WO2017061125A1 - Thérapie, diagnostic, et criblage utilisant card14 - Google Patents

Thérapie, diagnostic, et criblage utilisant card14 Download PDF

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WO2017061125A1
WO2017061125A1 PCT/JP2016/004521 JP2016004521W WO2017061125A1 WO 2017061125 A1 WO2017061125 A1 WO 2017061125A1 JP 2016004521 W JP2016004521 W JP 2016004521W WO 2017061125 A1 WO2017061125 A1 WO 2017061125A1
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card14
cells
expression
therapeutic
antibody
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PCT/JP2016/004521
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Japanese (ja)
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石井　健
麻優里 田中
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国立研究開発法人医薬基盤・健康・栄養研究所
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Priority to JP2017544375A priority Critical patent/JP6846808B2/ja
Publication of WO2017061125A1 publication Critical patent/WO2017061125A1/fr

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present invention relates to treatment, diagnosis and screening using CARD14. More specifically, the present invention relates to the use of CARD14 to treat or prevent psoriasis, multiple sclerosis, and ⁇ T cell mediated diseases and / or complications of immune intervention and their screening, Toll-like receptors (TLRs). ) Base / imiquimod (IMQ) / therapeutic or prophylactic agent for reducing side effects caused by immune stimulating adjuvant and screening thereof.
  • IMQ imiquimod
  • Non-Patent Document 1 Schon and Boehncke, 2005.
  • IMQ imiquimod
  • Non-patent document 2 Gilliet et al., 2004; Non-patent document 3: van der Fits et al., 2009.
  • TLR7-deficient mice showed hyperkeratosis of the epidermis comparable to wild type (WT) mice (Non-patent Document 4: Walter). et al., 2013). This suggests that a TLR7-independent mechanism may be important for pathogenesis.
  • Non-Patent Document 5 Johnson-Huang et al., 2012
  • Monoclonal antibodies against IL-23p19 showed significant clinical advantages over blocking the IL-12 / 23p40 subunit
  • Non-Patent Document 5 Johnson-Huang et al., 2012
  • Non-patent Reference 6 Sofen et al., 2014
  • Non-Patent Document 3 van der Fitts et al., 2009.
  • intradermal injection of recombinant IL-23 protein into mouse ears shared many features with psoriasis, such as epidermal hyperkeratosis via IL-22 upregulation and STAT3 activation. 7: Cai et al., 2011; Non-patent literature 8: Zheng et al., 2007).
  • caspase mobilization domain family member 14 also known as CARD14 [MIM60721], CARMA2
  • CARD14 MIM60721
  • CARMA2 psoriasis susceptibility locus 2
  • GWAS genome-wide association analysis
  • Non-patent Document 11 Bertin et al., 2001.
  • This evidence indicates that CARD14 is an important molecule in psoriasis and pore erythema, but the mechanism and correlation of immune responses are not known (Non-patent Document 12: Fuchs-Telem et al. ., 2012; Non-Patent Document 9: Jordan et al., 2012).
  • CARD14 is essential for the formation of psoriasis-like dermatitis, and based on this, it is possible to provide therapeutic agents and diagnostic agents and screen them.
  • the present invention was completed.
  • CARD14 in ⁇ T cells producing IL-17 and IL-22 was found to be essential for the formation of psoriatic-like dermatitis, a role related to psoriasis of CARD14 in hematopoietic cells, and the role of specific cells
  • the present invention was also completed.
  • CARD14 is an essential factor in the development of multiple sclerosis (MS), and concluded that therapeutic agents, diagnostic agents and screening thereof are possible based on this factor.
  • the present invention was completed upon the conclusion that provision of a therapeutic or prophylactic agent for multiple sclerosis using CARD14 and screening thereof were possible.
  • CARD14 in ⁇ T cells can be a potential therapeutic target for ⁇ T cell mediated diseases and complications of immune intervention, and complications of ⁇ T cell mediated diseases and / or immune interventions.
  • the present inventors completed the present invention by finding a conclusion that it is possible to provide a therapeutic or preventive agent and to screen them.
  • the present inventors have found that it is possible to provide therapeutic agents or preventive agents for reducing side effects caused by TLR-based / IMQ / immunostimulatory adjuvants and to screen them, and thus completed the present invention. .
  • CARD14 is a chronic inflammatory skin disease of unknown cause but has also been reported as a complication of imiquimod (IMQ) cream.
  • IMQ imiquimod
  • CARD14 deficient mice using two psoriasis-like models. The first model is induced by IMQ cream via Toll-like receptor (TLR) 7 and TLR9-mediated innate immune activation, and the other model is interleukins without TLR7 and TLR9 activation. Induced by (IL) -23.
  • CARD14 is expressed in and associated with epidermal ⁇ T cells that produce IL-17 and IL-22 in psoriatic skin lesions in these models.
  • Studies using bone marrow chimeric mice and gene-deficient mice reveal that CARD14, which is expressed in hematopoietic cells (especially ⁇ T cells) but not in non-hematopoietic cells, is important for the formation of psoriasis-like dermatitis. became.
  • the present invention provides the following. (1) A screening method for a therapeutic or prophylactic agent for psoriasis based on the expression of CARD14 in hematopoietic cells. (2) A screening method for a therapeutic or prophylactic agent for multiple sclerosis based on the expression of CARD14 in hematopoietic cells. (3) A screening method for a therapeutic or prophylactic agent for reducing side effects caused by TLR-based / IMQ / immunostimulatory adjuvant based on CARD14 expression in hematopoietic cells. (4) A screening method for a therapeutic or prophylactic agent for ⁇ T cell-mediated diseases and / or complications of immune intervention based on the expression of CARD14 in hematopoietic cells.
  • a therapeutic or prophylactic agent for ⁇ T cell-mediated diseases and / or complications of immune intervention comprising a hematopoietic cell-specific CARD14 inhibitor.
  • the therapeutic or prophylactic agent according to any one of items 11 to 14, wherein the hematopoietic cells are epidermal ⁇ T cells.
  • the therapeutic or prophylactic agent according to item 15 or 16, wherein the ⁇ T cell is a cell that produces IL-17 and IL-22.
  • the present invention also provides the following. (1A) a step of contacting a candidate substance with a hematopoietic cell, and a step of determining the expression of CARD14 in the hematopoietic cell, wherein the candidate substance suppresses or eliminates the expression of CARD14.
  • (2A) a step of bringing a candidate substance into contact with a hematopoietic cell, and a step of determining the expression of CARD14 in the hematopoietic cell, wherein the candidate substance suppresses or eliminates the expression of CARD14.
  • (3A) a step of contacting a candidate substance with a hematopoietic cell, and a step of determining the expression of CARD14 in the hematopoietic cell, wherein the candidate substance suppresses or eliminates the expression of CARD14.
  • / IMQ / by TLR-based / IMQ / immunostimulatory adjuvant based on the expression of CARD14 in hematopoietic cells including the step determined to be able to be used as a therapeutic or prophylactic agent to reduce the side effects of immunostimulatory adjuvant
  • a therapeutic agent or preventive agent for psoriasis based on the expression of CARD14 in hematopoietic cells a therapeutic agent or preventive agent for multiple sclerosis, and a therapeutic agent for reducing side effects caused by TLR base / IMQ / immunostimulatory adjuvant
  • prophylactic agents and therapeutic or prophylactic agents for ⁇ T cell-mediated diseases and / or complications of immune intervention are provided, and screening thereof is readily possible.
  • Both TLR7 and TLR9 are required for IMQ-induced psoriatic dermatitis, but not for IL-23-induced psoriatic dermatitis.
  • the bar represents 100 ⁇ m. Data are representative of 4 independent experiments.
  • C Ear thickness was monitored daily prior to IL-23 injection. Data are representative of two independent experiments and results are mean ⁇ s. d. All are Scheffe tests.
  • D H & E staining of skin of phosphate buffered saline (PBS) injected mice and IL-23 injected mice on the last day of the experiment. The bar represents 100 ⁇ m. Data are representative of two independent experiments. Generation of Card14 ⁇ / ⁇ mice. Card14 ⁇ / ⁇ mice were generated by replacing exon 2 and 3 of the Card14 gene with a neomycin (neo) resistance gene.
  • PBS phosphate buffered saline
  • the bar represents 100 ⁇ m. Data are representative of 4 independent experiments. While CARD14 in hematopoietic cells plays an important role in psoriasis, CARD14 in radioresistant skin resident cells is not.
  • (A) Reconstituted bone marrow chimera (WT BM ⁇ WT mouse (WT ⁇ WT), WT BM ⁇ Card14 ⁇ / ⁇ mouse (WT ⁇ KO), Card14 ⁇ / ⁇ BM ⁇ Card14 ⁇ / ⁇ mouse (KO ⁇ KO) And Card14 ⁇ / ⁇ BM ⁇ WT mice (KO ⁇ WT), n 4-6 mice / group) were treated with IMQ cream as described in FIG. Ear thickness was monitored daily.
  • a and B Card14 mRNA levels in some cells compared to GAPDH. Data are representative of 4 independent experiments.
  • C Cryosections from WT mice injected with IL-23 (left) and Card14 ⁇ / ⁇ mice (right) were subjected to immunofluorescent staining for DAPI (blue), CARD14 Ab (green), and GL3 ( Stained with anti-TCR ⁇ ) mAb (red). Merge is a diagram in which these are superimposed. The image was deconvoluted with a Z stack in 0.5 mm steps.
  • OVA ovalbumin
  • the left panel shows the evaluation over time (days) by EAE score, and the right panel shows the incidence. It was shown that CARD14 deficiency had a symptom reducing effect in an experimental demyelinating disease model, indicating that CARD14 plays an important role in multiple sclerosis.
  • CARD14 refers to a protein called Caspase recruitment domain-containing protein 14, CARD-containing MAGUK protein 2 (Carma 2) and a gene encoding the same. BIMP2; CARMA2; PRP; PSORS2; PSS1 may also be displayed.
  • the human amino acid sequence has an accession number of NP_001244899 (human), NP_570956 (mouse), and the mRNA sequence has an accession number of NM_001257970 (Human), NM_130886 (mouse).
  • the amino acid sequence of CARD14 is, for example, SEQ ID NO: 2 (human), 4 (mouse).
  • the nucleotide sequence of CARD14 mRNA is, for example, SEQ ID NO: 1 (human), 3 (mouse).
  • CARD14 has the activity of CARD14, its amino acid sequence is not limited. Therefore, as long as the specific purpose of the present invention is met, not only a protein (or a nucleic acid encoding it) having an amino acid sequence described in a specific SEQ ID NO or accession number, but also a functionally active analog thereof Or a functionally active fragment thereof, or a homologue thereof, or a variant encoded by a nucleic acid that hybridizes under high or low stringency conditions to a nucleic acid encoding this protein. It is understood that it can be used in the present invention.
  • a “derivative”, “analog” or “variant” is preferably a region substantially homologous to a protein of interest (eg, CARD14), although not intended to be limiting.
  • such molecules are at least when compared to sequences aligned over amino acid sequences of the same size or aligned by computer homology programs known in the art. Nucleic acids that are 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identical or that encode such molecules are subject to (highly) stringent conditions It can hybridize to a sequence encoding a component protein under moderately stringent or non-stringent conditions.
  • the representative nucleotide sequence of CARD14 is (A) a polynucleotide having the base sequence set forth in SEQ ID NO: 1 or 3 or a fragment sequence thereof; (B) a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or 4 or a fragment thereof; (C) a variant polypeptide or fragment thereof in which one or more amino acids have one mutation selected from the group consisting of substitution, addition and deletion in the amino acid sequence of SEQ ID NO: 2 or 4, A polynucleotide encoding a variant polypeptide having biological activity; (D) a polynucleotide which is a splice variant or allelic variant of the base sequence set forth in SEQ ID NO: 1 or 3 or a fragment thereof; (E) a polynucleotide encoding a species homologue of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or 4 or a
  • the biological activity typically means that CARD14 can be distinguished from other proteins present in the same organism as the activity or marker.
  • amino acid sequence of CARD14 (A) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 or 4 or a fragment thereof; (B) a polymorphism having one mutation selected from the group consisting of substitution, addition and deletion in the amino acid sequence of SEQ ID NO: 2 or 4, and having biological activity; peptide; (C) a polypeptide encoded by a splice variant or allelic variant of the base sequence set forth in SEQ ID NO: 1 or 3; (D) a polypeptide that is a species homologue of the amino acid sequence set forth in SEQ ID NO: 2 or 4; or (e) at least 70% identity to any one polypeptide of (a)-(d), at least A polypeptide having an amino acid sequence that is 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% and having
  • a substance that binds to CARD14 is a molecule or substance that binds to CARD14 at least temporarily.
  • a binding agent for CARD14 or “a molecule that interacts with CARD14” is a molecule or substance that binds to CARD14 at least temporarily.
  • a therapeutic agent is further bound.
  • substances that bind to CARD14 include antibodies, antisense oligonucleotides, siRNA, low molecular weight molecules (LMW), binding peptides, aptamers, ribozymes, and peptidomimetics.
  • a substance that binds to CARD14 or a CARD14-interacting molecule may be an inhibitor of CARD14, eg a binding protein or binding peptide directed against CARD14, in particular directed against the active site of CARD14, and Also included are nucleic acids directed against the CARD14 gene.
  • Nucleic acid for CARD14 refers to, for example, double-stranded or single-stranded DNA or RNA, or a modification or derivative thereof that inhibits CARD14 gene expression or CARD14 activity, and antisense nucleic acids, aptamers, siRNA (small molecule interference). RNA) and ribozymes.
  • binding protein or “binding peptide” for CARD14 refers to any protein or peptide that binds to CARD14 and is directed against CARD14 (eg, a polyclonal or monoclonal antibody), Including but not limited to antibody fragments and functional equivalents.
  • protein protein
  • polypeptide oligopeptide
  • peptide refers to a polymer of amino acids having an arbitrary length.
  • This polymer may be linear, branched, or cyclic.
  • the amino acid may be natural or non-natural and may be a modified amino acid.
  • the term can also encompass one assembled into a complex of multiple polypeptide chains.
  • the term also encompasses natural or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification (eg, conjugation with a labeling component).
  • amino acid is a general term for organic compounds having an amino group and a carboxyl group.
  • amino acid sequence may be chemically modified. Any amino acid in the amino acid sequence may form a salt or a solvate. Further, any amino acid in the amino acid sequence may be L-type or D-type.
  • the protein according to the embodiment of the present invention includes the above-mentioned “specific amino acid sequence”.
  • specific amino acid sequence examples include N-terminal modification (for example, acetylation, myristoylation, etc.), C-terminal modification (for example, amidation, glycosylphosphatidylinositol addition, etc.), or side chain Modifications (for example, phosphorylation, sugar chain addition, etc.) are known. As long as the object of the present invention is satisfied, it may be natural or non-natural.
  • polynucleotide As used herein, “polynucleotide”, “oligonucleotide”, and “nucleic acid” are used interchangeably herein and refer to a nucleotide polymer of any length. The term also includes “oligonucleotide derivatives” or “polynucleotide derivatives”. “Oligonucleotide derivatives” or “polynucleotide derivatives” refer to oligonucleotides or polynucleotides that include derivatives of nucleotides or that have unusual linkages between nucleotides, and are used interchangeably.
  • oligonucleotides include, for example, 2′-O-methyl-ribonucleotides, oligonucleotide derivatives in which phosphodiester bonds in oligonucleotides are converted to phosphorothioate bonds, and phosphodiester bonds in oligonucleotides.
  • oligonucleotide derivative in which ribose and phosphodiester bond in oligonucleotide are converted to peptide nucleic acid bond uracil in oligonucleotide is C- Oligonucleotide derivatives substituted with 5-propynyluracil, oligonucleotide derivatives where uracil in the oligonucleotide is substituted with C-5 thiazole uracil, and cytosine in the oligonucleotide substituted with C-5 propynylcytosine Nucleotide derivatives, oligonucleotide derivatives in which cytosine in the oligonucleotide is substituted with phenoxazine-modified cytosine, oligonucleotide derivatives in which ribose in DNA is substituted with 2'-O-propylribos
  • a particular nucleic acid sequence may also be conservatively modified (eg, degenerate codon substitutes) and complementary sequences, as well as those explicitly indicated. Is contemplated. Specifically, a degenerate codon substitute creates a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue. (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell .Probes 8: 91-98 (1994)).
