Method for identifying epitopes that bind to HLA with specific genotypes

Abstract

To provide a method for identifying epitopes that bind to HLA with specific genotypes.SOLUTION: Provided is a method for identifying an epitope that binds to an HLA having a specific genotype, comprising: determining that a site having the same amino acid sequence as a polypeptide commonly contained in at least one polypeptide obtained from dendritic cells prepared using classical monocytes derived from multiple human subjects having different HLA genotypes is an epitope, in an antigen protein, that binds to a first HLA having a first genotype.SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure relates to a method for identifying epitopes that bind to HLA having a specific genotype.

Background Art

[0002] In autoimmune diseases, the immune mechanism, which is originally a mechanism for removing foreign substances derived from outside the living body, abnormally activates the immune system by recognizing substances derived from inside the living body as antigens. Such abnormal activation of the immune system is known to cause various abnormalities in biological functions in the bodies of patients suffering from autoimmune diseases.

[0003] Human leukocyte antigen (HLA) is the major histocompatibility complex (MHC) in humans. In humoral immunity, HLA is exposed on the cell surface of antigen-presenting cells (APCs) in a state of binding an antigen, thereby driving the antibody production mechanism against the antigen. Depending on the HLA genotype, human leukocytes have tens of thousands of genotypes. The HLA genotype is one of the risk factors in autoimmune diseases, and it is known that humans having a genotype specific to HLA have a high possibility of developing autoimmune diseases.

[0004] In autoimmune diseases, HLA recognizes a part of an antigen and forms a bond with the antigen. Particularly when the antigen is a protein, the site recognized by HLA in the antigen protein is called an epitope. Since such epitopes have the potential as targets for the treatment and diagnosis of autoimmune diseases, their identification has been attempted in various autoimmune diseases.

[0005] Identifying the gene symbols of HLA to which an epitope binds and its genotype is important for using the epitope as a target for the treatment and diagnosis of autoimmune diseases. So far, the identification of such HLA gene symbols and their genotypes has been carried out by contacting a protein or its peptide fragment that may contain the epitope as part of the sequence with HLA proteins of specific genotypes, model animals or cells expressing them, and evaluating their binding or immune activation (for example, Non-Patent Document 1).

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] In the conventional identification of the gene symbols of HLA to which an epitope binds and its genotype, since it is necessary to contact a protein or its peptide fragment that may contain the epitope as part of the sequence with the expressing cells of HLA proteins with individual genotypes, expression model animals or purified proteins for evaluation, a lot of trial and error has been required for genotype identification.

[0008] In addition, in a human subject suffering from an autoimmune disease, a protein containing an epitope as part of its sequence exists not only in its full length but also as its peptide fragment, and such a peptide fragment may have a higher immunostimulatory effect from the viewpoints of affinity and pharmacokinetics. However, when peptide fragments produced in such a human subject are recovered from cells or the like, peptide fragments that bind to HLA of a plurality of gene symbols are recovered as a group, and thus, simply analyzing the recovered peptide fragments cannot identify the gene symbols and genotypes of HLA to which the peptide fragments bind. For example, since an anti-HLA-DR antibody binds to both HLA-DRB1 and HLA-DRB3, when peptide fragments produced in a human subject are recovered by a pull-down assay using the anti-HLA-DR antibody, it cannot be determined whether the recovered peptide fragments bind to HLA-DRB1 or HLA-DRB3. Then, even if the genotype of HLA in a human subject is known, it has been difficult to identify the gene symbols and genotypes of HLA to which the peptide fragments bind only from the results.

[0009] An object of the present disclosure is to provide a method for identifying an epitope that binds to HLA having a specific genotype.

Means for Solving the Problems

[0010] The present inventors contacted antigen proteins to dendritic cells prepared respectively using classical monocytes derived from a plurality of subjects, recovered polypeptides which are peptide fragments presented by those dendritic cells, and then, from the relationship between the genotype of each HLA protein in the subject used and the types of polypeptides included in the population of polypeptides presented by each dendritic cell, found that the gene symbols and genotypes of HLA to which each polypeptide binds can be identified, and completed the present disclosure.

[0011] The present disclosure relates to, for example, the following. [1]A method for identifying an epitope that binds to HLA having a specific genotype, comprising: contacting an antigen protein with a first dendritic cell prepared using classical monocytes derived from a subject in which a first HLA has a first genotype and a second HLA has a second genotype, and then obtaining from the first dendritic cell at least one first polypeptide that binds to a first HLA having the first genotype or a second HLA having the second genotype; contacting the antigen protein with a second dendritic cell prepared using classical monocytes derived from a subject in which a first HLA has a first genotype and a second HLA has a third genotype, and then obtaining from the second dendritic cell at least one second polypeptide that binds to a first HLA having the first genotype or a second HLA having the third genotype; and determining that a site having the same amino acid sequence as a polypeptide commonly contained in the at least one first polypeptide and the at least one second polypeptide is an epitope that binds to a first HLA having the first genotype in the antigen protein. A method comprising the above steps. [2]The method according to [1], wherein the first HLA is HLA-DRB1 and the second HLA is HLA-DRB3, or the first HLA is HLA-DRB3 and the second HLA is HLA-DRB1, or the first HLA is HLA-DQA1 and the second HLA is HLA-DQB1, or the first HLA is HLA-DQB1 and the second HLA is HLA-DQA1, or the first HLA is HLA-B and the second HLA is HLA-A or HLA-C, or the first HLA is HLA-A and the second HLA is HLA-B or HLA-C, or the first HLA is HLA-C and the second HLA is HLA-A or HLA-B. The method according to [1]. [3]Is the first HLA HLA-DRB1 and the second HLA HLA-DRB3, or Is the first HLA HLA-DRB3 and the second HLA HLA-DRB1, or Is the first HLA HLA-DQA1 and the second HLA HLA-DQB1, or Is the first HLA HLA-DQB1 and the second HLA HLA-DQA1, , the method according to [1] or [2]. [4] The method according to any one of [1] to [3], wherein the first HLA is HLA-DRB1, the second HLA is HLA-DRB3, and the first genotype is 03:01. [5] The method according to any one of [1] to [3], wherein the first HLA is HLA-DRB3, the second HLA is HLA-DRB1, and the first genotype is 01:01. [6] The method according to any one of [1] to [3], wherein the first HLA is HLA-DQA1, the second HLA is HLA-DQB1, and the first genotype is 05:01. [7] The method according to any one of [1] to [3], wherein the first HLA is HLA-DQB1, the second HLA is HLA-DQA1, and the first genotype is 02:01. [8] The method according to any one of [1] to [7], wherein the step of obtaining the first at least one polypeptide and the step of obtaining the second at least one polypeptide are performed using an antibody that binds to the first HLA and the second HLA. [9] A method for identifying an epitope that binds to an HLA having a specific genotype, For a first dendritic cell prepared using classical monocytes from a subject in which a first HLA has a first genotype, a second HLA has a second genotype, and a third HLA has a fourth genotype, after contacting with an antigen protein, obtaining from the first dendritic cell at least one first polypeptide that binds to a first HLA having the first genotype, a second HLA having the second genotype, or a third HLA having the fourth genotype; For a second dendritic cell prepared using classical monocytes from a subject in which a first HLA has a first genotype, a second HLA has a third genotype, and a third HLA has a fifth genotype, after contacting with the antigen protein, obtaining from the second dendritic cell at least one second polypeptide that binds to a first HLA having the first genotype, a second HLA having the third genotype, or a third HLA having the fifth genotype; and determining that a site having the same amino acid sequence as a polypeptide commonly contained in the at least one first polypeptide and the at least one second polypeptide is an epitope that binds to a first HLA having the first genotype in the antigen protein. A method comprising the above steps.

