA-DR binding epitope of an AChR

Abstract

An activator of regulatory T cells, an inhibitor of effector T cell activation, an inhibitor of B cell activation, a suppressor of humoral immunity, or a therapeutic or prophylactic agent for autoimmune diseases is disclosed, comprising a polypeptide having an amino acid sequence corresponding to an HLA-DR binding epitope of an AChR.

Description

Technology Field

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

[0002] In autoimmune diseases, the immune mechanism, which is normally designed to remove foreign substances originating from outside the body, comes to recognize substances originating from within the body as antigens, resulting in abnormal activation of the immune system. It is known that such abnormal activation of the immune system causes abnormalities in various biological functions within the body of patients suffering from autoimmune diseases.

[0003] Early-onset myasthenia gravis is a type of myasthenia gravis that occurs in individuals aged 10 to 49. Myasthenia gravis is designated as a designated intractable disease in Japan and is an organ-specific autoimmune disease 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) or muscle-specific receptor-type tyrosine kinases (MuSKs), molecules located on the postsynaptic membrane of the neuromuscular junction. As a result of these antibodies inhibiting neurotransmission, various symptoms occur. It is known that autoantibodies against AChRs account for about 80% of cases of myasthenia gravis.

[0005] In general humoral immunity, antigen-presenting cells present antigens to naive CD4 + By presenting to T cells, Naive CD4 + T cells effector CD4 + Antibody production occurs when T cells differentiate and these cells activate B cells; recently, as a new type of antigen-presenting cell, Naive CD4 +By presenting to T cells, Naive CD4 + T cells, effector CD4 + The existence of antigen-presenting cells capable of differentiating into regulatory T cells, rather than T cells, has been reported (Non-patent Literature 1, 2). Such antigen-presenting cells activate regulatory T cells by presenting an antigen, thereby [releasing] the effector CD4 on which the corresponding antigen is presented + Because it inhibits the activation of B cells by T cells, it has the effect of suppressing antibody production, contrary to the action of antigen-presenting cells known to date. In fact, clinical trials for autoimmune diseases using inhibitors of antibody production targeting this new type of antigen-presenting cell are being conducted (Non-patent Literature 3).

[0006] In autoimmune diseases, human leukocyte antigens (HLA), which are protein complexes responsible for presenting antigens on the surface of antigen-presenting cells, recognize parts of antigens and form bindings with them. In particular, when the antigen is a protein, the site on the antigen protein recognized by the HLA is called an epitope. Since such epitopes hold potential as targets for the treatment and diagnosis of autoimmune diseases, their identification is being attempted in various autoimmune diseases, and, for example, epitopes in early-onset myasthenia gravis have also been reported (Non-patent Literature 4). Prior art literature

[0007] Non-patent document 1: Laura Passerini and Silvia Gregori., "Induction of Antigen-Specific Tolerance in T Cell Mediated Diseases", Frontiers in Immunology 11(2194) (2020)Non-patent document 2: Matthias G. von Herrath and Leonard C.Harrison., "ANTIGEN-INDUCED REGULATORY T CELLS IN AUTOIMMUNITY", NATURE REVIEW I MMUNOLOGY 3: 223-232 (2003)) Non-patent document 3: 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).Non-patent document 4: 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 Immunol 56(1-2): 28-38 (1997). The problem to be solved

[0008] The present disclosure aims to provide a formulation containing a polypeptide having an amino acid sequence corresponding to an AChR epitope, which may be applicable to the treatment or diagnosis of autoimmune diseases. means of solving the problem

[0009] The inventors identified a peptide fragment of AChR that is secreted as a complex with human leukocyte type antigen (HLA) from human dendritic cells into which AChR has been introduced, by using a Natural Antigen Processing Assay (NAPA) method with dendritic cells prepared from human-derived classical monocytes. Furthermore, the inventors confirmed that this peptide fragment contains an amino acid sequence corresponding to an epitope of AChR, and that a preparation containing a polypeptide containing such an amino acid sequence has the potential to be applied to the treatment or diagnosis of autoimmune diseases, thereby completing the present disclosure.

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

[0011] [1] (A) One or more polypeptides having an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1, and a total length of 8 to 20 residues;

[0012] (B) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 2, and having a total length of 12 residues or more and 21 residues or less;

[0013] (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4, and having a total length of 7 residues or more and 17 residues or less; and

[0014] (E) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 5, and having a total length of 10 or more and 19 or less residues;

[0015] An activator of regulatory T cells or a composition for activation comprising at least one polypeptide selected from the group consisting of

[0016] [2] (A) One or more polypeptides having an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1, and a total length of 8 to 20 residues;

[0017] (B) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 2, and having a total length of 12 residues or more and 21 residues or less;

[0018] (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4, and having a total length of 7 residues or more and 17 residues or less; and

[0019] (E) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 5, and having a total length of 10 or more and 19 or less residues;

[0020] An inhibitor of effector T cell activity or a composition for inhibiting activity, comprising at least one polypeptide selected from the group consisting of

[0021] [3] (A) One or more polypeptides having an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1, and a total length of 8 to 20 residues;

[0022] (B) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 2, and having a total length of 12 residues or more and 21 residues or less;

[0023] (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4, and having a total length of 7 residues or more and 17 residues or less; and

[0024] (E) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 5, and having a total length of 10 or more and 19 or less residues;

[0025] A B cell activation inhibitor or a composition for inhibiting activation, comprising at least one polypeptide selected from the group consisting of

[0026] [4] (A) One or more polypeptides having an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1, and a total length of 8 to 20 residues;

[0027] (B) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 2, and having a total length of 12 residues or more and 21 residues or less;

[0028] (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4, and having a total length of 7 residues or more and 17 residues or less; and

[0029] (E) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 5, and having a total length of 10 or more and 19 or less residues;

[0030] An inhibitor of humoral immunity or a composition for inhibiting it, comprising at least one polypeptide selected from the group consisting of

[0031] [5] (A) One or more polypeptides having an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1, and a total length of 8 to 20 residues;

[0032] (B) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 2, and having a total length of 12 residues or more and 21 residues or less;

[0033] (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4, and having a total length of 7 residues or more and 17 residues or less; and

[0034] (E) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 5, and having a total length of 10 or more and 19 or less residues;

[0035] A therapeutic agent, prophylactic agent, therapeutic composition, or prophylactic composition for an autoimmune disease comprising at least one polypeptide selected from the group consisting of

[0036] [6] (A) One or more polypeptides having an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1, and a total length of 8 to 20 residues;

[0037] (B) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 2, and having a total length of 12 residues or more and 21 residues or less;

[0038] (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4, and having a total length of 7 residues or more and 17 residues or less; and

[0039] (E) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 5, and having a total length of 10 or more and 19 or less residues;

[0040] A method for treating or preventing an autoimmune disease, comprising administering at least one polypeptide selected from the group consisting of to a target in need thereof.

[0041] [7] For use in the treatment or prevention of autoimmune diseases,

[0042] (A) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1, and having a total length of 8 or more and 20 or less residues;

[0043] (B) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 2, and having a total length of 12 residues or more and 21 residues or less;

[0044] (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4, and having a total length of 7 residues or more and 17 residues or less; and

[0045] (E) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 5, and having a total length of 10 or more and 19 or less residues;

[0046] At least one polypeptide selected from the group consisting of

[0047] [8] In the manufacture of a therapeutic agent, preventive agent, therapeutic composition or preventive composition for autoimmune diseases,

[0048] (A) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1, and having a total length of 8 or more and 20 or less residues;

[0049] (B) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 2, and having a total length of 12 residues or more and 21 residues or less;

[0050] (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4, and having a total length of 7 residues or more and 17 residues or less; and

[0051] (E) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 5, and having a total length of 10 or more and 19 or less residues;

[0052] Use of at least one polypeptide selected from the group consisting of

[0053] [9] For use in the manufacture of a therapeutic agent, preventive agent, therapeutic composition or preventive composition for autoimmune diseases,

[0054] (A) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1, and having a total length of 8 or more and 20 or less residues;

[0055] (B) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 2, and having a total length of 12 residues or more and 21 residues or less;

[0056] (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4, and having a total length of 7 residues or more and 17 residues or less; and

[0057] (E) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 5, and having a total length of 10 or more and 19 or less residues;

[0058] At least one polypeptide selected from the group consisting of

[0059]

[10] As a diagnostic kit for autoimmune diseases,

[0060] (A) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1, and having a total length of 8 or more and 20 or less residues;

[0061] (B) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 2, and having a total length of 12 residues or more and 21 residues or less;

[0062] (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4, and having a total length of 7 residues or more and 17 residues or less; and

[0063] (E) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 5, and having a total length of 10 or more and 19 or less residues;

[0064] A kit comprising at least one polypeptide selected from the group consisting of, and a human leukocyte type antigen that binds to said at least one polypeptide.

[0065]

[11] A kit described in

[10] that further comprises a labeling dye that binds to the above human leukocyte type antigen.

[0066]

[12] A diagnostic agent for autoimmune diseases comprising a complex having a polypeptide, a human leukocyte type antigen and a labeling dye, wherein the polypeptide is

[0067] (A) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1, and having a total length of 8 or more and 20 or less residues;

[0068] (B) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 2, and having a total length of 12 residues or more and 21 residues or less;

[0069] (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4, and having a total length of 7 residues or more and 17 residues or less; and

[0070] (E) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 5, and having a total length of 10 or more and 19 or less residues;

[0071] A diagnostic agent, which is at least one polypeptide selected from the group consisting of

[0072]

[13] (A) One or more polypeptides having an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1, and a total length of 8 to 20 residues;

[0073] (B) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 2, and having a total length of 12 residues or more and 21 residues or less;

[0074] (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4, and having a total length of 7 residues or more and 17 residues or less; and

[0075] (E) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 5, and having a total length of 10 or more and 19 or less residues;

[0076] 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 a group consisting of

[0077]

[14] (A) One or more polypeptides having an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1, and a total length of 8 to 20 residues;

[0078] (B) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 2, and having a total length of 12 residues or more and 21 residues or less;

[0079] (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4, and having a total length of 7 residues or more and 17 residues or less; and

[0080] (E) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 5, and having a total length of 10 or more and 19 or less residues;

[0081] A method for acquiring data for the diagnosis of an autoimmune disease, comprising detecting T cells having antigen specificity for at least one polypeptide selected from a group consisting of

[0082]

[15] At least one polypeptide,

[0083] (A) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1, and having a total length of 8 or more and 20 or less residues;

[0084] An activator or composition described in [1], an activity inhibitor or composition described in [2], an activity inhibitor or composition described in [3], an inhibitor or composition described in [4], a therapeutic agent, a preventive agent or composition described in [5], a method described in [6], at least one polypeptide described in [7], a use described in [8], at least one polypeptide described in [9], a kit described in

[10] or

[11] , a diagnostic agent described in

[12] , or a method described in

[13] or

[14] .

[0085]

[16] At least one polypeptide,

[0086] (B) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 2, and having a total length of 12 residues or more and 21 residues or less;

[0087] An activator or composition described in [1], an activity inhibitor or composition described in [2], an activity inhibitor or composition described in [3], an inhibitor or composition described in [4], a therapeutic agent, a preventive agent or composition described in [5], a method described in [6], at least one polypeptide described in [7], a use described in [8], at least one polypeptide described in [9], a kit described in

[10] or

[11] , a diagnostic agent described in

[12] , or a method described in

[13] or

[14] .

[0088]

[17] At least one polypeptide,

[0089] (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4, and having a total length of 7 or more and 17 or less residues;

[0090] An activator or composition described in [1], an activity inhibitor or composition described in [2], an activity inhibitor or composition described in [3], an inhibitor or composition described in [4], a therapeutic agent, a preventive agent or composition described in [5], a method described in [6], at least one polypeptide described in [7], a use described in [8], at least one polypeptide described in [9], a kit described in

[10] or

[11] , a diagnostic agent described in

[12] , or a method described in

[13] or

[14] .