  • nucleic acid is also used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
  • nucleotide may be natural or non-natural.
  • gene refers to a factor that defines a genetic trait
  • gene may refer to “polynucleotide”, “oligonucleotide”, and “nucleic acid”.
  • homology of a gene refers to the degree of identity of two or more gene sequences to each other, and generally “having homology” means that the degree of identity or similarity is high. Say. Therefore, the higher the homology between two genes, the higher the sequence identity or similarity. Whether two genes have homology can be determined by direct sequence comparison or, in the case of nucleic acids, hybridization methods under stringent conditions. When directly comparing two gene sequences, the DNA sequence between the gene sequences is typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% , 95%, 96%, 97%, 98% or 99% are identical, the genes have homology.
  • a “homolog” or “homologous gene product” is a protein in another species, preferably a mammal, that performs the same biological function as the protein component of the complex further described herein. Means. Such homologues may also be referred to as “ortholog gene products”. It will be understood that such homologues, homologous gene products, orthologous gene products and the like can be used as long as they meet the objectives of the present invention.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides can also be referred to by the generally recognized one letter code.
  • comparison of similarity, identity and homology between amino acid sequences and base sequences is calculated using default parameters using BLAST, which is a sequence analysis tool.
  • the identity search can be performed, for example, using NCBI's BLAST 2.2.28 (issued 2013.4.2).
  • the identity value usually refers to a value when the above BLAST is used and aligned under default conditions. However, if a higher value is obtained by changing the parameter, the highest value is set as the identity value. When identity is evaluated in a plurality of areas, the highest value among them is set as the identity value. Similarity is a numerical value calculated for similar amino acids in addition to identity.
  • “several” may be, for example, 10, 8, 6, 5, 4, 3, or 2, and may be any value or less. It is known that polypeptides that have been deleted, added, inserted, or replaced by other amino acids at one or several amino acid residues maintain their biological activity (Mark et al., Proc Natl Acad Sci US A.1984 Sep; 81 (18): 5662-5666., Zoller et al., Nucleic Acids Res. 1982 Oct 25; 10 (20): 6487-6500., Wang et al., Science. 1984 Jun 29; 224 (4656): 1431-1433.).
  • Antibodies with deletions and the like can be prepared by, for example, site-specific mutagenesis, random mutagenesis, or biopanning using an antibody phage library.
  • site-specific mutagenesis method for example, KOD-Plus-Mutagenesis Kit (TOYOBO CO., LTD.) Can be used. It is possible to select an antibody having the same activity as that of the wild type from mutant antibodies into which deletion or the like has been introduced by performing various characterizations such as FACS analysis and ELISA.
  • “90% or more” may be, for example, 90, 95, 96, 97, 98, 99, or 100% or more, and is within the range of any two values thereof. Also good.
  • the above-mentioned “homology” may be calculated according to a method known in the art, based on the ratio of the number of amino acids homologous in two or more amino acid sequences. Before calculating the ratio, the amino acid sequences of the group of amino acid sequences to be compared are aligned, and a gap is introduced into a part of the amino acid sequence when necessary to maximize the ratio of the same amino acids.
  • polynucleotide hybridizing under stringent conditions refers to well-known conditions commonly used in the art.
  • a polynucleotide can be obtained by using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method or the like using a polynucleotide selected from among the polynucleotides of the present invention as a probe.
  • hybridization was performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which colony or plaque-derived DNA was immobilized, and then a 0.1 to 2-fold concentration was obtained.
  • SSC saline-sodium citrate
  • composition of 1-fold concentration of SSC solution is 150 mM sodium chloride, 15 mM sodium citrate
  • stringent conditions for example, the following conditions can be adopted.
  • Use low ionic strength and high temperature for washing eg, 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate (SDS) at 50 ° C.
  • high Use denaturing agents such as formamide during hybridization (eg, at 42 ° C., 50% (v / v) formamide and 0.1% bovine serum albumin / 0.1% ficoll / 0.1% polyvinylpyrrolidone / 50 mM pH 6.5 phosphate Sodium buffer, and 750 mM sodium chloride, 75 mM sodium citrate), or (3) 20% formamide, 5 ⁇ SSC, 50 mM sodium phosphate (pH 7.6), 5 ⁇ Denhardt's solution, 10% dextran sulfate, and 20 mg / Incubate overnight at 37 ° C.
  • a polypeptide used in the present invention is encoded by a nucleic acid molecule that hybridizes under highly or moderately stringent conditions to a nucleic acid molecule encoding a polypeptide specifically described in the present invention. are also included.
  • a “purified” substance or biological factor refers to a substance from which at least a part of the factor naturally associated with the substance or biological factor has been removed. .
  • the purity of a biological agent in a purified biological agent is higher (ie, enriched) than the state in which the biological agent is normally present.
  • the term “purified” as used herein is preferably at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight, Means the presence of the same type of biological agent.
  • the substance or biological agent used in the present invention is preferably a “purified” substance.
  • an “isolated” substance or biological agent is substantially free of the factors that naturally accompany the substance or biological agent. Say things.
  • the term “isolated” as used herein does not necessarily have to be expressed in purity, as it will vary depending on its purpose, but is preferably at least 75% by weight, more preferably if necessary. Means that there is at least 85%, more preferably at least 95%, and most preferably at least 98% by weight of the same type of biological agent.
  • the materials used in the present invention are preferably “isolated” materials or biological agents.
  • a “corresponding” amino acid or nucleic acid or moiety refers to a predetermined amino acid or nucleotide or moiety in a polypeptide or polynucleotide to be compared in a polypeptide molecule or polynucleotide molecule (eg, CARD14).
  • Amino acids or nucleotides that have or are expected to have a similar action especially in the case of enzyme molecules, amino acids that are present at similar positions in the active site and that contribute similarly to catalytic activity, In the case of a complex molecule, it refers to a corresponding part (for example, a transmembrane domain).
  • an antisense molecule can be a similar part in an ortholog corresponding to a particular part of the antisense molecule.
  • Corresponding amino acids for example, cysteinylation, glutathioneation, SS bond formation, oxidation (eg methionine side chain oxidation), formylation, acetylation, phosphorylation, glycosylation, myristylation, etc.
  • the corresponding amino acid can be an amino acid responsible for dimerization.
  • Such “corresponding” amino acids or nucleic acids may be regions or domains spanning a range. Thus, in such cases, it is referred to herein as a “corresponding” region or domain. Such corresponding regions or domains are useful when designing complex molecules in the present invention.
  • a “corresponding” gene eg, a polynucleotide sequence or molecule
  • a “corresponding” gene has, or is expected to have, in a species, the same effect as a given gene in a species to which comparison is made. It refers to a gene (for example, a polynucleotide sequence or a molecule), and when there are a plurality of genes having such an action, those having the same origin in evolution.
  • a gene corresponding to a gene can be an ortholog of that gene.
  • each human CARD14 can find the corresponding CARD14 in other animals (especially mammals).
  • Such corresponding genes can be identified using techniques well known in the art.
  • a corresponding gene in an animal is a query sequence that is a sequence of a gene serving as a reference for the corresponding gene (for example, CARD14 or the like (SEQ ID NO: 1, 3 or SEQ ID NO: 2, 4 or the like)). And can be found by searching a database containing the animal's sequence.
  • fragment refers to a polypeptide or polynucleotide having a sequence length of 1 to n ⁇ 1 with respect to a full-length polypeptide or polynucleotide (length is n).
  • the length of the fragment can be appropriately changed according to the purpose.
  • the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, Examples include 15, 20, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg, 11 etc.) are also suitable as lower limits obtain.
  • examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides.
  • Non-integer lengths may also be appropriate as a lower limit.
  • the full-length fragment functions as a marker, therapeutic agent or target molecule, as long as the fragment itself also functions as a marker, therapeutic agent or target molecule, the present invention It is understood that it falls within the scope of
  • the term “activity” refers herein to the function of a molecule in the broadest sense. Activity is not intended to be limiting, but generally includes the biological function, biochemical function, physical function or chemical function of a molecule. Activity activates, promotes, stabilizes, inhibits, suppresses or destabilizes, for example, enzyme activity, the ability to interact with other molecules, and the function of other molecules Ability, stability, and ability to localize to specific intracellular locations. Where applicable, the term also relates to the function of the protein complex in the broadest sense.
  • biological function refers to a specific function that a gene, nucleic acid molecule or polypeptide may have in vivo when referring to a gene or a nucleic acid molecule or polypeptide related thereto.
  • the antibody include, but are not limited to, generation of specific antibodies, enzyme activity, and imparting resistance.
  • CARD14 belongs to the membrane-bound guanylate kinase (MAGUK) family and functions as a molecular scaffold that forms an assembly of multiprotein complexes specialized in the plasma membrane region.
  • MAGUK membrane-bound guanylate kinase
  • biological activity refers to activity that a certain factor (eg, polynucleotide, protein, etc.) may have in vivo, and exhibits various functions (eg, transcription promoting activity). For example, an activity in which another molecule is activated or inactivated by interaction with one molecule.
  • a certain factor eg, polynucleotide, protein, etc.
  • the biological activity can be the binding between the two molecules and the resulting biological change, and for example, one molecule was precipitated using an antibody
  • the two molecules are considered to be linked. Therefore, seeing such coprecipitation is one of the judgment methods.
  • a factor is an enzyme
  • the biological activity includes the enzyme activity.
  • an agent is a ligand
  • the ligand includes binding to the corresponding receptor.
  • Such biological activity can be measured by techniques well known in the art.
  • “activity” indicates or reveals binding (either direct or indirect); affects the response (ie, has a measurable effect in response to some exposure or stimulus), refers to various measurable indicators, such as the affinity of a compound that directly binds to a polypeptide or polynucleotide of the invention, or the amount of protein upstream or downstream after some stimulation or event or other A measure of similar function.
  • the “value” of CARD14 may be any value directly or indirectly related to CARD14, such as the amount of CARD14 protein, the expression level of mRNA, and the enzyme activity. This “value” is used in the present invention as an indicator of reducing side effects from psoriasis, multiple sclerosis, ⁇ T cell mediated diseases and / or immune intervention complications, and / or TLR-based / IMQ / immunostimulatory adjuvants be able to.
  • expression of a gene, polynucleotide, polypeptide or the like means that the gene or the like undergoes a certain action in vivo to take another form.
  • a gene, a polynucleotide or the like is transcribed and translated into a polypeptide form.
  • transcription and production of mRNA are also an aspect of expression. Therefore, “expression product” as used herein includes such a polypeptide or protein, or mRNA. More preferably, such polypeptide forms may be post-translationally processed.
  • the expression level of CARD14 can be determined by any method.
  • the level of CARD14 expression can be determined by evaluating the amount of CARD14 mRNA, the amount of CARD14 protein, and the biological activity of CARD14 protein. Such measurements can be used in companion diagnostics.
  • the amount of CARD14 mRNA or protein can be determined by the methods detailed elsewhere in this specification or other methods known in the art.
  • the “functional equivalent” refers to any object having the same target function but different structure from the target original entity. Accordingly, the functional equivalent of “CARD14” or its antibody is not CARD14 or its antibody itself, but CARD14 or its variant or variant (for example, amino acid sequence variant, etc.), which CARD14 has. Those that have biological effects, as well as those that can change to CARD14 or its antibody itself or a variant or variant of this CARD14 or its antibody at the time of action (eg, CARD14 or its antibody itself or CARD14 or It is understood that nucleic acids encoding the antibody variants or variants, and vectors, cells, etc. containing the nucleic acids) are encompassed.
  • a functional equivalent of CARD14 or an antibody thereof can be used similarly to CARD14 or an antibody thereof, even if not specifically mentioned.
  • Functional equivalents can be found by searching a database or the like.
  • search refers to finding another nucleobase sequence having a specific function and / or property using a nucleobase sequence electronically or biologically or by other methods.
  • Electronic search includes BLAST (Altschul et al., J.Mol.Biol.
  • Bio searches include stringent hybridization, macroarrays with genomic DNA affixed to nylon membranes, microarrays affixed to glass plates (microarray assays), PCR, and in situ hybridization. It is not limited to. In the present specification, it is intended that the gene used in the present invention should include a corresponding gene identified by such an electronic search or biological search.
  • an amino acid sequence having one or more amino acid insertions, substitutions or deletions, or those added to one or both ends can be used.
  • “insertion, substitution or deletion of one or a plurality of amino acids in the amino acid sequence, or addition to one or both ends thereof” is a well-known technical method such as site-directed mutagenesis. It means that a modification has been made by substitution of a plurality of amino acids to the extent that can occur naturally by a method or by natural mutation.
  • the modified amino acid sequence has, for example, 1 to 30, preferably 1 to 20, more preferably 1 to 9, further preferably 1 to 5, particularly preferably 1 to 2 amino acid insertions, substitutions or deletions. Lost or added to one or both ends thereof.
  • the modified amino acid sequence is preferably an amino acid sequence having one or more (preferably one or several, or 1, 2, 3, or 4) conservative substitutions in the amino acid sequence of CARD14. May be.
  • conservative substitution means substitution of one or more amino acid residues with another chemically similar amino acid residue so as not to substantially alter the function of the protein. For example, when a certain hydrophobic residue is substituted by another hydrophobic residue, a certain polar residue is substituted by another polar residue having the same charge, and the like. Functionally similar amino acids that can make such substitutions are known in the art for each amino acid.
  • non-polar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine.
  • polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine.
  • positively charged (basic) amino acids include arginine, histidine, and lysine.
  • negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • an “inhibitor” or “inhibitor” or “inhibitor” refers to a target entity (eg, receptor or cell) of the receptor or cell.
  • a substance or factor that inhibits a biological action eg, receptor or cell
  • the inhibitor of CARD14 of the present invention include a factor that can temporarily or permanently reduce or eliminate the action or function of CARD14 or CARD14-expressing cells of interest.
  • factors include, but are not limited to, antibodies, antigen-binding fragments thereof, derivatives thereof, functional equivalents, nucleic acids in the form of RNAi factors such as antisense and siRNA, and the like.
  • the term “agonist” refers to a substance that expresses or enhances the biological action of a target entity (for example, a receptor).
  • a target entity for example, a receptor
  • synthetic antagonists and modified ones can be mentioned.
  • an antagonist can be included in the concept of an inhibitor (suppressor or inhibitor) or an inhibitory or suppressive factor because it suppresses or inhibits a physiological phenomenon. Accordingly, as used herein, an antagonist is used interchangeably with “inhibitor”.
  • antibody broadly refers to polyclonal antibodies, monoclonal antibodies, multispecific antibodies, chimeric antibodies, and anti-idiotype antibodies, and fragments thereof such as Fv fragments, Fab ′ fragments, F (ab ′) 2 and Fab fragments, and other recombinantly produced conjugates or functional equivalents (eg, chimeric antibodies, humanized antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single chains Antibody, scFV, diabody, sc (Fv) 2 (single chain (Fv) 2), scFv-Fc).
  • antibodies may be covalently linked or recombinantly fused to enzymes such as alkaline phosphatase, horseradish peroxidase, alpha galactosidase, and the like.
  • the anti-CARD14 antibody used in the present invention may be bound to the CARD14 protein, and its origin, type, shape, etc. are not limited. Specifically, known antibodies such as non-human animal antibodies (eg, mouse antibodies, rat antibodies, camel antibodies), human antibodies, chimeric antibodies, and humanized antibodies can be used. In the present invention, monoclonal or polyclonal antibodies can be used as antibodies, but monoclonal antibodies are preferred.
  • the binding of the antibody to the CARD14 protein is preferably specific binding.
  • the antibody includes an antibody modified product or an antibody unmodified product. In the modified antibody, an antibody and various molecules such as polyethylene glycol may be bound.
