WO2021029238A1 - Agent for preventing or treating th17 cell-induced disease and screening method for agents for preventing or treating th17 cell-induced disease - Google Patents

Agent for preventing or treating th17 cell-induced disease and screening method for agents for preventing or treating th17 cell-induced disease Download PDF

Info

Publication number
WO2021029238A1
WO2021029238A1 PCT/JP2020/029535 JP2020029535W WO2021029238A1 WO 2021029238 A1 WO2021029238 A1 WO 2021029238A1 JP 2020029535 W JP2020029535 W JP 2020029535W WO 2021029238 A1 WO2021029238 A1 WO 2021029238A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
cells
disease
agonist
induced
Prior art date
Application number
PCT/JP2020/029535
Other languages
French (fr)
Japanese (ja)
Inventor
洋一郎 岩倉
正承 村山
貴裕 小野
眞子 松岡
Original Assignee
学校法人東京理科大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 学校法人東京理科大学 filed Critical 学校法人東京理科大学
Publication of WO2021029238A1 publication Critical patent/WO2021029238A1/en

Links

Images

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • 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
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

Definitions

  • the present invention relates to a preventive or therapeutic agent for Th17 cell-induced disease and a screening method for a preventive or therapeutic agent for Th17 cell-induced disease.
  • Th17 cells are effector T cells that produce cytokines such as IL-17 and IL-22 and activate neutrophils and epithelial cells.
  • Th17 cells are autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and inflammatory bowel disease; allergic diseases such as asthma, contact hypersensitivity, and delayed hypersensitivity; psoriatic arthritis, psoriatic arthritis, tonic spondylosis; etc. It is known to play an important role in the pathogenesis of various diseases.
  • CTRP3 C1q / TNF-related protein 3
  • CTRP3 is a soluble secretory protein consisting of a short N-terminal variable region, collagen domain, and C-terminal complement C1q domain, and has a crystal structure similar to TNF and complement C1q.
  • the receptor for CTRP3 and the cells expressing the receptor have not yet been identified.
  • the C1q domain also has the secretory protein adiponectin.
  • Adiponectin is known to exert an action by binding to adiponectin receptor 1 (AdipoR1), adiponectin receptor 2 (AdipoR2), etc., and it has been reported that the C1q domain is important for the binding. ..
  • adiponectin is involved in the differentiation of naive T cells into Th17 cells.
  • adiponectin promotes differentiation into Th17 cells (see, for example, Non-Patent Documents 1 and 2)
  • it suppresses the differentiation see, for example, Non-Patent Documents 3 and 4). ..
  • the role of adiponectin in the differentiation mechanism into Th17 cells has not been clarified yet, and there is a problem that it is not easy to apply adiponectin to a preventive or therapeutic agent for Th17 cell-induced diseases.
  • the present invention has been proposed in view of the above, and is a novel prophylactic or therapeutic agent preferably used for the prevention or treatment of Th17 cell-induced diseases, and a method for screening a prophylactic or therapeutic agent for Th17 cell-induced diseases.
  • the purpose is to provide.
  • the present inventors have shown that an agonist that does not show agonist activity on adiponectin receptor 1 and that shows agonist activity on adiponectin receptor 2 suppresses the differentiation of naive T cells into Th17 cells.
  • the present invention provides the following.
  • the adiponectin receptor 1 (AdipoR1) and the adiponectin receptor 2 (AdiponR2) are receptors classified into Class I of the PAQR (progestin and AdiponQ receptors) family, and are also referred to as PAQR1 and PAQR2, respectively.
  • a prophylactic or therapeutic agent for Th17 cell-induced diseases which contains an agonist as an active ingredient, which does not exhibit agonist activity on adiponectin receptor 1 (PAQR1) and exhibits agonist activity on adiponectin receptor 2 (PAQR2).
  • ⁇ 2> The agent for preventing or treating Th17 cell-induced disease according to ⁇ 1>, wherein the agonist is any of the following (a) to (d).
  • B A protein consisting of the gC1q domain of the amino acid sequence shown in SEQ ID NO: 1.
  • C It has 80% or more sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 1, does not show agonist activity on adiponectin receptor 1 (PAQR1), and is an agonist on adiponectin receptor 2 (PAQR2).
  • PAQR1 adiponectin receptor 1
  • PAQR2 adiponectin receptor 2
  • (D) It has 80% or more sequence identity with respect to the gC1q domain of the amino acid sequence shown in SEQ ID NO: 1, does not show agonist activity on adiponectin receptor 1 (PAQR1), and has adiponectin receptor 2 (PAQR2). ), A protein that exhibits agonist activity.
  • PAQR1 adiponectin receptor 1
  • PAQR2 adiponectin receptor 2
  • Th17 cell-induced disease according to ⁇ 1> or ⁇ 2>, wherein the Th17 cell-induced disease is not rheumatoid arthritis or multiple sclerosis.
  • ⁇ 4> The preventive or therapeutic agent for a Th17 cell-induced disease according to ⁇ 1> or ⁇ 2>, wherein the Th17 cell-induced disease is not an autoimmune disease.
  • Th17 cell-induced diseases are psoriasis, psoriatic arthritis, tonic spondylosis, optic neuromyelitis, Alzheimer's disease, Huntington's disease, Parkinson's disease, type 1 diabetes, type 2 diabetes, gout, leukoplakia, and melanitis. , Acute and chronic nephritis, acute and chronic pain, inflammatory bowel disease, liver fibrosis, pancreatic fibrosis, inflammatory lung disease, or allergic disease, Th17 cell-inducible according to ⁇ 1> or ⁇ 2>. Preventive or therapeutic agent for diseases.
  • ⁇ 6> Use of the above-mentioned agonist for producing a prophylactic or therapeutic agent for Th17 cell-induced disease according to any one of ⁇ 1> to ⁇ 5>.
  • a prophylactic or therapeutic agent for Th17 cell-induced diseases which comprises screening a candidate agonist that does not show agonist activity on adiponectin receptor 1 (PAQR1) and shows agonist activity on adiponectin receptor 2 (PAQR2). Screening method.
  • PAQR1 adiponectin receptor 1
  • PAQR2 adiponectin receptor 2
  • the present invention it is possible to provide a novel preventive or therapeutic agent preferably used for the prevention or treatment of Th17 cell-induced disease, and a screening method for the preventive or therapeutic agent for Th17 cell-induced disease.
  • IL-17 + T cell Th17 cell
  • .. 3 is a graph showing the ratio of IL-17 + T cells (Th17 cells) when CTRP3 was added to induce differentiation into Th17 cells with respect to naive T cells purified from C1qtnf3 ⁇ / ⁇ mice.
  • 3 is a graph showing the amount of IL-17 protein in the culture supernatant when CTRP3 is added to induce differentiation into Th17 cells from naive T cells purified from C1qtnf3 ⁇ / ⁇ mice.
  • FIG. 1 is a graph showing the population of IFN- ⁇ + T cell (Th1 cell) when the differentiation induction into Th1 cell was performed with respect to the naive T cell purified from C1qtnf3 ⁇ / ⁇ mouse.
  • 3 is a graph showing the ratio of IFN- ⁇ + T cells (Th1 cells) when differentiation induction into Th1 cells was performed on naive T cells purified from C1qtnf3 ⁇ / ⁇ mice.
  • 6 is a graph showing the amount of IFN- ⁇ protein in the culture supernatant when induction of differentiation into Th1 cells was performed on naive T cells purified from C1qtnf3 ⁇ / ⁇ mice.
  • IL-17 + T cells when CTRP3 was added to induce differentiation into Th17 cells and AdipoR1 blocker or AdipoR2 blocker was added to naive T cells purified from C1qtnf3 ⁇ / ⁇ mice. It is a figure which shows the population of Th17 cell).
  • IL-17 + T cells when CTRP3 was added to induce differentiation into Th17 cells and AdipoR1 blocker or AdipoR2 blocker was added to naive T cells purified from C1qtnf3 ⁇ / ⁇ mice. It is a graph which shows the ratio of Th17 cells).
  • C1qtnf3 ⁇ / ⁇ To naive T cells purified from mice, IL- in the culture supernatant when CTRP3 was added when inducing differentiation into Th17 cells, and AdipoR1 blocker or AdipoR2 blocker was added. 17 is a graph showing the amount of protein. 6 is a graph showing swelling (ear thickness) of the ears of mice when Bethelna cream was applied to C1qtnf3 ⁇ / ⁇ mice and WT mice.
  • the prophylactic or therapeutic agent for Th17 cell-induced disease does not show an agonist activity against adiponectin receptor 1 (AdipoR1) and shows an agonist activity against adiponectin receptor 2 (AdipoR2) (hereinafter, "" Also referred to as "specific agonist”) as an active ingredient.
  • AdipoR1 adiponectin receptor 1
  • AdipoR2 adiponectin receptor 2
  • AdipoRon which is an agonist of the above-mentioned adiponectin and adiponectin receptor, is known to exhibit agonist activity against both AdipoR1 and AdipoR2. As shown in Examples described later, AdipoRon suppressed the differentiation into both Th17 cells and Th1 cells, whereas the specific agonist suppressed only the differentiation into Th17 cells and affected the differentiation into Th1 cells. There wasn't. From these facts, it was clarified that the activation of AdiponR1 suppresses the differentiation into Th1 cells.
  • the active ingredient contained in the preventive or therapeutic agent for Th17 cell-induced diseases is a selective agonist that does not suppress the differentiation into Th1 cells but only suppresses the differentiation into Th17 cells. Since the specific agonist does not show an agonist activity against AdiponR1 but shows an agonist activity only against AdiponR2 (in other words, it suppresses only the differentiation into Th17 cells and not the differentiation into Th1 cells), the above-mentioned selective agonist It is useful as.
  • Th17 cell-induced disease refers to a disease induced by Th17 cells.
  • the Th17 cell-induced disease according to the present embodiment is not particularly limited, but in one embodiment, it is a disease that is not rheumatoid arthritis and multiple sclerosis, and in another embodiment, it is a disease that is not an autoimmune disease.
  • Specific examples of such Th17 cell-induced diseases include psoriasis, psoriatic arthritis, tonic spondylosis, optic neuromyelitis, Alzheimer's disease, Huntington's disease, Parkinson's disease, type 1 diabetes, type 2 diabetes, gout, and white spots.
  • Inflammatory bowel diseases such as vasculitis, acute and chronic nephritis, acute and chronic pain, ulcerative colitis; liver fibrosis, pancreatic fibrosis, inflammatory lung disease, or asthma and contact dermatitis, delayed hypersensitivity Allergic diseases such as illness; etc.
  • the specific agonist is not particularly limited as long as it does not show an agonist activity against AdiponR1 and shows an agonist activity against AdiponR2.
  • CTRP3 which is one of the proteins belonging to the CTRP (C1q / TNF-related protein) family, does not show an agonist activity against AdipoR1 and shows an agonist activity against AdiponR2. was confirmed. Therefore, CTRP3 is mentioned as an example of the specific agonist in this embodiment.
  • the origin of CTRP3 is not particularly limited and may be derived from humans or other animals such as mice, but it is preferably derived from humans.
  • the amino acid sequence of CTRP3 the information registered in the GenBank database of the National Center for Biotechnology Information (NCBI) can be referred to.
  • accession numbers AAI12926 and the like are registered as amino acid sequences of human CTRP3
  • accession numbers AAY21928 and the like are registered as amino acid sequences of mouse CTRP3 in the GenBank database.
  • a typical amino acid sequence is as follows. The underlined portion of the amino acid sequence indicates a spherical domain (gC1q domain).
  • the overall homology in the amino acid sequences of human CTRP3 and mouse CTRP3 is as high as 96%, and the spherical domain (gC1q domain) portion has even higher homology as 99%.
  • CTRP3 may be a commercially available product, or may be produced by a gene recombination method or a chemical synthesis method.
  • CTRP3 is produced by the gene recombination method, for example, if a gene encoding CTRP3 is introduced into microorganisms such as Escherichia coli and yeast, plant cells, insect cells, or animal cells using an expression vector to express the protein. Good.
  • human CTRP3 or mouse CTRP3 mammalian cells are preferably used from the viewpoint of maintaining the three-dimensional structure and post-translational modification.
  • CTRP3 is produced by a chemical synthesis method, it may be produced by the liquid phase method, the solid phase method, the Boc method, the Fmoc method or the like alone or in combination.
  • CTRP3 does not show agonistic activity against AdiponR1 and shows agonistic activity against AdiponR2, from the amino acid sequence in which one or several amino acid residues are substituted, deleted, or added in the amino acid sequence of wild-type CTRP3. It may be a mutant CTRP3.
  • sequence identity between each mutant amino acid sequence and each wild-type amino acid sequence may be, for example, 80% or more, 85% or more, 90% or more, 93. It may be% or more, and may be 95% or more.
  • sequence identity of the spherical domain portion described later is preferably 80% or more, more preferably 90% or more, further preferably 93% or more, and particularly preferably 95% or more. It is preferably 100%, which is extremely preferable.
  • sequence identity means the matching between sequences when two amino acid sequences are aligned, and for example, a BLAST program (https://www.ncbi.nlm.nih.gov/Blast/). It can be calculated using cgi).
  • the protein belonging to the CTRP family exists in the living body only in the spherical domain (gC1q domain), and the spherical domain is considered to be a functional site (Trends. Immunol., 25 (10): 551-61). (2004)). Therefore, the specific agonist may be a peptide constituting the spherical domain of CTRP3.
  • each mutant amino acid sequence and each wild-type amino acid sequence may be, for example, 80% or more, 85% or more, or 90% or more. It may be 93% or more, or 95% or more.
  • Suitable examples of the specific agonist include those selected from the following (a) to (d).
  • A A protein consisting of the amino acid sequence shown in SEQ ID NO: 1.
  • B A protein consisting of the gC1q domain of the amino acid sequence shown in SEQ ID NO: 1.
  • C It has 80% or more sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 1, does not show agonist activity against adiponectin receptor 1 (AdipoR1), and is an agonist against adiponectin receptor 2 (AdipoR2). A protein that exhibits activity.
  • (D) It has 80% or more sequence identity with respect to the gC1q domain of the amino acid sequence shown in SEQ ID NO: 1, does not show agonist activity on adiponectin receptor 1 (AdipoR1), and has adiponectin receptor 2 (AdipoR2). ), A protein that exhibits agonist activity.
  • the specific agonist may be in a form other than a protein or peptide (for example, a low molecular weight compound) as long as it does not show an agonist activity against AdipoR1 and shows an agonist activity against AdipoR2.
  • the preventive or therapeutic agent for Th17 cell-induced disease may contain a component other than the specific agonist, if necessary.
  • Ingredients other than specific agonists include excipients, disintegrants, binders, lubricants, surfactants, buffers, solubilizers, stabilizers, tonicity agents, suspending agents, emulsifiers, buffers. Agents, solvents; etc.
  • the dosage form of the preventive or therapeutic agent for Th17 cell-induced disease according to the present embodiment is not particularly limited and can be selected according to the intended use.
  • the dosage form of the Th17 cell-induced disease preventive or therapeutic agent according to the present embodiment includes parenteral preparations such as injection liquid preparations and lyophilized powder preparations for injection; tablets, granules, capsules, liquid preparations and the like. Oral preparations; etc.
  • the preventive or therapeutic agent for Th17 cell-induced disease according to the present embodiment can be produced by any method adopted in the field of pharmaceuticals or a method with appropriate improvements.
  • the dose of the preventive or therapeutic agent for Th17 cell-induced disease according to the present embodiment is appropriately determined according to the administration target, administration route, target disease, symptom and the like. Further, the number of administrations, the administration interval, and the like are not particularly limited, and the administration may be a single administration or a plurality of administrations.
  • the animal species to which the Th17 cell-induced disease prophylaxis or therapeutic agent according to the present embodiment is administered is not particularly limited, and humans may be administered, and monkeys, pigs, cows, dogs, cats, mice and the like may be administered. Non-human animals may be the subject of administration.
  • prevention includes not only preventing the onset but also delaying the onset time.
  • treatment also includes suppressing the degree of progression of symptoms.
  • the use of a specific agonist for producing a prophylactic or therapeutic agent for Th17 cell-induced disease is also provided.
  • a method for preventing or treating Th17 cell-induced disease which comprises administering a drug containing a specific agonist to a subject in need of prevention or treatment of Th17 cell-induced disease. Will be done.
  • the administration target and the number of administrations in the method the above-mentioned administration target and the number of administrations for the preventive or therapeutic agent for Th17 cell-induced disease can be adopted.
  • the preventive or therapeutic agent for Th17 cell-induced disease according to the present embodiment may be administered in combination with a drug or the like containing another active ingredient.
  • the method for screening a prophylactic or therapeutic agent for a Th17 cell-induced disease is a candidate agonist that does not show an agonist activity against adiponectin receptor 1 (AdipoR1) and shows an agonist activity against adiponectin receptor 2 (AdipoR2).
  • AdipoR1 adiponectin receptor 1
  • AdiponR2 adiponectin receptor 2
  • a prophylactic or therapeutic agent for Th17 cell-induced disease can be screened by using the presence or absence of activation that does not show agonist activity against AdiponR1 and shows agonist activity against AdiponR2 as an index.
  • the method for evaluating the activation of AdipoR1 and AdipoR2 is not particularly limited, and any method can be adopted.
  • mice were bred under SPF conditions in a clean room with a lighting cycle from 8:00 to 20:00. Mice were fed a normal ⁇ -ray sterilized F1 diet (Funabashi Farm) and acidified tap water (adjusted to pH 2.5 with 0.002N HCl). The following tests used mice of the same age (8-12 weeks) and gender.
  • Naive T cells were purified from the spleens of C1qtnf3 ⁇ / ⁇ mice and WT mice using Naive CD 4+ T cell Isolation Kit (Miltenyi Biotec).
  • Naive T cells were plate-coated with 4 ⁇ g / mL anti-CD3 (Clone 145-2C11, BioLegend) and soluble 1 ⁇ g / mL anti-CD28 (Clone 37.51, BioLegend), 10 ⁇ g / mL anti-CD4 (Clone 11B11). , BioLegend), and X-VIVO 20 medium (Lonza) containing 5 ng / mL recombinant murine IL-12 (PeproTech) and containing 10% FBS for 3 days stimulation. Cultures were performed in recombinant human CTRP3 (Aviscera Bioscience), AdipoRon (Cayman Chemical), AdipoR1 blocker (Alpha Diagnostic), and in the presence of AdipoR2 blocker (Alpha).
  • Naive T cells were plate coated with 4 ⁇ g / mL anti-CD3 (clone 145-2C11, BioLegend) and soluble 1 ⁇ g / mL anti-CD28 (clone 37.51, BioLegend), 10 ⁇ g / mL anti-IFN ⁇ (clone R4).
  • ELISA ELISA
  • IL-17 ELISA MAX standard R & D Systems
  • mouse IFN- ⁇ ELISA MAX standard R & D Systems
  • the differentiation of naive T cells of C1qtnf3 ⁇ / ⁇ mice and WT mice into Th17 cells was induced by the method described above, and the population of IL-17 + T cells (Th17 cells) was analyzed by flow cytometry.
  • the amount of IL-17 protein in the culture supernatant was analyzed by ELISA.
  • the population of IL-17 + T cells was significantly increased in C1qtnf3 ⁇ / ⁇ mice as compared with WT mice.
  • the amount of IL-17 protein in the culture supernatant was significantly increased in C1qtnf3 ⁇ / ⁇ mice as compared with WT mice. From these results, it was found that CTRP3 deficiency promotes the differentiation of naive T cells into Th17 cells.
  • the population of IL-17 + T cells was significantly reduced in a concentration-dependent manner of CTRP3.
  • the amount of IL-17 protein in the culture supernatant was significantly decreased depending on the concentration of CTRP3. From these results, it was found that CTRP3 suppresses the differentiation of naive T cells into Th17 cells.
  • CTRP3 does not affect the differentiation of naive T cells into Th1 cells.
  • Test Example 4 In Test Example 4, the effect on the differentiation into Th17 cells and Th1 cells when AdipoRon, which is an agonist of the adiponectin receptor, was confirmed.
  • AdipoRon was added to the medium to a concentration of 1, 2, 3 ⁇ g / mL and cultured, and IL-17 + T cells were cultured.
  • Populations of (Th17 cells) and IFN- ⁇ + T cells (Th1 cells) were analyzed by flow cytometry.
  • the amount of IL-17 protein and the amount of IFN- ⁇ protein in the culture supernatant were analyzed by ELISA.
  • the same experiment was performed 4 times, and the average value and standard deviation were calculated.
  • the results of flow cytometry for Th17 cells are shown in FIGS. 4A and 4B, and the results of ELISA are shown in FIG. 4C.
  • the results of flow cytometry for Th1 cells are shown in FIGS. 4D and 4E, and the results of ELISA are shown in FIG. 4F.
  • the population of IL-17 + T cells was significantly reduced in a concentration-dependent manner of AdipoRon.
  • the amount of IL-17 protein in the culture supernatant was significantly decreased depending on the concentration of AdipoRon.
  • the population of IFN- ⁇ + T cells was significantly reduced in a concentration-dependent manner of AdipoRon.
  • the amount of IFN- ⁇ protein in the culture supernatant was significantly decreased depending on the concentration of AdipoRon. From these results, it was found that AdipoRon suppresses the differentiation into both Th17 cells and Th1 cells.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Neurology (AREA)
  • Biomedical Technology (AREA)
  • Neurosurgery (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Diabetes (AREA)
  • Biochemistry (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Biophysics (AREA)
  • Pain & Pain Management (AREA)
  • Psychology (AREA)
  • Pulmonology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Rheumatology (AREA)
  • Epidemiology (AREA)
  • Hospice & Palliative Care (AREA)
  • Hematology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Endocrinology (AREA)
  • Biotechnology (AREA)