[0091]

[18] At least one polypeptide,

[0092] (E) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 5, and having a total length of 10 or more and 19 or less residues;

[0093] An activator or composition described in [1], an activity inhibitor or composition described in [2], an activity inhibitor or composition described in [3], an inhibitor or composition described in [4], a therapeutic agent, a preventive agent or composition described in [5], a method described in [6], at least one polypeptide described in [7], a use described in [8], at least one polypeptide described in [9], a kit described in

[10] or

[11] , a diagnostic agent described in

[12] , or a method described in

[13] or

[14] .

[0094]

[19] At least one polypeptide,

[0095] (A) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1, and having a total length of 8 to 20 residues; and

[0096] (B) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 2, and having a total length of 12 residues or more and 21 residues or less;

[0097] An activator or composition described in [1], an activity inhibitor or composition described in [2], an activity inhibitor or composition described in [3], an inhibitor or composition described in [4], a therapeutic agent, a preventive agent or composition described in [5], a method described in [6], at least one polypeptide described in [7], a use described in [8], at least one polypeptide described in [9], a kit described in

[10] or

[11] , a diagnostic agent described in

[12] , or a method described in

[13] or

[14] .

[0098]

[20] At least one polypeptide,

[0099] (A) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1, and having a total length of 8 to 20 residues; and

[0100] (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4, and having a total length of 7 or more and 17 or less residues;

[0101] An activator or composition described in [1], an activity inhibitor or composition described in [2], an activity inhibitor or composition described in [3], an inhibitor or composition described in [4], a therapeutic agent, a preventive agent or composition described in [5], a method described in [6], at least one polypeptide described in [7], a use described in [8], at least one polypeptide described in [9], a kit described in

[10] or

[11] , a diagnostic agent described in

[12] , or a method described in

[13] or

[14] .

[0102]

[21] At least one polypeptide,

[0103] (A) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1, and having a total length of 8 to 20 residues; and

[0104] (E) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 5, and having a total length of 10 or more and 19 or less residues;

[0105] An activator or composition described in [1], an activity inhibitor or composition described in [2], an activity inhibitor or composition described in [3], an inhibitor or composition described in [4], a therapeutic agent, a preventive agent or composition described in [5], a method described in [6], at least one polypeptide described in [7], a use described in [8], at least one polypeptide described in [9], a kit described in

[10] or

[11] , a diagnostic agent described in

[12] , or a method described in

[13] or

[14] .

[0106]

[22] At least one polypeptide above,

[0107] (B) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 2, and having a total length of 12 residues or more and 21 residues or less; and

[0108] (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4, and having a total length of 7 or more and 17 or less residues;

[0109] An activator or composition described in [1], an activity inhibitor or composition described in [2], an activity inhibitor or composition described in [3], an inhibitor or composition described in [4], a therapeutic agent, a preventive agent or composition described in [5], a method described in [6], at least one polypeptide described in [7], a use described in [8], at least one polypeptide described in [9], a kit described in

[10] or

[11] , a diagnostic agent described in

[12] , or a method described in

[13] or

[14] .

[0110]

[23] At least one polypeptide above,

[0111] (B) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 2, and having a total length of 12 residues or more and 21 residues or less; and

[0112] (E) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 5, and having a total length of 10 or more and 19 or less residues;

[0113] An activator or composition described in [1], an activity inhibitor or composition described in [2], an activity inhibitor or composition described in [3], an inhibitor or composition described in [4], a therapeutic agent, a preventive agent or composition described in [5], a method described in [6], at least one polypeptide described in [7], a use described in [8], at least one polypeptide described in [9], a kit described in

[10] or

[11] , a diagnostic agent described in

[12] , or a method described in

[13] or

[14] .

[0114]

[24] At least one polypeptide,

[0115] (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4, and having a total length of 7 residues or more and 17 residues or less; and

[0116] (E) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 5, and having a total length of 10 or more and 19 or less residues;

[0117] An activator or composition described in [1], an activity inhibitor or composition described in [2], an activity inhibitor or composition described in [3], an inhibitor or composition described in [4], a therapeutic agent, a preventive agent or composition described in [5], a method described in [6], at least one polypeptide described in [7], a use described in [8], at least one polypeptide described in [9], a kit described in

[10] or

[11] , a diagnostic agent described in

[12] , or a method described in

[13] or

[14] .

[0118]

[25] At least one polypeptide,

[0119] (A) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1, and having a total length of 8 or more and 20 or less residues;

[0120] (B) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 2, and having a total length of 12 residues or more and 21 residues or less; and

[0121] (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4, and having a total length of 7 or more and 17 or less residues;

[0122] An activator or composition described in [1], an activity inhibitor or composition described in [2], an activity inhibitor or composition described in [3], an inhibitor or composition described in [4], a therapeutic agent, a preventive agent or composition described in [5], a method described in [6], at least one polypeptide described in [7], a use described in [8], at least one polypeptide described in [9], a kit described in

[10] or

[11] , a diagnostic agent described in

[12] , or a method described in

[13] or

[14] .

[0123]

[26] At least one polypeptide,

[0124] (A) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1, and having a total length of 8 or more and 20 or less residues;

[0125] (B) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 2, and having a total length of 12 residues or more and 21 residues or less; and

[0126] (E) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 5, and having a total length of 10 or more and 19 or less residues;

[0127] An activator or composition described in [1], an activity inhibitor or composition described in [2], an activity inhibitor or composition described in [3], an inhibitor or composition described in [4], a therapeutic agent, a preventive agent or composition described in [5], a method described in [6], at least one polypeptide described in [7], a use described in [8], at least one polypeptide described in [9], a kit described in

[10] or

[11] , a diagnostic agent described in

[12] , or a method described in

[13] or

[14] .

[0128]

[27] At least one polypeptide,

[0129] (A) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1, and having a total length of 8 or more and 20 or less residues;

[0130] (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4, and having a total length of 7 residues or more and 17 residues or less; and

[0131] (E) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 5, and having a total length of 10 or more and 19 or less residues;

[0132] An activator or composition described in [1], an activity inhibitor or composition described in [2], an activity inhibitor or composition described in [3], an inhibitor or composition described in [4], a therapeutic agent, a preventive agent or composition described in [5], a method described in [6], at least one polypeptide described in [7], a use described in [8], at least one polypeptide described in [9], a kit described in

[10] or

[11] , a diagnostic agent described in

[12] , or a method described in

[13] or

[14] .

[0133]

[28] At least one polypeptide above,

[0134] (B) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 2, and having a total length of 12 residues or more and 21 residues or less;

[0135] (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4, and having a total length of 7 residues or more and 17 residues or less; and

[0136] (E) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 5, and having a total length of 10 or more and 19 or less residues;

[0137] An activator or composition described in [1], an activity inhibitor or composition described in [2], an activity inhibitor or composition described in [3], an inhibitor or composition described in [4], a therapeutic agent, a preventive agent or composition described in [5], a method described in [6], at least one polypeptide described in [7], a use described in [8], at least one polypeptide described in [9], a kit described in

[10] or

[11] , a diagnostic agent described in

[12] , or a method described in

[13] or

[14] .

[0138]

[29] At least one polypeptide,

[0139] (A) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1, and having a total length of 8 or more and 20 or less residues;

[0140] (B) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 2, and having a total length of 12 residues or more and 21 residues or less;

[0141] (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4, and having a total length of 7 residues or more and 17 residues or less; and

[0142] (E) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 5, and having a total length of 10 or more and 19 or less residues;

[0143] An activator or composition described in [1], an activity inhibitor or composition described in [2], an activity inhibitor or composition described in [3], an inhibitor or composition described in [4], a therapeutic agent, a preventive agent or composition described in [5], a method described in [6], at least one polypeptide described in [7], a use described in [8], at least one polypeptide described in [9], a kit described in

[10] or

[11] , a diagnostic agent described in

[12] , or a method described in

[13] or

[14] .

[0144]

[30] For use in subjects with HLA-DRB3 genotype 01:01 and / or HLA-DRB1 genotype 03:01, wherein at least one polypeptide is,

[0145] (A) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1, and having a total length of 8 or more and 20 or less residues;

[0146] An activator, composition, activity inhibitor, activity inhibitor, inhibitor, therapeutic agent, preventive agent, method, at least one polypeptide, use, kit, or diagnostic agent comprising, as described in any one of [1] to

[29] .

[0147]

[31] For use in subjects with an HLA-DRB3 genotype of 01:01, the above at least one polypeptide,

[0148] (B) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 2, and having a total length of 12 residues or more and 21 residues or less;

[0149] An activator, composition, activity inhibitor, activation inhibitor, inhibitor, therapeutic agent, preventive agent, method, at least one polypeptide, use, kit, or diagnostic agent comprising [1] to

[30] .

[0150]

[32] For use in subjects with HLA-DRB3 genotype 01:01 and / or HLA-DRB1 genotype 09:01, wherein at least one polypeptide is,

[0151] (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4, and having a total length of 7 or more and 17 or less residues;

[0152] An activator, composition, activity inhibitor, activity inhibitor, inhibitor, therapeutic agent, preventive agent, method, at least one polypeptide, use, kit, or diagnostic agent comprising [1] to

[31] as described in any one of [1] to

[31] .

[0153]

[33] For use in subjects with HLA-DRB3 genotype 01:01 and / or HLA-DRB1 genotype 03:01, wherein at least one polypeptide is,

[0154] (E) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 5, and having a total length of 10 or more and 19 or less residues;

[0155] An activator, composition, activity inhibitor, activity inhibitor, inhibitor, therapeutic agent, preventive agent, method, at least one polypeptide, use, kit, or diagnostic agent comprising [1] to

[32] .

[0156]

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

[33] , comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in (A) SEQ ID NO. 1 and having a total length of 8 or more and 20 or less, and having 90% or more sequence identity with an amino acid sequence selected from the group consisting of (A') SEQ ID NOs 6 to 15.

[0157]

[35] One or more polypeptides having a total length of 12 residues or more and having a sequence identity of 90% or more with the amino acid sequence described in (B) SEQ ID NO. 2, and one or more polypeptides having a sequence identity of 90% or more with the amino acid sequence described in (B') SEQ ID NO. 16, and one or more polypeptides having a sequence identity of 90% or more with the amino acid sequence described in any one of [1] to

[34] , an activator, composition, activity inhibitor, activity inhibitor, inhibitor, therapeutic agent, preventive agent, method, at least one polypeptide, use, kit or diagnostic agent.

[0158]

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

[35] , comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in (D) SEQ ID NO. 4 and having a total length of 7 or more and 17 or less, and having 90% or more sequence identity with an amino acid sequence selected from the group consisting of (D') SEQ ID NOs 22 to 32.

[0159]

[37] One or more polypeptides having a total length of 10 residues or more and having a sequence identity of 90% or more with the amino acid sequence described in (E) SEQ ID NO. 5, and having a total length of 19 residues or more, and one or more polypeptides having a sequence identity of 90% or more with an amino acid sequence selected from the group consisting of (E') SEQ ID NOs 33 to 35, which is an activator, composition, activity inhibitor, activation inhibitor, inhibitor, therapeutic agent, preventive agent, method, at least one polypeptide, use, kit or diagnostic agent described in any one of [1] to

[36] .

[0160]

[38] The above-mentioned autoimmune disease is myasthenia gravis, [5] to

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

[0161]

[39] The above-mentioned autoimmune disease is early-onset myasthenia gravis, [5] to

[38] a therapeutic agent, prophylactic agent, composition, method, at least one polypeptide, use, kit, or diagnostic agent. Effects of the invention

[0162] According to the present disclosure, a formulation is provided that contains a polypeptide comprising an amino acid sequence corresponding to an epitope of an AChR and has the potential to be applied to the treatment or diagnosis of an autoimmune disease.