[10] The method according to [9], wherein the first HLA, the second HLA, and the third HLA are HLAs belonging to HLA class I.

[11] The method according to [9] or

[10] , wherein the first HLA is HLA-B, the second HLA is HLA-A, the third HLA is HLA-C, and the first genotype is 08:01.

[12] The method according to [9] or

[10] , wherein the first HLA is HLA-A, the second HLA is HLA-B, the third HLA is HLA-C, and the first genotype is 01:01.

[13] The method according to [9] or

[10] , wherein the first HLA is HLA-C, the second HLA is HLA-A, the third HLA is HLA-B, and the first genotype is 07:01.

[14] After the step of obtaining the first at least one polypeptide and the step of obtaining the second at least one polypeptide, the method according to any one of [1] to

[13] , comprising a step of determining the amino acid sequences of the polypeptides included in the first at least one polypeptide and the second at least one polypeptide.

[15] The method according to any one of [1] to

[14] , wherein the epitope is an epitope involved in myasthenia gravis.

[16] A method for producing a polypeptide that binds to HLA having a specific genotype, comprising a step of identifying an epitope according to the method according to any one of [1] to

[15] , and a step of producing a polypeptide having the same amino acid sequence as the identified epitope. [Advantages of the Invention]

[0012] According to the present disclosure, a method for identifying an epitope that binds to HLA having a specific genotype is provided.

[0013] According to the identification method according to the present disclosure, an epitope that binds to HLA having a specific genotype can be identified without performing a trial and error of contacting a polypeptide with an HLA protein-expressing cell, an expression model animal, or a purified protein having an individual genotype.

[0014] According to the identification method according to the present disclosure, the genotype of the HLA protein to which the peptide fragment presented by the dendritic cells prepared using classical monocytes derived from a subject binds can be identified, whereby an epitope that binds to HLA having a specific genotype in an antigen protein can be identified.

[0015] In the conventional method for identifying epitopes, in order to determine the epitopes that bind to HLA having a specific genotype at the amino acid residue level (the level corresponding to which positions in the amino acid sequence of the antigen protein), as done in Non-Patent Document 1, in order to determine the epitopes that bind to HLA having a specific genotype at the amino acid residue level (the level corresponding to which positions in the amino acid sequence of the antigen protein), a large number of peptide fragments corresponding to a part of the full length of the protein are synthetically prepared as peptide fragments that may contain the epitope in a part of the sequence, and each of those peptides needs to be contacted with and evaluated against cells expressing the HLA protein, an expression model animal, or a purified protein. In contrast, according to the identification method according to the present disclosure, since the population of peptide fragments presented by dendritic cells prepared using classical monocytes derived from a subject contains a plurality of polypeptides having different lengths at the amino acid residue level while containing the sequences of the same epitope, the epitope can be determined at the amino acid residue level without synthetically preparing a large number of peptide fragments.

Brief Description of the Drawings

[0016]

Figure 1

Modes for Carrying Out the Invention

[0017] The modes for carrying out the present disclosure will be described below, but the present disclosure is not limited to the following embodiments.

[0018] Human leukocyte antigen (HLA) is the major histocompatibility complex (MHC) in humans. In humoral immunity, HLA is exposed on the cell surface of antigen-presenting cells (APCs) such as dendritic cells in a state of binding an antigen, thereby driving the antibody production mechanism against the antigen. HLA belongs to HLA-A, HLA-C, HLA-B, etc., and is classified into HLA class I responsible for cellular immunity and HLA class II belonging to HLA-DR, HLA-DQ, HLA-DP, etc., and responsible for humoral immunity.

[0019] According to the notation defined by the WHO Nomenclature Committee for Factors of the HLA System, the HLA genotype (allele) is denoted in the form of "HLA-(gene symbol)*(Region 1):(Region 2):(Region 3):(Region 4)". Here, Regions 1 to 4 are each represented by two-digit numerical values. Region 1 represents the HLA specificity (antigen type), Region 2 represents non-synonymous substitution (variation in amino acid sequence within the same antigen type), Region 3 represents synonymous substitution (variation in the exon region that does not result in a change in the encoded amino acid sequence), and Region 4 represents nucleotide substitution outside the coding region (variation in the intron region sequence), respectively. In these notations, the gene symbol, Region 1, and Region 2 are information that directly affects the amino acid sequence of HLA and is clinically important.