Abstract

Provided are: an agent for preventing or treating Th17 cell-induced disease, the agent containing as an active ingredient an agonist that exhibits agonist activity against adiponectin receptors 2 (PAQR2) without exhibiting agonist activity against adiponectin receptors 1 (PAQR1); and a screening method for agents that prevent or treat Th17 cell-induced disease, the method including screening candidate agonists that exhibit agonist activity against adiponectin receptors 2 (PAQR2) without exhibiting agonist activity against adiponectin receptors 1 (PAQR1).

Description

Ｔｈ１７細胞誘導性疾患の予防又は治療剤、及びＴｈ１７細胞誘導性疾患の予防又は治療剤のスクリーニング方法A method for screening a preventive or therapeutic agent for Th17 cell-induced disease and a preventive or therapeutic agent for Th17 cell-induced disease.
　本発明は、Ｔｈ１７細胞誘導性疾患の予防又は治療剤、及びＴｈ１７細胞誘導性疾患の予防又は治療剤のスクリーニング方法に関する。 The present invention relates to a preventive or therapeutic agent for Th17 cell-induced disease and a screening method for a preventive or therapeutic agent for Th17 cell-induced disease.
　Ｔｈ１７細胞は、ＩＬ－１７やＩＬ－２２等のサイトカインを産生し、好中球や上皮細胞を活性化させるエフェクターＴ細胞である。Ｔｈ１７細胞は、関節リウマチ、多発性硬化症、炎症性腸疾患等の自己免疫疾患；喘息や接触性過敏症、遅延型過敏症等のアレルギー疾患；乾癬、乾癬性関節炎、強直性脊椎症；等の様々な疾患の病態形成において重要な役割を果たしていることが知られている。 Th17 cells are effector T cells that produce cytokines such as IL-17 and IL-22 and activate neutrophils and epithelial cells. Th17 cells are autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and inflammatory bowel disease; allergic diseases such as asthma, contact hypersensitivity, and delayed hypersensitivity; psoriatic arthritis, psoriatic arthritis, tonic spondylosis; etc. It is known to play an important role in the pathogenesis of various diseases.
　本発明者らは、以前、Ｃ１ｑ／ＴＮＦ－ｒｅｌａｔｅｄ　ｐｒｏｔｅｉｎ　３（ＣＴＲＰ３）が関節リウマチや多発性硬化症に関与していることを報告した（例えば、特許文献１参照）。ＣＴＲＰ３は、短いＮ末端可変領域、コラーゲンドメイン、及びＣ末端補体Ｃ１ｑドメインからなる可溶性分泌タンパク質であり、ＴＮＦ及び補体Ｃ１ｑに類似した結晶構造を有する。しかし、ＣＴＲＰ３の受容体及びその受容体を発現する細胞は未だ同定されていない。 The present inventors have previously reported that C1q / TNF-related protein 3 (CTRP3) is involved in rheumatoid arthritis and multiple sclerosis (see, for example, Patent Document 1). CTRP3 is a soluble secretory protein consisting of a short N-terminal variable region, collagen domain, and C-terminal complement C1q domain, and has a crystal structure similar to TNF and complement C1q. However, the receptor for CTRP3 and the cells expressing the receptor have not yet been identified.
　Ｃ１ｑドメインは、分泌タンパク質のアディポネクチンも有している。アディポネクチンは、アディポネクチン受容体１（ＡｄｉｐｏＲ１）及びアディポネクチン受容体２（ＡｄｉｐｏＲ２）等へ結合することで作用を及ぼすことが知られているが、Ｃ１ｑドメインはその結合に重要であることが報告されている。 The C1q domain also has the secretory protein adiponectin. Adiponectin is known to exert an action by binding to adiponectin receptor 1 (AdipoR1), adiponectin receptor 2 (AdipoR2), etc., and it has been reported that the C1q domain is important for the binding. ..
　アディポネクチンが、ナイーブＴ細胞からＴｈ１７細胞への分化に関与していることは多数報告されている。しかし、アディポネクチンがＴｈ１７細胞への分化を促進するという報告（例えば、非特許文献１及び２参照）がある一方で、該分化を抑制するという報告（例えば、非特許文献３及び４参照）もある。このように、Ｔｈ１７細胞への分化メカニズムにおけるアディポネクチンの役割は未だ明確に解明されておらず、Ｔｈ１７細胞誘導性疾患の予防又は治療剤へのアディポネクチンの応用が容易でないという問題がある。 Many reports have been made that adiponectin is involved in the differentiation of naive T cells into Th17 cells. However, while there are reports that adiponectin promotes differentiation into Th17 cells (see, for example, Non-Patent Documents 1 and 2), there are also reports that it suppresses the differentiation (see, for example, Non-Patent Documents 3 and 4). .. As described above, the role of adiponectin in the differentiation mechanism into Th17 cells has not been clarified yet, and there is a problem that it is not easy to apply adiponectin to a preventive or therapeutic agent for Th17 cell-induced diseases.
国際公開第２０１５／０７２５４４号International Publication No. 2015/072544
　本発明は、上記に鑑みて提案されたものであり、Ｔｈ１７細胞誘導性疾患の予防又は治療に好適に用いられる新規な予防又は治療剤、及びＴｈ１７細胞誘導性疾患の予防又は治療剤のスクリーニング方法を提供することを目的とする。 The present invention has been proposed in view of the above, and is a novel prophylactic or therapeutic agent preferably used for the prevention or treatment of Th17 cell-induced diseases, and a method for screening a prophylactic or therapeutic agent for Th17 cell-induced diseases. The purpose is to provide.
　本発明者らは、上記目的について鋭意研究した結果、アディポネクチン受容体１に対するアゴニスト活性を示さず、且つ、アディポネクチン受容体２に対するアゴニスト活性を示すアゴニストが、ナイーブＴ細胞からＴｈ１７細胞への分化を抑制すること、及びＴｈ１７細胞誘導性疾患の予防又は治療に有効であることを見出し、本発明を完成するに至った。より具体的には、本発明は、以下のようなものを提供する。なお、アディポネクチン受容体１（ＡｄｉｐｏＲ１）及びアディポネクチン受容体２（ＡｄｉｐｏＲ２）は、ＰＡＱＲ（ｐｒｏｇｅｓｔｉｎ　ａｎｄ　ＡｄｉｐｏＱ　ｒｅｃｅｐｔｏｒｓ）ファミリーのＣｌａｓｓ　Ｉに分類される受容体であり、それぞれＰＡＱＲ１、ＰＡＱＲ２とも称される。 As a result of diligent research on the above objectives, the present inventors have shown that an agonist that does not show agonist activity on adiponectin receptor 1 and that shows agonist activity on adiponectin receptor 2 suppresses the differentiation of naive T cells into Th17 cells. We have found that it is effective for the prevention or treatment of Th17 cell-induced diseases, and have completed the present invention. More specifically, the present invention provides the following. The adiponectin receptor 1 (AdipoR1) and the adiponectin receptor 2 (AdiponR2) are receptors classified into Class I of the PAQR (progestin and AdiponQ receptors) family, and are also referred to as PAQR1 and PAQR2, respectively.
＜１＞　アディポネクチン受容体１（ＰＡＱＲ１）に対するアゴニスト活性を示さず、且つ、アディポネクチン受容体２（ＰＡＱＲ２）に対するアゴニスト活性を示すアゴニストを有効成分として含有する、Ｔｈ１７細胞誘導性疾患の予防又は治療剤。 <1> A prophylactic or therapeutic agent for Th17 cell-induced diseases, which contains an agonist as an active ingredient, which does not exhibit agonist activity on adiponectin receptor 1 (PAQR1) and exhibits agonist activity on adiponectin receptor 2 (PAQR2).
＜２＞　上記アゴニストが下記（ａ）～（ｄ）のいずれかである、＜１＞に記載のＴｈ１７細胞誘導性疾患の予防又は治療剤。
（ａ）配列番号１で示されるアミノ酸配列からなるタンパク質。
（ｂ）配列番号１で示されるアミノ酸配列のｇＣ１ｑドメインからなるタンパク質。
（ｃ）配列番号１で示されるアミノ酸配列に対して８０％以上の配列同一性を有し、アディポネクチン受容体１（ＰＡＱＲ１）に対するアゴニスト活性を示さず、且つ、アディポネクチン受容体２（ＰＡＱＲ２）に対するアゴニスト活性を示すタンパク質。
（ｄ）配列番号１で示されるアミノ酸配列のｇＣ１ｑドメインに対して８０％以上の配列同一性を有し、アディポネクチン受容体１（ＰＡＱＲ１）に対するアゴニスト活性を示さず、且つ、アディポネクチン受容体２（ＰＡＱＲ２）に対するアゴニスト活性を示すタンパク質。 <2> The agent for preventing or treating Th17 cell-induced disease according to <1>, wherein the agonist is any of the following (a) to (d).
(A) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1.
(B) A protein consisting of the gC1q domain of the amino acid sequence shown in SEQ ID NO: 1.
(C) It has 80% or more sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 1, does not show agonist activity on adiponectin receptor 1 (PAQR1), and is an agonist on adiponectin receptor 2 (PAQR2). A protein that exhibits activity.
(D) It has 80% or more sequence identity with respect to the gC1q domain of the amino acid sequence shown in SEQ ID NO: 1, does not show agonist activity on adiponectin receptor 1 (PAQR1), and has adiponectin receptor 2 (PAQR2). ), A protein that exhibits agonist activity.
＜３＞　上記Ｔｈ１７細胞誘導性疾患が関節リウマチ及び多発性硬化症でない、＜１＞又は＜２＞に記載のＴｈ１７細胞誘導性疾患の予防又は治療剤。 <3> The prophylactic or therapeutic agent for Th17 cell-induced disease according to <1> or <2>, wherein the Th17 cell-induced disease is not rheumatoid arthritis or multiple sclerosis.
＜４＞　上記Ｔｈ１７細胞誘導性疾患が自己免疫疾患でない、＜１＞又は＜２＞に記載のＴｈ１７細胞誘導性疾患の予防又は治療剤。 <4> The preventive or therapeutic agent for a Th17 cell-induced disease according to <1> or <2>, wherein the Th17 cell-induced disease is not an autoimmune disease.
＜５＞　上記Ｔｈ１７細胞誘導性疾患が、乾癬、乾癬性関節炎、強直性脊椎症、視神経脊髄炎、アルツハイマー病、ハンチントン病、パーキンソン病、１型糖尿病、２型糖尿病、痛風、白斑、ぶどう膜炎、急性及び慢性腎炎、急性及び慢性疼痛、炎症性腸疾患、肝線維症、膵線維症、炎症性肺疾患、又はアレルギー性疾患である、＜１＞又は＜２＞に記載のＴｈ１７細胞誘導性疾患の予防又は治療剤。 <5> The Th17 cell-induced diseases are psoriasis, psoriatic arthritis, tonic spondylosis, optic neuromyelitis, Alzheimer's disease, Huntington's disease, Parkinson's disease, type 1 diabetes, type 2 diabetes, gout, leukoplakia, and melanitis. , Acute and chronic nephritis, acute and chronic pain, inflammatory bowel disease, liver fibrosis, pancreatic fibrosis, inflammatory lung disease, or allergic disease, Th17 cell-inducible according to <1> or <2>. Preventive or therapeutic agent for diseases.
＜６＞　＜１＞～＜５＞のいずれか一つに記載のＴｈ１７細胞誘導性疾患の予防又は治療剤を製造するための、上記アゴニストの使用。 <6> Use of the above-mentioned agonist for producing a prophylactic or therapeutic agent for Th17 cell-induced disease according to any one of <1> to <5>.
＜７＞　アディポネクチン受容体１（ＰＡＱＲ１）に対するアゴニスト活性を示さず、且つ、アディポネクチン受容体２（ＰＡＱＲ２）に対するアゴニスト活性を示す候補アゴニストをスクリーニングすることを含む、Ｔｈ１７細胞誘導性疾患の予防又は治療剤のスクリーニング方法。 <7> A prophylactic or therapeutic agent for Th17 cell-induced diseases, which comprises screening a candidate agonist that does not show agonist activity on adiponectin receptor 1 (PAQR1) and shows agonist activity on adiponectin receptor 2 (PAQR2). Screening method.
　本発明によれば、Ｔｈ１７細胞誘導性疾患の予防又は治療に好適に用いられる新規な予防又は治療剤、及びＴｈ１７細胞誘導性疾患の予防又は治療剤のスクリーニング方法を提供することができる。 According to the present invention, it is possible to provide a novel preventive or therapeutic agent preferably used for the prevention or treatment of Th17 cell-induced disease, and a screening method for the preventive or therapeutic agent for Th17 cell-induced disease.
