WO2007010692A1 - Pharmaceutical composition for treatment of inflammatory bowel disease which targets at hvem and btla, and therapeutic method - Google Patents

Pharmaceutical composition for treatment of inflammatory bowel disease which targets at hvem and btla, and therapeutic method Download PDF

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WO2007010692A1
WO2007010692A1 PCT/JP2006/312217 JP2006312217W WO2007010692A1 WO 2007010692 A1 WO2007010692 A1 WO 2007010692A1 JP 2006312217 W JP2006312217 W JP 2006312217W WO 2007010692 A1 WO2007010692 A1 WO 2007010692A1
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inflammatory bowel
hvem
bowel disease
btla
disease
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PCT/JP2006/312217
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French (fr)
Japanese (ja)
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Kazuo Sugamura
Naoto Ishii
Masao Ono
Kazuko Murata
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Japan Science And Technology Agency
Tohoku University
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Publication of WO2007010692A1 publication Critical patent/WO2007010692A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • composition for treating inflammatory bowel disease targeting HVEM and BTLA and therapeutic method comprising:
  • the present invention relates to a method for treating inflammatory bowel disease by enhancing a BTLA receptor signal on T cells, a pharmaceutical composition therefor, and the like.
  • HVEM Herpes Virus Entry Mediator
  • HSV simple herpesvirus
  • Glycoprotein Identified as a D-binding substance and known as a molecule that regulates HSV cell infection.
  • T cells, B cells, monocytes and rod cells are known as HVEM-expressing cells in vivo, and lymphocyte-mediated immune response control has been reported as a function of HVEM.
  • HVEM lymphotoxin
  • LT lymphotoxin
  • BTLA lymphotoxin
  • 8 receptor decoy receptor 3
  • HVEM decoy receptor 3
  • HVEM HVEM
  • TNF receptor I TNF receptor II
  • HVEM 3 The seed is known. Only HVEM has been proven as a BTLA receptor is there. Thus, the ligand-receptor system related to HVEM forms a complex network, and the details of their functional relevance in vivo are not clear.
  • BTLA the third ligand of HVEM
  • HVEM binds to BTLA to strongly suppress T cell activation
  • Nat Immunol, 6 90, 2004
  • HVEM-BTLA binding in vivo is important for immunosuppression
  • HVEM signal promotes the development of inflammatory bowel disease (Wang et al, J. Immunol.l74: 8173, 2005) 0
  • Non-Patent Document 2 Sedy et al, Nat Immunol, 6: 90-98, 2005, Gonzalez et al, Proc Natl A cad Sci U S A. 102: 1116-21, 2005
  • Non-Patent Document 3 Wang et al., The critical role of LIGHT in promoting intestinal inflam mation and Crohn 's disease, J. Immunol. 2005 Jun 15; 174 (12): 8173-82
  • HVEM gene mutation and expression are one of the causes of inflammatory diseases, particularly inflammatory bowel disease in animal models, and HV EM signals are associated with the onset of inflammatory bowel disease. First revealed that he was involved. The present invention has been made based on strong knowledge.
  • the present invention relates to the following aspects.
  • a pharmaceutical composition comprising a BTLA receptor ligand as an active ingredient.
  • a pharmaceutical composition comprising an BTLA receptor agonist as an active ingredient.
  • the pharmaceutical composition according to any one of 8 to 10 above which is a pharmaceutical composition for treating inflammatory bowel disease.
  • a method for measuring a mutation in the HVEM gene associated with the onset of inflammatory bowel disease by detecting the oligonucleotide or polynucleotide according to 13 above.
  • a kit for measuring a mutation in the HVEM gene associated with the onset of inflammatory bowel disease for carrying out the measurement method according to 14 above.
  • FIG.1 N2-OX40L- produced by crossing disease-susceptible strain (C57BL / 6) and disease-resistant strain (BALB / c) (C57BL / 6 X (C57BL / 6 x BALB / c))
  • the result of the QTL analysis by each genotype about the inflammatory bowel disease severity of each Tg mouse individual is shown.
  • the vertical axis represents the Lod score, and the horizontal axis represents the locus.
  • FIG.2 Produced by crossing disease-susceptible strain (C57BL / 6) and disease-resistant strain (BALB / c) (C57BL / 6 X (C57BL / 6 x BALB / c)) N2- OX40L- Tg mice
  • the results of QTL analysis by individual genotypes for individual colon weight Z body weight ratio are shown.
  • the vertical axis represents the Lod score, and the horizontal axis represents the gene locus.
  • FIG. 3 shows the correspondence between mouse genomic gene sequences and human genomic gene sequences. Part indicated by bar These are the mouse inflammatory bowel disease susceptibility locus identified in the present invention and the human inflammatory bowel disease susceptibility locus IBD7 (1 ⁇ 36). Both are in perfect agreement.
  • FIG. 4-1 Comparison of HVEM genomic gene base sequences of disease-susceptible strain C57BL / 6 (B6) and disease-resistant strain BALB / c (BALB). A polymorphism (mutation) was identified in the boxed area.
  • FIG. 5 shows C57BL / 6-specific amino acid mutations identified by analysis of HVEM cDNA sequences of disease-susceptible strain C57BL / 6 and disease-resistant strains BALB / c, 129 / Sv, and FVB. Norin in the HVE M transmembrane region is replaced with alanine.
  • FIG. 6 The upper part of FIG. A shows the semi-quantitative RT-PCR result of ⁇ -actin in the lower part of HVEM. Numbers indicate mouse strains (l: 129 / SvJ, 2: NOD, 3: C57BL / 6, 4: C57BL / 6xBALB / c Fl, 5: DBA 2, 6: FVB, 7: BALB / c) 0 C57BL / HVEM mRNA can hardly be detected in the spleen and intestinal tract of 6 origin.
  • Fig. B HVEM and ⁇ -actin mRNA levels were quantified by real-time PCR using TaqMan probe (Applied Biosystems).
  • Relative HVEM mRNA levels were calculated using the ⁇ -actin mRNA level as an internal reference and shown in the graph.
  • the amount of HVEM mRNA in the spleen and intestine of C57BL / 6 mice was less than 1/50 that of BALB / c mice.
  • T cells isolated from mouse spleen were stimulated with an anti-CD3 antibody immobilized on a plate, and the amount of 3 H-thymidine incorporation 72 hours after stimulation was measured. At that time, the amount of uptake was compared between soluble HVEM (10 mg / ml) added (white) and non-added (black). Addition of soluble HVEM suppressed T cell activation ability.
  • HVEM transgenic mice (Virology, 2004 Mar 15; 320 (2): 267-75) were crossed with OX40L-Tg mice to produce HVEM-OX40L double transgenic mice.
  • the mean value and standard deviation (error bar) of intestinal weight for each genotype are shown.
  • the present invention is that the HVEM gene polymorphism (abnormal) causes HVEM mRNA expression failure, and as a result, an immunosuppressive signal that should be originally supplied from BTLA.
  • the method for treating inflammatory bowel disease of the present invention is characterized by enhancing the BTLA signal.
  • Any method known to those skilled in the art can be used as a method for enhancing the BTLA signal.
  • a ligand for a BTLA receptor such as soluble HVEM or an antigen for a BTLA receptor such as an anti-BTLA antibody can react (bind) with BTLA to enhance the BTLA signal. .
  • Examples of main treatment targets of the method of the present invention include higher organisms such as mammals including humans.
  • an anti-BTLA antibody can be prepared by any method known to those skilled in the art.
  • the antibody may be a monoclonal antibody or a polyclonal antibody.
  • various chimeric antibodies (humanized antibodies) having anti-BTLA activity are prepared by genetic engineering recombination techniques known to those skilled in the art. It is also possible to do.
  • Preferable examples of the pharmaceutical carrier or diluent that can be used in the pharmaceutical composition of the present invention in combination with an active ingredient that is a ligand or an agonist for the BTLA receptor include: salt sodium salt; salt salt magnesium Salt, zinc, darcos, saccharose, lactose, ethyl alcohol, glycerin, mannitol, sorbitol, pentaerythritol, jetylene glycol, propylene glycolol, dipropylene glycolol, polyethylene glycol 400, other polyethylene glycols; fatty acid compounds and triglycerides such as glyceryl trilaurate and glyceryl distearate; pectin; starch; arginic acid; xylose; talc; Oils and fats such as corn oil, safflower oil, wheat germ oil, sesame oil, sesame oil, castor oil and cod liver oil; gelatin; lecithin; silic
  • the pharmaceutically effective amount and administration method or administration means of the compound of the present invention include the type of parasitic protozoa that causes infection, the parasitic site of the protozoa, the severity of the disease state, the treatment policy, the age of the patient, A person skilled in the art can select as appropriate according to weight, sex, general health condition, and (genetic) racial background of the patient.
  • the dosage of the compounds of the present invention is 1 to 10, 0 OOmgZ day Z body weight 70 kg, more typically 50 to 2000 mg Z day Z body weight 70 kg.
  • the pharmaceutical composition of the present invention can have any shape known to those skilled in the art depending on the administration method and the administration route. They can be administered by any suitable method. For example, liquids, powders, colloids, etc., can be injected intravenously, intraperitoneally, subcutaneously, or administered orally with the carrier or diluent described above. Is mentioned. still, The active ingredient is a force that can be contained in an appropriate amount depending on the purpose, object, shape, etc. of the pharmaceutical composition of the present invention. Usually, lmg to 10, OOOmg, preferably lOmg to 3, Contains about OOOmg.