[0020] In the present disclosure, when the first region is YY and the second region is ZZ in the genotype of the HLA gene with the gene symbol XXX (that is, up to the second region in the above notation is represented by 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 in the genotype of the HLA gene (HLA-DRB3 gene) with the gene symbol DRB3, the first region is 01 and the second region is 01, that is, the genotype is HLA-DRB3*01:01. Also, for example, "the genotype of HLA-DRB1 is 03:01" means that in the genotype of the HLA gene (HLA-DRB1 gene) with the gene symbol DRB1, the first region is 03 and the second region is 01, that is, the genotype is HLA-DRB1*03:01.

[0021] The present disclosure relates to a method for identifying an epitope that binds to an HLA having a specific genotype, the method comprising the steps of: contacting an antigen protein with a first dendritic cell prepared using classical monocytes derived from a subject in which a first HLA has a first genotype and a second HLA has a second genotype; obtaining at least one first polypeptide that binds to the first HLA having the first genotype or the second HLA having the second genotype from the first dendritic cell (first obtaining step); and a step of determining a site having the same amino acid sequence as a polypeptide commonly contained in the at least one first polypeptide and the at least one second polypeptide, the site being an epitope in the antigen protein that binds to the first HLA having the first genotype.

[0022] In the first and second acquisition steps of the identification method according to the present disclosure, dendritic cells prepared using classical monocytes derived from a subject are used. The subject may be a human subject, for example, a healthy person, a human suffering from an autoimmune disease, or a human suffering from myasthenia gravis. In addition, these human subjects may be human subjects whose genotypes of each HLA protein (for example, at least the genotypes of the first and second HLA) are known. In addition, when the genotypes of each HLA protein in a human subject are unknown, the identification method according to one embodiment of the present disclosure may further include a step of identifying the genotypes of each HLA protein of the human subject (HLA typing step), which may be, for example, by entrusting the identification to an external testing company, or by using a commercially available kit such as an HLA-DNA typing kit (VERITAS) to identify the genotype according to the protocol described in the attached document of the kit.

[0023] As classical monocytes derived from a subject, those commercially available as classical monocytes derived from a human subject may be used, or those isolated from a cell population commercially available that contains classical monocytes derived from a human subject may also be used, or those isolated from a sample collected from a human subject may also be used. The sample used for isolating classical monocytes is not particularly limited as long as it contains classical monocytes and classical monocytes can be isolated therefrom, and may be, for example, blood (e.g., peripheral blood) or serum. The cell population used for isolating classical monocytes is not particularly limited as long as it contains classical monocytes and classical monocytes can be isolated therefrom, and may be, for example, human peripheral blood mononuclear cells (human PBMC). Human PBMC is a cell population containing various mononuclear cells that make up the immune system, such as lymphocytes, monocytes, and dendritic cells, which are separated and obtained from human peripheral blood. Separation of human PBMC from blood (e.g., peripheral blood) collected from a human subject can be performed by a method commonly used by those skilled in the art, and can be performed by removing plasma components, red blood cells, platelets, and polymorphonuclear granulocytes (neutrophils, basophils, eosinophils, etc.) from peripheral blood.

[0024] Isolation of classical monocytes from the above cell population or sample can be performed by a method commonly used by those skilled in the art. Classical monocytes are known to highly express the CD14 (Cluster of Differentiation 14) protein and lowly express the CD16 (Cluster of Differentiation 16) protein among mononuclear cells involved in innate immunity. Therefore, isolating classical monocytes from a cell population of multiple types of mononuclear cells involved in innate immunity, such as human PBMC, can be performed, for example, by isolating monocytes among mononuclear cells (monocytes) that highly express CD14 and lowly express CD16 (CD14 + CD16 - ). Such a method for isolating CD14 + CD16 - monocytes is not particularly limited. For example, CD14 + CD16 -Cells other than monocytes may be labeled and subjected to negative selection using flow cytometry. For example, CD14 contained in a cell population + CD16 - Monocytes may be labeled and subjected to positive selection using flow cytometry. Such selection can be performed, for example, by a method using magnetic beads labeled with an anti-CD14 antibody or an anti-CD16 antibody. More specifically, CD14 contained in a cell population + CD16 - Labeling of cells other than monocytes can be performed using, for example, EasySep Human Monocyte Isolation Kit (VERITAS) or Dynabeads (registered trademark) Untouched (registered trademark) human monocytes (Thermo Fisher Scientific). Also, CD14 contained in a cell population + CD16 - Labeling of monocytes can be performed using, for example, Dynabeads (registered trademark) CD14 (Thermo Fisher Scientific).

[0025] Preparation of dendritic cells using classical monocytes can be performed by methods commonly used by those skilled in the art. It is known that classical monocytes are induced to differentiate into dendritic cells when cultured in the presence of a predetermined cytokine. For example, it is known that when cultured in the presence of IL-4 (interleukin-4) and GM-CSF (granulocyte-macrophage colony-stimulating factor, also known as CSF2), differentiation into dendritic cells is induced. Therefore, dendritic cells prepared using classical monocytes according to one embodiment of the present disclosure may be dendritic cells induced by culturing classical monocytes in the presence of a predetermined cytokine (for example, IL-4 and GM-CSF). Also, a specific method according to one embodiment of the present disclosure may include a step of inducing differentiation into dendritic cells (differentiation induction step) by culturing classical monocytes in the presence of a predetermined cytokine (for example, IL-4 and GM-CSF) before the first acquisition step and the second acquisition step.

[0026] When culturing classical monocytes to induce differentiation into dendritic cells, the concentration of cytokines to be present may be selected as the concentration usually used by those skilled in the art according to the type of cytokine. For example, GM-CSF may be 10 ng / mL to 200 ng / mL (for example, 50 ng / mL), and IL-4 may be 10 ng / mL to 200 ng / mL (for example, 50 ng / mL). Also, the period for culturing classical monocytes in the presence of cytokines may be, for example, 2 days or more and 20 days or less, or 4 days or more and 10 days or less (for example, 5 days, 7 days).