Ｃ１ｑｔｎｆ３－／－マウス及びＷＴマウスから精製したナイーブＴ細胞に対して、Ｔｈ１７細胞への分化誘導を行った場合の、ＩＬ－１７＋Ｔ細胞（Ｔｈ１７細胞）のポピュレーションを示す図である。It is a figure which shows the population of IL-17 + T cell (Th17 cell) when the differentiation induction into Th17 cell was performed with respect to the naive T cell purified from C1qtnf3 − / − mouse and WT mouse. Ｃ１ｑｔｎｆ３－／－マウス及びＷＴマウスから精製したナイーブＴ細胞に対して、Ｔｈ１７細胞への分化誘導を行った場合の、ＩＬ－１７＋Ｔ細胞（Ｔｈ１７細胞）の割合を示すグラフである。It is a graph which shows the ratio of IL-17 + T cell (Th17 cell) when the differentiation induction into Th17 cell was performed with respect to the naive T cell purified from C1qtnf3 − / − mouse and WT mouse. Ｃ１ｑｔｎｆ３－／－マウス及びＷＴマウスから精製したナイーブＴ細胞に対して、Ｔｈ１７細胞への分化誘導を行った場合の、培養上清中のＩＬ－１７タンパク質量を示す図である。It is a figure which shows the amount of IL-17 protein in the culture supernatant when the differentiation induction into Th17 cell was performed with respect to the naive T cell purified from C1qtnf3 − / − mouse and WT mouse. Ｃ１ｑｔｎｆ３－／－マウスから精製したナイーブＴ細胞に対して、Ｔｈ１７細胞への分化誘導を行う際にＣＴＲＰ３を添加した場合の、ＩＬ－１７＋Ｔ細胞（Ｔｈ１７細胞）のポピュレーションを示す図である。It is a figure which shows the population of IL-17 + T cell (Th17 cell) when CTRP3 is added at the time of inducing differentiation into Th17 cell to the naive T cell purified from C1qtnf3 − / − mouse. .. Ｃ１ｑｔｎｆ３－／－マウスから精製したナイーブＴ細胞に対して、Ｔｈ１７細胞への分化誘導を行う際にＣＴＲＰ３を添加した場合の、ＩＬ－１７＋Ｔ細胞（Ｔｈ１７細胞）の割合を示すグラフである。3 is a graph showing the ratio of IL-17 + T cells (Th17 cells) when CTRP3 was added to induce differentiation into Th17 cells with respect to naive T cells purified from C1qtnf3 − / − mice. Ｃ１ｑｔｎｆ３－／－マウスから精製したナイーブＴ細胞に対して、Ｔｈ１７細胞への分化誘導を行う際にＣＴＲＰ３を添加した場合の、培養上清中のＩＬ－１７タンパク質量を示すグラフである。3 is a graph showing the amount of IL-17 protein in the culture supernatant when CTRP3 is added to induce differentiation into Th17 cells from naive T cells purified from C1qtnf3 − / − mice. Ｃ１ｑｔｎｆ３－／－マウスから精製したナイーブＴ細胞に対して、Ｔｈ１細胞への分化誘導を行った場合の、ＩＦＮ－γ＋Ｔ細胞（Ｔｈ１細胞）のポピュレーションを示す図である。It is a figure which shows the population of IFN-γ + T cell (Th1 cell) when the differentiation induction into Th1 cell was performed with respect to the naive T cell purified from C1qtnf3 − / − mouse. Ｃ１ｑｔｎｆ３－／－マウスから精製したナイーブＴ細胞に対して、Ｔｈ１細胞への分化誘導を行った場合の、ＩＦＮ－γ＋Ｔ細胞（Ｔｈ１細胞）の割合を示すグラフである。3 is a graph showing the ratio of IFN-γ + T cells (Th1 cells) when differentiation induction into Th1 cells was performed on naive T cells purified from C1qtnf3 − / − mice. Ｃ１ｑｔｎｆ３－／－マウスから精製したナイーブＴ細胞に対して、Ｔｈ１細胞への分化誘導を行った場合の、培養上清中のＩＦＮ－γタンパク質量を示すグラフである。6 is a graph showing the amount of IFN-γ protein in the culture supernatant when induction of differentiation into Th1 cells was performed on naive T cells purified from C1qtnf3 − / − mice. ＷＴマウスから精製したナイーブＴ細胞に対して、Ｔｈ１７細胞への分化誘導を行う際にＡｄｉｐｏＲｏｎを添加した場合の、ＩＬ－１７＋Ｔ細胞（Ｔｈ１７細胞）のポピュレーションを示す図である。It is a figure which shows the population of IL-17 + T cell (Th17 cell) when AdipoRon was added to induce differentiation into Th17 cell to the naive T cell purified from WT mouse. ＷＴマウスから精製したナイーブＴ細胞に対して、Ｔｈ１７細胞への分化誘導を行う際にＡｄｉｐｏＲｏｎを添加した場合の、ＩＬ－１７＋Ｔ細胞（Ｔｈ１７細胞）の割合を示すグラフである。It is a graph which shows the ratio of IL-17 + T cell (Th17 cell) when AdipoRon was added when inducing differentiation into Th17 cell with respect to naive T cell purified from WT mouse. ＷＴマウスから精製したナイーブＴ細胞に対して、Ｔｈ１７細胞への分化誘導を行う際にＡｄｉｐｏＲｏｎを添加した場合の、培養上清中のＩＬ－１７タンパク質量を示すグラフである。It is a graph which shows the amount of IL-17 protein in the culture supernatant when AdipoRon was added to induce the differentiation into Th17 cell with respect to the naive T cell purified from WT mouse. ＷＴマウスから精製したナイーブＴ細胞に対して、Ｔｈ１細胞への分化誘導を行う際にＡｄｉｐｏＲｏｎを添加した場合の、ＩＦＮ－γ＋Ｔ細胞（Ｔｈ１細胞）のポピュレーションを示す図である。It is a figure which shows the population of IFN-γ + T cell (Th1 cell) when AdipoRon was added to induce differentiation into Th1 cell to the naive T cell purified from WT mouse. ＷＴマウスから精製したナイーブＴ細胞に対して、Ｔｈ１細胞への分化誘導を行う際にＡｄｉｐｏＲｏｎを添加した場合の、ＩＦＮ－γ＋Ｔ細胞（Ｔｈ１細胞）の割合を示すグラフである。It is a graph which shows the ratio of IFN-γ + T cells (Th1 cells) when AdipoRon was added to induce differentiation into Th1 cells with respect to naive T cells purified from WT mice. ＷＴマウスから精製したナイーブＴ細胞に対して、Ｔｈ１細胞への分化誘導を行う際にＡｄｉｐｏＲｏｎを添加した場合の、培養上清中のＩＦＮ－γタンパク質量を示すグラフである。It is a graph which shows the amount of IFN-γ protein in the culture supernatant when AdipoRon was added to induce differentiation into Th1 cell with respect to naive T cell purified from WT mouse. Ｃ１ｑｔｎｆ３－／－マウスから精製したナイーブＴ細胞に対して、Ｔｈ１７細胞への分化誘導を行う際にＣＴＲＰ３を添加し、且つ、ＡｄｉｐｏＲ１ブロッカー又はＡｄｉｐｏＲ２ブロッカーを添加した場合の、ＩＬ－１７＋Ｔ細胞（Ｔｈ１７細胞）のポピュレーションを示す図である。IL-17 + T cells (when CTRP3 was added to induce differentiation into Th17 cells and AdipoR1 blocker or AdipoR2 blocker was added to naive T cells purified from C1qtnf3 − / − mice. It is a figure which shows the population of Th17 cell). Ｃ１ｑｔｎｆ３－／－マウスから精製したナイーブＴ細胞に対して、Ｔｈ１７細胞への分化誘導を行う際にＣＴＲＰ３を添加し、且つ、ＡｄｉｐｏＲ１ブロッカー又はＡｄｉｐｏＲ２ブロッカーを添加した場合の、ＩＬ－１７＋Ｔ細胞（Ｔｈ１７細胞）の割合を示すグラフである。IL-17 + T cells (when CTRP3 was added to induce differentiation into Th17 cells and AdipoR1 blocker or AdipoR2 blocker was added to naive T cells purified from C1qtnf3 − / − mice. It is a graph which shows the ratio of Th17 cells). Ｃ１ｑｔｎｆ３－／－マウスから精製したナイーブＴ細胞に対して、Ｔｈ１７細胞への分化誘導を行う際にＣＴＲＰ３を添加し、且つ、ＡｄｉｐｏＲ１ブロッカー又はＡｄｉｐｏＲ２ブロッカーを添加した場合の、培養上清中のＩＬ－１７タンパク質量を示すグラフである。C1qtnf3 − / − To naive T cells purified from mice, IL- in the culture supernatant when CTRP3 was added when inducing differentiation into Th17 cells, and AdipoR1 blocker or AdipoR2 blocker was added. 17 is a graph showing the amount of protein. Ｃ１ｑｔｎｆ３－／－マウス及びＷＴマウスにベセルナクリームを塗布した際の、マウスの耳の腫れ（耳の厚さ）を示したグラフである。6 is a graph showing swelling (ear thickness) of the ears of mice when Bethelna cream was applied to C1qtnf3 − / − mice and WT mice.
　以下、本発明の実施形態について説明する。ただし、本発明は以下の実施形態に限定されるものではない。 Hereinafter, embodiments of the present invention will be described. However, the present invention is not limited to the following embodiments.
＜Ｔｈ１７細胞誘導性疾患の予防又は治療剤＞
　本実施形態に係るＴｈ１７細胞誘導性疾患の予防又は治療剤は、アディポネクチン受容体１（ＡｄｉｐｏＲ１）に対するアゴニスト活性を示さず、且つ、アディポネクチン受容体２（ＡｄｉｐｏＲ２）に対するアゴニスト活性を示すアゴニスト（以下、「特定アゴニスト」ともいう。）を有効成分として含有する。 <Preventive or therapeutic agent for Th17 cell-induced diseases>
The prophylactic or therapeutic agent for Th17 cell-induced disease according to the present embodiment does not show an agonist activity against adiponectin receptor 1 (AdipoR1) and shows an agonist activity against adiponectin receptor 2 (AdipoR2) (hereinafter, "" Also referred to as "specific agonist") as an active ingredient.
　上述したアディポネクチンやアディポネクチン受容体のアゴニストであるＡｄｉｐｏＲｏｎは、ＡｄｉｐｏＲ１及びＡｄｉｐｏＲ２の双方に対してアゴニスト活性を示すことが知られている。後述する実施例で示すように、ＡｄｉｐｏＲｏｎはＴｈ１７細胞及びＴｈ１細胞の双方への分化を抑制したのに対し、特定アゴニストはＴｈ１７細胞への分化のみ抑制し、Ｔｈ１細胞への分化には影響を及ぼさなかった。これらのことより、ＡｄｉｐｏＲ１の活性化が、Ｔｈ１細胞への分化を抑制することが明らかとなった。 AdipoRon, which is an agonist of the above-mentioned adiponectin and adiponectin receptor, is known to exhibit agonist activity against both AdipoR1 and AdipoR2. As shown in Examples described later, AdipoRon suppressed the differentiation into both Th17 cells and Th1 cells, whereas the specific agonist suppressed only the differentiation into Th17 cells and affected the differentiation into Th1 cells. There wasn't. From these facts, it was clarified that the activation of AdiponR1 suppresses the differentiation into Th1 cells.
　ここで、Ｔｈ１細胞は細胞性免疫において非常に重要な役割を果たしているため、Ｔｈ１細胞への分化が抑制されると、免疫機構の正常な働きが妨げられることとなる。したがって、Ｔｈ１７細胞誘導性疾患の予防又は治療剤に含まれる有効成分は、Ｔｈ１細胞への分化は抑制せず、Ｔｈ１７細胞への分化のみを抑制する選択的アゴニストであることが好ましい。特定アゴニストは、ＡｄｉｐｏＲ１に対するアゴニスト活性は示さず、ＡｄｉｐｏＲ２に対してのみアゴニスト活性を示す（換言すると、Ｔｈ１７細胞への分化のみ抑制し、Ｔｈ１細胞への分化は抑制しない）ことから、上記選択的アゴニストとして有用である。 Here, Th1 cells play a very important role in cell-mediated immunity, so if differentiation into Th1 cells is suppressed, the normal functioning of the immune system will be hindered. Therefore, it is preferable that the active ingredient contained in the preventive or therapeutic agent for Th17 cell-induced diseases is a selective agonist that does not suppress the differentiation into Th1 cells but only suppresses the differentiation into Th17 cells. Since the specific agonist does not show an agonist activity against AdiponR1 but shows an agonist activity only against AdiponR2 (in other words, it suppresses only the differentiation into Th17 cells and not the differentiation into Th1 cells), the above-mentioned selective agonist It is useful as.
　本明細書において、「Ｔｈ１７細胞誘導性疾患」とは、Ｔｈ１７細胞によって誘導される疾患のことをいう。本実施形態に係るＴｈ１７細胞誘導性疾患は特に限定されないが、一実施形態では関節リウマチ及び多発性硬化症でない疾患であり、また、他の一実施形態では自己免疫疾患でない疾患である。このようなＴｈ１７細胞誘導性疾患の具体例としては、乾癬、乾癬性関節炎、強直性脊椎症、視神経脊髄炎、アルツハイマー病、ハンチントン病、パーキンソン病、１型糖尿病、２型糖尿病、痛風、白斑、ぶどう膜炎、急性及び慢性腎炎、急性及び慢性疼痛、潰瘍性大腸炎等の炎症性腸疾患；肝線維症、膵線維症、炎症性肺疾患、又は喘息や接触性皮膚炎、遅発性過敏症等のアレルギー性疾患；等が挙げられる。 As used herein, the term "Th17 cell-induced disease" refers to a disease induced by Th17 cells. The Th17 cell-induced disease according to the present embodiment is not particularly limited, but in one embodiment, it is a disease that is not rheumatoid arthritis and multiple sclerosis, and in another embodiment, it is a disease that is not an autoimmune disease. Specific examples of such Th17 cell-induced diseases include psoriasis, psoriatic arthritis, tonic spondylosis, optic neuromyelitis, Alzheimer's disease, Huntington's disease, Parkinson's disease, type 1 diabetes, type 2 diabetes, gout, and white spots. Inflammatory bowel diseases such as vasculitis, acute and chronic nephritis, acute and chronic pain, ulcerative colitis; liver fibrosis, pancreatic fibrosis, inflammatory lung disease, or asthma and contact dermatitis, delayed hypersensitivity Allergic diseases such as illness; etc.
　特定アゴニストは、ＡｄｉｐｏＲ１に対するアゴニスト活性を示さず、且つ、ＡｄｉｐｏＲ２に対するアゴニスト活性を示すものであれば特に限定されない。本発明者らが鋭意研究を重ねた結果、ＣＴＲＰ（Ｃ１ｑ／ＴＮＦ－ｒｅｌａｔｅｄ　ｐｒｏｔｅｉｎ）ファミリーに属するタンパク質の一つであるＣＴＲＰ３が、ＡｄｉｐｏＲ１に対するアゴニスト活性を示さず、且つ、ＡｄｉｐｏＲ２に対するアゴニスト活性を示すことが確認された。したがって、本実施形態における特定アゴニストの一例としては、ＣＴＲＰ３が挙げられる。 The specific agonist is not particularly limited as long as it does not show an agonist activity against AdiponR1 and shows an agonist activity against AdiponR2. As a result of diligent research by the present inventors, CTRP3, which is one of the proteins belonging to the CTRP (C1q / TNF-related protein) family, does not show an agonist activity against AdipoR1 and shows an agonist activity against AdiponR2. Was confirmed. Therefore, CTRP3 is mentioned as an example of the specific agonist in this embodiment.
　ＣＴＲＰ３の由来は特に制限されず、ヒト由来であってもマウス等の他の動物由来であってもよいが、ヒト由来であることが好ましい。ＣＴＲＰ３のアミノ酸配列は、米国生物工学情報センター（ＮＣＢＩ）のＧｅｎＢａｎｋデータベースに登録されている情報を参照することができる。例えば、ＧｅｎＢａｎｋデータベースには、ヒトＣＴＲＰ３のアミノ酸配列としてアクセッション番号ＡＡＩ１２９２６等が、マウスＣＴＲＰ３のアミノ酸配列としてアクセッション番号ＡＡＹ２１９２８等が登録されている。典型的なアミノ酸配列は以下のとおりである。アミノ酸配列の下線部分は球状ドメイン（ｇＣ１ｑドメイン）を示す。 The origin of CTRP3 is not particularly limited and may be derived from humans or other animals such as mice, but it is preferably derived from humans. For the amino acid sequence of CTRP3, the information registered in the GenBank database of the National Center for Biotechnology Information (NCBI) can be referred to. For example, accession numbers AAI12926 and the like are registered as amino acid sequences of human CTRP3, and accession numbers AAY21928 and the like are registered as amino acid sequences of mouse CTRP3 in the GenBank database. A typical amino acid sequence is as follows. The underlined portion of the amino acid sequence indicates a spherical domain (gC1q domain).