  • the DNA sequence shown in SEQ ID NO: 1 is the genomic base sequence of the HVEM gene of mice with inflammatory bowel disease (C57BL / 6 strain), and various mutations (monobasic substitutions) that are unique to this gene. (Adapted length oligonucleotides or polynucleotide fragments containing similar mutations in the HVE M gene of other mammals such as humans corresponding to each position) Can be used to detect HVEM mutations associated with The nucleotide numbers indicating the positions of various mutations in SEQ ID NO: 1 are the HVEM gene genome sequences of inflammatory bowel disease developing mice (C57BL / 6 strain) and disease resistant strain BALB / c mice. Is assigned based on the base number in the genome sequence of the HVEM gene of disease resistant strain BALB / c mice when aligned so as to maximize.
  • the mutations in the HVEM gene of other mammals corresponding to the positions of the various mutations in SEQ ID NO: 1 are the same as the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of the HVEM gene in other animals.
  • the HVEM genomic genes of both susceptibility lines C57BL / 6 (B6), such as the 1,300th base and the disease resistant line BALB / c (BALB), 1,305 are shown. In comparisons between base sequences, the bases enclosed in the squares can be said to be the bases at the corresponding positions.
  • oligonucleotide sequencing sequence method
  • Taqman method Taqman PCR
  • invader method Invader method
  • RCA method RCA method
  • MALDI-TOFZMS method DNA chip method
  • DNA chip method etc.
  • the specific DNA length of the oligonucleotide or polynucleotide of the present invention can be appropriately selected by those skilled in the art according to such a detection method.
  • 10 ⁇ It consists of a continuous DNA sequence of L00. or,
  • the kit of the present invention can have an appropriate configuration depending on the specific method for measuring the mutation in the above base sequence.
  • the kit of the present invention can contain a probe or primer consisting of a base sequence of an appropriate length used for amplifying the oligonucleotide or polynucleotide of the present invention, which is shown in FIG.
  • a probe or primer consisting of a base sequence of an appropriate length used for amplifying the oligonucleotide or polynucleotide of the present invention, which is shown in FIG.
  • these polynucleotides or oligonucleotides may be labeled with an appropriate labeling substance such as a radioactive substance, a fluorescent substance, or a dye.
  • the above kit contains other elements or components known to those skilled in the art, such as various reagents, enzymes, buffers, reaction plates (containers), etc., depending on the composition and purpose of use.
  • OX40L-Tg An OX40 ligand gene-transferred mouse (hereinafter referred to as “OX40L-Tg”) produced by the present inventors spontaneously develops inflammatory bowel disease very similar to human ulcerative colitis with aging (special Open 2003-102330). Inflammatory bowel disease seen in OX40L-Tg mice occurs only in C57BL / 6 mice and not in BALB / c, DBA / 2, DBA / 1J strains. That is, the presence of a disease susceptibility gene in C5 7BL / 6 strain was assumed. Thus, a susceptible strain (C57BL / 6 strain) and a resistant strain (BALB / c) were crossed to produce 339 cross-bred OX40L-Tg mice.
  • Fig. 4 shows the polymorphisms (mutations) seen in the genomic nucleotide sequence (SEQ ID NO: 1) of the HVEM gene in mice with inflammatory bowel disease (C57BL / 6 strain).
  • the mouse spleen cell RNA of each strain was extracted according to a conventional method, and the HVEM cDNA was amplified by RT-PCR using the obtained RNA as a sample, and the HVEM cDNA base sequence of each strain was analyzed by the direct sequence method. It was determined.
  • spleen and intestinal fistula mouse strains (C57BL / 6) in each mouse showed that HVEM mRNA expression level in spleen lymphocytes was higher than that in disease resistant mouse strains (BALB / c). The ratio was less than 1/50.
  • HVEM gene polymorphism abnormality
  • HVEM mRNA expression deficiency and therefore lacked the immunosuppressive signal that should originally be supplied from BTLA. It is assumed that inflammatory bowel disease develops. Therefore, we concluded that the HVEM gene is a susceptibility gene for the development of mouse inflammatory bowel disease.
  • T cells isolated from the mouse spleen were stimulated with an anti-CD3 antibody (clone name: 145-2C11 (BD Biosciences)) immobilized on a plate according to a conventional method. After 72 hours, the amount of 3 H-thymidine incorporation was measured.
  • mouse soluble HVE M corresponding to amino acid 1st to 204th region (extracellular region) of mouse HVEM protein
  • HVEM gene-transduced mouse Virology, 2004 Mar 15; 320 (2): 267-75
  • OX40L-Tg mouse which are produced in a large amount in a soluble HVEM organism
  • HV EM-OX40L double transgenic mice were prepared.
  • gut weight and histological colitis severity which are one of the indicators of severity of inflammatory bowel disease. The severity was measured according to the [Severity Score] above.
  • In vivo expression of HVEM significantly suppressed intestinal weight increase and intestinal inflammatory response in OX40L-Tg mice (Fig. 8). From the above results, it was concluded that T cells can suppress the onset of inflammatory bowel disease by artificially supplying BTLA signals with soluble HVEM or anti-BTLA antibodies.
  • a ligand or agonist for a BTLA receptor such as soluble HVEM or anti-BTLA antibody
  • a BTLA receptor such as soluble HVEM or anti-BTLA antibody
  • HVEM-BTLA signal By administering a ligand or agonist for a BTLA receptor such as soluble HVEM or anti-BTLA antibody to an inflammatory bowel disease model mouse and controlling the HVEM-BTLA signal, the onset of inflammatory bowel disease can be suppressed. Disease treatment is possible. Furthermore, these substances can be clinically applied as pharmaceuticals.

Abstract

Disclosed are an effective therapeutic method and an effective pharmaceutical composition for an inflammatory bowel disease such as ulcerative colitis or Crohn's disease. A method for treatment of an inflammatory bowel disease such as ulcerative colitis or Crohn's disease, the method comprising reacting a BTLA receptor with its ligand (e.g., soluble HVEM) or agonist (e.g., anti-BTLA antibody) to increase the BTLA receptor signal on a T cell; and a pharmaceutical composition, particularly for the treatment of an inflammatory bowel disease, comprising a ligand or agonist of a BTLA receptor as an active ingredient.

Description

明 細 書  Specification
HVEM及び BTLAを標的とした炎症性腸疾患治療用医薬組成物及び 治療方法  Pharmaceutical composition for treating inflammatory bowel disease targeting HVEM and BTLA and therapeutic method
技術分野  Technical field
[0001] 本発明は、 T細胞上の BTLA受容体シグナルを増強することによる、炎症性腸疾患 治療方法、及びそのための医薬組成物等に関する。  [0001] The present invention relates to a method for treating inflammatory bowel disease by enhancing a BTLA receptor signal on T cells, a pharmaceutical composition therefor, and the like.
背景技術  Background art
[0002] ヒト炎症性腸疾患 (潰瘍性大腸炎及びクローン病）は原因不明の重篤な疾患であり、 先進国において近年患者数の急増が見られる。本疾患の発症感受性遺伝子座は少 なくとも IBD1〜IBD8までの 8つが知られており、一部のクローン病患者で、 IBD5領域 に位置する NOD2 (IBD5)遺伝子変異が明らかにされた。しかし、感受性遺伝子として NOD2は、そのコードする蛋白が細胞内分子であることなどから、それ自身を標的とし た治療法開発には困難を伴い、実際、 NOD2を標的にした治療法は存在しない。他 方、炎症性サイト力インである腫瘍壊死因子 (以下「TNF」と称する）の機能を阻害す る抗 TNF抗体がクローン病の治療薬として臨床応用されている。  [0002] Human inflammatory bowel disease (ulcerative colitis and Crohn's disease) is a serious disease of unknown cause, and the number of patients has been rapidly increasing in developed countries in recent years. There are at least eight susceptibility loci from IBD1 to IBD8 for the onset of this disease. In some patients with Crohn's disease, mutations in the NOD2 (IBD5) gene located in the IBD5 region have been revealed. However, NOD2 as a susceptibility gene is difficult to develop a therapeutic method targeting itself because the protein encoded by it is an intracellular molecule, and there is actually no therapeutic method targeting NOD2. On the other hand, anti-TNF antibodies that inhibit the function of tumor necrosis factor (hereinafter referred to as “TNF”), which is an inflammatory site force-in, have been clinically applied as therapeutic agents for Crohn's disease.