[0163] The polypeptide containing an amino acid sequence corresponding to an AChR epitope, which is contained in the formulation according to the present disclosure, was identified by the NAPA method using dendritic cells prepared from human-derived classical monocytes and is highly likely to actually exist in the body of a patient with an autoimmune disease; therefore, it is more likely to be applied to the treatment or diagnosis of autoimmune diseases than polypeptides with epitope-corresponding sequences identified so far. Brief explanation of the drawing

[0164] Figure 1 is a diagram showing the specified HLA-binding peptide in Example 1 as coordinates on human AChRα1. Figure 2 is a diagram showing the results of ICS (top) and ELISA (bottom) in which 6 independent specific T-cell induction ability evaluation tests were performed for each peptide using normal human-derived PBMC in Example 2. Figure 3 is a figure showing the results of an ELISA analysis in which the presence or absence of SEQ ID NO. 17-specific Th1 cells was evaluated using 5 samples of PBMC derived from MG patients containing DRB3*01:01, the expected binding allel of SEQ ID NO. 17, in Example 3. Figure 4 is a diagram showing the results of ICS (left) and ELISA (right) in which the specific Th1 cell induction ability of SEQ ID NO. 17 was evaluated using normal-derived PBMCs that have HLA-DRB1*09:01, which has been reported to be associated with MG in Asians, and do not have DRB1*03:01 or DRB3*01:01. Specific details for implementing the invention

[0165] The following describes forms for carrying out the present disclosure, but the present disclosure is not limited to the following embodiments.

[0166] Order Identity

[0167] In the present disclosure, "sequence identity" means the ratio (%) of identical residues to residues of overlapping amino acid sequences in the optimal alignment when two amino acid sequences are aligned. In the present disclosure, variations in amino acid sequences that do not have 100% sequence identity with respect to a certain amino acid sequence may be, for example, substitution, deletion, or insertion of amino acid residues, and more specifically, for example, substitution of one residue, deletion of one residue, or insertion of one residue, and such deletion or insertion may occur at the end of the amino acid sequence or may occur outside the end.

[0168] Human Leukocyte Type Antigens

[0169] Human leukocyte type antigens (HLA) are the major histocompatibility complex (MHC) in humans. In humoral immunity, HLA is exposed to the cell surface of antigen-presenting cells (APCs) in a bound state, thereby activating the mechanism for producing antibodies against said antigens. HLA is classified into HLA Class I, which includes HLA-A, HLA-C, and HLA-B and is responsible for cellular immunity, and HLA Class II, which includes HLA-DR, HLA-DQ, and HLA-DP and is responsible for humoral immunity.

[0170] Human leukocytes have tens of thousands of genotypes based on HLA genotypes. HLA genotypes are one of the risk factors for autoimmune diseases, and it is known that humans with specific HLA genotypes have a high probability of developing autoimmune diseases. In particular, in early-onset myasthenia gravis, having an HLA-DRB3 genotype of 01:01 or an HLA-DRB1 genotype of 03:01 is known as a risk factor.

[0171] According to the notation established by the WHO Nomenclature Committee for Factors of the HLA System, the HLA genotype (allel) is expressed in the format "HLA-(gene symbol)*(Zone 1):(Zone 2):(Zone 3):(Zone 4)". Here, Zones 1 through 4 are each represented by two-digit numbers; Zone 1 represents HLA specificity (antigen type), Zone 2 represents non-synonymic substitutions (variations in the amino acid sequence within the same antigen type), Zone 3 represents synonymic substitutions (variations in the exon region that do not alter the coding amino acid sequence), and Zone 4 represents extracoding base substitutions (variations in the intron region sequence). In this notation, the gene symbol, Zone 1, and Zone 2 represent information that directly affects the HLA amino acid sequence and are clinically important.

[0172] In the present disclosure, regarding the genotype of an HLA gene with gene symbol XXX, the first region is YY and the second region is ZZ (i.e., up to the second region in the above notation is indicated as HLA-XXX*YY:ZZ), it is also described as "the genotype of HLA-XXX is YY:ZZ." That is, for example, "the genotype of HLA-DRB3 is 01:01" means that the genotype of an HLA gene (HLA-DRB3 gene) with gene symbol DRB3 has the first region as 01 and the second region as 01, i.e., the genotype is HLA-DRB3*01:01. In addition, for example, "the genotype of HLA-DRB1 is 03:01" means that the genotype of the HLA gene (HLA-DRB1 gene) with the gene symbol DRB1 has a first region of 03 and a second region of 01, that is, the genotype is HLA-DRB1*03:01.

[0173] Acetylcholine receptors

[0174] Acetylcholine receptors (AChRs) are receptors that perform an essential function in neurotransmission by binding to acetylcholine secreted extracellularly. In autoimmune diseases where antibodies against acetylcholine receptors are overproduced, various symptoms develop because a portion of neurotransmission mediated by acetylcholine receptors is inhibited. Furthermore, among acetylcholine receptors, the α1 subunit of the human acetylcholine receptor (human AChRα1) is known to be recognized as an autoantigen in approximately 80% of patients with early-onset myasthenia gravis. Human AChRα1 has an amino acid sequence (sequence number 36) with Ref seq accession number NP_000070.1 according to the National Center for Biotechnology Information (NCBI).

[0175] <Activator of regulatory T cells or composition for activation>

[0176] The first aspect of the present disclosure is as follows (A), (B), (D) and (E):

[0177] (A) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1, and having a total length of 8 or more and 20 or less residues;

[0178] (B) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 2, and having a total length of 12 residues or more and 21 residues or less;

[0179] (D) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4, and having a total length of 7 residues or more and 17 residues or less; and

[0180] (E) One or more polypeptides comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 5, and having a total length of 10 or more and 19 or less residues;

[0181] The present invention relates to an activator of regulatory T cells comprising at least one polypeptide selected from the group consisting of

[0182] A formulation according to one embodiment may include (A). A formulation according to one embodiment may include (B). A formulation according to one embodiment may include (D). A formulation according to one embodiment may include (E). A formulation according to one embodiment may include (A) and (B). A formulation according to one embodiment may include (A) and (D). A formulation according to one embodiment may include (A) and (E). A formulation according to one embodiment may include (B) and (D). A formulation according to one embodiment may include (B) and (E). A formulation according to one embodiment may include (D) and (E). A formulation according to one embodiment may include (A), (B), and (D). A formulation according to one embodiment may include (A), (B), and (E). A formulation according to one embodiment may include (A), (D), and (E). A formulation according to one embodiment may include (B), (D), and (E). A formulation according to one embodiment may include (A), (B), (D), and (E).

[0183] In the case where the polypeptide represented by (A), (B), (D) and (E) comprises an amino acid sequence having 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 a sequence represented by SEQ ID NO. 37) or is composed of said amino acid sequence, the amino acid sequence may have more than 90% sequence identity with said predetermined sequence, 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%. In addition, when the polypeptide represented by (A), (B), (D) and the above one or more types comprises an amino acid sequence having 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 a sequence represented by SEQ ID NO. 37) or is composed of said amino acid sequence, said amino acid sequence may be, for example, an amino acid sequence comprising a variation of one residue, two residues, or three residues in said predetermined sequence, and in a preferred embodiment, may be an amino acid sequence comprising a variation of one residue in said predetermined sequence or said predetermined sequence.

[0184] The amino acid sequence described in SEQ ID NO. 1 corresponds to the 121st to 131st amino acid sequence of the α1 subunit of the human acetylcholine receptor (SEQ ID NO. 36), whose Ref seq accession number is NP_000070.1 according to the National Center for Biotechnology Information (NCBI). Additionally, the amino acid sequence described in SEQ ID NO. 1 corresponds to the 101st to 111th amino acid sequence (SEQ ID NO. 37), excluding the 1st to 20th amino acid sequences corresponding to the signal peptide from the amino acid sequence shown in SEQ ID NO. 36.

[0185] Sequence No. 1: AIVKFTKVLLQ

[0186] In one embodiment of the present disclosure, the total length of one or more polypeptides of (A) may be, for example, 8 residues or more, 9 residues or more, 10 residues or more, 11 residues or more, 12 residues or more, 13 residues or more, or 14 residues or more, and may be 20 residues or less, 19 residues or less, 18 residues or less, 17 residues or less, 16 residues or more, 15 residues or less, 14 residues or less, 13 residues or less, 12 residues or less, or 11 residues or less. In addition, in one embodiment of the present disclosure, the total length of one or more polypeptides of (A) may be, for example, 8 residues or more and 20 residues or less, 9 residues or more and 19 residues or less, 10 residues or more and 18 residues or less, 11 residues or more and 17 residues or less, 12 residues or more and 17 residues or less, 12 residues or more and 15 residues or less, 12 residues or more and 13 residues or less, or 12 residues. In a preferred embodiment, the total length of one or more polypeptides of (A) may be 12 residues or more and 17 residues or less.

[0187] In one embodiment of the present disclosure, the polypeptide of (A) or a plurality thereof comprises an amino acid sequence having sequence identity of 90% or more, 92% or more, 95% or more, 98% or more, or 100% with respect to the amino acid sequence described in SEQ ID NO. 1. In one embodiment of the present disclosure, the polypeptide of (A) or a plurality thereof comprises an amino acid sequence described in SEQ ID NO. 1 or an amino acid sequence having a variation of one residue, two residues, or three residues in said amino acid sequence.

[0188] In one embodiment of the present disclosure, the polypeptide of (A) or a plurality thereof is 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 113th, 99th to 113th, 100th to It may include an amino acid sequence having 90% or more sequence identity with at least one selected from the group consisting of the 113th, 101st to 113th, 98th to 112th, 99th to 112th, 100th to 112th, 101st to 112th, 98th to 111th, 99th to 111th, 100th to 111th, and 101st to 111th, and may be composed of an amino acid sequence having 90% or more sequence identity with said at least one, and may be composed of said at least one amino acid sequence. For example, the 98th to 117th of the amino acid sequence shown in SEQ ID NO. 37 is the amino acid sequence shown in SEQ ID NO. 3. In one embodiment of the present disclosure, one or more polypeptides of (A) may include an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO. 3, may be composed of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO. 3, and may be composed of said amino acid sequence.

[0189] Sequence No. 3: GDFAIVKFTKVLLQYTGHIT

[0190] In one embodiment of the present disclosure, the one or more polypeptides of (A) may be one or more polypeptides having a sequence identity of 90% or more with an amino acid sequence selected from the group consisting of amino acid sequences described in (A') SEQ ID NOs 6 to 15, and may be one or more polypeptides consisting of an amino acid sequence selected from the group consisting of amino acid sequences described in SEQ ID NOs 6 to 15. The amino acid sequence indicated in SEQ ID NO. 6 corresponds to the 98th to 111th amino acid sequence indicated in SEQ ID NO. 37. The amino acid sequence indicated in SEQ ID NO. 7 corresponds to the 98th to 113th amino acid sequence indicated in SEQ ID NO. 37. The amino acid sequence indicated in SEQ ID NO. 8 corresponds to the 98th to 114th amino acid sequence indicated in SEQ ID NO. 37. The amino acid sequence indicated in SEQ ID NO. 9 corresponds to the 99th to 111th amino acid sequence indicated in SEQ ID NO. 37. The amino acid sequence indicated in SEQ ID NO. 10 corresponds to the 99th to 113th amino acid sequence indicated in SEQ ID NO. 37. The amino acid sequence indicated in SEQ ID NO. 11 corresponds to the 99th to 114th amino acid sequence indicated in SEQ ID NO. 37. The amino acid sequence indicated in SEQ ID NO. 12 corresponds to the 100th to 111th amino acid sequence indicated in SEQ ID NO. 37. The amino acid sequence indicated in SEQ ID NO. 13 corresponds to the 100th to 114th amino acid sequence indicated in SEQ ID NO. 37. The amino acid sequence indicated in SEQ ID NO. 14 corresponds to the 101st to 114th amino acid sequence indicated in SEQ ID NO. 37. The amino acid sequence indicated in SEQ ID NO. 15 corresponds to the 101st to 117th amino acid sequence indicated in SEQ ID NO. 37.