（ヒトＣＴＲＰ３のアミノ酸配列（配列番号１））
ＭＬＷＲＱＬＩＹＷＱＬＬＡＬＦＦＬＰＦＣＬＣＱＤＥＹＭＥＳＰＱＴＧＧＬＰＰＤＣＳＫＣＣＨＧＤＹＳＦＲＧＹＱＧＰＰＧＰＰＧＰＰＧＩＰＧＮＨＧＮＮＧＮＮＧＡＴＧＨＥＧＡＫＧＥＫＧＤＫＧＤＬＧＰＲＧＥＲＧＱＨＧＰＫＧＥＫＧＹＰＧＩＰＰＥＬＱＩＡＦＭＡＳＬＡＴＨＦＳＮＱＮＳＧＩＩＦＳＳＶＥＴＮＩＧＮＦＦＤＶＭＴＧＲＦＧＡＰＶＳＧＶＹＦＦＴＦＳＭＭＫＨＥＤＶＥＥＶＹＶＹＬＭＨＮＧＮＴＶＦＳＭＹＳＹＥＭＫＧＫＳＤＴＳＳＮＨＡＶＬＫＬＡＫＧＤＥＶＷＬＲＭＧＮＧＡＬＨＧＤＨＱＲＦＳＴＦＡＧＦＬＬＦＥＴＫ (Amino acid sequence of human CTRP3 (SEQ ID NO: 1))
MLWRQLIYWQLLALFFLPFCLCQDEYMESPQTGGLPPDCSKCCHGDYSFRGYQGPPGPPGPPGIPGNHGNNGNNGATGHEGAKGEKGDKGDLGPRGERGQHGPKGEKGYPGIPPELQI AFMASLATHFSNQNSGIIFSSVETNIGNFFDVMTGRFGAPVSGVYFFTFSMMKHEDVEEVYVYLMHNGNTVFSMYSYEMKGKSDTSSNHAVLKLAKGDEVWLRMGNGALHGDHQRFSTFAGFL LFETK
（マウスＣＴＲＰ３のアミノ酸配列（配列番号２））
ＭＬＧＲＱＲＩＷＷＨＬＬＰＬＬＦＬＰＦＣＬＣＱＤＥＹＭＥＳＰＱＡＧＧＬＰＰＤＣＳＫＣＣＨＧＤＹＧＦＲＧＹＱＧＰＰＧＰＰＧＰＰＧＩＰＧＮＨＧＮＮＧＮＮＧＡＴＧＨＥＧＡＫＧＥＫＧＤＫＧＤＬＧＰＲＧＥＲＧＱＨＧＰＫＧＥＫＧＹＰＧＶＰＰＥＬＱＩＡＦＭＡＳＬＡＴＨＦＳＮＱＮＳＧＩＩＦＳＳＶＥＴＮＩＧＮＦＦＤＶＭＴＧＲＦＧＡＰＶＳＧＶＹＦＦＴＦＳＭＭＫＨＥＤＶＥＥＶＹＶＹＬＭＨＮＧＮＴＶＦＳＭＹＳＹＥＴＫＧＫＳＤＴＳＳＮＨＡＶＬＫＬＡＫＧＤＥＶＷＬＲＭＧＮＧＡＬＨＧＤＨＱＲＦＳＴＦＡＧＦＬＬＦＥＴＫ (Amino acid sequence of mouse CTRP3 (SEQ ID NO: 2))
MLGRQRIWWHLLPLLFLPFCLCQDEYMESPQAGGLPPDCSKCCHGDYGFRGYQGPPGPPGPPGIPGNHGNNGNNGATGHEGAKGEKGDKGDLGPRGERGQHGPKGEKGYPGVPPELQI AFMASLATHFSNQNSGIIFSSVETNIGNFFDVMTGRFGAPVSGVYFFTFSMMKHEDVEEVYVYLMHNGNTVFSMYSYETKGKSDTSSNHAVLKLAKGDEVWLRMGNGALHGDHQRFSTFAGFL LFETK
　このように、ヒトＣＴＲＰ３とマウスＣＴＲＰ３のアミノ酸配列における全体の相同性は９６％と高く、球状ドメイン（ｇＣ１ｑドメイン）部分においては、９９％と更に高い相同性を有している。 As described above, the overall homology in the amino acid sequences of human CTRP3 and mouse CTRP3 is as high as 96%, and the spherical domain (gC1q domain) portion has even higher homology as 99%.
　ＣＴＲＰ３は、市販品を使用してもよく、遺伝子組換え法又は化学合成法により製造してもよい。遺伝子組換え法によりＣＴＲＰ３を製造する場合、例えば、発現ベクターを用いて大腸菌、酵母等の微生物、植物細胞、昆虫細胞、又は動物細胞にＣＴＲＰ３をコードする遺伝子を導入し、タンパク質を発現させればよい。ヒトＣＴＲＰ３又はマウスＣＴＲＰ３を製造する場合、立体構造の保持及び翻訳後修飾の観点から、哺乳動物細胞が好ましく用いられる。化学合成法によりＣＴＲＰ３を製造する場合、液相法、固相法、Ｂｏｃ法、Ｆｍｏｃ法等を単独で又は組み合わせて製造すればよい。 CTRP3 may be a commercially available product, or may be produced by a gene recombination method or a chemical synthesis method. When CTRP3 is produced by the gene recombination method, for example, if a gene encoding CTRP3 is introduced into microorganisms such as Escherichia coli and yeast, plant cells, insect cells, or animal cells using an expression vector to express the protein. Good. When producing human CTRP3 or mouse CTRP3, mammalian cells are preferably used from the viewpoint of maintaining the three-dimensional structure and post-translational modification. When CTRP3 is produced by a chemical synthesis method, it may be produced by the liquid phase method, the solid phase method, the Boc method, the Fmoc method or the like alone or in combination.
　ＣＴＲＰ３は、ＡｄｉｐｏＲ１に対するアゴニスト活性を示さず、且つ、ＡｄｉｐｏＲ２に対するアゴニスト活性を示す限り、野生型ＣＴＲＰ３のアミノ酸配列において１個又は数個のアミノ酸残基が置換、欠失、又は付加されたアミノ酸配列からなる変異型ＣＴＲＰ３であってもよい。 As long as CTRP3 does not show agonistic activity against AdiponR1 and shows agonistic activity against AdiponR2, from the amino acid sequence in which one or several amino acid residues are substituted, deleted, or added in the amino acid sequence of wild-type CTRP3. It may be a mutant CTRP3.
　変異型の各アミノ酸配列と野生型の各アミノ酸配列との配列同一性は、例えば、８０％以上であってもよく、８５％以上であってもよく、９０％以上であってもよく、９３％以上であってもよく、９５％以上であってもよい。この場合、後述する球状ドメイン部分の配列同一性が８０％以上であることが好ましく、９０％以上であることがより好ましく、９３％以上であることが更に好ましく、９５％以上であることが特に好ましく、１００％であることが極めて好ましい。
　ここで、「配列同一性」とは、２つのアミノ酸配列をアラインメントした場合の配列間の一致性を意味し、例えば、ＢＬＡＳＴプログラム（ｈｔｔｐｓ：／／ｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／Ｂｌａｓｔ／ｃｇｉ）を使用して算出することができる。 The sequence identity between each mutant amino acid sequence and each wild-type amino acid sequence may be, for example, 80% or more, 85% or more, 90% or more, 93. It may be% or more, and may be 95% or more. In this case, the sequence identity of the spherical domain portion described later is preferably 80% or more, more preferably 90% or more, further preferably 93% or more, and particularly preferably 95% or more. It is preferably 100%, which is extremely preferable.
Here, "sequence identity" means the matching between sequences when two amino acid sequences are aligned, and for example, a BLAST program (https://www.ncbi.nlm.nih.gov/Blast/). It can be calculated using cgi).
　なお、ＣＴＲＰファミリーに属するタンパク質は、その球状ドメイン（ｇＣ１ｑドメイン）のみでも生体内に存在し、球状ドメインが機能部位であると考えられている（Ｔｒｅｎｄｓ．　Ｉｍｍｕｎｏｌ．，２５（１０）：５５１－６１（２００４））。そこで、特定アゴニストは、ＣＴＲＰ３の球状ドメインを構成するペプチドであってもよい。 In addition, the protein belonging to the CTRP family exists in the living body only in the spherical domain (gC1q domain), and the spherical domain is considered to be a functional site (Trends. Immunol., 25 (10): 551-61). (2004)). Therefore, the specific agonist may be a peptide constituting the spherical domain of CTRP3.
　また、特定アゴニストは、ＡｄｉｐｏＲ１に対するアゴニスト活性を示さず、且つ、ＡｄｉｐｏＲ２に対するアゴニスト活性を示す限り、ＣＴＲＰ３の球状ドメインのアミノ酸配列において１個又は数個のアミノ酸残基が置換、欠失、又は付加されたアミノ酸配列からなる変異型の球状ドメインを構成するペプチドであってもよい。このとき、変異型の各アミノ酸配列と野生型の各アミノ酸配列との配列同一性は、例えば、８０％以上であってもよく、８５％以上であってもよく、９０％以上であってもよく、９３％以上であってもよく、９５％以上であってもよい。 In addition, as long as the specific agonist does not show agonistic activity against AdipoR1 and shows agonistic activity against AdiponR2, one or several amino acid residues are substituted, deleted, or added in the amino acid sequence of the spherical domain of CTRP3. It may be a peptide constituting a mutant spherical domain consisting of an amino acid sequence. At this time, the sequence identity between each mutant amino acid sequence and each wild-type amino acid sequence may be, for example, 80% or more, 85% or more, or 90% or more. It may be 93% or more, or 95% or more.
　特定アゴニストとして好適なものとしては、例えば、下記（ａ）～（ｄ）から選択されるものが挙げられる。
（ａ）配列番号１で示されるアミノ酸配列からなるタンパク質。
（ｂ）配列番号１で示されるアミノ酸配列のｇＣ１ｑドメインからなるタンパク質。
（ｃ）配列番号１で示されるアミノ酸配列に対して８０％以上の配列同一性を有し、アディポネクチン受容体１（ＡｄｉｐｏＲ１）に対するアゴニスト活性を示さず、且つ、アディポネクチン受容体２（ＡｄｉｐｏＲ２）に対するアゴニスト活性を示すタンパク質。
（ｄ）配列番号１で示されるアミノ酸配列のｇＣ１ｑドメインに対して８０％以上の配列同一性を有し、アディポネクチン受容体１（ＡｄｉｐｏＲ１）に対するアゴニスト活性を示さず、且つ、アディポネクチン受容体２（ＡｄｉｐｏＲ２）に対するアゴニスト活性を示すタンパク質。 Suitable examples of the specific agonist include those selected from the following (a) to (d).
(A) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1.
(B) A protein consisting of the gC1q domain of the amino acid sequence shown in SEQ ID NO: 1.
(C) It has 80% or more sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 1, does not show agonist activity against adiponectin receptor 1 (AdipoR1), and is an agonist against adiponectin receptor 2 (AdipoR2). A protein that exhibits activity.
(D) It has 80% or more sequence identity with respect to the gC1q domain of the amino acid sequence shown in SEQ ID NO: 1, does not show agonist activity on adiponectin receptor 1 (AdipoR1), and has adiponectin receptor 2 (AdipoR2). ), A protein that exhibits agonist activity.
　特定アゴニストは、ＡｄｉｐｏＲ１に対するアゴニスト活性を示さず、且つ、ＡｄｉｐｏＲ２に対するアゴニスト活性を示すものであれば、タンパク質やペプチド以外の形態（例えば、低分子化合物等）であってもよい。 The specific agonist may be in a form other than a protein or peptide (for example, a low molecular weight compound) as long as it does not show an agonist activity against AdipoR1 and shows an agonist activity against AdipoR2.
　本実施形態に係るＴｈ１７細胞誘導性疾患の予防又は治療剤は、必要に応じて、特定アゴニスト以外の成分を含有していてもよい。特定アゴニスト以外の成分としては、賦形剤、崩壊剤、結合剤、滑沢剤、界面活性剤、緩衝剤、溶解補助剤、安定化剤、等張化剤、懸濁化剤、乳化剤、緩衝剤、溶剤；等が挙げられる。 The preventive or therapeutic agent for Th17 cell-induced disease according to the present embodiment may contain a component other than the specific agonist, if necessary. Ingredients other than specific agonists include excipients, disintegrants, binders, lubricants, surfactants, buffers, solubilizers, stabilizers, tonicity agents, suspending agents, emulsifiers, buffers. Agents, solvents; etc.
　本実施形態に係るＴｈ１７細胞誘導性疾患の予防又は治療剤の剤形は特に制限されず、用途に応じて選択することができる。例えば、本実施形態に係るＴｈ１７細胞誘導性疾患の予防又は治療剤の剤形としては、注射用液剤、注射用凍結乾燥粉末剤等の非経口剤；錠剤、顆粒剤、カプセル剤、液剤等の経口剤；等が挙げられる。 The dosage form of the preventive or therapeutic agent for Th17 cell-induced disease according to the present embodiment is not particularly limited and can be selected according to the intended use. For example, the dosage form of the Th17 cell-induced disease preventive or therapeutic agent according to the present embodiment includes parenteral preparations such as injection liquid preparations and lyophilized powder preparations for injection; tablets, granules, capsules, liquid preparations and the like. Oral preparations; etc.
　本実施形態に係るＴｈ１７細胞誘導性疾患の予防又は治療剤は、医薬品の分野において採用される任意の方法や適当な改良を加えた方法によって製造することができる。 The preventive or therapeutic agent for Th17 cell-induced disease according to the present embodiment can be produced by any method adopted in the field of pharmaceuticals or a method with appropriate improvements.
　本実施形態に係るＴｈ１７細胞誘導性疾患の予防又は治療剤の投与量は、投与対象、投与経路、対象疾患、症状等に応じて適宜決定される。また、投与回数や投与間隔等も特に制限されず、単回投与であっても、複数回投与であってもよい。 The dose of the preventive or therapeutic agent for Th17 cell-induced disease according to the present embodiment is appropriately determined according to the administration target, administration route, target disease, symptom and the like. Further, the number of administrations, the administration interval, and the like are not particularly limited, and the administration may be a single administration or a plurality of administrations.
　本実施形態に係るＴｈ１７細胞誘導性疾患の予防又は治療剤の投与対象となる動物種は特に限定されず、ヒトを投与対象としてもよいし、サル、ブタ、ウシ、イヌ、ネコ、マウス等の非ヒト動物を投与対象としてもよい。 The animal species to which the Th17 cell-induced disease prophylaxis or therapeutic agent according to the present embodiment is administered is not particularly limited, and humans may be administered, and monkeys, pigs, cows, dogs, cats, mice and the like may be administered. Non-human animals may be the subject of administration.