  • the modified antibody can be obtained by chemically modifying the antibody using a known technique.
  • a “polyclonal antibody” refers to, for example, mammals (eg, rats, mice, rabbits, cows, monkeys, etc.), birds, etc. in order to induce the production of polyclonal antibodies specific to the antigen. It can be generated by administering an immunogen containing the antigen of interest. Administration of the immunogen may involve infusion of one or more immunizing agents and, if desired, an adjuvant.
  • An adjuvant may be used to increase the immune response and may include Freund's adjuvant (complete or incomplete), mineral gel (such as aluminum hydroxide), or a surfactant (such as lysolecithin). .
  • Immunization protocols are known in the art and may be performed by any method that elicits an immune response, depending on the host organism chosen (Protein Experiment Handbook, Yodosha (2003): 86-91). .).
  • a “monoclonal antibody” is an antibody in which the individual antibodies constituting the population substantially correspond to a single epitope, except for antibodies having mutations that can naturally occur in small amounts. Including the case of Alternatively, the individual antibodies that make up the population may be antibodies that are substantially identical except for antibodies that have mutations that can occur naturally in small amounts. Monoclonal antibodies are highly specific and differ from normal polyclonal antibodies, which typically include different antibodies corresponding to different epitopes. In addition to its specificity, monoclonal antibodies are useful in that they can be synthesized from hybridoma cultures that are not contaminated by other immunoglobulins.
  • the form “monoclonal” may be characterized as being derived from a substantially homogeneous population of antibodies, but does not mean that the antibodies must be produced in any particular way.
  • the monoclonal antibody may be prepared by a method similar to the hybridoma method described in "Kohler G, Milstein C., Nature. 1975 Aug 7; 256 (5517): 495-497.”
  • monoclonal antibodies may be made by methods similar to recombinant methods such as those described in US Pat. No. 4,816,567.
  • the monoclonal antibody is “Clackson et al., Nature. 1991 Aug 15; 352 (6336): 624-628.” Or “Marks et al., J Mol Biol.
  • a “humanized antibody” has, for example, one or more CDRs derived from a non-human species, a framework region (FR) derived from a human immunoglobulin, and a constant region derived from a human immunoglobulin. And an antibody that binds to the desired antigen.
  • Antibody humanization can be performed using various techniques known in the art (Almagro et al., FRont Biosci. 2008 Jan 1: 13: 1619-1633.). For example, CDR grafting (Ozaki et al., Blood. 1999 Jun 1:93 (11): 3922-3930.), Re-surfacing (roguska et al., Proc Natl Acad Sci U S A.
  • FR shuffle (Damschroder et al., Mol Immunol. 2007 Apr; 44 (11): 3049-3060. Epub 2007 Jan 22.).
  • amino acid residues in the human FR region may be substituted with corresponding residues from the CDR donor antibody. This FR substitution can be performed by methods well known in the art (Riechmann et al., Nature. 1988 Mar 24; 332 (6162): 323-327.).
  • FR residues important for antigen binding may be identified by modeling the interaction of CDR and FR residues.
  • an unusual FR residue at a particular position may be identified by sequence comparison.
  • a “human antibody” is derived from a gene encoding human immunoglobulin, for example, the variable region and the constant region of the heavy chain and the region including the variable region and the constant region of the light chain that constitute the antibody.
  • the main production methods include a transgenic mouse method for producing human antibodies, a phage display method, and the like.
  • human antibodies having various antigen-binding abilities can be produced instead of mouse antibodies.
  • a human monoclonal antibody can be obtained by a conventional hybridoma method.
  • phage display method a foreign gene is fused to the N-terminal side of the coat protein (g3p, g10p, etc.) of filamentous phages such as M13 and T7, which are typically E. coli viruses. It is a system for expressing as a protein. For example, it can be produced by the method described in “Vaughan et al., Nat Biotechnol. 1996 Mar; 14 (3): 309-314.”
  • the “anti-CARD14 antibody” includes an antibody having binding ability to CARD14.
  • the method for producing this anti-CARD14 antibody is not particularly limited.
  • the anti-CARD14 antibody may be produced by immunizing mammals or birds with CARD14.
  • an antibody against CARD14 (anti-CARD14 antibody) or a fragment thereof” is, for example, in the case of an antibody, an antibody itself having a CARD14 binding activity, and if necessary, an inhibitory activity, and a fragment thereof.
  • the anti-CARD14 antibody according to one embodiment of the present invention is preferably an anti-CARD14 antibody that specifically binds to a specific epitope of CARD14 from the viewpoint of particularly strongly suppressing the disease.
  • the anti-CARD14 antibody according to one embodiment of the present invention may be a monoclonal antibody. If it is a monoclonal antibody, it can be made to act with respect to CARD14 efficiently compared with a polyclonal antibody. From the viewpoint of efficiently producing an anti-CARD14 monoclonal antibody, it is preferable to immunize CARD14 to chickens.
  • the antibody class of the anti-CARD14 antibody according to an embodiment of the present invention is not particularly limited, and may be, for example, IgM, IgD, IgG, IgA, IgE, or IgY.
  • the anti-CARD14 antibody according to one embodiment of the present invention may be an antibody fragment having an antigen binding activity (hereinafter also referred to as “antigen-binding fragment”).
  • antigen-binding fragment an antibody fragment having an antigen binding activity
  • there are effects such as an increase in stability or antibody production efficiency.
  • the anti-CARD14 antibody according to one embodiment of the present invention may be a fusion protein.
  • This fusion protein may be a polypeptide or oligopeptide bound to the N- or C-terminus of the anti-CARD14 antibody.
  • the oligopeptide may be a His tag.
  • the fusion protein may be a fusion of a mouse, human or chicken antibody partial sequence. Such fusion proteins are also included in one form of the anti-CARD14 antibody according to this embodiment.
  • the anti-CARD14 antibody according to an embodiment of the present invention may be, for example, an antibody obtained through a step of immunizing an organism with purified CARD14, CARD14-expressing cells, or a CARD14-containing lipid membrane. From the viewpoint of enhancing the therapeutic effect on CARD14-positive diseases, it is preferable to use CARD14-expressing cells for immunization.
  • the anti-CARD14 antibody according to an embodiment of the present invention may be an antibody having a CDR set of antibodies obtained through a step of immunizing an organism with purified CARD14, CARD14-expressing cell vesicles, or a CARD14-containing lipid membrane. From the viewpoint of enhancing the therapeutic effect on CARD14-positive diseases, it is preferable to use CARD14-expressing cells for immunization.
  • a CDR set is a set of heavy chain CDRs 1, 2, and 3 and light chain CDRs 1, 2, and 3.
  • the anti-CARD14 antibody according to an embodiment of the present invention may have any binding force as long as the object is achieved, for example, at least 1.0 ⁇ 10 6 or more, 2.0 ⁇ 10 6 or more. , 5.0 ⁇ 10 6 or more, 1.0 ⁇ 10 7 or more, but is not limited thereto. Usually, even when the dissociation constant (KD) value is 1.0 ⁇ 10 7 or more. Good.
  • KD dissociation constant
  • the anti-CARD14 antibody may be an antibody that binds to a wild type or mutant type of CARD14. Variants include those resulting from differences in DNA sequences between individuals.
  • the amino acid sequence of wild-type or mutant CARD14 is preferably 80% or more, more preferably 90% or more, more preferably 95% or more, particularly preferably 98% or more with respect to the amino acid sequence shown in SEQ ID NO: 2 or 4. Have homology.
  • CDR complementarity determining region
  • Fv variable region: including heavy chain variable region (VH) and light chain variable region (VL)
  • CDR1, CDR2, and CDR3 consisting of about 5 to 30 amino acid residues.
  • CDR3 is known to have the highest contribution in binding of an antibody to an antigen.
  • Kabat definition Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)
  • Chothia definition Chothia et al., J. Mol. , 1987; 196: 901-9157
  • the definition of Kabat is adopted as a preferred example, but is not necessarily limited thereto.
  • the overlapping part of the CDR according to each definition or the part including both the CDRs according to each definition It can also be a CDR.
  • antigen refers to any substrate that can be specifically bound by an antibody molecule.
  • immunogen refers to an antigen capable of initiating lymphocyte activation that produces an antigen-specific immune response.
  • epitope or “antigenic determinant” refers to a site in an antigen molecule to which an antibody or lymphocyte receptor binds. Methods for determining epitopes are well known in the art, and such epitopes can be determined by those skilled in the art using such well known techniques once the primary sequence of the nucleic acid or amino acid is provided. It will be understood that the antibodies of the present invention can be used in the same manner even if they have the same epitope, even antibodies having other sequences.
  • the antibody used in the present specification may be any specific antibody as long as undesirable effects such as side reactions are reduced. Therefore, the antibody used in the present invention may be a polyclonal antibody or a monoclonal antibody.
  • the “subject (person)” refers to a subject to be diagnosed or detected or treated according to the present invention (for example, an organism such as a human or a cell, blood, serum, etc. removed from the organism). .
  • drug drug
  • drug may also be a substance or other element (eg energy such as light, radioactivity, heat, electricity).
  • Such substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, DNA such as cDNA, genomic DNA, RNA such as mRNA), poly Saccharides, oligosaccharides, lipids, small organic molecules (for example, hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (for example, small molecule ligands, etc.)) , These complex molecules are included, but not limited thereto.
  • a factor specific for a polynucleotide is a polynucleotide having complementarity with a certain sequence homology to the sequence of the polynucleotide (eg, 70% or more sequence identity), Examples include, but are not limited to, a polypeptide such as a transcription factor that binds to the promoter region.
  • Factors specific for a polypeptide typically include an antibody specifically directed against the polypeptide or a derivative or analog thereof (eg, a single chain antibody), and the polypeptide is a receptor.
  • specific ligands or receptors in the case of ligands, and substrates thereof when the polypeptide is an enzyme include, but are not limited to.
  • treatment refers to prevention of worsening of a disease or disorder when it becomes such a condition or disease (eg, allergy), preferably maintenance of the current state, more preferably Means to reduce, more preferably to eliminate, and includes the ability to exert a symptom-improving effect or a preventive effect on one or more symptoms associated with a patient's disease or disease. Diagnosing in advance and performing appropriate treatment is referred to as “companion treatment”, and the diagnostic agent therefor is sometimes referred to as “companion diagnostic agent”.
  • therapeutic agent refers to any agent that can treat a target condition (for example, a disease such as allergy) in a broad sense.
  • the “therapeutic agent” may be a pharmaceutical composition comprising an active ingredient and one or more pharmacologically acceptable carriers.
  • the pharmaceutical composition can be produced by any method known in the technical field of pharmaceutics, for example, by mixing the active ingredient and the carrier.
  • the form of use of the therapeutic agent is not limited as long as it is a substance used for treatment, and it may be an active ingredient alone or a mixture of an active ingredient and an arbitrary ingredient.
  • the shape of the carrier is not particularly limited, and may be, for example, a solid or a liquid (for example, a buffer solution).
  • the allergy therapeutic agent includes a drug (preventive agent) used for preventing allergy, or an allergy suppressor.
  • prevention means that a certain disease or disorder (for example, allergy) is prevented from becoming such a state before it becomes such a state. Diagnosis can be performed using the drug of the present invention, and for example, allergies can be prevented using the drug of the present invention, or countermeasures for prevention can be taken as necessary.
  • a certain disease or disorder for example, allergy
  • prophylactic agent refers to any agent that can prevent a target condition (for example, a disease such as allergy) in a broad sense.
  • a “factor” (or drug, detection agent, etc.) that interacts (or binds) “specifically” to a biological agent such as a polynucleotide or a polypeptide is defined as that
  • the affinity for a biological agent such as a nucleotide or polypeptide thereof is typically equal or greater than the affinity for other unrelated (especially less than 30% identity) polynucleotides or polypeptides. Includes those that are high or preferably significantly (eg, statistically significant). Such affinity can be measured, for example, by hybridization assays, binding assays, and the like.
  • a first substance or factor interacts (or binds) “specifically” to a second substance or factor means that the first substance or factor has a relationship to the second substance or factor. Interact (or bind) with a higher affinity than a substance or factor other than the second substance or factor (especially other substances or factors present in the sample containing the second substance or factor) That means.
  • Specific interactions (or bindings) for substances or factors include, for example, hybridization in nucleic acids, antigen-antibody reactions in proteins, enzyme-substrate reactions, etc., nucleic acid and protein reactions, protein-lipid interactions, nucleic acid-lipids Examples include, but are not limited to, interactions.
  • the first substance or factor “specifically interacts” with the second substance or factor means that the first substance or factor has the second substance Or having at least a part of complementarity to the factor.
  • both substances or factors are proteins
  • the fact that the first substance or factor interacts (or binds) “specifically” to the second substance or factor is, for example, by antigen-antibody reaction Examples include, but are not limited to, interaction by receptor-ligand reaction, enzyme-substrate interaction, and the like.
  • the first substance or factor interacts (or binds) “specifically” to the second substance or factor by means of an antibody and its antigen Interaction (or binding) between is included.
  • an object in a sample can be detected or quantified.
  • detection or “quantification” of polynucleotide or polypeptide expression includes mRNA measurement and immunoassay methods, including, for example, binding or interaction with a detection agent, test agent or diagnostic agent. It can be achieved using any suitable method.
  • molecular biological measurement methods include Northern blotting, dot blotting, and PCR.
  • immunological measurement methods include ELISA using a microtiter plate, radioimmunoassay (RIA) method, fluorescent antibody method, luminescence immunoassay (LIA) method, immunoprecipitation method (IP), and immunodiffusion method. (SRID), immunoturbidimetry (TIA), Western blotting, immunohistochemical staining, and the like.
  • Examples of the quantification method include ELISA method and RIA method. It can also be performed by a gene analysis method using an array (eg, DNA array, protein array).
  • the DNA array has been extensively outlined in (edited by Shujunsha, separate volume of cell engineering "DNA microarray and the latest PCR method”).
  • Examples of gene expression analysis methods include, but are not limited to, RT-PCR, RACE method, SSCP method, immunoprecipitation method, two-hybrid system, in vitro translation and the like.
  • expression level refers to the amount of polypeptide or mRNA expressed in a target cell, tissue or the like. Such expression level is evaluated by any appropriate method including immunoassay methods such as ELISA, RIA, fluorescent antibody, Western blot, and immunohistochemical staining using the antibody of the present invention.
  • the expression level of the peptide at the mRNA level can be mentioned.
  • “Change in expression level” means expression at the protein level or mRNA level of the polypeptide used in the present invention evaluated by any appropriate method including the above immunological measurement method or molecular biological measurement method. Means the amount increases or decreases. By measuring the expression level of a certain marker, various detection or diagnosis based on the marker can be performed.
  • “reduction” or “suppression” or synonyms for activity, expression products (eg, proteins, transcripts (RNA, etc.)) or synonyms are reductions in the quantity, quality or effect of a particular activity, transcript or protein. Or activity to decrease. When “disappears” of the decrease, it means that the activity, the expression product, etc. are below the detection limit, and in particular, it is sometimes referred to as “disappearance”. As used herein, “disappearance” is encompassed by “decrease” or “suppression”.
  • “increase” or “activation” of an activity, expression product eg, protein, transcript (RNA, etc.) or a synonym thereof refers to a quantity, quality or effect of a particular activity, transcript or protein. An activity that increases or increases.
  • the “label” refers to a presence (for example, a substance, energy, electromagnetic wave, etc.) for distinguishing a target molecule or substance from others.
  • a labeling method include RI (radioisotope) method, fluorescence method, biotin method, chemiluminescence method and the like.
  • the labeling is performed with fluorescent substances having different fluorescence emission maximum wavelengths. The difference in the maximum fluorescence emission wavelength is preferably 10 nm or more.
  • Alexa TM Fluor is desirable as a fluorescent substance.
  • Alexa TM Fluor is a water-soluble fluorescent dye obtained by modifying coumarin, rhodamine, fluorescein, cyanine, etc., and is a series corresponding to a wide range of fluorescent wavelengths. It is stable, bright and has low pH sensitivity.