[0191] Sequence No. 6: GDFAIVKFTKVLLQ

[0192] Sequence No. 7: GDFAIVKFTKVLLQYT

[0193] Sequence No. 8: GDFAIVKFTKVLLQYTG

[0194] Sequence No. 9: DFAIVKFTKVLLQ

[0195] Sequence No. 10: DFAIVKFTKVLLQYT

[0196] Sequence No. 11: DFAIVKFTKVLLQYTG

[0197] Sequence No. 12: FAIVKFTKVLLQ

[0198] Sequence No. 13: FAIVKFTKVLLQYTG

[0199] Sequence No. 14: AIVKFTKVLLQYTG

[0200] Sequence No. 15: AIVKFTKVLLQYTGHIT

[0201] The inventors have confirmed that one or more of the polypeptides of (A) bind to HLA-DRB3*01:01 and HLA-DRB1*03:01 among human HLAs. Accordingly, a formulation according to one embodiment of the present disclosure is intended for use on a subject having an HLA-DRB3 genotype of 01:01 and / or an HLA-DRB1 genotype of 03:01, and the at least one polypeptide may be a formulation comprising one or more of the polypeptides of (A).

[0202] The amino acid sequence described in SEQ ID NO. 2 corresponds to the 110th to 124th amino acid sequence of the α1 subunit of the human acetylcholine receptor (SEQ ID NO. 36), whose Ref seq accession number is NP_000070.1 according to the National Center for Biotechnology Information (NCBI). Additionally, the amino acid sequence described in SEQ ID NO. 2 corresponds to the 90th to 104th amino acid sequence of the amino acid sequence (SEQ ID NO. 37), excluding the 1st to 20th amino acid sequences corresponding to the signal peptide from the amino acid sequence shown in SEQ ID NO. 36.

[0203] Sequence No. 2: LVLYNNADGDFAIVK

[0204] In one embodiment of the present disclosure, the total length of one or more polypeptides of (B) may be, for example, 12 residues or more, 13 residues or more, 14 residues or more, 15 residues or more, 16 residues or more, 17 residues or more, or 18 residues or more, and may be 21 residues or less, 20 residues or more, 19 residues or less, 18 residues or less, 17 residues or less, 16 residues or less, or 15 residues or less. Additionally, in one embodiment of the present disclosure, the total length of one or more polypeptides of (B) may be, for example, 12 residues or more and 21 residues or less, 13 residues or more and 21 residues or less, 14 residues or more and 21 residues or less, 15 residues or more and 21 residues or less, 15 residues or more and 19 residues or less, 15 residues or more and 17 residues or less, 15 residues or more and 16 residues or less, or 15 residues. In a preferred embodiment, the total length of one or more polypeptides of (B) may be 15 residues or more and 16 residues or less.

[0205] In one embodiment of the present disclosure, one or more polypeptides of (B) comprises an amino acid sequence having sequence identity of 90% or more, 93% or more, 95% or more, 98% or more, or 100% with respect to the amino acid sequence described in SEQ ID NO. 2. In one embodiment of the present disclosure, one or more polypeptides of (B) comprises an amino acid sequence described in SEQ ID NO. 2 or an amino acid sequence having a variation of one residue, two residues, or three residues in said amino acid sequence. In a preferred embodiment, one or more polypeptides of (B) comprises an amino acid sequence having sequence identity of 93% or more with respect to the amino acid sequence described in SEQ ID NO. 2.

[0206] In one embodiment of the present disclosure, the polypeptide of (B) or a plurality thereof may comprise an amino acid sequence having at least 90% sequence identity with at least one selected from the group consisting of the 87th to 107th, 88th to 107th, 89th to 107th, 90th to 107th, 87th to 106th, 88th to 106th, 89th to 106th, 90th to 106th, 87th to 105th, 88th to 105th, 89th to 105th, 90th to 105th, 87th to 104th, 88th to 104th, 89th to 104th, and 90th to 104th, and the amino acid having at least 90% sequence identity with said at least one. It can be composed of a sequence and can be composed of at least one amino acid sequence.

[0207] In one embodiment of the present disclosure, the one or more polypeptides of (B) may be one or more polypeptides having a sequence identity of 90% or more with an amino acid sequence selected from the group consisting of the amino acid sequences described in (B') SEQ ID NO. 16, and may be a polypeptide consisting of the amino acid sequence described in SEQ ID NO. 16. The amino acid sequence indicated in SEQ ID NO. 16 corresponds to the 90th to 104th amino acid sequence indicated in SEQ ID NO. 37.

[0208] Sequence No. 16: LVLYNNADGDFAIVK

[0209] The inventors have confirmed that one or more polypeptides of (B) bind to HLA-DRB3*01:01 among human HLAs. Accordingly, a formulation according to one embodiment of the present disclosure is intended for use on a target having an HLA-DRB3 genotype of 01:01, and the at least one polypeptide may be a formulation comprising one or more polypeptides of (B).

[0210] The amino acid sequence described in SEQ ID NO. 4 corresponds to the 334th to 343rd amino acid sequence of the α1 subunit of the human acetylcholine receptor (SEQ ID NO. 36), whose Ref seq accession number is NP_000070.1 according to the National Center for Biotechnology Information (NCBI). Additionally, the amino acid sequence described in SEQ ID NO. 4 corresponds to the 314th to 323rd amino acid sequence (SEQ ID NO. 37), excluding the 1st to 20th amino acid sequences corresponding to the signal peptide from the amino acid sequence shown in SEQ ID NO. 36.

[0211] Sequence No. 4: KVFIDTIPNI

[0212] In one embodiment of the present disclosure, the total length of one or more polypeptides of (D) may be, for example, 7 residues or more, 8 residues or more, 9 residues or more, 10 residues or more, 11 residues or more, 12 residues or more, 13 residues or more, or 14 residues or more, and may be 17 residues or less, 16 residues or more, 15 residues or less, 14 residues or less, 13 residues or less, 12 residues or less, or 11 residues or less. Additionally, in one embodiment of the present disclosure, the total length of one or more polypeptides of (D) may be, for example, 7 residues or more and 17 residues or less, 8 residues or more and 17 residues or less, 9 residues or more and 17 residues or less, 10 residues or more and 17 residues or less, 11 residues or more and 16 residues or less, 11 residues or more and 14 residues or less, 11 residues or more and 13 residues or less, 11 residues or more and 12 residues or less, or 11 residues. In a preferred embodiment, the total length of one or more polypeptides of (D) may be 11 residues or more and 16 residues or less.

[0213] In one embodiment of the present disclosure, one or more polypeptides of (D) comprises an amino acid sequence having sequence identity of 90% or more, 95% or more, 98% or more, or 100% with the amino acid sequence described in SEQ ID NO. 4. In one embodiment of the present disclosure, one or more polypeptides of (D) comprises an amino acid sequence described in SEQ ID NO. 4 or an amino acid sequence having a variation of one residue, two residues, or three residues in said amino acid sequence.

[0214] In one embodiment of the present disclosure, one or more polypeptides of (D) may comprise an amino acid sequence having 90% or more sequence identity with at least one selected from the group consisting of the 311th to 327th, 312th to 327th, 313th to 327th, 314th to 327th, 311th to 326th, 312th to 326th, 313th to 326th, 314th to 326th, 311th to 325th, 312th to 325th, 313th to 325th, 314th to 325th, 311th to 324th, 312th to 324th, 313th to 324th, and 314th to 324th. There is, and it may be composed of an amino acid sequence having 90% or more sequence identity with at least one of the above, and may be composed of at least one amino acid sequence. For example, the 311th to 327th amino acid sequences shown in SEQ ID NO. 37 are amino acid sequences shown in SEQ ID NO. 17. In one embodiment of the present disclosure, one or more polypeptides of (D) may include an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO. 17, may be composed of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO. 17, and may be composed of said amino acid sequence.

[0215] Sequence No. 17: WVRKVFIDTIPNIMFFS

[0216] In one embodiment of the present disclosure, the one or more polypeptides of (D) may be one or more polypeptides having a sequence identity of 90% or more with an amino acid sequence selected from the group consisting of amino acid sequences described in (D') SEQ ID NOs 22 to 32, and may be one or more polypeptides consisting of an amino acid sequence selected from the group consisting of amino acid sequences described in SEQ ID NOs 22 to 32. The amino acid sequence indicated in SEQ ID NO 22 corresponds to the 311th to 323rd amino acid sequence indicated in SEQ ID NO 37. The amino acid sequence indicated in SEQ ID NO 23 corresponds to the 311th to 324th amino acid sequence indicated in SEQ ID NO 37. The amino acid sequence indicated in SEQ ID NO 24 corresponds to the 312th to 324th amino acid sequence indicated in SEQ ID NO 37. The amino acid sequence indicated in SEQ ID NO 25 corresponds to the 312th to 325th amino acid sequence indicated in SEQ ID NO 37. The amino acid sequence indicated in SEQ ID NO. 26 corresponds to the 312th to 327th amino acid sequence indicated in SEQ ID NO. 37. The amino acid sequence indicated in SEQ ID NO. 27 corresponds to the 313th to 324th amino acid sequence indicated in SEQ ID NO. 37. The amino acid sequence indicated in SEQ ID NO. 28 corresponds to the 313th to 325th amino acid sequence indicated in SEQ ID NO. 37. The amino acid sequence indicated in SEQ ID NO. 29 corresponds to the 313th to 326th amino acid sequence indicated in SEQ ID NO. 37. The amino acid sequence indicated in SEQ ID NO. 30 corresponds to the 313th to 327th amino acid sequence indicated in SEQ ID NO. 37. The amino acid sequence indicated in SEQ ID NO. 31 corresponds to the 314th to 324th amino acid sequence indicated in SEQ ID NO. 37. The amino acid sequence indicated in SEQ ID NO. 32 corresponds to the 314th to 326th amino acid sequence indicated in SEQ ID NO. 37.

[0217] Sequence No. 22: WVRKVFIDTIPNI

[0218] Sequence No. 23: WVRKVFIDTIPNIM

[0219] Sequence No. 24: VRKVFIDTIPNIM

[0220] Sequence No. 25: VRKVFIDTIPNIMF

[0221] Sequence No. 26: VRKVFIDTIPNIMFFS

[0222] Sequence No. 27: RKVFIDTIPNIM

[0223] Sequence No. 28: RKVFIDTIPNIMF

[0224] Sequence No. 29: RKVFIDTIPNIMFF

[0225] Sequence No. 30: RKVFIDTIPNIMFFS

[0226] Sequence No. 31: KVFIDTIPNIM

[0227] Sequence No. 32: KVFIDTIPNIMFF

[0228] The inventors have confirmed that one or more of the polypeptides of (D) bind to HLA-DRB3*01:01 and HLA-DRB1*09:01 among human HLAs. Accordingly, a formulation according to one embodiment of the present disclosure is intended for use on a subject having an HLA-DRB3 genotype of 01:01 and / or an HLA-DRB1 genotype of 09:01, and the at least one polypeptide may be a formulation comprising one or more of the polypeptides of (D).

[0229] The amino acid sequence described in 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 is NP_000070.1 according to the National Center for Biotechnology Information (NCBI). Additionally, the amino acid sequence described in SEQ ID NO. 5 corresponds to the 145th to 157th amino acid sequence (SEQ ID NO. 37), excluding the 1st to 20th amino acid sequences corresponding to the signal peptide from the amino acid sequence shown in SEQ ID NO. 36.