　なお、本明細書において「予防」には、発症を防ぐことのほか、発症の時期を遅らせることも含まれる。また、「治療」には、症状を消失又は軽減させることのほか、症状の進行の度合いを抑制することも含まれる。 In addition, in the present specification, "prevention" includes not only preventing the onset but also delaying the onset time. In addition to eliminating or alleviating symptoms, "treatment" also includes suppressing the degree of progression of symptoms.
　また、本実施形態によれば、Ｔｈ１７細胞誘導性疾患の予防又は治療剤を製造するための特定アゴニストの使用もまた提供される。 Also, according to the present embodiment, the use of a specific agonist for producing a prophylactic or therapeutic agent for Th17 cell-induced disease is also provided.
＜Ｔｈ１７細胞誘導性疾患の予防又は治療方法＞
　本実施形態によれば、特定アゴニストを含む薬剤を、Ｔｈ１７細胞誘導性疾患の予防又は治療を必要とする対象に投与することを含む、Ｔｈ１７細胞誘導性疾患を予防又は治療するための方法が提供される。該方法における投与対象や投与回数等は、Ｔｈ１７細胞誘導性疾患の予防又は治療剤について上述した投与対象や投与回数等を採用することができる。また、本実施形態に係るＴｈ１７細胞誘導性疾患の予防又は治療剤は、他の有効成分を含む医薬品等と併用して投与してもよい。 <Prevention or treatment method for Th17 cell-induced diseases>
According to the present embodiment, there is provided a method for preventing or treating Th17 cell-induced disease, which comprises administering a drug containing a specific agonist to a subject in need of prevention or treatment of Th17 cell-induced disease. Will be done. As the administration target and the number of administrations in the method, the above-mentioned administration target and the number of administrations for the preventive or therapeutic agent for Th17 cell-induced disease can be adopted. In addition, the preventive or therapeutic agent for Th17 cell-induced disease according to the present embodiment may be administered in combination with a drug or the like containing another active ingredient.
＜Ｔｈ１７細胞誘導性疾患の予防又は治療剤のスクリーニング方法＞
　本実施形態に係るＴｈ１７細胞誘導性疾患の予防又は治療剤のスクリーニング方法は、アディポネクチン受容体１（ＡｄｉｐｏＲ１）に対するアゴニスト活性を示さず、且つ、アディポネクチン受容体２（ＡｄｉｐｏＲ２）に対するアゴニスト活性を示す候補アゴニストをスクリーニングすることを含む。上述のとおり、ＡｄｉｐｏＲ１に対するアゴニスト活性を示さず、且つ、ＡｄｉｐｏＲ２に対するアゴニスト活性を示す特定アゴニストは、Ｔｈ１７細胞への分化を抑制する。したがって、ＡｄｉｐｏＲ１に対するアゴニスト活性を示さず、且つ、ＡｄｉｐｏＲ２に対するアゴニスト活性を示すような活性化の有無を指標とすることにより、Ｔｈ１７細胞誘導性疾患の予防又は治療剤をスクリーニングすることができる。 <Method of screening for preventive or therapeutic agents for Th17 cell-induced diseases>
The method for screening a prophylactic or therapeutic agent for a Th17 cell-induced disease according to the present embodiment is a candidate agonist that does not show an agonist activity against adiponectin receptor 1 (AdipoR1) and shows an agonist activity against adiponectin receptor 2 (AdipoR2). Includes screening. As described above, a specific agonist that does not show agonist activity against AdiponR1 and shows agonist activity against AdiponR2 suppresses differentiation into Th17 cells. Therefore, a prophylactic or therapeutic agent for Th17 cell-induced disease can be screened by using the presence or absence of activation that does not show agonist activity against AdiponR1 and shows agonist activity against AdiponR2 as an index.
　ＡｄｉｐｏＲ１及びＡｄｉｐｏＲ２の活性化を評価する方法は特に制限されず、任意の方法を採用することができる。 The method for evaluating the activation of AdipoR1 and AdipoR2 is not particularly limited, and any method can be adopted.
　以下に実施例によって本発明をより具体的に説明するが、本発明はこれら実施例によって制限されるものではない。なお、統計解析はスチューデントの両側ｔ検定により行った（＊Ｐ＜０．０５；＊＊Ｐ＜０．０１；＊＊＊Ｐ＜０．００１）。 Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples. Statistical analysis was performed by Student's two-sided t-test ( * P <0.05; ** P <0.01; *** P <0.001).
＜材料及び方法＞（マウス）
　ＣＴＲＰ３をコードする遺伝子であるＣ１ｑｔｎｆ３をノックアウトしたＫＯマウス（Ｃ１ｑｔｎｆ３－／－マウス）を作製した。Ｃ１ｑｔｎｆ３－／－マウスは、Ｃ５７ＢＬ／６マウスをもとに既報（Ｂｉｏｃｈｅｍ．　Ｂｉｏｐｈｙｓ．　Ｒｅｓ．　Ｃｏｍｍｕｎ．，４４３：４２（２０１４））に従って作製した。野生型（ＷＴ）のＣ５７ＢＬ／６マウスは、日本エスエルシー株式会社から購入した。 <Materials and methods> (mouse)
A KO mouse (C1qtnf3 − / − mouse) in which C1qtnf3, which is a gene encoding CTRP3, was knocked out was prepared. C1qtnf3 − / − mice were prepared based on C57BL / 6 mice according to a previously reported report (Biochem. Biophys. Res. Commun., 443: 42 (2014)). Wild-type (WT) C57BL / 6 mice were purchased from Nippon SLC Co., Ltd.
　マウスは、８：００～２０：００の照明サイクルのクリーンルーム内でＳＰＦ条件下にて飼育した。マウスには、γ線滅菌した通常のＦ１食餌（船橋農場社）及び酸性化水道水（０．００２ＮのＨＣｌでｐＨ２．５に調整）を与えた。以下の試験には、週齢（８～１２週齢）及び性別を揃えたマウスを使用した。 Mice were bred under SPF conditions in a clean room with a lighting cycle from 8:00 to 20:00. Mice were fed a normal γ-ray sterilized F1 diet (Funabashi Farm) and acidified tap water (adjusted to pH 2.5 with 0.002N HCl). The following tests used mice of the same age (8-12 weeks) and gender.
（ナイーブＴ細胞の精製）
　Ｃ１ｑｔｎｆ３－／－マウス及びＷＴマウスの脾臓から、Ｎａｉｖｅ　ＣＤ４＋　Ｔ　ｃｅｌｌ　Ｉｓｏｌａｔｉｏｎ　Ｋｉｔ（Ｍｉｌｔｅｎｙｉ　Ｂｉｏｔｅｃ社）を用いて、ナイーブＴ細胞の精製を行った。 (Purification of naive T cells)
Naive T cells were purified from the spleens of C1qtnf3 − / − mice and WT mice using Naive CD 4+ T cell Isolation Kit (Miltenyi Biotec).
（ナイーブＴ細胞からＴｈ１細胞への分化誘導）
　ナイーブＴ細胞を、プレートコートした４μｇ／ｍＬの抗ＣＤ３（クローン１４５－２Ｃ１１、ＢｉｏＬｅｇｅｎｄ社）並びに可溶性１μｇ／ｍＬの抗ＣＤ２８（クローン３７．５１、ＢｉｏＬｅｇｅｎｄ社）、１０μｇ／ｍＬの抗ＣＤ４（クローン１１Ｂ１１、ＢｉｏＬｅｇｅｎｄ社）、及び５ｎｇ／ｍＬのｒｅｃｏｍｂｉｎａｎｔ　ｍｕｒｉｎｅ　ＩＬ－１２（ＰｅｐｒｏＴｅｃｈ社）を含み、１０％ＦＢＳを含有するＸ－ＶＩＶＯ　２０　ｍｅｄｉｕｍ（Ｌｏｎｚａ社）中で３日間培養して刺激した。培養はｒｅｃｏｍｂｉｎａｎｔ　ｈｕｍａｎ　ＣＴＲＰ３（Ａｖｉｓｃｅｒａ　Ｂｉｏｓｃｉｅｎｃｅ社）、ＡｄｉｐｏＲｏｎ（Ｃａｙｍａｎ　Ｃｈｅｍｉｃａｌ社）、ＡｄｉｐｏＲ１ブロッカー（Ａｌｐｈａ　Ｄｉａｇｎｏｓｔｉｃ社）、及びＡｄｉｐｏＲ２ブロッカー（Ａｌｐｈａ　Ｄｉａｇｎｏｓｔｉｃ）の非存在下又は存在下で行った。 (Induction of differentiation from naive T cells to Th1 cells)
Naive T cells were plate-coated with 4 μg / mL anti-CD3 (Clone 145-2C11, BioLegend) and soluble 1 μg / mL anti-CD28 (Clone 37.51, BioLegend), 10 μg / mL anti-CD4 (Clone 11B11). , BioLegend), and X-VIVO 20 medium (Lonza) containing 5 ng / mL recombinant murine IL-12 (PeproTech) and containing 10% FBS for 3 days stimulation. Cultures were performed in recombinant human CTRP3 (Aviscera Bioscience), AdipoRon (Cayman Chemical), AdipoR1 blocker (Alpha Diagnostic), and in the presence of AdipoR2 blocker (Alpha).
（ナイーブＴ細胞からＴｈ１７細胞への分化誘導）
　ナイーブＴ細胞を、プレートコートした４μｇ／ｍＬの抗ＣＤ３（クローン１４５－２Ｃ１１、ＢｉｏＬｅｇｅｎｄ社）並びに可溶性１μｇ／ｍＬの抗ＣＤ２８（クローン３７．５１、ＢｉｏＬｅｇｅｎｄ社）、１０μｇ／ｍＬの抗ＩＦＮγ（クローンＲ４－６Ａ２、ＢｉｏＬｅｇｅｎｄ社）、１０μｇ／ｍＬの抗ＣＤ４（クローン１１Ｂ１１、ＢｉｏＬｅｇｅｎｄ社）、２０ｎｇ／ｍＬのｒｅｃｏｍｂｉｎａｎｔ　ｍｏｕｓｅ　ＩＬ－６（ＰｅｐｒｏＴｅｃｈ社）、５ｎｇ／ｍＬのｒｅｃｏｍｂｉｎａｎｔ　ｈｕｍａｎ　ＴＧＦ－β１（ＰｅｐｒｏＴｅｃｈ社）、及び１０ｎｇ／ｍＬのｒｅｃｏｍｂｉｎａｎｔ　ｍｏｕｓｅ　ＩＬ－１α（ＰｅｐｒｏＴｅｃｈ社）を含み、１０％ＦＢＳを含有するＸ－ＶＩＶＯ　２０　ｍｅｄｉｕｍ（Ｌｏｎｚａ社）中で４又は５日間培養して刺激した。培養はｒｅｃｏｍｂｉｎａｎｔ　ｈｕｍａｎ　ＣＴＲＰ３（Ａｖｉｓｃｅｒａ　Ｂｉｏｓｃｉｅｎｃｅ社）、ＡｄｉｐｏＲｏｎ（Ｃａｙｍａｎ　Ｃｈｅｍｉｃａｌ社）、ＡｄｉｐｏＲ１ブロッカー（Ａｌｐｈａ　Ｄｉａｇｎｏｓｔｉｃ社）、及びＡｄｉｐｏＲ２ブロッカー（Ａｌｐｈａ　Ｄｉａｇｎｏｓｔｉｃ）の非存在下又は存在下で行った。 (Induction of differentiation from naive T cells to Th17 cells)
Naive T cells were plate coated with 4 μg / mL anti-CD3 (clone 145-2C11, BioLegend) and soluble 1 μg / mL anti-CD28 (clone 37.51, BioLegend), 10 μg / mL anti-IFNγ (clone R4). -6A2, BioLegend, 10 μg / mL anti-CD4 (clone 11B11, BioLegend), 20 ng / mL recombinant mouse IL-6 (PeproTech), 5 ng / mL recombinant human TGF-β1 It was stimulated by culturing for 4 or 5 days in X-VIVO 20 medium (Lonza) containing 10 ng / mL recombinant mouse IL-1α (PeproTech) and containing 10% FBS. Cultures were performed in recombinant human CTRP3 (Aviscera Bioscience), AdipoRon (Cayman Chemical), AdipoR1 blocker (Alpha Diagnostic), and in the presence of AdipoR2 blocker (Alpha).
（フローサイトメトリー）
　細胞を５０ｎｇ／ｍＬのＰＭＡ（Ｓｉｇｍａ社）、５００ｎｇ／ｍＬのイオノマイシン（Ｓｉｇｍａ社）、及び２μＭのモネンシン（Ｓｉｇｍａ社）で４時間刺激した。刺激後、細胞を染色バッファー（２％ＦＣＳ及び０．１％アジ化ナトリウムを含有するＨＢＳＳ）中の抗マウスＣＤ１６／ＣＤ３２モノクローナル抗体（２．４Ｇ２、ハイブリドーマ培養上清から精製したもの）で処理して、ＦｃＲ結合をブロックした。次に、マウスＩＬ－１７Ａ抗体（ＴＣ１１－１８Ｈ１０．１）、マウスＩＦＮ－γ抗体（ＸＭＧ１．２）、及びマウスＣＤ４抗体（ＲＭ４－５）を用いて、４℃で３０分間、既報（Ｎａｔ．Ｃｏｍｍｕｎ．，６：７４６４（２０１５））に従って染色した。なお、これらの抗体は、Ｂｉｏｌｅｇｅｎｄ社から入手した。その後、ＦＡＣＳ　Ｃａｎｔｏ　ＩＩサイトメーター、及びＣｅｌｌＱｕｅｓｔソフトウェア（Ｂｅｃｔｏｎ　Ｄｉｃｋｉｎｓｏｎ社）又はＦｌｏｗＪｏソフトウェア（Ｔｒｅｅ　Ｓｔａｒ社）により解析した。 (Flow cytometry)
Cells were stimulated with 50 ng / mL PMA (Sigma), 500 ng / mL ionomycin (Sigma), and 2 μM monensin (Sigma) for 4 hours. After stimulation, cells were treated with anti-mouse CD16 / CD32 monoclonal antibody (2.4G2, purified from hybridoma culture supernatant) in staining buffer (HBSS containing 2% FCS and 0.1% sodium azide). FcR binding was blocked. Next, using mouse IL-17A antibody (TC11-18H10.1), mouse IFN-γ antibody (XMG1.2), and mouse CD4 antibody (RM4-5), previously reported (Nat. Stained according to Commun., 6: 7464 (2015)). These antibodies were obtained from BioLegend. The analysis was then performed using a FACS Canto II cytometer and CellQuest software (Becton Dickinson) or FlowJo software (Tree Star).
（ＥＬＩＳＡ）
　培養上清中のＩＬ－１７及びＩＦＮ－γのタンパク質量は、ＩＬ－１７　ＥＬＩＳＡ　ＭＡＸ　ｓｔａｎｄａｒｄ（Ｒ＆Ｄ　Ｓｙｓｔｅｍｓ社）及びｍｏｕｓｅ　ＩＦＮ－γ　ＥＬＩＳＡ　ＭＡＸ　ｓｔａｎｄａｒｄ（Ｒ＆Ｄ　Ｓｙｓｔｅｍｓ社）を用いてそれぞれ解析した。 (ELISA)
The protein amounts of IL-17 and IFN-γ in the culture supernatant were analyzed using IL-17 ELISA MAX standard (R & D Systems) and mouse IFN-γ ELISA MAX standard (R & D Systems), respectively.
＜試験例１＞
　試験例１では、ＣＴＲＰ３欠損による、Ｔｈ１７細胞への分化における影響を確認した。上述した方法で、Ｃ１ｑｔｎｆ３－／－マウス及びＷＴマウスのナイーブＴ細胞からＴｈ１７細胞への分化誘導を行い、ＩＬ－１７＋Ｔ細胞（Ｔｈ１７細胞）のポピュレーションを、フローサイトメトリーにより解析した。また、培養上清中のＩＬ－１７タンパク質量を、ＥＬＩＳＡにより解析した。 <Test Example 1>
In Test Example 1, the effect of CTRP3 deficiency on the differentiation into Th17 cells was confirmed. The differentiation of naive T cells of C1qtnf3 − / − mice and WT mice into Th17 cells was induced by the method described above, and the population of IL-17 + T cells (Th17 cells) was analyzed by flow cytometry. In addition, the amount of IL-17 protein in the culture supernatant was analyzed by ELISA.
　同様の実験を３回行い、平均値と標準偏差を求めた。フローサイトメトリーの結果を図１Ａ及び図１Ｂに示し、ＥＬＩＳＡの結果を図１Ｃに示す。 The same experiment was performed 3 times, and the average value and standard deviation were calculated. The results of flow cytometry are shown in FIGS. 1A and 1B, and the results of ELISA are shown in FIG. 1C.
　図１Ａ及び図１Ｂに示されるように、Ｃ１ｑｔｎｆ３－／－マウスにおいては、ＷＴマウスと比較して、ＩＬ－１７＋Ｔ細胞のポピュレーションが有意に増加していた。また、図１Ｃに示されるように、Ｃ１ｑｔｎｆ３－／－マウスにおいては、ＷＴマウスと比較して、培養上清中のＩＬ－１７タンパク質量が有意に増加していた。これらの結果より、ＣＴＲＰ３欠損により、ナイーブＴ細胞からＴｈ１７細胞への分化が亢進することがわかった。 As shown in FIGS. 1A and 1B, the population of IL-17 + T cells was significantly increased in C1qtnf3 − / − mice as compared with WT mice. In addition, as shown in FIG. 1C, the amount of IL-17 protein in the culture supernatant was significantly increased in C1qtnf3 − / − mice as compared with WT mice. From these results, it was found that CTRP3 deficiency promotes the differentiation of naive T cells into Th17 cells.
