JPWO2017061125A1 - Treatment, diagnosis and screening with CARD14 - Google Patents

Abstract

The present invention provides a therapeutic or prophylactic agent for reducing side effects caused by psoriasis, multiple sclerosis, TLR base / IMQ / immunostimulatory adjuvant. CARD14 has been found to be associated with reduced side effects from psoriasis, multiple sclerosis, TLR-based / IMQ / immunostimulatory adjuvant, and provides therapeutic agents, diagnostic agents and screening thereof based on this. In particular, CARD14 has been found to be strongly associated with psoriasis, multiple sclerosis, TLR-based / IMQ / immunostimulatory adjuvant side effects for its expression in hematopoietic cells, particularly γδT cells, especially epidermal γδT cells. Providing therapeutic agents, diagnostic agents and screening thereof.

Description

  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 γδ 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.

  Psoriasis is a local T cell-mediated chronic inflammatory skin disorder characterized mainly by chronic erythematous plaques with very clear boundaries (Schon and Boehncke, 2005). The latest findings also suggest that topical application of imiquimod (IMQ) cream causes psoriasis-like skin inflammation in humans and mice (non-patent document 2: Gilliet et al., 2004; non-patent document). 3: van der Fits et al., 2009). IMQ is a specific agonist of mouse Toll-like receptor (TLR) 7, but 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.

  The important role of interleukin (IL) -17 and IL-23 in the pathogenesis of psoriasis has recently been clarified (Non-Patent Document 5: Johnson-Huang et al., 2012). Monoclonal antibodies against IL-23p19 (guselcoumab and tildrakizumab) showed significant clinical advantages over blocking the IL-12 / 23p40 subunit (Non-Patent Document 5: Johnson-Huang et al., 2012; Reference 6: Sofen et al., 2014). In this experimental setting, the absence of IL-23p19 in vivo suppresses IMQ cream-induced psoriasis-like skin inflammation (Non-Patent Document 3: van der Fits et al., 2009). Furthermore, intradermal injection of recombinant IL-23 protein in mice ears shared many features with psoriasis (eg, epikeratosis via IL-22 up-regulation and STAT3 activation) (non-patent literature). 7: Cai et al., 2011; Non-patent document 8: Zheng et al., 2007).

  Recently, caspase mobilization domain family member 14 (CARD14 [MIM60721], also known as CARMA2) at psoriasis susceptibility locus 2 (PSORS2) located in human chromosomal region 17q25.3 has a unique gain-of-function mutation. (Non-Patent Document 9: Jordan et al., 2012). This finding was further confirmed by genome-wide association analysis (GWAS) of PSORS2, which identified several risk-related variants including CARD14 (Non-Patent Document 10: Tsui et al., 2012). CARD14 has been shown to activate nuclear factor kappa B (NF-κB) via an IκB kinase complex in response to upstream stimuli (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 unknown (Non-Patent Document 12: Fuchs-Telem et al. , 2012; Non-Patent Document 9: Jordan et al., 2012).

Schon and Boehncke, 2005, The New England Journal of Medicine. 352: 1899-1912. Gilliet et al., 2004, Archives of Dermatology. 140: 1490-1495. van der Fits et al., 2009, Journal of Immunology. 182: 5836-5845. Walter et al., 2013, Nature Communications. 4: 1560. Johnson-Huang, L.M. et al., 2012, Disease Models & Mechanisms. 5: 423-433. Sofen et al., 2014, The Journal of Allergy and Clinical Immunology. 133: 1032-1040. Cai et al., 2011, Immunity. 35: 596-610. Zheng et al., 2007, Nature.445: 648-651. Jordan, C.T., et al., 2012, American Journal of Human Genetics. 90: 784-795. Tsoi et al., 2012, Nature Genetics. 44: 1341-1348. Bertin et al., 2001, The Journal of Biological Chemistry.276: 11877-11882. Fuchs-Telem et al., 2012, American Journal of Human Genetics. 91: 163-170.

  As a result of diligent research, the present inventors have found that 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. In particular, CARD14 in γδT cells producing IL-17 and IL-22 has been found to be essential for the formation of psoriasis-like dermatitis, a role associated with psoriasis of CARD14 in hematopoietic cells, and the role of specific cells The present invention was also completed.

  In addition, the present inventors have found that 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.

  Furthermore, the inventors have found that CARD14 in γδT cells can be a potential therapeutic target for γδT cell mediated diseases and complications of immune intervention, and complications of γδT 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.

  In addition, 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. .

Psoriasis is a chronic inflammatory skin disease of unknown cause but has also been reported as a complication of imiquimod (IMQ) cream. In recent years, CARD14 has been identified as a psoriasis susceptibility gene, but the immunological role in psoriasis pathogenesis in vivo remains unclear. Here, we evaluated CARD14 deficient (Card14 ^{− / −} ) 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. In both models, Card14 ^{− / −} mice showed significantly less increase in skin thickness and hyperkeratinization compared to wild type mice. CARD14 is expressed in and associated with epidermal γδ 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 γδT cells) but not in non-hematopoietic cells, is important for the formation of psoriasis-like dermatitis. became. The vaccine adjuvant effect of IMQ cream on co-administered protein antigens was not altered by CARD14 deficiency, which was inhibited by inhibiting CARD14 without affecting its adjuvant activity. Suggests that it can be reduced. From these findings, the present inventors have found that CARD14 in γδT cells strongly suggests that it may be a potential therapeutic target for γδT cell mediated diseases and complications of immune intervention.

As described above, 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 γδ T cell-mediated diseases and / or complications of immune intervention based on the expression of CARD14 in hematopoietic cells.
(5) The method according to any one of items 1 to 4, wherein the hematopoietic cells are γδT cells.
(6) The method according to any one of items 1 to 4, wherein the hematopoietic cells are epidermal γδ T cells.
(7) The method according to item 5 or 6, wherein the γδT cell is a cell that produces IL-17 and IL-22.
(8) The method according to any one of items 1 to 7, further based on non-expression of CARD14 in non-hematopoietic cells.
(9) The method according to item 8, wherein the non-hematopoietic cells include skin resident cells.
(10) The method according to any one of Items 1 to 9, wherein the disease includes IMQ-induced psoriasis and IL-23-induced psoriasis.
(11) A therapeutic or prophylactic agent for psoriasis comprising a hematopoietic cell-specific CARD14 inhibitor.
(12) A therapeutic or prophylactic agent for multiple sclerosis comprising a hematopoietic cell-specific CARD14 inhibitor.
(13) A therapeutic or prophylactic agent for reducing side effects caused by a TLR-based / IMQ / immunostimulatory adjuvant containing a hematopoietic cell-specific CARD14 inhibitor.
(14) A therapeutic or prophylactic agent for γδT cell-mediated diseases and / or complications of immune intervention, comprising a hematopoietic cell-specific CARD14 inhibitor.
(15) The therapeutic or prophylactic agent according to any one of items 11 to 14, wherein the hematopoietic cells are γδT cells.
(16) The therapeutic or prophylactic agent according to any one of items 11 to 14, wherein the hematopoietic cells are epidermal γδ T cells.
(17) The therapeutic or prophylactic agent according to item 15 or 16, wherein the γδ T cell is a cell that produces IL-17 and IL-22.
(18) The therapeutic or prophylactic agent according to any one of items 11 to 17, wherein the hematopoietic cell-specific CARD14 inhibitor is achieved by the method according to any one of items 1 to 10.
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. A method for screening a therapeutic or prophylactic agent for psoriasis based on the expression of CARD14 in hematopoietic cells, comprising a step determined to be usable as an agent or prophylactic agent.
(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. A method for screening a therapeutic or prophylactic agent for multiple sclerosis based on the expression of CARD14 in hematopoietic cells, which comprises a step determined to be usable as a therapeutic or prophylactic agent for the disease.
(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 screening method for a therapeutic or prophylactic agent for reducing side effects.
(4A) 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; Γδ T cell-mediated disease and / or immune intervention based on the expression of CARD14 in hematopoietic cells, comprising a step determined to be usable as a therapeutic or prophylactic agent for complications of mediated diseases and / or immune interventions Screening method for therapeutic or preventive agent for complications.
(5A) The method according to any one of Items 1A to 4A, wherein the hematopoietic cells are γδT cells.
(6A) The method according to any one of items 1A to 4A, wherein the hematopoietic cells are epidermal γδ T cells.
(7A) The method according to item 5A or 6A, wherein the γδT cells are cells that produce IL-17 and IL-22.
(8A) The method according to any one of items 1A to 7A, further comprising a step of determining that CARD14 is not expressed in non-hematopoietic cells.
(9A) The method according to items 1A to 8A, further comprising a step of bringing the candidate substance into contact with non-hematopoietic cells and selecting the candidate substance as the therapeutic agent or prophylactic agent when CARD14 has no substantial effect on expression. The method according to any one of the above.
(10A) The method according to item 8A or 9A, wherein the non-hematopoietic cells include skin resident cells.
(11A) A method according to any one of items 1A or 3A-10A, wherein the disease comprises IMQ-induced psoriasis and IL-23-induced psoriasis.
(12A) A therapeutic or prophylactic agent for psoriasis comprising a hematopoietic cell-specific CARD14 inhibitor.
(13A) A therapeutic or prophylactic agent for multiple sclerosis comprising a hematopoietic cell-specific CARD14 inhibitor.
(14A) A therapeutic or prophylactic agent for reducing side effects caused by a TLR-based / IMQ / immunostimulatory adjuvant containing a hematopoietic cell-specific CARD14 inhibitor.
(15A) A therapeutic or prophylactic agent for γδT cell-mediated diseases and / or complications of immune intervention, comprising a hematopoietic cell-specific CARD14 inhibitor.
(16A) The therapeutic or prophylactic agent according to any one of items 12A to 15A, wherein the hematopoietic cells are γδT cells.
(17A) The therapeutic or prophylactic agent according to any one of Items 12A to 15A, wherein the hematopoietic cells are epidermal γδ T cells.
(18A) The therapeutic or prophylactic agent according to item 16A or 17A, wherein the γδT cells are cells that produce IL-17 and IL-22.

  In the present invention, it is contemplated that one or more of the above features may be provided in further combinations in addition to the express combinations. Still further embodiments and advantages of the invention will be recognized by those of ordinary skill in the art upon reading and understanding the following detailed description as needed.