  • Examples of combinations of fluorescent dyes having a fluorescence maximum wavelength of 10 nm or more include a combination of Alexa TM 555 and Alexa TM 633, a combination of Alexa TM 488 and Alexa TM 555, and the like.
  • any nucleic acid can be used as long as it can bind to the base moiety, but cyanine dyes (eg, CyDyeTM series Cy3, Cy5, etc.), rhodamine 6G reagent, 2-acetylaminofluorene (AAF) ), AAIF (iodine derivative of AAF) or the like is preferably used.
  • cyanine dyes eg, CyDyeTM series Cy3, Cy5, etc.
  • AAF 2-acetylaminofluorene
  • AAIF iodine derivative of AAF
  • the fluorescent substance having a difference in maximum fluorescence emission wavelength of 10 nm or more include a combination of Cy5 and rhodamine 6G reagent, a combination of Cy3 and fluorescein, a combination of rhodamine 6G reagent and fluorescein, and the like.
  • the target object by using such a label, the target object can be modified so that it can be detected by the detection means
  • the “kit” is a unit provided with a portion to be provided (eg, a test agent, a diagnostic agent, a therapeutic agent, an antibody, a label, instructions, etc.) usually divided into two or more compartments.
  • a portion to be provided eg, a test agent, a diagnostic agent, a therapeutic agent, an antibody, a label, instructions, etc.
  • This kit form is preferred when it is intended to provide a composition that should not be provided in admixture for stability or the like, but preferably used in admixture immediately before use.
  • Such kits preferably include instructions or instructions that describe how to use the provided parts (eg, test agents, diagnostic agents, therapeutic agents, or how the reagents should be processed).
  • the kit when the kit is used as a reagent kit, the kit usually contains instructions including usage of test agents, diagnostic agents, therapeutic agents, antibodies, etc. Is included.
  • the “instruction sheet” describes the method for using the present invention for a doctor or other user.
  • This instruction manual includes a word indicating that the detection method of the present invention, how to use a diagnostic agent, or administration of a medicine or the like is given.
  • the instructions may include a word indicating that the administration site is oral or esophageal administration (for example, by injection).
  • This instruction is prepared in accordance with the format prescribed by the national supervisory authority (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States, etc.) It is clearly stated that it has been received.
  • the instruction sheet is a so-called package insert and is usually provided in a paper medium, but is not limited thereto, and is in a form such as an electronic medium (for example, a homepage or an e-mail provided on the Internet). But it can be provided.
  • CARD14 inhibitor A CARD14 inhibitor that can be used in the present invention refers to any substance that inhibits the action of CARD14, and its mechanism of action includes inhibiting the expression of CARD14, inhibiting the function, and the like. Therefore, “CARD14 inhibitor” refers to a substance that inhibits the expression of CARD14, a substance that inhibits the function of CARD14, and any substance that ultimately inhibits the action of CARD14 even if the mechanism of action is unknown. Including, but not limited to, antibodies, antibody fragments, derivatives thereof, nucleic acids, small molecules, other antagonists, and the like. Such various antibodies and the like are described elsewhere in this specification. Preferably, such a CARD14 inhibitor is advantageously one that specifically inhibits CARD14. This is because side effects due to influence on factors other than CARD14 can be reduced.
  • the “substance inhibiting CARD14 expression” means any level of transcription level of nucleic acid encoding CARD14 (CARD14 gene), post-transcriptional regulation level, translation level into CARD14 protein, post-translational modification level, etc. It may be one that works.
  • a substance that inhibits the expression of CARD14 for example, a substance that inhibits transcription of the CARD14 gene (eg, antigene), a substance that inhibits processing of the initial transcription product into mRNA, and inhibition of transport of mRNA to the cytoplasm
  • a substance that inhibits transcription of the CARD14 gene eg, antigene
  • a substance that inhibits processing of the initial transcription product into mRNA e.g., antigene
  • a substance that inhibits processing of the initial transcription product into mRNA eg, antigene
  • a substance that inhibits processing of the initial transcription product into mRNA eg, antigene
  • a substance that inhibits processing of the initial transcription product into mRNA eg, antigene
  • a substance that inhibits processing of the initial transcription product into mRNA eg, antigene
  • a substance that inhibits processing of the initial transcription product into mRNA e.g, antigene
  • a preferable example of the transcription product is mRNA.
  • a substance that specifically inhibits translation of CARD14 gene from mRNA to CARD14 preferably a base sequence complementary to or substantially complementary to the base sequence of these mRNAs or a part thereof
  • the nucleic acid containing is mentioned.
  • the nucleotide sequence substantially complementary to the nucleotide sequence of CARD14 gene mRNA binds to the target sequence of mRNA under physiological conditions of CARD14-producing cells (eg, neutrophils) in the mammal to be administered.
  • a base sequence having a degree of complementarity that can inhibit the translation (or cleave the target sequence) specifically, for example, a base sequence that is completely complementary to the base sequence of the mRNA (ie, , The base sequence of the complementary strand of mRNA) and a base sequence having a homology of about 90% or more, preferably about 95% or more, more preferably about 97% or more with respect to the overlapping region.
  • the base sequence complementary or substantially complementary to the base sequence of mRNA of the CARD14 gene is represented by the following (k) or (l): (k) SEQ ID NO: 1 or 3.
  • the stringent conditions are as described above.
  • CARD14 is not limited to these, but includes, for example, the function of membrane-bound guanylate kinase (MAGUK), the function as a molecular scaffold for assembling multiple protein complexes in specific regions of the plasma membrane, Since it has a caspase-related recruitment domain, it functions as a positive regulator of caspase-related recruitment function BCL10, NF- ⁇ B activation and cell apoptosis, NF- ⁇ B activation, and BCL10 phosphorylation induction function Etc.
  • MAGUK membrane-bound guanylate kinase
  • mRNA of CARD14 gene human CARD14 mRNA containing the base sequence represented by SEQ ID NO: 1 (Genbank Accession No. NM_052972), or their orthologs in other mammals (for example, mouse CARD14 (SEQ ID NO: 3, Genbank Accession No. NM — 029796)), and their splice variants, allelic variants, polymorphic variants, and the like.
  • the nucleotide sequence of the CARD14 gene mRNA and the “part of the complementary or substantially complementary nucleotide sequence” are capable of specifically binding to the mRNA of the CARD14 gene and translating the protein from the mRNA.
  • the length and position are not particularly limited as long as they can inhibit (or degrade the mRNA), but at least 10 bases are complementary or substantially complementary to the target sequence from the viewpoint of sequence specificity. As mentioned above, it preferably contains about 15 bases or more.
  • the nucleic acid comprising any one of the following (1) to (3) is preferably exemplified as a nucleic acid comprising a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of mRNA of the CARD14 gene.
  • a nucleic acid comprising a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of mRNA of the CARD14 gene Is: (1) an antisense nucleic acid against mRNA of the CARD14 gene, (2) a ribozyme nucleic acid against mRNA of the CARD14 gene, (3) A nucleic acid having RNAi activity against mRNA of CARD14 gene or a precursor thereof.
  • Antisense nucleic acid against mRNA of CARD14 gene includes a base sequence complementary to or substantially complementary to the base sequence of the mRNA or a part thereof. It is a nucleic acid and has a function of suppressing protein synthesis by forming a specific and stable duplex with a target mRNA.
  • Antisense nucleic acids include polydeoxyribonucleotides containing 2-deoxy-D-ribose, polyribonucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, Other polymers with non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence specific nucleic acid polymers) or other polymers containing special linkages, provided that the polymer is a base as found in DNA or RNA And a nucleotide having a configuration that allows attachment of a base).
  • RNA double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, DNA: RNA hybrids, unmodified polynucleotides (or unmodified oligonucleotides), known modifications Additions, such as those with labels known in the art, capped, methylated, one or more natural nucleotides replaced with analogs, intramolecular nucleotide modifications Eg, having uncharged bonds (eg, methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), having charged or sulfur-containing bonds (eg, phosphorothioates, phosphorodithioates, etc.) Things such as proteins (eg, nucleases, nuclease inhibitors, toxins, antibodies, Nalpeptide, poly-L-lysine, etc.) and sugars (eg, monosaccharides), etc., side chain groups, intercurrent compounds (eg, acridine, p
  • nucleoside may include not only purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleosides and modified nucleotides may also be modified at the sugar moiety, for example, one or more hydroxyl groups are replaced by halogens, aliphatic groups, etc., or functional groups such as ethers, amines, etc. It may be converted.
  • the antisense nucleic acid may be DNA or RNA, or may be a DNA / RNA chimera.
  • the RNA DNA hybrid formed by the target RNA and the antisense DNA can be recognized by endogenous RNase H and cause selective degradation of the target RNA. Therefore, in the case of antisense DNA directed to degradation by RNase H, the target sequence may be not only the sequence in mRNA but also the sequence of the intron region in the initial translation product of CARD14 gene.
  • the intron sequence can be determined by comparing the genomic sequence with the cDNA base sequence of the CARD14 gene using a homology search program such as BLAST or FASTA.
  • the target region of the antisense nucleic acid of the present invention is not particularly limited in length as long as the antisense nucleic acid hybridizes, and as a result, the translation into protein: CARD14 is inhibited.
  • the entire sequence or partial sequence of mRNA may be a short sequence of about 10 bases and a long sequence of mRNA or the initial transcript.
  • an oligonucleotide consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases is preferred, but is not limited thereto.
  • An 'end untranslated region, 3' end palindromic region or 3 'end hairpin loop or the like may be selected as a preferred target region of an antisense nucleic acid, but is not limited thereto.
  • the antisense nucleic acid of the present invention not only hybridizes with the mRNA of the CARD14 gene and the initial transcription product to inhibit translation into proteins, but also binds to these genes that are double-stranded DNA to form triple strands ( (Antigene) that can form a triplex) and inhibit transcription to RNA.
  • Antigene double-stranded DNA to form triple strands
  • the nucleotide molecules constituting the antisense nucleic acid may be natural DNA or RNA, but various chemicals may be used to improve stability (chemical and / or enzyme) and specific activity (affinity with RNA). Modifications can be included.
  • the phosphate residue (phosphate) of each nucleotide constituting the antisense nucleic acid is chemically modified, for example, phosphorothioate (PS), methylphosphonate, phosphorodithionate, etc. It can be substituted with a phosphate residue.
  • PS phosphorothioate
  • methylphosphonate methylphosphonate
  • phosphorodithionate etc. It can be substituted with a phosphate residue.
  • the hydroxyl group at the 2 ′ position of the sugar (ribose) of each nucleotide is represented by —OR (R ⁇ CH 3 (2′-O-Me), CH 2 CH 2 OCH 3 (2′-O-MOE), CH 2 (CH 2 NHC (NH) NH 2 , CH 2 CONHCH 3 , CH 2 CH 2 CN, etc.) may be substituted.
  • the base moiety pyrimidine, purine
  • RNA conformation of RNA is predominantly C2'-endo (S-type) and C3'-endo (N-type), and single-stranded RNA exists as an equilibrium between the two. Is fixed to the N type. Therefore, in order to give strong binding ability to the target RNA, a crosslinked nucleic acid (BNA) (BNA) (RNA derivative in which the conformation of the sugar moiety is fixed to N-type by crosslinking the 2 ′ oxygen and the 4 ′ carbon. Locked Nucleic Acid (LNA)) (Imanishi, T. et al., Chem.
  • the antisense oligonucleotide of the present invention determines the target sequence of mRNA or initial transcript based on the cDNA sequence or genomic DNA sequence of the CARD14 gene, and is a commercially available DNA / RNA automatic synthesizer (Applied Biosystems, Beckman) Etc.) can be prepared by synthesizing a complementary sequence thereto.
  • any of the above-described antisense nucleic acids containing various modifications can be chemically synthesized by a method known per se.
  • Ribozyme Nucleic Acid for mRNA of CARD14 Gene As another preferred example of a nucleic acid comprising a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of CARD14 gene mRNA or a part thereof, the mRNA is used as a coding region. Examples include ribozyme nucleic acids that can be cleaved specifically inside. “Ribozyme” refers to RNA having an enzyme activity that cleaves nucleic acids in a narrow sense, but in this specification, it is used as a concept including DNA as long as it has sequence-specific nucleic acid cleaving activity.
  • the most versatile ribozyme nucleic acid is self-splicing RNA found in infectious RNA such as viroid and virusoid, and hammerhead type and hairpin type are known.
  • the hammerhead type exhibits enzyme activity at about 40 bases, and a few bases at both ends adjacent to the part having the hammerhead structure (about 10 bases in total) are made into a sequence complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA.
  • This type of ribozyme nucleic acid has the additional advantage of not attacking genomic DNA because it uses only RNA as a substrate.
  • the target sequence is made single-stranded by using a hybrid ribozyme linked with an RNA motif derived from a viral nucleic acid that can specifically bind to an RNA helicase.
  • a hybrid ribozyme linked with an RNA motif derived from a viral nucleic acid that can specifically bind to an RNA helicase [Proc. Natl. Acad. Sci. USA, 98 (10): 5572-5577 (2001)].
  • the ribozyme is used in the form of an expression vector containing the DNA encoding the ribozyme, in order to promote the transfer of the transcription product to the cytoplasm, the ribozyme should be a hybrid ribozyme further linked with a tRNA-modified sequence. [Nucleic Acids Res., 29 (13): 2780-2788 (2001)].
  • siRNA RNA interference
  • RNAi RNA interference
  • mRNA complementary to the RNA has been known in nematodes, insects, plants, etc. Since this phenomenon has been confirmed to occur widely in animal cells [Nature, 411 (6836): 494-498 (2001)], it has been widely used as an alternative technique for the above-mentioned antisense nucleic acids and ribozymes.
  • siRNA is based on the cDNA sequence information of the target gene, for example, Elbashir et al. (Genes Dev., 15, 188-200 (2001)), Teramoto et al. (FEBS Lett. 579 (13): p2878-82 (2005)). Can be designed according to the rules proposed by The target sequence of siRNA has a length of 15 to 50 bases, preferably 19 to 49 bases, more preferably 19 to 27 bases in principle. For example, AA + (N) 19 (following AA, 19 Base sequence), AA + (N) 21 (base sequence of 21 bases following AA) or A + (N) 21 (base sequence of 21 bases following A).
  • the nucleic acid of the present invention may have an additional base at the 5 'or 3' end.
  • the length of the additional base is usually about 2 to 4 bases, and the total length of siRNA is 19 bases or more.
  • the additional base may be DNA or RNA, but the use of DNA may improve the stability of the nucleic acid.
  • Examples of such an additional base sequence include ug-3 ′, uu-3 ′, tg-3 ′, tt-3 ′, ggg-3 ′, guuu-3 ′, gttt-3 ′, and tttttt-3. Examples of such sequences include, but are not limited to, ', uuuu-3'.
  • siRNA may have an overhang
  • the siRNA may have a different number of bases in the sense strand and the antisense strand, for example, “aiRNA” in which the antisense strand has a protruding portion sequence (overhang) at the 3 ′ end and the 5 ′ end.
  • aiRNA in which the antisense strand has a protruding portion sequence (overhang) at the 3 ′ end and the 5 ′ end.
  • a typical aiRNA has an antisense strand consisting of 21 bases, a sense strand consisting of 15 bases, and has an overhang structure of 3 bases at both ends of the antisense strand (Sun, X. et al., Nature Biotechnology Vol26 No. .12 p1379, International Publication No. WO2009 / 029688 pamphlet).
  • the position of the target sequence is not particularly limited, but it is desirable to select the target sequence from the 5'-UTR and the start codon to about 50 bases, and from regions other than the 3'-UTR.
  • BLAST http: //www.ncbi.nlm Investigate using homology search software such as .nih.gov / BLAST /
  • a sense strand having a 3 'end overhang of TT or UU at 19-21 bases after AA (or NA), a sequence complementary to the 19-21 bases and TT or A double-stranded RNA consisting of an antisense strand having a 3 ′ terminal overhang of UU may be designed as siRNA.