＜試験例２＞
　試験例２では、ＣＴＲＰ３を添加した場合の、Ｔｈ１７細胞への分化における影響を確認した。Ｃ１ｑｔｎｆ３－／－マウスから精製したナイーブＴ細胞からＴｈ１７細胞への分化誘導において、ｒｅｃｏｍｂｉｎａｎｔ　ｈｕｍａｎ　ＣＴＲＰ３を５０、１００、２００ｎｇ／ｍＬの濃度となるように培地に添加して培養を行い、ＩＬ－１７＋Ｔ細胞（Ｔｈ１７細胞）のポピュレーションを、フローサイトメトリーにより解析した。また、培養上清中のＩＬ－１７タンパク質量を、ＥＬＩＳＡにより解析した。 <Test Example 2>
In Test Example 2, the effect of adding CTRP3 on the differentiation into Th17 cells was confirmed. C1qtnf3 − / − In the induction of differentiation of naive T cells purified from mice into Th17 cells, recombinant human CTRP3 was added to the medium to a concentration of 50, 100, 200 ng / mL and cultured, and IL-17 + was performed. Population of T cells (Th17 cells) was analyzed by flow cytometry. In addition, the amount of IL-17 protein in the culture supernatant was analyzed by ELISA.
　同様の実験を４回行い、平均値と標準偏差を求めた。フローサイトメトリーの結果を図２Ａ及び図２Ｂに示し、ＥＬＩＳＡの結果を図２Ｃに示す。 The same experiment was performed 4 times, and the average value and standard deviation were calculated. The results of flow cytometry are shown in FIGS. 2A and 2B, and the results of ELISA are shown in FIG. 2C.
　図２Ａ及び図２Ｂに示されるように、ＣＴＲＰ３の濃度依存的に、ＩＬ－１７＋Ｔ細胞のポピュレーションが有意に減少していた。また、図２Ｃに示されるように、ＣＴＲＰ３の濃度依存的に、培養上清中のＩＬ－１７タンパク質量が有意に減少していた。これらの結果より、ＣＴＲＰ３は、ナイーブＴ細胞からＴｈ１７細胞への分化を抑制することがわかった。 As shown in FIGS. 2A and 2B, the population of IL-17 + T cells was significantly reduced in a concentration-dependent manner of CTRP3. In addition, as shown in FIG. 2C, the amount of IL-17 protein in the culture supernatant was significantly decreased depending on the concentration of CTRP3. From these results, it was found that CTRP3 suppresses the differentiation of naive T cells into Th17 cells.
＜試験例３＞
　試験例３では、ＣＴＲＰ３を添加した場合の、Ｔｈ１細胞への分化における影響を確認した。Ｃ１ｑｔｎｆ３－／－マウスから精製したナイーブＴ細胞からＴｈ１細胞への分化誘導において、ｒｅｃｏｍｂｉｎａｎｔ　ｈｕｍａｎ　ＣＴＲＰ３を５０、１００、２００ｎｇ／ｍＬの濃度となるように培地に添加して培養を行い、ＩＦＮ－γ＋Ｔ細胞（Ｔｈ１細胞）のポピュレーションを、フローサイトメトリーにより解析した。また、培養上清中のＩＦＮ－γタンパク質量を、ＥＬＩＳＡにより解析した。 <Test Example 3>
In Test Example 3, the effect of adding CTRP3 on the differentiation into Th1 cells was confirmed. C1qtnf3 − / − In the induction of differentiation of naive T cells purified from mice into Th1 cells, recombinant human CTRP3 was added to the medium to a concentration of 50, 100, 200 ng / mL and cultured, and IFN-γ + Population of T cells (Th1 cells) was analyzed by flow cytometry. In addition, the amount of IFN-γ protein in the culture supernatant was analyzed by ELISA.
　同様の実験を４回行い、平均値と標準偏差を求めた。フローサイトメトリーの結果を図３Ａ及び図３Ｂに示し、ＥＬＩＳＡの結果を図３Ｃに示す。 The same experiment was performed 4 times, and the average value and standard deviation were calculated. The results of flow cytometry are shown in FIGS. 3A and 3B, and the results of ELISA are shown in FIG. 3C.
　図３Ａ及びＢに示されるように、ＣＴＲＰ３をいずれの濃度で添加しても、ＩＦＮ－γ＋Ｔ細胞のポピュレーションへの影響は示されなかった。同様に、図３Ｃに示されるように、ＣＴＲＰ３をいずれの濃度で添加しても、ＩＦＮ－γタンパク質量への影響は示されなかった。これらの結果より、ＣＴＲＰ３は、ナイーブＴ細胞からＴｈ１細胞への分化には影響しないことがわかった。 As shown in FIGS. 3A and 3B, the addition of CTRP3 at any concentration did not show any effect on the population of IFN-γ + T cells. Similarly, as shown in FIG. 3C, the addition of CTRP3 at any concentration did not show any effect on the amount of IFN-γ protein. From these results, it was found that CTRP3 does not affect the differentiation of naive T cells into Th1 cells.
＜試験例４＞
　試験例４では、アディポネクチン受容体のアゴニストであるＡｄｉｐｏＲｏｎを添加した場合の、Ｔｈ１７細胞及びＴｈ１細胞への分化における影響を確認した。ＷＴマウスから精製したナイーブＴ細胞からＴｈ１７細胞及びＴｈ１細胞への分化誘導において、ＡｄｉｐｏＲｏｎを１、２、３μｇ／ｍＬの濃度となるように培地に添加して培養を行い、ＩＬ－１７＋Ｔ細胞（Ｔｈ１７細胞）及びＩＦＮ－γ＋Ｔ細胞（Ｔｈ１細胞）のポピュレーションを、フローサイトメトリーにより解析した。また、培養上清中のＩＬ－１７タンパク質量及びＩＦＮ－γタンパク質量を、ＥＬＩＳＡにより解析した。 <Test Example 4>
In Test Example 4, the effect on the differentiation into Th17 cells and Th1 cells when AdipoRon, which is an agonist of the adiponectin receptor, was confirmed. In the induction of differentiation of naive T cells purified from WT mice into Th17 cells and Th1 cells, AdipoRon was added to the medium to a concentration of 1, 2, 3 μg / mL and cultured, and IL-17 + T cells were cultured. Populations of (Th17 cells) and IFN-γ + T cells (Th1 cells) were analyzed by flow cytometry. In addition, the amount of IL-17 protein and the amount of IFN-γ protein in the culture supernatant were analyzed by ELISA.
　同様の実験を４回行い、平均値と標準偏差を求めた。Ｔｈ１７細胞に関するフローサイトメトリーの結果を図４Ａ及び図４Ｂに示し、ＥＬＩＳＡの結果を図４Ｃに示す。また、Ｔｈ１細胞に関するフローサイトメトリーの結果を図４Ｄ及び図４Ｅに示し、ＥＬＩＳＡの結果を図４Ｆに示す。 The same experiment was performed 4 times, and the average value and standard deviation were calculated. The results of flow cytometry for Th17 cells are shown in FIGS. 4A and 4B, and the results of ELISA are shown in FIG. 4C. The results of flow cytometry for Th1 cells are shown in FIGS. 4D and 4E, and the results of ELISA are shown in FIG. 4F.
　図４Ａ及び図４Ｂに示されるように、ＡｄｉｐｏＲｏｎの濃度依存的に、ＩＬ－１７＋Ｔ細胞のポピュレーションが有意に減少していた。また、図４Ｃに示されるように、ＡｄｉｐｏＲｏｎの濃度依存的に、培養上清中のＩＬ－１７タンパク質量が有意に減少していた。また、図４Ｄ及び図４Ｅに示されるように、ＡｄｉｐｏＲｏｎの濃度依存的に、ＩＦＮ－γ＋Ｔ細胞のポピュレーションが有意に減少していた。また、図４Ｆに示されるように、ＡｄｉｐｏＲｏｎの濃度依存的に、培養上清中のＩＦＮ－γタンパク質量が有意に減少していた。これらの結果より、ＡｄｉｐｏＲｏｎは、Ｔｈ１７細胞及びＴｈ１細胞の双方への分化を抑制することがわかった。 As shown in FIGS. 4A and 4B, the population of IL-17 + T cells was significantly reduced in a concentration-dependent manner of AdipoRon. In addition, as shown in FIG. 4C, the amount of IL-17 protein in the culture supernatant was significantly decreased depending on the concentration of AdipoRon. Also, as shown in FIGS. 4D and 4E, the population of IFN-γ + T cells was significantly reduced in a concentration-dependent manner of AdipoRon. In addition, as shown in FIG. 4F, the amount of IFN-γ protein in the culture supernatant was significantly decreased depending on the concentration of AdipoRon. From these results, it was found that AdipoRon suppresses the differentiation into both Th17 cells and Th1 cells.
＜試験例５＞
　試験例５では、Ｔｈ１７細胞への分化における、ＡｄｉｐｏＲ１及びＡｄｉｐｏＲ２に対するＣＴＲＰ３のアゴニスト活性を確認した。Ｃ１ｑｔｎｆ３－／－マウスから精製したナイーブＴ細胞からＴｈ１７細胞への分化誘導において、ｒｅｃｏｍｂｉｎａｎｔ　ｈｕｍａｎ　ＣＴＲＰ３を２００ｎｇ／ｍＬとなるように培地に添加し、更に、ＡｄｉｐｏＲ１に対するブロッカー（ＡｄｉｐｏＲ１　ｂｌｏｃｋｅｒ）又はＡｄｉｐｏＲ２に対するブロッカー（ＡｄｉｐｏＲ２　ｂｌｏｃｋｅｒ）を１０μｇ／ｍＬとなるように培地に添加して培養を行い、ＩＬ－１７＋Ｔ細胞（Ｔｈ１７細胞）のポピュレーションを、フローサイトメトリーにより解析した。また、培養上清中のＩＬ－１７タンパク質量を、ＥＬＩＳＡにより解析した。 <Test Example 5>
In Test Example 5, the agonist activity of CTRP3 against AdipoR1 and AdipoR2 in the differentiation into Th17 cells was confirmed. C1qtnf3 − / − In the induction of differentiation of naive T cells purified from mice into Th17 cells, recombinant human CTRP3 was added to the medium to a concentration of 200 ng / mL, and a blocker for AdiponR1 (blocker for AdipoR1) or a blocker for AdiponR2 AdiponR2 blocker) was added to the medium to a concentration of 10 μg / mL and cultured, and the population of IL-17 + T cells (Th17 cells) was analyzed by flow cytometry. In addition, the amount of IL-17 protein in the culture supernatant was analyzed by ELISA.
　同様の実験を４回行い、平均値と標準偏差を求めた。フローサイトメトリーの結果を図５Ａ及び図５Ｂに示し、ＥＬＩＳＡの結果を図５Ｃに示す。 The same experiment was performed 4 times, and the average value and standard deviation were calculated. The results of flow cytometry are shown in FIGS. 5A and 5B, and the results of ELISA are shown in FIG. 5C.
　図５Ａ及び図５Ｂに示されるように、ＡｄｉｐｏＲ１ブロッカーを添加した場合には、ＩＬ－１７＋Ｔ細胞のポピュレーションへの影響は示されず、ＡｄｉｐｏＲ２ブロッカーを添加した場合には、ＩＬ－１７＋Ｔ細胞のポピュレーションが有意に増加していた。また、図５Ｃに示されるように、ＡｄｉｐｏＲ１ブロッカーを添加した場合には、培養上清中のＩＬ－１７タンパク質量には変化がみられなかったが、ＡｄｉｐｏＲ２ブロッカーを添加した場合には、培養上清中のＩＬ－１７タンパク質量が有意に増加していた。これらの結果より、ＣＴＲＰ３によるＴｈ１７細胞への分化抑制作用は、ＡｄｉｐｏＲ１に対するアゴニスト活性によるものではなく、ＡｄｉｐｏＲ２に対するアゴニスト活性によるものであることがわかった。 As shown in FIGS. 5A and 5B, in the case of adding the AdipoR1 blocker effects on population of IL-17 + T cells is not shown, the addition of AdipoR2 blockers, IL-17 + T The population of cells was significantly increased. Further, as shown in FIG. 5C, when the AdipoR1 blocker was added, the amount of IL-17 protein in the culture supernatant was not changed, but when the AdipoR2 blocker was added, the culture was not changed. The amount of IL-17 protein in Kiyonaka was significantly increased. From these results, it was found that the inhibitory effect of CTRP3 on Th17 cells is not due to the agonist activity on AdipoR1 but due to the agonist activity on AdiponR2.
＜試験例６＞
　試験例６では、ＣＴＲＰ３がＴｈ１７細胞誘導性疾患である乾癬に及ぼす影響を確認した。Ｃ１ｑｔｎｆ３－／－マウス及びＷＴマウス（ｎ＝６ずつ）の耳の腹側表面に、１４ｍｇのベセルナクリーム（５％イミキモド；持田製薬社）を毎日塗布した。耳の厚さは、マイクロメーターカリパスによって測定した。乾癬症状は耳の腫れを指標として確認し、塗布０日目の耳と比較した耳の厚さの百分率として示し、平均値と標準偏差を求めた。結果を図６に示す。 <Test Example 6>
In Test Example 6, the effect of CTRP3 on psoriasis, which is a Th17 cell-induced disease, was confirmed. 14 mg of Bethelna cream (5% imiquimod; Mochida Pharmaceutical Co., Ltd.) was applied daily to the ventral surface of the ears of C1qtnf3 − / − mice and WT mice (n = 6 each). Ear thickness was measured with a micrometer caliper. The psoriasis symptom was confirmed by using the swelling of the ear as an index, and was shown as a percentage of the thickness of the ear compared with the ear on day 0 of application, and the mean value and standard deviation were calculated. The results are shown in FIG.
　図６に示されるように、ＷＴマウスに比べて、Ｃ１ｑｔｎｆ３－／－マウスでは、耳の腫れが経時的に大きくなる症状がみられた。これらの結果より、ＣＴＲＰ３欠損によりＴｈ１７細胞誘導性疾患が重篤化することがわかった。 As shown in FIG. 6, the swelling of the ear was observed to increase with time in the C1qtnf3 − / − mouse as compared with the WT mouse. From these results, it was found that Th17 cell-induced disease is exacerbated by CTRP3 deficiency.
　２０１９年８月９日に出願された日本出願２０１９－１４８０５４の開示はその全体が参照により本明細書に取り込まれる。
　本明細書に記載された全ての文献、特許出願、及び技術規格は、個々の文献、特許出願、及び技術規格が参照により取り込まれることが具体的且つ個々に記された場合と同程度に、本明細書中に参照により取り込まれる。 The disclosure of Japanese application 2019-148504, filed August 9, 2019, is incorporated herein by reference in its entirety.
All documents, patent applications, and technical standards described herein are to the same extent as if the individual documents, patent applications, and technical standards were specifically and individually stated to be incorporated by reference. Incorporated herein by reference.