  According to the present invention, 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 Alternatively, prophylactic agents and therapeutic or prophylactic agents for γδT 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. Both ears from WT and Tlr7 ^{− / −} Tlr9 ^{− / −} mice (n = 4 mice / group) were treated with IMQ for 6 consecutive days or injected with 500 ng IL-23 every other day for 5 days. . (A) Ear thickness was measured daily before injection. Data are representative of 4 independent experiments, results are shown as mean ± standard deviation (sd), ^** indicates P <0.01; all Scheffe test. (B) H & E staining on the skin of control mice and IMQ mice on the last day of each group. The bar represents 100 μm. 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 μm. 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. (A) Structure of mouse Card14 locus (WT allele), Card14 targeting vector, and predicted mutant Card14 gene (mutated allele). Exons 2 and 3 of the Card14 gene were replaced with a neo resistance gene. Restriction enzymes: E, EcoRI; B, BamHI. (B) Card14 PCR genotyping of WT and mutant alleles using primers P1, P2 and P3. Genomic DNA was extracted from the mouse tail. The WT product was 350 bp and the knockout product was 250 bp. Lane M represents a base pair marker. (C) Card14 mRNA levels in epidermal cells and dermal cells from WT and Card14 ^{− / −} mice. Data is analyzed using real-time qPCR and shows Card14 expression compared to glyceraldehyde triphosphate dehydrogenase (GAPDH) (n = 4 mice / group). 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. Ears from WT mice and Card14 ^{− / −} mice (n = 4 mice / group) were treated with IMQ cream for 6 consecutive days or injected with IL-23 every other day for 5 days. (A) Ear thickness was measured daily before injection. Data are representative of at least 5 independent experiments and results are mean ± sd. d. ^* Indicates P <0.05, ^** indicates P <0.01; all are Scheffe tests. (B) H & E staining, keratin 5 staining, and Ki67 staining of the skin on the last day of mice receiving control and IMQ. The bar represents 100 μm. Data are representative of at least 5 independent experiments. (C) Ear thickness was monitored daily prior to IL-23 injection. Data are representative of 4 independent experiments and results are mean ± sd. d. ^** indicates P <0.01; all are Scheffe tests. (D) H & E staining, keratin 5 staining, and Ki67 staining of the skin on the last day of the experiment in PBS injected mice and IL-23 injected mice. The bar represents 100 μm. 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. Data are representative of 3 independent experiments and results are mean ± sd. d. ^** indicates P <0.01; all are Scheffe tests. (B) WT mice, Rag2 ^{− / −} mice, Rag2 ^{− / −} Il2rg ^{− / −} mice, and Tcrd ^{− / −} mice (n = 4 mice / group) were treated with IMQ cream for 6 consecutive days. Ear thickness was measured daily before cream treatment. Data are representative of 3 independent experiments and results are mean ± sd. d. ^* Indicates P <0.05, ^** indicates P <0.01; all are Scheffe tests. CARD14 is expressed in γδT cells and is required for the production of IL-17 and IL-22 by γδT cells. Ears from WT and Card14 ^{− / −} mice (n = 4 mice / group) were treated with IMQ or injected with IL-23 every other day for 5 days. (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-TCRγ) mAb (red). Merge is a diagram in which these are superimposed. The image was deconvoluted with a Z stack in 0.5 mm steps. The image was magnified 100 times. Data are representative of two independent experiments. (D and E) Epidermal cell suspensions from WT and Card14 ^{− / −} mice in IMQ model or IL-23 model were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin to induce intracellular IL− Expression of 17 and IL-22 was analyzed by flow cytometry. Local flow plots gated on CD45 ⁺ cells are representative of 3 independent experiments. Data are mean ± s. d. ^* Indicates P <0.05; Scheffe test. CARD14 does not interfere with the adjuvant effect of IMQ on co-administered antigens. For ovalbumin (OVA) immunization, WT, Tlr7 ^{− / −} Tlr9 ^{− / −} and Card14 ^{− / −} mice (n = 5-6 mice / group) were treated on day 0 and day 7. 100 μg of OVA protein was injected intradermally into the ear (20 μL / ear). On day 14, blood was collected and the antibody titer was measured. For the induction of psoriatic dermatitis, mouse ears were treated with IMQ cream for 6 consecutive days as previously described. (A) Ear thickness was measured daily prior to cream treatment and injection. Data are representative of 2 independent experiments and results are mean ± sd. d. ^* Indicates P <0.05; all Scheffe test. (B) and the OVA-specific IgG ₁ and _{IgG 2c} in the serum were measured using ELISA. Data are representative of 2 independent experiments and results are mean ± sd. d. ^** indicates P <0.01; all are Scheffe tests. FIG. 7 shows the symptom reduction effect of an experimental demyelinating disease model due to CARD14 deficiency. EAE scores of wild type (n = 8) and Card14 ^{− / −} mice (n = 9) after immunization with MOG / CFA / HKMTB in the presence of pertussis toxin (PTX) on day 0 and day 2. It is a result. Data are shown as mean ± SEM. 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.

  The present invention will be described below. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. Thus, it should be understood that singular articles (eg, “a”, “an”, “the”, etc. in the case of English) also include the plural concept unless otherwise stated. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

  In the present specification, “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 indicated. 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). As long as 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.

  As used herein, a “derivative”, “analog” or “variant” is preferably a region substantially homologous to a protein of interest (eg, CARD14), although not intended to be limiting. In various embodiments, 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. This is a product of a naturally occurring protein modified by amino acid substitutions, deletions and additions, respectively, and a derivative thereof that does not necessarily have the same degree of biological function of the naturally occurring protein. means. For example, the biological function of such proteins can be examined by suitable and available in vitro assays described herein or known in the art. As used herein, “functionally active” as used herein refers to the structural function of a protein, such as biological activity, according to the embodiment to which the polypeptide of the invention, ie, fragment or derivative, relates. It refers to a polypeptide, ie a fragment or derivative, having a regulatory function or a biochemical function. In the present invention, humans are mainly discussed for CARD14, but since many animals other than humans are known to express CARD14, these animals, particularly mammals, are also within the scope of the present invention. Is understood to enter.

Thus, 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 fragment thereof;
(F) a polynucleotide that hybridizes to the polynucleotide of any one of (a) to (e) under stringent conditions and encodes a polypeptide having biological activity; or (g) (a) to At least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one polynucleotide of (e) or its complementary sequence And a polynucleotide encoding a polypeptide having a biological activity. Here, the biological activity typically means that CARD14 can be distinguished from other proteins present in the same organism as the activity or marker.
As the 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 biological activity . Here, the biological activity can typically be distinguished from other proteins present in the same organism as the activity or marker of CARD14 (for example, it can function as a specific epitope when used as an antigen) Including the area).

  In the context of the present invention, “a substance that binds to CARD14”, “a binding agent for CARD14” or “a CARD14-interacting molecule” is a molecule or substance that binds to CARD14 at least temporarily. For detection purposes, preferably it is possible to indicate that it has been bound (eg, labeled or in a labelable state), and for therapeutic purposes, it is further advantageous that a therapeutic agent is further bound. . Examples of substances that bind to CARD14 include antibodies, antisense oligonucleotides, siRNAs, 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. As used herein, “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.

  As used herein, “protein”, “polypeptide”, “oligopeptide” and “peptide” are used interchangeably herein and refer to a polymer of amino acids of any 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). This definition also includes, for example, polypeptides containing one or more analogs of amino acids (eg, including unnatural amino acids, etc.), peptidomimetic compounds (eg, peptoids) and other modifications known in the art. Is done. In this specification, “amino acid” is a general term for organic compounds having an amino group and a carboxyl group. When an antibody according to an embodiment of the present invention includes a “specific amino acid sequence”, any amino acid in the 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. Even in such a case, it can be said that the protein according to the embodiment of the present invention includes the above-mentioned “specific amino acid sequence”. Examples of chemical modifications that amino acids contained in proteins undergo in vivo 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.

  As used herein, “polynucleotide”, “oligonucleotide” and “nucleic acid” are used interchangeably herein and refer to a polymer of nucleotides 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. Specific examples of such 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. Derivative converted to N3′-P5 ′ phosphoramidate bond, oligonucleotide derivative in which ribose and phosphodiester bond in oligonucleotide are converted to peptide nucleic acid bond, uracil in oligonucleotide is C- 5 Oligonucleotide derivatives substituted with propynyluracil, Oligonucleotide derivatives where uracil in the oligonucleotide is substituted with C-5 thiazole uracil, Oligonucleotide where cytosine in the oligonucleotide is 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-propylribose, and in oligonucleotides An oligonucleotide derivative in which the ribose is replaced with 2′-methoxyethoxyribose is exemplified. Unless otherwise indicated, 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)). As used herein, “nucleic acid” is also used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. In the present specification, the “nucleotide” may be natural or non-natural.

  As used herein, “gene” refers to a factor that defines a genetic trait, and “gene” may refer to “polynucleotide”, “oligonucleotide”, and “nucleic acid”.

  As used herein, “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. Thus, as used herein, 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. In the present specification, 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). In the present specification, 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.

  In one embodiment of the present invention, “several” may be, for example, 10, 8, 6, 5, 4, 3, or 2, or may be less than or equal to any of these values. Polypeptides that have been deleted, added, inserted, or replaced by other amino acids at one or several amino acid residues are known to 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. As a 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.

  In one embodiment of the present invention, “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. Methods for alignment, percentage calculation, comparison methods, and related computer programs are well known in the art (eg, BLAST, GENETYX, etc.). In the present specification, “homology” can be expressed by a value measured by NCBI BLAST unless otherwise specified. Blastp can be used as the default algorithm for comparing amino acid sequences with BLAST. Measurement results are quantified as Positives or Identities.

  As used herein, “polynucleotide hybridizing under stringent conditions” refers to well-known conditions commonly used in the art. Such 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. Specifically, 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. Means a polynucleotide that can be identified by washing the filter under conditions of 65 ° C using SSC (saline-sodium citrate) solution (composition of 1x concentrated SSC solution is 150 mM sodium chloride, 15 mM sodium citrate) To do. As the “stringent conditions”, for example, the following conditions can be adopted. (1) 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.), (2) 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. in a solution containing ml of denatured sheared salmon sperm DNA, then wash the filter with 1 × SSC at approximately 37-50 ° C. It should be noted that the formamide concentration may be 50% or more. The wash time may be 5, 15, 30, 60, or 120 minutes, or longer. Multiple factors such as temperature and salt concentration can be considered as factors affecting the stringency of hybridization reactions. For details, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995). . Examples of “highly stringent conditions” are 0.0015 M sodium chloride, 0.0015 M sodium citrate, 65-68 ° C., or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide, 42 ° C. Methods described in experiments such as hybridization, Molecular Cloning 2nd ed., Current Protocols in Molecular Biology, Supplement 1-38, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995) It can be performed according to. Here, a sequence that contains only the A sequence or only the T sequence is preferably excluded from the sequences that hybridize under stringent conditions. Moderate stringent conditions can be readily determined by those skilled in the art based on, for example, the length of the DNA, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Vol. 1, 7.42-7.45 Cold Spring Harbor Laboratory Press, 2001, and for nitrocellulose filters, 5 × SSC, 0.5% SDS, 1.0 mM ethylenediaminetetraacetic acid (EDTA) (pH 8.0) Other pre-wash solutions, such as about 50% formamide at about 40-50 ° C., 2 × SSC-6 × SSC (or Star's solution in about 50% formamide at about 42 ° C. The same hybridization solution), and the use of washing conditions of about 60 ° C., 0.5 × SSC, 0.1% SDS. Thus, 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.

  As used herein, a “purified” substance or biological factor (such as a nucleic acid or protein) refers to a substance from which at least a part of the factor naturally associated with the substance or biological factor has been removed. . Thus, typically, 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. As used herein, an “isolated” substance or biological agent (such as a nucleic acid or protein) 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.

