JP2021097682A - Therapy, diagnosis and screening using card14 - Google Patents

Abstract

To provide treatment, diagnosis and screening using CARD14.SOLUTION: The present invention provides a therapeutic agent or prophylactic agent for psoriasis, multiple sclerosis, and reduction of side effects caused by TLR-base/IMQ/immunostimulatory adjuvants. It was found that CARD14 is associated with psoriasis, multiple sclerosis, and side effects caused by TLR-base/IMQ/immunostimulatory adjuvants. On the basis of this finding, provided are a therapeutic agent and a diagnostic agent, and screening therefor. In particular, it was found that, in expression in hematopoietic cells, particularly γδT cells (more particularly, epidermal γδT cells), CARD14 is strongly associated with psoriasis, multiple sclerosis, and side effects of TLR-base/IMQ/immunostimulatory adjuvants. Based on this finding, a therapeutic agent and a diagnostic agent, and screening for them are provided.SELECTED DRAWING: None

Description

The present invention relates to treatment, diagnosis and screening using CARD14. More specifically, the present invention uses CARD14 as a therapeutic or prophylactic agent for psoriasis, multiple sclerosis, and γδT cell-mediated diseases and / or complications of immune intervention and their screening, Toll-like receptors (TLRs). ) Base / Imikimod (IMQ) / Therapeutic or prophylactic agents for reducing the side effects of immunostimulatory adjuvants and their screening.

Psoriasis is a locally T-cell-mediated chronic inflammatory skin disorder characterized by chronic erythematous plaques with very well-defined borders (Non-Patent Document 1: 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 similar epidermal hyperkeratosis as 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 roles of interleukins (IL) -17 and IL-23 in the pathogenesis of psoriasis have been elucidated in recent years (Non-Patent Document 5: Johnson-Huang et al., 2012). I
Monoclonal antibodies against L-23p19 (guselkumab and tildrakizumab) showed significant clinical advantages over blocking the IL-12 / 23p40 subunit (Non-Patent Document 5: Johnson-Huang et al., 2012; Non-Patent). Reference 6: Sofen et al., 2014). In this experimental setting, due to the absence of IL-23p19 in vivo
IMQ cream-induced psoriasis-like skin inflammation is suppressed (Non-Patent Document 3: van der Fits et
al., 2009). In addition, intradermal injection of recombinant IL-23 protein into the ears of mice shared many features with psoriasis (eg, epidermal hyperkeratosis via IL-22 upregulation and STAT3 activation) (non-patent literature). 7: Cai et al., 2011; Non-Patent Document 8: Zheng et al., 2007).

In recent years, psoriasis susceptibility locus 2 (PSORS2) located in the human chromosomal region 17q25.3
) Caspase Mobilization Domain Family Member 14 (CARD14 [MIM607]
211], also known as CARMA2), has been shown to have a unique gain-of-function mutation (Non-Patent Document 9: Jordan et al., 2012). This finding was further confirmed by a genome-wide association study (GWAS) of PSORS2, which identified several risk-related variants, including CARD14 (Non-Patent Document 10: Tsui et al., 20).
12). CARD14 has been shown to activate the nuclear factor kappa B (NF-κB) via the 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 keratosis pilaris, but the correlation between these mechanisms and the immune response is 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. 4:15 60. 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 to screen them. The present invention was completed by finding the conclusion. In particular, IL-17 and IL-
We found that CARD14 in 22-producing γδ T cells is essential for the formation of psoriasis-like dermatitis, found the role of CARD14 in hematopoietic cells related to psoriasis, and found that the role of specific cells is also important. Completed the invention.

In addition, the present inventors have found that CARD14 is an essential factor in the development of multiple sclerosis (MS), and conclude that therapeutic agents, diagnostic agents and their screening are possible based on this. The present invention was completed by finding the conclusion that it is possible to provide a therapeutic or preventive agent for multiple sclerosis and to screen them using CARD14.

Furthermore, we have found that CARD14 in γδ T cells can be a potential therapeutic target for γδ T cell-mediated disease and complications of immune intervention, and complications of γδ T cell-mediated disease and / or immune intervention. We have completed the present invention by finding the conclusion that it is possible to provide therapeutic or prophylactic agents for these diseases and to screen them.

In addition, the present inventors have completed the present invention by concluding that it is possible to provide therapeutic or prophylactic agents for reducing the side effects of TLR-based / IMQ / immunostimulatory adjuvants and to screen them. ..

Psoriasis is a chronic inflammatory skin disease of unknown cause, but has also been reported as a complication of imiquimod (IMQ) cream. Although CARD14 has recently been identified as a psoriasis susceptibility gene, its immunological role in psoriasis pathogenesis in vivo remains unclear. As used herein, we use two psoriasis-like models to use CARD14.
Deficient (Card14 ^{− / −} ) mice were evaluated. 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 interleukin without activation of TLR7 and TLR9. Induced by (IL) -23. In both models, Card14 ^{− / −} mice showed significantly less increase in skin thickness and hyperkeratosis than wild-type mice. CARD14 is IL-17 and IL-2 in these models of psoriatic skin lesions.
It is expressed in epidermal γδ T cells that produce 2 and is associated with these epidermal γδ T cells. Studies using bone marrow chimeric mice and gene-deficient mice reveal that CARD14, which is expressed in hematopoietic cells (particularly γδ T 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 the deficiency of CARD14, which indicates that
It is suggested that inhibition of CARD14 can reduce IMQ-induced psoriasis-like dermatitis without affecting its adjuvant activity. From these findings, the present inventors have γδ.
We have found that CARD14 in T cells strongly suggests that it may be a potential therapeutic target for γδ T cell-mediated diseases and complications of immune intervention.

From the above, the present invention provides the following.
(1) A method for screening a therapeutic or prophylactic agent for psoriasis based on the expression of CARD14 in hematopoietic cells.
(2) A method for screening a therapeutic or prophylactic agent for multiple sclerosis based on the expression of CARD14 in hematopoietic cells.
(3) A method for screening a therapeutic or prophylactic agent for reducing side effects of a TLR-based / IMQ / immunostimulatory adjuvant based on the expression of CARD14 in hematopoietic cells.
(4) A method for screening 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 cell is a γδ T cell.
(6) The method according to any one of items 1 to 4, wherein the hematopoietic cell is an epidermal γδ T cell.
(7) The method according to item 5 or 6, wherein the γδ T cells are cells that produce IL-17 and IL-22.
(8) The method according to any one of items 1 to 7, based on the 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 disease includes IMQ-induced psoriasis and IL-23-induced psoriasis, items 1 to 2.
The method according to any one of 9.
(11) A therapeutic or prophylactic agent for psoriasis, which comprises a hematopoietic cell-specific CARD14 inhibitor.
(12) A therapeutic or prophylactic agent for multiple sclerosis, which comprises a hematopoietic cell-specific CARD14 inhibitor.
(13) A therapeutic or prophylactic agent for reducing side effects of a TLR-based / IMQ / immunostimulatory adjuvant containing a hematopoietic cell-specific CARD14 inhibitor.
(14) A therapeutic or prophylactic agent for γδ T cell-mediated diseases and / or complications of immune intervention, including hematopoietic cell-specific CARD14 inhibitors.
(15) The therapeutic or prophylactic agent according to any one of items 11 to 14, wherein the hematopoietic cell is a γδ T cell.
(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 γδ T cell is a cell that produces IL-17 and IL-22, item 15
Or the therapeutic or prophylactic agent according to 16.
(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 1-10.
The present invention also provides:
(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, where the candidate substance suppresses or eliminates the expression of CARD14, the candidate substance is a treatment for psoriasis. A method of screening a therapeutic or prophylactic agent for psoriasis based on the expression of CARD14 in hematopoietic cells, comprising the steps determined to be able to be used as an agent or prophylactic agent.
(2A) 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 when the candidate substance suppresses or eliminates the expression of CARD14, the candidate substance is multiple sclerosis. 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 when the candidate substance suppresses or eliminates the expression of CARD14, the candidate substance is TLR-based. By TLR-based / IMQ / immunostimulatory adjuvant based on expression of CARD14 in hematopoietic cells, including steps determined that it can be used as a therapeutic or prophylactic agent to reduce the side effects of / IMQ / immunostimulatory adjuvant. A method of screening a therapeutic or prophylactic agent to reduce 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 when the candidate substance suppresses or eliminates the expression of CARD14, the candidate substance is a γδ T cell. Γδ T cell-mediated disease and / or immune intervention based on the expression of CARD14 in hematopoietic cells, including steps determined to be able to be used as therapeutic or prophylactic agents for mediated diseases and / or complications of immune intervention. How to screen for therapeutic or prophylactic agents of complications.
(5A) The method according to any one of items 1A to 4A, wherein the hematopoietic cell is a γδT cell.
(6A) The method according to any one of items 1A to 4A, wherein the hematopoietic cell is an epidermal γδ T cell.
(7A) The γδ T cell is a cell that produces IL-17 and IL-22, item 5A.
Or the method according to 6A.
(8A) The method according to any one of items 1A to 7A, further comprising the step of determining that CARD14 is not expressed in non-hematopoietic cells.
(9A) Item 1A-8A, further comprising contacting the non-hematopoietic cells with the candidate substance and selecting the candidate substance as the therapeutic or prophylactic agent if CARD14 has no substantial effect on expression. The method according to any one item.
(10A) The method according to item 8A or 9A, wherein the non-hematopoietic cells include skin resident cells.
(11A) The disease comprises IMQ-induced psoriasis and IL-23-induced psoriasis, item 1.
The method according to any one of A or 3A to 10A.
(12A) A therapeutic or prophylactic agent for psoriasis, including a hematopoietic cell-specific CARD14 inhibitor.
(13A) A therapeutic or prophylactic agent for multiple sclerosis, including a hematopoietic cell-specific CARD14 inhibitor.
(14A) A therapeutic or prophylactic agent for reducing side effects of a TLR-based / IMQ / immunostimulatory adjuvant, including a hematopoietic cell-specific CARD14 inhibitor.
(15A) Therapeutic or prophylactic agents for γδ T cell-mediated diseases and / or complications of immune intervention, including hematopoietic cell-specific CARD14 inhibitors.
(16A) The therapeutic or prophylactic agent according to any one of items 12A to 15A, wherein the hematopoietic cells are γδ T 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 γδ T cell is a cell that produces IL-17 and IL-22, item 1.
The therapeutic or prophylactic agent according to 6A or 17A.

In the present invention, it is intended that the one or more features described above may be provided in combination in addition to the specified combinations. Further embodiments and advantages of the present invention will be appreciated by those skilled in the art upon reading and understanding the following detailed description, if necessary.

According to the present invention, a therapeutic or prophylactic agent for psoriasis based on the expression of CARD14 in hematopoietic cells, a therapeutic or prophylactic agent for multiple sclerosis, and a therapeutic agent for reducing side effects of TLR-based / IMQ / immunostimulant adjuvant. Alternatively, prophylactic agents and therapeutic or prophylactic agents for γδ T cell-mediated diseases and / or complications of immune intervention are provided, and their screening 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 Thrr7 ^{− / −} Trr9 ^{− / −} 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. The data are representative of four independent experiments, showing the results in mean ± standard deviation (s.d.), where ^** indicates P <0.01; all Scheffe's test. (B) H & E staining in the skin of control mice and IMQ mice on the last day of each group. The bar indicates 100 μm. The data are representative of four independent experiments. (C) Ear thickness was monitored daily prior to injection of IL-23. The data are representative of two independent experiments, averaging the results ± s. d. Indicated by; All Scheffe's test. (D) H & E staining of the skin of phosphate buffered saline (PBS) -injected mice and IL-23-injected mice on the final day of the experiment. The bar indicates 100 μm. The data are representative of two independent experiments. Creation of Card14 ^{− / −} mice. Card14 ^{− / −} mice were generated by replacing exons 2 and 3 of the Card14 gene with a neomycin (neo) resistance gene. (A) Structure of the mouse Card14 locus (WT allele), the Card14 targeting vector, and the predicted mutant Card14 gene (mutated allele). Exons 2 and 3 of the Card14 gene were replaced with the neo resistance gene. Restriction enzymes: E, EcoRI; B, BamHI. (B) Card14 PCR genotyping of WT alleles and mutant alleles using primers P1, P2 and P3. Genomic DNA was extracted from the tail of the mouse. 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 and dermal cells from ^{WT and Card14 − / − mice.} Data are analyzed using real-time qPCR and show Card14 expression compared to glyceraldehyde triphosphate dehydrogenase (GAPDH) (n = 4 mice / group). CARD14 is essential for the development of skin inflammation in both IMQ-induced psoriasis-like models and IL-23-induced psoriasis-like models. Ears from WT and Card 14 ^{− / −} 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. The data are representative of at least 5 independent experiments and the results are averaged ± s. d. , ^* Indicates P <0.05, ^** indicates P <0.01; all Scheffe's tests. (B) H & E staining, keratin 5 staining, and Ki67 staining of the skin of mice that received control and IMQ on the last day. The bar indicates 100 μm. The data are representative of at least 5 independent experiments. (C) Ear thickness was monitored daily prior to injection of IL-23. The data are representative of four independent experiments, with the results averaging ± s. d. Indicated by, ^** indicates P <0.01; all Scheffe's test. (D) H & E staining, keratin 5 staining, and Ki67 staining of skin on the final day of the experiment in PBS-injected mice and IL-23-injected mice. The bar indicates 100 μm. The data are representative of four independent experiments. CARD14 in hematopoietic cells plays an important role in psoriasis, but not in radiation-resistant skin-resident cells. (A) Reconstructed bone marrow chimera (WT BM → WT mouse (WT → WT), WT BM → Card14 ^{− / −} mouse (WT → KO), Card14 ^{− / −} BM → Card14 ^{− / −} mouse (KO → KO) And Card 14 ^{− / −} BM → WT mice (KO → WT), n = 4-6 mice / group) were treated with IMQ cream as described in FIG. Ear thickness was monitored daily. The data are representative of three independent experiments, averaging ± s. d. Indicated by, ^** indicates P <0.01; all Scheffe's test. (B) WT mice, Rag2 ^{- / - mice, Rag2 - / - Il2rg - /} - mice, and TCRD ^{- / -} were treated with mouse (n = 4 mice / group) for 6 days IMQ cream continuously. Ear thickness was measured daily prior to cream treatment. The data are representative of three independent experiments, averaging ± s. d. , ^* Indicates P <0.05, ^** indicates P <0.01; all Scheffe's tests. CARD14 is expressed in γδ T cells and is required for the production of IL-17 and IL-22 by γδ T 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. The data are representative of four independent experiments. (C) Frozen sections from IL-23-injected WT mice (left) and Card14 ^{− / −} mice (right) were subjected to DAPI (blue), CARD14 Ab (green), and GL3 (green) for immunofluorescence staining. Stained with anti-TCRγ) mAb (red). Merge is a superposed view of these. The image was deconvolved in Z stack in 0.5 mm steps. The image was magnified 100 times. The data are representative of two independent experiments. ^{(D and E) Suspensions of epidermal cells derived from WT and Card 14 − / −} mice in IMQ or IL-23 models are stimulated with phorbol 12-millistart 13-acetate (PMA) and ionomycin and intracellular IL- The expression of 17 and IL-22 was analyzed by flow cytometry. Local flow plots gated with CD45 ⁺ cells are representative of three independent experiments. Average data ± s. d. Indicated by, ^* indicates P <0.05;Scheffe's test. CARD14 does not interfere with the adjuvant effect of IMQ on co-administered antigens. For ovalbumin (OVA) immunization, WT mice, Thrr7 ^{− / −} Trr9 ^{− / −} mice, and Card14 ^{− / −} mice (n = 5-6 mice / group) were injected on days 0 and 7. 100 μg of OVA protein was injected intradermally into the ear (20 μL / ear). Blood was collected on day 14 and antibody titers were measured. To induce 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. The data are representative of two independent experiments, averaging ± s. d. Indicated by, ^* indicates P <0.05; all Scheffe's test. (B) OVA-specific IgG ₁ and IgG _2c in serum were measured using ELISA. The data are representative of two independent experiments, averaging ± s. d. Indicated by, ^** indicates P <0.01; all Scheffe's test. FIG. 7 shows the symptomatology-relieving effect of an experimental demyelinating disease model due to CARD14 deficiency. ^{EAE scores of wild-type mice (n = 8) and Card14 − / −} mice (n = 9) after immunization with MOG / CFA / HKMTB in the presence of pertussis toxin (PTX) on days 0 and 2. The result. Data are shown as mean ± SEM. The left panel shows the evaluation over time (days) based on the EAE score, and the right panel shows the incidence rate. It was shown that CARD14 deficiency had a symptom-relieving effect in an experimental demyelinating disease model, indicating that CARD14 plays an important role in multiple sclerosis.

Hereinafter, the present invention will be described. Throughout the specification, it should be understood that the singular representation also includes its plural concept, unless otherwise stated. Therefore, the singular article (
For example, in the case of English, "a", "an", "the", etc.) should be understood to include the plural notion unless otherwise noted. It should also be understood that the terms used herein are used in the meaning commonly used in the art unless otherwise noted. Thus, unless otherwise defined, all terminology 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, this specification (including definitions) takes precedence.

As used herein, the term "CARD 14" refers to Caspase recruitment domain-containing.
Protein 14, CARD-containing MAGUK protein 2 (Carma 2) is also called a protein and a gene encoding it. It may also be displayed as BIMP2; CARMA2; PRP; PSORS2; PSS1, and for its human amino acid sequence, the accession number is NP_0.
For 01244899 (human), NP_570956 (mouse), and mRNA sequences, the accession numbers are NM_001257970 (human), NM_130886 (mouse). The amino acid sequence of CARD14 is, for example, SEQ ID NO: 2 (human) and 4 (mouse). The nucleotide sequence of CARD14 mRNA is, for example, SEQ ID NO: 1 (human) and 3 (mouse). CARD14 is CARD1
As long as it has the activity of 4, its amino acid sequence is not limited. Therefore, as long as it meets the specific object of the present invention, not only a protein having an amino acid sequence described in a specific sequence number or accession number (or a nucleic acid encoding the same), but also a functionally active similarity thereof. A body or derivative, or a fragment thereof that is functionally active, or a homologue thereof, or a variant encoded by a nucleic acid that hybridizes to a nucleic acid encoding this protein under high stringency or low stringency conditions. , It is understood that it can be used in the present invention.

