JP2008120740A - Cd8t cell activation-inhibiting agent, and therapeutic agent for rheumatism and dna vaccine for treating rheumatism comprising the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a CD8T cell activation-inhibiting agent, and a therapeutic agent for rheumatism and a DNA vaccine for treating rheumatism. <P>SOLUTION: The CD8T cell activation-inhibiting agent comprises a Jagged 1 gene or a Jagged 1 protein. The CD8T cell activation-inhibiting agent inhibits activation of a CD8T cell and accordingly makes prevention or treatment of rheumatism possible. Preferably, the gene above is particularly a structural gene in an extracellular region of the Jagged 1 protein. Preferably, a recombinant vector having the structural gene inserted therein is used as the DNA vaccine. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a CD8 T cell activation inhibitor, a rheumatic drug, and a DNA vaccine for rheumatic therapy.

  Rheumatoid arthritis is a chronic persistent polyarthritis that is painful due to inflammation and swelling in many joints of the body, and this condition progresses over a long period of time, and it is accompanied by joint deformation and dysfunction It is a refractory disease. It is known that there are nearly 1 million patients in Japan. The mechanism by which rheumatoid arthritis develops is thought to be due to the following immune abnormalities. That is, for some reason, the proliferation of synovial cells covering the inner surface of the joint cavity and the increase of joint blood vessels occur, and leukocytes such as lymphocytes and macrophages migrate from the blood vessels to the joint synovial tissue. For this reason, an immune response is generated locally in the joint, and an inflammatory reaction is caused by the action of cytokines produced by lymphocytes and macrophages, resulting in rheumatic diseases. However, the cause of such immune abnormalities has not yet been clarified.

In collagen diseases such as rheumatic diseases, it is generally considered that the activation of T cells recognizing a patient's own protein (self-antigen) forms a major pathological condition. Therefore, various types of drugs including immunosuppressive drugs are currently used as therapeutic agents for rheumatoid arthritis. Specifically, neutralizing antibodies targeting TNFγ that are excessively present in rheumatic diseases were used. Therapeutic drugs, therapeutic drugs that supplement TFN receptors (soluble TNF receptors only in the extracellular domain), and the like are on the market. However, a highly effective therapeutic agent has not yet been found.
Rovensky J, Svik K. Stancikova M, Istok R, Effect of immunostimulatory ribomunyl on preventive treatment of rat adjuvant arthritis with cyclosporine and methotrexate.J Rheumatol. 2003 Sep; 30 (9): 2027-32. Matsuura M, Imayoshi T, Chiba K, Okumoto T. Effect of FTY720, a novel immunosuppressant, on adjuvant-induced arthritis in rats.Inflamm Res. 2000 Aug; 49 (8): 404-10.

  Therefore, an object of the present invention is to provide a new therapeutic agent for rheumatism.

  To achieve the above object, the CD8 T cell activation inhibitor of the present invention is a CD8 T cell activation inhibitor containing either a gene or a protein, wherein the gene is a Jagged1 gene, and the protein Is a Jagged1 protein. Hereinafter, in the present invention, the Jagged1 protein is referred to as “Jag1 protein”, and the Jagged1 gene is referred to as “Jag1 gene”.

  In general, it is known that CD4 T cells (CD4 positive cells) and CD8 T cells (CD8 positive cells) have an important role in immunity against self. And polymyositis, which is an autoimmune disease like rheumatism, is mainly because killer T cells called cytotoxic CD8 T cells are moistened in inflamed muscles, so that their own myocytes are the target of CD8 T cells. Thus, it is considered that organizational destruction has occurred. On the other hand, unlike CD4 T cells, CD8 T cells are known to proliferate upon receiving antigen presentation in the lymph nodes, not the target tissue, and are transferred to the target tissue via peripheral blood, and proliferate even with non-specific stimulation. easy. For this reason, there are many CD8 T cells that attack external enemies such as viruses even in peripheral blood of healthy persons. In consideration of such characteristics of CD8 T cells, it is presumed that peripheral blood of polymyositis patients has clonal increase of CD8 T cells more frequently than healthy individuals. Therefore, the present inventor, in the same way, for rheumatic patients who are one of the autoimmune diseases, by suppressing (inactivating) the activation of CD8 T cells, the CD8 T cells are It was speculated that targeting of autologous cells (self tissue) could be suppressed, and as a result, rheumatism could be prevented and treated. As a result of intensive studies, the present inventor inhibited the activation of CD8 T cells by the interaction of Notch protein, which is a receptor for T cells, and Jag1 protein, which is its ligand (inactivation). The present invention has been completed.

  That is, according to the CD8 T cell activation inhibitor of the present invention, the activation of CD8 T cells is suppressed by the interaction of Jag1 protein (including Jag1 protein translated from Jag1 gene) and Notch protein. Inactivation of CD8 T cells, for example, cell responsiveness of T cells, cytokine production from T cells, proliferation of T cells, and the like are suppressed, and as a result, rheumatism can be prevented or treated. . Moreover, since it suppresses the activation of CD8 T cells which are considered to be involved in rheumatism, it is different from conventional drugs that suppress immunity, for example, it reduces side effects and is mainly involved in rheumatism, which is a therapeutic purpose. It is considered possible to suppress autoimmunity. Therefore, the CD8 T cell activation inhibitor of the present invention is extremely useful as a new therapeutic agent for rheumatism.

  As described above, the CD8 T cell activation inhibitor of the present invention contains either a gene or a protein, wherein the gene is a Jag1 gene, and the protein is a Jag1 protein.

  The reason why CD8 T cell activation is suppressed by the CD8 T cell activation inhibitor of the present invention is considered to be due to the interaction between Jag1 protein and Notch protein, which is a receptor for T cells, as described above. However, this estimation mechanism does not limit the present invention in any way. That is, the CD8 T cell activation inhibitor of the present invention only needs to contain at least one of the Jag1 protein and the Jag1 gene, and can suppress the activation of CD8 T cells regardless of the mechanism. In the examples described below, it has been proved that the CD8 T cell activation inhibitor of the present invention actually inactivates CD8 T cells and suppresses rheumatic symptoms.

  In the present invention, the component contained for suppressing activation of CD8 T cells may be any of the genes and proteins as described above. Below, the CD8 T cell activation inhibitor containing the said gene as 1st Embodiment and the CD8 T cell activation inhibitor containing the said protein as 2nd Embodiment are demonstrated.