  As used herein, 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). For example, an antisense molecule can be a similar part in an ortholog corresponding to a particular part of the antisense molecule. Corresponding amino acids are, for example, cysteinylated, glutathioneated, S—S bond formed, oxidized (eg, methionine side chain oxidation), formylation, acetylation, phosphorylation, glycosylation, myristylation, etc. Of amino acids. Alternatively, 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.

  As used herein, a “corresponding” gene (eg, a polynucleotide sequence or molecule) 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. Thus, a gene corresponding to a gene can be an ortholog of that gene. Thus, 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. Therefore, for example, a corresponding gene in an animal (for example, a mouse) 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.

  As used herein, “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.For example, in the case of a polypeptide, 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. In the case of polynucleotides, examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. Non-integer lengths (eg, 11 etc.) may also be appropriate as a lower limit. In the present specification, for example, when 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

  According to the present invention, 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.

  As used herein, “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. Examples of the antibody include, but are not limited to, generation of specific antibodies, enzyme activity, and imparting resistance. In the present invention, 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. And this protein belongs to the CARD family, has a specific caspase-related recruitment domain (CARD), specific interaction with BCL10 and phosphorylation of BCL, NF-κB Since it is also known to function as a positive regulator of activation and cell apoptosis, it can include, but is not limited to, its association with these functions. As used herein, a biological function can be exerted by “biological activity”. As used herein, “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. When two factors interact, 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 Sometimes, when other molecules co-precipitate, the two molecules are considered to be linked. Therefore, seeing such coprecipitation is one of the judgment methods. For example, when a factor is an enzyme, the biological activity includes the enzyme activity. In another example, when 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. Thus, “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.

  In the present specification, 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, γδ T cell mediated diseases and / or immune intervention complications, and / or TLR-based / IMQ / immunostimulatory adjuvants be able to.

  In the present specification, “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. Preferably, a gene, a polynucleotide or the like is transcribed and translated into a polypeptide form. However, transcription and production of mRNA are also an aspect of expression. Accordingly, “expression product” as used herein includes such a polypeptide or protein, or mRNA. More preferably, such polypeptide forms may be post-translationally processed. For example, the expression level of CARD14 can be determined by any method. Specifically, 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.

  In this specification, the “functional equivalent” refers to any target having the same target function but a different structure from the original target 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. In the present invention, it is understood that 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. As used herein, “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. Say. Electronic searches include BLAST (Altschul et al., J. Mol. Biol. 215: 403-410 (1990)), FASTA (Pearson & Lipman, Proc. Natl. Acad. Sci., USA 85: 2444- 2448 (1988)), Smith and Waterman method (Smith and Waterman, J. Mol. Biol. 147: 195-197 (1981)), and Needleman and Wunsch method (Needleman and Wunsch, J. Mol. Biol. 48: 443). -453 (1970)) and the like. Biological 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.

  As the functional equivalent of the present invention, 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. In the present specification, “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. As used herein, “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. Specific examples include non-polar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of positively charged (basic) amino acids include arginine, histidine, and lysine. Examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

In the present specification, an “inhibitor” or “inhibitor” or “inhibitor” (both in English corresponds to an inhibitor) refers to a target entity (eg, receptor or cell) of the receptor or cell. A substance or factor that inhibits a biological action. Examples of 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. Examples of such 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.

  As used herein, an “agonist” refers to a substance that expresses or enhances the biological action of a target entity (for example, a receptor). In addition to natural agonists (also called ligands), synthesized ones and modified ones can be mentioned. As used herein, the term “antagonist” refers to a substance that suppresses or inhibits the expression of a biological action of a target entity (for example, a receptor). In addition to natural antagonists, synthetic antagonists and modified ones can be mentioned. In addition to those that inhibit or inhibit competitively with agonists (or ligands), there are those that inhibit or inhibit non-competitively. It can also be obtained by modifying the agonist. 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”.

  As used herein, “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). In addition, such 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.

  In one embodiment of the present invention, 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). .).

  In one embodiment of the present invention, 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. For example, 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.”. Alternatively, monoclonal antibodies may be made by methods similar to recombinant methods such as those described in US Pat. No. 4,816,567. Alternatively, monoclonal antibodies can be obtained from "Clackson et al., Nature. 1991 Aug 15; 352 (6336): 624-628." Or "Marks et al., J Mol Biol. 1991 Dec 5; 222 (3): 581 It may be isolated from a phage antibody library using methods similar to those described in US Pat. Alternatively, the protein may be prepared by the method described in “Protein Experiment Hanbook, Yodosha (2003): 92-96”.

  In one embodiment of the present invention, 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 a variety of 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 US A. 1994 Feb 1; 91 ( 3): 969-973.), Or FR shuffle (Damschroder et al., Mol Immunol. 2007 Apr; 44 (11): 3049-3060. Epub 2007 Jan 22.). To alter (preferably improve) antigen binding, 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.). For example, FR residues important for antigen binding may be identified by modeling the interaction of CDR and FR residues. Alternatively, an unusual FR residue at a particular position may be identified by sequence comparison.

  In one embodiment of the present invention, 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. Antibody. The main production methods include a transgenic mouse method for producing human antibodies, a phage display method, and the like. In the transgenic mouse method for producing human antibodies, if a functional human Ig gene is introduced into a mouse in which endogenous Ig has been knocked out, human antibodies having various antigen-binding abilities can be produced instead of mouse antibodies. Furthermore, if this mouse is immunized, a human monoclonal antibody can be obtained by a conventional hybridoma method. For example, it can be prepared by the method described in “Lonberg et al., Int Rev Immunol. 1995; 13 (1): 65-93.”. In the 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 prepared by the method described in "Vaughan et al., Nat Biotechnol. 1996 Mar; 14 (3): 309-314."

  In one embodiment of the present invention, “anti-CARD14 antibody” includes an antibody having binding ability to CARD14. The method for producing this anti-CARD14 antibody is not particularly limited. For example, the anti-CARD14 antibody may be produced by immunizing mammals or birds with CARD14.

  The “functional equivalent” of “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. In addition to itself, chimeric antibody, humanized antibody, multifunctional antibody, bispecific or oligospecific antibody, single chain antibody, scFV, diabody, sc (Fv) 2 (single chain (Fv) 2) It is understood that scFv-Fc and the like are also included.

  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 an embodiment of the present invention may be an antibody fragment having an antigen binding activity (hereinafter also referred to as “antigen-binding fragment”). In this case, 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. Here, 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 one 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 an antibody obtained through a step of immunizing an organism with purified CARD14, CARD14-expressing cell vesicle or 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.

  The anti-CARD14 antibody according to one embodiment of the present invention 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.

  In one embodiment of the present invention, a “CDR (complementarity determining region)” is a region in an antibody that is actually in contact with an antigen to form a binding site. In general, the CDRs are located on the Fv (variable region: including heavy chain variable region (VH) and light chain variable region (VL)) of the antibody. In general, there are CDR1, CDR2, and CDR3 consisting of about 5 to 30 amino acid residues. In particular, it is known that the CDR of the heavy chain contributes to the binding of the antibody to the antigen. Among CDRs, CDR3 is known to have the highest contribution in binding of an antibody to an antigen. For example, "Willy et al., Biochemical and Biophysical Research Communications Volume 356, Issue 1, 27 April 2007, Pages 124-128" describes that the antibody binding ability was increased by modifying heavy chain CDR3. Has been. The Fv region other than CDR is called a framework region (FR) and consists of FR1, FR2, FR3 and FR4, and is relatively well conserved among antibodies (Kabat et al., “Sequence of Proteins of Immunological Interest”. US Dept. Health and Human Services, 1983.). That is, it can be said that the factor characterizing the reactivity of the antibody is the CDR, and particularly the heavy chain CDR.

  Several methods have been reported for determining the definition and location of CDRs. For example, Kabat definition (Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) or Chothia definition (Chothia et al., J. Mol. Biol , 1987; 196: 901-917) may be employed. In one embodiment of the present invention, the definition of Kabat is adopted as a preferred example, but is not necessarily limited thereto. In some cases, it may be determined in consideration of both Kabat definition and Chothia definition. It can also be a CDR. As a specific example of such a method, the method of Martin et al. Using Prox. Natl. Acad. Sci. USA, 1989; : 9268-9272). Such CDR information can be used to produce mutants that can be used in the present invention. In such antibody variants, one or several of the original antibody framework (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10) ) Substitutions, additions or deletions, but the CDRs can be produced without mutations.

  As used herein, “antigen” refers to any substrate that can be specifically bound by an antibody molecule. As used herein, “immunogen” refers to an antigen capable of initiating lymphocyte activation that produces an antigen-specific immune response. As used herein, “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.

  It will be understood that antibodies of any specificity may be used 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.

  In the present specification, 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). .

  In this specification, “drug”, “agent” or “factor” (both corresponding to “agent” in English) are used interchangeably in a broad sense, and so long as they can achieve their intended purpose. It 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. Typically, 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. Alternatively, specific ligands or receptors in the case of ligands, and substrates thereof when the polypeptide is an enzyme include, but are not limited to.

  As used herein, “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”.

  As used herein, “therapeutic agent (agent)” refers to any drug that can treat a target condition (for example, a disease such as an allergy) in a broad sense. In one embodiment of the present invention, 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. In addition, 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.

  As used herein, “prevention” refers to preventing a certain disease or disorder (eg, allergy) from becoming such a condition before it becomes such a condition. 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.

  As used herein, the term “prophylactic agent (agent)” refers to any agent that can prevent a target condition (for example, a disease such as allergy) in a broad sense.

  Therefore, in the present specification, 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.

  As used herein, 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, nucleic acid and protein reactions, protein-lipid interactions, nucleic acid-lipids Examples include, but are not limited to, interactions. Thus, when both a substance or factor is a nucleic acid, 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. Also, for example, when 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, interactions by receptor-ligand reactions, enzyme-substrate interactions, and the like. When the two substances or factors include proteins and nucleic acids, 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. By utilizing such a specific interaction or binding reaction, an object in a sample can be detected or quantified.

  As used herein, “detection” or “quantification” of polynucleotide or polypeptide expression includes mRNA measurement and immunological measurement methods, including, for example, binding or interaction with a detection agent, test agent or diagnostic agent. It can be achieved using any suitable method. Examples of molecular biological measurement methods include Northern blotting, dot blotting, and PCR. Examples of 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"). For protein arrays, see Nat Genet. 2002 Dec; 32 Suppl: 526-532. 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. Such further analysis methods are described in, for example, Genome Analysis Experimental Method / Yusuke Nakamura Laboratory Manual, Editing / Yusuke Nakamura Yodosha (2002), etc., all of which are incorporated herein by reference. Is done.

  In the present specification, the “expression level” refers to an amount at which a polypeptide or mRNA is 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 polypeptide of the present invention at the protein level, or the poly-polysaccharide used in the present invention evaluated by any suitable method including molecular biological measurement methods such as Northern blotting, dot blotting, and PCR. 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 that 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.

  As used herein, “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”.

  As used herein, “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.

