JP2014204675A - Peptide and pharmaceutical composition comprising the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To identify an additional antigen peptide which can suppress unusual activation of immunity through activation of a regulatory T cell, and to provide a medicine used for prevention and/or treatment of HLA-DR4 positive autoimmune diseases, such as rheumatoid arthritis using an epitope concerned.SOLUTION: Provided is a peptide which activates a regulatory T cell with a peptide consisting of specific amino acid sequences, a nucleic acid encoding the peptide and an expression vector comprising the nucleic acid. Provided is a pharmaceutical composition comprising any one of selected from the group consisting of the peptide, the nucleic acid, and the expression vector. An HLA-DR4 positive autoimmune disease is selected from the group consisting of rheumatoid arthritis, polyarthritis type of juvenile idiopathic arthritis, autoimmune thyroiditis, autoimmune hepatitis, insulin-dependent diabetes mellitus (type 1 diabetes mellitus), and multiple sclerosis in the pharmaceutical composition.

Description

  The present invention relates to peptides and the like used for treatment or prevention of autoimmune diseases.

Rheumatoid arthritis (RA) is an autoimmune disease that leads to destruction of bone and cartilage as a result of abnormally activated immunity and persistent systemic arthritis.
Current therapeutic strategies for RA are mainly anti-inflammatory therapies that suppress inflammation, and steroids and methotrexate, which are immunosuppressants, are used. In recent years, anti-cytokine therapy using biological products has also been used. However, since these methods also suppress normal immune functions, complications such as infections are likely to occur, and a sufficient therapeutic effect cannot be obtained in about 30% of patients.

Regarding the cause of RA development, attention is paid to the association with the polymorphism of the HLA-DR gene, and it is known that HLA-DR4 is involved in the development of RA.
The HLA-DR molecule is one of human major histocompatibility complex (MHC) class II molecules. MHC class II molecules are glycoproteins that are expressed in antigen-presenting cells and present antigen peptides to T cells. This antigenic peptide is also called an epitope. When the THC receptor of the CD4 positive T cell recognizes the MHC class II molecule presenting the epitope, the CD4 positive T cell is activated.
CD4-positive T cells proliferate when stimulated by antigens, and effector T cells that activate immunity through the production of inflammatory cytokines such as IL-10 and B cell autoantibody production There are regulatory T cells that are less responsive and suppress immunity through inhibition of the proliferation of other T cells. In autoimmune diseases, the balance between effector T cells and regulatory T cells is lost, and the activation of autoreactive effector T cells is thought to be excessive.

In fact, even in RA, it is known that CD4-positive T cells infiltrate many inflamed joints and are deeply involved in the pathology. In RA, there is an epitope derived from some self-antigen that is likely to be bound and presented to the HLA-DR4 molecule, and the hypothesis that autoreactive effector T cells that recognize the epitope are activated and arthritis develops is supported. ing.
Therefore, it was suggested that identifying an epitope that is presented on the HLA-DR4 molecule and binds to the T cell receptor can be used to prevent or treat RA by generating immune tolerance against that epitope.

Immunoglobulin binding protein (BiP; GenBank AF188611) is known as an RA-related autoantigen. The present inventors have reported that anti-BiP antibodies are increasing in the serum of RA patients (Non-patent Document 1). In addition, there has been a report that experimental arthritis is suppressed by BiP administration (Non-patent Document 2).
As a result of repeated research focusing on BiP, the present inventors have found that a BiP-derived peptide (BiP _336-355 ) becomes an epitope of HLA-DR4, and CD4-positive T cells that recognize BiP _336-355 have a pathological condition of RA. It was confirmed that IL-17 is strongly produced. Furthermore, it has been reported that arthritis can be prevented and treated by inducing immune tolerance to BiP _336-355 and its application to RA treatment (Patent Document 1).
However, since BiP _336-355 is an epitope of effector T cells, immune tolerance is induced by administration, but the risk of activating effector T cells and exacerbating RA cannot be denied. Therefore, development of a safer method has been desired.

International Publication No. 2011/071039

Shoda H. et al, Arthritis Res Ther, 13: R191, 2011 Brownlie RJ. Et al., Arthritis Rheum 54: 854, 2006

  It is an object of the present invention to identify an epitope capable of suppressing abnormal activation of immunity in autoimmune disease by activating regulatory T cells, and to provide a drug using such epitope And