[0230] Sequence No. 5: KLGTWTYDGSVVA

[0231] In one embodiment of the present disclosure, the total length of one or more polypeptides of (E) may be, for example, 10 residues or more, 11 residues or more, 12 residues or more, 13 residues or more, 14 residues or more, 15 residues or more, or 16 residues or more, and may be 19 residues or less, 18 residues or more, 17 residues or less, 16 residues or less, 15 residues or less, 14 residues or less, or 13 residues or less. Additionally, in one embodiment of the present disclosure, the total length of one or more polypeptides of (E) may be, for example, 10 residues or more and 19 residues or less, 11 residues or more and 19 residues or less, 12 residues or more and 19 residues or less, 13 residues or more and 19 residues or less, 13 residues or more and 17 residues or less, 13 residues or more and 15 residues or less, 13 residues or more and 14 residues or less, or 13 residues. In a preferred embodiment, the total length of one or more polypeptides of (E) may be 13 residues or more and 17 residues or less.

[0232] In one embodiment of the present disclosure, one or more polypeptides of (E) comprises an amino acid sequence having sequence identity of 90% or more, 92% or more, 95% or more, 98% or more, or 100% with respect to the amino acid sequence described in SEQ ID NO. 5. In one embodiment of the present disclosure, one or more polypeptides of (E) comprises an amino acid sequence described in SEQ ID NO. 5 or an amino acid sequence having a variation of one residue, two residues, or three residues in said amino acid sequence. In a preferred embodiment, one or more polypeptides of (E) comprises an amino acid sequence having sequence identity of 92% or more with respect to the amino acid sequence described in SEQ ID NO. 5.

[0233] In one embodiment of the present disclosure, one or more polypeptides of (E) are 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, 142nd to 158th, 143rd to 158th, 144th to 158th, 145th to 158th, 142nd to 157th, 143rd to 158th It may include an amino acid sequence having at least 90% sequence identity with at least one selected from the group consisting of the 157th, 144th to 157th and 145th to 157th, and may be composed of an amino acid sequence having at least 90% sequence identity with said at least one, and may be composed of said at least one amino acid sequence.

[0234] In one embodiment of the present disclosure, the one or more polypeptides of (E) may be one or more polypeptides having a sequence identity of 90% or more with an amino acid sequence selected from the group consisting of amino acid sequences described in (E') SEQ ID NOs 33 to 35, and may be one or more polypeptides consisting of an amino acid sequence selected from the group consisting of amino acid sequences described in SEQ ID NOs 33 to 35. The amino acid sequence indicated in SEQ ID NO. 33 corresponds to the 145th to 157th amino acid sequence indicated in SEQ ID NO. 37. The amino acid sequence indicated in SEQ ID NO. 34 corresponds to the 145th to 160th amino acid sequence indicated in SEQ ID NO. 37. The amino acid sequence indicated in SEQ ID NO. 35 corresponds to the 145th to 161st amino acid sequence indicated in SEQ ID NO. 37.

[0235] Sequence No. 33: KLGTWTYDGSVVA

[0236] Sequence No. 34: KLGTWTYDGSVVAINP

[0237] Sequence No. 35: KLGTWTYDGSVVAINPE

[0238] The inventors have confirmed that one or more polypeptides of (E) bind to HLA-DRB3*01:01 and HLA-DRB1*03:01 among human HLAs. Accordingly, a formulation according to one embodiment of the present disclosure is intended for use on a subject having an HLA-DRB3 genotype of 01:01 and / or an HLA-DRB1 genotype of 03:01, and the at least one polypeptide may be a formulation comprising one or more polypeptides of (E).

[0239] At least one polypeptide selected from the group consisting of (A), (B), (D), and (E) binds to HLA-DR. Therefore, although not wishing to be bound by any theory, by bringing at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) into contact with an antigen-presenting cell having an action that promotes the activation (differentiation) of regulatory T cells as described in Non-Patent Literature 1, and causing it to be phagocytosed, the at least one polypeptide bound to HLA-DR, and Naive CD4 + It is thought that it is presented to T cells and can consequently lead to the activation (differentiation) of regulatory T cells.

[0240] A formulation according to one embodiment may additionally contain one or more types of HLA-DRs in addition to at least one polypeptide selected from the group consisting of (A), (B), (D), and (E), and it is preferable that the HLA-DR is capable of binding to at least one polypeptide selected from the group consisting of (A), (B), (D), and (E), and it is also preferable that the genotype of the HLA-DR matches the genotype of the HLA-DR of the subject being administered. A formulation according to one embodiment may additionally contain an additive commonly used in the field of formulation technology in addition to at least one polypeptide selected from the group consisting of (A), (B), (D), and (E), and the additive may be, for example, at least one selected from the group consisting of excipients, buffers, stabilizers, antioxidants, binders, disintegrants, fillers, emulsifiers, and flow rate regulators.

[0241] The component contained in the formulation according to one embodiment may be encapsulated in a drug delivery vehicle intended for delivery into a cell. Examples of such drug delivery vehicles include metal nanoparticles (e.g., gold nanoparticles), liposomes, and capsids.

[0242] The formulation of the preparation 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 preparation according to one embodiment is contained in a drug delivery vehicle intended for delivery into a cell, the formulation is preferably a liquid.

[0243] The form of administration of the preparation according to one embodiment is not particularly limited and may include, for example, intramedullary administration, intravenous injection, subcutaneous injection, intramuscular injection, oral administration, or spinal injection. As an example of a specific dosage, for example, when administered to an adult human male (weight 60 kg), the daily dosage of the preparation may be 0.001 μg to 1000 mg / day / person in terms of the amount of the active ingredient.

[0244] In a formulation according to one embodiment, the content of at least one polypeptide selected from the group consisting of (A), (B), (D) and (E) is not particularly limited, and, for example, the sum of the contents may be 0.001 to 100 mass% based on the total amount of the formulation.

[0245] The subject of administration of the preparation according to one embodiment may be, for example, a human, or a human who has developed an autoimmune disease, is likely to have developed it, or has genetic factors that make one susceptible to developing it. The autoimmune disease may be, for example, an autoimmune disease caused by the overproduction of antibodies against human AChRα1 or its peptide fragments, and it is preferably myasthenia gravis, and more preferably early-onset myasthenia gravis. Meanwhile, myasthenia gravis is broadly classified into early-onset myasthenia gravis, which develops in individuals aged 10 to 49 years, and late-onset myasthenia gravis, which develops in individuals aged 50 years or older, and the genotypes of HLA that can be risk factors differ in some respects between the two.

[0246] At least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above is identified by the NAPA method using dendritic cells prepared from human-derived classical monocytes, and since it is highly likely to actually exist in the body of a patient suffering from an autoimmune disease, it is more likely to be applied to the treatment or diagnosis of autoimmune diseases than polypeptides with sequences corresponding to epitopes identified so far. Here, the fact that at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) is more likely to be applied to the treatment or diagnosis of autoimmune diseases than polypeptides with sequences corresponding to epitopes identified so far (other polypeptides) can be evaluated, for example, by the fact that the at least one polypeptide exhibits a higher T-cell activation effect when in contact with T cells than the other polypeptides. Such T-cell activation effect can be evaluated, for example, by a T-cell activation test. A T-cell activation test is a test that confirms whether T cells present in PBMCs are activated by exposure to polypeptides using an ELISA such as IFN-γ. When a polypeptide with a sequence corresponding to an identified epitope is added to human PBMCs, it is presented as an epitope by antigen-presenting cells within the PBMCs. T cells equipped with a TCR capable of capturing this HLA-binding polypeptide receive the signal and release activating cytokines such as IFN-γ. Activation is determined by measuring these released cytokines. Such an assay can be performed using commercially available kits, for example, using the BD OptEIA (Trademark) Human IFN-γ ELISA Set (BD Biosciences, Cat. No. 555142).

[0247] <Inhibitor of effector T cell activity or composition for inhibiting activity>

[0248] A second phase of the present disclosure relates to an inhibitor of effector T cell activity comprising at least one polypeptide selected from the group consisting of (A), (B), (D), and (E). In one embodiment, the second phase of the present disclosure may be a composition for inhibiting the activity of effector T cells comprising at least one polypeptide. As an inhibitor of effector T cell activity according to the second phase of the present disclosure, the same as that described in the activator of regulatory T cells according to the first phase of the present disclosure may be used, and the description and definitions described in the section on the activator of regulatory T cells according to the first phase of the present disclosure are also applied to the inhibitor of effector T cell activity according to the second phase of the present disclosure.

[0249] Although we do not wish to be bound by any theory, it is believed that an effector T cell activity 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 because it promotes the activation (differentiation) of regulatory T cells through the action of antigen-presenting cells as described in Non-Patent Document 1, as described in the first aspect of the present disclosure.

[0250] <B세포의 활성화 저해제 또는 활성화 저해용 조성물>

[0251] The third phase 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). In one embodiment, the third phase of the present disclosure may be a composition for inhibiting B cell activation comprising at least one polypeptide. As the inhibitor of B cell activation according to the third phase of the present disclosure, the same as that described in the regulatory T cell activator according to the first phase of the present disclosure may be used, and the description and definitions described in the section on the regulatory T cell activator according to the first phase of the present disclosure are also applied to the inhibitor of B cell activation according to the third phase of the present disclosure.

[0252] Although we do not wish 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 activation of effector T cells through the action of antigen-presenting cells as described in Non-Patent Document 1, as described in the second phase of the present disclosure, and thus can inhibit the activation of B cells accordingly.

[0253] <Inhibitors of humoral immunity or compositions for inhibition>

[0254] A fourth phase of the present disclosure relates to an inhibitor of humoral immunity (e.g., an antibody production inhibitor) comprising at least one polypeptide selected from the group consisting of (A), (B), (D), and (E). In one embodiment, a fourth phase of the present disclosure may be a composition for inhibiting humoral immunity comprising at least one polypeptide. As an inhibitor of humoral immunity according to the fourth phase of the present disclosure, the same as that described in the regulatory T cell activator according to the first phase of the present disclosure may be used, and the description and definitions described in the item regarding the regulatory T cell activator according to the first phase of the present disclosure are also applied to the inhibitor of humoral immunity according to the fourth phase of the present disclosure.

[0255] Although we do not wish to be bound by any theory, it is believed that an inhibitor of humoral immunity comprising at least one polypeptide selected from the group consisting of (A), (B), (D) and (E) above can inhibit the activation of B cells through the action of antigen-presenting cells as described in Non-Patent Document 1, as described in the third phase of the present disclosure, and thus can inhibit humoral immunity accordingly.

[0256] <Therapeutic agent, preventive agent, therapeutic composition or preventive composition for autoimmune diseases>

[0257] A fifth aspect of the present disclosure relates to a therapeutic agent or prophylactic agent for an autoimmune disease comprising at least one polypeptide selected from the group consisting of (A), (B), (D), and (E). A fifth aspect of the present disclosure may, in one embodiment, be a composition for treating or prophylacticating an autoimmune disease comprising the at least one polypeptide. A fifth aspect of the present disclosure may, in one embodiment, be a method for treating or prophylacticating an autoimmune disease comprising administering the at least one polypeptide to a subject in need thereof. A fifth aspect of the present disclosure may, in one embodiment, be the at least one polypeptide for use in the treatment or prevention of an autoimmune disease. A fifth aspect of the present disclosure may, in one embodiment, be the use of the at least one polypeptide in the manufacture of a therapeutic agent, prophylactic agent, therapeutic composition, or prophylactic composition for an autoimmune disease. In one embodiment, the fifth aspect of the present disclosure may be at least one polypeptide for use in the manufacture of a therapeutic agent, a prophylactic agent, a therapeutic composition, or a prophylactic composition for an autoimmune disease. As a therapeutic agent or prophylactic agent for an autoimmune disease according to the fifth aspect of the present disclosure, the same as that described in the regulatory T cell activator according to the first aspect of the present disclosure may be used, and the description and definitions described in the item regarding the regulatory T cell activator according to the first aspect of the present disclosure are also applied to the therapeutic agent or prophylactic agent for an autoimmune disease according to the fifth aspect of the present disclosure.