In this specification, the “label” refers to a presence (for example, a substance, energy, electromagnetic wave, etc.) for distinguishing a target molecule or substance from others. Examples of such a labeling method include RI (radioisotope) method, fluorescence method, biotin method, chemiluminescence method and the like. When a plurality of markers of the present invention or a factor or means for capturing them are labeled by the fluorescence method, 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. When labeling a ligand, any substance that does not affect the function can be used, but Alexa ^™ 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 ^™ 555 and Alexa ^™ 633, a combination of Alexa ^™ 488 and Alexa ^™ 555, and the like. Any nucleic acid can be used as long as it can bind to its 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. Examples of 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. In the present invention, by using such a label, the target object can be modified so that it can be detected by the detection means used. Such modifications are known in the art, and those skilled in the art can appropriately carry out such methods depending on the label and the target object.

  In this specification, 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. Say. 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). In the present specification, 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.

  In the present specification, the “instruction” describes a 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. In addition, 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 instructions are so-called package inserts and are usually provided in a paper medium, but are not limited thereto, and are 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.

  In the present invention, 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. Therefore, as 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 Substances that inhibit translation of CARD14 from mRNA (eg, antisense nucleic acid, miRNA), substances that degrade mRNA (eg, siRNA, ribozyme), and substances that inhibit post-translational modification of the initial translation product . Whichever stage is used can be preferably used.

  Here, a preferable example of the transcription product is mRNA.

  As a substance that specifically inhibits translation of CARD14 gene from mRNA to CARD14 (or degrades mRNA), 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 homology” in the present invention uses a homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool), and the following conditions (expected value = 10; allow gap; filtering = ON; Match score = 1; mismatch score = -3).

  More specifically, 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. (1) a base sequence that hybridizes under stringent conditions with a complementary strand sequence of the base sequence represented by SEQ ID NO: 1 or 3, and And a sequence encoding a protein having at least one function of CARD14 and a complementary or substantially complementary base sequence. The stringent conditions are as described above. The function of 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-κB activation and cell apoptosis, NF-κB activation, and BCL10 phosphorylation induction function Etc.

  As a preferable example of mRNA of CARD14 gene, human CARD14 mRNA containing the nucleotide 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 the like, and also 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.

Specifically, the nucleic acid containing any one of the following (1) to (3) is preferably exemplified as 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:
(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.

(1) Antisense nucleic acid against mRNA of CARD14 gene “Antisense nucleic acid against mRNA of CARD14 gene” in the present invention 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 are 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). They may be 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 Such as those having uncharged bonds (eg methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged bonds or sulfur-containing bonds (eg phosphorothioates, phosphorodithioates, etc.) Things such as proteins (eg, nucleases, nuclease inhibitors, toxins, antibodies, Null peptide, poly-L-lysine, etc.) and sugars (eg, monosaccharides), etc., side chain groups, intercurrent compounds (eg, acridine, psoralen, etc.), chelate compounds (eg, , Metals, radioactive metals, boron, oxidizing metals, etc.), alkylating agents, and modified bonds (eg, alpha anomeric nucleic acids) Also good. Here, the “nucleoside”, “nucleotide” and “nucleic acid” 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.

  As described above, the antisense nucleic acid may be DNA or RNA, or may be a DNA / RNA chimera. When the antisense nucleic acid is DNA, 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. In view of easiness of synthesis, antigenicity, intracellular migration, and the like, an oligonucleotide consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases is preferable, but not limited thereto. Specifically, the 5 ′ end hairpin loop, 5 ′ end 6-base pair repeat, 5 ′ end untranslated region, translation start codon, protein coding region, open reading frame (ORF) translation stop codon of CARD14 gene, 3 ′ 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.

  Furthermore, 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 ( A triplex) that can inhibit transcription to RNA (antigene).

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. For example, in order to prevent degradation by a hydrolase such as nuclease, 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. In addition, 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. Furthermore, 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, etc. Is mentioned.

  The conformation of the sugar part of RNA is dominated by C2′-endo (S type) and C3′-endo (N type). In single-stranded RNA, it exists as an equilibrium between the two, but double-stranded 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. Commun., 1653-9, 2002; Jepsen, JS et al., Oligonucleotides, 14, 130-46, 2004) and ENA (Morita, K. et al., Nucleosides Nucleotides Nucleic Acids, 22, 1619-21, 2003) can also be preferably used.

  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. In addition, any of the above-described antisense nucleic acids containing various modifications can be chemically synthesized by a method known per se.

(2) 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. When the mRNA of the CARD14 gene has a double-stranded structure by itself, 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. [Proc. Natl. Acad. Sci. USA, 98 (10): 5572-5577 (2001)]. Furthermore, when 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)].

(3) siRNA against CARD14 mRNA
In the present specification, a double-stranded RNA consisting of an oligo RNA complementary to the CARD14 gene mRNA and its complementary strand, so-called siRNA, is also complementary or substantially complementary to the nucleotide sequence of the CARD14 gene mRNA. Defined as encompassed by nucleic acid containing base sequence or part thereof. When a short double-stranded RNA is introduced into a cell, a phenomenon called RNA interference (RNAi), in which mRNA complementary to the RNA is degraded, 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 basically has a length of 15 to 50 bases, preferably 19 to 49 bases, more preferably 19 to 27 bases. 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 additional base sequences include ug-3 ′, uu-3 ′, tg-3 ′, tt-3 ′, ggg-3 ′, guuu-3 ′, gttt-3 ′, and tttt-3. Examples of such sequences include, but are not limited to, ', uuuu-3'.

  Further, siRNA may have a protruding portion sequence (overhang) at the 3 ′ end, and specifically includes those to which dTdT (dT represents a deoxythymidine residue of deoxyribonucleic acid) is added. . Further, it may be a blunt end (blunt end) without end addition.

  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. Can be mentioned. 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. For a candidate group of target sequences selected based on the above-mentioned rules and others, whether or not there is homology in a continuous sequence of 16-17 bases in mRNA other than the target is determined by BLAST (http: //www.ncbi.nlm Investigate using homology search software such as .nih.gov / BLAST /) to confirm the specificity of the selected target sequence. For the target sequence confirmed to be specific, 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. In addition, for the short hairpin RNA (shRNA) that is a precursor of siRNA, an arbitrary linker sequence (for example, about 5-25 bases) capable of forming a loop structure is appropriately selected, and the sense strand and the antisense strand are combined with each other. It can be designed by linking via a linker sequence.

The sequence of siRNA and / or shRNA can be searched using search software provided free of charge on various web sites. Examples of such 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 the 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. However, in the case of siRNA, RNAi activity may be lost when all ribonucleoside molecules in natural RNA are replaced with a modified form. Therefore, the minimum modified nucleoside capable of functioning RNA-induced silencing complex (RISC) Need to be introduced.

Specifically, in order to improve a part of the nucleotide molecule constituting siRNA, natural DNA, stability (chemical and / or enzyme) and specific activity (affinity with RNA) as the modification. In addition, it can be substituted with RNA having various chemical modifications (see Usman and Cedergren, 1992, TIBS 17,34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163). For example, in order to prevent degradation by a hydrolase such as nuclease, a phosphate residue (phosphate) of each nucleotide constituting siRNA is changed to a chemically modified phosphate such as phosphorothioate (PS), methylphosphonate, phosphorodithionate, etc. It can be substituted with a residue. In addition, 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.) or a fluorine atom (—F) may be substituted. Furthermore, 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. In addition, the method for modifying an antisense nucleic acid described in (1) above can be used. Or you may give the chemical modification (2'-deoxylation, 2'-H) which substitutes a part of RNA in siRNA with DNA. Alternatively, an artificial nucleic acid (LNA: Locked 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 may be used.

  In addition, 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.

  For siRNA, the sense strand and the antisense strand of the 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.

  In the present specification, 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 containing 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 (for example, about 5 to 25 bases) long enough to form a suitable loop structure. It can be prepared by designing RNA and synthesizing it with an automatic DNA / RNA synthesizer. There are tandem type and stem loop (hairpin) type in vectors expressing shRNA. In the former, 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). It is. On the other hand, the latter is one in which an shRNA expression cassette is inserted into a vector, in which shRNA is expressed in cells and processed by dicer to form dsRNA. As a promoter, a pol II promoter (for example, a CMV immediate early promoter) can be used, but a pol III promoter is generally used in order to cause transcription of a short RNA accurately. Examples of 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.

  The siRNA or shRNA expression cassette thus constructed is then inserted into a plasmid vector or viral vector. As such vectors, virus vectors such as retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes virus, Sendai virus, animal cell expression plasmids and the like are used.

  The 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. Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, Usman et al., 1987 J. Am. Chem. Soc., 109, 7845, Scaringe et al., 1990 Nucleic Acids Res., 18, 5433, and the method described in US Pat. No. 6,00131111. Specifically, it can be synthesized using a nucleic acid protecting group (for example, dimethoxytrityl group at the 5 'end) and a coupling group (for example, phosphoramidite at the 3' end) known to those skilled in the art. That is, 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. In the case of siRNA containing modified RNA or DNA, a modified RNA (for example, 2′-O-methyl nucleotide, 2′-deoxy-2′-fluoronucleotide) may be used as a raw material, and the coupling reaction These conditions can be adjusted as appropriate. When introducing a phosphorothioate bond in which the phosphate binding moiety is modified, a borage reagent (3H-1,2-benzodithiol-3-one 1,1-dioxide) can be used.

  Alternatively, oligonucleotides may be synthesized separately and joined together after synthesis, for example by ligation (Moore et al., 1992, Science 256,9923; Draper et al. International Publication WO 93/23569; Shabarova et al. , 1991, Nucleic Acids Research 19,4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16,951; Bellon et al., 1997, Nucleosides & Nucleotides, Bellon et al., 1997, Bioconjugate Chem. 8, 204), or They may be joined together by hybridization after deprotection. siRNA molecules can also be synthesized by tandem synthesis. That is, 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.

  Another preferred example of 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 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. Examples of 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. Examples of 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. Such 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.

  In the present invention, the “substance that inhibits the function of CARD14” may be any substance as long as CARD14 once produced functionally inhibits its function.

Specifically, 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. In addition to a complete antibody molecule, for example, Fab, Fab ′, F (Ab ′) ₂ such as a fragment, scFv, scFv-Fc, a conjugate molecule prepared by genetic engineering such as a minibody, diabody, or a molecule having a protein stabilizing action such as polyethylene glycol (PEG) They may be modified derivatives thereof.

  In a preferred embodiment, since an antibody against CARD14 is used as a pharmaceutical for human administration, the antibody (preferably a monoclonal 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. In addition, 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 weight compound suitable for Lipinski's Rule. Such a compound can be obtained, for example, using the screening method of the present invention described later.

  According to the present invention, a CARD14 inhibitor, such as a substance that inhibits the expression or function of CARD14, is a therapeutic or preventive 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 γδ T cell-mediated diseases and / or immune intervention.

(Preferred embodiment)
Hereinafter, preferred embodiments of the present invention will be described. The embodiments provided below are provided for a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention with reference to the description in the present specification. It will also be appreciated that the following embodiments of the invention may be used alone or in combination.