In order to solve the above-mentioned problems, the present inventors administered various BiP-derived peptides to peripheral blood mononuclear cells of HLA-DR4-positive RA patients, and can serve as an index of activation of regulatory T cells. When the concentration was measured, a peptide BiP _reg that highly induced IL-10 production was found.
Since BiP _reg does not induce IL-10 secretion in the presence of anti-HLA-DR antibody, the induction of IL-10 secretion by BiP _reg is induced by a reaction via HLA-DR and T cell receptor. It was thought that there was. In addition, the induction of T cell proliferation by BiP _336-355 is significantly suppressed by the addition of BiP _reg , and the suppression of proliferation induction by BiP _reg is not seen in the presence of anti-IL-10 antibody, The inhibitory action is thought to be _mediated by IL-10, and the above results strongly suggested that BiP _reg activates regulatory T cells that produce IL-10.
In addition, when BiP _reg is orally administered to a mouse arthritis model, the arthritis score and the frequency of onset are improved; in collagen-induced arthritic mice, serum anti-type II collagen antibody titer is significantly decreased by administration of BiP _reg , etc. As a result, the present invention was completed.
That is, the present invention
[1] Any of the following peptides:
(i) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1;
(ii) a peptide in which one or several amino acids are deleted, added or substituted in the amino acid sequence represented by SEQ ID NO: 1, and which activates regulatory T cells; and
(iii) a peptide having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 and activating regulatory T cells;
[2] The peptide of the above-mentioned [1], which is the peptide of (ii) or (iii) and includes the following sequence:
Val-Xaa ₁ -Xaa ₂ -Lys-Xaa ₃ -Xaa ₄ -Xaa ₅ -Xaa ₆ -Xaa ₇ -Arg (SEQ ID NO: 6)
[Wherein, Xaa _{1 to} Xaa ₇ each independently represents an arbitrary amino acid. ];
[3] Any of the following peptides:
(iv) a peptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 2 to 5;
(v) a peptide in which one or several amino acids are deleted, added or substituted in the amino acid sequence represented by any one of SEQ ID NOs: 2 to 5, and which activates regulatory T cells; as well as
(vi) a peptide having 80% or more sequence identity with the amino acid sequence represented by any one of SEQ ID NOs: 2 to 5 and activating regulatory T cells;
[4] A nucleic acid encoding the peptide according to any one of [1] to [3] above;
[5] An expression vector comprising the nucleic acid according to [4] above;
[6] Any one selected from the group consisting of the peptide according to any one of [1] to [3] above, the nucleic acid according to [4] above, and the expression vector according to [5] above A pharmaceutical composition comprising one;
[7] The pharmaceutical composition according to the above [6], which is used for treatment or prevention of an HLA-DR4 positive autoimmune disease; and [8] the HLA-DR4 positive autoimmune disease is rheumatoid arthritis, idiopathic The drug according to [7] above, selected from the group consisting of polyarthritis type of juvenile arthritis, autoimmune thyroiditis, autoimmune hepatitis, insulin-dependent diabetes (type 1 diabetes), and multiple sclerosis Composition,
About.

Since the peptide according to the present invention is selected from the BiP protein only for the portion that activates regulatory T cells, it activates the regulatory T cells efficiently and suppresses abnormal immunity without suppressing normal immunity. Suppresses immune activation.
Therefore, the peptide according to the present invention is useful as a preventive or therapeutic agent for autoimmune diseases.

FIG. 1A shows the results of examining IL-10 production induction from PBMC of RA patients by various BiP-derived peptide stimuli. FIG. 1B shows the results of examining the induction of IL-10 production from PBMC in healthy individuals (HD) and RA patients (RA) by BiP _reg . FIG. 1C shows the results of examining IL-10 production induction by BiP _reg in the presence or absence of anti-HLA-DR antibody. FIG. 2A shows the results of examining the proliferation-inducing ability of PBMC by BiP _336-355 and the inhibitory effect on this proliferation-inducing ability by BiP _reg . FIG. 2B shows the results of examining the inhibitory effect of BiP _{reg on} the ability of BiP _336-355 to induce PBMC proliferation in the presence of various concentrations of anti-IL-10 inhibitory antibodies. FIG. 3A shows the results of measuring the 50% inhibitory concentration (IC ₅₀ ) of the binding between BiP _reg or a peptide in which one of the amino acids of BiP _reg is substituted with Ala and the HLA-DRB1 * 0405 molecule. FIG. 3B shows the results of measuring the binding between BiP _reg and HLA-DRB1 * 0405 molecules by HPLC. A shows the result of orally administering BiP _reg to collagen-induced arthritis (CIA) mice and measuring the arthritis score. B shows the results of measuring the incidence of rheumatoid arthritis by orally administering BiP _reg to CIA mice. FIG. 5 shows the results of oral administration of BiP _reg to CIA mice and measuring the anti-type II collagen antibody titer in serum. A shows the result of proliferating the CIA mouse spleen CD4-positive T cells restimulated with BiP and type II collagen and measuring the proliferation reaction. B shows results of restimulation of CIA mouse spleen CD4-positive T cells with BiP, BiP _reg and type II collagen and measuring supernatant IL-10.

(peptide)
The peptide according to the present invention activates regulatory T cells, and is any of the following (i) to (iii).
(i) a peptide comprising an amino acid sequence represented by the following sequence;
DNQPTVTIKVYEGERPLTKD (SEQ ID NO: 1)
EDKKEDVGTVVGIDLGTTYS (SEQ ID NO: 2)
KGTGNKNKITITNDQNRLTP (SEQ ID NO: 3)
NRLTPEEIERMVNDAEKFAE (SEQ ID NO: 4)
GSAGPPPTGEEDTAELHHHH (SEQ ID NO: 5)
(ii) a peptide in which one or several amino acids are deleted, added or substituted in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 5; and
(iii) A peptide having 80% or more sequence identity with the amino acid sequence represented by any one of SEQ ID NOs: 1 to 5.

  Regulatory T cells are T cells characterized by suppressing immune activation. Typical regulatory T cells include CD4 positive CD25 positive FOXP3 positive regulatory T cells and Tr1 cells that produce IL-10 Etc. have been reported. It is known that when regulatory T cells are removed from a living body, various autoimmune diseases develop, and when regulatory T cells are administered at that time, the onset of autoimmune diseases is prevented. Patients with autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, autoimmune hepatitis, autoimmune polyendocrinopathy syndrome (APS) type-II, psoriasis, myasthenia gravis, Kawasaki disease, ANCA-related vasculitis, and mouse autoimmunity Also in disease models, functional or quantitative abnormalities of regulatory T cells are observed.