[0027] Also, in one aspect when a specific method according to an embodiment of the present disclosure includes a differentiation induction step, the specific method according to the present disclosure may further include a step of isolating classical monocytes from a cell population containing classical monocytes (isolation step) according to the method described above, before the differentiation induction step.

[0028] The cell culture performed in the specific method according to the present disclosure may be carried out according to the method usually performed by those skilled in the art. For example, it may be cultured in a medium containing about 10% by volume of a serum component such as inactivated fetal bovine serum (FBS) in a cell culture medium usually used by those skilled in the art, such as RPMI medium (Roswell Park Memorial Institute medium) and DMEM medium (Dulbecco’s Modified Eagle Medium), under the conditions of 37 °C, 5% CO2, and 100% humidity. Also, in the culture, the medium may be replaced with a fresh one once every 1 to 3 days.

[0029] In the first acquisition step and the second acquisition step of the specific method according to the present disclosure, the antigen protein is brought into contact with dendritic cells prepared using classical monocytes derived from a subject prepared as described above, whereby the antigen protein is phagocytosed by the dendritic cells, and the polypeptide, which is a fragment thereof, is presented by the dendritic cells. In this way, the method of presenting an antigen by an antigen-presenting cell and analyzing the presented polypeptide fragment is called the NAPA (Natural antigen processing assay) method. According to the NAPA method, it is possible to analyze the amino acid sequence of a peptide fragment (polypeptide) that is considered to actually occur in a living body, rather than an artificially synthesized peptide as in the conventional method, and thereby identify an epitope.

[0030] Next, an embodiment of the specific method according to the present disclosure will be described by taking as an example the case where the first HLA is HLA-DRB1, the second HLA is HLA-DRB3, the first genotype is 03:01, the second genotype is 01:01, the third genotype is 02:02, and the antigen protein is an acetylcholine receptor. At this time, the human subject from whom the classical monocytes used for the preparation of the first dendritic cells are derived has genotypes of HLA-DRB1*03:01 and HLA-DRB3*01:01. In addition, the human subject from whom the classical monocytes used for the preparation of the second dendritic cells are derived has genotypes of HLA-DRB1*03:01 and HLA-DRB3*02:02.

[0031] Note that the acetylcholine receptor (AChR) is a receptor that has an essential function in information transmission between nerve cells through the binding of acetylcholine secreted extracellularly in neurotransmission. In autoimmune diseases in which antibodies against the acetylcholine receptor are overproduced, some of the neurotransmission mediated by the acetylcholine receptor is inhibited, resulting in various symptoms. Among the acetylcholine receptors, in particular, the α1 subunit of the human acetylcholine receptor (human AChRα1) is known to be recognized as an autoantigen in about 80% of patients with early-onset myasthenia gravis. Human AChRα1 has an amino acid sequence (SEQ ID NO: 36) with an accession number of NP_000070.1 in RefSeq by the National Center for Biotechnology Information (NCBI) of the United States. The 1st to 20th amino acids in the amino acid sequence shown in SEQ ID NO: 36 correspond to the signal peptide, and the amino acid sequence excluding them is shown in SEQ ID NO: 37.

[0032] In addition, in early-onset myasthenia gravis, which is one of the autoimmune diseases, it is known that the genotype of HLA-DRB3 is 01:01 or the genotype of HLA-DRB1 is 03:01 as its risk factors. Early-onset myasthenia gravis is a type of myasthenia gravis that develops in patients aged 10 to 49 years. Myasthenia gravis is designated as a refractory disease in Japan and is an organ-specific autoimmune disease against molecules on the postsynaptic membrane of the neuromuscular junction, with muscle weakness as the main symptom. In myasthenia gravis, autoantibodies are produced in the patient's body against molecules present on the postsynaptic membrane of the neuromuscular junction, such as the acetylcholine receptor (AChR) and muscle-specific receptor tyrosine kinase (MuSK). The inhibition of neurotransmission by these antibodies results in various symptoms.

[0033] In the first acquisition step, acetylcholine receptor (antigen protein) is brought into contact with first dendritic cells prepared using classical monocytes derived from a subject having a genotype of HLA-DRB1 (first HLA) of 03:01 (first genotype) and a genotype of HLA-DRB3 (second HLA) of 01:01 (second genotype). Then, from the first dendritic cells, at least one first polypeptide that binds to HLA-DRB1*03:01 (first HLA having the first genotype) or HLA-DRB3*01:01 (second HLA having the second genotype) is obtained.

[0034] Contacting the antigen protein with the first dendritic cells is not particularly limited as long as it is contact under conditions that enable the dendritic cells to present a polypeptide that is a peptide fragment of the antigen protein. For example, it may be culturing the first dendritic cells in a medium containing the antigen protein. In this case, the concentration of the antigen protein may be, for example, 1 μg / mL to 1000 μg / mL, 5 μg / mL to 500 μg / mL, or 10 μg / mL to 250 μg / mL. Also, the time for contacting the antigen protein with the first dendritic cells may be, for example, 1 hour to 168 hours, 4 hours to 72 hours, or 12 hours to 48 hours.

[0035] Obtaining at least one first polypeptide from the first dendritic cells can be performed by recovering HLA (HLA-polypeptide complex) in a state where the polypeptide is bound from the first dendritic cells using an antibody against HLA or a modified form thereof, and then isolating the polypeptide from the HLA-polypeptide complex. That is, in one embodiment, the step of obtaining at least one first polypeptide and obtaining at least one second polypeptide may be performed using an antibody that binds to the first HLA and the second HLA.