<Therapeutic or preventive agent for psoriasis, and screening method>
In one aspect, the present invention provides a screening method for a therapeutic or prophylactic agent for psoriasis based on the expression of CARD14 in hematopoietic cells. Although 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 γδT cells, especially epidermal γδT cells. It is not provided for the first time in the present invention. Although not wishing to be bound by theory, in particular, CARD inhibitors such as agents that suppress or eliminate CARD14 expression in hematopoietic cells, particularly γδT cells, and preferably epidermal γδT 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.

  Examples of 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.

  In one embodiment, the targeted γδ T cells are cells that produce interleukin-17 (IL-17) and interleukin-22 (IL-22).

  In the screening method of the present invention, 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 γδT cell, particularly preferably epidermal γδT cell). For example, it is preferable to confirm that CARD14 is not expressed in skin resident cells). Here, in a preferred embodiment, it is desirable that hematopoietic cells and non-hematopoietic cells are derived from the same allogeneic strain, more preferably from the same individual. In one preferred embodiment, 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. In this specification, “there is no substantial influence” means that the CARD 14 can exert its function even if there is an influence.

  Psoriasis targeted by the present invention can include, for example, imomomid (IMQ) -induced psoriasis and IL-23-induced psoriasis.

  Therefore, the therapeutic agent or prophylactic agent of the present invention is preferably delivered specifically to hematopoietic cells (particularly γδT cells, particularly preferably epidermal γδT cells).

  In the present invention, specific delivery to a hematopoietic cell is achieved by a delivery system led by a specific molecule such as an antibody specific to the hematopoietic cell (for example, γδT cell).

<Therapeutic or preventive agent for multiple sclerosis and screening method thereof>
In one aspect, 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. Although 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 γδT cells, especially epidermal γδT cells. It has not been issued and is provided for the first time in the present invention. Although not wishing to be bound by theory, in particular, CARD inhibitors such as agents that suppress or eliminate CARD14 expression in hematopoietic cells, in particular γδT cells, and preferably epidermal γδT cells, are used to treat multiple sclerosis. It is understood that it can be used as an agent or prophylactic agent. In the present invention, 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. 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.

  Examples of 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.

  In one embodiment, the targeted γδ T cells are cells that produce interleukin-17 (IL-17) and interleukin-22 (IL-22).

  In the screening method of the present invention, in order to provide a better therapeutic or prophylactic agent, cells that are not hematopoietic cells (particularly γδT cells, particularly epidermal γδT cells) (“non-hematopoietic cells”). It is preferable to confirm that CARD14 is not expressed in the skin resident cells). Here, in a preferred embodiment, it is desirable that hematopoietic cells and non-hematopoietic cells are derived from the same allogeneic strain, more preferably from the same individual. In one preferred embodiment, 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.

  Therefore, the therapeutic agent or prophylactic agent of the present invention is preferably delivered specifically to hematopoietic cells (particularly γδT cells, particularly preferably epidermal γδT cells).

  In the present invention, specific delivery to a hematopoietic cell is achieved by a delivery system led by a specific molecule such as an antibody specific to the hematopoietic cell (for example, γδT cell).

<Therapeutic or preventive agent for reducing side effects caused by TLR base / IMQ / immunostimulatory adjuvant and screening method thereof>
In one aspect, 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. Although 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 γδT cells, particularly epidermal γδT 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. Although not wishing to be bound by theory, in particular, CARD inhibitors such as agents that suppress or eliminate CARD14 expression in hematopoietic cells, particularly γδT cells, and preferably epidermal γδT 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. As demonstrated herein, both 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. While it is still unclear which ligands or stimuli are responsible for TLR7- and TLR9-mediated innate immune activation in the development of IMQ-induced dermatitis, the above findings suggest that IMQ-induced psoriasis-like models and IL- A clear difference in innate immunity signal between the 23-induced psoriasis-like model will be demonstrated. Through such knowledge, a therapeutic or prophylactic agent for reducing side effects caused by TLR base / IMQ / immunostimulatory adjuvant can be screened. It can be an excellent target for reducing the side effects caused by IMQ-based adjuvants or other TLR-based adjuvants or immunostimulatory adjuvants. This will inhibit innate immune activation without suppressing the adaptive immune response specific to the co-administered antigen. 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.

  As used herein, “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 immunostimulatory adjuvant (such as CpG) is administered. Examples of such 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 from TLR-based / IMQ / immunostimulatory adjuvants. As introduced in this paper, the disease of psoriasis can occur as a side effect as an adjuvant of TLR7 ligand, and topical application of imiquimod (IMQ) cream causes psoriasis-like skin inflammation in humans and mice It has also been reported that Thus, it is understood that side effects due to TLR-based / IMQ / immunostimulatory adjuvants can be prevented or treated with the CARD14 inhibitors of the present invention.

  Examples of 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.

  In one embodiment, the targeted γδ T cells are cells that produce interleukin-17 (IL-17) and interleukin-22 (IL-22).

  In the screening method of the present invention, in order to provide a better therapeutic or preventive agent, cells that are not hematopoietic cells (especially γδT cells, particularly epidermal γδT cells) (“non-hematopoietic cells”, for example, It is preferable to confirm that CARD14 is not expressed in the skin resident cells). Here, in a preferred embodiment, it is desirable that hematopoietic cells and non-hematopoietic cells are derived from the same allogeneic strain, more preferably from the same individual. In one preferred embodiment, 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.

  Therefore, the therapeutic agent or prophylactic agent of the present invention is preferably delivered specifically to hematopoietic cells (particularly γδT cells, particularly preferably epidermal γδT cells).

  In the present invention, specific delivery to a hematopoietic cell is achieved by a delivery system led by a specific molecule such as an antibody specific to the hematopoietic cell (for example, γδT cell).

<Therapeutic or preventive agent for γδT cell-mediated diseases and / or complications of immune intervention and screening method thereof>
In one aspect, 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. Although CARD14 is a conventionally known molecule, its expression or expression in hematopoietic cells, particularly γδT cells, particularly epidermal γδT cells, can be used to treat or prevent γδT 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. Without wishing to be bound by theory, in particular, CARD inhibitors such as agents that suppress or eliminate CARD14 expression in hematopoietic cells, particularly γδT cells, and preferably epidermal γδT cells, are γδT 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.

  Examples of 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.

  In one embodiment, the targeted γδ T cells are cells that produce interleukin-17 (IL-17) and interleukin-22 (IL-22).

  In the screening method of the present invention, in order to provide a better therapeutic or prophylactic agent, cells that are not hematopoietic cells (particularly γδT cells, particularly epidermal γδT cells) (“non-hematopoietic cells”). It is preferable to confirm that CARD14 is not expressed in the skin resident cells). Here, in a preferred embodiment, it is desirable that hematopoietic cells and non-hematopoietic cells are derived from the same allogeneic strain, more preferably from the same individual. In one preferred embodiment, 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.

  Therefore, the therapeutic agent or prophylactic agent of the present invention is preferably delivered specifically to hematopoietic cells (particularly γδT cells, particularly preferably epidermal γδT cells).

  In the present invention, specific delivery to a hematopoietic cell is achieved by a delivery system led by a specific molecule such as an antibody specific to the hematopoietic cell (for example, γδT cell).

  As used herein, “γδT cell-mediated disease” refers to any disease mediated by γδT cells. Examples of “γδT cell-mediated disease” include psoriasis, multiple sclerosis, inflammation, and cytokineemia. But are not limited to these.

  In this specification, “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.

<Details of screening method>
When screening for a compound that inhibits CARD14 expression or function, 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). . In particular, such cells are preferably hematopoietic cells, preferably γδ T cells, more preferably γδ T cells of the epidermis. Alternatively, genetically modified cells may be used. Specifically, 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. DNA containing a base sequence encoding a polypeptide that hybridizes under a gentle condition and has a function equivalent to that of the protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or 4, preferably SEQ ID NO: 2, It can be prepared by ligating downstream of a promoter in an expression vector and introducing it into a host animal cell.

  In one embodiment, 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. Reduced amount of test substance to reduce side effects due to psoriasis, multiple sclerosis, TLR-based / IMQ / immunostimulatory adjuvant, or to treat complications of γδ T cell mediated diseases and / or immune interventions and // things used for prevention Including selecting as a quality candidate. In one embodiment, 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.

  In another embodiment, the present invention provides (A) a step of contacting CARD14 with a cell membrane fraction of hematopoietic cells (particularly γδT cells, particularly epidermal γδT 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 γδ T cell mediated diseases and / or immune interventions The process of selecting as is included.

  In a further embodiment, the present invention provides for the treatment of psoriasis, multiple sclerosis, TLR-based / IMQ / immunostimulatory adjuvant side effects, or the treatment of complications of γδ T cell mediated diseases and / or immune interventions and / or Alternatively, 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 γδ 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 γδ T cell mediated diseases and / or complications of immune intervention Further comprising.

  In another embodiment, the present invention provides (A) contacting hematopoietic cells (particularly γδT cells, particularly preferably epidermal γδT 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 γδ 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 γδT cell mediated diseases and / or immune interventions, psoriasis, multiple sclerosis, TLR-based in the absence of CARD14 A test substance that has reduced the side effects of / IMQ / immunostimulatory adjuvant or that has not suppressed the response of γδ T cell mediated diseases and / or complications of immune intervention has been shown to inhibit the function of CARD14, As a candidate for a substance used to reduce side effects due to multiple sclerosis, TLR-based / IMQ / immunostimulatory adjuvant, or for the treatment and / or prevention of complications of γδ T cell mediated diseases and / or immune interventions Including a step of selecting.

  In conducting the screening of the present invention, examples of the test substance 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.

  In addition, when selecting a substance that decreases the expression level of CARD14 or CARD14 gene, or a substance that decreases the function of CARD14, a control cell that is not contacted with a test substance can also be used as a comparative control. Here, “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. The 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 μM, for example. Examples of the incubation time include about 10 minutes to about 24 hours.

  When the CARD14-producing cells are provided in the form of a non-human mammal individual, the state of the animal individual is not particularly limited. For example, a psoriasis disease model animal in which inflammation has been induced by, for example, a drug or genetic modification (for example, implementation) IMQ-induced psoriatic dermatitis exemplified in Example 1, IL-23-induced psoriatic dermatitis, etc.), multiple sclerosis model animals (for example, experimental autoimmune encephalomyelitis used in Example 2) (Experimental autoimmune encephalomyelitis: EAE model, etc.), model animals of side effects caused by TLR-based / IMQ / immunostimulatory adjuvant (IMQ-induced psoriatic dermatitis of Example 1, IL-23-induced psoriatic dermatitis, etc.), Or a model animal of γδ T cell-mediated disease and / or complication of immune intervention (IMQ-induced psoriatic dermatitis of Example 1, IL-23-induced psoriatic dermatitis, etc. It may be equal. 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, preferably 1 to 3 times a day, 1 to 14 days. .

  Alternatively, 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. Provided is 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”). Alternatively, 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”).