Claims (7)

  1. 　アディポネクチン受容体１（ＰＡＱＲ１）に対するアゴニスト活性を示さず、且つ、アディポネクチン受容体２（ＰＡＱＲ２）に対するアゴニスト活性を示すアゴニストを有効成分として含有する、Ｔｈ１７細胞誘導性疾患の予防又は治療剤。 A prophylactic or therapeutic agent for Th17 cell-induced diseases, which contains an agonist as an active ingredient, which does not show an agonist activity for adiponectin receptor 1 (PAQR1) and shows an agonist activity for adiponectin receptor 2 (PAQR2).
  2. 　前記アゴニストが下記（ａ）～（ｄ）のいずれかである、請求項１に記載のＴｈ１７細胞誘導性疾患の予防又は治療剤。
    （ａ）配列番号１で示されるアミノ酸配列からなるタンパク質。
    （ｂ）配列番号１で示されるアミノ酸配列のｇＣ１ｑドメインからなるタンパク質。
    （ｃ）配列番号１で示されるアミノ酸配列に対して８０％以上の配列同一性を有し、アディポネクチン受容体１（ＰＡＱＲ１）に対するアゴニスト活性を示さず、且つ、アディポネクチン受容体２（ＰＡＱＲ２）に対するアゴニスト活性を示すタンパク質。
    （ｄ）配列番号１で示されるアミノ酸配列のｇＣ１ｑドメインに対して８０％以上の配列同一性を有し、アディポネクチン受容体１（ＰＡＱＲ１）に対するアゴニスト活性を示さず、且つ、アディポネクチン受容体２（ＰＡＱＲ２）に対するアゴニスト活性を示すタンパク質。     The preventive or therapeutic agent for a Th17 cell-induced disease according to claim 1, wherein the agonist is any of the following (a) to (d).
    (A) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1.
    (B) A protein consisting of the gC1q domain of the amino acid sequence shown in SEQ ID NO: 1.
    (C) It has 80% or more sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 1, does not show agonist activity on adiponectin receptor 1 (PAQR1), and is an agonist on adiponectin receptor 2 (PAQR2). A protein that exhibits activity.
    (D) It has 80% or more sequence identity with respect to the gC1q domain of the amino acid sequence shown in SEQ ID NO: 1, does not show agonist activity on adiponectin receptor 1 (PAQR1), and has adiponectin receptor 2 (PAQR2). ), A protein that exhibits agonist activity.
  3. 　前記Ｔｈ１７細胞誘導性疾患が関節リウマチ及び多発性硬化症でない、請求項１又は２に記載のＴｈ１７細胞誘導性疾患の予防又は治療剤。 The prophylactic or therapeutic agent for a Th17 cell-induced disease according to claim 1 or 2, wherein the Th17 cell-induced disease is not rheumatoid arthritis or multiple sclerosis.
  4. 　前記Ｔｈ１７細胞誘導性疾患が自己免疫疾患でない、請求項１又は２に記載のＴｈ１７細胞誘導性疾患の予防又は治療剤。 The preventive or therapeutic agent for a Th17 cell-induced disease according to claim 1 or 2, wherein the Th17 cell-induced disease is not an autoimmune disease.
  5. 　前記Ｔｈ１７細胞誘導性疾患が、乾癬、乾癬性関節炎、強直性脊椎症、視神経脊髄炎、アルツハイマー病、ハンチントン病、パーキンソン病、１型糖尿病、２型糖尿病、痛風、白斑、ぶどう膜炎、急性及び慢性腎炎、急性及び慢性疼痛、炎症性腸疾患、肝線維症、膵線維症、炎症性肺疾患、又はアレルギー性疾患である、請求項１又は２に記載のＴｈ１７細胞誘導性疾患の予防又は治療剤。 The Th17 cell-induced diseases include psoriasis, psoriatic arthritis, tonic spondylosis, optic neuromyelitis, Alzheimer's disease, Huntington's disease, Parkinson's disease, type 1 diabetes, type 2 diabetes, gout, leukoplakia, melanitis, acute and Prevention or treatment of Th17 cell-induced disease according to claim 1 or 2, which is chronic nephritis, acute and chronic pain, inflammatory bowel disease, liver fibrosis, pancreatic fibrosis, inflammatory lung disease, or allergic disease. Agent.
  6. 　請求項１～５のいずれか一項に記載のＴｈ１７細胞誘導性疾患の予防又は治療剤を製造するための、前記アゴニストの使用。 Use of the agonist for producing a prophylactic or therapeutic agent for Th17 cell-induced disease according to any one of claims 1 to 5.
  7. 　アディポネクチン受容体１（ＰＡＱＲ１）に対するアゴニスト活性を示さず、且つ、アディポネクチン受容体２（ＰＡＱＲ２）に対するアゴニスト活性を示す候補アゴニストをスクリーニングすることを含む、Ｔｈ１７細胞誘導性疾患の予防又は治療剤のスクリーニング方法。 A method for screening a prophylactic or therapeutic agent for Th17 cell-induced disease, which comprises screening a candidate agonist that does not show agonist activity on adiponectin receptor 1 (PAQR1) and shows agonist activity on adiponectin receptor 2 (PAQR2). ..
PCT/JP2020/029535 2019-08-09 2020-07-31 Agent for preventing or treating th17 cell-induced disease and screening method for agents for preventing or treating th17 cell-induced disease WO2021029238A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2019148054A JP2021028310A (en) 2019-08-09 2019-08-09 Agents for preventing or treating th17 cell-induced diseases and methods for screening agents for preventing or treating th17 cell-induced diseases
JP2019-148054 2019-08-09