<First Embodiment>
This embodiment is an example of a CD8 T cell activation inhibitor containing a gene. In the present embodiment, the CD8 T cell activation inhibitor only needs to contain the Jag1 gene. The Jag1 gene in the present invention may be, for example, a Jag1 gene containing a non-translated portion (for example, a full-length sequence), or a Jag1 protein structural gene containing only a translated portion (hereinafter referred to as “Jag1 structural gene”). May be. Furthermore, the present invention is not limited to this, and as described later, it may be a structural gene of a region where the intracellular region in Jag1 protein is partially or completely removed (for example, the extracellular region of Jag1 protein). In the present invention, “structural gene” means a gene (translation region) that undergoes transcription / translation and defines the amino acid sequence of a protein or polypeptide.

First, examples of the Jag1 structural gene include any one of DNAs shown in the following (a) to (c).
(A) DNA consisting of the base sequence of SEQ ID NO: 3
(B) DNA comprising the base sequence of SEQ ID NO: 8
(C) DNA encoding a protein having a base sequence in which one or several amino acids are deleted, substituted or added in the above (a) to (b) and having the same function as the Jag1 protein

  The DNA of (a) is a structural gene (CDS region) of mouse Jag1 protein, and encodes the amino acid sequence (1218 amino acid residues) of mouse Jag1 protein shown in SEQ ID NO: 2. The DNA of (b) is a structural gene (CDS region) of human Jag1 protein and encodes the amino acid sequence (1218 amino acid residues) of human Jag1 protein shown in SEQ ID NO: 7. The Jag1 structural gene is not limited to human or mouse-derived sequences, and may be, for example, a Jag1 structural gene from other mammals.

  SEQ ID NO: 1 shows the entire sequence of the mouse Jag1 gene, and SEQ ID NO: 6 shows the entire sequence of the human Jag1 gene (full-length sequence including an untranslated region). In addition, mouse Jag1 gene, mouse Jag1 structural gene (CDS region) and mouse Jag1 protein are NCBI accession: NM_013822, human Jag1 gene, human Jag1 structural gene (CDS region) and human Jag1 protein are NCBI accession: No . Each is registered in MN_00214.

  The Jag1 structural gene is not limited to the base sequence of SEQ ID NO: 3 in (a), and may be, for example, a DNA consisting of another base sequence encoding the amino acid sequence of the mouse Jag1 protein shown in SEQ ID NO: 2. . Similarly, it is not limited to the base sequence of SEQ ID NO: 8 in (b), and may be, for example, DNA consisting of another base sequence encoding the amino acid sequence of human sJag1 protein shown in SEQ ID NO: 7.

  Furthermore, the Jag1 structural gene in the present invention may be DNA as shown in (c) above. In (c), the number of bases that can be deleted, substituted or added is not particularly limited as long as the protein encoded by the DNA does not lose the function of the Jag1 protein. The number of bases is not particularly limited, but is preferably 1 to 6, more preferably 1 to 5, even more preferably 1 to 4, and particularly preferably 1 to 3 with respect to 50 bases. is there. In the present invention, “having the same function as Jag1 protein” means having the same function of inhibiting CD8 T cell activation as Jag1 protein (hereinafter the same). Therefore, the gene of the present invention may be a partial sequence of DNA of (a) or (b) as long as it has this function.

  In addition, the DNA shown in (c) is, for example, a DNA that hybridizes under stringent conditions with any of the DNAs of (a) and (b) as long as the function of the Jag1 protein is not lost. Alternatively, it may be a DNA having 90% or more homology with any of the above DNAs. The stringent conditions for hybridization include, for example, standard conditions in the technical field. For example, the temperature condition is ± 5 ° C., preferably ± 5 ° C. of the Tm value of the base sequence indicated by each SEQ ID NO. 2 ° C., more preferably ± 1 ° C. Specific examples of conditions include hybridization at 65 ° C. in 5 × SSC solution, 10 × Denhardt solution, 100 μg / ml salmon sperm DNA and 1% SDS, 65 ° C., 10 in 0.2 × SSC and 1% SDS. Minute washing (twice). The homology is, for example, 90% or more, preferably 95% or more, more preferably 97.5% or more.

  Next, the gene in the present invention may be a partial sequence of the Jag1 structural gene as described above. That is, the Jag1 protein has an intracellular region that is a transmembrane domain and an extracellular region, and the gene in the present invention is, for example, a structural gene of a region in which the intracellular region in the Jag1 protein is partially or completely removed. It may be. As a specific example, there is a structural gene of the extracellular region of Jag1 protein from which all the intracellular region has been removed. The extracellular region of this Jag1 protein is a region excluding the intracellular region rich in hydrophobicity, and exhibits solubility, so that it can be referred to as a solubilized Jag1 protein (hereinafter referred to as “sJag1 protein”). Called). The structural gene of the sJag1 protein is hereinafter referred to as “sJag1 structural gene”. Since such sJag1 protein is excellent in solubility, for example, it is easy to move in the living body, and as described above, it can be easily introduced into blood or the like in which a large amount of CD8T cells are present. Activation can be suppressed efficiently. Accordingly, the gene is particularly preferably an sJag1 structural gene that expresses the sJag1 protein.

Examples of the sJag1 structural gene include any one of DNAs shown in the following (d) to (f).
(D) DNA consisting of the base sequence of SEQ ID NO: 4
(E) DNA consisting of the base sequence of SEQ ID NO: 9
(F) DNA encoding a protein having a base sequence in which one or several bases are deleted, substituted or added in (d) to (e) and having the same function as the sJag1 protein

  The DNA of (d) is a structural gene of mouse sJag1 protein, and encodes the 1-1067th region (SEQ ID NO: 5) in the amino acid sequence (1218 amino acid residues) of mouse Jag1 protein shown in SEQ ID NO: 2. The DNA of (e) is a structural gene of human sJag1 protein and encodes the 1st to 930th regions (SEQ ID NO: 10) in the amino acid sequence (1218 amino acid residues) of human Jag1 protein shown in SEQ ID NO: 7. To do. The sJag1 structural gene is not limited to human or mouse-derived sequences, and may be, for example, an sJag1 structural gene from other mammals.