As used herein, “activate regulatory T cells” means that at least one of the immunoregulatory effects by regulatory T cells is enhanced. Examples of immunoregulatory effects by regulatory T cells include, but are not limited to, secretion of immunosuppressive cytokines such as TGF-β and IL-10.
“Immunoregulatory action is increased” includes a case where it is higher than a normal state and a case where a lowered state is recovered. Moreover, in addition to the case where it becomes statistically significant, the case where it can be judged that those skilled in the art tend to be high is included.

  Further, the peptide according to the present invention is a peptide in which one or several amino acids are deleted, added or substituted in the amino acid sequence represented by any of SEQ ID NOs: 1 to 5, or SEQ ID NOs: 1 to 1 A peptide having 80% or more sequence identity with the amino acid sequence represented by any one of 5 may be presented to HLA-DR4 in an antigen-presenting cell, that is, bind to HLA-DR4. Whether or not it binds to HLA-DR4 can be confirmed by a known method for detecting an interaction between proteins. Examples include, but are not limited to, affinity chromatography methods, protein microarrays, immunoprecipitation methods, surface plasmon resonance methods, and the like.

  Further, the peptide according to the present invention is a peptide in which one or several amino acids are deleted, added or substituted in the amino acid sequence represented by any of SEQ ID NOs: 1 to 5, or SEQ ID NOs: 1 to 1 A peptide having 80% or more sequence identity with the amino acid sequence represented by any one of 5 may bind to the T cell receptor of regulatory T cells. Whether or not it binds to the T cell receptor of regulatory T cells can be confirmed by a known method for detecting the interaction between proteins, such as affinity chromatography, protein microarray, immunoprecipitation Examples thereof include, but are not limited to, surface plasmon resonance.

As used herein, a peptide refers to a compound in which two or more amino acids are linked by peptide bonds.
In the present specification, “amino acid” is used in its broadest sense, and includes derivatives and artificial amino acids in addition to natural amino acids. As used herein, amino acids include naturally occurring proteinaceous L-amino acids; unnatural amino acids; chemically synthesized compounds having characteristics known in the art that are characteristic of amino acids. Examples of non-natural amino acids include α, α-disubstituted amino acids (such as α-methylalanine), N-alkyl-α-amino acids, D-amino acids, β-amino acids, α- Hydroxy acids, amino acids whose side chain structure is different from the natural type (norleucine, homohistidine, etc.), amino acids with extra methylene in the side chain (such as “homo” amino acids, homophenylalanine, homohistidine, etc.), and carvone in the side chain Examples include, but are not limited to, amino acids in which the acid functional amino acid is substituted with a sulfonic acid group (such as cysteic acid).

  In the present specification, amino acids may be represented by conventional one-letter code or three-letter code. Amino acids represented by one-letter code or three-letter code may include respective mutants and derivatives.

  In the present specification, when referring to “a peptide in which one or several amino acids are deleted, added, or substituted”, the number of amino acids to be deleted is particularly limited as long as the peptide activates regulatory T cells. Although not limited, for example, it can be 1, 2, 3, 4, or 5. The position of deletion, addition, or substitution may be at any end of the peptide, in the middle, at one place, or at two or more places. Two or more of deletion, addition, and substitution may occur.

  In the present specification, when the phrase “has 80% or more sequence identity with the amino acid sequence represented by any of SEQ ID NOs: 1 to 5”, the sequence identity is 80% or more, 85% or more, 90% or more, Including cases of 95% or more and 98% or more. Sequence identity can be examined using various sequence alignment algorithms (eg, BLAST).

Moreover, the peptide according to the present invention is the peptide of the above (ii) or (iii) according to SEQ ID NO: 1, and may include the following sequences.
Val-Xaa ₁ -Xaa ₂ -Lys-Xaa ₃ -Xaa ₄ -Xaa ₅ -Xaa ₆ -Xaa ₇ -Arg (SEQ ID NO: 6)
[Wherein, Xaa _{1 to} Xaa ₇ each independently represents an arbitrary amino acid. ].
The N-terminal Val of SEQ ID NO: 6 corresponds to Val at position 6 of SEQ ID NO: 1. As shown in the Examples described later, the 6th position Val, the 9th position Lys, and the 15th position Arg of SEQ ID NO: 1 are important for binding to HLA-DR4.

  The C-terminal of the peptide of the present invention may be an amide or ester as well as a carboxyl group or a carboxylate group. The peptides of the present invention also include peptide salts. As salts of peptides, salts with physiologically acceptable bases and acids are used. For example, addition salts of inorganic acids (hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc.), organic salts Addition salts of acids (p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carboxylic acid, succinic acid, citric acid, benzoic acid, acetic acid, etc.), inorganic bases (ammonium hydroxide or alkali or Alkaline earth metal hydroxides, carbonates, bicarbonates, etc.), amino acid addition salts, and the like.

The peptide of the present invention may be a fusion peptide with another functional peptide.
For example, a chimeric peptide with another peptide can be used for the purpose of inhibiting degradation by a host-derived protease, imparting membrane permeability, and the like.
When imparting membrane permeability, it may be fused with a membrane-penetrating peptide (CPP). CPP is a generic name for peptides with affinity for cell membranes and translocation into cells, and is a domain consisting of 11 amino acids of the trans-activator of transcription protein (TAT protein) expressed by HIV-1 virus. Protein-transduction domain (PTD), Drosophila Antennapedia, herpesvirus-derived VP22, oligoarginine and the like are known. In general, many CPPs have a high content of basic amino acids such as arginine, lysine and histidine.