[0258] The autoimmune disease according to the fifth phase of the present disclosure may be, for example, an autoimmune disease caused by the overproduction of antibodies against human AChRα1 or its peptide fragments, 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) is a peptide fragment comprising an amino acid sequence corresponding to an epitope of human AChRα1 known to be recognized as an autoantigen in myasthenia gravis and early-onset myasthenia gravis, according to a formulation according to one embodiment of the fifth phase of the present disclosure, myasthenia gravis and early-onset myasthenia gravis can preferably be treated and prevented.

[0259] A therapeutic agent or prophylactic agent according to one embodiment of the fifth phase of the present disclosure may be intended for administration to a human, for example, a human who has developed an autoimmune disease, is likely to have developed it, or has genetic factors that make him / her susceptible to developing it. The autoimmune disease may be, for example, an autoimmune disease caused by the overproduction of antibodies against human AChRα1 or peptide fragments thereof, preferably myasthenia gravis, and more preferably early-onset myasthenia gravis. Furthermore, if the agent according to one embodiment of the fifth phase of the present disclosure is a therapeutic agent, the subject of administration may be, for example, a human who has developed an autoimmune disease or is likely to have developed it. Furthermore, if the agent according to one embodiment of the fifth phase of the present disclosure is a prophylactic agent, the subject of administration may be a human who is likely to have developed an autoimmune disease or has genetic factors that make him / her susceptible to developing it.

[0260] Although we do not wish to be bound by any theory, it is believed that an autoimmune disease treatment or preventive agent comprising at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) above can suppress humoral immunity through the action of antigen-presenting cells as described in Non-Patent Document 1, as described in the fourth aspect of the present disclosure, and thus can treat and prevent autoimmune diseases, and, for example, by suppressing the production of anti-AChRα1 antibodies, it can suppress the degradation of AChRα1 and thus treat and prevent myasthenia gravis, such as early-onset myasthenia gravis.

[0261] <Diagnostic assistance methods for autoimmune diseases, methods for acquiring data for diagnosis, diagnostic methods>

[0262] A sixth phase of the present disclosure is 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), (B), (D), and (E). In one embodiment, the sixth phase of the present disclosure may be a method for acquiring data for 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), (B), (D), and (E). In one embodiment, the sixth phase of the present disclosure may be a method for diagnosing an 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). In such cases, the autoimmune disease may be, for example, an autoimmune disease caused by the overproduction of antibodies against human AChRα1 or its peptide fragments, preferably myasthenia gravis, and more preferably early-onset myasthenia gravis. In such cases, the at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) may be the same as that described in the regulatory T cell activator according to the first phase of the present disclosure, and the description and definition of the at least one polypeptide described in the regulatory T cell activator according to the first phase of the present disclosure are also applied to the at least one polypeptide in the method according to the sixth phase of the present disclosure.

[0263] In subjects afflicted with an autoimmune disease (e.g., an autoimmune disease caused by the overproduction of antibodies against human AChRα1 or its peptide fragments, preferably myasthenia gravis, more preferably early-onset myasthenia gravis), it becomes easier to produce T cells having antigen specificity for at least one polypeptide selected from the group consisting of (A), (B), (D), and (E), that is, T cells expressing antibodies on their cell surfaces that recognize at least one polypeptide selected from the group consisting of (A), (B), (D), and (E). Accordingly, by using the amount 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, acquire data for diagnosis, or perform diagnosis.

[0264] The subject to which the method according to one embodiment of the sixth phase of the present disclosure is utilized may be, for example, a human, or a human who has a possibility of being afflicted with an autoimmune disease or who has genetic factors that make him susceptible to such disease, and the said autoimmune disease may be, for example, an autoimmune disease caused by the overproduction of antibodies against human AChRα1 or its peptide fragments, preferably myasthenia gravis, and more preferably early-onset myasthenia gravis. In addition, the subject to which the method according to one embodiment of the sixth phase of the present disclosure is utilized may be a human who does not have a possibility of being afflicted with such an autoimmune disease or who has genetic factors that make him susceptible to such disease, and in that case, the method according to one embodiment may be a method for comprehensive exploration of the possibility of disease affliction (so-called screening test).

[0265] In one embodiment of the sixth phase 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) are detected in a sample taken from a subject, the subject may be assisted in diagnosis, data acquisition for diagnosis, or diagnosis may be performed, in which case the subject is highly likely to be suffering from an autoimmune disease (e.g., an autoimmune disease caused by the overproduction of antibodies against human AChRα1 or its peptide fragments, preferably myasthenia gravis, more preferably early-onset myasthenia gravis). In addition, in one embodiment of the sixth phase of the present disclosure, in a sample taken from a subject, 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) is greater than that of a sample taken from a control group (e.g., a normal person) that is not affected by an autoimmune disease (e.g., an autoimmune disease caused by the overproduction of antibodies against human AChRα1 or its peptide fragments, preferably myasthenia gravis, more preferably early-onset myasthenia gravis), then the subject may be assisted in diagnosis, data acquisition for diagnosis, or diagnosis may be performed, such that the subject is highly likely to be affected by said autoimmune disease.

[0266] 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) is not limited to any sample that can be obtained from a subject and can assist in the diagnosis of autoimmune disease, obtain data for diagnosis, or perform 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), and may be, for example, bone marrow fluid, cerebrospinal fluid, lymph fluid, blood, plasma, serum, or saliva, and in a preferred embodiment, may be bone marrow fluid, and said bone marrow fluid may be, for example, collected from an ilium or sternum.

[0267] 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 said T cells, but may be the intensity of a signal that can be used as an indicator of said T cell quantity. For example, when said T cells are labeled with a polypeptide, said quantity may be, for example, the intensity of a signal that can evaluate the quantity of said labeled polypeptide, and for example, when the polypeptide is fluorescently labeled, it may be the fluorescence intensity detected from the T cells. Furthermore, when said T cells are labeled with a polypeptide-human leukocyte type antigen complex, said quantity may be, for example, the intensity of a signal that can evaluate the quantity of said labeled complex, and for example, when the human leukocyte type antigen complex is fluorescently labeled, it may be the fluorescence intensity detected from the T cells. In such cases, the fluorescence intensity may be, for example, the fluorescence intensity measured by a microwell plate reader or a flow cytometer.

[0268] The detection method for detecting T cells having antigen specificity for at least one polypeptide selected from the group consisting of (A), (B), (D) and (E) is not particularly limited as long as it is a method capable of detecting said T cells, and can be performed, for example, by labeling said T cells with a fluorescently labeled polypeptide or a complex of a polypeptide and a fluorescently labeled human leukocyte type antigen, and then measuring the fluorescence intensity of said fluorescent dye in the cells included in the sample.

[0269] In a preferred embodiment, detecting T cells having antigen specificity for at least one polypeptide selected from the group consisting of (A), (B), (D), and (E) can be performed by labeling the T cells with a multimer (e.g., a tetramer) of a complex of a fluorescently labeled polypeptide and a human leukocyte type antigen (polypeptide-human leukocyte type antigen complex), and then measuring the fluorescence intensity of the fluorescent dye in the cells included in the sample. When the T cells are labeled with a multimer of the polypeptide-human leukocyte type antigen complex, the complex and the T cells can form a stable bond, so more accurate detection can be performed by removing the non-specifically bound complex through washing operations, etc. Such a polypeptide-human leukocyte type antigen complex can be prepared, for example, by using at least one polypeptide selected from the group consisting of (A), (B), (D) and (E) above, and a commercially available kit (e.g., Quick Switch (registered trademark) series, MBLlife science) that can be used to prepare a multimer of the polypeptide-human leukocyte type antigen complex as described above.

[0270] Diagnostic kit for autoimmune diseases

[0271] A seventh aspect of the present disclosure is a kit for diagnosing autoimmune diseases, comprising at least one polypeptide selected from the group consisting of (A), (B), (D), and (E), and a human leukocyte type antigen 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 human leukocyte type antigen. In one embodiment, the kit according to the seventh aspect of the present disclosure may comprise the human leukocyte type antigen and the labeling dye as a complex, may comprise the human leukocyte type antigen and the labeling dye as a complex, may comprise a polypeptide, the human leukocyte type antigen and the labeling dye as a complex, may comprise a multimer of the human leukocyte type antigen and the labeling dye as a complex, and may comprise a multimer of the polypeptide-human leukocyte type antigen complex and the labeling dye as a complex. That is, the seventh aspect of the present disclosure may, in one embodiment, be a diagnostic agent for an autoimmune disease comprising a complex having a polypeptide, a human leukocyte type antigen, and a labeling dye. In this case, the labeling dye may be a fluorescent dye. In this case, 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.

[0272] A kit according to the seventh phase of the present disclosure may be used for the diagnosis of an autoimmune disease according to a method according to an embodiment of the sixth phase of the present disclosure. In one embodiment, the kit according to the seventh phase of the present disclosure may further include an attached document, said attached document may be digitized, and said attached document may contain a protocol corresponding to a method according to an embodiment of the sixth phase of the present disclosure.

[0273] <Variation Example>

[0274] The eighth aspect of the present disclosure relates to formulations and kits that, with respect to the formulations, methods, and kits described above, contain a molecule (hereinafter also referred to as "molecule according to the eighth aspect") comprising, as a partial structure, at least one polypeptide selected from the group consisting of (A), (B), (D), and (E), instead of at least one polypeptide selected from the group consisting of (A), (B), (D), and (E). Such formulations and kits may be carried out in the same manner as described above with respect to the formulations according to the first through fifth aspects and the kit according to the seventh aspect, except that they contain the 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.

[0275] The molecule according to the eighth phase may be, for example, a molecule (tandem molecule) formed by directly or indirectly linking at least two polypeptides selected from the group consisting of (A), (B), (D), and (E) by peptide bonds. The tandem molecule may be chain-shaped or ring-shaped. The tandem molecule may be a molecule (tandem molecule) formed by directly or indirectly linking, for example, two, three, four, or five or more polypeptides selected from the group consisting of (A), (B), (D), and (E) by peptide bonds. Tandem molecules in which at least two polypeptides are directly joined by peptide bonds include, for example, a polypeptide consisting of two consecutive amino acid sequences of the amino acid sequence represented by SEQ ID NO. 17 as shown in SEQ ID NO. 18, a polypeptide consisting of a consecutive amino acid sequence of the amino acid sequence represented by SEQ ID NO. 17 and the amino acid sequence represented by SEQ ID NO. 3 as shown in SEQ ID NO. 19, or a cyclic polypeptide having peptide bonds formed at both ends thereof. Tandem molecules in which at least two polypeptides are indirectly joined by peptide bonds may, for example, be a molecule in which at least two polypeptides are joined by a peptide linker. As the above peptide linker, examples disclosed in International Application No. 2019 / 233842, etc., may be used, and in detail, GGGS (SEQ No. 38), GGGGS (SEQ No. 39), and self-digesting peptides T2A (EGRGSLLTCGDVEENPGP (SEQ No. 40) and GSGEGRGSLLTCGDVEENPGP (SEQ No. 41)) may be examples.Tandem molecules in which at least two polypeptides are indirectly joined by peptide bonds include, for example, a polypeptide consisting of an amino acid sequence in which GGGS is inserted between two amino acid sequences represented by 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 represented by SEQ ID NO. 17 and the amino acid sequence represented by SEQ ID NO. 3, and between the amino acid sequence represented by SEQ ID NO. 3 and the amino acid sequence represented by SEQ ID NO. 17, as shown in SEQ ID NO. 21.