[0036] For example, the anti-HLA-DR antibody binds to both HLA-DRB1 and HLA-DRB3. Thus, after lysing the first dendritic cells, when proteins are recovered from the lysate using an anti-HLA-DR antibody that has been subjected to recoverable modification (for example, modification with magnetic beads), both the HLA-DRB1*03:01-polypeptide complex and the HLA-DRB3*01:01-polypeptide complex are included in the recovered sample (the first at least one polypeptide). Thus, obtaining an HLA-polypeptide complex from the first dendritic cells may, in one embodiment, include lysing the first dendritic cells and recovering the HLA-polypeptide complex from the lysate using an antibody that binds to the first HLA and the second HLA and has been subjected to recoverable modification. In this case, the method for lysing the first dendritic cells is not particularly limited and can be carried out by a method commonly used by those skilled in the art (for example, a method of adding to a solution containing a surfactant). Also, the recoverable modification in the antibody is not particularly limited as long as it can recover the HLA-polypeptide complex, and may be, for example, modification with magnetic beads, and the recovery method at this time may be recovery by capturing the magnetic beads with a magnet. Also, the recoverable modification in the antibody may be, for example, beads having a particle size and mass larger than those of biopolymers such as antibodies, and the recovery method at this time may also be recovery by centrifugation or filtration.

[0037] Isolating the polypeptide from the HLA-polypeptide complex can be carried out according to a method that is not particularly limited as long as it is a method capable of isolating the polypeptide. For example, after adding an acid (e.g., 0.1% trifluoroacetic acid (TFA)) to release the polypeptide from the HLA-polypeptide complex, a method of recovering a solution containing the polypeptide as a filtrate by filtration using a microfilter may be used, but it is not limited thereto. Note that isolating the polypeptide from the HLA-polypeptide complex includes isolating the polypeptide as its solution or a composition containing the polypeptide, and is not limited to recovering the polypeptide in a solid state. Further, the solution containing the recovered polypeptide may have its solvent removed by freeze-drying or the like and stored as a solid, or may be used in the determination step as a solid.

[0038] In the second acquisition step, acetylcholine receptor (antigen protein) is brought into contact with a second dendritic cell prepared using classical monocytes derived from a subject having a genotype of HLA-DRB1 (first HLA) of 03:01 (first genotype) and a genotype of HLA-DRB3 (second HLA) of 02:02 (third genotype), and then at least one second polypeptide that binds to HLA-DRB1*03:01 (first HLA having the first genotype) or HLA-DRB3*02:02 (third HLA having the second genotype) is obtained from the second dendritic cell. Obtaining the polypeptide in the second acquisition step can be carried out in the same manner as described in the first acquisition step.

[0039] In one embodiment of the specific method of the present disclosure, after the first acquisition step and the second acquisition step, a step of determining the amino acid sequences of the polypeptides included in the at least one polypeptide of the first and the at least one polypeptide of the second may be included. In this case, in one embodiment of the specific method of the present disclosure, the amino acid sequences of the polypeptides included in the at least one polypeptide of the first and the polypeptides included in the at least one polypeptide of the second may be determined. In these cases, the method for determining the amino acid sequence of the polypeptide is not particularly limited as long as it can determine the amino acid sequence. For example, it may be determined based on mass by mass spectrometry, or it may be determined based on elution time by liquid chromatography. As a combined method of both, for example, it may be determined based on the measurement results of LC-MS or LC-MS / MS.

[0040] In the determination step, a site having the same amino acid sequence as the polypeptide commonly included in the at least one polypeptide of the first and the at least one polypeptide of the second is determined to be an epitope that binds to the first HLA having the first genotype in the antigen protein.

[0041] In the determination step, the polypeptides contained in each of the first at least one polypeptide and the second at least one polypeptide are compared, and the polypeptide commonly contained in both of them can be determined to be one that binds to the first HLA having the first genotype. In the example used for the explanation, the first at least one polypeptide contains a polypeptide that binds to HLA-DRB1*03:01 or HLA-DRB3*01:01, and the second at least one polypeptide contains a polypeptide that binds to HLA-DRB1*03:01 or HLA-DRB3*02:02. Therefore, the polypeptide contained in both of them can be determined to be a polypeptide that binds to HLA-DRB1*03:01. And thereby, a site having the same amino acid sequence as the polypeptide that binds to HLA-DRB1*03:01 can be determined to be an epitope that binds to HLA-DRB1*03:01 in the acetylcholine receptor (antigen protein).

[0042] In the example described above, the case where each of the first and second human subjects used for the preparation of dendritic cells has only one genotype for the first HLA and the second HLA was explained. However, these human subjects may have two or more genotypes for the HLA protein as long as they do not have a common genotype among human subjects. For example, in the above example, assume that the human subject used for the preparation of the first dendritic cell has, as the genotype of HLA-DRB1 (the first HLA), in addition to 03:01 (the first genotype), also 08:01 (the fourth genotype). In this case, the first at least one polypeptide contains a polypeptide that binds to HLA-DRB1*03:01, HLA-DRB1*08:01, or HLA-DRB3*01:01, and the second at least one polypeptide contains a polypeptide that binds to HLA-DRB1*03:01 or HLA-DRB3*02:02. Therefore, as the polypeptide common to both, a polypeptide that binds to HLA-DRB1*03:01 can be found.

[0043] In addition, in the examples described above, dendritic cells prepared from classical monocytes derived from two subjects, namely the first dendritic cell and the second dendritic cell, were used. However, when the fourth genotype as described above exists, at least one polypeptide recovered from those dendritic cells using a third or more dendritic cells may be used to determine an epitope based on a polypeptide commonly contained in the polypeptide. For example, the first at least one polypeptide contains a polypeptide that binds to HLA-DRB1*03:01, HLA-DRB1*08:01, or HLA-DRB3*01:01, and the second at least one polypeptide contains a polypeptide that binds to HLA-DRB1*03:01, HLA-DRB1*13:01, or HLA-DRB3*01:01. When the third at least one polypeptide contains a polypeptide that binds to HLA-DRB1*03:01, HLA-DRB1*08:01, HLA-DRB3*03:01, or HLA-DRB3*02:02, a polypeptide that binds to HLA-DRB1*03:01 can be found as a polypeptide common to all of these.