  Therefore, more specifically, 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 γδ 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 The amount of the protein under both conditions is measured and compared using the detection antibody of the present invention, psoriasis, multiple sclerosis, TLR Methods of screening for substances that can be used to reduce side effects due to base / IMQ / immunostimulatory adjuvants or to prevent and / or treat complications of γδT cell mediated diseases and / or immune interventions are provided.

  That is, screening for a substance that changes the expression level of CARD14 can be performed as follows.

  (I) Normal or disease model (eg, to reduce side effects from psoriasis, multiple sclerosis, TLR-based / IMQ / immunostimulatory adjuvant, or complications of γδ T cell-mediated disease and / or immune intervention) A test substance is administered to a human mammal (for example, 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 (for example, 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 conventional method, or can be quantified by a Northern blot analysis known per se. On the other hand, the protein amount of CARD14 can be quantified using Western blot analysis or various immunoassay methods described in detail below.

  (Ii) 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 (i) above Can be quantified and analyzed.

  Detection and quantification of the expression level of the CARD14 gene (mRNA) 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. Such a probe or primer can be designed using, for example, primer 3 (http://primer3.sourceforge.net/) or vector NTI (manufactured by Infomax) based on the base sequence of the CARD14 gene. .

When using Northern blotting, 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 After hybridization, 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 ( A method of detecting and measuring with BAS-1800II (manufactured by Fuji Film) or a fluorescence detector can be exemplified. In addition, using the AlkPhos Direct Labeling and Detection System (manufactured by GE Healthcare), 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 (manufactured by GE Healthcare) can also be used.

  When using the RT-PCR method, a 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 in which a primer (a normal strand that binds to the cDNA (-strand), a reverse strand that binds to a + strand) is hybridized with this, and a PCR method is performed according to a conventional method, and the resulting amplified double-stranded DNA is detected Can be illustrated. In addition, 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. For example, 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). Also, prepare an RT-PCR reaction solution according to the protocol with SYBR Green RT-PCR Reagents (Applied Biosystems), and react 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 a substance that changes the expression level of CARD14 can also be performed by a reporter gene assay using the transcriptional regulatory region of CARD14 gene. Here, the “transcriptional regulatory region” usually refers to a range from several kb to several tens of kb upstream of the chromosomal gene. For example, (i) 5′-race method (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.), oligocap method, S1 primer mapping; The 5′-upstream region can be obtained using a method manufactured by the same company, etc., and the obtained upstream region can be identified by a technique including a step of measuring promoter activity.

  A reporter protein expression vector is constructed by linking a nucleic acid encoding a reporter protein (hereinafter referred to as “reporter gene”) in a functional manner downstream of the transcriptional regulatory region of the CARD14 gene. The vector may be prepared by a method known to those skilled in the art. For example, it is cut out according to the usual genetic engineering technique 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. The transcriptional regulatory region of the CARD14 gene can be incorporated on a plasmid containing a reporter gene.

  Reporter proteins include β-glucuronidase (GUS), luciferase, chloramphenicol transacetylase (CAT), β-galactosidase (GAS), green fluorescent protein (GFP), yellow fluorescent protein (YFP), blue fluorescent protein ( CFP), red fluorescent protein (RFP) and the like.

  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.

  In addition, as a method for measuring the expression level of the reporter gene, a method corresponding to each reporter gene may be used. For example, when a luciferase gene is 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. In this case, a commercially available luciferase reaction detection kit such as Picker Gene Dual Kit (registered trademark; manufactured by Toyo Ink) can be used.

  As a method for measuring the amount of protein of CARD14, specifically, for example, (i) 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 using a radiation measuring instrument (BAI-1800II: manufactured by Fuji Film Co., Ltd.), a fluorescence detector or the like. Moreover, after using the antibody which recognizes CARD14 as a primary antibody, it detects according to this protocol using ECL Plus Western Blotting Detection System (made by GE Healthcare), and uses multi-bio-magerium STORM860 (made by GE Healthcare). It can also be measured.

  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.

  Methods for producing these antibodies are already well known, and the antibodies of the present invention can also be produced according to these conventional methods (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.12-11.13).

  In the above quantification method (ii), it is desirable that the two antibodies recognize different portions of CARD14. For example, if 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.

As 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. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are used. As the enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used. Furthermore, 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.

  In the insolubilization of the antigen or antibody, physical adsorption may be used, or a chemical bond usually used for insolubilizing and immobilizing proteins or enzymes may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass.

  In the sandwich method, 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. By measuring the amount or activity of the labeling agent, 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. Further, in the immunoassay by the sandwich method, 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.

  In the competition method, 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. In this reaction method, 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), and 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).

  In the immunometric 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. After reacting with an excess amount of labeled antibody, solid phase CARD14 is added to bind the unreacted labeled antibody to the solid phase, the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to quantify the amount of antigen in the sample solution.

  In nephrometry, the amount of insoluble precipitate produced as a result of the antigen-antibody reaction in a 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.

  In applying these individual immunological measurement methods to the quantification method of the present invention, special conditions, operations and the like are not required to be set. What is necessary is just to construct | assemble the measurement system of CARD14, adding the usual technical consideration of those skilled in the art to the usual conditions and operation method in each method. For details of these general technical means, it is possible to refer to reviews, books and the like.

  For example, Hiroshi Irie “Radioimmunoassay” (Kodansha, published in 1974), Hiroshi Irie “Continue Radioimmunoassay” (published in Kodansha, 1974), “Enzyme Immunoassay” edited by Eiji Ishikawa et al. 53)), edited by Eiji Ishikawa et al. “Enzyme Immunoassay” (2nd edition) (Medical Shoin, published in 1982), edited by Eiji Ishikawa et al. 62), “Methods in ENZYMOLOGY” Vol. 70 (Immunochemical Techniques (Part A)), Id. Vol. 73 (Immunochemical Techniques (Part B)), Id. Vol. 74 (Immunochemical Techniques (Part C)), Id. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), ibid.Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid.Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal A) ntibodies)) (published by Academic Press).

  As described above, the amount of CARD14 in a cell can be quantified with high sensitivity by using the detection antibody of the present invention.

  For example, in the above screening method, 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. When more than about 50% is inhibited, 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 γδ 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.

  Alternatively, in the screening method described above, 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. Such 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. When such cells are used, 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. In addition, this protein belongs to the CARD family and has a specific caspase-related recruitment domain (CARD), specific interaction with BCL10 and phosphorylation of BCL, NF-κB 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.

In yet another embodiment of the invention, the in vitro psoriasis, multiple sclerosis, TLR-based / IMQ / immunostimulatory adjuvant for reducing side effects or for γδ 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 γδ 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 γδ 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 γδ 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. 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 γδ T cell-mediated disease and / or a complication of immune intervention) or the like.

<Pharmaceutical>
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.

  As a composition for parenteral administration, for example, injections, suppositories and the like are used. 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. As 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. As 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))) etc. May be. As the 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 a suitable ampoule. Suppositories used for rectal administration may be prepared by mixing the nucleic acid with a normal suppository base.

  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. As the carrier and excipient for tablets, for example, lactose, starch, sucrose, and magnesium stearate are used.

  The above parenteral or oral pharmaceutical compositions are conveniently prepared in dosage unit form to suit the dosage of the active ingredient. Examples of the 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.).

(General technology)
The molecular biological techniques, biochemical techniques, and microbiological techniques used herein are well known and commonly used in the art, and include those already cited, for example, Sambrook J. et al. al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, FM (1987) Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, FM (1989) .Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience; Innis, MA (1990) .PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, FM (1992) .Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub.Associates; Ausubel, FM (1995) .Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. A ssociates; Innis, MA et al. (1995) .PCR Strategies, Academic Press; Ausubel, FM (1999) .Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, JJ et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, a separate volume of experimental medicine "Gene Transfer & Expression Analysis Experimental Method" Yodosha, 1997, etc. The part to be (which may be all) is incorporated by reference.

  For DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, see, for example, Gait, MJ (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, MJ (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991) .Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, RL et al. (1992) .The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, GM et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, GT (I996). Described in Bioconjugate Techniques, Academic Press, etc. Which are incorporated herein by reference in the relevant part.

  For example, it is possible herein to synthesize the oligonucleotides of the invention by standard methods known in the art, for example, by using automated DNA synthesizers (such as those commercially available from Biosearch, Applied Biosystems, etc.). is there. For example, phosphorothioate oligonucleotides can be synthesized by the method of Stein et al. (Stein et al., 1988, Nucl. Acids Res. 16: 3209) or controlled pore glass polymer supports (Sarin et al. , 1988, Proc. Natl. Acad. Sci. USA 85: 7448-7451), etc., can also be used to prepare methylphosphonate oligonucleotides.

  In this specification, “or” is used when “at least one or more” of the items listed in the text can be adopted. The same applies to “or”. In this specification, when “in the range of two values” is specified, the range includes the two values themselves.

  References such as scientific literature, patents and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as if each was specifically described.

  The present invention has been described with reference to the preferred embodiments for easy understanding. In the following, the present invention will be described based on examples, but the above description and the following examples are provided only for the purpose of illustration, not for the purpose of limiting the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described in the present specification, but is limited only by the scope of the claims.

(Example 1: Evaluation of immunological function in CARD14-deficient mice)
In this example, CARD14-deficient mice were generated and two mouse psoriasis-like models (one is a model induced by IMQ cream and the other is the recombinant IL-23 protein into the mouse ear). Was used to assess their immunological function.

  Because little is known about the immunological role of CARD14 in psoriasis-like dermatitis in vivo, CARD14 causes TLR7-mediated and / or other innate immunity that results in the activation of NF-κB 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.

(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.

(Generation of knockout mice and genotyping)
Card14 ^{− / −} mice were generated by Lexicon Pharmaceuticals (The Woodlands, TX, USA) using targeting vectors designed to remove exon 2 and a portion of exon 3 by homologous recombination. PCR was used for routine mouse genotyping. The following primers were used.
P1, 5'-GGGTGTTCCTCTGACTCTCCCAGTTGGATG-3 '(SEQ ID NO: 5)
P2, 5'-GCTGACCGCTTCCTCGTGCTTTACGGTATC-3 '(SEQ ID NO: 6)
P3, 5'-CAGTGACTCAAGGAGGGGCAAACGCCTATG-3 '(SEQ ID NO: 7)
Card14 ^{− / −} targeted mice were backcrossed at least 8 times against the C57BL / 6 background and determined using a 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).

(Psoriasis-like dermatitis model)
As previously described, for the induction of psoriatic dermatitis, Beselna containing 62.5 mg of 5% IMQ for 6 consecutive days (sold as Aldara ^™ outside Japan; 3M Pharmaceticals, St Paul Minn) cream (purchased from Mochida Pharmaceutical Co., Ltd.) was used to treat both ears of mice (Non-Patent Document 3 (van der Fits et al., 2009)).

  Control 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 μl PBS were used every other day using a 30 gauge needle. The test was performed on both ears of anesthetized mice for 5 days (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.

(Bone marrow chimera generation)
BM cells (5 × 10 ⁶ ) were obtained by flushing the femur and tibia of donor WT and donor Card14 ^{− / −} mice. Mice (WT and Card14 ^{− / −} ) 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.

