WO2015072544A1 - 自己免疫疾患の治療薬及び治療方法 - Google Patents
自己免疫疾患の治療薬及び治療方法 Download PDFInfo
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Classifications
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- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
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- A—HUMAN NECESSITIES
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- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
Definitions
- the present invention relates to a therapeutic agent for autoimmune disease, which contains C1q / TNF-related protein 3 (CTRP3).
- C1q / TNF-related protein 3 C1q / TNF-related protein 3 (CTRP3).
- RA Rheumatoid arthritis
- Inflammatory cytokines such as IL-1, IL-6, TNF- ⁇ , and IL-17 play an important role in the development of RA, and antibodies and inhibitors against these cytokines have been used to treat RA. Since high titer autoantibodies are detected in the serum of RA patients, antibodies against B cells are also useful for the treatment of RA. These approaches have greatly improved the effectiveness of RA treatment. However, the development of new therapies is still awaited because some patients are ineffective with these treatments, or are ineffective during these treatments.
- the inventors previously made two RA models.
- One is a type I human T cell leukemia virus (HTLV-I) gene transfer (Tg) mouse and the other is an IL-1 receptor antagonist (IL-1Ra) deficient (KO) mouse, both of which are autoimmune arthritis Spontaneously develop.
- HTLV-I human T cell leukemia virus
- IL-1Ra IL-1 receptor antagonist
- KO IL-1 receptor antagonist
- autoimmune arthritis Spontaneously develop Since multiple genes have been implicated in the development of autoimmune diseases, the inventors searched for new disease-related genes using DNA microarray technology.
- WT wild type mice
- the C1qtnf3 gene is one of those genes and encodes CTRP3 (also called C1q / TNF-related protein ⁇ 3, CORS-26, cartducin and cartnectin) and is well expressed in both models .
- CTRP3 is a soluble secreted protein consisting of a short N-terminal variable region, a collagen domain and a C-terminal complement C1q domain (R. Ghai et al., Immunobiology 212 (2007) pp253-266).
- CTRP3 belongs to the C1q / TNF-related protein (CTRP) family member (L. Shapiro et al., Curr Biol 8 (1998) pp 335-338), and has a crystal structure similar to TNF and complement C1q (JR Dunkerberger et al., Cell Res 20 (2010) pp34-50).
- the C1q domain of complement C1q is important for antigen binding IgM or IgG recognition and binding to the C1q receptor (C1qR), and the C1q domain of adiponectin is important for binding to adiponectin receptors (adipoR1 and adipoR2)
- C1qR C1q receptor
- adipoR1 and adipoR2 adiponectin receptors
- CTRP3 has been identified as a growth factor and promotes the proliferation of chondrogenic progenitor cells and chondrocytes (T. Maeda et al., J Cell Physiol 206 (2006) pp537-544.).
- CTRP3 has been reported to reduce the secretion of inflammatory cytokines from human adipocytes, monocytes and fibroblasts in vitro (A. Kopp et al., Endocrinology 151 (2010) pp5267-5278, J. Weigert et al., FEBS Lett 579 (2005) pp5565-5570, C. Hofmann et al., Inflamm Bowel Dis 17 (2011) pp2462-2471).
- CTRP3 the physiological role of CTRP3 in vivo has not yet been elucidated.
- CTRP3 in autoimmune diseases such as RA was also unclear.
- One of the objects of the present invention is to provide a novel therapeutic agent for autoimmune diseases.
- the inventors prepared a mouse (C1qtnf3 ⁇ / ⁇ mouse) in which C1qtnf3, which is a gene encoding CTRP3, was knocked out, and examined the effect of CTRP3 on the development of autoimmune arthritis. Furthermore, CTRP3 was administered to a disease model animal to confirm its effect.
- the present invention includes the following aspects.
- ⁇ 1> A therapeutic drug for autoimmune diseases, comprising CTRP3.
- ⁇ 2> The autoimmune disease therapeutic agent according to ⁇ 1>, wherein the autoimmune disease is rheumatoid arthritis or multiple sclerosis.
- ⁇ 3> A method for treating an autoimmune disease, comprising administering an autoimmune disease therapeutic agent containing CTRP3 to a subject in need of treatment for the autoimmune disease.
- ⁇ 4> The method for treating an autoimmune disease according to ⁇ 3>, wherein the autoimmune disease is rheumatoid arthritis or multiple sclerosis.
- ⁇ 5> Use of CTRP3 as an autoimmune disease therapeutic agent according to ⁇ 1> or ⁇ 2>.
- ⁇ 6> Use of CTRP3 in the manufacture of a therapeutic agent for autoimmune diseases according to ⁇ 1> or ⁇ 2>.
- ⁇ 7> A model animal for rheumatoid arthritis or multiple sclerosis, characterized by lacking the C1qtnf3 gene.
- a novel therapeutic agent for autoimmune disease a novel method for treating autoimmune disease, a novel autoimmune disease model animal, and the like can be provided.
- Exons are represented by black boxes.
- a diphtheria toxin gene (DT) was attached to the 5 'end of the genomic fragment for negative selection. SacII (S) was used for linearization.
- C Homologous recombination of the C1qtnf3 locus was performed by Southern blot hybridization using a 5 ′ probe (upper) or 3 ′ probe (lower) in the genome digested with EcoRI (E) or HindIII (H), respectively. It was investigated.
- D Absence of C1qtnf3 transcript was confirmed by RT-PCR using the primers ( ⁇ ⁇ ) in FIG.
- FIG. 3 shows that inflammation is increased in C1qtnf3 ⁇ / ⁇ mice.
- the seventh day after the induction of arthritis refers to the seventh day when the day on which the first intradermal injection of IIC was performed is defined as day 0.
- C, D On day 7 after induction of arthritis, inguinal LN cells were removed and cultured with IIC (0, 100, 200 ⁇ g / ml). Next, IIC-specific proliferative responses were measured by [ 3 H] TdR incorporation (C).