[0044] In addition, in the examples described above, the case where the first HLA is HLA-DRB1 and the second HLA is HLA-DRB3 has been described. However, the first HLA and the second HLA are not limited to these. In one embodiment, the first HLA and the second HLA may be HLAs belonging to HLA class II. The HLA belonging to HLA class II is an HLA responsible for the immune mechanism through antigen presentation. Therefore, when the first HLA and the second HLA are HLAs belonging to HLA class II, they can be suitably used for identifying an epitope of an autoimmune disease, which is a disease caused by a self-antigen. In addition, in one embodiment, the first HLA and the second HLA may be HLAs belonging to HLA class I.

[0045] In a preferred embodiment, for the first HLA and the second HLA, the first HLA may be HLA-DRB1 and the second HLA may be HLA-DRB3, or the first HLA may be HLA-DRB3 and the second HLA may be HLA-DRB1, or the first HLA may be HLA-DQA1 and the second HLA may be HLA-DQB1, or the first HLA may be HLA-DQB1 and the second HLA may be HLA-DQA1, or the first HLA may be HLA-B and the second HLA may be HLA-A or HLA-C, or the first HLA may be HLA-A and the second HLA may be HLA-B or HLA-C, or the first HLA may be HLA-C and the second HLA may be HLA-A or HLA-B. In a more preferred embodiment, for the first HLA and the second HLA, the first HLA may be HLA-DRB1 and the second HLA may be HLA-DRB3, or the first HLA may be HLA-DRB3 and the second HLA may be HLA-DRB1, or the first HLA may be HLA-DQA1 and the second HLA may be HLA-DQB1, or the first HLA may be HLA-DQB1 and the second HLA may be HLA-DQA1. Among these HLAs, both HLA-DR and HLA-DQ are HLAs belonging to HLA class II and are known to be involved in various autoimmune diseases (e.g., myasthenia gravis). On the other hand, anti-HLA-DR antibodies or anti-HLA-DQ antibodies, which are specific antibodies against them, bind to HLAs with a plurality of gene symbols, namely HLA-DRB1 and HLA-DRB3, HLA-DQA1 and HLA-DQB1. Therefore, when peptide fragments produced in a human subject are recovered by a pull-down assay using these antibodies, it is impossible to determine which gene symbol of HLA the recovered peptide fragments bind to, and it has been difficult to identify the gene symbol and genotype of HLA to which the peptide fragments bind only from the results.In contrast, according to a specific method of the present disclosure, by comparing a plurality of results recovered by a pull-down assay using these antibodies, it is possible to identify the gene symbols and genotypes of HLA to which the polypeptide binds. HLA-B is an HLA belonging to HLA class I and is known to be involved in various autoimmune diseases (e.g., myasthenia gravis). On the other hand, the anti-HLA-A, B, C antibody, which is a specific antibody against it, binds to HLA with a plurality of gene symbols, namely HLA-B, HLA-A, and HLA-C. According to the method according to an embodiment of the present disclosure, it is possible to identify the gene symbols and genotypes of HLA to which the polypeptide binds in the same manner as described above.

[0046] In one embodiment, the first HLA may be HLA-DRB1, the second HLA may be HLA-DRB3, and the first genotype may be 03:01. At this time, the second genotype may be 01:01. At this time, the third genotype may be 02:02.

[0047] In one embodiment, the first HLA may be HLA-DRB3, the second HLA may be HLA-DRB1, and the first genotype may be 01:01. At this time, the second genotype may be 03:01. At this time, the third genotype may be 01:01.

[0048] In one embodiment, the first HLA may be HLA-DQA1, the second HLA may be HLA-DQB1, and the first genotype may be 05:01. At this time, the second genotype may be 02:01.

[0049] In one embodiment, the first HLA may be HLA-DQB1, the second HLA may be HLA-DQA, and the first genotype may be 02:01. At this time, the second genotype may be 05:01.

[0050] In one embodiment, the first HLA may be HLA-B, the second HLA may be HLA-A or HLA-C, and the first genotype may be 08:01. In one embodiment, the first HLA may be HLA-A, the second HLA may be HLA-B or HLA-C, and the first genotype may be 01:01. In one embodiment, the first HLA may be HLA-C, the second HLA may be HLA-A or HLA-B, and the first genotype may be 07:01.

[0051] In the examples described above, the case where the genotypes of the first HLA are common and the genotypes of the second HLA are different for the subject from which the classical monocytes specifically used are derived has been described. However, in one embodiment of the present disclosure, the genotype of the third HLA may be even more different. That is, one embodiment of the present disclosure is a method for identifying an epitope that binds to an HLA having a specific genotype, wherein a first dendritic cell prepared using classical monocytes derived from a subject in which a first HLA has a first genotype, a second HLA has a second genotype, and a third HLA has a fourth genotype is contacted with an antigen protein, and then, from the first dendritic cell, a first at least one polypeptide that binds to a first HLA having a first genotype, a second HLA having a second genotype, or a third HLA having a fourth genotype is obtained; a second dendritic cell prepared using classical monocytes derived from a subject in which a first HLA has a first genotype, a second HLA has a third genotype, and a third HLA has a fifth genotype is contacted with the antigen protein, and then, from the second dendritic cell, a second at least one polypeptide that binds to a first HLA having a first genotype, a second HLA having a third genotype, or a third HLA having a fifth genotype is obtained; and a step of determining that a site having the same amino acid sequence as a polypeptide commonly included in the first at least one polypeptide and the second at least one polypeptide is an epitope that binds to a first HLA having a first genotype in the antigen protein may be included.

[0052] In one embodiment where the genotype of the third HLA is further different as described above, the first HLA, the second HLA, and the third HLA may be HLAs belonging to HLA class I. While anti-HLA-A,B,C antibodies bind to HLAs with multiple gene symbols such as HLA-B, HLA-A, and HLA-C, according to the method according to one embodiment of the present disclosure, it is possible to identify the gene symbols and genotypes of the HLAs to which the polypeptide binds. For example, in one embodiment, the first HLA may be HLA-B, the second HLA may be HLA-A, and the third HLA may be HLA-C, or the first HLA may be HLA-A, the second HLA may be HLA-B, and the third HLA may be HLA-C, or the first HLA may be HLA-C, the second HLA may be HLA-A, and the third HLA may be HLA-B.