  • siRNA short hairpin RNA
  • an arbitrary linker sequence for example, about 5-25 bases
  • the sense strand and the antisense strand are combined with each other. It can be designed by linking via a linker sequence.
  • siRNA and / or shRNA can be searched using search software provided free of charge on various web sites.
  • sites include the insert design tool for siRNA Target Finder (http://www.ambion.com/jp/techlib/misc/siRNA_finder.html) and pSilencer (Registered Trademark) Expression Vector provided by Ambion ( http://www.ambion.com/techlib/misc/psilencer_converter.html), GeneSeer provided by RNAi Codex (http://codex.cshl.edu/scripts/newsearchhairpin.cgi) Not.
  • the ribonucleoside molecule constituting siRNA may also be modified in the same manner as in the above-described antisense nucleic acid in order to improve stability, specific activity and the like.
  • RNAi activity may be lost if all ribonucleoside molecules in natural RNA are replaced with a modified form. Therefore, the minimum modified nucleoside that can function as an RNA-induced silencing complex (RISC) Need to be introduced.
  • RISC RNA-induced silencing complex
  • RNA having various chemical modifications see Usman and Cedergren, 1992, TIBS 17,34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163.
  • a phosphate residue (phosphate) of each nucleotide constituting siRNA is changed to a chemically modified phosphate such as phosphorothioate (PS), methylphosphonate, phosphorodithionate, etc.
  • hydroxyl group at the 2 ′ position of the sugar (ribose) of each nucleotide is represented by —OR (R ⁇ CH 3 (2′-O-Me), CH 2 CH 2 OCH 3 (2′-O-MOE), CH 2 CH 2 NHC (NH) NH 2 , CH 2 CONHCH 3 , CH 2 CH 2 CN, etc.) or a fluorine atom (—F) may be substituted.
  • the base moiety (pyrimidine, purine) may be chemically modified, for example, introduction of a methyl group or a cationic functional group at the 5-position of the pyrimidine base, or substitution of the carbonyl group at the 2-position with thiocarbonyl. Is mentioned.
  • the method for modifying an antisense nucleic acid described in (1) above can be used.
  • chemical modification (2′-deoxylation, 2′-H) for substituting a part of RNA in siRNA with DNA may be performed.
  • LNA Locked Nucleic Acid
  • ribose ribose
  • N-type an artificial nucleic acid in which the 2′-position and 4′-position of sugar (ribose) are cross-linked with —O—CH 2 — and the conformation is fixed to N-type
  • the sense strand and antisense strand constituting siRNA are linked via a linker to a ligand, peptide, sugar chain, antibody, lipid, positive charge, or molecular structure that specifically recognizes a receptor present on the cell surface. It may be chemically bonded to oligoarginine, Tat peptide, Rev peptide, Ant peptide or the like that adsorbs and penetrates the surface layer.
  • a sense strand and an antisense strand of a target sequence on mRNA are respectively synthesized by a DNA / RNA automatic synthesizer, denatured at about 90 to about 95 ° C. for about 1 minute in an appropriate annealing buffer, It can be prepared by annealing at about 30 to about 70 ° C. for about 1 to about 8 hours. It can also be prepared by synthesizing a short hairpin RNA (shRNA) serving as a precursor of siRNA and cleaving it with a dicer.
  • shRNA short hairpin RNA
  • a nucleic acid designed to generate siRNA against CARD14 gene mRNA in vivo also includes a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of CARD14 gene mRNA or one of the nucleotide sequences. Defined as encompassed by a nucleic acid containing a moiety. Examples of such a nucleic acid include an expression vector constructed so as to express the above-mentioned shRNA and siRNA.
  • the shRNA is an oligo comprising a base sequence in which a sense sequence and an antisense strand of a target sequence on mRNA are connected by inserting a spacer sequence having a length (for example, about 5 to 25 bases) that can form an appropriate loop structure.
  • RNA can be prepared by designing RNA and synthesizing it with an automatic DNA / RNA synthesizer.
  • RNA synthesizer There are tandem type and stem loop (hairpin) type in vectors expressing shRNA.
  • siRNA sense strand expression cassette and antisense strand expression cassette are connected in tandem, and each strand is expressed and annealed in the cell to form double stranded siRNA (dsRNA).
  • dsRNA double stranded siRNA
  • shRNA expression cassette is inserted into a vector, in which shRNA is expressed in cells and processed by dicer to form dsRNA.
  • a pol II promoter for example, a CMV immediate early promoter
  • a pol III promoter is generally used in order to cause transcription of a short RNA accurately.
  • the polIII promoter include mouse and human U6-snRNA promoter, human H1-RNasePRNA promoter, human valine-tRNA promoter and the like. Further, a sequence in which 4 or more Ts are continuous is used as a transcription termination signal.
  • siRNA or shRNA expression cassette constructed in this way is then inserted into a plasmid vector or virus vector.
  • virus vectors such as retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes virus, Sendai virus, animal cell expression plasmids and the like are used.
  • siRNA is based on nucleotide sequence information, for example, 394 Applied Biosystems, Inc. It can be chemically synthesized according to a conventional method using an automatic DNA / RNA synthesizer such as a synthesizer. For example, Caruthers et al., 1992, Methods in Enzymology 211, 3-19, Thompson et al., International Publication WO99 / 54459, Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al. , 1997, Methods Mol.
  • nucleic acid protecting group for example, dimethoxytrityl group at the 5 'end
  • a coupling group for example, phosphoramidite at the 3' end
  • the protecting group at the 5 'end is deprotected with an acid such as TCA (trichloroacetic acid) and a coupling reaction is performed. Then, after capping with an acetyl group, the next nucleic acid condensation reaction is performed.
  • a modified RNA for example, 2′-O-methyl nucleotide, 2′-deoxy-2′-fluoronucleotide
  • a cage reagent 3H-1,2-benzodithiol-3-one 1,1-dioxide
  • oligonucleotides may be synthesized separately and linked together after synthesis, for example, by ligation (Moore et al., 1992, Science 256, 9923; Draper et al. International Publication WO93 / 23569; Shabarova et al. , 1991, Nucleic Acids Research 19,4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16,951; Bellon et al., 1997, Nucleosides & Nucleotides, Belon et al., 1997, BioconjugateChem. They may be joined together by hybridization after deprotection. siRNA molecules can also be synthesized by tandem synthesis.
  • both siRNA strands are synthesized as a single continuous oligonucleotide separated by a cleavable linker, which is then cleaved to generate separate siRNA fragments that are hybridized and purified.
  • the linker may be a polynucleotide linker or a non-nucleotide linker.
  • the synthesized siRNA molecules can be purified using methods known to those skilled in the art. For example, a method of purification by gel electrophoresis or a method of purification using high performance liquid chromatography (HPLC) can be mentioned.
  • HPLC high performance liquid chromatography
  • nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of mRNA of the CARD14 gene or a part thereof is microRNA (miRNA) targeting the mRNA.
  • miRNA can also be prepared according to the method described for siRNA.
  • a nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of mRNA of the CARD14 gene or a part thereof is provided in a special form such as a liposome or a microsphere, or other molecules are added. It can be provided in the form.
  • additional forms that can be used include polycationic substances such as polylysine that act to neutralize the charge of the phosphate group skeleton, and lipids that enhance interaction with cell membranes and increase nucleic acid uptake. Hydrophobic substances such as (eg, phospholipid, cholesterol, etc.) can be mentioned.
  • Preferred lipids for addition include cholesterol and derivatives thereof (eg, cholesteryl chloroformate, cholic acid, etc.).
  • Such can be attached to the 3 'or 5' end of the nucleic acid and can be attached via a base, sugar, intramolecular nucleoside linkage.
  • the other group include a cap group specifically arranged at the 3 'end or 5' end of a nucleic acid, which prevents degradation by nucleases such as exonuclease and RNase.
  • capping groups include, but are not limited to, hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
  • the CARD14 expression inhibitory activity of these nucleic acids is examined using a transformant introduced with a nucleic acid encoding CARD14, an in vivo or in vitro CARD14 gene expression system, or an in vivo or in vitro CARD14 protein translation system. Can do.
  • the substance that inhibits the expression of CARD14 in the present invention is not limited to a nucleic acid comprising a base sequence complementary to or substantially complementary to the base sequence of mRNA of the CARD14 gene as described above, or a CARD14 production.
  • Other substances such as low molecular weight compounds may be used as long as they are directly or indirectly inhibited. Such a substance can be obtained, for example, by the screening method of the present invention described later.
  • the “substance that inhibits the function of CARD14” may be any substance as long as CARD14 once functionally produced inhibits its function.
  • examples of the substance that suppresses the function of CARD14 include an antibody against CARD14.
  • the antibody may be a polyclonal antibody or a monoclonal antibody. These antibodies can be produced according to per se known antibody or antiserum production methods.
  • the isotype of the antibody is not particularly limited, but preferably includes IgG, IgM or IgA, and particularly preferably IgG.
  • the antibody is not particularly limited as long as it has at least a complementarity determining region (CDR) for specifically recognizing and binding to a target antigen.
  • CDR complementarity determining region
  • Fab, Fab ′, F (Ab ′) 2 such as a fragment, scFv, scFv-Fc, a genetically engineered conjugate molecule such as a minibody or diabody, or a molecule having a protein stabilizing action such as polyethylene glycol (PEG)
  • PEG polyethylene glycol
  • the antibody preferably a monoclonal antibody
  • the antibody has an reduced risk of showing antigenicity when administered to a human.
  • Specific examples include fully human antibodies, humanized antibodies, mouse-human chimeric antibodies, and particularly preferably fully human antibodies.
  • Humanized antibodies and chimeric antibodies can be produced by genetic engineering according to conventional methods.
  • a fully human antibody can be produced from a human-human (or mouse) hybridoma, but in order to provide a large amount of antibody stably and at low cost, a human antibody-producing mouse or phage display is produced. It is desirable.
  • Another preferred substance that suppresses the function of CARD14 is a low molecular compound commensurate with Lipinski's Rule. Such a compound can be obtained, for example, using the screening method of the present invention described later.
  • a CARD14 inhibitor such as a substance that inhibits the expression or function of CARD14, is a therapeutic or prophylactic agent for psoriasis, multiple sclerosis, a therapeutic agent for reducing side effects caused by TLR-based / IMQ / immunostimulatory adjuvant, or It is useful as a prophylactic agent, a therapeutic agent or a prophylactic agent for complications of ⁇ T cell-mediated diseases and / or immune intervention.
  • the present invention provides a screening method for a therapeutic or prophylactic agent for psoriasis based on the expression of CARD14 in hematopoietic cells.
  • CARD14 is a conventionally known molecule, it has been found that a therapeutic or preventive agent for psoriasis can be screened by observing its expression in hematopoietic cells, particularly ⁇ T cells, especially epidermal ⁇ T cells. It is not provided for the first time in the present invention.
  • CARD inhibitors such as agents that suppress or eliminate CARD14 expression in hematopoietic cells, particularly ⁇ T cells, and preferably epidermal ⁇ T cells, are therapeutic or preventive agents for psoriasis. It is understood that it can be used as an agent. Therefore, the method of the present invention comprises the steps of bringing a candidate substance into contact with a hematopoietic cell, and determining the expression of CARD14 in the hematopoietic cell, wherein the candidate substance suppresses or eliminates CARD14 expression. Including a step where it is determined that the substance can be used as a therapeutic or prophylactic agent for psoriasis.
  • CARD inhibitors such as agents that suppress or eliminate CARD14 expression include inhibitors against CARD14 expression products, such as antibodies, antibody fragments, functional equivalents, or CARD14 antisense nucleic acids and RNAi functions. And the like.
  • the targeted ⁇ T cells are cells that produce interleukin-17 (IL-17) and interleukin-22 (IL-22).
  • non-hematopoietic cell in order to provide a better therapeutic or preventive agent, it is also referred to as a cell (“non-hematopoietic cell”) that is not a hematopoietic cell (particularly ⁇ T cell, particularly preferably epidermal ⁇ T cell).
  • ⁇ T cell particularly preferably epidermal ⁇ T cell
  • CARD14 it is preferable to confirm that CARD14 is not expressed in skin resident cells).
  • it is desirable that hematopoietic cells and non-hematopoietic cells are derived from the same allogeneic strain, more preferably from the same individual.
  • the method of the present invention comprises contacting the candidate substance with non-hematopoietic cells, and selecting the candidate substance as the therapeutic or prophylactic agent when CARD14 has no substantial effect on expression.
  • “there is no substantial influence” means that the CARD 14 can exert its function even if there is an influence.
  • the psoriasis targeted by the present invention can include, for example, imomomid (IMQ) -induced psoriasis and IL-23-induced psoriasis.
  • IMQ imomomid
  • the therapeutic agent or prophylactic agent of the present invention is specifically delivered to hematopoietic cells (particularly ⁇ T cells, preferably epidermal ⁇ T cells).
  • specific delivery to hematopoietic cells is achieved by a delivery system that leads a specific molecule such as an antibody specific to the hematopoietic cells (for example, ⁇ T cells).
  • the present invention provides a screening method for a therapeutic or prophylactic agent for multiple sclerosis based on the expression of CARD14 in hematopoietic cells.
  • CARD14 is a conventionally known molecule, it can be seen that a therapeutic or prophylactic agent for multiple sclerosis can be screened by observing its expression in hematopoietic cells, particularly ⁇ T cells, especially epidermal ⁇ T cells. It has not been issued and is provided for the first time in the present invention.
  • CARD inhibitors such as agents that suppress or eliminate CARD14 expression in hematopoietic cells, in particular ⁇ T cells, and preferably epidermal ⁇ T cells, are used to treat multiple sclerosis. It is understood that it can be used as an agent or prophylactic agent.
  • CARD14 since the role of CARD14 was clearly shown in an experimental autoimmune encephalomyelitis (EAE) model of a multiple sclerosis model (autoimmune disease model), it is based on CARD14. It is believed that the mechanism can be used to prevent or treat multiple sclerosis.
  • EAE experimental autoimmune encephalomyelitis
  • the method of the present invention comprises the steps of bringing a candidate substance into contact with a hematopoietic cell, and determining the expression of CARD14 in the hematopoietic cell, wherein the candidate substance suppresses or eliminates CARD14 expression. Including a step where it is determined that the substance can be used as a therapeutic or prophylactic agent for psoriasis.
  • CARD inhibitors such as agents that suppress or eliminate CARD14 expression include inhibitors against CARD14 expression products, such as antibodies, antibody fragments, functional equivalents, or CARD14 antisense nucleic acids and RNAi functions. And the like.
  • the targeted ⁇ T cells are cells that produce interleukin-17 (IL-17) and interleukin-22 (IL-22).
  • non-hematopoietic cells cells that are not hematopoietic cells (particularly ⁇ T cells, particularly epidermal ⁇ T cells) (“non-hematopoietic cells”). It is preferable to confirm that CARD14 is not expressed in the skin resident cells).
  • the method of the present invention comprises contacting the candidate substance with non-hematopoietic cells, and selecting the candidate substance as the therapeutic or prophylactic agent when CARD14 has no substantial effect on expression. Is further included.
  • the therapeutic agent or prophylactic agent of the present invention is specifically delivered to hematopoietic cells (particularly ⁇ T cells, preferably epidermal ⁇ T cells).
  • specific delivery to hematopoietic cells is achieved by a delivery system that leads a specific molecule such as an antibody specific to the hematopoietic cells (for example, ⁇ T cells).
  • the present invention provides a screening method for a therapeutic or prophylactic agent for multiple sclerosis based on the expression of CARD14 in hematopoietic cells.
  • CARD14 is a conventionally known molecule
  • a therapeutic agent for reducing side effects caused by TLR-based / IMQ / immunostimulatory adjuvant by examining its expression in hematopoietic cells, particularly ⁇ T cells, particularly epidermal ⁇ T cells, or It has not been found that a prophylactic agent can be screened, and is provided for the first time in the present invention.
  • CARD inhibitors such as agents that suppress or eliminate CARD14 expression in hematopoietic cells, particularly ⁇ T cells, and preferably epidermal ⁇ T cells, are TLR-based / IMQ / immune It will be appreciated that it can be used as a therapeutic or prophylactic agent to reduce the side effects of stimulating adjuvants.