As used herein, a "derivative", "similar" or "mutant" is preferably a region that is not intended to be limiting but is substantially homologous to the protein of interest (eg, CARD14). Such molecules include, in various embodiments, at least when compared to sequences aligned over identical size amino acid sequences or by computer homology programs known in the art. 30%, 40%, 50
Nucleic acids that are%, 60%, 70%, 80%, 90%, 95% or 99% identical or encode such molecules are (highly) stringent conditions, moderately stringent. It can hybridize to sequences encoding component proteins under conditions or non-stringent conditions. This is due to amino acid substitutions, deletions and additions, respectively.
A modified product of a naturally occurring protein, the derivative of which means a protein that still exhibits the biological function of the naturally occurring protein, although not necessarily to the same extent. For example
It is also possible to investigate the biological function of such proteins by appropriate and available in vitro assays described herein or known in the art. As used herein, "functionally active" is the structural function of a protein, such as biological activity, according to the embodiments in which the polypeptide of the invention, ie, a fragment or derivative, is associated herein. Refers to a polypeptide, i.e. a fragment or derivative, having a regulatory or biochemical function. In the present invention, humans are mainly discussed about CARD14, but since it is known that many animals other than humans express CARD14, these animals, especially mammals, are also within the scope of the present invention. It is understood to enter.

Therefore, a representative nucleotide sequence of CARD14 is
(A) A polynucleotide having the nucleotide 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) In the amino acid sequence set forth in SEQ ID NO: 2 or 4, one or more amino acids are substituted.
A polynucleotide that encodes a modified polypeptide having one mutation selected from the group consisting of additions and deletions or a fragment thereof and having biological activity;
(D) A polynucleotide that is a splice variant or allelic variant of the nucleotide sequence set forth in SEQ ID NO: 1 or 3 or a fragment thereof;
(E) A polynucleotide encoding a species homologue or fragment thereof of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or 4.
(F) A polynucleotide that hybridizes to any one of the polynucleotides (a) to (e) under stringent conditions and encodes a polypeptide having biological activity;
Or (g) identity to any one of the polynucleotides (a) to (e) or its complementary sequence is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, It can be a polynucleotide that comprises a base sequence of at least 98%, or at least 99%, and encodes a polypeptide having biological activity. Here, the biological activity typically means that it can be distinguished from other proteins existing in the same organism as the activity or marker of CARD14.
The amino acid sequence of CARD14 is
(A) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or 4 or a fragment thereof;
(B) In the amino acid sequence set forth in SEQ ID NO: 2 or 4, one or more amino acids have one mutation selected from the group consisting of substitutions, additions and deletions, and have biological activity. peptide;
(C) A polypeptide encoded by a splice or allelic variant of the nucleotide 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 at least 70%, at least 70%, identity to any one of the polypeptides of (e) (a)-(d). It has 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
It can be a polypeptide, which has biological activity. Here, the biological activity can be typically distinguished from other proteins present in the same organism as the activity or marker of CARD14 (eg, 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 "binder" or "CARD14 interacting molecule" is a molecule or substance that binds to CARD14 at least temporarily. For detection purposes, it is preferably to be able to indicate that it is bound (eg, labeled or in a labelable state), and for therapeutic purposes it is further advantageous that a therapeutic agent is bound. .. Examples of substances that bind to CARD14 include antibodies, antisense oligonucleotides, siRNAs, low molecular weight molecules (LMW), binding peptides, aptamers, ribozymes and peptidomimetics. The substance or CARD14 interacting molecule that binds to CARD14 may be an inhibitor of CARD14, eg, a binding protein or binding peptide directed at CARD14, particularly against the active site of CARD14, as well as Also included are nucleic acids directed against the CARD14 gene. Nucleic acid for CARD14 is, for example, CARD.
Double-stranded or single-stranded DNA or R that inhibits the expression of 14 genes or the activity of CARD14
Refers to NA, or a modification or derivative thereof, and antisense nucleic acid, aptamer, si
Includes, but is not limited to, RNA (small interfering RNA) and ribozymes. As used herein, the term "binding protein" or "binding peptide" for CARD 14 refers to CARD 1.
Refers to any protein or peptide that binds to 4, and includes, but is not limited to, antibodies directed against CARD14 (eg, polyclonal or monoclonal antibodies), antibody fragments and functional equivalents.

As used herein, "protein", "polypeptide", "oligopeptide" and "peptide" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear, branched or cyclic. The amino acid may be natural or non-natural, or may be a modified amino acid. The term may also include those assembled into a complex of multiple polypeptide chains. The term also includes naturally 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) is included. This definition also includes, for example, polypeptides containing one or more analogs of amino acids (including, for example, unnatural amino acids), peptide-like compounds (eg, peptoids) and other modifications known in the art. Will be done. In the present specification, "
"Amino acid" is a general term for organic compounds having an amino group and a carboxyl group. When the antibody according to the embodiment of the present invention contains a "specific amino acid sequence", any amino acid in the amino acid sequence may be chemically modified. Moreover, 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 cases, it can be said that the protein according to the embodiment of the present invention contains the above-mentioned "specific amino acid sequence". Chemical modifications that amino acids contained in proteins undergo in vivo include, for example, N-terminal modification (eg, acetylation, myristoylation, etc.), C-terminal modification (eg, amidation, glycosylphosphatidylinositol addition, etc.), or side chain. Modifications (eg, phosphorylation, glycosylation, etc.) are known. It may be natural or non-natural as long as it satisfies the object of the present invention.

In the present specification, "polynucleotide", "oligonucleotide" and "nucleic acid" are referred to as "nucleic acid".
As used interchangeably herein, it refers to a polymer of nucleotides of any length. The term also includes "oligonucleotide derivatives" or "polynucleotide derivatives". The "oligonucleotide derivative" or "polynucleotide derivative" refers to an oligonucleotide or polynucleotide containing a derivative of a nucleotide or having an unusual bond between nucleotides, and is used interchangeably. Specifically, as such an oligonucleotide, for example, 2'-O-methyl-ribonucleotide, an oligonucleotide derivative in which a phosphodiester bond in the oligonucleotide is converted into a phosphorothioate bond,
An oligonucleotide derivative in which a phosphate diester bond in an oligonucleotide is converted into an N3'-P5'phosphoroamidate bond, an oligonucleotide derivative in which ribose and a phosphate diester bond in an oligonucleotide are converted into a peptide nucleic acid bond, The uracil in the oligonucleotide is replaced with C-5 propynyl uracil, the oligonucleotide derivative in which the uracil in the oligonucleotide is replaced with C-5 thiazole uracil, and the cytosine in the oligonucleotide is C-5 propynylcitosine. Substituted oligonucleotide derivative, citosine in the oligonucleotide is phenoxazine-modified citosine (
Oligonucleotide derivatives substituted with phenoxazine-modified cytosine), oligonucleotide derivatives with ribose in DNA substituted with 2'-O-propylribose, and oligonucleotides with ribose in oligonucleotides substituted with 2'-methoxyethoxyribose Examples include nucleotide derivatives. Unless otherwise indicated, certain nucleic acid sequences are also conservatively modified variants (eg, degenerate codon substitutions) and complementary sequences, as are the explicitly indicated sequences. Is intended to be included. Specifically, the degenerate codon substitution product 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. Can be achieved by (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Bi
ol.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 genes, cDNAs, mRNAs, oligonucleotides, and polynucleotides. As used herein, the "nucleotide" may be natural or non-natural.

As used herein, the term "gene" refers to a factor that defines a genetic trait, and the term "gene" means "gene".
It may refer to "polynucleotide", "oligonucleotide" and "nucleic acid".

In the present specification, "homology" of a gene means the degree of identity of two or more gene sequences to each other, and generally, having "homology" means a high degree of identity or similarity. Say. Therefore, the higher the homology of two genes, the higher the identity or similarity of their sequences. Whether or not the two genes are homologous can be examined by direct sequence comparison or, in the case of nucleic acids, hybridization under stringent conditions.
When comparing two gene sequences directly, the DNA sequences are typically at least 50% identical, preferably at least 70% identical, and more preferably at least 80%, 90%. , 95%, 96%, 97%, 98% or 99% if they are identical, the genes are homologous. Therefore, in the present specification, "homologous" or "homologous gene product" is referred to as "homologous" or "homologous gene product".
It exerts the same biological functions as the protein components of the complex described further herein.
Means a protein in another species, preferably a mammal. Such homologues are also
Sometimes referred to as the "ortholog gene product." It is understood that such homologues, homologous gene products, ortholog gene products and the like can also be used as long as they meet the object of the present invention.

Amino acids are either their commonly known three-letter symbols or IUPAC-IUB Biochemical Nomenc.
It may be referred to herein by any of the one-letter symbols recommended by the lature Commission. Nucleotides can also be referred to by the generally accepted one-letter code. In the present specification, comparison of similarity, identity and homology of amino acid sequence and base sequence is calculated using default parameters using BLAST, which is a tool for sequence analysis. The identity search can be performed using, for example, NCBI's BLAST 2.2.28 (issued April 2013). The value of identity in the present specification usually refers to the value when the above BLAST is used and aligned under the 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 multiple regions, the highest value among them is set as the identity value. Similarity is a number that takes into account similar amino acids in addition to identity.

In one embodiment of the present invention, the "several pieces" may be, for example, 10, 8, 6, 5, 4, 3, or two pieces, or may be less than or equal to any one of them. Polypeptides that have been deleted, added, inserted, or replaced by other amino acids with one or several amino acid residues are known to maintain their biological activity (Mark et al., Proc). Natl Acad Sci USA
.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): 1431-1
433.). The deleted antibody can be produced, for example, by a site-specific mutagenesis method, a random mutagenesis method, biopanning using an antibody phage library, or the like. 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 the wild type from the mutant antibody into which the 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 of them. May be good. The above-mentioned "homology" may be calculated by calculating the ratio of the number of amino acids homologous in an amino acid sequence between two or a plurality of amino acids according to a method known in the art. Before calculating the proportion, align the amino acid sequences of the amino acid sequences to be compared and introduce a gap in part of the amino acid sequence if necessary to maximize the proportion of the same amino acid. Methods for alignment, method of calculating percentages, methods of comparison, and related computer programs are well known in the art (eg, BLAST, GENETYX, etc.). As used herein, "homology" can be expressed as a value measured by NCBI's BLAST unless otherwise specified. Blastp can be used by default for algorithms when comparing amino acid sequences with BLAST. The measurement result is Po
It is quantified as sitives or Identities.

As used herein, the term "polynucleotide that hybridizes under stringent conditions" refers to well-known conditions commonly used in the art. Such a polynucleotide can be obtained by using a polynucleotide selected from the polynucleotides of the present invention as a probe and using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like. In particular,
0.7-1.0 M Na using a filter with DNA immobilized from colonies or plaques
After hybridization at 65 ° C. in the presence of Cl, SSC (saline-sod) at a concentration of 0.1 to 2 times.
It means a polynucleotide that can be identified by washing the filter under 65 ° C. conditions using an ium citrate) solution (the composition of the 1-fold SSC solution is 150 mM sodium chloride, 15 mM sodium citrate). For the "stringent condition", for example, the following conditions can be adopted. (1) Use low ionic strength and high temperature for cleaning (eg 50)
At ° C, 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate (SDS)), (2) use a denaturing agent such as formamide during hybridization (2)
For example, at 42 ° C, 50% (v / v) formamide and 0.1% bovine serum albumin / 0.1% Ficoll / 0
.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5, and 750 mM
Sodium chloride, 75 mM sodium citrate), or (3) 20% formamide, 5 x SSC, 50
mM sodium phosphate (pH 7.6), 5 x denhard solution, 10% dextran sulfate, and 20 mg / ml
Incubate overnight at 37 ° C in a solution containing denatured sheared salmon sperm DNA, then about 37-50
Clean the filter with 1 x SSC at ° C. The formamide concentration may be 50% or more. The wash time may be 5, 15, 30, 60, or 120 minutes, or longer. Temperature is a factor that affects the stringency of hybridization reactions.
Multiple factors such as salinity are possible, see Ausubel et al., Current Protocols in
See Molecular Biology, Wiley Interscience Publishers, (1995). An example of "highly stringent conditions" is 0.0015M sodium chloride, 0.
.. 0015M sodium citrate, 65-68 ° C, or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide, 42 ° C. Hybridization, Molecular Cloning 2nd ed., Current Protocols in M
olecular Biology, Supplement 1-38, DNA Cloning 1: Core Techniques, A Pract
It can be performed according to the method described in experimental books such as ical Approach, Second Edition, Oxford University Press (1995). Here, sequences containing only the A sequence or only the T sequence are preferably excluded from the sequences that hybridize under stringent conditions. Moderate stringent conditions can be easily determined by one of ordinary skill in the art, eg, based on DNA length, Sambrook et al., Molecular Cloning: A Laboratory.
Manual, 3rd edition, Vol. 1,7.42-7.45 Cold Spring Harbor Laboratory
Shown in Press, 2001, and with respect to nitrocellulose filters, 5 × SSC, 0.
Pre-wash solution of 5% SDS, 1.0 mM ethylenediaminetetraacetic acid (EDTA) (pH 8.0), about 50% formamide at about 40-50 ° C, 2 x SSC-6 x SSC (or at about 42 ° C). Stark's wash in about 50% formamide
Hybridization conditions of other similar hybridization solutions), such as on),
And use of cleaning conditions of about 60 ° C., 0.5 × SSC, 0.1% SDS is included. Thus, the polypeptides used in the present invention are encoded by nucleic acid molecules that hybridize under highly or moderately stringent conditions to the nucleic acid molecules encoding the polypeptides specifically described in the present invention. Polypeptides are also included.

As used herein, a "purified" substance or biological factor (eg, nucleic acid or protein) is one in which at least some of the factors naturally associated with the substance or biological factor have been removed. .. Therefore, the purity of the biological factor in the purified biological factor is usually higher (ie, enriched) than in the state in which the biological factor 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. It means that the same type of biological factor is present. The substance or biological factor used in the present invention is preferably a "purified" substance. As used herein, an "isolated" substance or biological factor (eg, nucleic acid or protein) is substantially depleted of a factor naturally associated with that substance or biological factor. Say something. The term "isolated" as used herein varies depending on its purpose and therefore does not necessarily have to be expressed in purity, but if necessary, preferably at least 75.
%%, more preferably at least 85% by weight, even more preferably at least 95% by weight
, And most preferably at least 98% by weight, which means that there is an isomorphic biological factor. The substance used in the present invention is preferably an "isolated" substance or biological factor.

As used herein, a "corresponding" amino acid or nucleic acid or moiety is a predetermined amino acid or nucleotide or moiety in a polypeptide or polynucleotide that is a reference for comparison in a polypeptide molecule or polynucleotide molecule (eg, CARD14). Amino acids or nucleotides that have or are expected to have similar effects, especially in the case of enzyme molecules, amino acids that are present at similar positions in the active site and make similar contributions to catalytic activity. In the case of a complex molecule, it means a corresponding portion (for example, a transmembrane domain). For example, if it is an antisense molecule, it can be a similar part in the ortholog corresponding to a particular part of the antisense molecule. The corresponding amino acids are, for example,
Cysteinization, glutathione formation, SS bond formation, oxidation (eg, oxidation of the methionine side chain)
, Formylation, acetylation, phosphorylation, glycosylation, myristylation, etc. can be specific amino acids. Alternatively, the corresponding amino acid can be the amino acid responsible for dimerization.
Such "corresponding" amino acids or nucleic acids may be regions or domains over a range. Thus, such cases are referred to herein as "corresponding" regions or domains. 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 some species, similar effects to a given gene in the species of reference for comparison. A gene (for example, a polynucleotide sequence or a molecule), which has the same evolutionary origin when there are a plurality of genes having such an action. Therefore, the gene corresponding to a gene can be the ortholog of that gene. Thus, each human CARD 14 can find a corresponding CARD 14 in other animals, especially mammals. Such corresponding genes can be identified using techniques well known in the art. Thus, for example, the corresponding gene in an animal (eg, mouse) is the reference gene for the corresponding gene (eg, CAR).
It can be found by searching the database containing the sequence of the animal using the sequence of D14 etc. (SEQ ID NO: 1, 3 etc. or SEQ ID NO: 2, 4 etc.) as the query sequence.

As used herein, the term "fragment" refers to a polypeptide or polynucleotide having a sequence length from 1 to n-1 with respect to a full-length polypeptide or polynucleotide (length n). The length of the fragment can be appropriately changed according to its purpose. For example, in the case of a polypeptide, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, 15, 20
, 25, 30, 40, 50 and more amino acids, and lengths represented by integers not specifically listed here (eg, 11) may also be appropriate as lower bounds. In the case of polynucleotides, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides are mentioned, and are specifically listed here. A length represented by a non-integer (eg, 11) can also be a suitable lower bound. As used herein, such fragments, for example, where the full length functions as a marker, therapeutic agent or target molecule, the present invention as long as the fragment itself also functions as a marker, therapeutic agent or target molecule. It is understood that it falls within the range of.

According to the present invention, the term "activity" refers to the function of a molecule in the broadest sense herein. Activities are not intended to be limiting, but generally include the biological, biochemical, physical or chemical functions of the molecule. The activity, for example, activates, promotes, stabilizes, inhibits, suppresses, or destabilizes enzymatic activity, the ability to interact with other molecules, and the function of other molecules. Includes the ability to do, stability, and the ability to localize to specific intracellular locations. Where applicable, the term also relates to the function of protein complexes in the broadest sense.

As used herein, the term "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 include, but are not limited to, the production of specific antibodies, the impartation of enzyme activity, resistance, and the like. In the present invention, CARD14 belongs to the membrane-associated guanylate kinase (MAGUK) family and functions as a molecular scaffold that specializes in the region of the plasma membrane to form an assembly of a multiprotein complex. And also, this protein belongs to the CARD family, has a specific caspase-related recruitment domain (CARD), has a 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, associations with, but are not limited to, these functions can be mentioned. As used herein, biological function can be exerted by "biological activity". "Biological activity" herein
Is an activity that a certain factor (for example, polynucleotide, protein, etc.) can have in a living body, and includes an activity that exerts various functions (for example, transcription promoting activity), for example, a certain molecule. Also included is an activity in which another molecule is activated or inactivated by interaction with. When two factors interact, their biological activity can be the binding between the two molecules and the resulting biological changes, and, for example, one molecule was precipitated with an antibody. Sometimes when other molecules also coprecipitate, the two molecules are considered to be bound.
Therefore, seeing such coprecipitation is one of the judgment methods. For example, if a factor is an enzyme, its biological activity comprises that enzymatic activity. In another example, when a factor is a ligand, the ligand involves 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). Various measurable indicators, such as the affinity of a compound that directly binds to a polypeptide or polynucleotide of the invention, or, for example, the amount of upstream or downstream protein or other after some stimulation or event. A similar functional scale can be mentioned.

As used herein, the "value" of CARD14 is the amount of protein in CARD14, mRN.
Any value directly or indirectly with respect to CARD14, such as the expression level of A and the enzyme activity, may be used. This "value" is used in the present invention as an indicator of reducing psoriasis, multiple sclerosis, γδ T cell-mediated disease and / or complications of immune intervention, and / or side effects of TLR-based / IMQ / immunostimulatory adjuvants. be able to.