- (F) B cells were cultured with anti-IgM Ab (0-10 ⁇ g / ml) and CTRP3 (0, 10, 100 ng / ml). The proliferative response was then measured by [ 3 H] TdR incorporation (wild type, C1qtnf3 ⁇ / ⁇ : n 3).
- (G) IL-1 ⁇ production from neutrophils following C5a stimulation (0-1 ng / ml) in the presence of CTRP3 (0-500 ng / ml) was measured by ELISA (wild type, C1qtnf3 ⁇ / - : N 3 respectively). All data was reproducible in another independent experiment. * P ⁇ 0.05, ** P ⁇ 0.01. Mean values and standard errors are shown.
- CTRP3 has been difficult to elucidate the exact role of CTRP3 because the receptor for CTRP3 and the cells expressing the receptor have not yet been identified. Nevertheless, the inventors generated C1qtnf3 ⁇ / ⁇ mice and investigated the effect of CTRP3 on the development of autoimmune arthritis. The inventors have administered CTRP3 to a model animal of autoimmune arthritis and found that the incidence and severity of autoimmune arthritis are improved, and CTRP3 may reduce the onset and severity of autoimmune arthritis. Clearly shown. CTRP3 has been shown to be useful as a medicament for the treatment of autoimmune diseases such as rheumatoid arthritis (RA) acting by a new mechanism.
- RA rheumatoid arthritis
- the term “process” is not limited to an independent process, and is included in the term if the intended purpose of the process is achieved even when it cannot be clearly distinguished from other processes. .
- a numerical range indicated using “to” indicates a range including the numerical values described before and after “to” as the minimum value and the maximum value, respectively.
- the amount of each component in the composition is the total amount of the plurality of substances present in the composition unless there is a specific indication when there are a plurality of substances corresponding to each component in the composition. means.
- embodiments of the present invention will be described.
- Autoimmune diseases are diseases in which the immune system attacks the cells / tissues of itself and causes inflammation / damage of the cells / tissues.
- autoimmune disease examples include rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus (Lupus), inflammatory bowel diseases such as Crohn's disease and inflammatory bowel disease, and Behcet's disease.
- the autoimmune disease to which the therapeutic agent for autoimmune disease or the method for treating autoimmune disease according to the present invention is applied is particularly preferably rheumatoid arthritis or multiple sclerosis.
- Rheumatoid arthritis is an autoimmune disease and causes tissue damage mediated by cytokines, chemokines, metalloproteases and the like. In rheumatoid arthritis, the joint is inflamed and often causes progressive destruction of the joint structure.
- MS Multiple Sclerosis
- CNS myelin antigen that causes inflammation in the brain, spinal cord, optic nerve, etc., and repeatedly worsens neurological symptoms such as motor paralysis and sensory impairment. is there.
- Rituximab has been reported to be effective in the treatment of MS, and B cells are thought to be involved in the pathology of MS.
- CTRP3 is composed of 246 amino acid residues in the case of human CTRP3, and typical amino acid sequences and nucleic acid sequences are as shown below.
- accession numbers: AAI12926 and Q9BXJ4 are registered as amino acid sequences of human CTRP3, and accession numbers: BC112925, EU399231, EU399232, and the like are registered as nucleic acid sequences encoding human CTRP3.
- CTRP3 derived from organisms other than humans may be used, and the amino acid sequence and gene sequence of CTRP3 from organisms other than humans can also be obtained from databases such as the Genebank database or published papers.
- ⁇ Amino acid sequence of human CTRP3 (SEQ ID NO: 1)> MLWRQLIYWQLLALFFLPFCLCQDEYMESPQTGGLPPDCSKCCHGDYSFRGYQGPPGPPGPPGIPGNHGNNGNNGATGHEGAKGEKGDKGDLGPRGERGQHGPKGEKGYPGIPPELQIAFMASLATHFSNQNSGIIFSSVETNIGNFFDVMTGRFGAPVSGVYFFTFSMMKHEDVEEVYVYLMHNGNTVFSMYSYEMKGKSDTSSNHAVLKLAKGDEVWLRMGNGALHGDHQRFSTFAGFLLFETK
- One embodiment of the present invention is a therapeutic agent for autoimmune diseases containing CTRP3.
- the method for producing CTRP3 is not particularly limited, and may be obtained by a genetic recombination method, a synthesis method, or the like based on a known amino acid sequence or polynucleotide sequence, or a commercially available product may be used.
- CTRP3 When CTRP3 is produced by a genetic recombination method, for example, a gene encoding CTRP3 may be introduced into a microorganism such as Escherichia coli or yeast, a plant cell, an insect cell, or an animal cell using an expression vector to express CTRP3. .
- a gene encoding CTRP3 When human CTRP3 is produced, mammalian cells are particularly preferably used from the viewpoint of maintaining the three-dimensional structure and post-translational modification.
- CTRP3 may be produced by a chemical synthesis method using a liquid phase method, a solid phase method, a Boc method, an Fmoc method or the like alone or in combination.
- recombinant human CTRP3 As a commercial item, recombinant human CTRP3 (Aviscera bioscience company, USA) is mentioned, for example.
- CTRP3 may be a mutant CTRP3 in which a part of amino acid residues is added, substituted, or deleted to a known amino acid sequence as long as its physiological function is not impaired. It may be a mutant CTRP3 in which is added, substituted or deleted. CTRP3 may be a derivative of CTRP3. There is no restriction
- CTRP3 derivatives include CTRP3 combined with sugar chains, oligonucleotides, polynucleotides, polyethylene glycol, and other pharmaceutically acceptable additives or treatment agents.
- the administration method of the autoimmune disease therapeutic agent according to the present invention is not particularly limited, and may be either parenteral administration or oral administration.
- parenteral administration When administered parenterally by intra-articular injection, intramuscular injection, intravenous injection, or subcutaneous injection, prepare a sterile solution with solutes such as sodium chloride and glucose added as isotonic agents as pharmaceutical additives.