  • TLR7 and TLR9 signaling in combination is required to fully develop IMQ-induced psoriatic dermatitis, while IL-23-induced psoriasis
  • the dermatitis model has been demonstrated to directly activate downstream of TLR7 and TLR9-mediated innate immune activation.
  • the method of the present invention comprises the steps of bringing a candidate substance into contact with a hematopoietic cell, and determining the expression of CARD14 in the hematopoietic cell, wherein the candidate substance suppresses or eliminates CARD14 expression. Including a step where it is determined that the substance can be used as a therapeutic or prophylactic agent for psoriasis.
  • a side effect due to TLR base / IMQ / immunostimulatory adjuvant refers to any side effect that occurs when an adjuvant based on TLR, imiquimod (IMQ) or an immune stimulating adjuvant (for example, CpG) is administered.
  • side effects include, but are not limited to, psoriasis, inflammation such as psoriasis-like skin inflammation, cytokineemia and the like. See, for example, Non-Patent Document 2 (Gilliet et al., 2004) for information on side effects of TLR base / IMQ / immunostimulatory adjuvants.
  • CARD inhibitors such as agents that suppress or eliminate CARD14 expression include inhibitors against CARD14 expression products, such as antibodies, antibody fragments, functional equivalents, or CARD14 antisense nucleic acids and RNAi functions. And the like.
  • the targeted ⁇ T cells are cells that produce interleukin-17 (IL-17) and interleukin-22 (IL-22).
  • non-hematopoietic cells cells that are not hematopoietic cells (especially ⁇ T cells, particularly epidermal ⁇ T cells) (“non-hematopoietic cells”, for example, It is preferable to confirm that CARD14 is not expressed in the skin resident cells).
  • non-hematopoietic cells it is desirable that hematopoietic cells and non-hematopoietic cells are derived from the same allogeneic strain, more preferably from the same individual.
  • the method of the present invention comprises contacting the candidate substance with non-hematopoietic cells, and selecting the candidate substance as the therapeutic or prophylactic agent when CARD14 has no substantial effect on expression. Is further included.
  • the therapeutic agent or prophylactic agent of the present invention is specifically delivered to hematopoietic cells (particularly ⁇ T cells, preferably epidermal ⁇ T cells).
  • specific delivery to hematopoietic cells is achieved by a delivery system that leads a specific molecule such as an antibody specific to the hematopoietic cells (for example, ⁇ T cells).
  • the present invention provides a screening method for a therapeutic or prophylactic agent for multiple sclerosis based on the expression of CARD14 in hematopoietic cells.
  • CARD14 is a conventionally known molecule, its expression or expression in hematopoietic cells, particularly ⁇ T cells, particularly epidermal ⁇ T cells, can be used to treat or prevent ⁇ T cell-mediated diseases and / or complications of immune intervention. It has not been found that agents can be screened and is provided for the first time in the present invention.
  • CARD inhibitors such as agents that suppress or eliminate CARD14 expression in hematopoietic cells, particularly ⁇ T cells, and preferably epidermal ⁇ T cells, are ⁇ T cell-mediated diseases and It is understood that it can be used as a therapeutic or prophylactic agent for complications of immune intervention. Therefore, the method of the present invention comprises the steps of bringing a candidate substance into contact with a hematopoietic cell, and determining the expression of CARD14 in the hematopoietic cell, wherein the candidate substance suppresses or eliminates CARD14 expression. Including a step where it is determined that the substance can be used as a therapeutic or prophylactic agent for psoriasis.
  • CARD inhibitors such as agents that suppress or eliminate CARD14 expression include inhibitors against CARD14 expression products, such as antibodies, antibody fragments, functional equivalents, or CARD14 antisense nucleic acids and RNAi functions. And the like.
  • the targeted ⁇ T cells are cells that produce interleukin-17 (IL-17) and interleukin-22 (IL-22).
  • non-hematopoietic cells cells that are not hematopoietic cells (particularly ⁇ T cells, particularly epidermal ⁇ T cells) (“non-hematopoietic cells”). It is preferable to confirm that CARD14 is not expressed in the skin resident cells).
  • the method of the present invention comprises contacting the candidate substance with non-hematopoietic cells, and selecting the candidate substance as the therapeutic or prophylactic agent when CARD14 has no substantial effect on expression. Is further included.
  • the therapeutic agent or prophylactic agent of the present invention is specifically delivered to hematopoietic cells (particularly ⁇ T cells, preferably epidermal ⁇ T cells).
  • specific delivery to hematopoietic cells is achieved by a delivery system that leads a specific molecule such as an antibody specific to the hematopoietic cells (for example, ⁇ T cells).
  • ⁇ T cell-mediated disease refers to any disease mediated by ⁇ T cells.
  • examples of “ ⁇ T cell-mediated disease” include psoriasis, multiple sclerosis, inflammation, and cytokineemia. But are not limited to these.
  • “complication of immune intervention” refers to any complication of immune intervention, and examples of “complication of immune intervention” include inflammation, cytokineemia, etc. It is not limited to.
  • the screening method involves culturing cells capable of producing CARD14 in the presence and absence of a test substance, and expression of CARD14 under both conditions. Comparing the amount or degree of function.
  • a compound that inhibits the function of CARD14 can also be screened by testing the ability to bind to purified CARD14 protein and the binding inhibitory activity between CARD14 protein and its binding protein (for example, CARD14 receptor).
  • such cells are preferably hematopoietic cells, preferably ⁇ T cells, more preferably ⁇ T cells of the epidermis.
  • genetically modified cells may be used.
  • such a cell contains a DNA encoding CARD14 (ie, SEQ ID NO: 1 or 3, preferably the nucleotide sequence represented by SEQ ID NO: 1 or a nucleotide sequence complementary to the nucleotide sequence and a string.
  • the screening method includes (A) contacting a cell containing a nucleic acid encoding a CARD14 gene or a reporter protein under the control of the transcriptional regulatory region of the gene with a test substance; ) A step of measuring the expression level of CARD14 gene or CARD14 protein or reporter protein in the cell; and (C) expression of CARD14 gene or CARD14 protein or reporter protein as compared with the case where measurement is performed in the absence of the test substance.
  • CARD14 can be brought into contact with a test substance, and a test substance capable of binding to CARD14 can be selected as a candidate substance used for the treatment and / or prevention of psoriasis.
  • the present invention provides (A) a step of contacting CARD14 with a cell membrane fraction of hematopoietic cells (particularly ⁇ T cells, particularly epidermal ⁇ T cells) in the presence of a test substance; Measuring the amount of CARD14 bound to the minute; and (C) reducing the amount of CARD14 bound to the cell membrane fraction compared to when measured in the absence of the test substance, psoriasis, Candidates for use in reducing multiple side effects due to multiple sclerosis, TLR-based / IMQ / immunostimulatory adjuvants, or for the treatment and / or prevention of complications of ⁇ T cell mediated diseases and / or immune interventions
  • the present invention provides for the treatment of psoriasis, multiple sclerosis, TLR-based / IMQ / immunostimulatory adjuvant side effects, or the treatment of complications of ⁇ T cell mediated diseases and / or immune interventions and / or
  • test substances selected as candidates for substances used for prevention may be used to reduce side effects caused by psoriasis, multiple sclerosis, TLR-based / IMQ / immunostimulatory adjuvants, and / or ⁇ T cell-mediated diseases and / or Or to apply to models of complications of immune intervention (eg, including model animals described in the Examples) to reduce side effects due to psoriasis, multiple sclerosis, TLR-based / IMQ / immunostimulatory adjuvant in the model Whether to suppress the response of ⁇ T cell mediated diseases and / or complications of immune intervention Further comprising a Rukoto.
  • the present invention provides (A) contacting hematopoietic cells (particularly ⁇ T cells, particularly preferably epidermal ⁇ T cells) with a test substance in the presence and absence of CARD14; (B) Measure the degree of complications of psoriasis, multiple sclerosis, TLR-based / IMQ / immunostimulatory adjuvant side effects, or ⁇ T cell mediated diseases and / or immune interventions in the cells under each condition And (C) to reduce side effects due to psoriasis, multiple sclerosis, TLR-based / IMQ / immunostimulatory adjuvant in the presence of CARD14 as compared to when measured in the absence of the test substance, Or suppresses the response of complications of ⁇ T cell mediated diseases and / or immune intervention, and in the absence of CARD14, psoriasis, multiple sclerosis, TLR A test substance that reduces the side effects caused by S / IMQ / immunostimulatory adjuvant or that does
  • test substance examples include proteins, peptides, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. These substances may be novel or may be known ones.
  • a control cell that is not brought into contact with a test substance can also be used as a comparative control.
  • “do not contact the test substance” means that when the same amount of solvent (blank) as the test substance is added instead of the test substance, the expression level of the CARD14 or CARD14 gene or the function of CARD14 is affected. The case where a negative control substance not given is added is also included.
  • the contact of the test substance with the cells is, for example, the above-mentioned medium or various buffers (for example, HEPES buffer, phosphate buffer, phosphate buffered saline, Tris-HCl buffer, borate buffer, acetic acid).
  • the test substance can be added to a buffer solution or the like, and the cells can be incubated for a certain time.
  • concentration of the test substance to be added varies depending on the type of compound (solubility, toxicity, etc.), but is appropriately selected within the range of about 0.1 nM to about 100 ⁇ M, for example. Examples of the incubation time include about 10 minutes to about 24 hours.
  • the state of the animal individual is not particularly limited.
  • Example 1 Or a model animal of ⁇ T cell-mediated disease and / or complication of immune intervention (IMQ-induced psoriatic dermatitis of Example 1, IL-23-induced psoriatic skin It may be a etc.), and the like.
  • IMQ-induced psoriatic dermatitis of Example 1, IL-23-induced psoriatic skin It may be a etc. There are no particular restrictions on the breeding conditions of the animals to be used, but it is preferable that the animals are raised in an environment of SPF grade or higher.
  • Contact of the test substance with the cells is carried out by administering the test substance to the animal individual.
  • the administration route is not particularly limited, and examples thereof include intravenous administration, intraarterial administration, subcutaneous administration, intradermal administration, intraperitoneal administration, oral administration, intratracheal administration, and rectal administration.
  • the dose is not particularly limited. For example, a dose of about 0.5 to 20 mg / kg can be administered 1 to 5 times a day,
  • the above screening method can be performed by contacting a test substance with an extract of the cells or CARD14 isolated and purified from the cells, instead of the cells having the ability to produce CARD14.
  • the present invention is used for prevention and / or treatment of psoriasis characterized by comparing the expression of the protein (gene) in cells capable of producing CARD14 in the presence and absence of a test substance.
  • a method for screening a substance that can be used. The cells used in this method, the type of test substance, the mode of contact between the test substance and cells, etc. are the same as described above.
  • the expression level of CARD14 is a nucleic acid capable of hybridizing under stringent conditions with the above-mentioned DNA encoding CARD14, that is, under the stringent conditions with the nucleotide sequence represented by SEQ ID NO: 1 or a complementary nucleotide sequence thereto.
  • the CARD14 gene mRNA can be detected at a RNA level using a nucleic acid (DNA) that can be hybridized with (hereinafter, sometimes referred to as “the nucleic acid for detection of the present invention”).
  • the expression level can also be measured at the protein level by detecting these proteins using the above-mentioned antibody against CARD14 (hereinafter sometimes referred to as “the detection antibody of the present invention”).
  • the present invention relates to (a) culturing cells having the ability to produce CARD14 in the presence and absence of a test substance, and the amount of mRNA encoding the protein under both conditions For reducing side effects caused by psoriasis, multiple sclerosis, TLR-based / IMQ / immunostimulatory adjuvant, or ⁇ T cell-mediated disease, characterized in that And / or a method for screening a substance that can be used for prevention and / or treatment of complications of immune intervention, and (b) culturing cells having the ability to produce CARD14 in the presence and absence of the test substance And the amount of the protein under both conditions is measured and compared using the detection antibody of the present invention, psoriasis, multiple sclerosis, TL Methods of screening for substances that can be used to reduce side effects from R-based / IMQ / immunostimulatory adjuvants or to prevent and / or treat complications of ⁇ T cell mediated diseases and / or immune interventions are
  • screening for a substance that changes the expression level of CARD14 can be performed as follows.
  • test substance is administered to a human mammal (eg, mouse, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc.), and after a certain period of time (30 minutes to 3 days later, preferably After 1 hour to 2 days, more preferably after 1 hour to 24 hours, blood, a specific organ (eg, brain, etc.), or a tissue or cell isolated from the organ is obtained.
  • a human mammal eg, mouse, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc.
  • a certain period of time (30 minutes to 3 days later, preferably After 1 hour to 2 days, more preferably after 1 hour to 24 hours, blood, a specific organ (eg, brain, etc.), or a tissue or cell isolated from the organ is obtained.
  • CARD14 mRNA can be quantified by extracting mRNA from cells or the like by a usual method, or can be quantified by Northern blot analysis known per se.
  • the protein amount of CARD14 can be quantified using Western blot analysis or various immunoassay methods described in detail below.
  • a cell that expresses the CARD14 gene (for example, a transformant introduced with CARD14) is prepared according to the above method, and a test substance is added to the medium or buffer when culturing according to a conventional method for a certain period of time. After incubation (1 day to 7 days later, preferably 1 day to 3 days later, more preferably 2 days to 3 days later), CARD14 or mRNA encoding the same contained in the cell culture is the same as in (i) above. Can be quantified and analyzed.
  • the detection and quantification of the expression level of the CARD14 gene can be performed by a known method such as Northern blotting or RT-PCR using RNA prepared from the cells or a complementary polynucleotide transcribed therefrom. Specifically, by using a polynucleotide having at least 15 bases continuous in the base sequence of the CARD14 gene and / or its complementary polynucleotide as a primer or a probe, the presence or absence of the expression of the CARD14 gene in RNA or its expression The level can be detected and measured.
  • a probe or primer can be designed based on the base sequence of the CARD14 gene using, for example, primer 3 (http://primer3.sourceforge.net/) or vector NTI (manufactured by Infomax). .
  • the primers or probes radioisotope (such as 32 P, 33 P: RI) or a fluorescent substance labeled with a, it, RNA transfer cells derived from a nylon membrane or the like according to a conventional method
  • the formed duplex of the primer or probe (DNA or RNA) and RNA is used as a signal derived from a label (RI or fluorescent substance) of the primer or probe as a radiation detector ( Examples thereof include a method of detecting and measuring with a BAS-1800II (manufactured by Fuji Film) or a fluorescence detector.
  • the probe is labeled in accordance with the protocol, hybridized with cell-derived RNA, and then the signal derived from the labeled substance of the probe is multi-bidirectional.
  • a method of detecting and measuring with an imager STORM860 can also be used.
  • cDNA is prepared from cell-derived RNA according to a conventional method, and using this as a template, a target CARD14 gene region can be amplified.
  • a method of detecting the amplified double-stranded DNA obtained by hybridizing a primer (a normal strand that binds to the cDNA ( ⁇ strand), a reverse strand that binds to the + strand) and performing a PCR method according to a conventional method can be illustrated.
  • the detection of the amplified double-stranded DNA was performed by a method for detecting the labeled double-stranded DNA produced by performing the PCR using a primer previously labeled with RI or a fluorescent substance.
  • a method may be used in which double-stranded DNA is transferred to a nylon membrane or the like according to a conventional method, and the labeled primer is used as a probe to hybridize with this to detect it.
  • the produced labeled double-stranded DNA product can be measured with an Agilent 2100 Bioanalyzer (manufactured by Yokogawa Analytical Systems).
  • RT-PCR reaction solution is prepared according to the protocol with SYBR Green RT-PCR Reagents (Applied Biosystems) and reacted with ABI PRIME 7900 Sequence Detection System (Applied Biosystems) to detect the reaction product. You can also.
  • the expression of the CARD14 gene in the cells to which the test substance is added is 2/3 times or less, preferably 1/2 times or less, more preferably 1/3 times the expression level in the control cells to which no test substance is added.