[0003] 単純へルぺスウィルス侵入仲介物質（以下、「Herpes Virus Entry Mediator: HVEM」 と称する）は、 TNF受容体ファミリーに属し、当初、単純へルぺスウィルス（以下「HSV」 と称する)糖タンパク D結合物質として同定され、 HSVの細胞感染を制御する分子とし て知られる。生体内の HVEM発現細胞として T細胞、 B細胞、単球、榭状細胞が知ら れ HVEMの機能としてリンパ球を介した免疫反応制御が報告されて ヽる。その機構の 一つとして、 HVEMがそのリガンドと結合すると、 HVEMからの細胞内シグナルが T細 胞活性ィ匕反応に促進的に働くことが明らかにされた (Kwon et al. J. Biol. Chem 272: 14272, 1997, Harrop et al. J. Immunol.161: 1786, 1998)。  [0003] A simple herpesvirus invasion mediator (hereinafter referred to as "Herpes Virus Entry Mediator: HVEM") belongs to the TNF receptor family and is initially referred to as a simple herpesvirus (hereinafter referred to as "HSV"). ) Glycoprotein Identified as a D-binding substance and known as a molecule that regulates HSV cell infection. T cells, B cells, monocytes and rod cells are known as HVEM-expressing cells in vivo, and lymphocyte-mediated immune response control has been reported as a function of HVEM. As one of the mechanisms, it has been clarified that when HVEM binds to its ligand, intracellular signals from HVEM act to promote T cell activity (Kwon et al. J. Biol. Chem). 272: 14272, 1997, Harrop et al. J. Immunol. 161: 1786, 1998).
[0004] HVEMのリガンドは LIGHTとリンフォトキシン（以下、「LT」と称する） a、 BTLAの 3つ あることが知られる。一方、 LIGHTの受容体は少なくとも、 LT |8受容体、デコイ受容 体 3および HVEMの 3種が知られ、同様に、 LT aの受容体は TNF受容体 I、 TNF受容 体 II、 HVEMの 3種が知られる。 BTLAの受容体として証明されたのは HVEMのみで ある。このように HVEMが関連したリガンド ·受容体系は複雑なネットワークを形成して おり、それらの生体内における機能的関連性の詳細は明らかではない。 [0004] It is known that there are three HVEM ligands: LIGHT, lymphotoxin (hereinafter referred to as “LT”) a, and BTLA. On the other hand, there are at least three receptors for LIGHT: LT | 8 receptor, decoy receptor 3 and HVEM. Similarly, LTa receptors are TNF receptor I, TNF receptor II and HVEM 3 The seed is known. Only HVEM has been proven as a BTLA receptor is there. Thus, the ligand-receptor system related to HVEM forms a complex network, and the details of their functional relevance in vivo are not clear.
[0005] これまでに、疾患との関連においては、アレルギーや自己免疫疾患の患者血清中に 、可溶型 HVEMが検出されること、動脈硬化病変部に HVEMを発現するマクロファー ジが存在することが報告された（Jung HW et al. Exp Mol Med. 35:501-508, 2003、 Le e WH et al, Arterioscler Thromb Vase Biol. 21; 2004—2010, 2001)。しかし、これらの 報告は HVEMを指標とした疾患重症度診断の可能性を示唆したに過ぎず、 HVEMを 標的とした疾患治療の可能性とは全く関連はない。又、動物実験において、可溶型 L Τ β受容体投与が LIGHT機能を抑制することにより、ラット特異的自己免疫性眼ブド ゥ膜炎およびマウス移植片対宿主病を抑制したとの報告がある（Shao H, et al, Eur J Immunol, 3: 1736, 2003, Tamada et al, Nat Med 6: 283, 2000, Tamada et al, J Cln I nvest 109: 549, 2002) 0これらの文献は LIGHTもしくは膜型 LT機能を抑制することに より、ある種の異常免疫反応を抑制できることを示唆する。 [0005] So far, in relation to diseases, soluble HVEM has been detected in sera of patients with allergies and autoimmune diseases, and macrophages that express HVEM exist in atherosclerotic lesions. (Jung HW et al. Exp Mol Med. 35: 501-508, 2003, Lee WH et al, Arterioscler Thromb Vase Biol. 21; 2004—2010, 2001). However, these reports only suggest the possibility of disease severity diagnosis using HVEM as an index, and have nothing to do with the possibility of disease treatment targeting HVEM. In animal experiments, it was reported that administration of soluble L 可溶 β receptor suppressed rat-specific autoimmune ocular bursitis and mouse graft-versus-host disease by suppressing LIGHT function. (Shao H, et al, Eur J Immunol, 3: 1736, 2003, Tamada et al, Nat Med 6: 283, 2000, Tamada et al, J Cln I nvest 109: 549, 2002) 0 This suggests that suppression of membrane LT function can suppress certain abnormal immune responses.
[0006] HVEMの第 3のリガンドである BTLAはそれ自身 T細胞上の受容体タンパクであり、 H VEMが BTLAに結合することにより、 T細胞活性化を強力に抑制する（Nat. Immunol, 6:90, 2004) o更に、 HVEM遺伝子欠損マウスの T細胞の活性化能が正常マウスの T 細胞よりも増強されていることが明らかにされ、生体内での HVEM- BTLA結合が免疫 抑制に重要である可能性が示唆された（Sedy et al, Nat Immunol, 6: 90-98, 2005, G onzalez et al, Proc Natl Acad Sci U S A. 102: 1116-21, 2005)。又、一方で、 HVEM シグナルが炎症性腸疾患発症を促進するとの結果が報告されている (Wang et al, J. Immunol.l74:8173, 2005) 0 [0006] BTLA, the third ligand of HVEM, is itself a receptor protein on T cells, and HVEM binds to BTLA to strongly suppress T cell activation (Nat. Immunol, 6 : 90, 2004) oIn addition, it was revealed that the T cell activation ability of HVEM gene-deficient mice is enhanced compared to that of normal mice, and HVEM-BTLA binding in vivo is important for immunosuppression (Sedy et al, Nat Immunol, 6: 90-98, 2005, Gonzalez et al, Proc Natl Acad Sci US A. 102: 1116-21, 2005). On the other hand, it has been reported that HVEM signal promotes the development of inflammatory bowel disease (Wang et al, J. Immunol.l74: 8173, 2005) 0
非特許文献 l : Nat. Immunol, 6:90, 2004  Non-patent literature l: Nat. Immunol, 6:90, 2004
非特許文献 2 : Sedy et al, Nat Immunol, 6: 90-98, 2005, Gonzalez et al, Proc Natl A cad Sci U S A. 102:1116-21, 2005  Non-Patent Document 2: Sedy et al, Nat Immunol, 6: 90-98, 2005, Gonzalez et al, Proc Natl A cad Sci U S A. 102: 1116-21, 2005
非特許文献 3 : Wang et al., The critical role of LIGHT in promoting intestinal inflam mation and Crohn' s disease, J. Immunol. 2005 Jun 15; 174 (12):8173-82  Non-Patent Document 3: Wang et al., The critical role of LIGHT in promoting intestinal inflam mation and Crohn 's disease, J. Immunol. 2005 Jun 15; 174 (12): 8173-82
発明の開示  Disclosure of the invention
発明が解決しょうとする課題 [0007] 現在の処、炎症性腸疾患発症原因については未だ不明であり、 BTLAの炎症性腸 疾患発症に対する関与は全く明らかにされていない。更に、 HVEM- BTLA系特異的 にシグナルを制御した際の炎症性腸疾患発症抑制効果も不明である。 Problems to be solved by the invention [0007] At present, the cause of the onset of inflammatory bowel disease is still unknown, and the involvement of BTLA in the development of inflammatory bowel disease has not been clarified at all. Furthermore, the inhibitory effect on the development of inflammatory bowel disease when the signal is controlled specifically for the HVEM-BTLA system is unknown.
[0008] 本発明者は、動物モデルにおいて炎症性疾患、特に炎症性腸疾患の発症原因のひ とつが HVEMの遺伝子変異と発現不全であることを見出し、炎症性腸疾患発症に HV EMシグナルが関与することを初めて明らかにした。本発明は、力かる知見に基づき なされたものである。  [0008] The present inventor has found that HVEM gene mutation and expression are one of the causes of inflammatory diseases, particularly inflammatory bowel disease in animal models, and HV EM signals are associated with the onset of inflammatory bowel disease. First revealed that he was involved. The present invention has been made based on strong knowledge.
課題を解決するための手段  Means for solving the problem
[0009] 即ち、本発明は以下の各態様に係る。 That is, the present invention relates to the following aspects.
1. T細胞上の BTLA受容体シグナルを増強することによる、炎症性腸疾患治療方法  1. Method for treating inflammatory bowel disease by enhancing BTLA receptor signal on T cells
2. BTLA受容体とそのリガンドを反応させることにより、 T細胞上の BTLA受容体シ グナルを増強する、上記 1記載の方法。 2. The method according to 1 above, wherein the BTLA receptor signal on T cells is enhanced by reacting the BTLA receptor with its ligand.
3.リガンドが可溶型 HVEMである、上記 2記載の方法。  3. The method according to 2 above, wherein the ligand is soluble HVEM.
4. BTLA受容体とそのァゴニストを反応させることにより、 T細胞上の BTLA受容体 シグナルを増強する、上記 1記載の方法。  4. The method according to 1 above, wherein the BTLA receptor signal on T cells is enhanced by reacting the BTLA receptor with its agonist.
5.ァゴ-ストが抗 BTLA抗体である、上記 4記載の方法。  5. The method according to 4 above, wherein the antigen is an anti-BTLA antibody.
6.炎症性腸疾患が、潰瘍性大腸炎又はクローン病である、上記 1〜5のいずれか一 項に記載の方法。  6. The method according to any one of 1 to 5 above, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease.