- solutes such as sodium chloride and glucose added as isotonic agents
- pharmaceutical composition suitable for parenteral administration include injections, infusions, inhalants, transdermal absorption agents, transmucosal absorption agents, and the like.
- the autoimmune disease therapeutic agent according to the present invention is preferably administered as an injection by intraarticular injection or intravenous injection, but is not limited thereto.
- Suitable dosage forms for injections include aqueous injections, non-aqueous injections, suspension injections, emulsion injections, lyophilized injections, powder injections, filled injections and cartridges, and the like. It is done.
- Examples of the dosage form suitable for oral administration include tablets, capsules, powders, granules, liquids, elixirs and the like.
- the dose of the therapeutic agent for autoimmune diseases according to the present invention may be an amount effective as a therapeutic agent for autoimmune diseases.
- the dose of CTRP3 may be 0.1 ⁇ g / kg to 10 mg / kg / day.
- the dose is preferably increased or decreased as appropriate according to age, disease state, symptoms and the like. It may be administered multiple times. In the case of multiple administrations, the administration interval is preferably increased or decreased appropriately depending on the disease state, symptoms and the like.
- the autoimmune disease therapeutic drug according to the present invention may be administered in combination with other drugs.
- it may be combined with a steroid, high molecular hyaluron, or non-steroidal anti-inflammatory drug (NSAID) to form a combination or kit.
- NSAID non-steroidal anti-inflammatory drug
- Examples of the additive for preparation of the autoimmune disease therapeutic agent according to the present invention include liquid media such as water, physiological saline, dextrose or similar sugar solution, ethylene glycol, propylene glycol, polyethylene glycol, polypropylene glycol, etc. Glycols; antioxidants such as sulfites; pH regulators and buffers such as sodium citrate, sodium acetate, sodium phosphate; stabilizers such as sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid; sodium chloride And isotonic agents such as glucose; local anesthetics such as procaine hydrochloride and lidocaine hydrochloride; and surfactants such as dimethyl sulfoxide (DMSO).
- a known additive can be contained in accordance with the dosage form.
- the animal species to be administered is not particularly limited and can be appropriately selected depending on the purpose. For example, human, monkey, pig, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, rabbit , Birds and the like. Although it is desirable that the animal species to be administered coincide with the animal species from which CTRP3 contained in the autoimmune disease is derived, there is no limitation.
- the administration target is a human
- human CTRP3 or mutant CTRP3 derived from human CTRP3 or a derivative thereof should be used from the viewpoint of high efficacy as a drug and few side effects such as immune reaction. Is preferred.
- CTRP3 derived from other animals may be used as a therapeutic drug for autoimmune diseases targeting humans.
- One aspect of the present invention includes a method for treating an autoimmune disease comprising administering an agent comprising CTRP3 to a subject in need of treatment for the autoimmune disease.
- an agent comprising CTRP3 comprising CTRP3 to a subject in need of treatment for the autoimmune disease.
- an animal species used as administration object What is necessary is just to select suitably as needed.
- Humans may be administered, non-human animals including primates such as monkeys, mammals such as pigs, cows, sheep, goats, dogs, cats, mice, rats, guinea pigs and rabbits, and birds such as birds It is good.
- the autoimmune disease is preferably rheumatoid arthritis or multiple sclerosis.
- One embodiment of the present invention includes a model animal for autoimmune diseases such as rheumatoid arthritis, which is characterized by lacking the C1qtnf3 gene.
- the autoimmune disease model animal may be homozygous or heterozygous for the C1qtnf3 gene, but is preferably homozygous.
- the target animal species is not particularly limited, but mice or rats are preferably used.
- the relationship between CTRP3 and autoimmune disease is not known, and a new autoimmune disease model animal can be provided. In particular, it is suitable as a model animal for rheumatoid arthritis or multiple sclerosis.
- the sequence of the C1qtnf3 gene is known as described above.
- a method for producing a model animal in which the C1qtnf3 gene is deleted is known, and an existing method can be used.
- a method in which the C1qtnf3 gene of embryonic stem cells is deleted by homologous recombination method this is injected into a mouse blastocyst to produce a chimeric mouse, and a chimeric mouse having germ cells deficient in the gene is mated, etc. It can be illustrated.
- Example 1 Generation of C1qtnf3 ⁇ / ⁇ Mice The inventors previously based on comprehensive gene expression analysis using DNA microarrays of two RA models, HTLV-I Tg mice and IL-1Ra KO mice, C1qtnf3 was identified as a candidate autoimmune related gene.
- the qPCR technique was used to confirm the enhanced expression of C1qtnf3 in the joint region of RA model mice (HTLV-ITg mice and IL-1Ra KO mice) (FIG. 1A). It has been reported that the expression of C1qtnf3 is greatly enhanced in the joints of K / BxN mice in addition to HTLV-I Tg mice Il and IL-1Ra ⁇ / ⁇ mice (Reference Document 22). C1qtnf3 ⁇ / ⁇ mice were prepared to examine the effect of CTRP3 on the development of CIA.
- Genomic DNA containing the C1qtnf3 gene was isolated from EGR-101ES cells derived from C57BL / 6 embryos (reference document 14).
- the targeting vector is a 1.7 kb DNA fragment containing the neomycin resistance gene (Ned) under the phosphoglycerate kinase (PGK) 1 promoter sandwiched between loxP sequences and 170 bp containing exon 4 of the C1qtnf3 gene encoding the C1q domain. Constructed by replacing genomic fragments.
- the diphtheria toxin (DT) A gene under the MC1 promoter was ligated to the 3 ′ end of the targeting vector for negative selection of targeted ES clones.