[0276] Sequence No. 18: WVRKVFIDTIPNIMFFSWVRKVFIDTIPNIMFFS

[0277] Sequence No. 19: WVRKVFIDTIPNIMFFSGDFAIVKFTKVLLQYTGHIT

[0278] Sequence No. 20: WVRKVFIDTIPNIMFFSGGGSWVRKVFIDTIPNIMFFS

[0279] Sequence No. 21: WVRKVFIDTIPNIMFFSGGGGSGDFAIVKFTKVLLQYTGHITGGGGSWVRKVFIDTIPNIMFFS

[0280] A molecule according to the eighth phase may be a molecule comprising, for example, at least one polypeptide selected from the group consisting of (A), (B), (D), and (E); and a non-peptide structure as a substructure. Such a molecule may be a molecule in which at least two polypeptides selected from the group consisting of (A), (B), (D), and (E) are joined through a bond other than a peptide bond, and such a molecule may additionally have a non-peptide structure at a peptide end not joined to a linker. Such a bond other than a peptide bond may be a covalent bond (e.g., a carbon-carbon bond) or a bond formed by a non-covalent interaction, such as the interaction between biotin and streptavidin.

[0281] Examples

[0282] In the following, the present disclosure will be described in more detail using examples, but the present disclosure is not limited to the following examples.

[0283] [Example 1: Specificity of human AChRα1-derived antigen peptides binding to HLA-DR]

[0284] <Process 1: Clinical Sample Collection>

[0285] The peripheral blood mononuclear cells (hereinafter referred to as PBMC) used in this example were frozen samples from normal individuals and were purchased from VERITAS Inc. The genotypes of the purchased PMBCs in terms of HLA are shown in Table 1 below.

[0286]

[0287] Process 2: CD14 + CD16 - Simple principle of Dangu

[0288] Frozen PBMC was rapidly thawed in a 37°C water bath and washed twice in RPMI-1640 medium containing 10% FBS (RPMI-1640 / 10% FBS medium). Using the EasySep Human Monocyte Isolation Kit (VERITAS, Cat.No.ST-19359), CD14 according to the kit's attached documentation + CD16 - The single sphere was isolated by negative selection. Accordingly, the classical single sphere was isolated from the PBMC.

[0289] <Process 3: Preparation of Monocyte-Derived Dendritic Cells and Addition of Human AChRα1>

[0290] Danrihan CD14 + CD16 - Monocytes, 1.5 x 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 Culture was started at cells / mL and cultured for 7 days in a 5% CO2 incubator at 37°C to produce monocyte-derived dendritic cells. Then, human AChRα1 was added at a concentration of 100 μg / well and cultured again for 24 hours. Meanwhile, the human AChRα1 used in this process was prepared by performing gene synthesis based on the sequence information of AChRα, inserting the synthesized gene into a transfer vector, co-transfecting the expression vector into insect cells (SF9 cells), and then performing production and purification.

[0291] <Process 4: Isolation of HLA-DR Binding Peptides>

[0292] Regarding the samples after culture, the procedure was performed according to the protocol for Dynabeads Protein G (Invitrogen, Cat. No. 10004D). 5 μg of anti-HLA-DR antibody was conjugated to the beads. 0.1% TFA was added to the solution containing the HLA-DR conjugated with the obtained peptide, and the peptide was released from the HLA-DR by reacting at room temperature for 1 minute. A Microcon (registered trademark) Centrifugal Filter Device (Merck, Cat. No. MRCPRT010) capable of separating particles of 10 kD or less was attached to a dedicated 1.5 mL tube, the solution containing the recovered peptide and HLA-DR was added to it, and the HLA-DR and peptide were separated by centrifuging at 14,000 kJ for 30 minutes. The solvent was removed from the peptide solution in the tube using a freeze dryer, and the sample was preserved as a freeze-dried sample.

[0293] <Process 5: LC-MS / MS Analysis>

[0294] Purified water was added to the obtained freeze-dried sample and stirred to prepare a sample for measurement. This was analyzed using LC-MS / MS under the conditions shown in Table 2 below.

[0295]

[0296] <Process 6: LC-MS / MS Spectrum Data Analysis>

[0297] For the analysis of spectral data obtained from LC-MS / MS analysis, Mascot Server ver. 2.8 (Matrix Science) was used, and the analysis was performed under the conditions shown in Table 3 below. For sequence searches of NCBI prot and AChRα1, peptides satisfying a False Discovery Rate (FDR) p < 0.01 were selected as search targets, while for searches using only AChRα1 sequences, FDR calculation was not performed. Additionally, Skyline Ver. 22.2 (MacCoss Lab.) was used for quantitative analysis. After introducing the spectral data obtained from LC-MS / MS analysis and the identification results obtained from Mascot Server, the peak area values ​​in the peptide extraction ion chromatograms were calculated.

[0298]

[0299] <Process 7: HLA Allelic Identification by Sample Comparison>

[0300] Comparisons were performed for each donor to identify HLA allelates regarding the LC-MS / MS analysis data obtained in process 6. The peptide commonly identified in samples with DRB1*03:01 was designated as the DRB1*03:01 binding peptide, and the peptide commonly identified in samples with DRB3*01:01 was designated as the DRB3*01:01 binding peptide.

[0301] Table 4 shows the results of representing the HLA-DRB1*03:01 binding peptides identified in this manner, along with their corresponding positions in the amino acid sequence indicated in SEQ ID NO. 37. Table 5 shows the results of representing the HLA-DRB3*01:01 binding peptides identified in this manner, along with their corresponding positions in the amino acid sequence indicated in SEQ ID NO. 37. Figure 1 shows the results of representing the HLA binding peptides identified in this manner as coordinates on human AChRα1. In Figure 1, the clusters of the identified DRB binding peptides are illustrated in boxes, and clusters indicated by diagonal lines represent those derived from DRB1*03:01, clusters indicated by dot hatching represent those derived from DRB3*01:01, and clusters indicated in black represent those derived from other HLAs. Meanwhile, in Figure 1, the light gray region in the row below the amino acid number represents the AChRα1 sequence, and the dark gray region represents the transmembrane region.

[0302]

[0303]

[0304] From these results, in the subject-derived sample with an HLA-DRB3 genotype of 01:01 and the subject-derived sample with an HLA-DRB1 genotype of 03:01, the peptides shown in SEQ ID NOs 6 to 15 were commonly identified and 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 furthermore, the region 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.

[0305] In addition, from these results, the peptide shown in SEQ ID NO. 16 (SEQ ID NO. 2) was commonly identified in samples derived from subjects with the HLA-DRB3 genotype 01:01. Therefore, the peptide having the amino acid sequence shown in SEQ ID NO. 2 is a DRB3*01:01 binding peptide, and it was also revealed that the region in human AChRα1 having the amino acid sequence shown in SEQ ID NO. 2 is an epitope for DRB3*01:01.

[0306] In addition, from these results, in the subject-derived samples with an HLA-DRB3 genotype of 01:01, the peptides shown in SEQ ID NOs 22 to 32 were commonly identified, and they shared the amino acid sequence shown in SEQ ID NO. 4. Therefore, the peptide having the amino acid sequence shown in SEQ ID NO. 4 is a DRB3*01:01 binding peptide, and it was also revealed that the region in human AChRα1 having the amino acid sequence shown in SEQ ID NO. 4 is an epitope for DRB3*01:01.

[0307] From these results, in the subject-derived sample with an HLA-DRB3 genotype of 01:01 and the subject-derived sample with an HLA-DRB1 genotype of 03:01, the peptides shown in SEQ ID NOs 33 to 35 were commonly identified and shared the amino acid sequence shown in SEQ ID NO. 5. Therefore, 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 it was also revealed that the region 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.

[0308] [Example 2: Evaluation of Specific T-Cell Induction Ability Using Peripheral Blood-Derived Mononuclear Cells (PBMCs) from Normal Individuals]

[0309] We examined whether the peptide containing the epitope identified in Example 1 has the effect of inducing specific T cells from normal-derived PBMCs.

[0310] Preparation of Culture Media

[0311] Human AB type serum (GeminiBio, Cat:100-512) and fetal bovine serum (Nichirei, Cat:175012) were used after inactivation treatment at 56°C for 30 minutes and filtered through a 0.2 μm filter. AIM-V (Gibco, Cat:12055-091) containing 5% human AB type serum was used as the culture medium for PBMCs. Cell culture was performed under 37°C - 5% CO2- water vapor saturation conditions. D-PBS(-) containing 2% fetal bovine serum was used as the staining buffer.

[0312] <List of PBMC origins from normal people>

[0313] The HLA alleles of PBMCs derived from normal individuals and the peptides evaluated used in the review of this example are shown in Table 6. For each peptide, six induction ability evaluation experiments were performed. Each PBMC was purchased from Precision for Medicine or STEMCELLS. Bold italics indicate the expected binding alleles of peptides consisting of the amino acid sequences listed in SEQ ID NOs. 3, 16, 35, and 17, and underlined indicates the expected binding alleles of peptides consisting of the amino acid sequence listed in SEQ ID NO. 17. Meanwhile, samples marked with ※ in Table 6 were evaluated in two independent experiments.

[0314]

[0315] <List of Evaluated Peptides>

[0316] The evaluation peptides used in the review are shown in Table 7. These peptides were dissolved in DMSO (ATCC, Cat:4-X) to a concentration of 20 mg / mL.

[0317]

[0318] Induction of Peptide-Specific T Cells Using Normally Derived PBMCs

[0319] PBMC stored in the liquid nitrogen gas phase was removed and rapidly thawed in a 37°C water bath. It was washed once with AIM-V, resuspended in AIM-V containing 5% human AB serum (hereinafter referred to as the complete medium), and 1.5 x 10 6 24-well plates were seeded at a rate of 1 / well. Culture was started by adding peptide at a final concentration of 20 μg / mL and IL-7 at a final concentration of 10 ng / mL (PeproTech, Cat:AF-200-07-50µg) (Day 0, total medium volume: 2 mL / well).

[0320] One week after the start of culture, PBMC derived from the same donor as the PBMC in culture is thawed in the same manner as on Day 0, washed once with AIM-V, and 3 x 10 6 The solution was prepared at a concentration of 1 / mL or less, and the same peptide used for induction on Day 0 was added at a concentration of 20 μg / mL and incubated for 2 hours. Then, a final concentration of 50 μg / mL of Mitomycin C solution was added, and the cells were incubated again for 45 minutes. Afterward, the cells were washed three times with AIM-V to form antigen-presenting cells. Cells cultured for one week from Day 0 were harvested, and 1 to 1.5 x 10 6The cells were seeded into a new 24-well plate within the / well range, and an equal number of prepared antigen-presenting cells were seeded. Culture was then resumed by adding IL-7 at a final concentration of 10 ng / mL (Day 7, total medium volume: 2 mL / well). After 2 days, half of the culture medium was exchanged with complete medium containing 40 U / L IL-2, and culture was continued for one week while further exchanging half of the medium with complete medium containing 20 U / mL IL-2. During this time, when the cells reached confluence, the 2 mL / well culture medium was thoroughly suspended, 1 mL of which was seeded into a new well, and 20 U / mL IL-2 complete medium was added to each well to a volume of 1 mL / well (hereinafter referred to as subculturing). On Day 14, the cultured cells were harvested, and the <Intracellular Cytokine Staining (ICS)> and described later<ELISA에 의한 IFN-γ 생산능 평가> Used for.