7. BTLA受容体のリガンドを有効成分として含有する、医薬組成物。  7. A pharmaceutical composition comprising a BTLA receptor ligand as an active ingredient.
8.リガンドが可溶型 HVEMである、上記 7記載の医薬組成物。  8. The pharmaceutical composition according to 7 above, wherein the ligand is soluble HVEM.
9. BTLA受容体のァゴニストを有効成分として含有する、医薬組成物。  9. A pharmaceutical composition comprising an BTLA receptor agonist as an active ingredient.
10.ァゴ-ストが抗 BTLA抗体である、上記 9記載の医薬組成物。  10. The pharmaceutical composition according to 9 above, wherein the antigen is an anti-BTLA antibody.
11.炎症性腸疾患治療用医薬組成物である、上記 8〜10のいずれか一項に記載の 医薬組成物。  11. The pharmaceutical composition according to any one of 8 to 10 above, which is a pharmaceutical composition for treating inflammatory bowel disease.
12.炎症性腸疾患が、潰瘍性大腸炎又はクローン病である、上記 11記載の炎症性 腸疾患治療用医薬組成物。  12. The pharmaceutical composition for treating inflammatory bowel disease according to the above 11, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease.
13.配列番号 1に示される DNA配列力も成るポリヌクレオチドにおける、 333番目力 ら 336番目までの 4塩基欠損、 1305番目のグァニン、 1542番目のアデ-ン、 1669 番目のグァニン、 2606番目のアデニン、 4544番目のアデニン、 5681番目のグァニ ン、 5704番目のチミン、 5832番目の 1塩基欠損、 5867番目のシ卜シン、 5896番目 のチミン、 6295番目のチミン、 6566番目のシトシンと 6567番目のシトシン間へのグ ァニン 2塩基の挿入、 6567番目のシトシンと 6568番目のチミン間へのグァニンの揷 入、 6568番目のチミンと 6569番目のグァニン間へのアデニンーグァニン 2塩基の揷 入、又は 6579番目のシトシンに対応する塩基を含む 10〜100の連続した DNA配 列からなるオリゴヌクレオチド又はポリヌクレオチド。 13. The 333rd power in the polynucleotide having the DNA sequence power shown in SEQ ID NO: 1 Et al. 336, 4 base deletion, 1305th guanine, 1542th adenine, 1669th guanine, 2606th adenine, 4544th adenine, 5681th guanine, 5704th thymine, 5832th 1 base deletion, 5867th cytosine, 5896th thymine, 6295th thymine, insertion of 2 bases of guanine between 6566th cytosine and 6567th cytosine, between 6567th cytosine and 6568th thymine Guanine insertion into the 6568th thymine and 6569th guanine between the two bases of adenine-guanine, or the base sequence corresponding to the 6579th cytosine. An oligonucleotide or polynucleotide.
14.上記 13に記載のオリゴヌクレオチド又はポリヌクレオチドを検出することによる、 炎症性腸疾患の発症と関連する HVEM遺伝子の変異の測定法。  14. A method for measuring a mutation in the HVEM gene associated with the onset of inflammatory bowel disease by detecting the oligonucleotide or polynucleotide according to 13 above.
15.上記 14に記載の測定法を実施するための、炎症性腸疾患の発症と関連する H VEM遺伝子の変異の測定用キット。  15. A kit for measuring a mutation in the HVEM gene associated with the onset of inflammatory bowel disease for carrying out the measurement method according to 14 above.
発明の効果  The invention's effect
[0010] 動物モデルにおいて炎症性疾患、特に炎症性腸疾患の発症原因のひとつが HVEM の遺伝子変異と発現不全であることが解明された。従って、 HVEM— BTLAシグナル を制御することにより、炎症性腸疾患の治療が可能になる。具体的には、可溶型 HVE M投与あるいは抗 BTLA抗体投与等による人為的 BTLAシグナルの増強が炎症性 腸疾患治療に有効である。  [0010] It has been elucidated that one of the causes of inflammatory diseases, particularly inflammatory bowel diseases, in animal models is HVEM gene mutation and expression deficiency. Therefore, by controlling the HVEM-BTLA signal, it becomes possible to treat inflammatory bowel disease. Specifically, artificial BTLA signal enhancement by administration of soluble HVE M or anti-BTLA antibody is effective in treating inflammatory bowel disease.
図面の簡単な説明  Brief Description of Drawings
[0011] [図 1]疾患感受性系統 (C57BL/6)と疾患抵抗性系統 (BALB/c)の交配により作製し た（C57BL/6 X (C57BL/6 x BALB/c)) N2- OX40L- Tgマウス各個体の炎症性腸疾患 重症度についての各遺伝子型による QTL解析の結果を示す。縦軸は Lodスコア、横 軸は遺伝子座を表す。  [0011] [Fig.1] N2-OX40L- produced by crossing disease-susceptible strain (C57BL / 6) and disease-resistant strain (BALB / c) (C57BL / 6 X (C57BL / 6 x BALB / c)) The result of the QTL analysis by each genotype about the inflammatory bowel disease severity of each Tg mouse individual is shown. The vertical axis represents the Lod score, and the horizontal axis represents the locus.
[図 2]疾患感受性系統 (C57BL/6)と疾患抵抗性系統 (BALB/c)の交配により作製し た（C57BL/6 X (C57BL/6 x BALB/c)) N2- OX40L- Tgマウス各個体の大腸重量 Z体 重比について各遺伝子型による QTL解析の結果を示す。縦軸は Lodスコア、横軸は 遺伝子座を表す。  [Fig.2] Produced by crossing disease-susceptible strain (C57BL / 6) and disease-resistant strain (BALB / c) (C57BL / 6 X (C57BL / 6 x BALB / c)) N2- OX40L- Tg mice The results of QTL analysis by individual genotypes for individual colon weight Z body weight ratio are shown. The vertical axis represents the Lod score, and the horizontal axis represents the gene locus.
[図 3]マウスゲノム遺伝子配列とヒトゲノム遺伝子配列の対応を示す。棒線で示した部 位が、本発明で同定されたマウス炎症性腸疾患感受性遺伝子座、およびヒト炎症性 腸疾患感受性遺伝子座 IBD7 (1ρ36)である。両者は完全に一致する。 FIG. 3 shows the correspondence between mouse genomic gene sequences and human genomic gene sequences. Part indicated by bar These are the mouse inflammatory bowel disease susceptibility locus identified in the present invention and the human inflammatory bowel disease susceptibility locus IBD7 (1ρ36). Both are in perfect agreement.
[図 4-1]疾患感受性系統 C57BL/6 (B6)と疾患抵抗性系統 BALB/c (BALB)の HVEM ゲノム遺伝子塩基配列の比較を示す。四角で囲んだ箇所に多型 (変異）が同定され た。  [Fig. 4-1] Comparison of HVEM genomic gene base sequences of disease-susceptible strain C57BL / 6 (B6) and disease-resistant strain BALB / c (BALB). A polymorphism (mutation) was identified in the boxed area.
[図 4-2]続き。  [Figure 4-2] Continued.
[図 4-3]続き。 [Figure 4-3] Continued.
[図 4-4]続き。 [Figure 4-4] Continued.
[図 4-5]続き。 [Figure 4-5] Continued.
[図 4-6]続き。 [Figure 4-6] Continued.
[図 5]疾患感受性系統 C57BL/6と疾患抵抗性系統 BALB/c、 129/Sv、 FVBの HVEM c DNA塩基配列の解析により同定された、 C57BL/6特異的アミノ酸変異を示す。 HVE M膜貫通領域内のノリンがァラニンに置換して 、る。  FIG. 5 shows C57BL / 6-specific amino acid mutations identified by analysis of HVEM cDNA sequences of disease-susceptible strain C57BL / 6 and disease-resistant strains BALB / c, 129 / Sv, and FVB. Norin in the HVE M transmembrane region is replaced with alanine.
[図 6]図 A上段は HVEM下段は βァクチンの半定量的 RT-PCR結果を示す。数字は マウス系統を示す（l:129/SvJ, 2:NOD, 3:C57BL/6, 4:C57BL/6xBALB/c Fl, 5:DBA 2, 6:FVB, 7:BALB/c) 0 C57BL/6由来の脾臓と腸管では HVEM mRNAがほとんど検 出できない。図 B :TaqMan probe (アプライドバイオシステム社）を用いたリアルタイム P CRにより HVEMおよび βァクチン mRNA量を定量した。 βァクチン mRNA量を内的対 照として相対的 HVEM mRNA量を計算し、グラフに示した。 C57BL/6マウス脾臓およ び腸管における HVEM mRNA量は BALB/cマウスに比し、 50分の 1以下であった。 圆 7]マウス脾臓より単離した T細胞をプレートに固層化した抗 CD3抗体で刺激し、刺 激後 72時間後の3 H-チミジンの取り込み量を測定した。その際に、可溶型 HVEM (10 mg/ml)添加（白）と無添加（黒)でその取り込み量を比較した。可溶型 HVEM添加に より、 T細胞活性化能が抑制された。 [FIG. 6] The upper part of FIG. A shows the semi-quantitative RT-PCR result of β-actin in the lower part of HVEM. Numbers indicate mouse strains (l: 129 / SvJ, 2: NOD, 3: C57BL / 6, 4: C57BL / 6xBALB / c Fl, 5: DBA 2, 6: FVB, 7: BALB / c) 0 C57BL / HVEM mRNA can hardly be detected in the spleen and intestinal tract of 6 origin. Fig. B: HVEM and β-actin mRNA levels were quantified by real-time PCR using TaqMan probe (Applied Biosystems). Relative HVEM mRNA levels were calculated using the β-actin mRNA level as an internal reference and shown in the graph. The amount of HVEM mRNA in the spleen and intestine of C57BL / 6 mice was less than 1/50 that of BALB / c mice. [7] T cells isolated from mouse spleen were stimulated with an anti-CD3 antibody immobilized on a plate, and the amount of 3 H-thymidine incorporation 72 hours after stimulation was measured. At that time, the amount of uptake was compared between soluble HVEM (10 mg / ml) added (white) and non-added (black). Addition of soluble HVEM suppressed T cell activation ability.