  The sJag1 structural gene is not limited to the base sequence of SEQ ID NO: 4 in (d) above, and may be a DNA consisting of another base sequence encoding the amino acid sequence of the mouse sJag1 protein shown in SEQ ID NO: 5. Similarly, the base sequence of SEQ ID NO: 9 in (e) above is not limited, and for example, it may be a DNA consisting of another base sequence encoding the amino acid sequence of human sJag1 protein shown in SEQ ID NO: 10.

  Further, the sJag1 gene in the present invention may be DNA as shown in (f) above. In (f), the number of bases that can be deleted, substituted or added is not particularly limited as long as the protein encoded by the DNA does not lose the function of the sJag1 protein. For example, with respect to 50 bases, 1 to 6 are preferable, more preferably 1 to 5, further preferably 1 to 4, and particularly preferably 1 to 3. In the present invention, “having the same function as sJag1 protein” means having the same function of inhibiting CD8 T cell activation as sJag1 protein (hereinafter the same). Therefore, the gene in the present invention may be a partial sequence of DNA of (d) or (e) as long as it has this function.

  The DNA shown in (f) may be, for example, a DNA that hybridizes under stringent conditions with any of the DNAs of (d) to (e) as long as it does not lose the function of the sJag1 protein. Alternatively, it may be a DNA having 90% or more homology with any of the above DNAs. The homology is preferably 95% or more, more preferably 97.5% or more.

  Further, the gene in the present invention may be, for example, a sequence encoding a dimer of sJag1 protein.

  These genes may be natural products or synthetic products. For example, based on information such as the amino acid sequences of the known Jag1 protein and sJag1 protein, the base sequences of their structural genes, and the like, known in the art such as PCR and RT-PCR It is possible to clone by this method. In addition, the DNA in which the base shown in (c) or (f) is deleted can be easily obtained by applying a conventionally known method such as site-directed mutagenesis.

  Although it does not restrict | limit especially as a form of the CD8 T cell activation inhibitor in this embodiment, It is preferable that it is a DNA vaccine. The DNA vaccine of this invention should just contain genes, such as the above-mentioned Jag1 structural gene and sJag1 structural gene, The preparation method, a composition, a usage method, etc. are not restrict | limited at all.

  Hereinafter, a recombinant vector will be described as an example of a DNA vaccine. That is, it is a recombinant vector in which a gene such as the aforementioned Jag1 structural gene or sJag1 structural gene is inserted into a vector such as an expression vector. This is an example and does not limit the present invention.

  The vector is not particularly limited, and can be appropriately determined according to the target cell or tissue to which the DNA vaccine is administered, the administration method, and the like, and examples thereof include non-viral vectors and viral vectors. Examples of the non-viral vector include vectors capable of expressing the gene in cells or in vivo targeted for treatment or prevention. For example, pcDNA3.1 (Invitrogen), pZeoSV (Invitrogen), pBK- Expression vectors such as CMV (Stratagene) and pCAGGS (Gene108, 193-200 (1991)) can be exemplified. Usually, the gene may be inserted downstream of the promoter of these vectors so that it can be expressed. Examples of viral vectors include retrovirus vectors, lentivirus vectors, adenovirus vectors, adeno-associated vectors (AAV vectors; adeno associated viruses), herpes viruses, vaccinia viruses, pox viruses, polioviruses, synbis viruses, Sendai viruses. , SV40, DNA viruses such as immunodeficiency virus (HIV), and RNA viruses. A recombinant vector can be produced by inserting the gene into the above vector based on a conventionally known method.

  The recombinant vector may further include a regulatory sequence that regulates the expression of the gene. Examples of the regulatory sequence include a promoter derived from cytomegalovirus (CMV), rous sarcoma virus (RSV), simian virus-40 (SV-40), muscle β-actin promoter, herpes simplex virus (HSV) and the like. And tissue-specific promoters such as thymidine kinase promoter, growth hormone-regulated promoters, promoters under the control of the lac operon sequence, zinc-inducible metallothionein promoters and other inducible promoters and regulatory promoters. Such a regulatory sequence may be arranged or bound to a site capable of functionally regulating the expression of the gene based on a conventionally known method. In addition, for example, an enhancer sequence, a polyadenylation signal, an origin of replication sequence (ori) and the like may be included.

  The recombinant vector may have a sequence encoding a selection marker such as a drug resistance marker, a fluorescent protein marker, an enzyme marker, a cell surface receptor marker, and the like. In addition, the recombinant vector preferably has, for example, a CpG motif (unmethylated CpG motif) from the viewpoint of effective gene expression.

  Next, a method for introducing the recombinant vector into a subject as a DNA vaccine will be described. The method for introducing the DNA vaccine is not particularly limited. For example, a method for directly introducing the DNA vaccine into the body in vivo, a method for introducing the DNA vaccine ex vivo into a target cell or tissue taken out from the subject, and a cell after the introduction, etc. There is a method of returning to the body of the subject.

  In the case of the former in vivo method, for example, the recombinant vector may be suspended in a suitable solvent, appropriately sterilized, and then introduced into the subject. The introduction method is not particularly limited, and for example, a conventionally known method can be appropriately employed depending on the type of the target cell or tissue. Specific examples include intramuscular, intraperitoneal injection, oral administration, subcutaneous tissue administration, gene gun administration, immersion, and the like.

  In the case of the latter ex vivo method, for example, calcium phosphate method, polyethylene glycol method, lipofection method, electroporation method, ultrasonic nucleic acid introduction method, gene gun introduction method, DEAE-dextran method, direct using micro glass tube Examples thereof include an injection method and a method using a virus vector as described above.

  The composition of the DNA vaccine is not particularly limited, but may include, for example, an auxiliary agent in addition to the recombinant vector. The auxiliary agent is not particularly limited, and for example, a known auxiliary agent for DNA vaccine can be appropriately used.

  The administration site of the DNA vaccine is not particularly limited, and for example, it can be administered to a site where CD8 T cell activation has occurred or is likely to occur due to autoimmunity. In addition, administration to the target site may be direct administration or indirect administration. When administered indirectly, the DNA vaccine preferably contains the sJag1 structural gene as a gene. As described above, since sJag1 expressed from the sJag1 structural gene is soluble, the expressed sJag1 can be efficiently flowed to the target site even by indirect administration, for example. The administration target is not particularly limited, and examples thereof include mammals such as humans and mice, and these cells and tissues can be collected and administered in vitro.

  The dose of the DNA vaccine is not particularly limited, and can be appropriately determined depending on, for example, the subject to be administered, the administration method, the administration form, the symptoms, and the like. As a specific example, it is preferable to administer 50 to 100 μg of the recombinant vector to a human (adult).