[0053] In one embodiment where the genotype of the third HLA is further different as described above, the first HLA is HLA-B, the second HLA is HLA-A, the third HLA is HLA-C, and the first genotype may be 08:01. In one embodiment where the genotype of the third HLA is further different as described above, the first HLA is HLA-A, the second HLA is HLA-B, the third HLA is HLA-C, and the first genotype may be 01:01. In one embodiment where the genotype of the third HLA is further different as described above, the first HLA is HLA-C, the second HLA is HLA-A, the third HLA is HLA-B, and the first genotype may be 07:01.

[0054] In the examples described above, the example where the antigen protein is an acetylcholine receptor has been described. However, the antigen protein is not limited thereto, and any protein whose peptide fragment is presented by contacting the first dendritic cell and the second dendritic cell may be used, and it may be an antigen protein that can be an autoantigen in an autoimmune disease, for example, an acetylcholine receptor or a muscle-specific receptor-type tyrosine kinase.

[0055] In one embodiment, the identified epitope may be an epitope involved in myasthenia gravis. In this case, the antigen protein may be, for example, an acetylcholine receptor or a muscle-specific receptor tyrosine kinase. Also, for the first HLA and the second HLA, the first HLA is HLA-DRB1 and the second HLA is HLA-DRB3, or the first HLA is HLA-DRB3 and the second HLA is HLA-DRB1, or the first HLA is HLA-DQA1 and the second HLA is HLA-DQB1, or the first HLA is HLA-DQB1 and the second HLA is HLA-DQA1, or the first HLA is HLA-B and the second HLA is HLA-A or HLA-C, or the first HLA is HLA-B, the second HLA is HLA-A, and the third HLA is HLA-C.

[0056] As described above, the specific method according to the present disclosure attempts to identify an epitope that binds to an HLA having a specific genotype, using classical monocytes derived from a plurality of subjects (for example, derived from a plurality of humans) as a sample, and does not attempt to obtain any information including the HLA genotype for each individual subject. Therefore, the specific method according to the present disclosure is not a diagnostic method and can be said to be an in vitro method.

[0057] Also, one aspect of the specific method of the present disclosure may also be a method for producing a polypeptide that binds to an HLA having a specific genotype, including a step of identifying an epitope according to the specific method according to an embodiment of the present disclosure, and a step of producing a polypeptide having the same amino acid sequence as the identified epitope. The method for producing the polypeptide in this case is not particularly limited and may be produced by a method commonly used by those skilled in the art.

Examples

[0058] Hereinafter, the present disclosure will be described in more detail using examples, but the present disclosure is not limited to the following examples.

[0059] [Example 1: Identification of an antigenic peptide derived from human AChRα1 that binds to HLA-DR] <Step 1: Collection of clinical samples> The peripheral blood mononuclear cells (hereinafter referred to as PBMCs) used in this example were frozen samples from healthy individuals and were purchased from VERITAS Co., Ltd. The genotypes of the purchased PMBCs in HLA are shown in Table 1 below.

Table 1

[0060] <Step 2: Isolation of CD14 + CD16 - monocytes> The frozen PBMCs were rapidly thawed in a 37°C water bath and washed twice with RPMI-1640 medium containing 10% FBS (RPMI-1640 / 10% FBS medium). Using the EasySep Human Monocyte Isolation Kit (VERITAS, Cat. No. ST-19359), according to the kit's attached instructions, CD14 + CD16 - monocytes were isolated by negative selection. As a result, classical monocytes were isolated from PBMCs.

[0061] <Step 3: Generation of monocyte-derived dendritic cells and addition of human AChRα1> The isolated CD14 + CD16 - monocytes were cultured in RPMI-1640 / 10% FBS medium containing 50 ng / mL GM-CSF and 50 ng / mL IL-4 in 1 well of a 6-well dish at 1.5×10 6Cells were cultured starting at cells / mL and cultured for 7 days in a 5% CO2 incubator at 37°C to generate monocyte-derived dendritic cells. Subsequently, human AChRα1 was added to a concentration of 100 μg / well and cultured for an additional 24 hours. The human AChRα1 used in this step was prepared by performing gene synthesis based on the sequence information of AChRα, inserting the synthetic gene into a transfer vector, co-transfecting the expression vector into SF9 cells, which are insect cells, and then performing production and purification.

[0062] <Step 4: Isolation of HLA-DR Binding Peptides> For the samples after culture, it was carried out according to the protocol of Dynabeads Protein G (Invitrogen, Cat. No. 10004D). 5 μg of anti-HLA-DR antibody was bound to the beads. 0.1% TFA was added to the solution containing HLA-DR to which the obtained peptide was bound, and the peptide was released from HLA-DR by reacting at room temperature for 1 minute. Microcon (registered trademark) Centrifugal Filter Devices (Merck, Cat. No. MRCPRT010) capable of separating 10 kD or less was attached to a dedicated 1.5 mL tube, and the solution containing the recovered peptide and HLA-DR was added thereto, and centrifuged at 14,000×g for 30 minutes to separate HLA-DR and the peptide. The peptide solution in the tube was lyophilized to remove the solvent and stored as a lyophilized sample.

[0063] <Step 5: LC-MS / MS Analysis> Purified water was added to the obtained lyophilized sample and stirred to obtain a sample for measurement. This was subjected to LC-MS / MS analysis under the conditions shown in Table 2 below.

Table 2

[0064] <Step 6: LC-MS / MS Spectrum Data Analysis> For the analysis of the spectral data obtained by LC-MS / MS, Mascot Server ver.2.8 (Matrix Science) was used and the analysis was carried out under the conditions shown in Table 3 below. For the search of the sequences of NCBI prot and AChRα1, peptides satisfying False discovery rate (FDR) p<0.01 were targeted for the search, and FDR was not calculated for the search with only the sequence of AChRα1. Peptides satisfying False discovery rate (FDR) p<0.01 were targeted for the search. Also, Skyline Ver.22.2 (MacCoss Lab.) was used for the quantitative analysis. After importing the spectral data obtained by LC-MS / MS analysis and the identification results obtained from Mascot Sever, the peak area values in the extracted ion chromatogram of the peptides were calculated.

Table 3

[0065] <Step 7: HLA allele identification by sample comparison> For the purpose of identifying HLA alleles for the LC-MS / MS analysis data obtained in Step 6, comparisons were made for each Donor. Peptides commonly identified in the samples having DRB1*03:01 were defined as DRB1*03:01-binding peptides, and peptides commonly identified in the samples having DRB3*01:01 were defined as DRB3*01:01-binding peptides.

[0066] The results showing the HLA-DRB1*03:01 binding peptides thus identified in correspondence with their positions in the amino acid sequence shown in SEQ ID NO: 37 are shown in Table 4. The results showing the HLA-DRB3*01:01 binding peptides thus identified in correspondence with their positions in the amino acid sequence shown in SEQ ID NO: 37 are shown in Table 5. The results showing the HLA binding peptides thus identified as coordinates on human AChRα1 are shown in Figure 1. In Figure 1, the clusters of the identified DRB binding peptides are shown as boxes. The cluster shown with slashes is derived from DRB1*03:01, the cluster shown with dot hatching is derived from DRB3*01:01, and the cluster shown in black represents other HLA origins, respectively. In Figure 1, the light gray portion in the row below the amino acid numbers indicates the AChRα1 sequence, and the dark gray portion indicates the transmembrane portion.

[0067]

Table 4

[0068]

Table 5

[0069] From these results, in samples derived from subjects with HLA-DRB3 genotype 01:01 and samples derived from subjects with HLA-DRB1 genotype 03:01, the peptides shown in SEQ ID NOs: 6 to 15 were commonly found, and these shared the amino acid sequence shown in SEQ ID NO: 1. Therefore, it was clarified 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 the site having the amino acid sequence shown in SEQ ID NO: 1 in human AChRα1 is an epitope for DRB3*01:01 and DRB1*03:01.

[0070] Also, from these results, in the samples derived from subjects with the HLA-DRB3 genotype of 01:01, the peptides shown in SEQ ID NO: 16 (SEQ ID NO: 2) were commonly found. Therefore, the peptide having the amino acid sequence shown in SEQ ID NO: 2 is a DRB3*01:01-binding peptide, and it was revealed that the site having the amino acid sequence shown in SEQ ID NO: 2 in human AChRα1 is an epitope for DRB3*01:01.

[0071] Also, from these results, in the samples derived from subjects with the HLA-DRB3 genotype of 01:01, the peptides shown in SEQ ID NOs: 17 to 21 were commonly found, and these shared the amino acid sequence shown in SEQ ID NO: 3. Therefore, the peptide having the amino acid sequence shown in SEQ ID NO: 3 is a DRB3*01:01-binding peptide, and it was revealed that the site having the amino acid sequence shown in SEQ ID NO: 3 in human AChRα1 is an epitope for DRB3*01:01.

[0072] Also, from these results, in the samples derived from subjects with the HLA-DRB3 genotype of 01:01, the peptides shown in SEQ ID NOs: 22 to 32 were commonly found, and these 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 revealed that the site having the amino acid sequence shown in SEQ ID NO: 4 in human AChRα1 is an epitope for DRB3*01:01.

[0073] From these results, in the samples derived from the subjects with the HLA-DRB3 genotype of 01:01 and the samples derived from the subjects with the HLA-DRB1 genotype of 03:01, the peptides shown in SEQ ID NOs: 33 to 35 were commonly found, and these 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 revealed that the site having the amino acid sequence shown in SEQ ID NO: 5 in human AChRα1 is an epitope for DRB3*01:01 and DRB1*03:01.

Claims

**Claim 1** A method for identifying an epitope that binds to an HLA having a specific genotype, comprising: contacting an antigen protein with a first dendritic cell prepared using classical monocytes derived from a subject in which a first HLA has a first genotype and a second HLA has a second genotype, and then obtaining, from the first dendritic cell, at least one first polypeptide that binds to a first HLA having the first genotype or a second HLA having the second genotype; contacting the antigen protein with a second dendritic cell prepared using classical monocytes derived from a subject in which a first HLA has a first genotype and a second HLA has a third genotype, and then obtaining, from the second dendritic cell, at least one second polypeptide that binds to a first HLA having the first genotype or a second HLA having the third genotype; and determining that a site having the same amino acid sequence as a polypeptide commonly contained in the at least one first polypeptide and the at least one second polypeptide is an epitope that binds to a first HLA having the first genotype in the antigen protein. A method comprising the above steps. **Claim 2** The method according to claim 1, wherein the first HLA is HLA-DRB1 and the second HLA is HLA-DRB3, or the first HLA is HLA-DRB3 and the second HLA is HLA-DRB1. The method according to claim 1. **Claim 3** The method according to claim 1, wherein the first HLA is HLA-DRB1, the second HLA is HLA-DRB3, and the first genotype is 03:

01. **Claim 4** The method according to claim 1, wherein the first HLA is HLA-DRB3, the second HLA is HLA-DRB1, and the first genotype is 01:

01. **Claim 5** The method according to any one of claims 1 to 4, wherein the step of obtaining the at least one first polypeptide and the step of obtaining the at least one second polypeptide are performed using antibodies that bind to the first HLA and the second HLA. **Claim 6** After the step of obtaining the first at least one polypeptide and the step of obtaining the second at least one polypeptide, a method according to any one of claims 1 to 4, comprising a step of determining the amino acid sequences of the polypeptides contained in the first at least one polypeptide and the second at least one polypeptide.

7. The method according to any one of claims 1 to 4, wherein the epitope is an epitope involved in myasthenia gravis.

8. A method for producing a polypeptide that binds to HLA having a specific genotype, comprising a step of identifying an epitope according to the method according to any one of claims 1 to 4, and a step of producing a polypeptide having the same amino acid sequence as the identified epitope.

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