(Histopathology of ear skin)
On the last day of the experiment, mice were sacrificed and ears were collected and fixed with 10N formalin for subsequent embedding in paraffin. Paraffin tissue sections (4 μm 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. Using 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).

(Immunofluorescence staining)
Mice were sacrificed and their ears were collected. The ear skin tissue was snap frozen at −80 ° C. in mounting medium (Tissue-Tek OCT compound, Sakura Finetechnical. Co., Tokyo, Japan). Staining for CARD14 and γδTCR was performed on 6 μm sections of all mouse ears using a cryostat (Leica). Sections are typically fixed with 4% paraformaldehyde for 15 minutes at room temperature (RT) and blocked with 2% goat serum in PBS containing 1% BSA and 0.2% Triton X-100 for 1 hour at room temperature. And incubated with anti-mouse γδTCR (1:50; BD PharMingen, GL3, PE) and rabbit anti-mouse polyclonal antibody against CARD14 (1: 150) overnight at 4 ° C. After washing in PBS, the sections were incubated with AlexaFluor 488 conjugated donkey anti-rabbit IgG (H + L) (1: 250; Invitrogen) for 1 hour at room temperature, then washed in PBS and co-cultured using DAPI. Stained. Images were acquired using a LeicaTCS SP2 confocal microscope system and analyzed using Volocity (version 6.2.1).

(Preparation of ear cells)
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 μm 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 is in a total volume of 20 μL, 10 μL SYBR Green Master mix (Roche), forward and reverse primers (CARD14 and glyceraldehyde-3-phosphate dehydrogenase; 200 nM for GAPDH) and 2 μL 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 (ΔCt) detected during the threshold cycle was recorded by software installed on the instrument. In order to normalize target gene levels for variability in RNA and cDNA quality, GAPDH was amplified under the same conditions as the internal control.

(Flow cytometry analysis and cell sorting)
The following antibodies were purchased from BDPharMingen: anti-CD8a (Ly-2, APC); anti-CD45 (30-F11, PerCP / Cy5.5); anti-mouse γδ TCR (GL3, PE); anti-IL-17A (TC11-18H10) , PE); and anti-NK1.1 (PK136, PE). The following antibodies from BioLegend were used: 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-TCRβ (H57-597, PerCP / Cy5.5). The following antibodies were purchased from eBioscience: anti-CD19 (1D3, PacificBlue); anti-IL-22 (IL22JOP, APC); and anti-TCRγδ (GL3, APC). For 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. For 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. For intracellular staining, cells were fixed and permeabilized with a 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).

(Application of OVA + IMQ cream and measurement of antibody response)
Intradermal injections of 20 μl PBS were given on days 0 and 7, either alone or containing 100 μg LPS free OVA (Seikagaku). The antibody titer was measured on the 14th day after blood collection. Mouse ears were treated with IMQ for 6 consecutive days as previously described to induce psoriatic dermatitis. OVA specific antibodies were quantified using ELISA. Briefly, flat bottom 96-well MaxiSorp plates (Nunc) were coated with 50 μg / mL OVA in 0.5 M NaHCO ₃ . Plates were incubated overnight at 4 ° C., washed with PBS containing 0.05% Tween 20, and blocked for 1 hour at room temperature. After washing, 50 μL of diluted serum was incubated for 2 hours at room temperature. The plates were then washed and incubated with alkaline phosphate conjugated goat anti-mouse IgG ₁ antibody and IgG _2C antibody (1: 4000, Southern Biotech) for 2 hours at room temperature. Plates were washed and then bound antibody was visualized using TMB substrate solution (Kirkegaard & PerryLaboratories). The reaction was stopped by adding 2N H ₂ SO ₄ and the absorbance at 450 nm was read on a BioStack ^™ (BioTek Instruments Inc.) microplate reader using the Gen5 ELISA (Biotek®) program.

(Statistical analysis)
Mann-Whitney U test or Scheffe test was used to test for statistical significance of differences between groups. Statistical significance was accepted as P <0.05.

(Results and Discussion)
(Both TLR7 and TLR9 are required for IMQ-induced psoriatic dermatitis but not for IL-23-induced psoriatic dermatitis)
To evaluate the role of CARD14 in the innate immune system in psoriasis pathogenesis in vivo, we utilized two established mouse psoriasis-like models. One model is derived by topical application of Aldara ^™ cream containing IMQ to both ears for 6 days (Non-Patent Document 3 (van der Fits et al., 2009)). Since IMQ is a TLR7 agonist, we used TLR-deficient mice to assess whether TLR7 is essential or unnecessary in our experimental psoriasis model. Consistent with previous reports (Walter et al., 2013), TLR7 single deficient (Tlr7 ^{− / −} ) mice developed psoriatic dermatitis similar to WT mice (data is Not shown). Similarly, TLR9 single deficient (Tlr9 ^{− / −} ) mice showed the same phenotype as WT mice (data not shown). In marked contrast, Tlr7 ^{− / −} Tlr9 ^{− / −} mice were compared to WT mice when evaluating TLR7 and TLR9 double deficient (Tlr7 ^{− / −} Tlr9 ^{− / −} ) mice in the same experimental setup. Thus, the last 3 days of application showed a marked decrease in ear thickness, epidermal hyperkeratosis and dermal cell infiltration (FIGS. 1A and 1B). Although previously speculated by many groups (Pantelyushin et al., 2012, The Journal of Clinical Investigation. 122: 2252-2256; Non-Patent Document 4 (Walter et al., 2013)) The results showed for the first time evidence that IMQ-induced psoriatic dermatitis requires both TLR7 and TLR9.

  Next, we have used another established psoriatic dermatitis model (Zheng et al., 2007) induced by administration of IL-23, using TLR7 and / or Or the potential involvement of TLR9 was examined. Consistent with previous reports, injections of recombinant IL-23 on days 0, 2 and 4 significantly increased ear thickness with epidermal thickening and dermal inflammatory cell infiltration on day 5 Occurred. All of these changes were independent of TLR7 or TLR9 (FIGS. 1C and 1D). These results indicate that both TLR7 and TLR9 signaling in combination is necessary to fully develop IMQ-induced psoriatic dermatitis, while the IL-23-induced psoriatic dermatitis model is , Demonstrated direct activation downstream of TLR7 and TLR9-mediated innate immune activation. While it is still unclear which ligands or stimuli are responsible for TLR7- and TLR9-mediated innate immune activation in the development of IMQ-induced dermatitis, the above findings suggest that IMQ-induced psoriasis-like models and IL- A clear difference in the signal of innate immunity between the 23-induced psoriasis-like model was demonstrated.

(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)
In order to better characterize the role of CARD14 in vivo, we generated CARD14 deficient (Card14 ^{− / −} ) mice (FIG. 2), an IMQ-induced psoriasis-like model and an IL-23-induced psoriasis-like model. (Non-Patent Document 3 (van der Fits et al., 2009); Non-Patent Document 8 (Zheng et al., 2007)) were evaluated for the role of CARD14. We applied IMQ cream for 6 consecutive days or injected IL-23 every other day for 5 days. WT mice had worsened skin condition after IMQ application and increased skin thickness, but significantly protected Card14 ^{− / −} mice from skin inflammation (FIG. 3A). Histochemical experiments showed that hyperproliferating cells stained for keratin 5 and Ki67 in the ear skin of Card14 ^{− / −} mice were similar to those in control mice, while excess in WT mice that received IMQ application. Proliferating cells revealed a dramatic increase (FIG. 3B). Furthermore, IL-23-induced ear swelling was significantly suppressed in Card14 ^{− / −} mice (FIG. 3C). Epidermal hyperkeratosis (seen as hyperproliferating cells stained for keratin 5 and Ki67) and dermal inflammation (seen as infiltrating cells in H & E staining) are both reduced in Card23 ^{− / −} mice injected with IL-23. (FIG. 3D). These results indicate that CARD14 is essential for the formation of psoriatic dermatitis induced by application of IMQ cream and injection of IL-23. Thus, the inventors have found that CARD14 is required for signaling downstream of TLR7 and TLR9 and also required for signaling downstream of IL-23 that does not require TLR7 and TLR9. Rather, it was concluded that it is related to signaling pathways in adaptive immune cells.

(CARD14 in hematopoietic cells plays an important role in psoriasis, but CARD14 in radioresistant skin resident cells is not)
Although 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)), the present invention Their above results suggest a potential role for CARD14 in other cell types, suggesting that CARD14 functions downstream of IL-23 signaling. To further clarify this issue, we created a reciprocal bone marrow (BM) chimera and further assessed the role of CARD14 using an IMQ-induced psoriasis-like model. BM cells derived from WT mice and Card14 ^{− / −} mice were transplanted into Card14 ^{− / −} mice and WT mice irradiated with X-rays, 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). This indicates that CARD14 expression in hematopoietic BM-derived cells is essential for the pathogenesis of psoriasis in the IMQ-induced model, but CARD14 expression in radioresistant skin resident cells was not essential. In addition to the RAG gene, we have the potential contribution of T and B cells controlled by natural killer (NK) cells, natural killer T (NKT) cells, natural lymphocytes (ILC) and γδ T cells. Clarified about. In the T cell receptor δ deficient (Tcrd ^{− / −} ) mice lacking γδ T cells and in Rag2 ^{− / −} Il2rg ^{− / −} mice lacking T cells, NK cells, NKT cells and ILCs. We compared the severity of psoriasis-like models. Consistent with previous reports (Pantelyushin et al., 2012, The Journal of Clinical Investigation. 122: 2252-2256), Tcrd ^{− / −} mice and Rag2 ^{− / −} Il2rg ^{− / −} mice were treated with IMQ cream. Showed a marked decrease in skin swelling compared to WT mice (FIG. 4B). Although the possibility that 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 γδT cells, is IMQ-induced psoriasis. It strongly suggests that it is necessary for the onset of dermatitis.

(CARD14 is expressed in γδT cells and is required for the production of IL-17 and IL-22 by γδT cells)
To test CARD14 expression in several different cell types, cells from ear skin were isolated and mRNA was measured using real-time quantitative polymerase chain reaction (qPCR). Consistent with previous reports (Nordan et al., 2012), CARD14 mRNA was expressed at high levels in keratinocytes (FIG. 5A). Among immune cells, CARD14 mRNA in γδ T cells in ear skin was as high as keratinocytes and higher than other immune cell types (FIGS. 5A and 5B). To further elucidate the cell types that express CARD14, the inventors used an antibody against CARD14 and the T cell receptor γδ (TCRγδ) 5 days after the administration of IL-23 to co-relate the ear skin. Focus microscopy analysis was performed. A CARD14 positive signal was detected in the epidermal region of WT mice that received IL-23, whereas no CARD14 positive signal was detected in Card14 ^{− / −} mice that received IL-23. In both WT mice and Card14 ^{− / −} mice, a TCRγδ positive signal was detected in the peripheral region between the epidermis and the dermis, and a specific TCRγδ positive signal overlapped with a CARD14 positive signal expressed in the cytoplasm, On the other hand, no overlapping region was detected in Card14 ^{− / −} mice (FIG. 5C). These data indicate that CARD14 positive γδT details are present in psoriatic-like skin.