  • the test substance can be selected as a CARD14 gene expression inhibitor if it is below.
  • screening for substances that change the expression level of CARD14 can also be performed by a reporter gene assay using the transcriptional regulatory region of CARD14 gene.
  • the “transcriptional regulatory region” usually refers to a range from several kb to several tens of kb upstream of the chromosomal gene.
  • 5′-race method for example, 5 (ii) Genome Walker Kit (Clontech), which can be carried out using conventional methods such as' -full RaceCoreKit (manufactured by Takara Bio Inc.), oligo cap method, S1 primer mapping, etc .
  • the 5′-upstream region can be obtained using a method such as that manufactured by the same company, and the obtained upstream region can be identified by a technique including a step of measuring promoter activity.
  • reporter gene A nucleic acid encoding a reporter protein (hereinafter referred to as “reporter gene”) in a functional form downstream of the transcriptional regulatory region of the CARD14 gene is linked to construct a reporter protein expression vector.
  • the vector may be prepared by a method known to those skilled in the art. For example, it is extracted according to the usual genetic engineering methods described in Molecular Cloning: A Laboratory Manual 2nd edition (1989), Cold Spring Harbor Laboratory Press “Current Protocols In Molecular Biology” (1987), John Wiley & Sons, Inc., etc.
  • the transcriptional regulatory region of the CARD14 gene can be incorporated on a plasmid containing a reporter gene.
  • Reporter proteins include ⁇ -glucuronidase (GUS), luciferase, chloramphenicol transacetylase (CAT), ⁇ -galactosidase (GAS), green fluorescent protein (GFP), yellow fluorescent protein (YFP), blue fluorescent protein ( CFP), red fluorescent protein (RFP) and the like.
  • GUS ⁇ -glucuronidase
  • CAT chloramphenicol transacetylase
  • GAS ⁇ -galactosidase
  • GFP green fluorescent protein
  • YFP yellow fluorescent protein
  • CFP blue fluorescent protein
  • RFP red fluorescent protein
  • the reporter gene formed by linking the prepared CARD14 gene transcriptional regulatory region in a functional manner is inserted into a vector that can be used in a cell into which the reporter gene is to be introduced, using a normal genetic engineering technique, and a plasmid is inserted. And can be introduced into a suitable host cell. Stable transformed cells can be obtained by culturing in a medium with selection conditions according to the selection marker gene mounted on the vector. Alternatively, a reporter gene in which the transcriptional regulatory region of the CARD14 gene is operably linked may be transiently expressed in the host cell.
  • a method for measuring the expression level of the reporter gene a method corresponding to each reporter gene may be used.
  • a luciferase gene when used as a reporter gene, the transformed cell is cultured for several days, an extract of the cell is obtained, and then the extract is reacted with luciferin and ATP to cause chemiluminescence, and the luminescence intensity Promoter activity can be detected by measuring.
  • a commercially available luciferase reaction detection kit such as Picker Gene Dual Kit (registered trademark; manufactured by Toyo Ink) can be used.
  • a detection antibody of the present invention is reacted competitively with a sample solution and labeled CARD14, and a label bound to the antibody is labeled.
  • a method for quantifying CARD14 in a sample solution by detecting the converted protein (ii) the detection solution of the present invention insolubilized on the sample solution and the carrier, and another labeled detection method of the present invention Examples include a method of quantifying CARD14 in a sample solution by reacting an antibody simultaneously or continuously and then measuring the amount (activity) of the labeling agent on the insolubilized carrier.
  • the detection and quantification of the protein expression level of CARD14 can be quantified according to a known method such as Western blotting using an antibody that recognizes CARD14.
  • Western blotting uses an antibody that recognizes CARD14 as a primary antibody, and then binds to a primary antibody labeled with a radioisotope such as 125 I, a fluorescent substance, an enzyme such as horseradish peroxidase (HRP) as a secondary antibody. This is carried out by measuring the signal derived from these labeling substances with a radiation measuring instrument (BAI-1800II: manufactured by Fuji Film Co., Ltd.), a fluorescence detector, or the like.
  • a radiation measuring instrument BAI-1800II: manufactured by Fuji Film Co., Ltd.
  • the form of the above-described antibody is not particularly limited, and may be a polyclonal antibody having CARD14 as an immunogen or a monoclonal antibody, and is usually at least a continuous amino acid sequence constituting CARD14.
  • An antibody having an antigen binding property to a polypeptide consisting of 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids can also be used.
  • the two antibodies recognize different portions of CARD14.
  • one antibody recognizes the N-terminal part of CARD14, one that reacts with the C-terminal part of the protein can be used as the other antibody.
  • a labeling agent used in a measurement method using a labeling substance for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance or the like is used.
  • the radioisotope for example, [ 125 I], [ 131 I], [ 3 H], [ 14 C] and the like are used.
  • the above enzyme those which are stable and have high specific activity are preferable.
  • ⁇ -galactosidase, ⁇ -glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used.
  • the fluorescent substance for example, fluorescamine, fluorescein isothiocyanate and the like are used.
  • luminescent substance for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used.
  • a biotin- (strept) avidin system can also be used for binding of an antibody or antigen to a labeling agent.
  • the CARD14 quantification method using the detection antibody of the present invention is not particularly limited, and the amount of antibody, antigen or antibody-antigen complex corresponding to the amount of antigen in the sample solution is chemically or physically determined. Any measurement method may be used as long as it is a measurement method that is detected by means and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competition method, immunometric method and sandwich method are preferably used. In view of sensitivity and specificity, for example, the sandwich method described later is preferably used.
  • the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass.
  • the sample solution is reacted with the insolubilized detection antibody of the present invention (primary reaction), and further labeled with another detection antibody of the present invention (secondary reaction), and then on the insolubilized carrier.
  • primary reaction the insolubilized detection antibody of the present invention
  • secondary reaction another detection antibody of the present invention
  • CARD14 in the sample solution can be quantified.
  • the primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at different times.
  • the labeling agent and the insolubilization method can be the same as those described above.
  • the antibody used for the immobilized antibody or the labeled antibody is not necessarily one type, and a mixture of two or more types of antibodies is used for the purpose of improving measurement sensitivity. May be.
  • the detection antibody of the present invention can also be used in measurement systems other than the sandwich method, such as a competitive method, an immunometric method, or nephrometry.
  • CARD14 in a sample solution and labeled CARD14 are reacted competitively with an antibody, and then unreacted labeled antigen (F) and labeled antigen (B) bound to the antibody are separated.
  • B / F separation CARD14 in the sample solution is quantified by measuring the labeling amount of either B or F.
  • a soluble antibody is used as an antibody
  • B / F separation is performed using polyethylene glycol or a secondary antibody against the antibody (primary antibody)
  • a solid phase is used as the primary antibody. Either an antibody is used (direct method), or a primary antibody is soluble, and a solid phase antibody is used as a secondary antibody (indirect method).
  • the CARD14 in the sample solution and the immobilized CARD14 are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated, or the CARD14 in the sample solution is separated from the CARD14 in the sample solution.
  • solid phase CARD14 is added to bind the unreacted labeled antibody to the solid phase, the solid phase and the liquid phase are separated.
  • the amount of label in any phase is measured to quantify the amount of antigen in the sample solution.
  • the amount of insoluble precipitate produced as a result of antigen-antibody reaction in gel or solution is measured. Even when the amount of CARD14 in the sample solution is small and only a small amount of sediment can be obtained, laser nephrometry using laser scattering is preferably used.
  • the amount of CARD14 in a cell can be quantified with high sensitivity by using the detection antibody of the present invention.
  • the expression level of CARD14 (mRNA amount or protein amount) in the presence of the test substance is about 20% or more, preferably about 30%, compared to the case in the absence of the test substance.
  • the test substance is reduced to CARD14 expression inhibitory substance, and therefore, side effects reduced by psoriasis, multiple sclerosis, TLR base / IMQ / immunostimulatory adjuvant, or ⁇ T cells. It can be selected as a candidate for a substance that can be used for the prevention and / or treatment of mediated diseases and / or complications of immune intervention.
  • a cell containing a reporter gene under the control of the transcriptional regulatory region in the CARD14 gene can be used instead of the cell expressing the CARD14 gene.
  • a cell may be a cell, tissue, organ or individual of a transgenic animal into which a reporter gene (eg, luciferase, GFP, etc.) under the control of the transcriptional regulatory region of the CARD14 gene has been introduced.
  • a reporter gene eg, luciferase, GFP, etc.
  • the expression level of CARD14 can be evaluated by measuring the expression level of the reporter gene using a conventional method.
  • the screening method of the present invention can also be performed using as an index whether or not a test substance inhibits the function of CARD14.
  • CARD14 belongs to the membrane-bound guanylate kinase (MAGUK) family and functions as a molecular scaffold that forms an assembly of multiprotein complexes specifically for the plasma membrane region.
  • this protein belongs to the CARD family, has a specific caspase-related recruitment domain (CARD), specific interaction with BCL10 and phosphorylation of BCL, NF- ⁇ B activation and cell apoptosis Since it is also known to function as a positive regulator of inhibitors, inhibitors can be screened using such a function as an index.
  • CARD caspase-related recruitment domain
  • the in vitro psoriasis, multiple sclerosis, TLR-based / IMQ / immunostimulatory adjuvant for reducing side effects or for ⁇ T cell mediated diseases and / or immune interventions Using a model of complications to inhibit the function of CARD14 to reduce side effects from psoriasis, multiple sclerosis, TLR-based / IMQ / immunostimulatory adjuvants, or for ⁇ T cell mediated diseases and / or immune interventions Substances that act to treat or prevent complications can also be screened in one step.
  • the method includes the following steps (1) to (3): (1) Target cells for diseases (eg, psoriasis, multiple sclerosis, to reduce side effects due to TLR-based / IMQ / immunostimulatory adjuvants, or complications of ⁇ T cell-mediated diseases and / or immune interventions) (2) contacting the test substance in the presence and absence of CARD14 (2) diseases in the cells under each condition (for example, psoriasis, multiple sclerosis, TLR-based / IMQ / immune stimulating adjuvant side effects Response (eg psoriasis-specific response, multiple sclerosis-specific response, TLR-related response, complication-specific symptoms) to reduce or to complications of ⁇ T cell mediated disease and / or immune intervention)
  • Step (3) for measuring the degree of reaction CARD14 suppresses the reaction in the presence of CARD14 as compared to the case where measurement is performed in the absence of the test substance.
  • CARD14 In the absence of a test substance that did not suppress the reaction, the function of CARD14 is inhibited to reduce side effects caused by diseases (eg, psoriasis, multiple sclerosis, TLR base / IMQ / immunostimulatory adjuvant, Or a ⁇ T cell-mediated disease and / or a complication of immune intervention) or the like.
  • diseases eg, psoriasis, multiple sclerosis, TLR base / IMQ / immunostimulatory adjuvant, Or a ⁇ T cell-mediated disease and / or a complication of immune intervention
  • the medicament of the present invention may be administered per se or as an appropriate pharmaceutical composition.
  • the pharmaceutical composition used for administration may contain the medicament of the present invention and a pharmacologically acceptable carrier, diluent or excipient.
  • Such pharmaceutical compositions are provided as dosage forms suitable for oral or parenteral administration.
  • injections are dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, infusions, and the like. May be included.
  • Such an injection can be prepared according to a known method.
  • a method for preparing an injection it can be prepared, for example, by dissolving, suspending or emulsifying the nucleic acid of the present invention in a sterile aqueous liquid or oily liquid usually used for injection.
  • an aqueous solution for injection for example, an isotonic solution containing physiological saline, glucose and other adjuvants, and the like are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castoroil)) May be.
  • alcohol eg, ethanol
  • polyalcohol eg, Propylene glycol, polyethylene glycol
  • nonionic surfactants eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castoroil)
  • oily liquid for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent.
  • the prepared injection solution is preferably filled in
  • compositions for oral administration include solid or liquid dosage forms, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups Agents, emulsions, suspensions and the like.
  • Such a composition is produced by a known method and may contain a carrier, a diluent or an excipient usually used in the pharmaceutical field.
  • a carrier and excipient for tablets for example, lactose, starch, sucrose, and magnesium stearate are used.
  • the above parenteral or oral pharmaceutical composition is conveniently prepared in a dosage unit form suitable for the dose of the active ingredient.
  • dosage form of such a dosage unit include tablets, pills, capsules, injections (ampoules), and suppositories.
  • the medicament of the present invention has low toxicity and can be used as it is as a liquid or as a pharmaceutical composition of an appropriate dosage form for humans or mammals (eg, rats, rabbits, sheep, pigs, cattle, cats, dogs, monkeys, etc.). It can be administered orally or parenterally (eg, intravascular administration, subcutaneous administration, etc.).
  • humans or mammals eg, rats, rabbits, sheep, pigs, cattle, cats, dogs, monkeys, etc.
  • parenterally eg, intravascular administration, subcutaneous administration, etc.
  • the oligonucleotide of the present invention can be synthesized by standard methods known in the art, for example, by using an automated DNA synthesizer (commercially available from Biosearch, Applied Biosystems, etc.). is there.
  • an automated DNA synthesizer commercially available from Biosearch, Applied Biosystems, etc.
  • Example 1 Evaluation of immunological function in CARD14-deficient mice
  • CARD14-deficient mice were generated and two mouse psoriasis-like models (one is a model induced by IMQ cream and the other is recombinant IL-23 protein applied to the mouse ear). was used to assess their immunological function.
  • CARD14 causes TLR7-mediated and / or other innate immunity that results in the activation of NF- ⁇ B in the innate immune system of the skin This is based on the hypothesis of the present inventors that it may be involved in a receptor-mediated intracellular signal transduction pathway.
  • mice and methods (Materials and methods) (Mouse strain) Card14 ⁇ / ⁇ (details are described below and in FIG. 2), Tlr7 ⁇ / ⁇ , Tlr9 ⁇ / ⁇ and Tlr7 ⁇ / ⁇ Tlr9 ⁇ / ⁇ double deficient mice (Onishi et al., 2015) They were raised in these animal facilities and maintained under specific pathogen removal conditions.
  • Rag2 ⁇ / ⁇ Il2rg ⁇ / ⁇ mice were obtained from Taconic Biosciences.
  • Tcrd ⁇ / ⁇ mice were obtained from Jackson Laboratories.
  • C57BL / 6 (WT) mice were purchased from CLEA Japan, Inc. (Tokyo, Japan). All experiments were conducted in accordance with institutional ethics guidelines for the management and use of laboratory animals from the Institute of Pharmaceutical Sciences, Health and Nutrition.
  • mice Card14 ⁇ / ⁇ mice were generated by Lexicon Pharmaceuticals (The Woodlands, TX, USA) using a targeting vector designed to remove exon 2 and part of exon 3 by homologous recombination. PCR was used for routine mouse genotyping. The following primers were used.
  • mice were backcrossed at least 8 times against the C57BL / 6 background and determined using the marker-assisted speed congenic method. As a result, more than 99% of the B6 background genome was replaced in the N8 generation (Markel et al., 1997, Nature Genetics. 17: 280-284).
  • mice were not treated with cream. As previously described, either alone or containing 500 ng of recombinant mouse IL-23 (BioLegend), intradermal injections of 20 ⁇ l PBS were used every other day using a 30 gauge needle. The test was carried out for 5 days on both ears of anesthetized mice (Non-Patent Document 8 (Zheng et al., 2007)). Ear measurements were made at the center of the ear using a constant pressure thickness gauge (PG-16J, Teclock, Japan). Mice were sacrificed at various time points and tissues were collected.
  • PG-16J constant pressure thickness gauge
  • BM cells (5 ⁇ 10 6 ) were obtained by flushing the femur and tibia of donor WT and donor Card14 ⁇ / ⁇ mice.
  • Mice received a lethal dose of X-ray (9 Gy) and were then reconstituted by intravenous injection of BM cells via the orbital venous plexus.
  • the treatment with IMQ cream was performed 6 weeks after reconstitution.
  • mice were sacrificed and ears were collected and fixed with 10N formalin for subsequent embedding in paraffin.