- the targeting vector was introduced into ES cells by electroporation to select G418 resistant clones (Nacalai Tesque, Japan). Homologous recombinant ES clones were screened by PCR and Southern blotting (FIG. 1C). The following primers were used for PCR: 5′-GCAGTAACAATGGCAACAGCAG-3 ′ (SEQ ID NO: 3), 5′-GCTCGGTACCCCATCAAGCTTAT-3 (SEQ ID NO: 4).
- the 5 ′ probe used was a DNA fragment amplified with the following primers: 5′-TGAAGAAAGGGCTTGGGCATCTTT-3 ′ (SEQ ID NO: 5), 5′-AGAAAACCCTCCTCCCAGCTCCAA-3 ′ (SEQ ID NO: 6).
- As the 3 ′ probe a DNA fragment amplified with the following primers was used: 5′-GADATGAAGGATGTTGAAGTCGGG-3 ′ (SEQ ID NO: 7), 5′-TCTATGCAAAATCATCATCTTTGAGG-3 ′ (SEQ ID NO: 8). Chimeric mice were generated by agglutination after karyotype analysis of targeted ES clones (Reference 15).
- Genotype analysis of C1qtnf3 deficient mice was performed using the following PCR primers: Primer 1, 5′-GATGCAGAGCAACATACACAGAG-3 ′ (SEQ ID NO: 9); Primer 2, 5′-GTTGATTTCTGCATCTCCACCTG-3 ′ (SEQ ID NO: 10) Primer 3, 5'-GCTCGGTACCCCATCAAGCTTAT-3 '(SEQ ID NO: 11). Primers 1 and 2 were used to detect the wild type allele (336 bp) and primers 1 and 3 were used to detect the mutant allele (195 bp). The lack of C1qtnf3 transcript was confirmed by RT-PCR (FIG. 1D). C1qtnf3 ⁇ / ⁇ mice were fertile, were born at the expected Mendelian ratio and did not show clear abnormalities before the age of one.
- CTRP3 has been reported to promote the proliferation of chondrogenic progenitor cells and chondrocytes in vitro (Refs. 10 and 23), suggesting that CTRP3 is involved in cartilage formation. It has also been implicated in the development of mandibular condylar cartilage (Reference 23). However, no clear skeletal abnormalities were detected in C1qtnf3 ⁇ / ⁇ mice. In addition, C1qtnf3 ⁇ / ⁇ mice were fertile, were born at the expected Mendelian ratio, and had no obvious skeletal deformation. Since other CTRP family members such as adiponectin are also involved in the regulation of cartilage formation (Ref. 24), it is possible that the effects of CTRP3 deficiency are compensated by other CTRP family members.
- Example 2 Incidence and severity of collagen-induced arthritis (CIA) in C1qtnf3 ⁇ / ⁇ mice Induction of collagen-induced arthritis (CIA) To assess the role of CTRP3 in the development of autoimmune arthritis, CIA was performed using C1qtnf3 ⁇ / ⁇ mice. Female wild type mice or C1qtnf3 ⁇ / ⁇ mice were immunized with 100 ⁇ l of 2 mg / ml type II collagen (IIC) (Sigma, USA) emulsified with complete Freund's adjuvant (CFA).
- IIC 2 mg / ml type II collagen
- CFA complete Freund's adjuvant
- CFA consists of incomplete Freund's adjuvant (Thermo Scientific, USA) and 1.65 mg / ml Mycobacterium tuberculosis (H37Ra; Difco, USA) at day 0 Intradermal injections were made at three nearby locations. On day 21, mice were boosted with the same amount of IIC / CFA intradermally near the previous injection site.
- 1 thickening with slight inflammatory cell infiltration and synovial surface proliferation
- 2 first grade change and granulomatous lesions in subsynovial tissue
- 3 second grade Change, pannus formation and bone destruction.
- the arthritic index of the ankle joint was estimated from the average of the talus of each mouse and the surrounding bone grade including the tibia and ribs (Ref. 20).
- Example 3 Analysis of role of CTRP3 in RA Role in the complement system Since CTRP3 has a complement C1q domain, the possibility that the complement system is involved in CTRP3 function was investigated.
- Plasma was collected from mice 7 days after the first IIC / CFA immunization and the levels of complement active products C3a and C5a were measured by ELISA.
- Plasma C3a and C5a levels were determined by sandwich ELISA using capture antibody-coated plates and detection antibodies to C3a or C5a (BD Farmingen, USA), 10 mM from female mice (8-10 weeks old) EDTA chelated plasma was used. The results showed that the levels of C3a and C5a in C1qtnf3 ⁇ / ⁇ mice were similar to those in C1qtnf3 + / + mice (FIGS. 3A and 3B).
- Adiponectin a member of the CTRP family, is involved in the regulation of the complement classical pathway by activation of C1q (Ref. 25) and the regulation of the alternative pathway by collaboration with complement factor H (Ref. 26) Has been reported.
- C1qtnf3 ⁇ / ⁇ mice no abnormality of the complement system was detected in C1qtnf3 ⁇ / ⁇ mice. This suggests that CTRP3 is not involved in the regulation of the complement system.
- C3a and C5a levels and in vitro complement activation assay results indicate that CTRP3 is not a complement regulator.
- CTRP3 deficiency affects inflammatory cytokine production from synovial cells.
- Primary synovial cells were collected from the synovium of the kneecap and ankle of wild type mice or C1qtnf3 ⁇ / ⁇ mice and cultured in DMEM medium (Gibco) containing 10% FBS and 1% penicillin-streptomycin.
- DMEM medium Gibco
- IL-1 ⁇ 0., 1, 5 and 10 pg / ml
- IL-6 levels in the culture supernatant after 24 hours were measured using mouse IL-6 ELISA MAX TM Standard (Biolegend, USA).
- IL-6 derived from synovial cells of C1qtnf3 ⁇ / ⁇ mice is similar to that of wild-type mice, and exogenous CTRP3 does not suppress IL-6 release from synovial cells. (FIG. 3E).