It has been shown that γδT cells producing IL-17 and IL-22 play an important role in the pathogenesis of psoriasis (Non-patent Document 7 (Cai et al., 2011)). It has also been reported that the majority of IL-23 receptor (IL-23R) expressing cells are γδT cells and that mutations of IL-23R were found in certain psoriasis patients (Cai et al. , 2011); Non-Patent Document 10 (Tsoi et al., 2012)). 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). WT mice treated with either IMQ or IL-23 showed a significantly higher number of epidermal TCRγδ ⁺ IL-17 and TCRγδ ⁺ IL-22 cells than the control group. In contrast, both IMQ-treated Card14 ^{− / −} mice and IL-23 treated Card14 ^{− / −} mice had significantly fewer epidermal γδ T cells producing IL-17 and IL-22 (FIG. 5D and 5E). These data suggest that CARD14 may play an important role in γδ T cells in psoriatic skin epidermal lesions that secrete IL-17 and IL-22.

(CARD14 does not interfere with the adjuvant effect of IMQ on co-administered antigen)
Since IMQ is an agonist of TLR7, a vaccine adjuvant commonly used in many other applications, we have determined that CARD14 affects IMQ's adjuvant effect on co-administered antigens. It was targeted. To solve this question, we modified the experimental protocol of a psoriatic-like dermatitis model using IMQ cream to replace WT mice, Tlr7 ^{− / −} Tlr9 ^{− / −} mice and Card14 ^{− / −} mice. Immunized with ovalbumin (OVA) via intradermal injection in both ears, while IMQ cream was applied topically to the same ear. Even though the increase in ear thickness was observed in WT mice, while mice were injected with OVA protein, Tlr7 ^{− / −} Tlr9 ^{− / −} mice and Card14 ^{− / −} mice consistently had ears on day 7. It was shown that the thickness of was significantly thin (FIG. 6A). WT mice were treated with IMQ cream with OVA immunization and, as expected, had significantly higher OVA-specific immunoglobulin (IgG) ₁ and IgG _2c antibody responses compared to the untreated OVA immunization group. Shown (FIG. 6B). OVA-specific _{IgG 2c} antibody ^{titer, Tlr7 - / - Tlr9 - /} - was markedly reduced in mice, Card14 ^{- / -} mice showed levels of serum anti-OVA _{IgG 2c} comparable to WT mice (Fig. 6B). These results indicate that 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.

  The data thus strongly indicate that CARD14 is required for optimal production of IL-17 and IL-22 by epidermal γδT 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-κB 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 we first found that CARD14 is in epithelial stromal cells such as keratinocytes. We hypothesized to be a downstream signaling molecule for TLR7-mediated NF-κB activation. However, our results show that TLR7 and TLR9 are required for IMQ-induced psoriasis-like dermatitis but not for IL-23-induced psoriasis-like dermatitis (FIG. 1), while CARD14 is Demonstrating that it is important for both IMQ-induced psoriatic dermatitis and IL-23-induced psoriatic dermatitis (FIG. 3). These data suggest that our initial hypothesis was doubtful, rather that CARD14 is downstream of IL-23 and is more important for adaptive immune cells than innate immune cells Seem. Downstream of the signal transduction pathway initiated by administration of IL-23, cells expressing IL-23R should play an important role in CARD14-mediated pathogenesis of psoriatic dermatitis. It is not clear which cells express IL-23R, but most cells expressing IL-23R have been shown to be γδT cells (Awasthi et al., 2009, Journal of Immunology. 182: 5904). -5908.) Our results demonstrate that CARD14 is required for IMQ-mediated and IL-23-mediated production of IL-17 and IL-22 in TCRγδT cells (FIGS. 5D and 5E). ) Further studies are needed to elucidate how CARD14 is involved in these intracellular signaling pathways. We have shown that CARD14 is required for psoriatic lesion formation through suppression of IL-17 and IL-22 producing γδ T cells in two established psoriasis-like dermatitis models .

  The inventors have found that CARD14 is expressed in γδ T cells in psoriatic skin rashes 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 TCRγδ 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). Thus, the role of CARD14 in humans and mice (including animal models) 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 γδT 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. γδ T-cells have been used in 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)
Next, in this example, it was examined whether CARD14 deficiency shows a symptom reduction effect in an experimental demyelinating disease model of multiple sclerosis. In this example, Rui et al. (Yuxiang 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 ).

  Experimental autoimmune encephalomyelitis (EAE) used in this example is an autoimmune disease model induced by immunizing protein antigens and peptides derived from central nervous tissue, and is a multiple Since it shares many pathological conditions with multiple sclerosis (MS), it is commonly used in the pathological research of MS and the development of therapeutics and therapeutic agents.

(mouse)
CARD14 ^{− / −} mice were prepared by knocking out genes according to Rui et al.

(EAE mouse)
According to Rui et al., Mice were treated with 200 μg MOG _35-55 peptide (peptide corresponding to MOG _35-55 residue), complete Freund's adjuvant (CFA), and 250 μg heat-killed mycobacteria (HKMTB). Subcutaneous (sc) immunization with 0.2 mL of emulsion. In addition, mice received two intraperitoneal (ip) injections of 200 ng pertussis toxin (PTX) at the time of immunization and 48 hours later. In some experiments, suboptimal immunization conditions, excluding HKMTB, PTX, or both, were also used. 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 mean clinical score for each experimental group.

For restimulation assays, 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). In a volume of 1 mL, 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 μg / mL). Incubated with. The supernatant was collected after 3 days for cytokine analysis.

FIG. 7 shows wild-type mice (n = 8) and Card14 ^{− / −} mice (n = 9) after immunization with MOG / CFA / HKMTB in the presence of pertussis toxin (PTX) on day 0 and day 2. EAE score is shown (left of FIG. 7). On the right side, it was shown that the CARD14 deficiency in the experimental demyelinating disease model was reduced by the incidence of CARD14, indicating that CARD14 plays an important role in multiple sclerosis.

(Example 3: Screening)
In this example, 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 γδ 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.

  Administration of these inhibitors in the models of psoriasis, multiple sclerosis, side effects from TLR-based / IMQ / immunostimulatory adjuvants, or complications of γδ T cell mediated diseases and / or immune interventions described in Example 1 or 2 Then, the follow-up of symptoms and the like can be performed, and those whose symptoms can be prevented or treated can be identified as preventive or therapeutic agents for these diseases, disorders or symptoms.

(List of references: In addition, the documents in these lists are not listed for the purpose of admitting that they are prior art to the present invention.)
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As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, it is understood that the scope of this invention should be construed only by the claims. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood. This application claims priority to Japanese Patent Application No. 2015-200948 filed in Japan on October 9, 2015, the contents of which are hereby incorporated by reference in their entirety.

  The present invention finds industrial applicability in the biopharmaceutical industry and the pharmaceutical industry.

SEQ ID NO: 1 CARD14 nucleic acid sequence (human) NM_001257970
SEQ ID NO: 2: CARD14 amino acid sequence (human) NP_001244899
SEQ ID NO: 3: CARD14 nucleic acid sequence (mouse) NM_130886
SEQ ID NO: 4: CARD14 amino acid sequence (mouse) NP_570956
Sequence number 5: P1, 5'-GGGTGTTCCTCTGACTCTCCCAGTTGGATG-3 '
Sequence number 6: P2, 5'-GCTGACCGCTTCCTCGTGCTTTACGGTATC-3 '
Sequence number 7: P3, 5'-CAGTGACTCAAGGAGGGGCAAACGCCTATG-3 '
Sequence number 8: mCARD14, 5'-TGCATAGCTCCCGTTTCAC-3 '(forward)
Sequence number 9: mCARD14, 5'-GGAACTTCAGGCTTTCCAGA-3 '(reverse)
Sequence number 10: mGAPDH, 5'-CAAGATTGTCAGCAATGCATCC-3 '(forward)
Sequence number 11: mGAPDH, 5'-CCTTCCACAATGCCAAGTTG-3 '(reverse)

Claims (18)

Contacting a hematopoietic cell with a candidate substance, and determining the expression of CARD14 in the hematopoietic cell, wherein the candidate substance suppresses or eliminates CARD14 expression, the candidate substance is a therapeutic or preventive agent for psoriasis A method for screening a therapeutic or prophylactic agent for psoriasis based on the expression of CARD14 in hematopoietic cells, comprising a step determined to be usable as an agent. 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, wherein the candidate substance treats multiple sclerosis A method for screening a therapeutic or prophylactic agent for multiple sclerosis based on the expression of CARD14 in hematopoietic cells, comprising a step determined to be usable as an agent or prophylactic agent. Contacting a hematopoietic cell with a candidate substance, and determining the expression of CARD14 in the hematopoietic cell, wherein if the candidate substance suppresses or eliminates the expression of CARD14, the candidate substance is TLR-based / IMQ / Reducing side effects from TLR-based / IMQ / immunostimulatory adjuvant based on expression of CARD14 in hematopoietic cells, including steps determined to be usable as therapeutic or prophylactic agents to reduce side effects from immunostimulatory adjuvants A screening method for a therapeutic or preventive agent. 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, the candidate substance is a γδT cell-mediated disease And / or complications of γδ T cell-mediated diseases and / or immune interventions based on the expression of CARD14 in hematopoietic cells, comprising steps determined to be usable as therapeutic or preventive agents for complications of immune interventions Screening method for therapeutic or prophylactic agent. The method according to any one of claims 1 to 4, wherein the hematopoietic cells are γδT cells. The method according to any one of claims 1 to 4, wherein the hematopoietic cells are epidermal γδ T cells. The method according to claim 5 or 6, wherein the γδT cells are cells that produce IL-17 and IL-22. The method according to any one of claims 1 to 7, further comprising the step of determining that CARD14 is not expressed in non-hematopoietic cells. The method according to any one of claims 1 to 8, further comprising the step of contacting the candidate substance with a non-hematopoietic cell and selecting the candidate substance as the therapeutic agent or prophylactic agent when CARD14 has no substantial effect on expression. 2. The method according to item 1. The method according to claim 8 or 9, wherein the non-hematopoietic cells include skin resident cells. 11. The method of any one of claims 1 or 3-10, wherein the disease includes IMQ-induced psoriasis and IL-23-induced psoriasis. A therapeutic or prophylactic agent for psoriasis comprising a hematopoietic cell-specific CARD14 inhibitor. A therapeutic or prophylactic agent for multiple sclerosis comprising a hematopoietic cell-specific CARD14 inhibitor. A therapeutic or prophylactic agent for reducing side effects caused by a TLR-based / IMQ / immunostimulatory adjuvant comprising a hematopoietic cell-specific CARD14 inhibitor. A therapeutic or prophylactic agent for γδ 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 claims 12 to 15, wherein the hematopoietic cells are γδT cells. The therapeutic or prophylactic agent according to any one of claims 12 to 15, wherein the hematopoietic cells are epidermal γδ T cells. The therapeutic or prophylactic agent according to claim 16 or 17, wherein the γδ T cells are cells that produce IL-17 and IL-22.