[図 8]HVEM遺伝子導入マウス（Virology, 2004 Mar 15; 320 (2): 267- 75)と OX40L- T gマウスを交配し、 HVEM- OX40L二重遺伝子導入マウスを作製した。 20週齢の野生 型マウス（n= 9)、 HVEM遺伝子導入マウス（n= 14)、 OX40L- Tgマウス（n= 12)、 及び、 HVEM- OX40L二重遺伝子導入マウス (n= 12)における腸管重量 (左）及び 組織学的大腸炎重症度 (右)の結果を示す。尚、左図においては各遺伝子型毎の腸 管重量の平均値及び標準偏差 (エラーバー)を示す。 [FIG. 8] HVEM transgenic mice (Virology, 2004 Mar 15; 320 (2): 267-75) were crossed with OX40L-Tg mice to produce HVEM-OX40L double transgenic mice. Intestinal tract in 20-week-old wild type mice (n = 9), HVEM transgenic mice (n = 14), OX40L-Tg mice (n = 12), and HVEM-OX40L double transgenic mice (n = 12) Weight (left) and The results of histological colitis severity (right) are shown. In the left figure, the mean value and standard deviation (error bar) of intestinal weight for each genotype are shown.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0012] 以下の実施例に示されるように、本発明は、 HVEM遺伝子多型（異常）が HVEM mRN Aの発現不全を引き起こし、その結果、特に、本来 BTLAから供給されるはずの免疫 抑制シグナルが欠如するために炎症性腸疾患が発症する、という新たな考えに基づ くものである。従って、本発明の炎症性腸疾患治療方法は BTLAシグナルを増強さ せることを特徴とする。 BTLAシグナルの増強の方法としては、当業者に公知の任意 の手段を取ることが出来る。例えば、可溶型 HVEMのような BTLA受容体に対するリ ガンド、又は、抗 BTLA抗体のような BTLA受容体に対するァゴ-ストを BTLAと反 応 (結合)させることにより BTLAシグナル増強することができる。  [0012] As shown in the following examples, the present invention is that the HVEM gene polymorphism (abnormal) causes HVEM mRNA expression failure, and as a result, an immunosuppressive signal that should be originally supplied from BTLA. This is based on the new idea that inflammatory bowel disease develops due to lack of Therefore, the method for treating inflammatory bowel disease of the present invention is characterized by enhancing the BTLA signal. Any method known to those skilled in the art can be used as a method for enhancing the BTLA signal. For example, a ligand for a BTLA receptor such as soluble HVEM or an antigen for a BTLA receptor such as an anti-BTLA antibody can react (bind) with BTLA to enhance the BTLA signal. .
[0013] 本発明方法の主な治療対象としては、ヒトを含む哺乳動物等の高等生物を挙げるこ とが出来る。  [0013] Examples of main treatment targets of the method of the present invention include higher organisms such as mammals including humans.
[0014] 可溶型 HVEMは市販されており容易に入手可能である。具体的には、 HVEM (human ):Fc (human) Alexis社）等を挙げることが出来る。或いは、 http：〃 www.ncbi.nlm.nih.g ov/entrez/viewer.fcgi?db=nucleotide&val=23200040等にそのアミノ酸配列が記載さ れているので、それらに基づき当業者に公知の方法で作成することも可能である。  [0014] Soluble HVEM is commercially available and easily available. Specific examples include HVEM (human): Fc (human) Alexis). Alternatively, the amino acid sequence is described in http: 〃 www.ncbi.nlm.nih.g ov / entrez / viewer.fcgi? Db = nucleotide & val = 23200040, etc., and based on them, a method known to those skilled in the art. It is also possible to create it.
[0015] 一方、ヒト BTLAのアミノ酸配列は、例えば、 http://www.ncbi.nlm.nih.gov/entrez/vie wer.fcgi?db=nucleotide&val=54607121等に記載されて公知であるので、このような情 報に基づき、当業者に公知の任意の方法で抗 BTLA抗体を作成することが出来る。 該抗体はモノクローナル抗体、又は、ポリクローナル抗体でも良ぐ更に、これらに基 づき、当業者に公知の遺伝子工学的組換え技術で、抗 BTLA活性を有する各種キ メラ抗体 (ヒト型化抗体)を作成することも可能である。  On the other hand, the amino acid sequence of human BTLA is known and described in, for example, http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=54607121 Based on such information, an anti-BTLA antibody can be prepared by any method known to those skilled in the art. The antibody may be a monoclonal antibody or a polyclonal antibody. Based on these antibodies, various chimeric antibodies (humanized antibodies) having anti-BTLA activity are prepared by genetic engineering recombination techniques known to those skilled in the art. It is also possible to do.
[0016] BTLA受容体に対するリガンド又はァゴニストである有効成分と組合せて、本発明 の医薬組成物に用いることのできる医薬キャリアー又は希釈剤の好適な例としては、 塩ィ匕ナトリウム;塩ィ匕マグネシウム;塩ィ匕亜鉛、ダルコ一ス；サッカロ一ス；ラクト一ス；ェ チルアルコール；グリセリン；マンニトール；ソルビトール；ペンタエリスリトール；ジェチ レングリコール、プロピレングリコーノレ、ジプロピレングリコーノレ、ポリエチレングリコー ル 400、他のポリエチレングリコール；トリラウリン酸グリセリル、及びジステアリン酸グリ セリルの如き脂肪酸のモ入ジ及びトリグリセリド；ぺクチン；でんぷん；アルギニン酸； キシロース；タルク；石松子;ォリーブ油、ピーナツ油、ヒマシ油、コーン油、紅花油、 小麦麦芽油、ゴマ油、棉実油、ヒマヮリ油及びタラ肝油の如きオイル及び油脂；ゼラチ ン；レシチン；シリカ；セノレロース；メチルヒドロキシプロピルセルロース、メチルセル口 ス、ヒドロキシェチルセルロースの如きセルロース誘導体；ステアリン酸カルシウム、ラ ゥリン酸カルシウム、ォレイン酸マグレシゥム、パルミチン酸カルシウム、ベヘン酸カル シゥム及びステアリン酸マグネシウム等の 12〜22の炭素原子を有する脂肪酸の塩； シクロデキストリン類（例えば、 ひ シクロデキストリン、 j8—シクロデキストリン、 γ—シ クロデキストリン、ヒドロキシェチル一 /3—シクロデキストリン、ヒドロキシプロピル一 β —シクロデキストリン、ジヒドロキシプロピル一 13—シクロデキストリン、カルボキシメチ ルェチル一 /3—シクロデキストリン、シクロアヮォドリン、及びジメチル一 β—シクロデ キストリン等）；乳化剤 (例えば、 2〜22の炭素原子，特に 10〜18の炭素原子を有す る飽和及び不飽和の脂肪酸とグリコール、グリセリン、ジエチレングリコール、ペンタエ リスリトール、エチルアルコール、ブチルアルコール、ォクタデシルアルコールの如き 1〜20の炭素原子を有する一価の脂肪族アルコール又は多価アルコールとのエス テル;及びジメチルポリシロキサンの如きシリコーン等が挙げられる。更に、医薬組成 物に従来力 用いられてきた当業者に公知の任意の追加のキャリアーも本発明の医 薬組成物に使用することが出来る。 [0016] Preferable examples of the pharmaceutical carrier or diluent that can be used in the pharmaceutical composition of the present invention in combination with an active ingredient that is a ligand or an agonist for the BTLA receptor include: salt sodium salt; salt salt magnesium Salt, zinc, darcos, saccharose, lactose, ethyl alcohol, glycerin, mannitol, sorbitol, pentaerythritol, jetylene glycol, propylene glycolol, dipropylene glycolol, polyethylene glycol 400, other polyethylene glycols; fatty acid compounds and triglycerides such as glyceryl trilaurate and glyceryl distearate; pectin; starch; arginic acid; xylose; talc; Oils and fats such as corn oil, safflower oil, wheat germ oil, sesame oil, sesame oil, castor oil and cod liver oil; gelatin; lecithin; silica; cenorelose; methyl hydroxypropyl cellulose; methyl cellulose; Cellulose derivatives such as: salts of fatty acids having 12 to 22 carbon atoms such as calcium stearate, calcium laurate, magnesium oleate, calcium palmitate, calcium behenate and magnesium stearate; cyclodextrins (eg Cyclodextrin, j8-cyclodextrin, γ-cyclodextrin, hydroxyethyl 1 / 3-cyclodextrin, hydroxypropyl 1 β-cyclodextrin, dihydroxypropyl-1-13-cyclodextrin, carboxymethylethyl 1 / 3- Cyclodextrin, cycloazodrine, dimethyl mono-β-cyclodextrin, etc.); emulsifiers (eg saturated and unsaturated fatty acids and glycols, glycerin having 2 to 22 carbon atoms, especially 10 to 18 carbon atoms) , Esters with monohydric aliphatic or polyhydric alcohols having 1 to 20 carbon atoms such as diethylene glycol, pentaerythritol, ethyl alcohol, butyl alcohol, octadecyl alcohol; and silicones such as dimethylpolysiloxane. Cited That. Further, any additional carriers known to those skilled in the art that have been used conventionally force in the pharmaceutical compositions can also be used in a pharmaceutical composition of the present invention.