- CTRP3 has been reported to suppress the release of inflammatory cytokines in human monocytes (Reference 12). However, CTRP3 did not suppress cytokine production in synovial cells of arthritic joints.
- Hamster mAb (145-2C11) or rat mAb (RA3-6B2) for mouse CD3 and B220 was purchased from Biolegend (USA) and hamster mAb (HL3) for CD11c was purchased from BD Farmingen (USA).
- Cells were stained according to standard techniques and analyzed with a FACS Canto II cytometer and CellQuest software (Becton Dickinson, USA) or FlowJo software (Tree Star, USA).
- C1qtnf3 ⁇ / ⁇ mouse lymph nodes have more T and B cells than wild type mouse lymph nodes.
- the cell population was found to be equivalent between C1qtnf3 ⁇ / ⁇ mice and wild type mice (FIGS. 4-1A and 4-1B).
- LN cells were collected from the regional lymph nodes at the site of arthritis development in wild-type mice or C1qtnf3 ⁇ / ⁇ mice 7 days after IIC immunization. LN cells were cultured for 72 hours in the absence or presence of 100 or 200 ⁇ g / ml denatured IIC, followed by uptake of [ 3 H] thymidine (0.25 ⁇ Ci / ml) (Amersham, UK) for 6 hours. It was. The cells were then harvested with a micro 96 cell harvester (Skatron, Norway) and the radioactivity was measured with micro beta (Pharmacia Biotech, USA). The IFN- ⁇ level in the culture supernatant of the proliferation assay after 72 hours was measured with mouse IFN-gamma DuoSet (R & D Systems, USA).
- IIC-specific IgG in C1qtnf3 ⁇ / ⁇ mice was examined. Serum was collected on day 42 after the first IIC / CFA immunization, and IIC-specific IgG levels were measured by ELISA.
- the ELISA method uses a 20 ⁇ g / ml IIC-coated plate and alkaline phosphatase-conjugated polyclonal rabbit anti-mouse IgG antibody (Zymed, USA), using serum from mice 42 days after IIC / CFA immunization. IIC-specific IgG levels in were measured.
- IIC-specific IgG levels in the serum of C1qtnf3 ⁇ / ⁇ mice were significantly higher than those of wild type mice. It was shown that antibody production against IIC was significantly increased in C1qtnf3 ⁇ / ⁇ mice after induction of CIA. However, anti-IgM-induced B cell proliferation was comparable between C1qtnf3 ⁇ / ⁇ B cells and wild type B cells, and CTRP3 did not suppress B cell proliferation in vitro (FIG. 4-F).
- C1qtnf3 deficiency is responsible for the development of CIA in C1qtnf3 ⁇ / ⁇ mice.
- B cell proliferative response was normal after stimulation with anti-IgM, the functions inherent to B cells were also normal.
- no abnormality was detected in the recall proliferation response to IIC in C1qtnf3 ⁇ / ⁇ mouse T cells.
- IL-1 ⁇ production of C1qtnf3 ⁇ / ⁇ neutrophils was similar to that of wild-type mice, indicating that CTRP3 did not suppress IL-1 ⁇ release from neutrophils in vitro ( Fig. 4-2G).
- Cytokine production after neutrophil activation by complement component C5a was normal, and neutrophils, one of the CTRP3-producing cells, were not considered to be responsible for the increased inflammation in the joints of C1qtnf3 ⁇ / ⁇ mice .
- CTRP3 plays an important role in the development of autoimmune arthritis by regulating antibody production.
- a female wild type using 600 ⁇ g of MOG 35-55 peptide (MEVGWRSPFSRVVHLYRNK) (SEQ ID NO: 12) (manufactured by Scrun Japan), which is a part of myelin oligodendrocyte glycoprotein (MOG) emulsified with complete Freund's adjuvant (CFA) Mice or C1qtnf3 ⁇ / ⁇ mice were immunized.
- CFA consists of incomplete Freund's adjuvant (Thermo Scientific, USA) and 5 mg / ml Mycobacterium tuberculosis (H37Ra; Difco, USA). It was injected intradermally. On day 21, mice were boosted with the same amount of emulsion intradermally near the previous injection site.
- each mouse received 30 ⁇ L of recombinant human CTRP3 (300 ng / day, Aviscera Biosciences, USA) (SEQ ID NO: 1) in the joint space of the left knee joint as a control in the joint space of the right knee joint.
- PBS was injected once a day.
- the arthritis evaluation score was 1 or more, it was determined that arthritis occurred.
- the incidence of arthritis is shown in FIG. 6A, and the mean value and standard error of the arthritis evaluation score are shown in FIG. 6B.
- Statistical analysis was performed using the chi-square test for the incidence of arthritis and the Mann-Whitney U test for the clinical score.
- Administration of CTRP3 reduced the incidence of arthritis and clinical scores.
- CTRP3 was clearly shown to reduce the occurrence of autoimmune arthritis.
- CTRP3 has been shown to be useful as a medicament for the treatment of autoimmune diseases such as RA.
- mice 8- to 10-week-old homologous C57BL / 6 background mice were used. Mice were bred under specific pathogen removal conditions in the laboratory animal facilities of the University of Tokyo Institute of Medical Science System Disease Model Research Center and the Tokyo University of Science. All experiments were approved by the Animal Experiment Committee and conducted according to animal experiment ethics guidelines and genetic engineering experiment safety guidelines.