  In addition, the peptide of the present invention is a peptide to which modifications such as phosphorylation, methylation, acetylation, adenylylation, ADP ribosylation, and glycosylation are added as long as the problem of the present invention is solved. Also good. It may be fused with other peptides or proteins.

The peptide of the present invention is extracted from natural products and decomposed; liquid phase method, solid phase method, chemical synthesis method such as hybrid method combining liquid phase method and solid phase method; It can be produced by a peptide production method.
When extracting from natural products, it can be prepared from tissues or cells of, for example, humans or other animals (eg, mice, rats, rabbits, horses, sheep, dogs, monkeys, pigs, etc.). In this case, the tissue or cells are homogenized and then extracted with acid, etc., and the extract is purified by salting out, dialysis, gel filtration, reverse phase chromatography, ion exchange chromatography, affinity chromatography, etc. After release, it may be cleaved with protease or peptidase.

In the solid-phase method, for example, the hydroxyl group of a resin having a hydroxyl group is esterified with the carboxy group of the first amino acid (usually the C-terminal amino acid of the target peptide) in which the α-amino group is protected with a protecting group. . As the esterification catalyst, known dehydration condensing agents such as 1-mesitylenesulfonyl-3-nitro-1,2,4-triazole (MSNT), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI) and the like can be used.
Next, the α-amino group protecting group of the first amino acid is removed, and a second amino acid in which all functional groups other than the carboxy group of the main chain are protected is added to activate the carboxy group. , Combining the first and second amino acids. Further, the α-amino group of the second amino acid is deprotected, a third amino acid in which all functional groups other than the carboxy group of the main chain are protected is added, the carboxy group is activated, and the second and Bind a third amino acid. This is repeated, and when a peptide having a desired length is synthesized, all functional groups are deprotected.

Resins for solid phase synthesis include Merrifield resin, MBHA resin, Cl-Trt resin, SASRIN resin, Wang resin, Rink amide resin, HMFS resin, Amino-PEGA resin (Merck), HMPA-PEGA resin (Merck) Is mentioned. These resins can be used after being washed with a solvent (dimethylformamide (DMF), 2-propanol, methylene chloride, etc.).
Protecting groups for α-amino groups include benzyloxycarbonyl (Cbz or Z) group, tert-butoxycarbonyl (Boc) group, fluorenylmethoxycarbonyl (Fmoc) group, benzyl group, allyl group, allyloxycarbonyl (Alloc ) Group and the like. The Cbz group can be deprotected by hydrofluoric acid, hydrogenation, etc., the Boc group can be deprotected by trifluoroacetic acid (TFA), and the Fmoc group can be deprotected by treatment with piperidine.
For protecting the α-carboxy group, methyl ester, ethyl ester, benzyl ester, tert-butyl ester, cyclohexyl ester and the like can be used.
As other functional groups of amino acids, the hydroxy group of serine or threonine can be protected with a benzyl group or a tert-butyl group, and the hydroxy group of tyrosine is protected with a 2-bromobenzyloxycarbonyl dilute or tert-butyl group. The amino group of the lysine side chain and the carboxy group of glutamic acid or aspartic acid can be protected in the same manner as the α-amino group and α-carboxy group.

  The activation of the carboxy group can be performed using a condensing agent. Examples of condensing agents include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC or WSC), and (1H-benzotriazol-1-yloxy) tris. (Dimethylamino) phosphonium hexafluorophosphate (BOP), 1- [bis (dimethylamino) methyl] -1H-benzotriazolium-3-oxide hexafluorophosphate (HBTU) and the like.

The peptide chain can be cleaved from the resin by treatment with an acid such as TFA or hydrogen fluoride (HF).
When the peptide chain is long, the peptide synthesized by the ligation method (for example, the native chemical ligation method) can be linked after being synthesized to some extent by the solid phase method.

  A method for producing a polypeptide by a genetic recombination method can be performed using the nucleic acid of the present invention as described later.

(Nucleic acid)
The nucleic acid of the present invention is a nucleic acid encoding the peptide of the present invention, and may be DNA or RNA.
The nucleic acid of the present invention can be prepared by a known method or a method analogous thereto. For example, it can be synthesized by an automatic synthesizer. In order to insert the obtained DNA into a vector, a restriction enzyme recognition site may be added, or a base sequence encoding an amino acid sequence for cutting out the resulting peptide chain with an enzyme or the like may be incorporated.
When the peptide of the present invention is fused with another peptide, the nucleic acid of the present invention also includes a nucleic acid encoding the peptide to be fused.

(Expression vector)
The expression vector of the present invention contains the nucleic acid of the present invention. The nucleic acid of the present invention can be inserted as it is, after digestion with a restriction enzyme, or with a linker added, downstream of the promoter of the expression vector. As vectors, E. coli-derived plasmids (pBR322, pBR325, pUC12, pUC13, pUC18, pUC19, pUC118, pBluescript II, etc.), Bacillus subtilis-derived plasmids (pUB110, pTP5, pC1912, pTP4, pE194, pC194 etc.), yeast-derived plasmids ( pSH19, pSH15, YEp, YRp, YIp, YAC, etc., bacteriophage (λ phage, M13 phage, etc.), virus (retrovirus, vaccinia virus, adenovirus, adeno-associated virus (AAV), Sendai virus, cauliflower mosaic virus, Tobacco mosaic virus, baculovirus, etc.) and cosmids.