[0017] 本発明の化合物の薬学的な有効量及び投与方法又は投与手段は、感染症の原 因となる寄生原虫の種類、原虫の寄生部位、病状の重さ、治療方針、患者の年齢、 体重、性別、全般的な健康状態、及び患者の (遺伝的)人種的背景に応じて、当業 者が適宜選択することができる。一般的には、本発明の化合物の投与量は 1〜10, 0 OOmgZ日 Z体重 70kg、より一般的には 50〜2000mgZ日 Z体重 70kgである。  [0017] The pharmaceutically effective amount and administration method or administration means of the compound of the present invention include the type of parasitic protozoa that causes infection, the parasitic site of the protozoa, the severity of the disease state, the treatment policy, the age of the patient, A person skilled in the art can select as appropriate according to weight, sex, general health condition, and (genetic) racial background of the patient. Generally, the dosage of the compounds of the present invention is 1 to 10, 0 OOmgZ day Z body weight 70 kg, more typically 50 to 2000 mg Z day Z body weight 70 kg.
[0018] 本発明医薬組成物は投与方法'投与経路等に応じて当業者に公知の任意の形状 とすることが出来る。それらは適当な方法で投与することが出来る。例えば、形状とし ては、液体状、粉末状、及びコロイド状等があり、上記のキャリアー又は希釈剤を伴つ た形で、静脈内、腹腔内、皮下に注射するか、又は、経口投与等が挙げられる。尚、 有効成分は、本発明の医薬組成物の使用目的、対象、及び形状等に応じて、適当 な量で含有されることが出来る力 通常、 lmg〜10, OOOmg程度、好ましくは、 lOmg 〜3, OOOmg程度含有されている。 [0018] The pharmaceutical composition of the present invention can have any shape known to those skilled in the art depending on the administration method and the administration route. They can be administered by any suitable method. For example, liquids, powders, colloids, etc., can be injected intravenously, intraperitoneally, subcutaneously, or administered orally with the carrier or diluent described above. Is mentioned. still, The active ingredient is a force that can be contained in an appropriate amount depending on the purpose, object, shape, etc. of the pharmaceutical composition of the present invention. Usually, lmg to 10, OOOmg, preferably lOmg to 3, Contains about OOOmg.
[0019] 配列番号 1に示される DNA配列は、炎症性腸疾患発症マウス (C57BL/6系統）の H VEM遺伝子のゲノム塩基配列であり、この遺伝子に特有に見られる各種変異（一塩 基置換、塩基欠損、塩基挿入等)の各位置に対応するヒト等の他の哺乳動物の HVE M遺伝子における同様な変異を含む適応な長さのオリゴヌクレオチド又はポリヌクレ ォチド断片は、炎症性腸疾患の発症と関連する HVEM遺伝子の変異の検出に使用 することが出来る。尚、配列番号 1における各種変異の位置を示す塩基番号は、炎 症性腸疾患発症マウス (C57BL/6系統）と疾患抵抗性系統 BALB/cマウスの HVEM 遺伝子ゲノム配列をそれらの間の同一性が最大となるようにァライメントさせた場合の 、疾患抵抗性系統 BALB/cマウスの HVEM遺伝子ゲノム配列における塩基番号に 基づき付されたものである。  [0019] The DNA sequence shown in SEQ ID NO: 1 is the genomic base sequence of the HVEM gene of mice with inflammatory bowel disease (C57BL / 6 strain), and various mutations (monobasic substitutions) that are unique to this gene. (Adapted length oligonucleotides or polynucleotide fragments containing similar mutations in the HVE M gene of other mammals such as humans corresponding to each position) Can be used to detect HVEM mutations associated with The nucleotide numbers indicating the positions of various mutations in SEQ ID NO: 1 are the HVEM gene genome sequences of inflammatory bowel disease developing mice (C57BL / 6 strain) and disease resistant strain BALB / c mice. Is assigned based on the base number in the genome sequence of the HVEM gene of disease resistant strain BALB / c mice when aligned so as to maximize.
[0020] 尚、ここで、配列番号 1における各種変異の位置と対応する他の哺乳動物の HVEM 遺伝子における変異は、配列番号 1の塩基配列と他の動物における HVEM遺伝子の 塩基配列とを、それらの間の同一性 (identity)が最大となるようにァライメントさせた場 合、互いの塩基が同じ位置になることを意味し、必ずしも、対応する位置にある塩基 が夫々の遺伝子にお!、て 5 '末端力ゝらの同じ位置 (番目）存在することを意味するもの ではない。具体的には、図 4で、疾患感受性系統 C57BL/6 (B6)の第 1,300番目の塩 基と疾患抵抗性系統 BALB/c (BALB)の第 1,305番目のような、両者の HVEMゲノム 遺伝子の塩基配列の間の比較において四角で囲んだ塩基が夫々対応する位置に ある塩基といえる。  [0020] Here, the mutations in the HVEM gene of other mammals corresponding to the positions of the various mutations in SEQ ID NO: 1 are the same as the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of the HVEM gene in other animals. Means that the bases of each other are in the same position, and the bases in the corresponding positions are not necessarily in each gene! 5 'It does not mean that the same position (th) of the end force exists. Specifically, in Figure 4, the HVEM genomic genes of both susceptibility lines C57BL / 6 (B6), such as the 1,300th base and the disease resistant line BALB / c (BALB), 1,305, are shown. In comparisons between base sequences, the bases enclosed in the squares can be said to be the bases at the corresponding positions.
[0021] このような変異を検出する様々な方法は当業者に公知であり、例えば、塩基配列決 定（シークェンス法）、並びに、タクマン法（Taqman PCR)、侵入法（Invader method) 、 RCA法、 MALDI—TOFZMS法、及び DNAチップ法等のス -ップタイピングで 使用される各種方法を挙げることが出来る。従って、本発明のオリゴヌクレオチド又は ポリヌクレオチドの具体的な DNAの長さは、このような検出方法等に応じて、当業者 が適宜選択することが出来る。通常、 10〜： L00の連続した DNA配列からなる。又、 本発明のキットは、上記塩基配列における変異を測定するための具体的な方法'手 段に応じて、適当な構成をとることが出来る。例えば、本発明キットには、本発明のォ リゴヌクレオチド又はポリヌクレオチドを増幅するために使用される適当な長さの塩基 配列からなるプローブ又はプライマーを含有することが出来、それらは、図 4に示され た塩基配列情報に基づき当業者が適宜設計することが可能である。更に、必要に応 じて、これらポリヌクレオチド又はオリゴヌクレオチドは放射性物質、蛍光物質、色素 等の適当な標識物質によって標識されていても良い。更に、上記キットには、その構 成 ·使用目的などに応じて、当業者に公知の他の要素又は成分、例えば、各種試薬 、酵素、緩衝液、反応プレート (容器)等が含まれる。 [0021] Various methods for detecting such mutations are known to those skilled in the art. For example, nucleotide sequencing (sequence method), Taqman method (Taqman PCR), invader method (Invader method), RCA method , MALDI-TOFZMS method, DNA chip method, etc. Accordingly, the specific DNA length of the oligonucleotide or polynucleotide of the present invention can be appropriately selected by those skilled in the art according to such a detection method. Usually, 10 ~: It consists of a continuous DNA sequence of L00. or, The kit of the present invention can have an appropriate configuration depending on the specific method for measuring the mutation in the above base sequence. For example, the kit of the present invention can contain a probe or primer consisting of a base sequence of an appropriate length used for amplifying the oligonucleotide or polynucleotide of the present invention, which is shown in FIG. Those skilled in the art can appropriately design based on the indicated base sequence information. Furthermore, if necessary, these polynucleotides or oligonucleotides may be labeled with an appropriate labeling substance such as a radioactive substance, a fluorescent substance, or a dye. Furthermore, the above kit contains other elements or components known to those skilled in the art, such as various reagents, enzymes, buffers, reaction plates (containers), etc., depending on the composition and purpose of use.
実施例  Example
[0022] 以下に本発明を詳細に説明するために実施例を示す。尚、本発明の技術的範囲は これら実施例に限定されるものではない。  [0022] Examples are given below to explain the present invention in detail. The technical scope of the present invention is not limited to these examples.