Abstract
Description
<1> CTRP3を含有する、自己免疫疾患治療薬。
<2> 前記自己免疫疾患が関節リウマチ又は多発性硬化症である、<1>に記載の自己免疫疾患治療薬。
<3> CTRP3を含有する自己免疫疾患治療薬を、自己免疫疾患の治療を必要とする対象に投与すること、を含む自己免疫疾患を治療する方法。
<4> 前記自己免疫疾患が関節リウマチ又は多発性硬化症である、<3>に記載の自己免疫疾患を治療する方法。
<5> <1>または<2>に記載の自己免疫疾患治療薬としてのCTRP3の使用。
<6> <1>または<2>に記載の自己免疫疾患治療薬の製造におけるCTRP3の使用。
<7> C1qtnf3遺伝子を欠損していることを特徴とする、関節リウマチ又は多発性硬化症のモデル動物。
また本明細書において「~」を用いて示された数値範囲は、「~」の前後に記載される数値をそれぞれ最小値および最大値として含む範囲を示す。
さらに本明細書において組成物中の各成分の量は、組成物中に各成分に該当する物質が複数存在する場合、特に断らない限り、組成物中に存在する当該複数の物質の合計量を意味する。
以下、本発明の態様について説明する。
MLWRQLIYWQLLALFFLPFCLCQDEYMESPQTGGLPPDCSKCCHGDYSFRGYQGPPGPPGPPGIPGNHGNNGNNGATGHEGAKGEKGDKGDLGPRGERGQHGPKGEKGYPGIPPELQIAFMASLATHFSNQNSGIIFSSVETNIGNFFDVMTGRFGAPVSGVYFFTFSMMKHEDVEEVYVYLMHNGNTVFSMYSYEMKGKSDTSSNHAVLKLAKGDEVWLRMGNGALHGDHQRFSTFAGFLLFETK
ATGCTTTGGAGGCAGCTCATCTATTGGCAACTGCTGGCTTTGTTTTTCCTCCCTTTTTGCCTGTGTCAAGATGAATACATGGAGTCTCCACAAACCGGAGGACTACCCCCAGACTGCAGTAAGTGTTGTCATGGAGACTACAGCTTTCGAGGCTACCAAGGCCCCCCTGGGCCACCGGGCCCTCCTGGCATTCCAGGAAACCATGGAAACAATGGCAACAATGGAGCCACTGGTCATGAAGGAGCCAAAGGTGAGAAGGGCGACAAAGGTGACCTGGGGCCTCGAGGGGAGCGGGGGCAGCATGGCCCCAAAGGAGAGAAGGGCTACCCGGGGATTCCACCAGAACTTCAGATTGCATTCATGGCTTCTCTGGCAACCCACTTCAGCAATCAGAACAGTGGGATTATCTTCAGCAGTGTTGAGACCAACATTGGAAACTTCTTTGATGTCATGACTGGTAGATTTGGGGCCCCAGTATCAGGTGTGTATTTCTTCACCTTCAGCATGATGAAGCATGAGGATGTTGAGGAAGTGTATGTGTACCTTATGCACAATGGCAACACAGTCTTCAGCATGTACAGCTATGAAATGAAGGGCAAATCAGATACATCCAGCAATCATGCTGTGCTGAAGCTAGCCAAAGGGGATGAGGTTTGGCTGCGAATGGGCAATGGCGCTCTCCATGGGGACCACCAACGCTTCTCCACCTTTGCAGGATTCCTGCTCTTTGAAACTAAGTAA
経口投与に適した剤型としては、例えば、錠剤、カプセル剤、粉剤、顆粒剤、液剤、エリキシル剤等が挙げられる。
なお、特に記載がない場合には、統計解析はスチューデントの両側t検定により行われた。
発明者らは以前に、2つのRAモデルであるHTLV-I TgマウスとIL-1Ra KOマウスのDNAマイクロアレイを用いる網羅的遺伝子発現解析に基づいて、C1qtnf3を自己免疫関連遺伝子の候補として特定した。qPCR技術を用いてRAモデルマウス(HTLV-I Tgマウス及びIL-1Ra KOマウス)の関節局所におけるC1qtnf3の発現の亢進を確認した(図1A)。C1qtnf3の発現は、HTLV-I TgマウスIl及びIL-1Ra-/-マウスの他に、K/BxNマウスの関節において非常に亢進されることが報告されている(参考文献22)。C1qtnf3-/-マウスを作製し、CIAの発症におけるCTRP3の影響を調べた。
1.コラーゲン誘導性関節炎(CIA)の誘導
自己免疫性関節炎の発生におけるCTRP3の役割を評価するために、C1qtnf3-/-マウスを用いてCIAを実施した。
完全フロイントアジュバント(CFA)で乳化した100μlの2mg/mlのII型コラーゲン(IIC)(シグマ社、米国)を用いてメスの野生型マウス又はC1qtnf3-/-マウスに免疫した。CFAは不完全フロイントアジュバント(サーモ・サイエンティフィック社、米国)と1.65mg/mlの加熱殺菌した結核菌(Mycobacterium tuberculosis)(H37Ra;Difco社、米国)から成り、0日目に尾の基部近くの3か所に皮内注射された。21日目にマウスに同量のIIC/CFAを以前の注射部位の近くの皮内にブースター注射した。
肉眼による評価により関節炎の発生を判定した。それぞれの足における関節炎の発生を次のように等級付けした:0=変化無し;1=軽度の腫脹;2=明確な関節の腫脹;3=重度の関節の腫脹と強直性変化(個々のマウスについて最大で12ポイント)(参考文献19、20)。CIAの発症率をカイ二乗検定により評価した。
図2Aに示されているように、野生型マウスと比較してC1qtnf3-/-マウスにおける関節炎の発生率が上昇し、C1qtnf3-/-マウスの関節炎の臨床スコアが顕著に増加した(図2B)。臨床スコアはマン・ホイットニーのU検定により評価した。
エーテル麻酔下でマウスを殺処理し、病理組織診断のために後肢の足首の関節を取り出し、固定し、脱灰化し、パラフィン包埋した。距骨全体の2~3μm厚の連続切片を矢状に作製し、光学顕微鏡法による検査のためにH&Eで染色した。踵骨と足首の関節の前方と後方の滑膜組織を含んで病変を病理組織学的に評価した。各関節を0~3の尺度で等級付けした。その等級付けでは0=正常、1=わずかな炎症細胞浸潤を有する肥厚化と滑膜表層の増殖、2=第1等級の変化と滑膜下層組織における肉芽腫病変、および3=第2等級の変化とパンヌス形成と骨破壊である。足首の関節の関節炎インデックスを各マウスの距骨、および脛骨と踵骨を含む周りの骨の等級の平均から推定した(参考文献20)。
1.補体系における役割
CTRP3は補体C1qドメインを有するので、補体系がCTRP3機能に関与する可能性を調査した。最初のIIC/CFA免疫後7日目におけるマウスから血漿を採取し、ELISAにより補体活性産物C3aおよびC5aのレベルを測定した。血漿中のC3aとC5aレベルを、捕捉抗体被覆プレートとC3aまたはC5aに対する検出抗体(BDファーミンジェン社、米国)を使用するサンドイッチELISAにより、メスのマウス(8~10週齢)に由来する10mMのEDTAでキレートした血漿を使用して測定した。その結果、C1qtnf3-/-マウスにおけるC3aとC5aのレベルはC1qtnf3+/+マウスにおけるものと同様であることを示した(図3Aおよび3B)。