<Second Embodiment>
This embodiment is an example of a CD8 T cell activation inhibitor containing protein. In the present embodiment, the CD8 T cell activation inhibitor only needs to contain Jag1 protein. However, the present invention is not limited to this, and as described later, it may be a region where the intracellular region in Jag1 protein is partially or entirely removed (for example, the extracellular region of Jag1 protein).

First, examples of the Jag1 protein include any of the proteins shown in the following (A) to (C).
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2 (B) a protein comprising the amino acid sequence of SEQ ID NO: 7 (C) in the amino acid sequence of (A) or (B), wherein one or several amino acids are deleted, A protein comprising a substituted or added amino acid sequence and having the same function as Jag1 protein

  The protein (A) is a mouse Jag1 protein, and the protein (B) is a human Jag1 protein. In addition, it is not restrict | limited, Jag1 protein, such as another mammal animal, can also be used.

  In (C), the number of amino acid residues that can be deleted, substituted or added is particularly limited as long as it does not lose the function of the mouse sJag1 protein of (A) or the human Jag1 protein of (B). Not. The number of amino acid residues that can be deleted or the like is, for example, preferably 1 to 4, more preferably 1 to 3, and still more preferably 1 to 2, with respect to 50 amino acid residues. Moreover, the homology with respect to the amino acid sequence of sequence number 2 or sequence number 7 is 70% or more, for example. Therefore, the protein of the present invention may be the partial sequence (A) or (B) as long as it has the same function as the Jag1 protein.

  Next, the protein in the present invention is not limited to the Jag1 protein as described above, and may be a partial sequence thereof. That is, it may be a region in which the intracellular region in the Jag1 protein is completely or partially removed, and a specific example is the extracellular region of the Jag1 protein (sJag1 protein) from which the intracellular region has been completely removed. In the present invention, sJag1 protein is particularly preferred for the reasons described above.

Examples of the sJag1 protein include any of the following proteins (D) to (F).
(D) A protein comprising the amino acid sequence of SEQ ID NO: 5 (E) A protein comprising the amino acid sequence of SEQ ID NO: 10 (F) In the amino acid sequence of (D) or (F), one or several amino acids are deleted, A protein comprising a substituted or added amino acid sequence and having the same function as the sJag1 protein

  The protein (D) is a mouse sJag1 protein, and corresponds to the 1st to 1067th regions in the amino acid sequence (1218 amino acid residues) of the mouse-derived Jag1 protein shown in SEQ ID NO: 2. The protein (E) is a human sJag1 protein and corresponds to the 1st to 930th regions in the amino acid sequence (1218 amino acid residues) of the human-derived Jag1 protein shown in SEQ ID NO: 7. In addition, it is not restrict | limited, sJag1 protein, such as another mammal animal, can also be used.

  In (F), the number of amino acid residues that can be deleted, substituted or added is not particularly limited as long as it does not lose the function of the human sJag1 protein of (D) or the mouse sJag1 protein of (E). Not. For example, with respect to 50 amino acid residues, 1 to 4 are preferable, more preferably 1 to 3, and still more preferably 1 to 2. The homology with the amino acid sequences of SEQ ID NO: 5 and SEQ ID NO: 10 is, for example, 70% or more, preferably 80% or more, more preferably 90% or more, and particularly preferably 95% or more. Therefore, the protein of the present invention may be the partial sequence (D) or (E) as long as it has the same function as sJag1.

  The protein in the present invention may be, for example, a dimer of sJag1 protein.

  Such a protein may be, for example, a natural product or a synthetic product by chemical synthesis, but is preferably a protein expressed using a recombinant DNA technique because it can be easily prepared in large quantities. . A preparation method using such a recombinant DNA technique is not particularly limited. For example, it is preferable to use the recombinant vector described in the first embodiment. The protein can be expressed by introducing the recombinant vector into an appropriate host based on, for example, a host-vector system. The protein obtained by such a method can be subjected to purification treatment such as salt precipitation, centrifugation, column chromatography, etc., if necessary, and the recovered purified protein can be used as a vaccine.

  The method for administering the protein as a vaccine to a subject is not particularly limited, and conventionally known methods can be employed. For example, it can be appropriately employed depending on the type of target cells and tissues, and examples thereof include intramuscular injection, intraperitoneal injection, oral administration, subcutaneous tissue administration, gene gun administration, immersion, and the like.

  The protein is preferably used as a vaccine composition mixed with an adjuvant such as aluminum hydroxide. In addition, the target protein may be encapsulated in, for example, a liposome or joined to a polymer.

  The dose of the protein is not particularly limited and can be appropriately determined depending on, for example, the subject to be administered, the administration method, the dosage form, etc. For example, 500 to 1000 μg is preferable for humans (adults).

  Next, the therapeutic agent for rheumatism of the present invention comprises the CD8 T cell activation inhibitor of the present invention. According to the therapeutic agent for rheumatism of the present invention, activation of CD8 T cells can be suppressed, for example, by administration to humans or mammals other than humans, thereby preventing or treating rheumatism. In addition, the usage method similar to the above-mentioned CD8 T cell activation inhibitor can be employ | adopted for the therapeutic agent of rheumatism of this invention.

  Next, the DNA vaccine for treating rheumatism of the present invention is characterized in that it contains the Jag1 gene. As described above, the Jag1 gene may be a Jag1 gene including a non-translated region (for example, a full-length sequence), a Jag1 structural gene, or a partial sequence thereof (for example, an sJag1 structural gene). Further, the form and usage are the same as described above.

[Example]
Next, examples of the present invention will be described together with comparative examples. However, the present invention is not limited by the following examples and comparative examples. In addition, unless otherwise indicated, the commercially available reagent was used based on those protocols.

<Production of DNA vaccine>
As a DNA vaccine, a vector (Jagged1 / pcDNA3.1) in which a CDS sequence encoding mouse Jag1 protein is inserted, and a sequence encoding a protein portion (extracellular region: sJag1 protein) that lacks the intracellular region of mouse Jagged1 protein A vector (Soluable Jagged1 / pcDNA3.1) into which was inserted was prepared by the following method. 1A shows an outline of Jagged 1 / pcDNA 3.1, and FIG. 1B shows an outline of Solved Jagged 1 / pcDNA 3.1.