[0321] Intracellular Cytokine Staining (ICS)

[0322] Place the recovered cultured cells in a 2.0 x 10 96-well round-bottom plate. 62 to 6 wells were seeded one by one, and the peptide used for half-induction was added at a concentration of 20 μg / mL, while DMSO, a solvent, was added to the remaining half at a concentration of 0.1%, and the wells were incubated for 2 hours. Brefeldin A (BioLegend, Cat:420601) and Monensin (BioLegend, Cat:420701) at a final concentration of 1X were added, and the wells were incubated again for 4 hours. Cells were harvested, and Staining Buffer containing Fc Block (Miltenyi Biotec, Cat:130-059-901), 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, and the mixture was reacted at 4°C for 15 minutes. Cytofix Cytoperm Fix / Perm Solution (Beckton Dickinson, Cat:554714) was added, and the mixture was treated at 4°C for 20 minutes. Washed with Perm. / Wash Buffer (10X product enclosed in Cat: 554714, diluted 10-fold with distilled water), PE-labeled anti-human IFN-γ antibody (BioLegend, Cat: 502509) was added, reacted at 37°C for 30 minutes, and after washing with Perm. / Wash Buffer, washed with Staining Buffer and analyzed by FACS.

[0323] <ELISA에 의한 IFN-γ 생산능 평가>

[0324] Place the recovered cultured cells in a 1.0 x 10 96-well round-bottom plate. 5Six wells were seeded individually. 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 remaining half at a concentration of 0.1%. The wells were incubated for at least 16 hours. The culture supernatant was collected, appropriately diluted with an assay diluent, and prepared for ELISA analysis. IFN-γ concentration was measured using a BD OptEIA ELISA Set (human IFN-γ, Becton Dickinson, Cat: 555142) in accordance with the attached documentation. If the absorbance was lower than that of the blank well, the IFN-γ concentration was treated as 0.

[0325] <Result>

[0326] Figure 2 shows the results of ICS (top) and ELISA (bottom), which evaluated specific T-cell induction ability in six peptide-independent cycles using PBMCs derived from healthy individuals. Based on the ICS and ELISA results, samples in which peptide-specific T-cell induction was evident are indicated by solid lines, and other samples are indicated by dotted lines. Peptide-specific T-cell induction was confirmed in 1 out of 6 cases for SEQ ID NO. 16, 3 out of 6 cases for SEQ ID NOs. 3 and 35, and 1 out of 6 cases for SEQ ID NO. 17. It was revealed that helper T-cells possessing T-cell receptors (TCRs) that recognize HLA and each peptide complex exist in healthy individuals. In particular, regarding SEQ ID NO. 17, even from the ICS results, IFN-γ production upon peptide stimulation was observed in CD4 compared to the solvent control (solvent = DMSO). + It was found that the T-cell ratio was high and it has high potential.

[0327] [Example 3: Evaluation of Specific T-Cell Induction Ability Using Peripheral Blood-Derived Mononuclear Cells (PBMCs) from MG Patients]

[0328] Regarding the peptide represented by SEQ ID NO. 17, which was able to confirm peptide-specific T-cell induction in all examples of Example 2, we decided to perform an examination using PBMCs derived from MG to confirm the presence of peptide-specific Th1 cells among MG patients.

[0329] <List of PBMCs from Myasthenia Gravis Patients>

[0330] The HLA alleles of PBMCs derived from myasthenia gravis patients used in the review are shown in Table 8. The peptides evaluated were only those consisting of the amino acid sequence listed in SEQ ID NO. 17. All PBMCs were purchased from Eolas Bio. Bold italics are the expected binding alleles of SEQ ID NO. 17.

[0331]

[0332] <MG 환자 유래 PBMC를 이용한 펩티드 특이적 T세포의 유도>

[0333] Basically, induction was performed using the same method as in Example 2, <Induction of Peptide-Specific T Cells Using Normally Derived PBMCs>; however, since the objective was to amplify memory T cells or effector T cells within patient PBMCs, peptides and IL-7 were added on Day 0, and from two days later, the medium was changed to a complete medium containing IL-2 and subculture was performed, and the cells were prepared for ELISA analysis on Day 7. The ELISA analysis was performed according to Example 2.<ELISA에 의한 IFN-γ 생산 능력 평가> It was performed in the same way as.

[0334] <Result>

[0335] Figure 3 is a figure showing the results of an ELISA analysis evaluating the presence or absence of SEQ ID NO. 17-specific Th1 cells using 5 PBMC specimens derived from MG patients with DRB3*01:01, the expected binding allelin of SEQ ID NO. 17. As a result, the presence of SEQ ID NO. 17-specific Th1 cells was suggested for 3 out of 4 specimens. From the above results, SEQ ID NO. 17-specific Th1 cells are present in DRB3*01:01-positive MG patients, and their pathogenicity was suspected.

[0336] [Example 4: Examination of the possibility of additional restraint for other allelates]

[0337] In the results of the ICS of Example 2, the proportion of Sequence No. 17-specific Th1 cells (IFNγ-positive CD4 cells upon peptide stimulation and DMSO stimulation) + The T cell doubling rate was high, ranging from 0.70 to 7.47% (median: 2.3%). Although it is thought that normal individuals do not undergo priming by endogenous antigens, typically, naive T cells take 2 to 3 days to differentiate into Th1 cells upon antigen stimulation, and subsequently receive additional stimulation and clonal amplify in the presence of IL-2. The doubling time for activated T cells is approximately 6 to 8 hours, and one cell amplifies to approximately 1 million over the 5 days from Day 9, when IL-2 is added, to Day 14, when cells are harvested. On the other hand, TCR diversity is 10 8 to 10 9 It is thought that, if there is only one Naive T cell recognizing the HLA-peptide complex, the abundance is 10 -9 to 10 -8It is %, and it is thought to be about 0.1 to 1% after receiving the above clone amplification. Since the experimental value obtained in Example 2 was higher than this theoretical value, additional binding of SEQ ID NO. 17 to other allelates was suspected. Therefore, the specific Th1 cell induction ability of SEQ ID NO. 17 was evaluated using normal human-derived PBMCs that have HLA-DRB1*09:01, which has been reported to be associated with MG in Asians, and do not have DRB1*03:01 or DRB3*01:01.

[0338] <List of PBMC origins from normal people>

[0339] The HLA alleles of PBMC derived from normal individuals used in the review of Example 4 are as shown in Table 9. The peptides evaluated were only those consisting of the amino acid sequence listed in SEQ ID NO. 17. Bold italics indicate alleles reported to be associated with the development of myasthenia gravis in Asians.

[0340]

[0341] <Result>

[0342] Figure 4 shows the results of ICS (left) and ELISA (right) evaluating the specific Th1 cell induction ability of SEQ ID NO. 17 using normal-derived PBMCs that possess HLA-DRB1*09:01, which has been reported to be associated with MG in Asians, and do not possess DRB1*03:01 or DRB3*01:01. As shown in Figure 4, SEQ ID NO. 17-specific Th1 cells were induced in 7 out of 9 samples provided for the ICS analysis (left, solid line). Of the 9 samples provided for the ICS analysis, 5 samples (4 samples with SEQ ID NO. 17-specific Th1 cell induction, 1 sample without induction) were provided for the ELISA analysis; similarly, for the 4 samples in which induction was confirmed in the ICS, production of SEQ ID NO. 17-specific Th1 cell-derived IFNγ was detected (right, solid line). From the above results, it was found that Sequence No. 17 is bound to HLA-DRB1*09:01 in addition to the binding allel (DRB1*03:01, DRB3*01:01) that was originally assumed.

Claims

Claim 1 A therapeutic agent or preventive agent for an autoimmune disease comprising at least one polypeptide selected from the group consisting of: (D) one or more polypeptides having an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4 and a total length of 7 or more and 17 or less; (A) one or more polypeptides having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1 and a total length of 8 or more and 20 or less; (B) one or more polypeptides having 12 or more and 21 or less sequence identity with the amino acid sequence described in SEQ ID NO. 2 and a total length of 12 or more and 21 or less sequence identity with the amino acid sequence described in SEQ ID NO. 5 and a total length of 10 or more and 19 or less sequence identity with the amino acid sequence described in SEQ ID NO.

5. Claim 2 (D) one or more polypeptides having an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4 and a total length of 7 or more and 17 or less; (A) one or more polypeptides having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1 and a total length of 8 or more and 20 or less; (B) one or more polypeptides having 12 or more and 21 or less sequence identity with the amino acid sequence described in SEQ ID NO. 2 and a total length of 12 or more and 21 or less; and (E) one or more polypeptides having 10 or more and 19 or less sequence identity with the amino acid sequence described in SEQ ID NO. 5; comprising at least one polypeptide selected from the group consisting of Claim 3 (D) one or more polypeptides having an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4 and a total length of 7 or more and 17 or less; (A) one or more polypeptides having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1 and a total length of 8 or more and 20 or less; (B) one or more polypeptides having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 2 and a total length of 12 or more and 21 or less; and (E) one or more polypeptides having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 5 and a total length of 10 or more and 19 or less; an inhibitor of effector T cell activity comprising at least one polypeptide selected from the group consisting of these. Claim 4 (D) one or more polypeptides having an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4 and a total length of 7 or more and 17 or fewer residues; (A) one or more polypeptides having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1 and a total length of 8 or more and 20 or fewer residues; (B) one or more polypeptides having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 2 and a total length of 12 or more and 21 or fewer residues; and (E) one or more polypeptides having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 5 and a total length of 10 or more and 19 or fewer residues; an inhibitor of B cell activation comprising at least one polypeptide selected from the group consisting of these. Claim 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 having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4 and a total length of 7 or more and 17 or less; (A) one or more polypeptides having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1 and a total length of 8 or more and 20 or less; (B) one or more polypeptides having 12 or more and 21 or less sequence identity with the amino acid sequence described in SEQ ID NO. 2 and a total length of 12 or more and 21 or less sequence identity with the amino acid sequence described in SEQ ID NO. 5 and a total length of 10 or more and 19 or less sequence identity with the amino acid sequence described in SEQ ID NO.

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

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

5. Claim 9 A diagnostic agent for an autoimmune disease comprising a complex having a polypeptide, a human leukocyte type antigen, and a labeling dye, wherein the polypeptide comprises: (D) one or more polypeptides having an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4 and a total length of 7 residues or more and 17 residues or less; (A) one or more polypeptides having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1 and a total length of 8 residues or more and 20 residues or less; (B) one or more polypeptides having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 2 and a total length of 12 residues or more and 21 residues or less; and (E) at least one polypeptide selected from the group consisting of one or more polypeptides having an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 5 and a total length of 10 residues or more and 19 residues or less. Claim 10 A kit for diagnosing an autoimmune disease comprising at least one polypeptide selected from the group consisting of: (D) one or more polypeptides having an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4 and a total length of 7 residues or more and 17 residues or less; (A) one or more polypeptides having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1 and a total length of 8 residues or more and 20 residues or less; (B) one or more polypeptides having 12 residues or more and a total length of 21 residues or less, having an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 2; and (E) one or more polypeptides having 10 residues or more and a total length of 19 residues or less, having an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4; and a human leukocyte type antigen that binds to the at least one polypeptide. Claim 11 A kit according to claim 10, further comprising a labeling dye that binds to the human leukocyte type antigen. Claim 12 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: (D) one or more polypeptides having an amino acid sequence having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 4 and a total length of 7 or more and 17 or less; (A) one or more polypeptides having 90% or more sequence identity with the amino acid sequence described in SEQ ID NO. 1 and a total length of 8 or more and 20 or less; (B) one or more polypeptides having 12 or more and 21 or less sequence identity with the amino acid sequence described in SEQ ID NO. 2 and a total length of 12 or more and 21 or less sequence identity with the amino acid sequence described in SEQ ID NO. 4 and a total length of 10 or more and 19 or less sequence identity with the amino acid sequence described in SEQ ID NO. 4.

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