The promoter can be appropriately selected depending on the type of host. When the host is an animal cell, for example, a promoter derived from SV40 (simian virus 40) or a promoter derived from CMV (cytomegalovirus) can be used. When the host is Escherichia coli, trp promoter, T7 promoter, lac promoter, etc. may be used.
Expression vector encodes DNA replication origin (ori), selection marker (antibiotic resistance, auxotrophy, etc.), enhancer, splicing signal, poly A addition signal, tag (FLAG, HA, GST, GFP, etc.) Nucleic acid or the like may be incorporated.

  The expression vector of the present invention may be used to transform a host in vitro as described below, and to express the polypeptide of the present invention. Can also be used to express.

(Transformant)
The peptide of the present invention can be expressed by transforming a suitable host with the expression vector of the present invention. The host can be appropriately selected in relation to the vector, and for example, Escherichia coli, Bacillus subtilis, Bacillus genus, yeast, insect or insect cell, animal cell and the like are used. As animal cells, for example, HEK293T cells, CHO cells, COS cells, myeloma cells, HeLa cells, and Vero cells can be used. Transformation can be performed according to a known method such as a lipofection method, a calcium phosphate method, an electroporation method, a microinjection method, or a particle gun method, depending on the type of host.
By culturing the obtained transformant according to a conventional method, the desired polypeptide is expressed. What is necessary is just to select the culture medium used at this time according to a host suitably. For example, in the case of HEK293T cells, a DMEM medium supplemented with serum such as fetal calf serum can be used.

For peptide purification from transformant cultures, cultured cells are collected, suspended in an appropriate buffer, disrupted by sonication, freeze-thawing, etc., and roughly extracted by centrifugation or filtration. Obtain a liquid. When the peptide is secreted into the culture solution, the supernatant is collected.
Purification from a crude extract or culture supernatant is a known method or a method equivalent thereto (for example, salting out, dialysis method, ultrafiltration method, gel filtration method, SDS-PAGE method, ion exchange chromatography, affinity chromatography, Reversed phase high performance liquid chromatography).
The obtained peptide may be converted from a free form to a salt or from a salt to a free form by a known method or a method analogous thereto.

The translation synthesis system may be a cell-free translation system. Cell-free translation systems include, for example, ribosomal proteins, aminoacyl-tRNA synthetase (ARS), ribosomal RNA, amino acids, rRNA, GTP, ATP, translation initiation factor (IF) elongation factor (EF), termination factor (RF), and ribosome Includes regeneration factor (RRF), as well as other factors required for translation. In order to increase the expression efficiency, an E. coli extract or wheat germ extract may be added. In addition, a rabbit erythrocyte extract or an insect cell extract may be added.
By supplying energy continuously to a system containing these using dialysis, peptides of several hundred μg to several mg / mL can be produced. In order to perform transcription from DNA together, a system containing RNA polymerase may be used. As cell-free translation systems on the market, E. coli-derived systems such as RTS-100 (registered trademark) from Roche Diagnostics, PURESYSTEM (registered trademark) from PGI, etc. For example, those from Zoigene or Cell Free Science can be used.
According to the cell-free translation system, the expression product can be obtained in a highly purified form without purification.

(Pharmaceutical composition)
The pharmaceutical composition according to the present invention includes the above-described peptide, nucleic acid, or expression vector of the present invention. The pharmaceutical composition according to the present invention can induce immune tolerance and suppress abnormal activation of immunity through the activation of regulatory T cells. Therefore, the pharmaceutical composition according to the present invention is useful for autoimmune diseases.
As used herein, “immunological tolerance” refers to an immune tolerance in a narrow sense in which an antigen is administered to an individual in advance, such as orally, and suppresses an immune response to the antigen itself, and an antigen that is not pathogenic to an individual having a specific disease. And tolerant in a broad sense to suppress the disease by suppressing the immune response to other pathogenic antigens.
The target disease of the pharmaceutical composition of the present invention is not particularly limited as long as the pharmaceutical composition of the present invention exhibits a preventive or therapeutic effect. Here, prevention or treatment means that in addition to the cure or remission of the disease, at least one of prevention or delay of the onset, prevention or delay of the progression of the disease, alleviation of at least one symptom associated with the disease, etc. Say.