  Jagged 1 / pcDNA3.1 was prepared by inserting the sequence shown in SEQ ID NO: 3 into the EcoRI / NotI site of pcDNA3.1 / Zeocin (trade name, manufactured by Invitrogen). The sequence shown in SEQ ID NO: 3 is a cDNA sequence encoding amino acids 1-11218 of mouse Jag1 protein. This Jagged1 / pcDNA3.1 is hereinafter also referred to as “Jag1 / pcDNA”.

  Moreover, Soluble Jagged1 / pcDNA3.1 was prepared by inserting the sequence shown in SEQ ID NO: 4 into the EcoRV site of pcDNA3.1 / Zeocin (trade name, manufactured by Invitrogen). The sequence shown in SEQ ID NO: 4 is a sequence encoding the extracellular region of mouse Jag1 protein (amino acids 1 to 1067 of mouse Jag1 protein shown in SEQ ID NO: 2). This Soluble Jagged 1 / pcDNA 3.1 is hereinafter also referred to as “sJag1 / pcDNA”.

  The sJag1 protein expressed from the sJag1 / pcDNA prepared in Example 1 was contacted with a T cell in vitro, and it was confirmed whether or not the sJag1 protein interacts with the Notch protein that is a receptor of the T cell. .

<Reporter assay>
The day before transfection, Plat-E cells (transient expression packaging cells) were seeded in a 6-well plate to which RPMI 1640 medium was added so as to be 1 × 10 ⁶ cells / well. To this Plat-E cell, sJag1 / pcDNA (1 μg), which is a DNA vaccine, was introduced using an introduction reagent (trade name Fugene, manufactured by Roche), and after 24 hours, the medium in the 6-well plate was replaced with a new medium. Exchanged. Further, after culturing for a predetermined time (72 hours), the supernatant was recovered from the culture solution. As Comparative Example 1, pcDNA3.1 (trade name, manufactured by Invitrogen, the same applies hereinafter) in which the coding sequence of sJag1 was not inserted was introduced into Plat-E cells instead of sJag1 / pcDNA, and the same as described above. Culture and supernatant collection were performed. The culture temperature was 37 ° C. (hereinafter the same). Note that, by culturing the Plat-E cells into which sJag1 / pcDNA has been introduced, the collected supernatant contains packaged infectious virus.

On the other hand, Jurkat cells (T lymphocytes derived from human acute T lymphocytic leukemia) were cultured at a concentration of 3.4 × 10 ⁵ cells / well in a 24-well plate supplemented with RPMI 1640 medium. Then, 1 μg of reporter gene (TP1) and pGL4.74 vector (trade name, manufactured by Promega), which is a luciferase reporter vector, were introduced into the cultured cells using an introduction reagent (trade name DMRIE-C, manufactured by Invitrogen). 0.2 μg was introduced and cultured for 48 hours.

In a 96-well plate, feeder cells (OP9 cells, which are bone marrow stromal cells) are seeded at 1 × 10 ⁴ cells / well, and the above-mentioned Jurkat cells are added to the supernatant (100 μl / well). Then, the cells were cultured for 24 hours using RPMI 1640 medium. The Jurkat cells after culture were lysed by Dual-Luciferase Reporter Assay System (trade name, manufactured by Promega), and the intensity of the luminescence reaction by firefly luciferase and Renilla luciferase luciferase was measured with a luminometer. Then, a value (R1 / R2) obtained by dividing the intensity (R1) of firefly luciferase, which is a reporter, by the intensity (R2) of the control Renilla luciferase was obtained. The result is shown in FIG. The time (72 hr) in FIG. 2 is the culture time of the Plat-E cells after vector introduction.

  As shown in the figure, as a result of using the culture supernatant of Plat-E cells into which sJag1 / pcDNA was introduced, the value of R1 / R2 in Jurkat cells was remarkably increased. An increase in R1 / R2 means that sJag1 protein binds to Notch protein, which is a receptor of Jurkat cells, thereby causing signal transduction via Notch protein (positive Notch signal). Therefore, it was confirmed that the interaction with the Notch protein occurs only in the extracellular region (sJag1 protein) from which the intracellular region has been removed.

  The effect of DNA vaccines (Jag1 / pcDNA and sJag1 / pcDNA) in a collagen-induced rheumatoid arthritis model was confirmed.

  First, 4 days before the collagen induction test (day-4), PBS (manufactured by SIGMA) containing 0.25% bupivacaine (local anesthetic) was intramuscularly injected into the gastrocnemius muscles of both legs of DBA / 1J mice (male) ( 50 μl / one leg). Next, two days before the test (day-2), Jag1 / pcDNA or sJag1 / pcDNA, which is a DNA vaccine, was intramuscularly injected into the gastrocnemius muscle of both feet of the mouse (50 μg / 50 μl / one leg). As a comparative example, pcDNA3.1 (hereinafter referred to as “pcDNA”) into which the coding sequences of Jag1 and sJag1 were not inserted was injected instead of the DNA vaccine. Hereinafter, a group of mice injected with the Jag1 / pcDNA vector having the coding sequence (full length) of Jag1 protein is referred to as “Jag1 group”, and a group of mice injected with the sJag1 / pcDNA vector having the coding sequence of sJag1 protein is referred to as “sJag1 group”. A group of mice injected with pcDNA into which a coding sequence such as Jag1 has not been inserted is referred to as a “comparison group”.

  On the day of the collagen induction test (day 0), a collagen-containing liquid that is a rheumatoid inducer is emulsified with an equal amount of Freund's complete adjuvant (trade name: M. Tuberculosis H37Ra: manufactured by DIFCO), and this emulsion is added to the mouse. Vaccinated. Specifically, the Jag1 group was inoculated into the footpad of both legs under the condition of collagen 100 μg / 50 μl / one leg, and the sJag1 group was inoculated subcutaneously at the tail base under the condition of collagen 200 μg / 100 μl / animal. The comparison group is further divided into two groups. One is inoculated with footpad under the same conditions as the Jag1 group, and the other is inoculated subcutaneously at the base of the tail under the same conditions as the sJag1 group. Went. The collagen-containing solution was prepared by adding bovine collagen type II (collagen technology workshop) to 20 mg Tris / 150 mM NaCl (pH 8.0) to 4 mg / ml. Further, as a control for each group (collagen non-induction), PBS was used instead of the collagen-containing liquid, and the emulsion was administered in the same manner.