Publications (1)

Publication Number Publication Date
WO2021029238A1 true WO2021029238A1 (en) 2021-02-18

Family

ID=74570622

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2020/029535 WO2021029238A1 (en) 2019-08-09 2020-07-31 Agent for preventing or treating th17 cell-induced disease and screening method for agents for preventing or treating th17 cell-induced disease

Country Status (2)

Country Link
JP (1) JP2021028310A (en)
WO (1) WO2021029238A1 (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160175396A1 (en) * 2013-03-15 2016-06-23 The Johns Hopkins University Methods for treating or preventing fatty liver disease using ctrp3

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160175396A1 (en) * 2013-03-15 2016-06-23 The Johns Hopkins University Methods for treating or preventing fatty liver disease using ctrp3

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
C HENG, C. T. ET AL.: "CTRP3 attenuates hepatic stellate cell activation through transforming growth factor-beta/smad pathway", BIOMED PHARMACOTHE R, vol. 89, 2017, pages 1387 - 1391, XP029987207, DOI: 10.1016/j.biopha.2017.03.021 *
ELSAID, H. H. ET AL.: "Complement clq tumor necrosis factor-related protein 3 a novel adipokine, protect against diabetes mellitus in young adult egyptian s", DIABETES METAB SYND R, vol. 13, no. 1, 12 October 2018 (2018-10-12), pages 434 - 438, XP085577627 *
MOHAMADINARAB, M. ET AL.: "Serum levels of Clq/TNF- related protein-3 in inflammatory bowel disease patients and its inverse association with inflammatory cytokines and insulin resistance", IUBMB LIFE, vol. 72, no. 8, 20 April 2020 (2020-04-20), pages 1698 - 1704, XP055793194 *
XUE, K. ET AL.: "No. 309 A novel adipokine Clq/TNF-related proteins-3 decreases in psoriasis patients and ameliorates imiquimod-induced psoriasis-like skin lesions by inhibiting the proliferation and inflammatory cytokines expression of keratinocytes", J INVEST DERMATOL, vol. 137, no. 10, 2017, pages S245, XP055793323 *
YAMAUCHI TOSHIMASA ET AL: "Adiponectin receptors agonists", JOURNAL OF CLINICAL AND EXPERIMENTAL MEDICINE, vol. 252, no. 5, 2015, pages 633 - 637 *

Also Published As

Publication number Publication date
JP2021028310A (en) 2021-02-25

Similar Documents

Publication Publication Date Title
McGeachy et al. The interleukin 23 receptor is essential for the terminal differentiation of interleukin 17–producing effector T helper cells in vivo
Kulkarni et al. Role of chemokine receptors and intestinal epithelial cells in the mucosal inflammation and tolerance
JP6266486B2 (en) Compositions and methods for the treatment of microbial disorders
Giacomin et al. Epithelial-intrinsic IKKα expression regulates group 3 innate lymphoid cell responses and antibacterial immunity
AU2009244039B2 (en) Means for the treatment and/or prophylaxis of an autoimmune disease and for the formation of regulatory T-cells
JP6166858B2 (en) Compositions and methods for the treatment of intracellular damage
CA2604604A1 (en) Methods for treating and preventing fibrosis by il-21 / il-21r antagonists
AU2003301114B2 (en) Uses of mammalian cytokine; related reagents
JP3657271B2 (en) Composition for the treatment of mammals infected with pathogenic or opportunistic infectious organisms
Bachus et al. IL-6 prevents Th2 cell polarization by promoting SOCS3-dependent suppression of IL-2 signaling
WO2021029238A1 (en) Agent for preventing or treating th17 cell-induced disease and screening method for agents for preventing or treating th17 cell-induced disease
EP2046809B1 (en) Wsx-1/il-27 as a target for anti-inflammatory responses
Seier et al. Antigen-specific induction of osteopontin contributes to the chronification of allergic contact dermatitis
Kurohara et al. Low-dose warfarin functions as an immunomodulator to prevent cyclophosphamide-induced NOD diabetes
JP2016069314A (en) Immunosuppressant
Tanaka et al. Enhancement of T Helper2Response in the Absence of Interleukin (IL-) 6; An Inhibition of IL-4-Mediated T Helper2Cell Differentiation by IL-6
KR101145337B1 (en) Antibodies to IP-10 for Treating Bone Diseases with Bone Destruction
Sharapova et al. Nijmegen Breakage Syndrome (NBS1)
Perez-Lopez et al. CCL28 modulates neutrophil responses during infection with mucosal pathogens
Stark Aroeira et al. Evidence for B cell participation in the in vitro and in vivo maintenance of in vivo staphylococcal enterotoxin B-induced T cell anergy.
WO2005035576A2 (en) Method of resisting osteoclast formation
Etling The aging mucosal immune system in the interleukin-10-deficient mouse
Garg et al. Neonatal-Onset Multisystem Inflammatory Disease (NOMID)
WO2000027873A9 (en) Interleukin-2 binding protein from arthropods
Lin THE ROLE OF BP180 IN GRANULOPOIESIS AND NLRP3 INFLAMMASOME ACTIVATION IN BULLOUS PEMPHIGOID

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20853059

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20853059

Country of ref document: EP

Kind code of ref document: A1