  • Paraffin tissue sections (4 ⁇ m in diameter) were deparaffinized and rabbit anti-mouse polyclonal antibody against CARD14 (1: 100; Proteintech, Chicago, IL, USA), rabbit anti-mouse polyclonal antibody against keratin 5 (1: 400; Covance, Berkeley, CA, USA) and stained with a rabbit anti-mouse monoclonal antibody (Thermo Fisher Scientific) against Ki67. Sections were treated in 0.01 M citric acid (adjusted to pH 6.0) for 10 minutes at 80 ° C. before adding the primary antibody.
  • the secondary antibody used was a peroxidase labeled polymer (Dako) conjugated to goat anti-rabbit immunoglobulin.
  • Dako peroxidase labeled polymer conjugated to goat anti-rabbit immunoglobulin.
  • 3,3′-diaminobenzidine (Dako) as a chromogenic source resulted in brown staining (Uchio et al., 2002, Lab Invest. 82: 619-628). All slides were scanned at 20x absolute magnification using Aperio ScanScope (Aperio Technologies).
  • Epidermal cell suspensions were prepared by incubating the epidermis with trypsin-EDTA for 45 minutes at 37 ° C.
  • a dermal suspension was obtained using a buffer containing 1.6 mg / mL collagenase IV (Worthington) and 0.1 mg / mL Dnase-I (Sigma) in complete RPMI medium (Nacalai Tesque).
  • the cells were then filtered through a 70 ⁇ m pore size cell strainer (BDFalcon) to obtain a single cell suspension (Nakashima et al. The Journal of Allergy and Clinical Immunology. 134: 100-107, 2014; Tokura et al , 1994, The Journal of Investigative Dermatology. 102: 31-38).
  • RNA extraction and real-time quantitative PCR Stored cells were harvested by centrifugation at 400 xg for 5 minutes. RNA was isolated using the Qiagen RNeasy kit (Qiagen). Subsequently, RNA was reverse transcribed into cDNA using reverse transcriptase (ReverTra Ace, Toyobo, Osaka, Japan) and oligo dT primer.
  • the real-time PCR mix consists of a total volume of 20 ⁇ L, 10 ⁇ L SYBR Green Master mix (Roche), forward and reverse primers (CARD14 and glyceraldehyde-3-phosphate dehydrogenase; 200 nM for GAPDH) and 2 ⁇ L cDNA Consists of samples.
  • Primers for amplification are as follows. mCARD14, 5'-TGCATAGCTCCCGTTTCAC-3 '(forward) (SEQ ID NO: 8) 5'-GGAACTTCAGGCTTTCCAGA-3 '(reverse) (SEQ ID NO: 9) mGAPDH, 5′-CAAGATTGTCAGCAATGCATCC-3 ′ (forward) (SEQ ID NO: 10) 5′-CCTTCCACAATGCCAAGTTG-3 ′ (reverse) (SEQ ID NO: 11)
  • the PCR reaction was performed for 40 cycles (95 ° C. for 15 minutes, 60 ° C. for 1 minute) in a MicroAmp optical 384 well reaction plate from LightCycler 480 System II (Roche Life Science).
  • the fluorescence signal ( ⁇ Ct) detected during the threshold cycle was recorded by software installed on the instrument.
  • ⁇ Ct fluorescence signal
  • anti-CD8a Ly-2, APC
  • anti-CD45 (30-F11, PerCP / Cy5.5
  • anti-mouse ⁇ TCR GL3, PE
  • anti-IL-17A TC11-18H10
  • PE anti-NK1.1
  • BioLegend anti-CD3e (134-2C11, PerCP / Cy5.5); anti-CD4 (RM4-5, PE / Cy7); anti-CD11b (M1 / 70, FITC); anti-CD45 (30 -F11, PE / Cy7); and anti-TCR ⁇ (H57-597, PerCP / Cy5.5).
  • the following antibodies were purchased from eBioscience: anti-CD19 (1D3, PacificBlue); anti-IL-22 (IL22JOP, APC); and anti-TCR ⁇ (GL3, APC).
  • FACS analysis the fixed area of the ear was scrubbed using Trephine (8-mm, Kai Industries Co., Ltd.). Cells were preincubated with Fc-block (BioLegend) for 15 minutes and then labeled with the appropriate cell surface antibody for 30 minutes at 4 ° C.
  • Fc-block BioLegend
  • intracellular cytokine staining cells were stimulated with 50 ng / ml PMA (Sigma) and 500 ng / ml ionomycin (Sigma) and treated with GolgiStop (BD Bioscience) for 5 minutes at 37 ° C.
  • BD Cytofix / Cytoperm fixation / permeabilization kit (BDBiosciences) and then stained with antibodies at 4 ° C. for 30 minutes.
  • a BD LSR II flow cytometer was used for FACS analysis.
  • a FACSAria flow cytometer was used for cell sorting. Samples were then analyzed using FlowJo software (Treestar).
  • TLR7 single deficient mice developed psoriatic dermatitis similar to WT mice (data is Not shown).
  • TLR9 single deficient (Tlr9 ⁇ / ⁇ ) mice showed the same phenotype as WT mice (data not shown).
  • Tlr7 ⁇ / ⁇ Tlr9 ⁇ / ⁇ mice were compared to WT mice when TLR7 and TLR9 double deficient (Tlr7 ⁇ / ⁇ Tlr9 ⁇ / ⁇ ) mice were evaluated in the same experimental setup.
  • CARD14 is essential for the development of skin inflammation in both the IMQ-induced psoriasis-like model and the IL-23-induced psoriasis-like model
  • CARD14-deficient mice FIG. 2
  • IMQ-induced psoriasis-like model IL-23-induced psoriasis-like model.
  • Non-Patent Document 3 van der Fits et al., 2009
  • Non-Patent Document 8 Zheng et al., 2007
  • CARD14 in hematopoietic cells plays an important role in psoriasis, but CARD14 in radioresistant skin resident cells is not
  • CARD14 has been shown to be important for innate immunity of keratinocytes (Non-patent document 12 (Fuchs-Telem et al., 2012); Non-patent document 9 (Jordan et al., 2012)
  • Their above results suggest a potential role for CARD14 in other cell types, suggesting that CARD14 functions downstream of IL-23 signaling.
  • BM reciprocal bone marrow
  • BM cells from WT mice and Card14 ⁇ / ⁇ mice were transplanted into X-irradiated Card14 ⁇ / ⁇ mice and WT mice, respectively. Six weeks later, the mice were treated for 6 consecutive days with IMQ cream on both ears. Psoriasis-like inflammation was induced in Card14 ⁇ / ⁇ and WT mice transplanted with BM from WT mice, but induced in irradiated WT mice or Card14 ⁇ / ⁇ mice transplanted with Card14 ⁇ / ⁇ BM Not (FIG. 4A).
  • CARD14 in non-hematopoietic cells containing keratinocytes plays a role in psoriasis-like inflammation cannot be ruled out, the above results indicate that CARD14 in BM-derived cells, potentially ⁇ T cells, is IMQ-induced psoriasis. It strongly suggests that it is necessary for the onset of dermatitis.
  • CARD14 is expressed in ⁇ T cells and is required for the production of IL-17 and IL-22 by ⁇ T cells
  • qPCR real-time quantitative polymerase chain reaction
  • the present inventors have used the antibodies against CARD14 and T cell receptor ⁇ (TCR ⁇ ) 5 days after the administration of IL-23 to co-relate ear skin. Focus microscopy analysis was performed. A CARD14 positive signal was detected in the epidermal area of WT mice that received IL-23, while no CARD14 positive signal was detected in Card14 ⁇ / ⁇ mice that received IL-23.
  • Non-patent Document 7 (Cai et al., 2011)
  • IL-23R IL-23 receptor
  • IMQ-induced dermatitis and IL-23-induced dermatitis are closely related to the production of IL-17 and IL-23 (Cai et al., 2011); Pantelyushin et al. , 2012, The Journal of Clinical Investigation. 122: 2252-2256; Non-Patent Document 8 (Zheng et al., 2007)), we have developed WT mice and Card14 ⁇ / ⁇ that have received IMQ or IL-23. Cells producing IL-17 and IL-22 recovered from mouse epidermal sheets were examined using intracellular staining (FIGS. 5D and 5E).
  • CARD14 does not interfere with the adjuvant effect of IMQ on co-administered antigen
  • IMQ is an agonist of TLR7, a vaccine adjuvant commonly used in many other applications
  • CARD14 affects IMQ's adjuvant effect on co-administered antigens. It was targeted.
  • Immunized with ovalbumin (OVA) via intradermal injection in both ears, while IMQ cream was applied topically to the same ear.
  • OVA ovalbumin
  • IMQ cream has an adjuvant effect on the co-administered protein antigen and CARD14 does not affect the adjuvant effect. Since IMQ-induced psoriasis is a complication of IMQ cream, the development of CARD14 inhibitors may inhibit the pathogenesis of psoriatic dermatitis without affecting IMQ-induced innate immune function and its adjuvant effect is there.
  • CARD14 is required for optimal production of IL-17 and IL-22 by epidermal ⁇ T cells and is induced by both IMQ and IL-23.
  • CARD14 is responsible for most psoriasis-like dermatitis in mouse experimental models.
  • CARD14 and CARD11 were shown to be novel MAGUK family members that function as activators upstream of BCL10 and NF- ⁇ B signaling (Non-Patent Document 11 (Bertin et al., 2001)).
  • CARD14 is expressed in epithelial cells of skin lesions in patients with psoriasis (Harden et al., 2014, PloS One. 9: e111255), and the present inventors first identified CARD14 in epithelial stromal cells such as keratinocytes. We hypothesized to be a downstream signaling molecule for TLR7-mediated NF- ⁇ B activation.
  • CARD14 is expressed in ⁇ T cells in psoriatic-like skin rash in addition to its expression in epithelial keratinocytes (FIGS. 5A and 5C).
  • Experimental conditions differ between human patient samples and experimental mouse models, and the amount of TCR ⁇ cells is known to differ between humans and mice (Non-Patent Document 7 (Cai et al., 2011); Pasparakis et al., 2014, Nature reviews. Immunology. 14: 289-301).
  • the role of CARD14 in humans and mice may differ with respect to either CARD14-expressing cell types or experimental conditions. Further studies are needed to clarify the role of CARD14 in human ⁇ T cells in psoriasis.
  • CARD14 may be an excellent target for reducing the pathogenesis of psoriatic dermatitis and the side effects caused by IMQ-based adjuvants or other TLR-based adjuvants or immunostimulatory adjuvants. This inhibits innate immune activation without suppressing the adaptive immune response specific for the co-administered antigen.
  • the immunological significance of CARD14-mediated innate and adaptive immune responses should be studied.
  • Various immune disorders such as multiple sclerosis (Sutton et al., 2009, Immunity. 31: 331-341) and other infectious diseases (Carding and Egan, 2002, Nature reviews. Immunology. 2: 336-) 345 .; Sutton et al., 2012, European Journal of Immunology. 42: 2221-2231).
  • Example 2 Multiple sclerosis
  • Rui et al. Yamamoto Rui, Tasuku Honjo, and Shunsuke Chikuma.
  • Programmed cell death 1 inhibits inflammatory helper T-cell development through controlling the innate immune response.
  • PNAS 2013 110 40) 16073-16078 ).
  • EAE Experimental autoimmune encephalomyelitis
  • MS multiple sclerosis
  • mice CARD14 ⁇ / ⁇ mice were prepared by knocking out genes according to Rui et al.
  • mice were treated with 200 ⁇ g of MOG 35-55 peptide (peptide corresponding to MOG 35-55 residue), complete Freund's adjuvant (CFA), and 250 ⁇ g of heat-killed mycobacteria (HKMTB).
  • CFA complete Freund's adjuvant
  • HKMTB heat-killed mycobacteria
  • sc subcutaneous immunization with 0.2 mL of emulsion.
  • mice received two intraperitoneal (ip) injections of 200 ng pertussis toxin (PTX) at the time of immunization and 48 hours later.
  • PTX pertussis toxin
  • mice were observed daily, observed for clinical signs of disease, and EAE scores were assessed as follows. 0, no disease; 1, tail tail (limp tail); 2, hind limb weakness; 3, paralysis of one hind limb; 4, paralysis of both hind limbs; 5, limited movement; 6, Moribund or death.
  • the score is shown as the average clinical score for each experimental group.
  • spleens were harvested 8 or 30 days after immunization and CD4 + T cell enriched samples were obtained using anti-CD4 microbeads and autoMACS system (Miltenyi).
  • CD4 + T cell enriched samples were obtained using anti-CD4 microbeads and autoMACS system (Miltenyi).
  • 2 ⁇ 10 6 isolated CD4 + T cells were transformed into 2 ⁇ 10 6 mitomycin C-treated spleen cells from CARD-14 + / + mice in the presence of MOG 35-55 peptide (30 ⁇ g / mL). Incubated with. The supernatant was collected after 3 days for cytokine analysis.
  • PTX pertussis toxin
  • a therapeutic or prophylactic agent for psoriasis a therapeutic or prophylactic agent for multiple sclerosis, a therapeutic or prophylactic agent for reducing side effects caused by TLR-based / IMQ / immunostimulatory adjuvant, a ⁇ T cell-mediated disease and / or Or screen for agents to treat or prevent complications of immune intervention.
  • the inhibitory activity for CARD14 is measured by any method known in the art, the inhibitory activity of the candidate substance is measured, and those having inhibitory activity are identified as inhibitors.
  • CARD11 and CARD14 are novel caspase recruitment domain (CARD) / membrane-associated guanylate kinase (MAGUK) family members that interact with BCL10 and activate NF-kappa B.
  • CARD caspase recruitment domain
  • MAGUK membrane-associated guanylate kinase
  • the present invention finds industrial applicability in the biopharmaceutical industry and the pharmaceutical industry.

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Abstract

La présente invention concerne un agent thérapeutique ou un agent prophylactique pour le psoriasis, la sclérose en plaques, et la réduction des effets secondaires provoqués par les adjuvants à base de TLR/IMQ/immunostimulants. Il a été découvert que CARD14 est lié au psoriasis, à la sclérose en plaques, et à la réduction des effets secondaires provoqués par les adjuvants à base de TLR/IMQ/immunostimulants. Sur la base de cette découverte, est préparé un agent thérapeutique et un agent de diagnostic, et un criblage pour ces derniers. En particulier, il a été découvert que, dans l'expression dans les cellules hématopoïétiques, particulièrement les cellules γδT (plus particulièrement, les cellules épidermiques γδT), CARD14 est fortement lié au psoriasis, à la sclérose en plaques, et aux effets secondaires des adjuvants à base de TLR/IMQ/immunostimulants. Sur la base de cette découverte, sont fournis un agent thérapeutique et un agent de diagnostic, et leur criblage.
PCT/JP2016/004521 2015-10-09 2016-10-07 Thérapie, diagnostic, et criblage utilisant card14 WO2017061125A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060115455A1 (en) * 2004-10-22 2006-06-01 Reed Kenneth C Therapeutic RNAi agents for treating psoriasis

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060115455A1 (en) * 2004-10-22 2006-06-01 Reed Kenneth C Therapeutic RNAi agents for treating psoriasis

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CAI, YIHUA ET AL.: "Pivotal role of dermal IL-17-producing gammadelta T cells in skin inflammation", IMMUNITY, vol. 35, no. 4, 2011, pages 596 - 610, XP028330368 *
HARDEN, JAMIE L. ET AL.: "CARD14 expression in dermal endothelial cells in psoriasis", PLOS ONE, vol. 9, no. 11, 2014, pages 1 - 9, XP055376248 *
JORDAN, CATHERINE T. ET AL.: "PSORS2 is due to mutations in CARD14", AM. J. HUM. GENET., vol. 90, no. 5, 2012, pages 784 - 795, XP028481903 *
VAN DER FITS, LESLIE ET AL.: "Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL -23/ IL -17 axis", J. IMMUNOL., vol. 182, no. 9, 2009, pages 5836 - 5845, XP055376245 *

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