  After the administration of the emulsion, the DNA vaccine was intramuscularly injected (additional immunization) three times every 7 days under the same conditions as 2 days before the test (day 2). Specifically, the Jag1 group was injected with Jag1 / pcDNA, the sJag1 group with sJag1 / pcDNA, and the comparative group with pcDNA. On the 21st day from the start of the test (day 21), the collagen-containing solution was emulsified with an equal amount of Freund's incomplete adjuvant (trade name: M. Tuberculosis H37Ra: manufactured by DIFCO), and this emulsion was added to the tail of the mouse. Inoculated subcutaneously (collagen 200 μg / 100 μl / animal). As a control for each group, PBS was used in place of the collagen-containing solution, and the emulsion was administered in the same manner. Then, for these groups of mice, the symptoms of collagen-induced arthritis (CIA) were determined at predetermined intervals (0, 2, 5, 7, 9, 11, 14, 16, 18, 21, 23 days), and the clinical score was evaluated based on the following evaluation criteria.

(Evaluation criteria)
0 = no change 1 = local redness of the limb, or enlargement and redness of one toe 2 = mild enlargement and erythema of the limb, or enlargement of two or more toes 3 = limbs Marked hypertrophy and erythema 4 = Maximum limb enlargement and erythema, and subsequent ankylosia

  These results are shown in FIG. (A) shows the results of inoculating Footpad with Jag1 / pcDNA, and (B) shows the results of inoculating sJag1 / pcDNA at the tail. In addition, the clinical score shown in FIG. 3A is a value obtained by obtaining three paw clinical scores for each of three mice and summing them and dividing by 9. The clinical score shown in FIG. 5B is a value obtained by obtaining a clinical score (clinical score) of 4 legs for each mouse for 3 mice, summing them, and dividing by 3. The number of days in the figure is the number of days after the final immunization. In addition, the following table shows the types of mouse groups plotted in the figure. In the examples described later, the mouse groups are as follows.

  As shown in FIG. 5A, in the CII / Jag1 group using Jag1 / pcDNA (in the figure, ●: Example 3-1), the CII / pcDNA group using pcDNA having no coding sequence of Jag1 The clinical score decreased and clinical symptoms of CIA were reduced compared to (□ in the figure: Comparative Example 2-1). Further, as shown in FIG. 4B, CII / sJag1 group using sJag1 / pcDNA (in the figure, ■: Example 3-2) also used CII using pcDNA having no coding sequence of sJag1. The clinical score was significantly reduced and clinical symptoms of CIA were remarkably reduced as compared with the / pcDNA group (in the figure, ◯: Comparative Example 2-2). In particular, according to sJag1 / pcDNA, the clinical score, that is, the clinical symptoms of CIA could be further reduced as compared with the case where Jag1 / pcDNA was used.

  Collagen-induced rheumatoid arthritis model mice were inoculated with a DNA vaccine (sJag1 / pcDNA), and cytokine production ability was confirmed for the T cells of the model mice.

  In the same manner as in Example 3, DBA / 1J mice (male) were immunized using sJag1 / pcDNA. After booster immunization, about the group inoculated with sJag1 / pcDNA, the spleen was collected 25 days later (25 days after the final immunization was 0 days), and single cell suspension was prepared by trypsin treatment.

The prepared single cell suspension was cultured in the presence of a predetermined amount (0 μg / ml, 50 μg / ml) of collagen for 72 hours to induce collagen. Then, this cultured cell is coated on a 96-well plate coated with 1 ug / ml anti-CD3 antibody (2C11) and 1 ug / ml anti-CD28 antibody (37.51), respectively, so that the concentration becomes 5 × 10 ⁵ cells / well / 200 μl. Then, after further culturing for 24 hours, the culture supernatant was collected. As Comparative Example 3, the culture supernatant was also collected in the same manner for the mouse group (CII / pcDNA group) using pcDNA, and the controls for Example 4 and Comparative Example were the same as in Example 3. did.

On the other hand, 0.5 μg / ml purified anti-mouse IFNγ antibody (XMG1.2: manufactured by eBioscience) and 0.5 μg / ml purified anti-mouse IL-4 antibody (11B11: manufactured by eBioscience) were each in a 96-well plate ( (Trade name Maxisorp, manufactured by Nunc) and incubated at 4 ° C. overnight. The well plate on which this antibody was immobilized was washed 5 times with 0.05% PBST (PBS containing 0.05% Tween 80), PBS containing 1% BSA was added, and incubated at room temperature for 1 hour. After incubation, the plate was washed 5 times with 0.05% PBST. Then, the collected culture supernatant was added to this plate (100 μl / well) and incubated at room temperature for 1 hour. For the wells on which the purified anti-mouse IFNγ antibody is immobilized, the supernatant is diluted 10-fold, 20-fold or 40-fold, and the well on which the purified anti-mouse IL-4 antibody is immobilized is added. In addition, diluted 2-fold, 4-fold and 8-fold were added. In addition, the phosphate buffer solution was used for the dilution. Then, after the well plate was washed 5 times with 0.05% PBST, 0.5 μg / ml biotin-anti-mouse IFNγ antibody (R4-6A2: manufactured by eBioscience) was added to the well in which each antibody was immobilized. 0.5 μg / ml biotin-anti-mouse IL-4 antibody (BVD6-24G2: manufactured by eBioscience) was added at 100 μl / well and incubated at room temperature for 1 hour. The well plate was washed 5 times with 0.05% PBST, and 1/1000 (1000-fold diluted) avidin-horse radish peroxidase (HRP) (manufactured by eBioscience) was added to 100 μl / well. Incubated for 30 minutes at room temperature. After this well plate was washed 5 times with 0.05% PBST, a TMB substrate (manufactured by eBioscience) was added at 100 μl / well to perform a color reaction. After a predetermined time (5 to 15 min) from the addition of TMB, 0.5 MH ₂ SO ₄ was added at 50 μl / well to stop the color reaction, and the degree of color development was measured with a microplate reader.