プレート(Nunc社、デンマーク)を古典的経路(CP)、レクチン経路(LP)および副経路(AP)の補体活性化のアッセイのためにそれぞれOVA/抗OVA免疫複合体(OVA:シグマ社、米国、および抗OVA Ab:ミリポア社、ドイツ)、50μg/mlのマンナン類(シグマ社、米国)、または200μg/mlのLPS(シグマ社、米国)で被覆した(参考文献16、17、18)。オスのマウスから血清を得て、CP活性とLP活性のアッセイのためにはGVB++緩衝液で希釈し、AP活性のためにはGVB/Mg2+EGTA緩衝液で希釈した。希釈したマウス血清(10%)をプレート上に37℃で1時間保温し、冷20mM EDTA/PBSにより反応を停止させた。マウスC3に対するラットモノクローナル抗体(Abcam社、英国)によりC3bの沈着を検出した。その結果、CTRP3欠損はインビトロでは補体活性化に影響しないことを発見した(図3C)。
野生型マウス又はC1qtnf3-/-マウスへの最初のIIC/CFA免疫後42日目における関節局所でのサイトカインの発現を調査した。足首の関節におけるTNF-α、IL-1b、IL-6及びIL-10のメッセンジャーRNA発現を定法に従い半定量的PCRにより測定した。その結果、関節炎を生じた関節局所におけるTNF-α、IL-1b、IL-6及びIL-10の発現量は、野生型マウスよりもC1qtnf3-/-マウスにおいて増加することが示された(図3D)。
CTRP3がヒト単球における炎症性サイトカインの放出を抑制することが報告されている(参考文献12)。しかし、CTRP3は関節炎の関節の滑膜細胞のサイトカイン産生を抑制しなかった。
滑膜表層と関節周囲領域へのT細胞とB細胞の浸潤はRA患者ならびにRAモデルにおいて共通して観察される。そこで、最初のIIC/CFA免疫後42日目の野生型マウス又はC1qtnf3-/-マウスの所属リンパ節から採取したリンパ節細胞の細胞組成をフローサイトメトリー解析で調べた。定法に従い、細胞をパシフィックブルー複合体化モノクローナル抗体、FITC複合体化モノクローナル抗体、およびAPC複合体化モノクローナル抗体(mAb)で染色した(参考文献21)。マウスCD3とB220に対するハムスターmAb(145-2C11)またはラットmAb(RA3-6B2)をバイオレジェンド社(米国)から、CD11cに対するハムスターmAb(HL3)をBDファーミンジェン社(米国)から購入し、標準的な技術に従って細胞を染色し、FACS Canto IIサイトメーターとCellQuestソフトウェア(ベクトンディッキンソン社、米国)かFlowJoソフトウェア(ツリー・スター社、米国)により分析した。
IIC免疫後7日目の野生型マウス又はC1qtnf3-/-マウスの関節炎発症部位の所属リンパ節からLN細胞を回収した。100または200μg/mlの変性IICが存在しない、または存在する状態でLN細胞を72時間培養し、続いて[3H]チミジン(0.25μCi/ml)(アマーシャム社、英国)を6時間取り込ませた。次に、細胞をマイクロ96セル・ハーベスター(スカトロン社、ノルウェー)で回収し、放射活性をマイクロ・ベータ(ファルマシア・バイオテック社、米国)で測定した。72時間後の増殖アッセイの培養上清中のIFN‐γレベルをマウスIFN-ガンマ DuoSet(R&Dシステムズ社、米国)で測定した。
関節リウマチと同様に多発性硬化症も自己免疫疾患の1つであるため、多発性硬化症の誘導モデルである実験的自己免疫性脳脊髄炎(Experimental autoimmune encephalomyelitis;EAE)を誘導したマウスを用いて、多発性硬化症におけるCTRP3の影響を評価した。
完全フロイントアジュバント(CFA)で乳化した、ミエリンオリゴデンドロサイト糖蛋白質(MOG)の一部分であるMOG35-55 ペプチド (MEVGWYRSPFSRVVHLYRNGK) (配列番号12)(Scrun Japan社製)600μgを用いてメスの野生型マウス又はC1qtnf3-/-マウスに免疫した。CFAは不完全フロイントアジュバント(サーモ・サイエンティフィック社、米国)と5mg/mlの加熱殺菌した結核菌(Mycobacterium tuberculosis)(H37Ra;Difco社、米国)から成り、0日目に四肢近くの4か所に皮内注射された。21日目にマウスに同量のエマルジョンを以前の注射部位の近くの皮内にブースター注射した。
これらの結果から、EAE発症においてCTRP3は抑制的に機能していることが示された。
DBA/1Jマウス(メス、6~8週齢、n=6)を、フロインド完全アジュバント(ディフコ社、USA)と共にエマルジョンとした、2mg/mLのニワトリII型コラーゲン(シグマ社、USA)(IIC/CFA)を100μl用いて、0日目に尾の基部近くの3か所に皮内注射して免疫した。21日目に、マウスに同量のIIC/CFAを以前の注射部位の近くの皮内にブースター注射した。28日目からそれぞれのマウスに、左膝関節の関節腔に30μLの組換えヒトCTRP3(300ng/日、Avisceraバイオサイエンス社、USA)(配列番号1)を、右膝関節の関節腔に対照としてPBS、を1日1回注射した。
・関節炎の評価基準(臨床スコア)
0:変化なし。
1:紅斑及び軽微な腫脹が足根関節に認められた。
2:紅斑及び軽微な腫脹が足根関節から足指に広がっていた。
3:紅斑及び中等度の腫脹が中足骨関節から広がっていた。
4:紅斑及び重度の腫脹が足首、足及び足指に広がっていたか、又は、四肢に強直があった。
CTRP3が自己免疫性関節炎の発生を軽減することが明確に示された。CTRP3がRAなどの自己免疫疾患の治療のための医薬として有用であることを示している。
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[15] R. Horai, M. Asano, K. Sudo, H. Kanuka, M. Suzuki, M. Nishihara, M. Takahashi, Y. Iwakura, Production of mice deficient in genes for interleukin (IL)-1alpha, IL-1beta, IL-1alpha/beta, and IL-1 receptor antagonist shows that IL-1beta is crucial in turpentine-induced fever development and glucocorticoid secretion, J Exp Med 187 (1998) 1463-1475.