As used herein, the term "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,
Genes, polynucleotides, etc. are transcribed and translated into the form of polypeptides, but transcription to produce mRNA is also an aspect of expression. Therefore,
As used herein, the term "expression product" includes such a polypeptide or protein, or mRNA. More preferably, the form of such a polypeptide can be post-translationally processed. For example, the expression level of CARD14 can be determined by any method. Specifically, CAR by assessing the amount of CARD14 mRNA, the amount of CARD14 protein, and the biological activity of CARD14 protein.
The expression level of D14 can be known. Such measurements can be used in companion diagnostics. The amount of CARD 14 mRNA or protein can be determined by methods as detailed elsewhere herein or by other methods known in the art.

As used herein, the term "functional equivalent" refers to any entity that has the same target function but a different structure with respect to the original entity of interest. Thus, the functional equivalent of "CARD14" or its antibody is not CARD14 or its antibody itself, but a variant or variant of CARD14 or its antibody (eg, an amino acid sequence variant, etc.) and CAR.
Those having the biological action of D14, and at the time of action, CARD1
4 or its antibody itself or one that can be transformed into a variant or variant of this CARD14 or its antibody (eg, CARD14 or its antibody itself or CARD1)
4 or a nucleic acid encoding a variant or variant of an antibody thereof, and a vector containing the nucleic acid, a cell, etc.) is understood to be included. It is understood that in the present invention, the functional equivalent of CARD 14 or an antibody thereof can be used in the same manner as CARD 14 or an antibody thereof, without particular mention. Functional equivalents can be found by searching databases and the like. As used herein, the term "search" refers to finding another nucleobase sequence having a specific function and / or property by utilizing one nucleobase sequence electronically, biologically, or by some other method. Say. For electronic search, BLAST (A)
ltschul et al., J.Mol.Biol. 215: 403-410 (1990)), FASTA (Pearson & Lipman, P)
roc.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
The law (Needleman and Wunsch, J.Mol.Biol.48: 443-453 (1970)) is mentioned, but not limited to them. Biological searches include stringent hybridization, macroarrays in which genomic DNA is attached to a nylon membrane or the like, or microarrays (microarray assays) in which genomic DNA is attached to a glass plate, PCR and in situ hybridization, and the like. Not limited to. As used herein, it is intended that the genes used in the present invention should also include corresponding genes identified by such electronic and biological searches.

As the functional equivalent of the present invention, one or more amino acids inserted, substituted or deleted, or added to one or both ends of the amino acid sequence can be used. In the present specification, "insertion, substitution or deletion of one or more amino acids in an amino acid sequence, or addition to one or both ends" is a well-known technical technique such as a site-specific mutagenesis method. It means that the modification has been made by a method or by a natural mutation, such as by substituting a plurality of amino acids that can occur naturally. The modified amino acid sequence includes, for example, 1 to 30, preferably 1 to 20, more preferably 1 to 9, still more preferably 1 to 5, particularly preferably 1 to 2 amino acids inserted, substituted, or missing. It can be lost, or added to one or both ends. The modified amino acid sequence is preferably an amino acid sequence in which the amino acid sequence has one or more (preferably one or several or 1, 2, 3, or 4) conservative substitutions in the amino acid sequence of CARD14. You may. As used herein, "conservative substitution" means substituting 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, there is a case where one hydrophobic residue is replaced with another hydrophobic residue, a case where one polar residue is replaced with another polar residue having the same charge, and the like. Functionally similar amino acids capable of making 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. Polar (neutral) amino acids include glycine, serine,
Examples include threonine, tyrosine, glutamine, asparagine and cysteine. Examples of positively charged (basic) amino acids include arginine, histidine, lysine and the like. Examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

As used herein, the term "inhibitor" or "inhibitor" or "inhibitor" (both of which correspond to inhibitors in English) refers to a substance of interest (eg, a receptor or cell) of the receptor or cell. A substance or factor that inhibits a biological action. As the inhibitor of CARD14 of the present invention, the target CARD14 or CARD
Factors that can temporarily or permanently reduce or eliminate the action or function of cells expressing 14 and the like. Such factors include, but are not limited to, antibodies, antigen-binding fragments thereof, derivatives thereof, functional equivalents, antisense, nucleic acid forms such as RNAi factors such as siRNA, and the like.

As used herein, the term "agonist" refers to a substance that expresses or enhances the biological action of a receptor on a target entity (for example, a receptor). In addition to natural agonists (also called ligands), synthetic and modified ones can be mentioned. As used herein, the term "antagonist" refers to a substance that suppresses or inhibits the expression of the biological action of a receptor on a target entity (for example, a receptor).
In addition to natural antagonists, synthetic and modified ones can be mentioned. In addition to those that competitively suppress or inhibit agonists (or ligands), there are those that suppress or inhibit non-competitively. It can also be obtained by modifying the agonist. Antagonists can be included in the concept of inhibitors (inhibitors or inhibitors) or inhibitors or suppressors (inhibitors) because they suppress or inhibit physiological phenomena. Therefore, in the present specification, an antagonist is used substantially synonymously with "inhibitor".

In the present specification, "antibody" is broadly referred to as a polyclonal antibody, a monoclonal antibody, a multispecific antibody, a chimeric antibody, and an anti-idiotype antibody, and fragments thereof such as Fv fragment, Fab'fragment, and F (ab'). 2 and Fab fragments, as well as conjugates or functional equivalents produced by other recombination (eg, chimeric antibodies, humanized antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single chains Antibody, scFV, diabody, sc (Fv) 2 (single chain (Fv) 2), sc
Fv-Fc) is included. Further, such an antibody may be covalently linked or recombinantly fused to an enzyme such as alkaline phosphatase, horseradish peroxidase, alpha galactosidase, or the like. The anti-CARD14 antibody used in the present invention may bind to the protein of CARD14, 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, humanized antibodies and the like can be used. In the present invention, monoclonal or polyclonal can be used as an antibody, but a monoclonal antibody is preferable.
The binding of the antibody to the CARD14 protein is preferably a specific binding. Antibodies also include antibody-modified or antibody-unmodified products. The antibody-modified product may have various molecules such as polyethylene glycol bonded to the antibody. The antibody-modified product can be obtained by chemically modifying the antibody using a known method.

In one embodiment of the present invention, the "polyclonal antibody" is used in mammals (for example, rats, mice, rabbits, cows, monkeys, etc.), birds, etc. in order to induce the production of an antigen-specific polyclonal antibody. It can be produced by administering an immunogen containing the antigen of interest. The immunogen may be administered by injecting one or more immunogens and, if desired, an adjuvant. The 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 detergent (such as lysolecithin). .. Immune protocols are known in the art and may be carried out by any method that elicits an immune response, depending on the host organism of choice (Protein Experiment Handbook, Yodosha (2003): 86-
91.).

In one embodiment of the invention, a "monoclonal antibody" is an antibody that corresponds to a substantially single epitope, except for antibodies in which the individual antibodies that make up the population have mutations that can occur spontaneously in small amounts. Including the case where. Alternatively, the individual antibodies that make up the population may be substantially identical, except for antibodies that have mutations that can occur spontaneously in small amounts. Monoclonal antibodies are highly specific and differ from conventional polyclonal antibodies, which typically contain 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 with other immunoglobulins. The description "monoclonal" may indicate that it is obtained from a substantially homogeneous population of antibodies, but does not imply that the antibody must be produced in any particular way. For example, the monoclonal antibody "Kohle
r G, Milstein C., Nature. 1975 Aug 7; 256 (5517): 495-497. "Can be prepared by a method similar to the hybridoma method, or monoclonal antibodies can be made from the United States. It may be prepared by a method similar to the recombinant method described in Japanese Patent No. 4816567, or the monoclonal antibody may be prepared by "Clackson et al., Nature. 1991.
Aug 15; 352 (6336): 624-628. ", Or" Marks et al., J Mol Biol. 1991 Dec
It may be isolated from the phage antibody library using a technique similar to that described in 5; 222 (3): 581-597. "Or" Protein Experiments Hanbook, Yodosha ( 2003): 9
It may be prepared by the method described in 2-96. ".

In one embodiment of the invention, the "humanized antibody" is, for example, one or more CDRs from a non-human species.
, And a framework region (FR) derived from human immunoglobulin, and a constant region derived from human immunoglobulin, which is an antibody that binds to a desired antigen. Humanization of antibodies 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 graphing (Ozaki et al., Blood)
. 1999 Jun 1; 93 (11): 3922-3930.), Re-surfacing (roguska et al., Proc Nat
l Acad Sci US A. 1994 Feb 1; 91 (3): 969-973.), Or FR shuffle (Damsch)
roder et al., Mol Immunol. 2007 Apr; 44 (11): 3049-3060. Epub 2007 Jan 22
.) And so on. Human F to modify (preferably improve) antigen binding
The amino acid residue in the R region may be replaced with the corresponding residue from the CDR donor antibody. This FR substitution can be performed by methods well known in the art (Riechmann et al., Natur).
e. 1988 Mar 24; 332 (6162): 323-327.). For example, FR residues important for antigen binding may be identified by modeling the interaction between CDR and FR residues. Alternatively, sequence comparison may identify anomalous FR residues at specific positions.

In one embodiment of the present invention, the "human antibody" is derived from, for example, a gene in which a variable region and a constant region of a heavy chain and a region including a variable region and a constant region of a light chain constituting the antibody encode a human immunoglobulin. It is an antibody to be used. The main production methods include a transgenic mouse method for producing human antibodies and a phage display method. In the transgenic mouse method for producing human antibodies, if a functional human Ig gene is introduced into a mouse in which endogenous Ig is knocked out, a human antibody having various antigen-binding ability is produced instead of the mouse antibody. 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.". The phage display method typically fuses a foreign gene on the N-terminal side of the coat protein (g3p, g10p, etc.) of a fibrous phage such as M13 or T7, which is one of the Escherichia coli viruses, so as not to lose the infectivity of the phage. It is a system that is expressed 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 invention, the "anti-CARD14 antibody" comprises an antibody that has binding to CARD14. The method for producing this anti-CARD14 antibody is not particularly limited, but for example, CA
RD14 may be produced by immunizing mammals or birds.

Further, the "functional equivalent" of "antibody against CARD14 (anti-CARD14 antibody) or fragment thereof" is, for example, in the case of an antibody, the antibody itself having CARD14 binding activity and, if necessary, inhibitory activity and a fragment thereof. In addition to itself, chimeric antibodies, humanized antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single chain antibodies, s
cFV, diabody, sc (Fv) 2 (single chain (Fv) 2),
It is understood that scFv-Fc and the like are also included.

The anti-CARD 14 antibody according to one embodiment of the present invention is preferably an anti-CARD 14 antibody that specifically binds to a specific epitope of CARD 14 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. Monoclonal antibodies can act on CARD14 more efficiently than polyclonal antibodies. From the viewpoint of efficiently producing the anti-CARD14 monoclonal antibody, it is preferable to immunize the chicken from CARD14.

The antibody class of the anti-CARD14 antibody according to one 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, may be referred to as an “antigen-binding fragment”). In this case, there are effects such as increased 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 one in which a polypeptide or oligopeptide is bound to the N- or C-terminus of the anti-CARD14 antibody. Here, the oligopeptide may be a His tag. The fusion protein may also be a fusion of a mouse, human, or chicken antibody partial sequence. Fusion proteins such as these 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 includes, for example, purified CARD14, CAR.
It may be an antibody obtained through a step of immunizing an organism with a D14-expressing cell or a CARD14-containing lipid membrane. From the viewpoint of enhancing the therapeutic effect on CARD14 positive diseases, CAR
It is preferred to use D14 expressing cells for immunization.

The anti-CARD14 antibody according to one embodiment of the present invention is a C of an antibody obtained through a step of immunizing an organism with purified CARD14, CARD14-expressing cell or CARD14-containing lipid membrane.
It may be an antibody having a DR set. From the viewpoint of enhancing the therapeutic effect on CARD14-positive diseases, it is preferable to use CARD14-expressing cells for immunity. The CDR set is a set of heavy chain CDRs 1, 2, and 3, and light chain CDRs 1, 2, and 3.

Anti CARD14 antibody according to an embodiment of the present invention, as far as it achieves the object may have any binding force, for example, at least 1.0 × 10 ⁶ or more, 2.0 × 10 ⁶ or more , 5.0 × 10 ⁶ or more, there may be mentioned a 1.0 × 10 ⁷ or more without being limited to, usually, the dissociation constant (KD) values, even 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 form of CARD14. Mutants include those due to 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, and particularly preferably 98% or more, based on the amino acid sequence shown in SEQ ID NO: 2 or 4. Has homology.

In one embodiment of the invention, the "CDR (complementarity determining regions)" is a region of an antibody that is actually in contact with an antigen to form a binding site. Generally, CDR is the Fv of an antibody (variable region:
It is located on the heavy chain variable region (VH) and the light chain variable region (VL). Also generally
CDRs include CDR1, CDR2, and CDR3, which consist of about 5 to 30 amino acid residues. In particular, it is known that heavy chain CDRs contribute to the binding of antibodies to antigens. Also, among the CDRs, CDR3
Is known to have the highest contribution of antibodies to antigen binding. For example, "Willy et
al., Biochemical and Biophysical Research Communications Volume 356, I
ssue 1, 27 April 2007, Pages 124-128 "states that the binding ability of the antibody was increased by modifying the heavy chain CDR3. The Fv region other than the CDR is the framework region (F).
Called R), it consists of FR1, FR2, FR3 and FR4 and is relatively well conserved among the antibodies (K).
abat et al., "Sequence of Proteins of Immunological Interest" US Dept.
Health and Human Services, 1983.). That is, the factor that characterizes antibody reactivity is CDR.
It can be said that it is especially in heavy chain CDR.

Several methods have been reported to determine the definition of CDR and its location. For example, the definition of Kabat
(Sequences of Proteins of Immunological Interest, 5th ed., Public Healt
h Service, National Institutes of Health, Bethesda, MD. (1991)), or C
The definition of hothia (Chothia et al., J. Mol. Biol., 1987; 196: 901-917) may be adopted. In one embodiment of the invention, the definition of Kabat is adopted as a preferred example, but is not necessarily limited to this. In some cases, both the Kabat definition and the Chothia definition may be taken into consideration when deciding. For example, the overlapping part of the CDR according to each definition or the part including both the CDR according to each definition may be determined. It can also be a CDR. A concrete example of such a method is Oxford Molecular's AbM antibod, which is a compromise between Kabat's definition and Chothia's definition.
Martin et al.'S method using y modeling software (Proc.Natl.Acad.Sci.USA, 1989; 86:92)
68-9272). Such CDR information can be used to produce variants that can be used in the present invention. In such antibody variants, one or several (eg, 2, 3, 4, 5, 6, 7, 8, 9, 9, 10) in the original antibody framework. ) Is included, but the CDR can be produced without mutations.

As used herein, the term "antigen" refers to any substrate that can be specifically bound by an antibody molecule. As used herein, the term "immunogen" refers to an antigen capable of initiating lymphocyte activation that produces an antigen-specific immune response. As used herein, the term "epitope" or "antigen 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 one of ordinary skill in the art when the primary sequence of nucleic acid or amino acid is provided. It is understood that the antibody of the present invention can be similarly utilized even if the antibody has another sequence as long as the epitope is the same.

Antibodies used herein are used as long as undesired effects such as side reactions are diminished.
It is understood that antibodies of any specificity may be used. Therefore, the antibody used in the present invention may be a polyclonal antibody or a monoclonal antibody.

In the present specification, the "subject (person)" is a subject (for example, an organism such as a human being or cells taken from the organism, blood, serum, etc.) to be diagnosed, detected, or treated according to the present invention.
To say.

As used herein, "drug", "drug" or "factor" (both corresponding to agents in English) are used interchangeably in any sense as 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, for example.
cDNA, DNA such as genomic DNA, including RNA such as mRNA), polysaccharides, oligosaccharides, lipids, organic small molecules (eg, hormones, ligands, signaling substances, organic small molecules,
Molecules synthesized in combinatorial chemistry, small molecules that can be used as pharmaceuticals (for example, small molecule ligands, etc.), and complex molecules thereof include, but are not limited to. Factors specific to a polynucleotide typically include polynucleotides that have a certain degree of sequence homology (eg, 70% or more sequence identity) and complementarity to the sequence of the polynucleotide. Examples include, but are not limited to, polypeptides such as transcription factors that bind to the promoter region. Factors specific to polypeptides include
Typically, an antibody or derivative thereof or an analog thereof (eg, a single chain antibody) specifically directed to the polypeptide, a specific ligand or receptor when the polypeptide is a receptor or ligand. , When the polypeptide is an enzyme, its substrate and the like can be mentioned, but not limited thereto.

As used herein, the term "treatment" refers to the prevention of exacerbation of a disease or disorder (eg, allergy) in the event of such a condition, preferably maintaining the status quo, more preferably. Refers to alleviation, more preferably withdrawal, and includes the ability to exert a symptom-improving or preventive effect on a patient's disease or one or more symptoms associated with the disease. Diagnosis in advance and appropriate treatment is called "companion treatment".
The diagnostic agent for that purpose is sometimes called "companion diagnostic agent".

As used herein, the term "therapeutic agent (agent)" broadly refers to any agent capable of treating a desired condition (for example, a disease such as allergy). In one embodiment of the 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, for example, by mixing the active ingredient with the above carrier and using any method known in the technical field of pharmaceutics. Further, the therapeutic agent is not limited in the form of use as long as it is used for treatment, and 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 therapeutic agent for allergies includes a drug (preventive agent) used for preventing allergies or an allergy suppressant.

As used herein, the term "prevention" refers to preventing a disease or disorder (eg, allergy) from becoming such a condition before it becomes such a condition. The agent of the present invention can be used for diagnosis, and if necessary, the agent of the present invention can be used to prevent, for example, allergies, or to take preventive measures.

As used herein, the term "preventive drug (drug)" in a broad sense means any drug that can prevent a desired condition (for example, a disease such as allergy).

Thus, as used herein, a "factor" (or drug, detector, etc.) that "specifically" interacts with (or binds to) a biological factor, such as a polynucleotide or polypeptide, is that poly. Affinity for biological factors such as nucleotides or their polypeptides is typically equivalent or better than affinity for other unrelated (especially less than 30% identity) polynucleotides or polypeptides. High or preferably significant (eg,
Includes statistically significant) higher ones. Such affinity can be measured, for example, by hybridization assay, binding assay and the like.