The pharmaceutical composition according to the present invention is useful for the treatment or prevention of autoimmune diseases, and is considered to be particularly useful for the treatment or prevention of autoimmune diseases that have more than the amount or function of regulatory T cells. It is considered useful for the treatment or prevention of autoimmune diseases related to HLA-DR4.
Autoimmune diseases include rheumatoid arthritis (RA), systemic lupus erythematosus, scleroderma, polymyositis, Sjogren's syndrome, ANCA-related vasculitis, Behcet's disease, Kawasaki disease, mixed cryoglobulinemia, multiple sclerosis , Guillain-Barre syndrome, myasthenia, type 1 diabetes, Graves' disease, Hashimoto's disease, Addison's disease, IPEX, APS type-II, autoimmune myocarditis, interstitial pneumonia, bronchial asthma, autoimmune hepatitis, primary Examples include biliary cirrhosis, Crohn's disease, ulcerative colitis, psoriasis, atopic dermatitis, hemolytic anemia, autoimmune thyroiditis, idiopathic juvenile arthritic polyarthritis.
Among these, autoimmune diseases that are known to be associated with abnormalities of regulatory T cells include RA, systemic lupus erythematosus, Kawasaki disease, autoimmune hepatitis, APS type-II, psoriasis, severe muscle Examples include asthenia, Kawasaki disease, ANCA-related vasculitis, mixed cryoglobulinemia, multiple sclerosis, and IPEX.
Autoimmune diseases related to HLA-DR4 include RA, idiopathic juvenile arthritis polyarthritis, autoimmune thyroiditis, autoimmune hepatitis, type 1 diabetes, and multiple sclerosis. It is not limited.
The pharmaceutical composition of the present invention is particularly useful for suppressing inflammation of arthritis and inhibiting bone destruction in RA. The pharmaceutical composition of the present invention is also preferably administered to RA patients in which anti-BiP antibodies have appeared.
The pharmaceutical composition of the present invention may be used in combination with other drugs and treatments useful for the above diseases.

The dosage form of the pharmaceutical composition according to the present invention is not particularly limited, and it may be orally or parenterally administered. Parenteral administration includes, for example, intramuscular injection, intravenous injection, subcutaneous injection, transdermal administration, transmucosal administration (nasal, oral cavity, ocular, pulmonary, vaginal, transrectal) administration. Etc. are raised.
In particular, when the pharmaceutical composition according to the present invention is orally administered, the mechanism of intestinal tolerance is utilized to enhance the action of self-antigen BiP-specific regulatory T cells, and through the enhancement of IL-10 production, which is a regulatory cytokine. It is possible to improve autoimmune abnormalities.

When the pharmaceutical composition according to the present invention contains a peptide, various modifications can be made in view of the property of being easily metabolized and excreted. For example, polyethylene glycol (PEG) or a sugar chain can be added to a peptide to increase the residence time in blood and reduce antigenicity. In addition, biodegradable polymer compounds such as polylactic acid / glycol (PLGA), porous hydroxyapatite, liposomes, surface modified liposomes, emulsions prepared with unsaturated fatty acids, nanoparticles, nanospheres, etc. are sustained release bases It may be used as a polypeptide. In the case of transdermal administration, a weak current can be passed through the skin surface to penetrate the stratum corneum (iontophoresis method).
Further, when the pharmaceutical composition according to the present invention contains a peptide, it may be administered (for example, intravenously administered) in a state presented to dendritic cells having an inhibitory function.

  When the pharmaceutical composition according to the present invention contains a nucleic acid or an expression vector, various DNA introduction methods can be used. For example, a viral vector such as a retrovirus (such as a lentivirus), an adenovirus, an adeno-associated virus, or a Sendai virus is used as an expression vector, and this is injected into the affected tissue by injection, inhalation, application, or the patient's own For example, a method can be used in which a blood cell is once taken out of the body, the vector is introduced outside the body, and then returned to the patient.

The above-mentioned pharmaceutical composition may be used as it is, or may be formulated by adding pharmaceutically acceptable carriers, excipients, additives and the like. Examples of the dosage form include liquids (for example, injections), dispersions, suspensions, tablets, pills, powders, suppositories, powders, fine granules, granules, capsules, syrups, lozenges, Examples include inhalants, ointments, eye drops, nasal drops, ear drops, and poultices.
Formulation includes, for example, excipients, binders, disintegrants, lubricants, solubilizers, solubilizers, colorants, flavoring agents, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjustments It can be carried out by a conventional method using an agent, preservative, antioxidant and the like as appropriate.
Examples of ingredients used for formulation include purified water, saline, phosphate buffer, dextrose, glycerol, ethanol and other pharmaceutically acceptable organic solvents, animal and vegetable oils, lactose, mannitol, glucose, sorbitol, crystalline cellulose , Hydroxypropyl cellulose, starch, corn starch, silicic anhydride, magnesium magnesium silicate, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium carboxymethyl cellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch , Pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, tragacanth, casein, agar, polyethylene glycol, diglyse Emissions, glycerine, propylene glycol, vaseline, paraffin, octyldodecyl myristate, isopropyl myristate, higher alcohol, stearyl alcohol, stearic acid, human serum albumin, and the like without limitation.
When the pharmaceutical composition contains a peptide, surfactants such as polyoxyethylene lauryl ethers, sodium lauryl sulfate, and saponin are used as an absorption accelerator for improving absorption of a poorly absorbable drug that is difficult to be absorbed through the mucosa such as a peptide. Bile salts such as glycocholic acid, deoxycholic acid and taurocholic acid; chelating agents such as EDTA and salicylic acids; fatty acids such as caproic acid, capric acid, lauric acid, oleic acid, linoleic acid and mixed micelles; enamine derivatives N-acyl collagen peptides, N-acyl amino acids, cyclodextrins, chitosans, nitric oxide donors, and the like can be used.

Pills or tablets can also be coated with sugar coatings, gastric and enteric substances.
The injection can contain distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, alcohols and the like. Furthermore, wetting agents, emulsifiers, dispersants, stabilizers, solubilizers, solubilizers, preservatives and the like can be added.