[0023] 本発明者らが作製した OX40リガンド遺伝子導入 (以下、「OX40L-Tg」と記す)マウス は、ヒト潰瘍性大腸炎に極めて類似の炎症性腸疾患を加齢とともに自然発症する (特 開 2003-102330)。 OX40L-Tgマウスでみられる炎症性腸疾患は C57BL/6系統のマウ スでのみ発症し、 BALB/c、 DBA/2, DBA/1J系統では全く発症しない。すなわち、 C5 7BL/6系統に疾患感受性遺伝子の存在が想定された。そこで、感受性系統 (C57BL /6系統）と抵抗性系統 (BALB/c)を交配し、 339匹の交雑 OX40L-Tgマウスを作製し た。それら OX40L-Tgマウスが炎症性腸疾患を発症する 20週齢に達した時点で、各 個体の腸疾患重症度、腸管重量 Z体重比を測定すると共に、各種遺伝子マーカー を用いて遺伝子型を決定した。疾患の重症度は、以下に示すように、常法により判定 した。  [0023] An OX40 ligand gene-transferred mouse (hereinafter referred to as “OX40L-Tg”) produced by the present inventors spontaneously develops inflammatory bowel disease very similar to human ulcerative colitis with aging (special Open 2003-102330). Inflammatory bowel disease seen in OX40L-Tg mice occurs only in C57BL / 6 mice and not in BALB / c, DBA / 2, DBA / 1J strains. That is, the presence of a disease susceptibility gene in C5 7BL / 6 strain was assumed. Thus, a susceptible strain (C57BL / 6 strain) and a resistant strain (BALB / c) were crossed to produce 339 cross-bred OX40L-Tg mice. When these OX40L-Tg mice reach 20 weeks of age when they develop inflammatory bowel disease, each individual's intestinal disease severity, intestinal weight and weight-Z ratio are measured, and genotypes are determined using various genetic markers. did. The severity of the disease was determined by conventional methods as shown below.
[0024] 上行、横行結腸、直腸の 3点からの標本を評価し、一番重症度スコアの高いものをも つて個体の重症度とする。  [0024] Evaluate specimens from three points: the ascending, transverse colon, and rectum, and use the one with the highest severity score as the individual's severity.
[重症度スコア]  [Severity score]
0…粘膜炎症の徴候なし。  0… No signs of mucosal inflammation.
1 · · '粘膜炎症の徴候あり。白血球浸潤は軽度。  1 · · · Signs of mucosal inflammation. Leukocyte infiltration is mild.
2· · '粘膜炎症の徴候あり。白血球浸潤が中等度で腸管肥厚が認められる。 3 · ·，粘膜炎症の徴候あり。白血球浸潤が中等度以上で腸管肥厚が認められる。カロ えて、次の（1)〜（4)のいずれかの徴候を満たす。 2 ·· 'Signs of mucosal inflammation. There is moderate leukocyte infiltration and intestinal thickening. 3 ··· Signs of mucosal inflammation. Leukocyte infiltration is moderate or higher and intestinal thickening is observed. Calorie satisfies any of the following signs (1) to (4).
(1)陰窩膿瘍形成が目立つ。  (1) Formation of crypt abscess is conspicuous.
(2)びらん形成が認められる。  (2) Erosion is observed.
(3)粘膜壊死巣が認められる。  (3) Mucosal necrotic lesions are observed.
(4)高度の粘膜線維化が認められる。  (4) Severe mucosal fibrosis is observed.
[0025] その結果、マウス第 4染色体 80 cM付近が両ァリルとも感受性系統 (C57BL/6系統） の場合に高頻度に疾患の発症が見られ、それに対して 1ァリルが疾患非発症マウス 系統 (BALB/c)由来であればほとんど疾患が発症しないことが明らかになった。  [0025] As a result, the onset of disease was frequently observed when mouse chromosome 4 near 80 cM was susceptible to both strains (C57BL / 6 strain), whereas 1 aryl was a disease-free mouse strain ( It was clarified that the disease hardly develops if derived from BALB / c).
[0026] 更に、各個体の遺伝子型と腸疾患重症度（図 1)および腸管重量 Z体重比（図 2)とで QTL解析を行った結果、 HVEM遺伝子部位に一致して高 、ピーク（Lodスコアはそれ ぞれ 7.4 (図 1)、 4.1 (図 2) )を検出した。さらに、疾患重症群 (スコア 2以上）、疾患未 発症群 (スコア 0)と、 HVEM遺伝子多型の有無との相関を単点解析したところ χ 2検 定で ρ = 4.6 X 10— 8と疾患発症と遺伝子多型との間で極めて強い相関を認めた。以上 より、同部位が炎症性腸疾患感受性遺伝子座であることが明らかになった。この遺伝 子座はヒト炎症性腸疾患感受性遺伝子 IBD7 (1ρ36)に完全に一致して 、た（図 3)。 この事実はヒトにおいても同一の遺伝子が疾患感受性遺伝子である可能性を強く示 唆するものである。 [0026] Furthermore, as a result of QTL analysis using the genotype, severity of intestinal disease (Fig. 1) and intestinal weight Z body weight ratio (Fig. 2) of each individual, high and peak (Lod Scores 7.4 (Fig. 1) and 4.1 (Fig. 2) were detected. Furthermore, disease severity group (score 2 or more), the disease presymptomatic group (score 0), HVEM genetic polymorphism correlate single point analysis was place chi 2 - test at ρ = 4.6 X 10- 8 and disease and the presence or absence of There was a very strong correlation between onset and genetic polymorphism. From the above, it was revealed that this site is an inflammatory bowel disease susceptibility locus. This locus was in complete agreement with the human inflammatory bowel disease susceptibility gene IBD7 (1ρ36) (Figure 3). This fact strongly suggests that the same gene may be a disease susceptibility gene in humans.
[0027] 更に、当業者に公知のダイレクトシークェンス法を用いて、同遺伝子座に存在する候 補遺伝子のゲノム配列を解析したところ、疾患発症マウスが極めて高頻度に HVEM 遺伝子多型 (異常)を有することが明らかになった。図 4において、炎症性腸疾患発 症マウス（C57BL/6系統）の HVEM遺伝子のゲノム塩基配列（配列番号 1)に見られる 多型 (変異)を図 4に示す。  [0027] Furthermore, when the genome sequence of a candidate gene present at the same locus was analyzed using a direct sequence method known to those skilled in the art, HVEM gene polymorphisms (abnormalities) were found in disease-causing mice very frequently. It became clear to have. Fig. 4 shows the polymorphisms (mutations) seen in the genomic nucleotide sequence (SEQ ID NO: 1) of the HVEM gene in mice with inflammatory bowel disease (C57BL / 6 strain).
[0028] 次に、常法に従い、各系統のマウス脾臓細胞 RNAを抽出し、得られた RNAを検体とし て RT- PCR法により HVEMcDNAを増幅し、ダイレクトシークェンス法により各系統の H VEMcDNA塩基配列を決定した。  [0028] Next, the mouse spleen cell RNA of each strain was extracted according to a conventional method, and the HVEM cDNA was amplified by RT-PCR using the obtained RNA as a sample, and the HVEM cDNA base sequence of each strain was analyzed by the direct sequence method. It was determined.
こうして得られた cDNA配列より推定されるアミノ酸配列の比較により、炎症性腸疾患 発症マウスでは図 5に示すアミノ酸変異 (HVEM膜貫通領域内のパリンがァラニンに 置換)を有していることが明らかになった。この変異は疾患を発症しない BALB/c、 129 /Sv、 FVBの 3系統には検出されなかった。 By comparing the amino acid sequences deduced from the cDNA sequences thus obtained, the amino acid mutations shown in Fig. 5 were observed in mice with inflammatory bowel disease (parin in the HVEM transmembrane region was changed to alanine). It became clear to have (substitution). This mutation was not detected in the three strains of BALB / c, 129 / Sv, and FVB that did not cause disease.
[0029] 次に、常法に従い、疾患感受性系統 C57BL/6と疾患抵抗性系統 BALB/cマウス脾臓 細胞より RNAを抽出し、オリゴ dTプライマーを用いて cDNAを合成した。作製した cDN Aを铸型として半定量的 RT- PCRを行った（TaqMan Gene Expression Assays (ァプラ イドバイオシステム社））。更に、 TaqMan probe (アプライドバイオシステム社）を用い たリアルタイム PCRにより HVEMおよび j8ァクチン mRNA量を定量した。その結果、図 6に示すように、各マウスの脾臓及び腸菅の疾患発症マウス系統 (C57BL/6)では、 脾臓リンパ球における HVEM mRNAの発現量が疾患抵抗性マウス系統（BALB/c)に 比し 50分の 1以下であった。  [0029] Next, RNA was extracted from disease-susceptible strain C57BL / 6 and disease-resistant strain BALB / c mouse spleen cells according to a conventional method, and cDNA was synthesized using oligo dT primers. Semi-quantitative RT-PCR was performed using the prepared cDNA as a saddle type (TaqMan Gene Expression Assays (Applied Biosystems)). Furthermore, HVEM and j8 actin mRNA levels were quantified by real-time PCR using TaqMan probe (Applied Biosystems). As a result, as shown in FIG. 6, spleen and intestinal fistula mouse strains (C57BL / 6) in each mouse showed that HVEM mRNA expression level in spleen lymphocytes was higher than that in disease resistant mouse strains (BALB / c). The ratio was less than 1/50.