  These results are shown in FIG. In the figure, (A) is a graph showing the amount of IFNγ produced from T cells, and (B) is a graph showing the amount of IL-4 produced from T cells. As shown in each of FIGS. (A) and (B), Comparative Example 3 (CII / pcDNA) using pcDNA which does not contain the coding sequence of sJag1 was induced by collagen induction compared to control (PBS / pcDNA). The production of IFNγ and IL-4 was significantly increased. On the other hand, Example 4 (CII / sJag1 group) using sJag1 / pcDNA containing the coding sequence of sJag1 is shown in Comparative Example 3 (CII / s) as shown in FIGS. Compared with pcDNA), both IFNγ and IL-4 production were confirmed to be sufficiently reduced. Thus, since the production of IFNγ and IL-4 was suppressed by inoculation with sJag1 / pcDNA, it can be seen that sJag1 / pcDNA can suppress the activation of T cells related to autoimmunity.

  A collagen vaccine-induced rheumatoid arthritis model mouse was inoculated with a DNA vaccine (sJag1 / pcDNA), and the limbs of the model mouse were examined histologically.

  For DBA / 1J mice (male) immunized with sJag1 / pcDNA in Example 4, the limbs were collected 25 days later (25 days after the final immunization). Then, the limb sections were fixed with a 10% formalin solution according to a conventional method, and after disaggregation, a paraffin-embedded block was prepared and stained with hematoxylin and eosin. The CII / pcDNA in Comparative Example 3 and the control PBS / pcDNA were also stained in the same manner. The result of observing this section with an optical microscope (trade name CX31, manufactured by Olympus Corporation) is shown in FIG. In the same figure, (A) is a mouse using sJag1 / pcDNA (CII / sJag1), (B) is a mouse using pcDNA3.1 (CII / pcDNA3.1), and (C) is collagen-induced. It is a photograph which shows the result of the mouse | mouth (control PBS / pcDNA3.1) which is not shown and used pcDNA3.1. The following table shows the lesion site and the degree of wetting. In addition, the degree of wetting in the following table was evaluated as follows.

(Evaluation criteria for wetness)
2: Strong cell infiltration 1: Mild cell infiltration 0: No infiltration

  In the CII / pcDNA3.1 group not expressing sJag1, collagen-induced inflammatory cell wetting was confirmed, as shown in FIG. In contrast, in the CII / sJag1 group expressing sJag1 shown in FIG. 6A, inflammatory cell wetting was remarkably suppressed, and the results were the same as the control in FIG. This result is consistent with the reduction of clinical symptoms. This shows that sJag1 / pcDNA, which is a DNA vaccine, is useful for collagen-induced inflammation.

  The effect of DNA vaccine (Jag1 / pcDNA) on CD8 positive T cell response to substances other than collagen (ovalbumin: OVA) was confirmed.

  Three days before the test (day-3), PBS (manufactured by SIGMA) containing 0.25% 5% bupivacaine (local anesthetic) was intramuscularly injected into the gastrocnemius of both legs of C57B1 / 6 mice (female) (50 μl / one leg). ). Next, on the day of the test (day 0), 50 μg / 25 μl of DNA vaccine (Jag1 / pcDNA) and 50 μg / 25 μl of OVA expression vector (OVA / pCI) were mixed, and this mixture was injected intramuscularly into the gastrocnemius of both feet of the mouse. (Total volume 50 μl / one leg). Further, every 14 days, booster immunization was performed using a mixture of the DNA vaccine and the OVA expression vector under the same conditions (3 times in total including day 0 inoculation). After the third vaccine, on the 33rd day (day 33) from the start of the test, popliteal and inguinal lymph nodes were collected, and a single cell suspension was prepared by trypsinization. In addition, as a comparative example, a mixture of OVA / pCI and pcDNA, a mixture of OVA / pCI and Delta1 / pcDNA was used, and pcDNA was used as a control, and a single cell suspension was prepared in the same manner. . OVA / pCI is a vector in which the CDS sequence of the OVA protein is inserted into the EcoRI restriction enzyme site of the pCI vector (Promega). Delta1 / pcDNA is the same as EcoRV of the pcDNA3.1 vector (Invitrogen). This is a vector in which the CDS sequence of Delta1 protein is inserted into the EcoRI restriction enzyme site.

  The obtained single cell suspension was treated with an FcR blocking reagent (trade name 2.4G2, manufactured by Becton Dickinson) to block the Fc receptor (FcR) of mouse cells. Then, the blocked cells were stained with FITC-anti-mouse CD8 antibody (53-6.7: manufactured by eBioscience) and OVA tetramer-PE (manufactured by MBL). After these cells were washed with a buffer solution (trade name FACS (registered trademark) buffer, Sigma), the cells were further suspended in the buffer solution, and a flow cytometer (trade names FACS Calibur, Becton Dickinson) was used. The OVA antigen was detected.

  These results are shown in FIG. The figure is a flow cytometry diagram showing the number of T cells having a T cell antigen receptor that reacts with an OVA antigen. In the figure, the lower right is the result of the mouse inoculated with OVA / pCI and Jag1 / pcDNA, the upper right is the result of the mouse inoculated with OVA / pCI and pcDNA (OVA / Jag1), and the lower left is , The results of mice inoculated with OVA / pCI and Delta1 / pcDNA (OVA / DL1), and the upper left is a control that has not been induced by OVA and vaccinated with pCI and PBS (pCI / PBS) Indicates.

  As shown in the figure, in the mice not inoculated with Jag1 / pcDNA, that is, in the upper right OVA / Jag1 and the lower left OVA / DL1, the proportion of T cells having a T cell antigen receptor that reacts with the OVA antigen Was very high at 1.08% and 0.93%. In contrast, the mice inoculated with Jag1 / pcDNA were able to be suppressed to an extremely low value of 0.31% for the T cells. From this result, it was proved that the DNA vaccine having the coding sequence of Jag1 can reduce the CD8 positive T cell responsiveness to any antigen regardless of collagen.

  The effect of sJag1 protein on human T cells was confirmed.

Mononuclear cells were separated from human peripheral blood by specific gravity centrifugation and added to a 24-well plate at 1 × 10 ⁶ (500 μl). As the medium, RPMI 1640/10% FCS was used. Then, cells were stimulated by adding OKT3 antibody (Becton Dickinson) at a concentration of 1 μg / ml to the 24-well plate, and at the same time, a culture supernatant containing human-derived sJag1 protein prepared as follows was added. This supernatant was prepared by culturing and recovering 293T cells introduced with sJag1 / pcDNA for 48 hours using Fugene 6 (Roche). The amount of the culture supernatant added was 100 μl / well for a 24-well plate. At 60 hours after antibody stimulation, [3H] -thymidine was added to a 24-well plate at 1 μCi (20 μl) / well, and 12 hours later, [3H] -thymidine was taken up by a liquid scintillation counter. It was measured. In addition, as a control, uptake was measured in the same manner as in Example 6 except that the culture supernatant of 293T cells introduced with pcDNA3.1 into which no human sJag1 coding sequence was inserted was added without stimulation with antibodies. did. As a comparative example, uptake was measured in the same manner as in Example 6 except that the culture supernatant of 293T cells into which no human sJag1 coding sequence was introduced was added.