  When the pharmaceutical composition of the present invention is administered to mammals (eg, humans, mice, rats, guinea pigs, rabbits, dogs, horses, monkeys, pigs, etc.), particularly humans, the dosage may vary depending on symptoms, patient age, sex It varies depending on body weight, sensitivity difference, administration method, administration interval, active ingredient type, formulation type, and is not particularly limited, for example, 30 μg to 1000 mg, 100 μg to 500 mg, 100 μg to 100 mg divided into 1 time or several times Can be administered. In the case of injection administration, 1 μg / kg to 3000 μg / kg, 3 μg / kg to 1000 μg / kg may be administered once or divided into several times depending on the weight of the patient.

(Method of treatment)
The present invention also includes a method for treating or preventing an autoimmune disease comprising the step of administering a peptide, nucleic acid or expression vector according to the present invention to a subject.

  The disclosures of all patent and non-patent documents cited herein are hereby incorporated by reference in their entirety.

  EXAMPLES Hereinafter, although this invention is demonstrated concretely based on an Example, this invention is not limited to this at all. Those skilled in the art can change the present invention into various modes without departing from the meaning of the present invention, and such changes are also included in the scope of the present invention.

1. Screening for BiP-derived epitopes that induce IL-10 production
Peripheral blood from HLA-DR4-positive RA patients was collected by venous blood collection, and peripheral blood mononuclear cells (PBMC) were separated by density centrifugation with Ficoll-Paque, and then RPMI1640 medium supplemented with 10% FCS PBMC ( Gibco) was adjusted to 1 x 10 ⁶ / mL, BiP-derived peptide 10 mg / mL was added, and then cultured on a Corning 96 well plate at 37 ° C in a 5% CO ₂ incubator for 72 hours . The IL-10 concentration in the culture supernatant was measured by ELISA (eBioscience) according to the kit protocol. The results are shown in FIG. 1A. As shown in FIG. 1A, peptides having the following amino acid sequences most strongly induced IL-10 production in PBMCs (including T cells) of RA patients. This peptide was used as BiP _reg in subsequent experiments. As shown in FIG. 1B, BiP _reg peptide induced IL-10 production from PBMC in RA and healthy individuals (Healthy donor (HD)).
BiP _reg : DNQPTVTIKVYEGERPLTKD (SEQ ID NO: 1)
The following sequences also induced IL-10 production from PBMC.
BiP A: EDKKEDVGTVVGIDLGTTYS (SEQ ID NO: 2)
BiP F: KGTGNKNKITITNDQNRLTP (SEQ ID NO: 3)
BiP G: NRLTPEEIERMVNDAEKFAE (SEQ ID NO: 4)
BiP I: GSAGPPPTGEEDTAELHHHH (SEQ ID NO: 5)
Moreover, as shown in FIG. 1C, when an anti-HLA-DR inhibitory antibody (clone L243, 10 mg / mL) was added and cultured, induction of IL-10 production by BiP _reg was significantly suppressed. This strongly suggested that IL-10 secretion was induced by a reaction via HLA-DR and T cell receptor.

2. Induction of PBMC proliferation by BiP _336-355 peptide and inhibition of proliferation induction by BiP _reg
Peripheral blood mononuclear cells (PBMC) from HLA-DR4-positive RA patients, _add BiP _336-355 peptide and BiP _reg peptide 10 μg / mL to 1 x 10 ⁶ / mL, incubate for 72 hours, and add ³ H-Thymidine Proliferative response was measured by the uptake method 18 hours after the addition.
The results are shown in FIGS. As shown in FIG. 2A, BiP _336-355 (RSTMKPVQKVLEDSDLKKSD; SEQ ID NO: 7) induced PBMC proliferation. On the other hand, if in addition to the BiP _336-355 adding an equal volume of BiP _reg, proliferation of PBMC by BiP _336-355 was significantly suppressed.
In addition, as shown in FIG. 2B, this inhibitory effect by BiP _reg was neutralized in a dose-dependent manner by the addition of an anti-IL-10 inhibitory antibody (clone 25209). It was strongly suggested that BiP _reg activated IL-10 production regulatory T cells.

3. Binding of BiP _reg to HLA-DRB1 * 0405 molecule
A pMT vector of HLA-DRA * 0101, HLA-DRB1 * 0405 gene was prepared, and HLA-DRB1 * 0405 protein was prepared by introducing the gene into cell S2 cells using cellfectin (invitrogen). HLA-DRB1 * 0405 molecule, HA307-319 (PKYVKQNTLKLAT (SEQ ID NO: 8)), which has been previously known to bind to biotinylated peptide, and BiP _reg peptide with part of the sequence changed to an alanine residue After reacting at 37 degrees for 16 hours, streptavidin-alkaline phosphatase was reacted, and the enzyme activity was measured. That is, it is conceivable that the enzyme activity decreases in a concentration-dependent manner due to the binding of the BiP _reg peptide to the HLA-DR molecule, and the binding strength can be estimated by IC ₅₀ measurement.
The results are shown in FIG. 3A. As shown in FIG. 3A, BiP _{reg had} an IC ₅₀ of 336 nM, indicating moderate binding ability to HLA-DRB1 * 0405 molecules. As a result of measuring the binding ability by substituting each amino acid with Ala, the 6th position Val, the 9th position Lys, and the 15th position Arg of SEQ ID NO: 1 are important for binding to the HLA-DRB1 * 0405 molecule. It was suggested that
In addition, when the peptide bound to the HLA-DRB1 * 0405 molecule, the presence or absence of binding was confirmed by a shift in HPLC migration time due to the change in molecular weight.
The results are shown in FIG. 3B. As shown in FIG. 3B, as a result of measurement by HPLC, it was confirmed that the HLA-DRB1 * 0405 molecule and BiP _reg were actually bound.