[0030] 以上の結果から、 C57BL/6でみられた HVEM遺伝子多型（異常）が HVEM mRNAの 発現不全を引き起こし、そのために本来 BTLAから供給されるはずの免疫抑制シグナ ルが欠如するために炎症性腸疾患が発症することが推察される。従って、これらのこ とから、 HVEM遺伝子がマウス炎症性腸疾患発症の感受性遺伝子であると結論した  [0030] From the above results, the HVEM gene polymorphism (abnormality) observed in C57BL / 6 caused HVEM mRNA expression deficiency, and therefore lacked the immunosuppressive signal that should originally be supplied from BTLA. It is assumed that inflammatory bowel disease develops. Therefore, we concluded that the HVEM gene is a susceptibility gene for the development of mouse inflammatory bowel disease.
[0031] これを確認するために、常法に従い、マウス脾臓より単離した T細胞をプレートに固 層化した抗 CD3抗体（クローン名： 145-2C11 (BD Biosciences社)）で刺激し、刺激後 72時間後の3 H-チミジンの取り込み量を測定した。ここで、実際にマウス可溶型 HVE M (マウス HVEM蛋白のアミノ酸 1番目〜204番目の領域（細胞外領域）に一致）を T 細胞に添加すると、 T細胞免疫反応が抑制されることが明らかになった（図 7)。 [0031] In order to confirm this, the T cells isolated from the mouse spleen were stimulated with an anti-CD3 antibody (clone name: 145-2C11 (BD Biosciences)) immobilized on a plate according to a conventional method. After 72 hours, the amount of 3 H-thymidine incorporation was measured. Here, it is clear that T cell immune response is suppressed when mouse soluble HVE M (corresponding to amino acid 1st to 204th region (extracellular region) of mouse HVEM protein) is actually added to T cells. (Fig. 7).
[0032] 更に、可溶型 HVEM生体内に多量に産生する遺伝子改変マウス (HVEM遺伝子導 入マウス： Virology, 2004 Mar 15; 320 (2): 267- 75)と OX40L- Tgマウスを交配し、 HV EM- OX40L二重遺伝子導入マウスを作製した。 20週齢で炎症腸疾患重症度の指 標の一つである腸管重量と組織学的大腸炎重症度を測定した。重症度の測定は、 上記の [重症度スコア]に従った。 HVEMの生体内発現によつて OX40L-Tgマウスの腸 管重量増加と腸管炎症反応が有意に抑制された（図 8)。以上の結果から、可溶性 H VEMあるいは抗 BTLA抗体により、 BTLAシグナルを人為的に供与することにより、 T 細胞により炎症性腸疾患惹起を抑制できると結論された。 産業上の利用可能性 [0032] Furthermore, a gene-modified mouse (HVEM gene-transduced mouse: Virology, 2004 Mar 15; 320 (2): 267-75) and OX40L-Tg mouse, which are produced in a large amount in a soluble HVEM organism, HV EM-OX40L double transgenic mice were prepared. At 20 weeks of age, we measured gut weight and histological colitis severity, which are one of the indicators of severity of inflammatory bowel disease. The severity was measured according to the [Severity Score] above. In vivo expression of HVEM significantly suppressed intestinal weight increase and intestinal inflammatory response in OX40L-Tg mice (Fig. 8). From the above results, it was concluded that T cells can suppress the onset of inflammatory bowel disease by artificially supplying BTLA signals with soluble HVEM or anti-BTLA antibodies. Industrial applicability
可溶型 HVEMあるいは抗 BTLA抗体等の BTLA受容体に対するリガンド又はァゴ- ストを炎症性腸疾患モデルマウスに投与し、 HVEM-BTLAシグナルを制御することに より炎症性腸疾患発症が抑制でき、疾患治療が可能である。さらにそれらの物質を医 薬品として臨床応用できる。 By administering a ligand or agonist for a BTLA receptor such as soluble HVEM or anti-BTLA antibody to an inflammatory bowel disease model mouse and controlling the HVEM-BTLA signal, the onset of inflammatory bowel disease can be suppressed. Disease treatment is possible. Furthermore, these substances can be clinically applied as pharmaceuticals.

Claims

請求の範囲 The scope of the claims
[I] T細胞上の BTLA受容体シグナルを増強することによる、炎症性腸疾患治療方法。  [I] A method for treating inflammatory bowel disease by enhancing BTLA receptor signal on T cells.
[2] BTLA受容体とそのリガンドを反応させることにより、 T細胞上の BTLA受容体シグナ ルを増強する、請求項 1記載の方法。 [2] The method according to claim 1, wherein the BTLA receptor signal on T cells is enhanced by reacting the BTLA receptor with its ligand.
[3] リガンドが可溶型 HVEMである、請求項 2記載の方法。 [3] The method according to claim 2, wherein the ligand is soluble HVEM.
[4] BTLA受容体とそのァゴニストを反応させることにより、 T細胞上の BTLA受容体シグ ナルを増強する、請求項 1記載の方法。  [4] The method according to claim 1, wherein the BTLA receptor signal on T cells is enhanced by reacting the BTLA receptor with its agonist.
[5] ァゴニストが抗 BTLA抗体である、請求項 4記載の方法。 [5] The method according to claim 4, wherein the agonist is an anti-BTLA antibody.
[6] 炎症性腸疾患が、潰瘍性大腸炎又はクローン病である、請求項 1〜5のいずれか一 項に記載の方法。  [6] The method according to any one of claims 1 to 5, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease.
[7] BTLA受容体のリガンドを有効成分として含有する、医薬組成物。  [7] A pharmaceutical composition comprising a BTLA receptor ligand as an active ingredient.
[8] リガンドが可溶型 HVEMである、請求項 7記載の医薬組成物。 8. The pharmaceutical composition according to claim 7, wherein the ligand is soluble HVEM.
[9] BTLA受容体のァゴニストを有効成分として含有する、医薬組成物。 [9] A pharmaceutical composition comprising an BTLA receptor agonist as an active ingredient.
[10] ァゴ-ストが抗 BTLA抗体である、請求項 9記載の医薬組成物。 10. The pharmaceutical composition according to claim 9, wherein the antigen is an anti-BTLA antibody.
[II] 炎症性腸疾患治療用医薬組成物である、請求項 8〜10のいずれか一項に記載の医 薬組成物。  [II] The pharmaceutical composition according to any one of claims 8 to 10, which is a pharmaceutical composition for treating inflammatory bowel disease.
[12] 炎症性腸疾患が、潰瘍性大腸炎又はクローン病である、請求項 11記載の炎症性腸 疾患治療用医薬組成物。  12. The pharmaceutical composition for treating inflammatory bowel disease according to claim 11, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease.
[13] 配列番号 1に示される DNA配列力も成るポリヌクレオチドにおける、 333番目力 33 6番目までの 4塩基欠損、 1305番目のグァニン、 1542番目のアデニン、 1669番目 のグァニン、 2606番目のアデニン、 4544番目のアデニン、 5681番目のグァニン、 5 704番目のチミン、 5832番目の 1塩基欠損、 5867番目のシ卜シン、 5896番目のチ ミン、 6295番目のチミン、 6566番目のシトシンと 6567番目のシトシン間へのグァ- ン 2塩基の挿入、 6567番目のシトシンと 6568番目のチミン間へのグァニンの挿入、 6568番目のチミンと 6569番目のグァニン間へのアデニンーグァニン 2塩基の挿入、 又は 6579番目のシトシンに対応する塩基を含む 10〜100の連続した DNA配列か らなるオリゴヌクレオチド又はポリヌクレオチド。  [13] In the polynucleotide having the DNA sequence strength shown in SEQ ID NO: 1, the 333th force, 33 bases up to 6 base deletion, 1305th guanine, 1542th adenine, 1669th guanine, 2606th adenine, 4544 5th adenine, 5681th guanine, 5 704th thymine, 5832th 1 base deletion, 5867th thymine, 5896th thymine, 6295th thymine, 6566th cytosine and 6567th cytosine Insertion of 2 bases of guanine, insertion of guanine between 6567th cytosine and 6568th thymine, insertion of 2 bases of adenine-guanine between 6568th thymine and 6569th guanine, or 6579th An oligonucleotide or a polynucleotide comprising a continuous DNA sequence of 10 to 100 containing a base corresponding to cytosine.
[14] 請求項 13に記載のオリゴヌクレオチド又はポリヌクレオチドを検出することによる、炎 症性腸疾患の発症と関連する HVEM遺伝子の変異の測定法。 [14] A flame by detecting the oligonucleotide or polynucleotide according to claim 13. Of mutations in the HVEM gene associated with the onset of inflammatory bowel disease
請求項 14に記載の測定法を実施するための、炎症性腸疾患の発症と関連する HVE M遺伝子の変異の測定用キット。 A kit for measuring a mutation of the HVE M gene associated with the onset of inflammatory bowel disease for carrying out the measurement method according to claim 14.
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JP2016518436A (en) * 2013-05-17 2016-06-23 アンスティチュ ナショナル ドゥ ラ サンテ エ ドゥ ラ ルシェルシュ メディカル Antagonists of BTLA / HVEM interaction for use in therapy
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US11939367B2 (en) 2015-06-30 2024-03-26 Sanford Burnham Prebys Medical Discovery Institute BTLA fusion protein agonists and uses thereof

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