[16] P.J. Lachmann, Preparing serum for functional complement assays, J Immunol Methods 352 (2010) 195-197.
[17] M.A. Seelen, A. Roos, J. Wieslander, T.E. Mollnes, A.G. Sjoholm, R. Wurzner, M. Loos, F. Tedesco, R.B. Sim, P. Garred, E. Alexopoulos, M.W. Turner, M.R. Daha, Functional analysis of the classical, alternative, and MBL pathways of the complement system: standardization and validation of a simple ELISA, J Immunol Methods 296 (2005) 187-198.
[18] Y. Kimura, T. Miwa, L. Zhou, W.C. Song, Activator-specific requirement of properdin in the initiation and amplification of the alternative pathway complement, Blood 111 (2008) 732-740.
[19] D.E. Trentham, A.S. Townes, A.H. Kang, Autoimmunity to type II collagen an experimental model of arthritis, J Exp Med 146 (1977) 857-868.
[20] J.J. Inglis, E. Simelyte, F.E. McCann, G. Criado, R.O. Williams, Protocol for the induction of arthritis in C57BL/6 mice, Nat Protoc 3 (2008) 612-618.
[21] N. Fujikado, S. Saijo, T. Yonezawa, K. Shimamori, A. Ishii, S. Sugai, H. Kotaki, K. Sudo, M. Nose, Y. Iwakura, Dcir deficiency causes development of autoimmune diseases in mice due to excess expansion of dendritic cells, Nat Med 14 (2008) 176-180.
[22] S. Garcia, J. Forteza, C. Lopez-Otin, J.J. Gomez-Reino, A. Gonzalez, C. Conde, Matrix metalloproteinase-8 deficiency increases joint inflammation and bone erosion in the K/BxN serum-transfer arthritis model, Arthritis Res Ther 12 (2010) R224.
[23] T. Yokohama-Tamaki, T. Maeda, T.S. Tanaka, S. Shibata, Functional analysis of CTRP3/cartducin in Meckel's cartilage and developing condylar cartilage in the fetal mouse mandible, J Anat 218 (2011) 517-533.
[24] K.M. Tong, C.P. Chen, K.C. Huang, D.C. Shieh, H.C. Cheng, C.Y. Tzeng, K.H. Chen, Y.C. Chiu, C.H. Tang, Adiponectin increases MMP-3 expression in human chondrocytes through AdipoR1 signaling pathway, J Cell Biochem 112 (2011) 1431-1440.
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本明細書に記載された全ての文献、特許出願、および技術規格は、個々の文献、特許出願、および技術規格が参照により取り込まれることが具体的か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。
本発明に係る例示的実施形態についての以上の記載は例示および説明の目的でされたものであり、網羅的であることあるいは発明を開示されている形態そのものに限定することを意図するものではない。明らかなことではあるが、多くの改変あるいは変更が当業者には自明である。上記実施形態は発明の原理及び実用的応用を最もうまく説明し、想定される特定の用途に適するような種々の実施形態や種々の改変と共に他の当業者が発明を理解できるようにするために選択され、記載された。本発明に係る範囲の範囲は以下の請求項およびその均等物によって規定されることが意図されている。
Claims (4)
- CTRP3を含有する、自己免疫疾患治療薬。
- 前記自己免疫疾患が関節リウマチ又は多発性硬化症である、請求項1に記載の自己免疫疾患治療薬。
- CTRP3を含有する自己免疫疾患治療薬を、自己免疫疾患の治療を必要とする対象に投与すること、を含む自己免疫疾患を治療する方法。
- 自己免疫疾患が関節リウマチ又は多発性硬化症である、請求項3に記載の自己免疫疾患を治療する方法。
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JP2015547807A JP6551886B2 (ja) | 2013-11-15 | 2014-11-14 | 自己免疫疾患の治療薬及び治療方法 |
US15/036,518 US10383917B2 (en) | 2013-11-15 | 2014-11-14 | Method for the treatment of an autoimmune disease with an agent comprising CTRP3 |
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CN108904782A (zh) * | 2018-07-10 | 2018-11-30 | 中国人民解放军第四军医大学 | Ctrp3用于制备预防治疗心肌肥厚药物的应用 |
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JPWO2019159925A1 (ja) * | 2018-02-14 | 2021-03-04 | 学校法人東京理科大学 | 軟骨細胞増殖促進剤、軟骨細胞増殖促進方法、及び軟骨細胞増殖促進剤のスクリーニング方法 |
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