As used herein, a first substance or factor "specifically" interacts with (or binds to) a second substance or factor when the first substance or factor interacts with (or binds to) a second substance or factor. To interact (or bind) with a higher affinity for 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. Say that. Specific interactions (or bindings) with respect to a substance or factor include, for example, hybridization in nucleic acids, antigen-antibody reaction in proteins, and so on.
Nucleic acid-protein reactions, protein-lipid interactions, nucleic acids-, such as enzyme-substrate reactions
Examples include, but are not limited to, lipid interactions. Therefore, when a substance or factor is both nucleic acids, the first substance or factor "specifically interacts" with the second substance or factor means that the first substance or factor is the second substance. Alternatively, it includes having complementarity to at least a part of the factor. Also, for example, when a substance or factor is both proteins, the first substance or factor "specifically" interacts with (or binds to) the second substance or factor, for example, by an antigen-antibody reaction. Interaction, receptor-
Interactions by ligand reaction, enzyme-substrate interaction, etc. are included, but are not limited thereto. When two substances or factors include proteins and nucleic acids, the first substance or factor to "specifically" interact with (or bind to) the second substance or factor is an antibody and its antigen. Interactions (or bindings) with are included. By utilizing such a specific interaction or binding reaction, it is possible to detect or quantify an object in a sample.

"Detection" or "quantification" of polynucleotide or polypeptide expression herein.
Can be achieved using suitable methods, including methods for measuring mRNA and immunological measurement, including, for example, binding or interaction to a detection agent, test agent or diagnostic agent. Examples of the molecular biological measurement method include Northern blotting, dot blotting, PCR and the like. Examples of immunological measurement methods include ELISA method using a microtiter plate, radioimmunoassay (RIA) method, fluorescent antibody method, and luminescent immunoassay (LIA).
Examples thereof include methods, immunoprecipitation (IP), immunodiffusion (SRID), turbidimetry (TIA), Western blotting, and immunohistochemical staining. Moreover, as a quantitative method, an ELISA method, an RIA method and the like are exemplified. It can also be performed by a gene analysis method using an array (for example, a DNA array or a protein array). DNA arrays are widely outlined in (Shujunsha ed., Cell Engineering Separate Volume "DNA Microarrays and Latest PCR Methods"). For protein arrays, Nat
Genet. It is detailed in 2002 Dec; 32 Suppl: 526-532. In addition to the above, gene expression analysis methods include RT-PCR, RACE method, SSCP method, immunoprecipitation method, two-hybrid system, and in vi.
Examples include, but are not limited to, tro translations. Such further analytical methods are described, for example, in the Genome Analysis Experimental Method / Yusuke Nakamura Lab Manual, edited by Yusuke Nakamura Yodosha (2002), and all of these descriptions are incorporated herein by reference. Will be done.

As used herein, the term "expression level" refers to the amount at which a polypeptide, mRNA, or the like is expressed in a target cell, tissue, or the like. As such an expression level, ELIS using the antibody of the present invention
The expression level of the polypeptide of the present invention at the protein level, or Northern, evaluated by any suitable method including immunological measurement methods such as method A, RIA method, immunofluorescence method, Western blotting method, and immunohistochemical staining method. The polypeptide used in the present invention evaluated by any suitable method including molecular biological measurement methods such as blotting, dot blotting, PCR and the like.
The expression level 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 as evaluated by any suitable method including the above immunological measurement method or molecular biological measurement method. It means that the amount increases or decreases. By measuring the expression level of a certain marker, various detections or diagnoses based on the marker can be performed.

As used herein, the "activity, expression product (eg, protein, transcript (RNA, etc.))"
"Reduced" or "suppressed" or a synonym thereof refers to an activity that reduces or reduces a particular activity, the quantity, quality or effect of a transcript or protein. When "disappeared" out of the decrease, it means that the activity, expressed product, etc. are below the detection limit, and in particular, it may be referred to as "disappeared". As used herein, "disappearance" is included in "decrease" or "suppression".

As used herein, the "activity, expression product (eg, protein, transcript (RNA, etc.))"
"Increase" or "activation" or a synonym thereof refers to an activity that increases or increases in a particular activity, the quantity, quality or effect of a transcript or protein.

As used herein, the term "label" refers to an entity (eg, substance, energy, electromagnetic wave, etc.) for identifying a molecule or substance of interest from others. As such a labeling method, RI
(Radioisotope) method, fluorescence method, biotin method, chemiluminescence method and the like can be mentioned.
When a plurality of markers or factors or means for capturing the markers of the present invention are labeled by the fluorescence method, the markers are labeled with fluorescent substances having different fluorescence maximum wavelengths. The difference in fluorescence emission maximum wavelength is preferably 10 nm or more. When labeling the ligand, any one that does not affect the function can be used, but Alexa ^TM Fluor is preferable as the fluorescent substance. Alexa ^TM Fluor is a water-soluble fluorescent dye obtained by modifying coumarin, rhodamine, fluorescein, cyanine, etc., and is a series that supports a wide range of fluorescent wavelengths. Stable, bright and low pH sensitivity. As a combination of fluorescent dyes having a maximum fluorescence wavelength of 10 nm or more, a combination of Alexa ^TM 555 and Alexa ^TM 633,
The combination of Alexa ^TM 488 and Alexa ^TM 555 can be mentioned. When labeling nucleic acids
Any one that can bind to the base portion can be used, but cyanine pigments (for example, Cy3, Cy5, etc. of CyDyeTM series), rhodamine 6G reagent, 2-acetylaminofluorene (AAF), AAIF (iodine of AAF). Derivatives) and the like are preferably used. Examples of the fluorescent substance having a difference in fluorescence emission maximum 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, such a label can be used to modify the target object 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 implement such methods depending on the label and the subject of interest.

As used herein, a "kit" is a unit that is usually divided into two or more compartments and is provided with parts to be provided (for example, a test drug, a diagnostic drug, a therapeutic drug, an antibody, a label, an instruction manual, etc.). To say. The form of this kit is preferred when the purpose is to provide a composition that should not be mixed and provided for stability and the like, but is preferably mixed and used immediately before use.
Such kits are preferably instructions or instructions describing how to use the provided parts (eg, test agents, diagnostic agents, therapeutic agents, or how reagents should be treated). When the kit is used as a reagent kit in the present specification, the kit usually includes an instruction manual or the like that describes how to use a test drug, a diagnostic drug, a therapeutic drug, an antibody, or the like. Is included.

As used herein, the "instruction" describes the method of using the present invention to a doctor or another user. This instruction sheet contains words instructing the detection method of the present invention, how to use a diagnostic agent, or administration of a medicine or the like. In addition, the instruction sheet may include a wording instructing the administration site to be administered orally or to the esophagus (for example, by injection). This instruction is prepared in accordance with the format prescribed by the regulatory agency of the country in which the invention is implemented (for example, the Ministry of Health, Labor and Welfare in Japan, the Food and Drug Administration (FDA) in the United States, etc.) and approved by the regulatory agency. It is clearly stated that it has been received. Instructions are so-called package inserts, usually provided in paper media, but are not limited to, for example, in the form of electronic media (eg, homepages provided on the Internet, e-mail). But can be provided.

(CARD14 inhibitor)
The 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 its function, and the like. Therefore, the "CARD14 inhibitor" includes a substance that inhibits the expression of CARD14, a substance that inhibits the function of CARD14, and any other substance that finally inhibits the action of CARD14 even if the mechanism of action is unknown. It may include, but is not limited to, antibodies, antibody fragments, derivatives thereof, nucleic acids, small molecules, other antagonists and the like. Such various antibodies and the like will be described elsewhere herein. Preferably, it is advantageous that such a CARD14 inhibitor is one that specifically inhibits CARD14. This is because side effects due to the influence on factors other than CARD 14 can be reduced.

In the present invention, the "substance that inhibits the expression of CARD14" means any stage such as the transcription level of the nucleic acid encoding CARD14 (CARD14 gene), the level of posttranscriptional regulation, the level of translation into CARD14 protein, the level of post-translational modification, and the like. It may act on. Therefore, as substances that inhibit the expression of CARD14, for example, substances that inhibit transcription of the CARD14 gene (eg, antigene), substances that inhibit the processing of early transcripts to mRNA, and substances that inhibit the transport of mRNA to the cytoplasm. CARD from mRNA
14 include substances that inhibit translation (eg, antisense nucleic acid, miRNA) or that degrade mRNA (eg, siRNA, ribozyme), substances that inhibit post-translational modification of the initial translation product, and the like. Anything that acts at any stage can be preferably used.

Here, mRNA is mentioned as a preferable example of a transcript.

As a substance that specifically inhibits (or degrades) the translation of the CARD14 gene mRNA into CARD14, it is preferable that the base sequence is complementary to or substantially complementary to the base sequence of these mRNAs or a part thereof. Nucleic acid containing.

A base sequence that is substantially complementary to the base sequence of the mRNA of the CARD14 gene is a base sequence that is substantially complementary to the base sequence of the CARD14 gene under the physiological conditions of CARD14-producing cells (eg, neutrophils) in the mammal to be administered.
It means a base sequence having a degree of complementarity that can bind to the target sequence of the mRNA and inhibit its translation (or cleave the target sequence), and specifically, for example, completely with the base sequence of the mRNA. A base sequence having about 90% or more, preferably about 95% or more, more preferably about 97% or more homology with respect to a region that overlaps with the complementary base sequence (that is, the base sequence of the complementary strand of mRNA). Is. “Nucleotide sequence homology” in the present invention refers to the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Ba).
Using the sic Local Alignment Search Tool), it can be calculated under the following conditions (expected value = 10; gap allowed; filtering = ON; match score = 1; mismatch score = -3).

More specifically, the base sequence complementary or substantially complementary to the base sequence of the mRNA of the CARD14 gene is represented by the following (k) or (l): (k) SEQ ID NO: 1 or 3. Nucleotide sequence complementary or substantially complementary to the base sequence; (l) A base sequence that hybridizes with the complementary chain sequence of the base sequence represented by SEQ ID NO: 1 or 3 under stringent conditions, and A sequence encoding a protein having at least one function of CARD14 and a complementary or substantially complementary base sequence; can be mentioned. The stringent conditions are as described above. The function of CARD14 is not limited to these, but for example, the function of membrane-associated guanylate kinase (MAGUK), the function as a molecular scaffold for assembling multiple protein complexes in a specific region of the plasma membrane, and the like. Caspase-related recruitment function due to having a caspase-related recruitment function Function of interaction with BCL10, function as a positive regulator of NF-κB activation and cell apoptosis, N
Examples include activation of F-κB and a function of inducing phosphorylation of BCL10.

A preferred example of the mRNA of the CARD14 gene is a human CA containing the nucleotide sequence represented by SEQ ID NO: 1 (Genbank Accession No. NM_052972).
RD14 mRNA, or their orthologs in other mammals (eg, mouse CARD14 (SEQ ID NO: 3, Genbank Accession No. NM_0)
29996) etc.), and further include splicing variants, allelic variants, polymorphic variants and the like.

The base sequence of the mRNA of the CARD14 gene and "a part of the base sequence complementary or substantially complementary" can specifically bind to the mRNA of the CARD14 gene and translate the protein from the mRNA. As long as it can inhibit (or degrade the mRNA), its length and position are not particularly limited, but from the viewpoint of sequence specificity, at least 10 bases of a portion complementary or substantially complementary to the target sequence As described above, it preferably contains about 15 bases or more.

Specifically, any 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 the mRNA of the CARD14 gene or a part thereof. Be done:
(1) Antisense nucleic acid against mRNA of CARD14 gene,
(2) Ribozyme nucleic acid for mRNA of CARD14 gene,
(3) A nucleic acid having RNAi activity against the mRNA of the CARD14 gene or a precursor thereof.

(1) Antisense Nucleic Acid Against mRNA of CARD14 Gene The "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. A nucleic acid that forms and binds to a target mRNA by forming a specific and stable duplex.
It has a function of suppressing protein synthesis.

Antisense nucleic acids include polydeoxyribonucleotides containing 2-deoxy-D-ribose, polyribonucleotides containing D-ribose, and other types of polymers that are N-glycosides of purine or pyrimidine bases. Other polymers with a non-nucleotide skeleton (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special bindings (provided that the polymer is a base as found in DNA or RNA). (Contains a nucleotide having an arrangement that allows the pairing and attachment of bases). They may be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, DNA: RNA hybrids, as well as unmodified polynucleotides (or unmodified oligonucleotides), known modifications. Additions, such as those with a label known in the art, those with a cap, those that are methylated, 1
Two or more natural nucleotides substituted with relatives, those with intramolecular nucleotide modifications, such as those with uncharged bonds (eg, methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.), charge Or having a sulfur-containing bond (eg, phosphorothioate, phosphorodithioate, etc.), such as a protein (eg, nuclease, nuclease inhibitor, toxin, antibody, signal peptide, poly-L-lysine, etc.) or sugar (eg, nuclease, nuclease inhibitor, toxin, antibody, signal peptide, poly-L-lysine, etc.) Examples, those having side chain groups such as monosaccharides (eg, monosaccharides, etc.), those having intermediate compounds (eg, aclysine, solarene, etc.), chelating compounds (eg, metals, radioactive metals, boron, oxidizing) Metals, etc.)
Those containing an alkylating agent and those having a modified bond (for example, α-anomeric type nucleic acid) may be used. Here, the "nucleoside", "nucleotide" and "nucleic acid" may include those containing not only purine and pyrimidine bases but also modified other heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleosides and modified nucleotides may also have modified sugar moieties, for example, one or more hydroxyl groups substituted with halogens, aliphatic groups, etc., or functional groups such as ethers, amines, etc. It may have been converted.

As mentioned above, the antisense nucleic acid may be DNA, RNA, or D.
It may be an NA / RNA chimera. When the antisense nucleic acid is DNA, the RNA: DNA hybrid formed by the target RNA and the antisense DNA is endogenous RNAs.
It can be recognized by eH and cause selective degradation of target RNA. Therefore, R
In the case of antisense DNA directed to degradation by NaseH, the target sequence may be not only the sequence in mRNA but also the sequence of the intron region in the initial translation of the 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 length of the target region of the antisense nucleic acid of the present invention is not particularly limited as long as the hybridization of the antisense nucleic acid inhibits the translation into protein: CARD14, and CARD14 is encoded. The entire sequence or partial sequence of the mRNA to be used may be used, and the short one may be about 10 bases, and the long one may be the full sequence of the mRNA or the initial transcript. Considering the ease of synthesis, antigenicity, intracellular translocation, and the like, oligonucleotides consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases are preferable.
Not limited to that. Specifically, the 5'end hairpin loop of the CARD14 gene, the 5'end 6-base pair repeat, the 5'end untranslated region, the translation initiation codon, the protein coding region, the open reading frame (ORF) translation termination codon, 3 'End untranslated regions, 3'end parindrome regions or 3'end hairpin loops can be selected as preferred target regions for antisense nucleic acids, but are not limited thereto.

Furthermore, the antisense nucleic acid of the present invention not only hybridizes with the mRNA and early transcript of the CARD14 gene to inhibit translation into protein, but also binds to these genes, which are double-stranded DNA, to form a triple strand (triple strand). It may be one that can form a triplet) and inhibit transcription into RNA (antigene).

The nucleotide molecules that make up the antisense nucleic acid may be native DNA or RNA, but various chemistries to improve stability (chemical and / or anti-enzyme) and specific activity (affinity with RNA). Modifications can be included. For example, in order to prevent degradation by a hydrolyzing enzyme such as nuclease, the phosphate residue (phosphate) of each nucleotide constituting the antisense nucleic acid is chemically modified with, for example, phosphorothioate (PS), methylphosphonate, phosphorodithionate or the like. It can be replaced with a phosphoric acid residue. Further, the hydroxyl groups at the 2'position of the sugar (ribose) of each nucleotide are represented by -OR (R = CH ₃ (2'-O-Me), C
H ₂ CH ₂ OCH ₃ (2'-O-MOE), CH ₂ CH ₂ NHC (NH) NH ₂ , CH ₂
It _{may be replaced with CONHCH 3} , CH ₂ CH ₂ CN, etc.). Further, 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. Can be mentioned.

The conformation of the sugar portion of RNA is C2'-endo (S type) and C3'-endo (
(N type) is dominant, and in single-strand RNA, it exists as an equilibrium between the two, but when double-stranded is formed, it is fixed to N type. Therefore, cross-linked nucleic acid (BNA) (BNA), which is an RNA derivative in which the conformation of the sugar portion is fixed to N type by cross-linking 2'oxygen and 4'carbon in order to impart strong binding ability to the target RNA. Locked Nucleic Acid (LNA)) (Im
anishi, T. et al., Chem. Commun., 1653-9, 2002; Jepsen, JS et al.,
Oligonucleotides, 14, 130-46, 2004) and ENA (Morita, K. et al., Nucleosid)
es 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 early 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 used to prepare by synthesizing a sequence complementary to this. In addition, all of the antisense nucleic acids containing the above-mentioned various modifications can be chemically synthesized by a method known per se.

(2) Ribozyme Nucleic Acid for CARD14 Gene mRNA Another preferred example of a nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of CARD14 gene mRNA or a part thereof is the mRNA in the coding region. Examples thereof include ribozyme nucleic acids that can be specifically cleaved internally. In a narrow sense, "ribozyme" refers to RNA having an enzymatic activity of cleaving nucleic acid, but in the present 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 includes self-splicing RNA found in infectious RNAs such as viroids and virusoids, and hammerhead type and hairpin type are known. The hammer head type exerts enzymatic activity at about 40 bases, and several bases at both ends adjacent to the part having the hammer head structure (about 10 bases in total) are arranged in 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 in which an RNA motif derived from a viral nucleic acid capable of specifically binding to RNA helicase is linked. Can be [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 it, it should be a hybrid ribozyme in which tRNA-modified sequences are further linked in order to promote the transfer of the transcript to the cytoplasm. You can also [Nucleic Acids Res., 29 (13):
2780-2788 (2001)].

(3) siRNA against mRNA of CARD14 gene
In the present specification, a double-stranded RNA consisting of an oligo RNA complementary to the mRNA of the CARD 14 gene and its complementary strand, so-called siRNA, is also an mRN of the CARD 14 gene.
It is defined as being included in a nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of A or a part thereof. The so-called RNA interference (RNAi) phenomenon, in which mRNA complementary to that RNA is degraded when a short double-stranded RNA is introduced into a cell, has long been known in nematodes, insects, plants, etc. Since it was confirmed that this phenomenon occurs widely in animal cells [Nature, 411 (6836): 494-498 (2001)], it has been widely used as an alternative technology for the above-mentioned antisense nucleic acids and ribozymes.

The siRNA is based on, for example, Elbashir et al. (Gene) based on the cDNA sequence information of the target gene.
s Dev., 15, 188-200 (2001)), Teramoto et al. (FEBS Lett. 579 (13): p2878-82 (2005)
)) Can be designed according to the rules proposed. The target sequence of siRNA has a length of 15 to 50 bases, preferably 19 to 49 bases, more preferably 19 to 27 bases in principle, and for example, AA + (N) 19 (following AA, 19). Base sequence of base), AA + (N
) 21 (21 base sequence following AA) or A + (N) 21 (21 base sequence 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 the siRNA is 19 bases or more. The additional base may be DNA or RNA, but DNA may be used to improve the stability of the nucleic acid. Examples of such an additional base sequence include ug-3.
', uu-3', tg-3', tt-3', ggg-3', guu-3', gtt-
Sequences such as 3', tttttt-3', and uuuuu-3'can be mentioned, but are not limited thereto.

In addition, siRNA may have a protruding sequence (overhang) at the 3'end.
Specific examples thereof include those to which dTdT (dT represents a deoxythymidine residue of a deoxyribonucleic acid) is added. Further, it may be a blunt end without end addition.

Further, 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 sequence (overhang) at the 3'end and the 5'end. Can be mentioned. A typical aiRNA has two antisense strands
It consists of 1 base, the sense strand consists 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. WO 2009/029688 Pamphlet). The position of the target sequence is not particularly limited, but it is desirable to select the target sequence from 5'-UTR and about 50 bases from the start codon, and from regions other than 3'-UTR. For a candidate group of target sequences selected based on the above rules and others, BLAST (http://www.ncbi.nlm) is checked to see if there is homology in consecutive sequences of 16-17 bases in non-target mRNA. Check using homology search software such as .nih.gov / BLAST /) to confirm the specificity of the selected target sequence. For the target sequence whose specificity has been confirmed, TT or UU is added to 19-21 bases after AA (or NA).
A double-stranded R consisting of a sense strand having a 3'end overhang of TT or UU and an antisense strand having a sequence complementary to the 19-21 base and a 3'end overhang of TT or UU.
NA may be designed as siRNA. Also, short hairpin RNA (SHRNA), which is a precursor of siRNA, can be any linker sequence that can form a loop structure (eg, 5
It can be designed by appropriately selecting (about -25 bases) and linking the sense strand and the antisense strand via the linker sequence.

SiRNA and / or shRNA sequences can be searched using search software provided free of charge on various web sites. Such sites include, for example, Ambion.
SiRNA Target Finder (http://www.ambion.com/jp/techlib/misc/siRNA_fin) provided by
der.html) and pSilencer® Expression Vector Insert Design Tool (http://www.ambion.com/jp/techlib/misc/psilencer_converter.html), RNAi Code
There is a GeneSeer (http://codex.cshl.edu/scripts/newsearchhairpin.cgi) provided by x, but it is not limited to these.

The ribonucleoside molecule constituting siRNA may also undergo the same modifications as in the case of the antisense nucleic acid described above in order to improve stability, specific activity and the like. However, siR
In the case of NA, when all ribonucleoside molecules in native RNA are replaced with modified forms, R
Since NAi activity may be lost, the introduction of minimal modified nucleosides that allow RNA-induced silencing complex (RISC) to function is required.

Specifically, as the modification, in order to improve the stability (chemical and / or counter-enzyme) and specific activity (affinity with RNA) of a part of the nucleotide molecules constituting siRNA, as well as the natural DNA. Can be replaced with RNA that has undergone various chemical modifications (Usman and
See Cedergren, 1992, TIBS 17,34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31,163). For example, siRNA to prevent degradation by hydrolases such as nucleases
The phosphate residue (phosphate) of each nucleotide constituting the above can be replaced with a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, or phosphorodithionate. In addition, the hydroxyl group at the 2'position of the sugar (ribose) of each nucleotide is changed to -O.
R (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 replaced with a fluorine atom (-F). Further, the base moiety (pyrimidine, purine) may be chemically modified, for example, introduction of a methyl group or a cationic functional group to the 5-position of the pyrimidine base,
Alternatively, substitution of the carbonyl group at the 2-position with thiocarbonyl can be mentioned. Others above (
The modification method for antisense nucleic acid described in 1) can be used. Or
Chemical modification (2'-deoxydation, 2'to replace part of RNA in siRNA with DNA
-H) may be applied. Further, an artificial nucleic acid (LNA: Locked Nucleic Acid) in which the 2'position and the 4'position of the sugar (ribose) are _{crosslinked with −O—CH 2 − and the conformation is fixed to N type may be used.}

In addition, the sense strand and antisense strand that make up siRNA are ligands, peptides, sugar chains, antibodies, lipids, positive charges, and cell membranes that specifically recognize receptors present on the cell surface via a linker. Oligoarginine, Tat peptide, Re that adsorbs and penetrates the surface layer
It may be chemically bonded to a v peptide, an Ant peptide, or the like.

siRNA DNA / RN the sense and antisense strands of the target sequence on the mRNA
A Synthesize each with an automatic synthesizer, and in an appropriate annealing buffer, about 1 at about 90 to about 95 ° C.
It can be prepared by denaturing for about 1 minute and then annealing at about 30 to about 70 ° C. for about 1 to about 8 hours. In addition, short hairpin RNA (SH), which is a precursor of siRNA.
RNA) can also be prepared by synthesizing and cleaving it with a dicer.

As used herein, a nucleic acid designed to be capable of producing siRNA against the mRNA of the CARD14 gene in vivo is also a base sequence complementary or substantially complementary to the base sequence of the mRNA of the CARD14 gene or one of them. It is defined as being included in the nucleic acid containing the part. Examples of such nucleic acids include expression vectors constructed to express the above-mentioned shRNA and siRNA. The shRNA is long enough to form a suitable loop structure of the sense and antisense strands of the target sequence on the mRNA (eg 5-25).
It can be prepared by designing an oligo RNA containing a base sequence in which a spacer sequence (about a base) is inserted in between and linked, and synthesizing this with an automatic DNA / RNA synthesizer.
Vectors expressing shRNA include tandem type and stem loop (hairpin) type. The former is a tandem link between the expression cassette of the sense strand of siRNA and the expression cassette of the antisense strand, and each strand is expressed and annealed in the cell to form double-stranded siRNA (dsRNA). Is. On the other hand, the latter is a vector in which an shRNA expression cassette is inserted, and the shRNA is expressed intracellularly and processed by a dicer to form dsRNA. As the promoter, a polII-based promoter (for example, a pre-CMV early promoter) can be used, but it is common to use a polIII-based promoter in order to allow accurate transcription of short RNA. Mouse and human U6 as polIII promoters
-SnRNA promoter, human H1-RNase PRNA promoter, human valine-
Examples include the tRNA promoter. In addition, a sequence in which four or more Ts are continuous is used as the transcription termination signal.

The siRNA or shRNA expression cassette constructed in this manner is then inserted into a plasmid vector or viral vector. As such a vector, a viral vector such as a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, a herpesvirus, or a Sendai virus, an animal cell expression plasmid, or the like is used.

The siRNA is, for example, 394 Applier, based on the nucleotide sequence information.
d 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 a
l., 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 methods described in US Pat. No. 6,001311. Specifically, it can be synthesized using a nucleic acid protecting group known to those skilled in the art (for example, a dimethoxytrityl group at the 5'end) and a coupling group (for example, phosphoramidite at the 3'end). That is, the protecting group at the 5'end is deprotected with an acid such as TCA (trichloroacetic acid) to carry out a coupling reaction. Then, after capping with an acetyl group, the next nucleic acid condensation reaction is carried out. Modified RNA
In the case of siRNA containing DNA or DNA, RNA modified as a raw material (for example, 2'-O)
-Methyl nucleotide, 2'-deoxy-2'-fluoronucleotide) may be used, and the conditions of the coupling reaction can be adjusted as appropriate. In addition, when introducing a phosphorothioate bond in which the phosphate bond moiety is modified, a vocabulary reagent (3H-1,2-benzodithiol-3-one1,1-dioxide) can be used.

Alternatively, the 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., 19
97, Nucleosides & Nucleotides, Bellon et al., 1997, Bioconjugate Chem. 8,204
), Or may be coupled together by hybridization after synthesis and / or deprotection. The siRNA molecule can also be synthesized by tandem synthesis. That is, both siRNA strands are synthesized as a single contiguous oligonucleotide separated by a cleaving linker, which is then cleaved to produce separate siRNA fragments, which are hybridized and purified. .. The linker may be a polynucleotide linker or a non-nucleotide linker.

The synthesized siRNA molecule can be purified using a method known to those skilled in the art. For example, a method of purifying by gel electrophoresis or a method of purifying by using high performance liquid chromatography (HPLC) can be mentioned.

As another preferable example of a nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of the mRNA of the CARD14 gene or a part thereof, a micro that targets the mRNA.
RNA (miRNA) can be mentioned. The miRNA can also be prepared according to the method described for siRNA described above.

Nucleic acids containing a base sequence complementary to or substantially complementary to the base sequence of the mRNA of the CARD14 gene or a part thereof are provided in a special form such as liposomes and microspheres, or other molecules are added. Can be provided in form. Used in additional forms are polycations such as polylysine, which act to neutralize the charge of the phosphate skeleton, and lipids that enhance interaction with cell membranes and increase nucleic acid uptake. Hydrophobic ones such as (eg, phosphorlipids, cholesterol, etc.) can be mentioned. Preferred lipids to add include cholesterol and its derivatives (eg, cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3'or 5'ends of nucleic acids and can be attached via bases, sugars, intramolecular nucleoside bonds. Other groups include cap groups specifically located at the 3'or 5'ends of nucleic acids to prevent degradation by nucleases such as exonucleases and RNases. Examples of such cap groups include hydroxyl-protecting groups known in the art, such as glycols such as polyethylene glycol and tetraethylene glycol.
It is not limited to that.

The CARD14 expression inhibitory activity of these nucleic acids can be expressed by a transformant into which a nucleic acid encoding CARD14 has been introduced, an in vivo or in vitro CARD14 gene expression system, or an in vivo or in vitro C.
It can be investigated using a translation system for the ARD14 protein.

The substance that inhibits the expression of CARD14 in the present invention is not limited to the nucleic acid containing the base sequence complementary or substantially complementary to the base sequence of the mRNA of the CARD14 gene as described above or a part thereof, and the production of CARD14. It may be another substance such as a low molecular weight compound as long as it directly or indirectly inhibits. 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 CARD 14" may be any substance as long as it inhibits the function of CARD 14 once functionally produced.

Specifically, as a substance that suppresses the function of CARD14, for example, an antibody against CARD14 can be mentioned. The antibody may be either a polyclonal antibody or a monoclonal antibody. These antibodies can be produced according to a method for producing an antibody or antiserum known per se. The isotype of the antibody is not particularly limited, but is preferably IgG, IgM or I.
Examples include gA, particularly preferably IgG. Further, 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, and is not particularly limited as long as it has a complete antibody molecule, for example, Fab, Fab', F. (Ab') Fragments such as _{2, s}
Genetically engineered conjugated molecules such as cFv, scFv-Fc, minibody, and diabody, or derivatives thereof modified with molecules having a protein stabilizing effect such as polyethylene glycol (PEG). You may.

In a preferred embodiment, since the antibody against CARD14 is used as a human-targeted drug, the antibody (preferably a monoclonal antibody) is an antibody with a reduced risk of exhibiting antigenicity when administered to humans. Specifically, a fully human antibody, a humanized antibody, a mouse-human chimeric antibody, or the like, and particularly preferably a fully human antibody. Humanized antibodies and chimeric antibodies can be genetically engineered according to conventional methods. Further, 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 phagedis is produced. Is desirable.

Another preferred substance that suppresses the function of CARD14 is Lipinski's Rule.
It is a low molecular weight compound suitable for. Such a compound can be obtained, for example, by using the screening method of the present invention described later.

According to the present invention, CARD14 inhibitors such as substances that inhibit the expression or function of CARD14 are therapeutic or prophylactic agents for psoriasis, multiple sclerosis, therapeutic agents for reducing side effects due to TLR-based / IMQ / immunostimulant adjuvants, or It is useful as a prophylactic, therapeutic or prophylactic for γδ T cell-mediated diseases and / or complications of immune intervention.

(Preferable embodiment)
A preferred embodiment of the present invention will be described below. It is understood that the embodiments provided below are provided for a better understanding of the invention and that the scope of the invention should not be limited to the following description. Therefore, it is clear that a person skilled in the art can appropriately make modifications within the scope of the present invention in consideration of the description in the present specification. It is also understood that the following embodiments of the present invention may be used alone or in combination.

<Treatment or prevention of psoriasis and screening method>
In one aspect, the invention provides a method of screening 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 its expression in hematopoietic cells, especially γδ T cells, especially epidermal γδ T cells, can be used to screen for therapeutic or prophylactic agents for psoriasis. It is the first provided in the present invention. Although not bound by theory, CARD inhibitors, such as agents that suppress or eliminate CARD14 expression in hematopoietic cells, especially γδ T cells, preferably epidermal γδ T cells, are therapeutic or prophylactic agents for psoriasis. It is understood that it can be used as an agent. Therefore, the method of the present invention is 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. It is determined that the substance can be used as a therapeutic or prophylactic agent for psoriasis,
Including the process.

Examples of CARD inhibitors such as agents that suppress or eliminate the expression of CARD14 include, for example.
Suppressants for CARD14 expression products, such as antibodies, antibody fragments, functional equivalents, or CARD14 antisense nucleic acids, molecules with RNAi function, and the like can be mentioned.

In one embodiment, the target γδ T cells are interleukin-17 (IL-17).
And cells that produce 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 γδ T cells, particularly preferably epidermal γδ T cells) (also referred to as “non-hematopoietic cells”). For example, it is preferable to confirm that CARD14 is not expressed in skin-resident cells). Here, in a preferred embodiment, the hematopoietic cells and non-hematopoietic cells are preferably derived from the same allogeneic origin, and more preferably from the same individual. In one preferred embodiment, the method of the invention is a step of contacting a non-hematopoietic cell with the candidate substance and selecting the candidate substance as the therapeutic or prophylactic agent if CARD 14 has no substantial effect on expression. Is further included. As used herein, the term "substantially no effect" means that the CARD 14 can exert its function even if there is such an effect.

The psoriasis targeted by the present invention includes, for example, immokimid (IMQ) -induced psoriasis and IL-2.
It may include 3-induced psoriasis.

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

In the present invention, in order to specifically deliver to a hematopoietic cell, it is achieved by a delivery system led by a specific molecule such as an antibody specific to the hematopoietic cell (for example, γδ T cell).

<Therapeutic or preventive agents for multiple sclerosis and their screening methods>
In one aspect, the invention provides a method of screening 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, especially γδT cells, especially epidermal γδT cells. It has not been issued and is provided for the first time in the present invention. I do not want to be bound by theory, but in particular hematopoietic cells, especially γδ T cells, especially the epidermal γδ.
It is understood that CARD inhibitors, such as agents that suppress or eliminate CARD14 expression in T cells, can be used as therapeutic or prophylactic agents for multiple sclerosis. In the present invention, an experimental autoimmune encephalomyelitis (Expe) of a model of multiple sclerosis (autoimmune disease model)
Since the role of CARD14 was clearly shown in the rimental autoimmune encephalomyelitis (EAE) model, it is considered that multiple sclerosis can be prevented or treated by using a mechanism based on CARD14. Therefore, the method of the present invention is 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. Includes steps in which 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 the expression of CARD14 include, for example.
Suppressants for CARD14 expression products, such as antibodies, antibody fragments, functional equivalents, or CARD14 antisense nucleic acids, molecules with RNAi function, and the like can be mentioned.

In one embodiment, the target γδ T cells are interleukin-17 (IL-17).
And cells that produce 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 γδ T cells, particularly preferably epidermal γδ T cells) (“non-hematopoietic cells”, for example. It is preferable to confirm that CARD14 is not expressed in skin-resident cells). Here, in a preferred embodiment, the hematopoietic cells and non-hematopoietic cells are preferably derived from the same allogeneic origin, and more preferably from the same individual. In one preferred embodiment, the method of the invention is a step of contacting a non-hematopoietic cell with the candidate substance and selecting the candidate substance as the therapeutic or prophylactic agent if CARD 14 has no substantial effect on expression. Is further included.

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

In the present invention, in order to specifically deliver to a hematopoietic cell, it is achieved by a delivery system led by a specific molecule such as an antibody specific to the hematopoietic cell (for example, γδ T cell).

<Therapeutic or prophylactic agents for reducing side effects of TLR-based / IMQ / immunostimulant adjuvants and their screening methods>
In one aspect, the invention provides a method of screening 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 is a therapeutic agent for reducing the side effects of TLR-based / IMQ / immunostimulant adjuvants by observing its expression in hematopoietic cells, especially γδT cells, especially epidermal γδT cells. It has not been found that a prophylactic agent can be screened and is provided for the first time in the present invention. I don't want to be bound by theory,
In particular, CARD in hematopoietic cells, especially γδ T cells, particularly preferably epidermal γδ T cells.
It is understood that CARD inhibitors, such as agents that suppress or eliminate the expression of 14, can be used as therapeutic or prophylactic agents to reduce the side effects of TLR-based / IMQ / immunostimulatory adjuvants. As demonstrated herein, T in combination
Both LR7 and TLR9 signaling are required to completely develop IMQ-induced psoriasis-like dermatitis, while the IL-23-induced psoriasis-like dermatitis model is TLR7 and TLR9-mediated innate immunity. It has been demonstrated to directly activate downstream of 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 are based on IMQ-induced psoriasis-like models and IL-. A clear difference in innate immune signals from the 23-induced psoriasis-like model will be demonstrated. Through such findings, therapeutic or prophylactic agents for reducing the side effects of TLR-based / IMQ / immunostimulatory adjuvants 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 is 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. Includes steps in which it is determined that the substance can be used as a therapeutic or prophylactic agent for psoriasis.

As used herein, the term "TLR-based / IMQ / immunostimulatory adjuvant" refers to any side effect that occurs when an adjuvant based on TLR, imiquimod (IMQ) or immunostimulatory adjuvant (eg, CpG, etc.) is administered. Examples of such side effects include, but are not limited to, psoriasis, inflammation such as psoriasis-like skin inflammation, and cytokineemia. See, for example, Non-Patent Document 2 (Gilliet et al., 2004) for information on the side effects of TLR-based / IMQ / immunostimulatory adjuvants. As introduced in this paper, a disease called psoriasis can occur as a side effect of the TLR7 ligand as an adjuvant, and topical application of imiquimod (IMQ) cream causes psoriasis-like skin inflammation in humans and mice. It has also been reported that this is suggested. Therefore, it is understood that the side effects of TLR-based / IMQ / immunostimulatory adjuvants can be prevented or treated by the CARD14 inhibitors of the present invention.

Examples of CARD inhibitors such as agents that suppress or eliminate the expression of CARD14 include, for example.
Suppressants for CARD14 expression products, such as antibodies, antibody fragments, functional equivalents, or CARD14 antisense nucleic acids, molecules with RNAi function, and the like can be mentioned.

In one embodiment, the target γδ T cells are interleukin-17 (IL-17).
And cells that produce 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 γδ T cells, particularly preferably epidermal γδ T cells) (“non-hematopoietic cells”, for example, It is preferable to confirm that CARD14 is not expressed in skin-resident cells). Here, in a preferred embodiment, the hematopoietic cells and non-hematopoietic cells are preferably derived from the same allogeneic origin, and more preferably from the same individual. In one preferred embodiment, the method of the invention is a step of contacting a non-hematopoietic cell with the candidate substance and selecting the candidate substance as the therapeutic or prophylactic agent if CARD 14 has no substantial effect on expression. Is further included.

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

In the present invention, in order to specifically deliver to a hematopoietic cell, it is achieved by a delivery system led by a specific molecule such as an antibody specific to the hematopoietic cell (for example, γδ T cell).

<Therapeutic or prophylactic agents for γδ T cell-mediated diseases and / or complications of immune intervention and their screening methods>
In one aspect, the invention provides a method of screening 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 in hematopoietic cells, especially γδ T cells, especially epidermal γδ T cells, is used to treat or prevent γδ T cell-mediated diseases and / or complications of immune intervention. No agent has been found to be able to be screened and is the first provided in the present invention. Although not bound by theory, CARD inhibitors such as agents that suppress or eliminate the expression of CARD14 in hematopoietic cells, especially γδT cells, particularly preferably epidermal γδT cells, are γδT cell-mediated diseases and / Or 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 is a step of contacting a candidate substance with a hematopoietic cell and CA in the hematopoietic cell
A step of determining the expression of RD14, wherein if the candidate substance suppresses or eliminates the expression of CARD14, it is determined that the candidate substance can be used as a therapeutic or prophylactic agent for psoriasis. Include.

Examples of CARD inhibitors such as agents that suppress or eliminate the expression of CARD14 include, for example.
Suppressants for CARD14 expression products, such as antibodies, antibody fragments, functional equivalents, or CARD14 antisense nucleic acids, molecules with RNAi function, and the like can be mentioned.

In one embodiment, the target γδ T cells are interleukin-17 (IL-17).
And cells that produce 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 γδ T cells, particularly preferably epidermal γδ T cells) (“non-hematopoietic cells”, for example. It is preferable to confirm that CARD14 is not expressed in skin-resident cells). Here, in a preferred embodiment, the hematopoietic cells and non-hematopoietic cells are preferably derived from the same allogeneic origin, and more preferably from the same individual. In one preferred embodiment, the method of the invention is a step of contacting a non-hematopoietic cell with the candidate substance and selecting the candidate substance as the therapeutic or prophylactic agent if CARD 14 has no substantial effect on expression. Is further included.

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

In the present invention, in order to specifically deliver to a hematopoietic cell, it is achieved by a delivery system led by a specific molecule such as an antibody specific to the hematopoietic cell (for example, γδ T cell).

As used herein, the term "γδ T cell-mediated disease" refers to any disease mediated by γδ T cells, and the “γδ T cell-mediated disease” includes, for example, psoriasis, multiple sclerosis, inflammation, cytokineemia and the like. Can be mentioned, but not limited to them.

As used herein, the term "complications of immune intervention" refers to any complications of immune intervention, and is referred to as "complications of immune intervention."
Examples of "complications of immune intervention" include, but are not limited to, inflammation, cytokineemia, and the like.

<Details of screening method>
When screening for a compound that inhibits the expression or function of CARD14, the screening method involves culturing cells capable of producing CARD14 in the presence and absence of a test substance, and expressing CARD14 under both conditions. Includes comparing the amount or degree of function. In addition, compounds that inhibit the function of CARD14 include the ability to bind to purified CARD14 protein, CARD14 protein and its binding protein (for example, CARD1).
Screening can also be performed by testing the binding inhibitory activity with 4 receptors). In particular, as such cells, hematopoietic cells, preferably γδT cells, are advantageous, and more preferably, epidermal γδT cells are preferable. Alternatively, recombinant cells may be used.
Specifically, such a cell is a DNA encoding CARD14 (that is, a base sequence represented by SEQ ID NO: 1 or 3, preferably SEQ ID NO: 1, or a base sequence and a string having complementarity to the base sequence. Hybridize under gentle conditions and SEQ ID NO: 2 or 4,
A DNA containing a base sequence encoding a polypeptide having the same function as the protein consisting of the amino acid sequence represented by SEQ ID NO: 2 is preferably linked downstream of the promoter in an appropriate expression vector to host animal cells. It can be prepared by introduction.

In one embodiment, as a screening technique, (A) a step of contacting a cell containing a nucleic acid encoding the CARD14 gene or a reporter protein under the control of the transcriptional regulatory region of the gene with a test substance; (B). ) Step of measuring the expression level of CARD14 gene or CARD14 protein or reporter protein in the cells; and (C) expression of CARD14 gene or CARD14 protein or reporter protein as compared with the case of measuring in the absence of the test substance. Reduced doses of the test substance to reduce the side effects of psoriasis, multiple sclerosis, TLR-based / IMQ / immunostimulatory adjuvants, or the treatment of γδT cell-mediated disease and / or complications of immune intervention and / Or includes the step of selecting as a candidate for a substance used for prevention. In one embodiment, CARD 14 can be brought into contact with the test substance and the test substance capable of binding to CARD 14 can be selected as a candidate substance used for the treatment and / or prevention of psoriasis.

In another embodiment, the present invention is (A) a step of contacting CARD 14 with a cell membrane fraction of hematopoietic cells (particularly γδ T cells, particularly preferably epidermal γδ T cells) in the presence of a test substance; (B).
) Step of measuring the amount of CARD14 bound to the cell membrane fraction; and (C) the test substance having a reduced amount of CARD14 bound to the cell membrane fraction as compared with the case of measuring in the absence of the test substance. Used to reduce the side effects of psoriasis, multiple sclerosis, TLR-based / IMQ / immunostimulatory adjuvants, or for the treatment and / or prevention of γδ T cell-mediated diseases and / or complications of immune intervention Includes the step of selecting as a candidate for the substance.

In a further embodiment, the invention is to reduce the side effects of psoriasis, multiple sclerosis, TLR-based / IMQ / immunostimulatory adjuvant, or to treat and / or complications of γδT cell-mediated disease and / or immune intervention. Or test substances selected as candidates for substances used for prophylaxis to reduce side effects from psoriasis, multiple sclerosis, TLR-based / IMQ / immunostimulant adjuvants, or γδT cell-mediated diseases and / Or to apply to models of complications of immune intervention (eg, including model animals described in the Examples) to reduce psoriasis, multiple sclerosis, and side effects of TLR-based / IMQ / immunostimulant adjuvants in the model. Further includes testing whether or not to suppress the response of γδT cell-mediated disease and / or complications of immune intervention.

In another embodiment, the invention comprises (A) contacting hematopoietic cells (particularly γδ T cells, preferably epidermal γδ T cells) with a test substance in the presence and absence of CARD14; (B). To reduce the side effects of psoriasis, multiple sclerosis, TLR-based / IMQ / immunostimulatory adjuvants, or to measure the degree of complications of γδ T cell-mediated disease and / or immune intervention in said cells under each condition. Steps; and (C) to reduce psoriasis, multiple sclerosis, and side effects of TLR-based / IMQ / immunostimulatory adjuvants in the presence of CARD14, as compared to when measured in the absence of test material. Or to suppress the response of γδ T cell-mediated diseases and / or complications of immune intervention and reduce psoriasis, multiple sclerosis, TLR-based / IMQ / immunostimulatory adjuvant side effects in the absence of CARD14, or Test substances that did not suppress the response of γδ T cell-mediated diseases and / or complications of immune intervention, inhibited the function of CARD14, psoriasis, multiple sclerosis, TLR
To reduce side effects with base / IMQ / immunostimulant adjuvant, or γδT
Includes the process of selecting as a candidate for substances used for the treatment and / or prevention of cell-mediated diseases and / or complications of immune intervention.

In carrying out the screening of the present invention, the test substance may be, for example, a protein.
Examples thereof include peptides, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc., and these substances may be novel or known. You may.

In addition, a substance that reduces the expression level of CARD14 or CARD14 gene, or C
When selecting a substance that reduces the function of ARD14, control cells that are not in contact with the test substance can also be used as a comparative control. Here, "not contacting the test substance" means that when the same amount of solvent (blank) as the test substance is added instead of the test substance, or the expression level of the CARD14 or CARD14 gene or the function of CARD14 is affected. It also includes the case of adding a negative control substance that is not given.

The contact of the test substance with the cells is, for example, the above-mentioned medium or various buffer solutions (for example, HEPE).
By adding the test substance to S buffer, phosphate buffer, phosphate buffered saline, Tris-hydrochloric acid buffer, borate buffer, acetate buffer, etc.) and incubating the cells for a certain period of time. Can be carried out. The concentration of the test substance to be added is the type of compound (solubility, toxicity, etc.)
However, for example, it is appropriately selected in the range of about 0.1 nM to about 100 μM. The incubation time includes, for example, about 10 minutes to about 24 hours.

When the cells producing CARD14 are provided in the form of a non-human mammalian individual, the condition of the individual animal is not particularly limited, but for example, a psoriasis disease model animal in which inflammation is induced by a drug or genetic modification (for example, implementation). IMQ-induced psoriatic dermatitis exemplified in Example 1, IL-23
Inducible psoriatic dermatitis, etc.), Multiple sclerosis model animals (eg, Experimental autoimmune encephalomyelitis (EAE) model used in Example 2), TLR-based / IMQ / immunity Model animals for the onset of side effects due to stimulant adjuvants (IMQ-induced psoriatic dermatitis of Example 1, IL-23-induced psoriatic dermatitis, etc.), or γ
It may be a model animal of δT cell-mediated disease and / or complications of immune intervention (IMQ-induced psoriatic dermatitis of Example 1, IL-23-induced psoriatic dermatitis, etc.) and the like. There are no particular restrictions on the breeding conditions of the animals used, but it is preferable that the animals are bred in an environment of SPF grade or higher. Contact of the test substance with the cells is performed by administration of the test substance to the individual animal. The route of administration is not particularly limited, but for example, intravenous administration, intraarterial administration, subcutaneous administration,
Intradermal administration, intraperitoneal administration, oral administration, respiratory tract administration, rectal administration and the like can be mentioned. The dose is not particularly limited, but for example, about 0.5 to 20 mg / kg can be administered as a single dose 1 to 5 times a day, preferably 1 to 3 times a day for 1 to 14 days. ..

Alternatively, the above screening method can be performed by contacting the test substance with the 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 the prevention and / or treatment of psoriasis, which comprises 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 substances that can be used. The cells used in this method, the type of the test substance, the mode of contact between the test substance and the cells, and the like are the same as described above.

The expression level of CARD14 is a nucleic acid capable of hybridizing with the above-mentioned DNA encoding CARD14 under stringent conditions, that is, the base sequence represented by SEQ ID NO: 1 or a base sequence complementary thereto and stringent conditions. Nucleic acid that can hybridize with (DN
It can be measured at the RNA level by detecting the mRNA of the CARD14 gene using A) (hereinafter, may be referred to as “nucleic acid for detection of the present invention”). Alternatively, the expression level can be measured at the protein level by detecting these proteins using the antibody against CARD14 described above (hereinafter, may be referred to as “detection antibody of the present invention”).

Therefore, more specifically, the present invention comprises (a) culturing cells capable of producing CARD14 in the presence and absence of a test substance and the amount of mRNA encoding the protein under both conditions. To reduce the side effects of psoriasis, multiple sclerosis, TLR-based / IMQ / immunostimulatory adjuvants, or γδT cell-mediated diseases, characterized by measurement and comparison using the detection nucleic acids of the present invention. And / or prevention of complications of immune intervention and /
Alternatively, a method for screening a substance that can be used for treatment, and (b) CARD1.
A feature is that cells capable of producing 4 are cultured in the presence and absence of a test substance, and the amount of the protein under both conditions is measured and compared using the detection antibody of the present invention. Can be used to reduce the side effects of psoriasis, multiple sclerosis, TLR-based / IMQ / immunostimulatory adjuvants, or to prevent and / or treat γδT cell-mediated diseases and / or complications of immune intervention. Provided is a method for screening possible substances.

That is, the screening of substances that change the expression level of CARD14 can be performed as follows.

(I) Normal or disease models (eg, psoriasis, multiple sclerosis, TLR-based / IMQ)
/ Non-human mammals (eg, mice, rats, rabbits, sheep, pigs, cows, cats, dogs, etc.) to reduce the side effects of immunostimulatory adjuvants, or γδ T cell-mediated diseases and / or complications of immune intervention , Monkey, etc.), and after a certain period of time has passed (30 minutes to 3 days, preferably 1 to 2 days, more preferably 1 to 24 hours), blood , Or a specific organ (eg, brain, etc.), or a tissue or cell isolated from the organ.

The mRNA of CARD14 can be quantified by extracting mRNA from cells or the like by a usual method, or can be quantified by Northern blot analysis known per se. On the other hand, the protein amount of CARD14 can be quantified by using Western blot analysis or various immunoassay methods described in detail below.

(Ii) Cells expressing the CARD14 gene (for example, a transformant into which CARD14 has been introduced) are prepared according to the above method, and when the cells are cultured according to a conventional method, the test substance is added to a medium or a buffer solution for a certain period of time. After incubation (1-7 days, preferably 1-3)
After 1 day, more preferably 2 to 3 days later), CARD14 or mRNA encoding the same contained in the cell culture can be quantified and analyzed in the same manner as in (i) above.

The detection and quantification of the expression level of the CARD14 gene (mRNA) can be carried out by a known method such as Northern blotting or RT-PCR using RNA prepared from the cells or a complementary polynucleotide transcribed from the RNA. Specifically, the presence or absence of expression of the CARD14 gene in RNA and its expression by using a polynucleotide having at least 15 consecutive bases in the base sequence of the CARD14 gene and / or a polynucleotide complementary thereto as a primer or a probe. Levels can be detected and measured. Such probes or primers are, for example, primer3 (http://primer3.sourceforge.net/) or vector N based on the nucleotide sequence of the CARD14 gene.
It can be designed using TI (manufactured by Infomax).

When Northern blotting is used, the primer or probe is ^{labeled with a radioisotope (32} P, ³³ P, etc .: RI) or a fluorescent substance, and the RNA is transferred to a nylon membrane or the like according to a conventional method. After hybridizing with, the double strand of the formed primer or probe (DNA or RNA) and RNA is combined with RNA.
As a signal derived from the labeled substance (RI or fluorescent substance) of the primer or probe, a method of detecting and measuring with a radiation detector (BAS-1800II, manufactured by Fuji Film Co., Ltd.) or a fluorescence detector can be exemplified. Also, AlkPhos Direct Labeling and D
Using the etection System (manufactured by GE Healthcare), the probe is labeled according to the protocol, hybridized with cell-derived RNA, and then the signal derived from the label of the probe is signaled by the multibioimager STORM860 (GE Healthcare). It is also possible to use the method of detection and measurement by (Care).

When using the RT-PCR method, cDNA is prepared from cell-derived RNA according to a conventional method, and CARD can be used as a template to amplify the region of the target CARD14 gene.
A pair of primers prepared based on the sequence of 14 genes (a normal strand that binds to the above-mentioned cDNA (-chain) and a reverse strand that binds to the + strand) are hybridized with this, and the PCR method is performed according to a conventional method to obtain the obtained product. A method for detecting amplified double-stranded DNA can be exemplified. The amplified double-stranded DNA was detected by a method for detecting the labeled double-stranded DNA produced by performing the above PCR using a primer labeled with RI or a fluorescent substance in advance. A method can 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 the probe for detection. The generated labeled double-stranded DNA product can be measured with an Agilent 2100 bioanalyzer (manufactured by Yokogawa Analytical Systems) or the like. Also, SY
BR Green RT-PCR Reagents (manufactured by Applied Biosystems) according to the protocol RT
-Prepare the PCR reaction solution and prepare the ABI PRIME 7900 Sequence Detection System (Applied Bio).
The reaction product can also be detected by reacting with (manufactured by systems).

The expression of the CARD14 gene in the cells to which the test substance was added was 2/3 times or less, preferably 1/2 times or less, more preferably 1 time, as compared with the expression level in the control cells to which the test substance was not added.
If it is / 3 times or less, the test substance can be selected as a substance that suppresses the expression of the CARD14 gene.

In addition, screening of substances that change the expression level of CARD14 can also be performed by a reporter gene assay using the transcriptional regulatory region of the CARD14 gene. here,
The "transcriptional regulatory region" usually refers to the range of several kb to several tens of kb upstream of the chromosomal gene, for example, (i) 5'-race method (5'-RACE method) (for example, 5'-full). RaceCoreKit
(Can be carried out using Takara Bio Inc.), etc.), the step of determining the 5'end by a usual method such as oligocap method, S1 primer mapping; (ii) Genome Walker
A 5'-upstream region can be obtained using a Kit (manufactured by Clonetech) or the like, and the obtained upstream region can be identified by a method including a step of measuring promoter activity;

A nucleic acid encoding a reporter protein (hereinafter referred to as "reporter gene") is ligated downstream of the transcriptional regulatory region of the CARD14 gene in a functional manner to construct a reporter protein expression vector. The vector may be prepared by a method known to those skilled in the art. For example
Molecular Cloning: A Laboratory Manual 2nd edition (1989), Cold Spring Harbo
r Laboratory Press "Current Protocols In Molecular Biology" (1987), John W
CARD cut out according to the usual genetic engineering method described in iley & Sons, Inc., etc.
Transcriptional regulatory regions of 14 genes can be integrated on plasmids containing reporter genes.

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.

A reporter gene in which the transcriptional regulatory regions of the prepared CARD14 gene are operably linked is inserted into a vector that can be used in the cell into which the reporter gene is introduced by using a conventional gene engineering technique, and the plasmid is inserted. It can be prepared and introduced into a suitable host cell. Stable transformed cells can be obtained by culturing in a medium with selection conditions according to the selectable marker gene mounted on the vector. Alternatively, the reporter gene, which is operably linked to the transcriptional regulatory region of the CARD14 gene, may be transiently expressed in the host cell.

Further, 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 to obtain an extract of the cell, and then the extract is reacted with luciferin and ATP to chemiluminescent and the luminescence intensity thereof. The promoter activity can be detected by measuring. At this time, a commercially available luciferase reaction detection kit such as Pickagene Dual Kit (registered trademark; manufactured by Toyo Ink Co., Ltd.) can be used.

Specifically, as a method for measuring the amount of protein of CARD14, for example, (i) a labeling in which the detection antibody of the present invention is competitively reacted with a sample solution and labeled CARD14 and bound to the antibody. A method for quantifying CARD14 in a sample solution by detecting a modified protein, or (ii) for detecting a sample solution, a detection antibody of the present invention insolubilized on a carrier, and another labeled antibody of the present invention. Examples thereof include a method of quantifying CARD14 in a sample solution by measuring the amount (activity) of the labeling agent on the insolubilizing carrier after reacting with the antibody simultaneously or continuously.

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 the primary antibody and then ¹²⁵ I as the secondary antibody.
Label with an antibody that binds to a primary antibody labeled with a radioisotope such as, fluorescent substance, enzyme such as horseradish peroxidase (HRP), etc., and signals derived from these labeled substances are labeled with a radiation measuring instrument (BAI-1800II: Fuji). It can be carried out by measuring with a fluorescence detector (manufactured by Film Co., Ltd., etc.). In addition, after using an antibody that recognizes CARD14 as the primary antibody, ECL Plus Western Blotting Detection System (manufactured by GE Healthcare)
It can also be detected according to the protocol and measured with a multi-biometer STORM860 (manufactured by GE Healthcare).

The above-mentioned antibody is not particularly limited in its form, and may be a polyclonal antibody having CARD14 as an immunogen, a monoclonal antibody, and further, at least continuous among the amino acid sequences constituting CARD14, usually. Antibodies having 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 quantification method of (ii) above, it is desirable that the two antibodies recognize different parts of CARD14. For example, if one antibody recognizes the N-terminus of CARD14, the other antibody that reacts with the C-terminus of the protein can be used.

As the labeling agent used in the measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. Radioisotopes include, for example, [ ¹²⁵ I
], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are used. The enzyme is preferably stable and has a large specific activity. For example, β-galactosidase, β-glucosidase, etc.
Alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescamine isothiocyanate and the like are used. As the luminescent substance, for example, luminol, a luminol derivative, luciferin, lucigenin and the like are used. In addition, biotin is used to bind antibodies or antigens to labeling agents.
-(Streptavidin) Avidin system can also be used.

The method for quantifying CARD14 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 measuring method may be used as long as it is detected by means and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competitive method, immunometric method and sandwich method are preferably used. In terms of sensitivity and specificity, for example, it is preferable to use the sandwich method described later.

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

In the sandwich method, the sample solution is reacted with the insolubilized antibody for detection of the present invention (primary reaction), and another labeled antibody for detection of the present invention is reacted (secondary reaction), and then on the insolubilized carrier. CARD14 in the sample solution can be quantified by measuring the amount or activity of the labeling agent. The primary reaction and the secondary reaction may be carried out in the reverse order, at the same time, or at different times. Labeling agents and insolubilization methods can be similar to those described above. Further, in the immunoassay method by the sandwich method, the antibody used for the immobilized antibody or the labeled antibody does not necessarily have to be one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving the measurement sensitivity and the like. You may.

The detection antibody of the present invention can also be used in measurement systems other than the sandwich method, for example, competitive methods, immunometric methods, nephrometry and the like.

In the competitive method, CARD14 in the sample solution and labeled CARD14 are competitively reacted with the antibody, and then the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. By measuring the labeled amount of (B / F separation), B, or F, CARD1 in the sample solution
Quantify 4. In this reaction method, a soluble antibody is used as an antibody, and a liquid phase method in which B / F separation is performed using polyethylene glycol or a secondary antibody against the antibody (primary antibody), and a liquid phase method.
A solid-phase antibody is used as the primary antibody (direct method), or a soluble primary antibody is used and a solid-phase antibody is used as the secondary antibody (indirect method).

In the immunometric method, CARD14 in a sample solution and immobilized CARD14 are competitively reacted with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated, or CARD14 in the sample solution is used. The solid phase and the liquid phase are separated after reacting with an excess amount of the labeled antibody and then adding the immobilized CARD 14 to bind the unreacted labeled antibody to the solid phase. Next, the labeled amount of any phase is measured to quantify the amount of antigen in the sample solution.

In nephrometry, the amount of insoluble sediment produced as a result of the antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of CARD 14 in the sample liquid is small and only a small amount of sediment is obtained, laser nephrometry or the like utilizing laser scattering is preferably used.

In applying these individual immunological measurement methods to the quantification method of the present invention, it is not necessary to set special conditions, operations, or the like. The measurement system of CARD 14 may be constructed by adding the usual technical considerations of those skilled in the art to the usual conditions and operation methods in each method. For details of these general technical means, review articles, books, etc. can be referred to.

For example, "Radioimmunoassay" edited by Hiroshi Irie (Kodansha, published in 1974), edited by Hiroshi Irie "
"Radioimmunoassay" (Kodansha, published in 1979), "Enzyme immunoassay" edited by Eiji Ishikawa et al. (Igaku Shoin, published in 1978), "Enzyme immunoassay" edited by Eiji Ishikawa et al. Shoin, published in 1982), edited by Eiji Ishikawa et al., "Enzyme Immunoassay" (3rd edition) (Igaku Shoin, 1982)
Published in 2 years), "Methods in ENZY MOLOGY" Vol.70 (Immunochemical Techniques (Par)
t A)), immunochemical Techniques (Part B), ibid. Vol. 74
(Immunochemical Techniques (Part C)), ibid. Vol. 84 (Immunochemical Tech
niques (Part D: Selected Immunoassays)), ibid. Vol. 92 (Immunochemical Te)
chniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)
), Immunochemical Techniques (Part I: Hybridoma Technolog)
y and Monoclonal Antibodies)) (above, published by Academic Press), etc. can be referred to.

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

For example, in the above screening method, the expression level (mRNA amount or protein amount) of CARD14 in the presence of the test substance is higher than that in the absence of the test substance.
When it is inhibited by about 20% or more, preferably about 30% or more, more preferably about 50% or more.
The test substance is used as a CARD14 expression inhibitor, thus preventing psoriasis, multiple sclerosis, reduction of side effects by TLR-based / IMQ / immunostimulatory adjuvant or complications of γδ T cell-mediated disease and / or immune intervention. / Or can be selected as a candidate for a substance that can be used for treatment.

Alternatively, in the above screening method, instead of the cell expressing the CARD14 gene, a cell containing a reporter gene under the control of the transcriptional regulatory region inherent in the CARD14 gene can be used. Such cells may be cells, tissues, organs or individuals of transgenic animals 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 whether or not the test substance inhibits the function of CARD 14 as an index. CARD14 is a membrane-associated guanylate kinase (MAGUK).
) It belongs to the family and functions as a molecular scaffold that specializes in the region of the plasma membrane to form the assembly of the multiprotein complex. Therefore, the relationship with these functions and this protein belongs to the CARD family. It is also known to have a specific caspase-related recruitment domain (CARD) and to function as a specific regulator of BCL10 specific interaction and BCL phosphorylation, NF-κB activation and cell apoptosis. Therefore, inhibitors can be screened using such a function as an index.

In yet another embodiment of the invention, the in vitro psoriasis, multiple sclerosis, TL.
To reduce the side effects of R-based / IMQ / immunostimulant adjuvants, or γδ
Using a model of T cell-mediated disease and / or complications of immune intervention, to inhibit the function of CARD14 to reduce psoriasis, multiple sclerosis, TLR-based / IMQ / immunostimulatory adjuvant side effects, or Substances that are effective in treating or preventing γδ T cell-mediated diseases and / or complications of immune intervention can also be screened in one step. The method is the following steps (1) to (3):
(1) Target cells for diseases (eg, psoriasis, multiple sclerosis, TLR-based / IMQ / immunostimulatory adjuvants to reduce side effects, or γδT cell-mediated diseases and / or complications of immune intervention) , Steps of contacting the test substance in the presence and absence of CARD14 (2) Diseases in the cells under each condition (eg, psoriasis, multiple sclerosis, side effects of TLR-based / IMQ / immunostimulatory adjuvants) Responses (eg, psoriasis-specific reactions, multiple sclerosis-specific reactions, TLR-related reactions, complication-specific symptoms) to reduce or complications of γδT cell-mediated disease and / or immune interventions. Step of measuring the degree (3) A test in which the reaction was suppressed in the presence of CARD14 and the reaction was not suppressed in the absence of CARD14, as compared with the case of measurement in the absence of the test substance. The substance, CA
Diseases that inhibit the function of RD14 (eg, psoriasis, multiple sclerosis, TLR-based / IMQ /
It comprises the step of selecting as a candidate for a substance that has a therapeutic or prophylactic effect to reduce the side effects of an immunostimulatory adjuvant or (such as γδ T cell-mediated disease and / or complications of immune intervention).

<Pharmaceutical>
The medicament of the present invention may be administered by itself or as a suitable pharmaceutical composition. The pharmaceutical composition used for administration may include the pharmaceutical of the present invention and a pharmacologically acceptable carrier, diluent or excipient. Such pharmaceutical compositions are provided in dosage forms suitable for oral or parenteral administration.

As the composition for parenteral administration, for example, injections, suppositories and the like are used, and the injections are dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections and drip injections. May be included. Such injections can be prepared according to known methods. As a method for preparing an injection, for example, the nucleic acid of the present invention is dissolved in a sterile aqueous solution or an oily solution usually used for an injection.
It can be prepared by suspending or emulsifying. As the aqueous solution for injection, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents are used, and suitable solubilizing agents such as alcohol (eg, ethanol) and polyalcohol (eg, eg) are used. Propylene glycol, polyethylene glycol), nonionic surfactants [eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydro of hardened castor oil)
It may be used in combination with genated castor oil))]. 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 above nucleic acids with a conventional suppository base.

Compositions for oral administration include solid or liquid dosage forms, specifically tablets (sugar-coated tablets,
Includes film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, emulsions, suspensions and the like. Such a composition may be produced by a known method and may contain a carrier, diluent or excipient commonly used in the pharmaceutical field. As the carrier and excipient for tablets, for example, lactose, starch, sucrose, and magnesium stearate are used.

It is convenient that the parenteral or oral pharmaceutical compositions described above are prepared in dosage forms of dosage units that are compatible with the dosage of the active ingredient. Dosage forms of such dosage units include, for example, tablets, pills, capsules, injections (ampoules), and suppositories.

The medicament of the present invention is low in toxicity and can be used as a liquid as it is or as a pharmaceutical composition of an appropriate dosage form for humans or mammals (eg, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). On the other hand, it can be administered orally or parenterally (eg, intravascular administration, subcutaneous administration, etc.).

(General technology)
The molecular biological, biochemical, and microbiological methods used herein are well known and commonly used in the art, including those already cited, eg, Sambrook J. et. al. (1989). Molecular Cloning: A Laboratory Manual, Col
d Spring Harbor and its 3rd Ed. (2001); Ausubel, FM (1987). Current Protoc
ols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience;
Ausubel, FM (1989). Short Protocols in Molecular Biology: A Compendiu
m of Methods from Current Protocols in Molecular Biology, Greene Pub. A
ssociates and Wiley-Interscience; Innis, MA (1990). PCR Protocols: A Guide
to Methods and Applications, Academic Press; Ausubel, FM (1992). Short P
rotocols in Molecular Biology: A Compendium of Methods from Current Pr
otocols in Molecular Biology, Greene Pub. Associates; Ausubel, FM (1995)
). Short Protocols in Molecular Biology: A Compendium of Methods from C
urrent Protocols in Molecular Biology, Greene Pub. Associates; 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, Aca
It is described in demic Press, separate volume experimental medicine "Gene transfer & expression analysis experimental method" Yodosha, 1997, etc., and these are incorporated by reference in the relevant parts (which may be all) in this specification.

For DNA synthesis techniques and nucleic acid chemistry for the production of artificially synthesized genes, see, for example, Gait, MJ (1985). Oligonucleotide Synthesis: A Practical Approach, IR.
L Press; Gait, MJ (1990). Oligonucleotide Synthesis: A Practical Approach
, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practica
l Approach, IRL Press; Adams, RL et al. (1992). The Biochemistry of th
e Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. (1994). Advanced Organ
ic Chemistry of Nucleic Acids, Weinheim; Blackburn, GMet al. (1996). Nuc
leic Acids in Chemistry and Biology, Oxford University Press; Hermanson
, GT (I996). Bioconjugate Techniques, Academic Press, etc., which are incorporated herein by reference.

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

As used herein, "or" is used when "at least one" of the matters listed in the text can be adopted. The same applies to "or". When the specification "within the range of two values" is specified in the present specification, the range also includes the two values themselves.

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

The present invention has been described above by showing preferred embodiments for ease of understanding. Hereinafter, the present invention will be described based on examples, but the above description and the following examples are provided for purposes of illustration only and not for the purpose of limiting the present invention. Therefore, 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 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 was an IMQ cream-induced model and the other was a recombinant IL-23 protein into the mouse ear. The injection-induced model) was used to assess their immunological function.

Because little is known about the immunological role of CARD14 in psoriasis-like dermatitis in vivo, CARD14 is TLR7-mediated and / or other innate immunity that results in activation of NF-κB in the skin's innate immune system. It is based on our hypothesis that it may be involved in receptor-mediated intracellular signaling pathways.

(Materials and methods)
(Mouse strain)
Card14 ^{− / −} (details are described below and in FIG. 2), Trr7 ^{− / −} , Trr9
^{− / −} And Trr7 ^{− / −} Trr9 ^{− / −} double-deficient mice (Onishi et al., 2015)
Was bred in our animal facility and maintained under specific pathogen-free conditions. Rag2 ^{− / −} I
l2rg ^{− / −} mice were obtained from Taconic Biosciences. Tcrd ^{− / −} Mouse Ja
Obtained from ckson Laboratories. C57BL / 6 (WT) mice CLEA Japan, Inc
Purchased from. (Tokyo, Japan). All experiments were performed in accordance with the Institutional Ethics Guidelines for the Management and Use of Laboratory Animals at the National Institutes of Biomedical Innovation, Health and Nutrition.

(Creation of knockout mice and genotyping)
Card14 ^{− / −} mice used Lexicon Pharmaceuticals (Th) using a targeting vector designed to remove parts of exon 2 and exon 3 by homologous recombination.
e Woodlands, TX, USA). PCR was used for conventional 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 a C57BL / 6 background and determined using marker-assisted speed congenic methods. 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, Beselna (sold as Aldara TM} outside Japan; 3M Pharmaceticals, St Paul) containing 62.5 mg of 5% IMQ for 6 consecutive days for the induction of psoriatic dermatitis. , Minn) Cream (Mochida Pharmaceutical C)
Both ears of mice were treated by topical administration of o., Ltd. (Non-Patent Document 3 (van der)).
Fits et al., 2009)).

Control mice were not treated with cream. As previously described, an intradermal injection of 20 μl PBS, either alone or containing 500 ng of recombinant mouse IL-23 (BioLegend), every other day using a 30 gauge needle. 5 days
It was applied to both ears of anesthetized mice (Non-Patent Document 8 (Zheng et al., 2007)). Ear measurements were performed in the center of the ear using a constant pressure film thickness meter (PG-16J, Teclock, Japan). Mice were sacrificed at various time points and tissues were collected.

(Generation of bone marrow chimera)
BM cells (5 × 10 ⁶⁾ donor WT mice and donor Card14 ^{- / -} was obtained by flushing the femurs and tibia of mice. Mouse (WT and Card14
^{− / −} ) received a lethal dose of X-ray (9 Gy) and was subsequently reconstituted by intravenous injection of BM cells via the orbital plexus. It was performed 6 weeks after reconstitution of the treatment with IMQ cream.

(Histopathology of ear skin)
Mice were sacrificed on the final day of the experiment, ears were harvested and fixed with 10N formalin for subsequent embedding in paraffin. A paraffin tissue section (4 μm in diameter) was deparaffinized and a 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 Ki67 were stained with a rabbit anti-mouse monoclonal antibody (Thermo Fisher Scientific). Sections 0. before adding the primary antibody.
Treated in 01 M citric acid (adjusted to pH 6.0) for 10 minutes at 80 ° C. The secondary antibody is a peroxidase-labeled polymer (Dako) conjugated to goat anti-rabbit immunoglobulin.
)It was used. When 3,3'-diaminobenzidine (Dako) is used as a dye source,
A brown stain occurred (Uchio et al., 2002, Lab Invest. 82: 619-628). Aperio
All slides were scanned at 20x absolute magnification using ScanScope (Aperio Technologies).

(Immunofluorescent staining)
Mice were sacrificed and their ears were recovered. Encapsulant (Tissue-Tek OC) this ear skin tissue
T compound, Sakura Finetechnical. Co., Tokyo, Japan) was flash frozen at -80 ° C. Staining for CARD14 and γδTCR, cryostat (Leic)
A) was used on a 6 μm section of the ear of all mice. Sections are typically fixed with 4% paraformaldehyde at room temperature (RT) for 15 minutes with 1% BSA and 0.2% Trit.
Rabbit anti-mouse polyclonal antibody against anti-mouse γδ TCR (1:50; BD PharMingen, GL3, PE) and CARD 14 (1: 150) blocked with 2% goat serum in PBS containing on X-100 for 1 hour at room temperature. Incubated with 4 ° C. overnight. After washing in PBS, the sections were subjected to Alexa Fluor 488 conjugated donkey anti-rabbit IgG (H + L).
) (1: 250; Invitrogen) was incubated with (1: 250; Invitrogen) for 1 hour at room temperature, then washed in PBS and co-stained using DAPI. Images were acquired using the LeicaTCS SP2 confocal microscopy 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. 1.6 mg / m in complete RPMI medium (Nacalai Tesque)
A dermis suspension was obtained using a buffer containing L collagenase IV (Worthington) and 0.1 mg / mL Dnase-I (Sigma). The cells were then filtered through a 70 μm pore-sized cell strainer (BDFalcon) to give a single cell suspension (Nakashima et al. The Jou).
rnal 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)
The stored cells were collected by centrifugation at 400 xg for 5 minutes. Qiag RNA
Isolated using the en RNeasy kit (Qiagen). Then RNA is reverse transcriptase (ReverTr)
a Ace, Toyobo, Osaka, Japan) and oligo dT primers were used for reverse transcription into cDNA. The real-time PCR mixture has a total volume of 20 μL and 10 μL of SYBR Green Mas.
It consists of ter mix (Roche), forward and reverse primers (CARD14 and glyceraldehyde-3-phosphate dehydrogenase; 200 nM for GAPDH) and 2 μL of cDNA sample. The 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)
PCR reaction, LightCycler480 System II (Roche Life Science) Micro Amp op
40 cycles (95 ° C. for 15 minutes, 60 ° C. for 1 minute) were performed on a tical 384-well reaction plate. The fluorescence signal (ΔCt) detected during the threshold cycle was recorded by the software installed on the device. GAPDH was amplified under the same conditions as internal controls to standardize target gene levels with respect to variability in RNA and cDNA quality.

(Flow cytometry analysis and cell sorting)
The following antibodies were purchased from BD Pharma Mingen: Anti-CD8a (Ly-2, APC); Anti-CD45 (30-F11, PerCP / Cy5)
.5); Anti-mouse γδTCR (GL3, PE); Anti-IL-17A (TC11-18H10, PE); and Anti-NK1.1 (PK136, PE). Bio
The following antibodies from Legend were used: anti-CD3e (134-2C11, PerCP / Cy5.5); anti-CD4 (RM4-5, PE / Cy)
7); 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, A)
PC); and anti-TCRγδ (GL3, APC). Trephine fixed areas of the ear for FACS analysis
It was hollowed out using (8-mm, Kai Industries Co., Ltd.). Fc-block (BioLege) cells
It was pre-incubated with nd) for 15 minutes and then labeled with the appropriate cell surface antibody at 4 ° C. for 30 minutes. 50 ng / ml PMA (S) cells for intracellular cytokine staining
Stimulate with igma) and 500 ng / ml ionomycin (Sigma) and GolgiStop (
BD Bioscience) was used for 5 minutes at 37 ° C. For intracellular staining, BD C
It was fixed and permeabilized with the ytofix / Cytoperm Immobilization / Permeation Treatment Kit (BD Biosciences) and then stained with antibody 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 performed on days 0 and 7, either alone or with 100 μg of LPS-free OVA (Seikagaku). After blood collection, antibody titers were measured on the 14th day. 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, a flat bottom 96-well MaxiSorp plate (Nunc) was coated with 50 μg / mL OVA in _{0.5 M NaHCO 3.} The 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. Wash the plate, then TMB substrate solution (Kirkegaard & Perry)
Laboratories) was used to visualize the bound antibody. The reaction was _{stopped by adding 2NH 2} SO ₄ and 450 n using the Gen5ELISA (Biotek®) program.
The absorbance of m ^{was read with a BioStack TM} (BioTek Instruments Inc.) microplate reader.

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

(Results and Discussion)
(Both TLR7 and TLR9 are required for IMQ-induced psoriatic dermatitis, but IL-23
Not required for inducible psoriatic dermatitis)
To evaluate the role of CARD14 in the innate immune system in in vivo psoriasis pathogenesis, we utilized two established mouse psoriasis-like models. One model is IMQ
^{It is induced by topical application of Aldara TM} cream containing the above 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 evaluate whether TLR7 is essential or unnecessary in our experimental psoriasis model. Consistent with previous reports (Non-Patent Document 4 (Walter et al., 2013)), TLR7 single deficiency (Trr7 ^{− / −}
) Mice developed psoriatic dermatitis similar to WT mice (data not shown). Similarly
TLR9 single-deficient (Tll9 ^{− / −} ) mice showed the same phenotype as WT mice (data not shown). In striking contrast, when evaluating TLR7 and TLR9 double deficient (Tlr7 ^{− / −} Trr9 ^{− / −} ) mice in ^{the same experimental setup, Trr7 − / −} Tl
r9 ^{− / −} mice showed marked reductions in ear thickness, epidermal hyperkeratosis and dermal cell infiltration during the last 3 days of application compared to WT mice (FIGS. 1A and 1B). Previously speculated by many groups (Pantelyushin et al., 2012, The Journal of Cl)
inical Investigation. 122: 2252-2256; Non-Patent Document 4 (Walter et al., 2013),
For the first time, our results provide evidence that IMQ-induced psoriatic dermatitis requires both TLR7 and TLR9.

Next, we used another established psoriatic dermatitis model induced by administration of IL-23 (Non-Patent Document 8 (Zheng et al., 2007)) to TLR7 and /. Or the potential involvement of TLR9 was investigated. Consistent with previous reports, injection of recombinant IL-23 on days 0, 2, and 4 markedly increased ear thickness with epidermal thickening and dermal inflammatory cell infiltration on day 5. Has occurred. All of these changes were independent of TLR7 or TLR9 (FIGS. 1C and 1D). These results show that both TLR7 and TLR9 signaling in combination is required to completely develop IMQ-induced psoriatic dermatitis, while IL-23-induced psoriatic dermatitis models , TLR7 and TLR9 mediated direct activation downstream of innate immune activation. While it is still unclear which ligand or stimulus is responsible for TLR7 and TLR9-mediated innate immune activation in the development of IMQ-induced dermatitis, the above findings are based on the IMQ-induced psoriasis-like model and IL-. Demonstrated a clear difference in innate immunity signals from the 23-induced psoriasis-like model.

(CARD14 is essential for the development of skin inflammation in both IMQ-induced psoriasis-like and IL-23-induced psoriasis-like models)
To better characterize the role of CARD14 in vivo, we present CARD.
D14-deficient (Card14 ^{− / −} ) mice were generated (Fig. 2), and IMQ-induced psoriasis-like model and IL-23-induced psoriasis-like model (Non-Patent Document 3 (van der Fits et al., 2009)).
); The role of CARD 14 in both Non-Patent Document 8 (Zheng et al., 2007)) was evaluated. We applied IMQ cream for 6 consecutive days or injected IL-23 every other day for 5 days. WT mice had worsened skin condition and increased skin thickness after application of IMQ, but ^{significantly protected Card14 − / −} mice from skin inflammation (
FIG. 3A). Histochemical experiments showed keratin 5 in the skin of the ear of ^{Card14 − / − mice.}
And Ki67-stained hyperproliferative cells were similar to those of control mice, while hyperproliferative cells in WT mice treated with IMQ were found to be significantly increased (Fig. 3B). In addition, IL-23-induced ear swelling was ^{significantly suppressed in Card14 − / −} mice (Fig. 3C). Epidermal hyperkeratosis (seen as hyperproliferative cells stained with keratin 5 and Ki67) and dermal inflammation (seen as infiltrating cells in H & E staining) were both reduced in ^{IL-23-injected Card14 − / − mice.} (Fig. 3D). These results show that CARD14 applies IMQ cream and IL-
It has been shown to be essential for the formation of psoriasis-like dermatitis induced by 23 injections. Therefore, we have found that CARD14 is required for signal transduction downstream of TLR7 and TLR9, and is also required for signal transduction downstream of IL-23, which does not require TLR7 and TLR9, and thus innate immune cells. 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 not in radiation-resistant skin-resident cells)
Although CARD14 has been shown to be important for the innate immunity of keratinocytes (Non-Patent Document 12 (Fuchs-Telem et al., 2012); Non-Patent Document 9 (Jordan et al., 2012)).
), The above results of the present inventors 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 evaluated the role of CARD14 using an IMQ-induced psoriasis-like model. W
BM cells derived from T-mouse and Card14 ^{− / −} mice were X-ray-irradiated Card1
4 ^{− / −} mice and WT mice were transplanted, respectively. After 6 weeks, mice were treated with IMQ cream on both ears for 6 consecutive days. Psoriasis-like inflammation was ^{induced in Card14 − / −} mice and WT mice transplanted with BM from WT mice, but Card14 ^−
/ -BM was not induced in irradiated WT or Card 14 ^{− / −} mice transplanted (Fig. 4A). This is because CARD14 expression in hematopoietic BM-derived cells is IM.
It is essential for the pathogenesis of psoriasis in the Q-induced model, but indicates that CARD14 expression in radiation-resistant skin-resident cells was not essential. In addition to the RAG gene, we have the potential contribution of T and B cells regulated by natural killer (NK) cells, natural killer T (NKT) cells, innate lymphoid cells (ILC) and γδ T cells. Clarified about. We have a T cell receptor δ that lacks γδ T cells.
The severity of the psoriasis-like model was compared between deficient (Tcrd ^{− / − ) mice and Rag2 − / −} Il2rg ^{− / −} mice lacking T cells, NK cells, NKT cells and ILC.
Previous Report (Pantelyushin et al., 2012, The Journal of Clinical Investiga
Consistent with statement.122: 2252-2256), Tcrd ^{− / −} mice and Rag2 ^{− / −} Il2r
g ^{− / −} mice showed a marked reduction in skin swelling compared to WT mice after application of IMQ cream (FIG. 4B). Although it cannot be ruled out that CARD14 in non-hematopoietic cells, including keratinocytes, may play a role in psoriasis-like inflammation, the above results show that B
CARD14 in M-derived cells, potentially γδ T cells, strongly suggests that it is required for the development of IMQ-induced psoriasis-like dermatitis.

(CARD14 is expressed in γδ T cells and IL-17 and I by γδ T cells
Necessary for the production of L-22)
To test CARD14 expression in several different cell types, cells from ear skin were isolated and mRNA was used using real-time quantitative polymerase chain reaction (qPCR).
Was measured. Consistent with previous reports (Non-Patent Document 9 (Jordan et al., 2012)), CA
RD14 mRNA was expressed at high levels in keratinocytes (Fig. 5A). Among immune cells, CARD14 mRNA in γδ T cells of ear skin was as high as keratinocytes and higher than other immune cell types (FIGS. 5A and 5B). To further clarify the cell type expressing CARD14, we used antibodies against CARD14 and the T cell receptor γδ (TCRγδ) 5 days after administration of IL-23 to confocal the ear skin. Confocal microscopic analysis was performed. A CARD14 positive signal was detected in the epidermal region of WT mice that received IL-23, while Card14 ^{− / − that received IL-23.}
No CARD14 positive signal was detected in mice. In addition, ^{in both WT and Card14 − / −} mice, TCRγδ-positive signals were detected in the peripheral region between the epidermis and dermis, and specific TCRγδ-positive signals and CA expressed in the cytoplasm were detected.
RD14 positive signals overlapped, while ^{overlapping regions were not detected in Card14 − / −} mice (FIG. 5C). These data indicate that CARD14-positive γδT details are present in psoriasis-like skin.

It has been shown that γδ T cells that produce IL-17 and IL-22 play an important role in the pathogenesis of psoriasis (Non-Patent Document 7 (Cai et al., 2011)). The majority of IL-23 receptor (IL-23R) expressing cells are γδ T cells, and IL-
It has also been reported that a mutation in 23R was found in a specific psoriasis patient (Non-Patent Document 7).
(Cai et al., 2011); Non-Patent Document 10 (Tsoi et al., 2012)). Since IMQ-induced dermatitis and IL-23-induced dermatitis are closely associated with the production of IL-17 and IL-23 (Non-Patent Document 7 (Cai et al., 2011); Pantelyushin et al. , 20
12, The Journal of Clinical Investigation. 122: 2252-2256; Non-Patent Document 8 (Zhe)
ng et al., 2007)), we found IL-17 and IL-22 recovered from the epidermal sheets of ^{WT and Card 14 − / − mice that received IMQ or IL-23.}
Cells producing the above were tested using intracellular staining (FIGS. 5D and 5E). WT mice treated with either IMQ or IL-23 showed significantly higher numbers of epidermal TCRγδ ⁺ IL-17 cells and TCRγδ ⁺ IL-22 cells than in the control group. In contrast, IMQ-treated Card14 ^{− / −} mice and IL-23 treated Card14
^{Both − / −} mice had significantly less epidermal γδ T cells producing IL-17 and IL-22 (FIGS. 5D and 5E). These data suggest that CARD14 may play an important role in γδ T cells in epidermal lesions of psoriatic skin that secrete IL-17 and IL-22.

(CARD14 does not interfere with the adjuvant effect of IMQ on co-administered antigens)
IMQ is a vaccine adjuvant commonly used in many other applications T
Being an agonist of LR7, we investigated whether CARD14 affects the adjuvant effect of IMQ on co-administered antigens. To answer this question, we modified the experimental protocol for a psoriasis-like dermatitis model using IMQ cream to WT mice, Thrr7 ^{− / −} Trr9 ^{− / −} mice and Card14 ^−.
/ -Mice were immunized with ovalbumin (OVA) via intradermal injection in both ears, while IMQ cream was applied topically to the same ear. Increased ear thickness was observed in WT mice, on the other hand, injection of OVA protein into mice also resulted in Trr7 ^{− / −} Trr9 ^−
/ -Mice and Card14 ^{− / −} mice showed consistently significantly thinner ears on day 7 (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 immunized group. Shown (Fig. 6B). OVA-specific _{IgG 2c} antibody ^{titer, Tlr7 - / - Tlr9 - /} - was markedly reduced in mice, Card14 ^{- / -} mice, the levels comparable to WT mouse serum anti-OVA IgG
_2c is shown (Fig. 6B). These results indicate that IMQ cream has an adjuvant effect on co-administered protein antigens and CARD14 does not affect the adjuvant effect. CARD because IMQ-induced psoriasis is a complication of IMQ cream
Development of 14 inhibitors may suppress the pathogenesis of psoriatic dermatitis without affecting IMQ-induced innate immune function and its adjuvant effect.

Therefore, the above data show that CARD14 is IL-17 and I by epidermal γδ T cells.
It is required for optimal production of L-22 and strongly indicates that it is induced by both IMQ and IL-23. CARD14 is responsible for most psoriasis-like dermatitis in experimental mouse models.

CARD 14 and CARD 11 have been shown to be novel MAGUK family members that function as upstream activators 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).
We first found that CARD14 was a T in epithelial stromal cells such as keratinocytes.
It was hypothesized to be a downstream signaling molecule for LR7 mediated NF-κB activation. However, our findings indicate that TLR7 and TLR9 are required for IMQ-induced psoriasis-like dermatitis but not for IL-23-induced psoriasis-like dermatitis (Fig. 1), while CAR.
D14 demonstrates that it is important for both IMQ-induced psoriasis-like dermatitis and IL-23-induced psoriasis-like dermatitis (Fig. 3). These data suggest that our first hypothesis was questionable, rather that CARD 14 is downstream of IL-23,
It appears to be more important for adaptive immune cells than innate immune cells. Downstream of the signaling pathway initiated by administration of IL-23, cells expressing IL-23R should play an important role in CARD14-mediated pathogenesis of psoriasis-like dermatitis. Which cell is I
It is not clear if it expresses L-23R, but most cells expressing IL-23R have been shown to be γδ T cells (Awasthi et al., 2009, Journal of Immunolog).
y.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 research is needed to elucidate how CARD14 is involved in these intracellular signaling pathways. We found that CARD14 was IL-17 in two established psoriasis-like dermatitis models.
And it was shown to be required for psoriasis lesion formation through suppression of IL-22-producing γδ T cells.

We have found that CARD14 is expressed in γδ T cells in psoriasis-like skin rashes, in addition to its expression in epithelial keratinocytes (FIGS. 5A and 5C).
.. It is known that the experimental conditions differ between the human patient sample and the experimental mouse model, and that the amount of TCRγδ cells differs between human and mouse (Non-Patent Document 7 (Cai et al., 201).
1); Pasparakis et al., 2014, Nature reviews. Immunology. 14: 289-301).
Therefore, the role of CARD 14 in humans and mice (including animal models) is CA.
It can differ with respect to either the RD14 expressing cell type or the experimental conditions. Further research is needed to elucidate the role of CARD14 in human γδ T cells of psoriasis.

CARD14 can 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. It inhibits innate immune activation without suppressing the adaptive immune response specific to the co-administered antigen. The immunological significance of CARD14-mediated innate and adaptive immune responses should be studied. γδ T cells have 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 .;
It plays an important role in Sutton et al., 2012, European Journal of Immunology. 42: 2221-2231).

(Example 2: Multiple sclerosis)
Next, in this example, it was investigated whether CARD14 deficiency had a symptomatological reduction effect in an experimental demyelinating disease model of multiple sclerosis. In this example, the literature of Rui et al. (Yuxiang) is basically used.
Rui, Tasuku Honjo, and Shunsuke Chikuma. Programmed cell death 1 inh
ibits inflammatory helper T-cell development through controlling the inn
ate immune response. PNAS 2013 110 (40) 16073-16078).

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

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

(EAE mouse)
_{200 μg of MOG 35-55} peptide (MOG35-) in mice according to the literature of Rui et al.
Peptide corresponding to 55 residues), complete Freund's adjuvant (CFA), and 250
μg of heat-killed mycobacterium;
It was subcutaneously (sc) immunized with a 0.2 mL emulsion containing HKMTB). In addition, mice were immunized with 200 ng of pertussis toxin (PTX) twice intraperitoneally (ip) at the time of immunization and 48 hours later.
) I gave an injection. In some experiments, suboptimal immune conditions excluding HKMTB, PTX, or both were also used. Mice were observed daily, observed for clinical signs of disease, and the EAE score was evaluated as follows.
0, no disease;
1, 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.

Scores are shown as mean clinical scores for each experimental group.

For the restimulation assay, the spleen is taken 8 or 30 days after immunization and a CD4 ⁺ T cell enriched sample is taken with anti-CD4 microbeads and the autoMACS system (Miltenyi).
) Was obtained. ^{2 × 10 6} isolated CD4 ⁺ T cells in a volume of 1 mL _{, MOG 35-}
2 × 10 from CARD-14 +/+ mice in the presence of _{55 peptides (30 μg / mL)}
⁶ Mitomycin C-treated spleen cells were cultured together. 3 for cytokine analysis of the supernatant
Collected a day later.

^{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 days 0 and 2. Shows EAE score (
FIG. 7 left). On the right side, it was shown that Incidence CARD14 deficiency had a symptom-relieving effect in an experimental demyelinating disease model, 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 the side effects of a TLR-based / IMQ / immunostimulatory adjuvant, a γδT cell-mediated disease and / Alternatively, screen for treatment or prophylaxis of complications of immune intervention.

The inhibitory activity of CARD14 is measured by any method known in the art, the inhibitory activity of the candidate substance is measured, and the inhibitory activity is identified as an inhibitor.

These inhibitors are administered in a model of psoriasis, multiple sclerosis, side effects from a TLR-based / IMQ / immunostimulatory adjuvant, or complications of γδT cell-mediated disease and / or immune intervention as described in Example 1 or 2. Then, follow-up of the symptoms and the like can be performed, and those for which the symptoms can be prevented or treated can be identified as a preventive agent or a therapeutic agent for these diseases, disorders or symptoms.

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..
As described above, the present invention has been illustrated using the preferred embodiments of the present invention, but it is understood that the scope of the present invention should be interpreted only by the scope of claims. The patents, patent applications and documents cited herein are to be incorporated by reference in their content as they are specifically described herein. Understood. This application claims priority to Japanese Patent Application No. 2015-200948 filed in Japan on October 9, 2015, the entire contents of which are incorporated herein by reference in its entirety.

The present invention finds industrial applicability in the biopharmacy 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
SEQ ID NO: 5: P1, 5'-GGGTGTTCCTCTGACTCTCCCAGTTGGATG-3'
SEQ ID NO: 6: P2, 5'-GCTGACCGCTTCCTCGTGCTTTACGGTATC-3'
SEQ ID NO: 7: P3, 5'-CAGTGACTCAAGGAGGGGCAAACGCCTATG-3'
SEQ ID NO: 8: mCARD14, 5'-TGCATAGCTCCCGTTTCAC-3' (forward)
SEQ ID NO: 9: mCARD14, 5'-GGAACTTCAGGCTTTCCAGA-3' (reverse)
SEQ ID NO: 10: mGAPDH, 5'-CAAGATTGTCAGCAATGCATCC-3' (forward)
SEQ ID NO: 11: mGAPDH, 5'-CCTTCCACAATGCCAAGTTG-3' (reverse)

Claims (1)

The invention described in the specification.

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