  These results are shown in FIG. As shown in the figure, the comparative system (OKT3 in the figure) to which no human-derived sJag1 protein was added showed very high thymidine incorporation, whereas human sJag1 protein was added. In the system of Example 1 (OKT3 + sJag1), the amount of uptake could be effectively suppressed. From this, it can be said that in the system to which human sJag1 was added, the proliferation of T cells was remarkably suppressed.

  As described above, in each Example, the suppression of CD8 lymphocyte activation and the onset of rheumatism in vitro and in model mice was confirmed for the mouse Jag1 structural gene, mouse sJag1 structural gene and human sJag1 structural gene. did. These results show that, for example, even when other Jag1 protein such as human, sJag1 protein, Jag1 structural gene, sJag1 structural gene, etc. are used, other humans and other animals are used. Obviously, the same effect can be obtained. In particular, those skilled in the art can easily understand that similar results can be obtained when the human Jag1 gene or the human sJag1 gene is administered to a human in vivo. Moreover, those skilled in the art can use, for example, other Jag1 proteins including humans, sJag1 protein, Jag1 structural gene, sJag1 structural gene, and the like without excessive trial and error. Activation inhibitors and rheumatic therapeutic agents can be prepared and used.

  As described above, according to the therapeutic agent for autoimmune diseases of the present invention, it is possible to prevent or treat autoimmune diseases such as rheumatism by suppressing the activation of T cells. Particularly for rheumatic diseases, since it is chronic persistent polyarthritis and is difficult to treat or prevent, the therapeutic agent for autoimmune diseases of the present invention is excellent in usefulness as a new therapeutic agent.

FIG. 1 is a schematic diagram showing an outline of a recombinant vector according to one embodiment of the present invention. FIG. 1A shows a Jagged 1 / pcDNA3.1 vector, and FIG. 1B shows a Soluble Jagged 1 / pcDNA 3.1. It is a vector. FIG. 2 is a graph showing the presence or absence of interaction between the sJag1 protein and the receptor Notch protein in another example of the present invention. FIG. 3 is a graph showing changes in CIA over time in a collagen-induced rheumatoid arthritis model supplemented with a DNA vaccine in still another example of the present invention, and FIG. As a result of using pcDNA, the same figure (B) shows the result of using sJag1 / pcDNA as a DNA vaccine. FIG. 4 is a graph showing the effects of IFNγ and IL-4 over time in a collagen-induced rheumatoid arthritis model supplemented with a DNA vaccine in still another example of the present invention. As a result, FIG. 5B shows the result of IL-4. FIG. 5 is a photograph of a tissue in a collagen-induced rheumatoid arthritis model supplemented with a DNA vaccine in still another example of the present invention. FIG. 5A shows the result of using sJag1 / pcDNA. B) shows the results of a comparative example using pcDNA, and FIG. 10C shows the results of a control without collagen induction. FIG. 6 is a flow cytometry result showing the effect of a DNA vaccine on CD8 positive T cell response to OVA in yet another example of the present invention. FIG. 7 is a graph showing the results of confirming the inhibition of T cell proliferation by human-derived sJag1 protein with the amount of thymidine incorporated in still another example of the present invention.

Claims (12)

A CD8 T cell activation inhibitor comprising either a gene or a protein,
The CD8 T cell activation inhibitor, wherein the gene is a Jagged1 gene and the protein is a Jagged1 protein.   The CD8 T cell activation inhibitor according to claim 1, wherein the gene is a structural gene of Jagged1 protein. The CD8 T cell activation inhibitor according to claim 2, wherein the Jagged 1 structural gene is any one of DNAs shown in (a) to (c) below.
(A) DNA consisting of the base sequence of SEQ ID NO: 3
(B) DNA comprising the base sequence of SEQ ID NO: 8
(C) DNA encoding a protein comprising a base sequence in which one or several amino acids are deleted, substituted or added in (a) and (b) and having the same function as the Jagged1 protein   The CD8 T cell activation inhibitor according to claim 1, wherein the gene is a structural gene of an extracellular region of Jagged1 protein. The CD8 T cell activation inhibitor according to claim 4, wherein the structural gene in the extracellular region is any one of DNAs shown in the following (d) to (f).
(D) DNA consisting of the base sequence of SEQ ID NO: 4
(E) DNA consisting of the base sequence of SEQ ID NO: 9
(F) A protein having a base sequence in which one or several bases are deleted, substituted or added in (d) and (e) and having the same function as the extracellular region of the Jagged1 protein DNA encoding   The CD8 T cell activation inhibitor according to any one of claims 1 to 5, further comprising a vector, wherein the gene is inserted into the vector.   The CD8 T cell activation inhibitor according to claim 6, wherein the vector is a viral vector or a non-viral vector. The CD8 T cell activation inhibitor according to claim 1, wherein the Jagged 1 protein is any one of the following proteins (A) to (C).
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2 (B) a protein comprising the amino acid sequence of SEQ ID NO: 7 (C) in the amino acid sequence of (A) or (B), wherein one or several amino acids are deleted, A protein consisting of a substituted or added amino acid sequence and having the same function as the Jagged1 protein   The CD8 T cell activation inhibitor according to claim 1, wherein the protein is an extracellular region of Jagged1 protein. The CD8 T cell activation inhibitor according to claim 1, wherein the extracellular region of the Jagged 1 protein is any one of the following proteins (D) to (F).
(D) a protein consisting of the amino acid sequence of SEQ ID NO: 5 (E) a protein consisting of the amino acid sequence of SEQ ID NO: 10 (F) in the amino acid sequence of (D) or (E), wherein one or several amino acids are deleted, A protein comprising a substituted or added amino acid sequence and having the same function as the extracellular region of the Jagged1 protein   A therapeutic agent for rheumatism, comprising the CD8 T cell activation inhibitor according to any one of claims 1 to 10, wherein the therapeutic agent is for rheumatism.   A DNA vaccine for treating rheumatism, comprising a Jagged 1 gene.

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