4). Experimental study on suppression of arthritis in mice by administration of BiP _reg
DBA / 1J mice (female, 6 weeks old) were immunized subcutaneously with bovine type II collagen 100 mg (Chondrex) and Complete Freund's adjuvant (Chondrex) 50 ml.On day 21, bovine type II collagen 100 mg and incomplete Freund's adjuvant (Chondrex) 50 ml In collagen-induced arthritis (CIA) boosting immunization, place the group that orally administers 200 mg BiP _reg peptide for 5 consecutive days from the 28th day immediately before the onset, and the group that orally administers PBS in an equal amount (control). The score and incidence were observed.
The results are shown in FIG. Compared to the control, both the arthritis score (FIG. 4A) and the onset frequency (FIG. 4B) were improved in the BiP _reg administration group.

5. Decrease of anti-type II collagen antibody by BiP _reg administration
In CIA mice, antibodies against type II collagen are thought to cause arthritis, and thus anti-type II collagen antibody titers were measured in the serum of mice administered with BiP _reg .
Serum was collected on the 42nd day after the first immunization of CIA mice, coated with 10 mg / mL type II collagen for 16 hours, and reacted with 100% diluted serum for 1 hour at room temperature on a plate blocked with 3% skim milk. The anti-mouse IgG-HRP antibody diluted 3000 times was allowed to react at room temperature for 1 hour, and the substrate was developed with TMB solution to measure the serum anti-type II collagen IgG antibody titer (ELISA method).
The results are shown in FIG. In the BiP _reg oral administration group (CIA BiP _reg OT +), a significant decrease in the anti-type II collagen antibody titer was observed. This suggests that BiP _reg suppresses abnormal activation of immunity through the action on regulatory T cells.

5. Effect of oral administration of BiP _{reg on} T cells
Spleen CD4-positive T cells on day 42 after the initial immunization with CIA mice were separated by negative selection by the MACS method and cultured at a concentration of 1 × 10 ⁵ / mL for 72 hours. At the same time, spleen cells after irradiation (1 × 10 ⁶ / mL) were added as antigen-presenting cells, and BiP, BiP _reg peptide, and type II collagen 10 mg / mL were added as antigens. After 72 hours, cell proliferation was measured by ³ H-thymidine method. The culture supernatant was collected and the IL-10 concentration was measured by ELISA. A group of mice that were not immunized (no immunized) was placed.
The results are shown in FIG. In BiP _reg oral administration group (CIA BiP _reg OT +), as compared to the control group (CIA), CD4-positive T-cell proliferation to type II collagen (BC2) and BiP protein is suppressed (FIG. 6A) BiP and BiP _reg Reacted IL-10 production was increased (FIG. 6B).

SEQ ID NO: 1 represents the amino acid sequence of BiP _reg .
SEQ ID NO: 2 represents the amino acid sequence of BiP A.
SEQ ID NO: 3 represents the amino acid sequence of BiPF.
SEQ ID NO: 4 represents the amino acid sequence of BiPG.
SEQ ID NO: 5 represents the amino acid sequence of BiP I.
SEQ ID NO: 6 is a general formula of an amino acid sequence of one embodiment of a peptide according to the present invention.
SEQ ID NO: 7 represents the amino acid sequence of BiP _336-355 .
SEQ ID NO: 8 represents the amino acid sequence of HA307-319.

Claims (8)

Any of the following peptides:
(i) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1;
(ii) a peptide in which one or several amino acids are deleted, added or substituted in the amino acid sequence represented by SEQ ID NO: 1, and which activates regulatory T cells; and
(iii) A peptide having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1 and activating regulatory T cells. The peptide according to claim 1, which is the peptide of (ii) or (iii) and comprises the following sequence:
Val-Xaa ₁ -Xaa ₂ -Lys-Xaa ₃ -Xaa ₄ -Xaa ₅ -Xaa ₆ -Xaa ₇ -Arg (SEQ ID NO: 6)
[Wherein, Xaa _{1 to} Xaa ₇ each independently represents an arbitrary amino acid. ]. Any of the following peptides:
(iv) a peptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 2 to 5;
(v) a peptide in which one or several amino acids are deleted, added or substituted in the amino acid sequence represented by any one of SEQ ID NOs: 2 to 5, and which activates regulatory T cells; as well as
(vi) A peptide having 80% or more sequence identity with the amino acid sequence represented by any one of SEQ ID NOs: 2 to 5 and activating regulatory T cells.   A nucleic acid encoding the peptide according to any one of claims 1 to 3.   An expression vector comprising the nucleic acid according to claim 4.   A pharmaceutical composition comprising any one selected from the group consisting of the peptide according to any one of claims 1 to 3, the nucleic acid according to claim 4, and the expression vector according to claim 5.   The pharmaceutical composition according to claim 6, which is used for treatment or prevention of an HLA-DR4 positive autoimmune disease.   The HLA-DR4 positive autoimmune diseases include rheumatoid arthritis, idiopathic juvenile arthritis polyarthritis, autoimmune thyroiditis, autoimmune hepatitis, insulin-dependent diabetes (type 1 diabetes), and multiple sclerosis The pharmaceutical composition according to claim 7, selected from the group consisting of: