JP2025077049A - Treatment, diagnosis and screening using CARD14 - Google Patents

Abstract

To provide therapy, diagnosis and screening using a CARD 14.SOLUTION: The invention provides a therapy agent or a prevention agent for reducing, adverse effects by psoriasis, multiple sclerosis, TLR base/IMQ/immune stimulation adjuvant, it has been found a fact that, a CARD 14 relates to reduction of adverse effects caused by psoriasis, multiple sclerosis, TLR base/IMQ/immune stimulation adjuvant, and on the basis of the fact, the therapy agent, a diagnosis agent, and screening of them are provided. Especially, the CARD 14 has strong relation to the adverse effects by psoriasis, multiple sclerosis, TLR base/IMQ/immune stimulation adjuvant, in expression in hematopoietic cells, especially expression in γδT cells, especially in epidermal γδT cells, and the therapy agent, the diagnosis agent and screening of them are provided.SELECTED DRAWING: None

Description

The present invention relates to treatment, diagnosis and screening using CARD14. More particularly, the present invention relates to a therapeutic or preventive agent for psoriasis, multiple sclerosis, and γδ T cell-mediated diseases and/or complications of immune intervention, and a screening thereof, and a therapeutic or preventive agent for reducing side effects caused by Toll-like receptor (TLR)-based/imiquimod (IMQ)/immunostimulatory adjuvants, and a screening thereof, using CARD14.

Psoriasis is a localized, T cell-mediated, chronic inflammatory skin disorder characterized primarily by chronic, well-defined erythematous plaques (Schon and Boehncke, 2005).
Recent findings also suggest that topical application of imiquimod (IMQ) cream induces psoriasis-like skin inflammation in humans and mice (Gilliet, et al., 2003).
et al., 2004; van der Fits et al., 2009). IMQ is a specific agonist of mouse Toll-like receptor (TLR) 7, and TLR7-deficient mice showed epidermal hyperkeratosis to the same extent as wild-type (WT) mice (Walter et al., 2004).
et al., 2013), suggesting that TLR7-independent mechanisms may be important in the pathogenesis.

The important role of interleukin (IL)-17 and IL-23 in the pathogenesis of psoriasis has been recently revealed (Johnson-Huang et al., 2012). Monoclonal antibodies against IL-23p19 (guselkumab and tildrakizumab) showed significant clinical benefits over blockade of IL-12/23p40 subunits (Johnson-Huang et al., 2012; Sofen et al., 2014). In this experimental setting, the absence of IL-23p19 in vivo abrogates IMQ cream-induced psoriasis-like skin inflammation (van der Fits et al., 2009). Furthermore, intradermal injection of recombinant IL-23 protein into mouse ears shared many characteristics with psoriasis, such as epidermal hyperkeratosis via IL-22 upregulation and STAT3 activation (Non-Patent Document 7: Cai et al., 2011; Non-Patent Document 8: Zheng et al., 2007).

Recently, caspase recruitment domain family member 14 (CARD14 [MIM607211], also known as CARMA2), located in the psoriasis susceptibility locus 2 (PSORS2) at human chromosomal region 17q25.3, was shown to harbor a unique gain-of-function mutation (Jordan et al., 2012). This finding was further confirmed by a genome-wide association study (GWAS) of PSORS2, which identified several risk-associated variants, including CARD14 (Tsoi et al., 2012). CARD14 was shown to activate nuclear factor kappa B (NF-κB) through the IκB kinase complex in response to upstream stimuli (Bertin et al., 2001). This evidence indicates that CARD14 is an important molecule in psoriasis and pityriasis rubra pilaris, but the mechanisms and correlation of these immune responses are unknown (Non-Patent Document 12: Fuchs-Telem et al., 2012; Non-Patent Document 9: Jordan et al., 2012).

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

As a result of intensive research, the inventors have found that CARD14 is essential for the formation of psoriasis-like dermatitis, and have concluded that it is possible to provide and screen for therapeutic and diagnostic agents based on this finding, thereby completing the present invention. In particular, they have found that CARD14 in γδT cells that produce IL-17 and IL-22 is essential for the formation of psoriasis-like dermatitis, discovered the role of CARD14 in hematopoietic cells in relation to psoriasis, and discovered that the role of specific cells is also important, thereby completing the present invention.

The inventors also discovered that CARD14 is an essential factor in the onset of multiple sclerosis (MS), and concluded that based on this, it is possible to develop therapeutic and diagnostic agents and screen for them. They also concluded that it is possible to provide therapeutic or preventive agents for multiple sclerosis using CARD14 and screen for them, thus completing the present invention.

Furthermore, the inventors have found that CARD14 in γδ T cells can be a potential therapeutic target for γδ T cell-mediated diseases and complications of immune intervention, and have concluded that it is possible to provide and screen therapeutic or preventive agents for γδ T cell-mediated diseases and/or complications of immune intervention, thus completing the present invention.

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

Psoriasis is a chronic inflammatory skin disease of unknown etiology, but has also been reported as a complication of imiquimod (IMQ) cream. Recently, CARD14 was identified as a psoriasis susceptibility gene, but its immunological role in psoriasis pathogenesis in vivo remains unclear. Herein, we evaluated CARD14-deficient (Card14 ^−/− ) mice using two psoriasis-like models. The first model is induced by IMQ cream through Toll-like receptor (TLR) 7- and TLR9-mediated innate immune activation, and the other model is induced by interleukin (IL)-23 without TLR7 and TLR9 activation. In both models, Card14 ^−/− mice showed significantly less increase in skin thickness and hyperkeratinization compared to wild-type mice. CARD14 is expressed in and associated with epidermal γδ T cells that produce IL-17 and IL-22 in psoriatic skin lesions in these models. Studies using bone marrow chimeric and gene-deficient mice revealed that CARD14, which is expressed in hematopoietic cells (especially γδ T cells) but not in non-hematopoietic cells, is important for the formation of psoriasiform dermatitis. The vaccine adjuvant effect of IMQ cream against co-administered protein antigens was not altered by CARD14 deficiency, suggesting that inhibition of CARD14 may reduce IMQ-induced psoriasiform dermatitis without affecting its adjuvant activity. From these findings, we found that CARD14 in γδ T cells strongly suggests that it may be a potential therapeutic target for γδ T cell-mediated diseases and complications of immune intervention.

Based on the above, the present invention provides the following.
(1) A method for screening a therapeutic or preventive agent for psoriasis based on the expression of CARD14 in hematopoietic cells.
(2) A method for screening a therapeutic or preventive agent for multiple sclerosis based on the expression of CARD14 in hematopoietic cells.
(3) A method for screening a therapeutic or preventive agent for reducing side effects caused by TLR-based/IMQ/immunostimulatory adjuvants based on the expression of CARD14 in hematopoietic cells. (4) A method for screening a therapeutic or preventive agent for γδ T cell-mediated diseases and/or complications of immune intervention based on the expression of CARD14 in hematopoietic cells.
(5) The method according to any one of items 1 to 4, wherein the hematopoietic cells are γδ T cells.
(6) The method according to any one of items 1 to 4, wherein the hematopoietic cells are epidermal γδ T cells. (7) The method according to item 5 or 6, wherein the γδ T cells are cells that produce IL-17 and IL-22.
(8) The method according to any one of items 1 to 7, further based on non-expression of CARD14 in non-hematopoietic cells.
(9) The method according to item 8, wherein the non-hematopoietic cells include skin resident cells.
(10) The method according to any one of items 1 to 9, wherein the disease includes IMQ-induced psoriasis and IL-23-induced psoriasis.
(11) A therapeutic or preventive agent for psoriasis, comprising a hematopoietic cell-specific CARD14 inhibitor.
(12) A therapeutic or preventive agent for multiple sclerosis, comprising a hematopoietic cell-specific CARD14 inhibitor. (13) A therapeutic or preventive agent for reducing side effects caused by a TLR-based/IMQ/immunostimulatory adjuvant, comprising a hematopoietic cell-specific CARD14 inhibitor.
(14) A therapeutic or preventive agent for a γδ T cell-mediated disease and/or complications of immune intervention, comprising a hematopoietic cell-specific CARD14 inhibitor.
(15) The therapeutic or prophylactic agent according to any one of items 11 to 14, wherein the hematopoietic cells are γδ T cells.
(16) The therapeutic or prophylactic agent according to any one of items 11 to 14, wherein the hematopoietic cells are epidermal γδ T cells.
(17) The therapeutic or prophylactic agent according to item 15 or 16, wherein the γδ T cells are cells that produce IL-17 and IL-22.
(18) The therapeutic or prophylactic agent according to any one of items 11 to 17, wherein the hematopoietic cell-specific CARD14 inhibitor is achieved by the method according to any one of items 1 to 10.
The present invention also provides the following:
(1A) A method for screening a therapeutic or preventive agent for psoriasis based on the expression of CARD14 in hematopoietic cells, comprising the steps of contacting a candidate substance with hematopoietic cells and determining the expression of CARD14 in the hematopoietic cells, 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 preventive agent for psoriasis.
(2A) A method for screening a therapeutic or preventive agent for multiple sclerosis based on the expression of CARD14 in hematopoietic cells, comprising the steps of contacting a candidate substance with hematopoietic cells and determining the expression of CARD14 in the hematopoietic cells, 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 preventive agent for multiple sclerosis.
(3A) A method for screening a therapeutic or prophylactic agent for reducing side effects caused by a TLR-based/IMQ/immunostimulatory adjuvant based on the expression of CARD14 in hematopoietic cells, comprising the steps of contacting a candidate substance with hematopoietic cells and determining the expression of CARD14 in the hematopoietic cells, 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 reducing side effects caused by a TLR-based/IMQ/immunostimulatory adjuvant.
(4A) A method for screening for a therapeutic or preventive agent for a γδ T cell-mediated disease and/or complications of immune intervention based on the expression of CARD14 in hematopoietic cells, comprising the steps of contacting a candidate substance with hematopoietic cells and determining the expression of CARD14 in the hematopoietic cells, 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 preventive agent for a γδ T cell-mediated disease and/or complications of immune intervention.
(5A) The method according to any one of items 1A to 4A, wherein the hematopoietic cells are γδ T cells.
(6A) The method according to any one of items 1A to 4A, wherein the hematopoietic cells are epidermal γδ T cells.
(7A) The method according to item 5A or 6A, wherein the γδ T cells are cells that produce IL-17 and IL-22.
(8A) The method according to any one of items 1A to 7A, further comprising determining that CARD14 is not expressed in non-hematopoietic cells.
(9A) The method according to any one of items 1A to 8A, further comprising the step of contacting the candidate substance with a non-hematopoietic cell, and selecting the candidate substance as the therapeutic or prophylactic agent if CARD14 expression is not substantially affected.
(10A) The method according to item 8A or 9A, wherein the non-hematopoietic cells comprise skin resident cells.
(11A) The method according to any one of items 1A or 3A to 10A, wherein the disease includes IMQ-induced psoriasis and IL-23-induced psoriasis.
(12A) A therapeutic or preventive agent for psoriasis, comprising a hematopoietic cell-specific CARD14 inhibitor.
(13A) A therapeutic or preventive agent for multiple sclerosis, comprising a hematopoietic cell-specific CARD14 inhibitor.
(14A) A therapeutic or prophylactic agent for reducing side effects caused by TLR-based/IMQ/immunostimulatory adjuvants comprising a hematopoietic cell-specific CARD14 inhibitor.
(15A) A therapeutic or preventive agent for a γδ T cell-mediated disease and/or complications of immune intervention, comprising a hematopoietic cell-specific CARD14 inhibitor.
(16A) The therapeutic or prophylactic agent according to any one of Items 12A to 15A, wherein the hematopoietic cells are 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 therapeutic or prophylactic agent according to item 16A or 17A, wherein the γδ T cells are cells that produce IL-17 and IL-22.

It is contemplated that one or more of the above features may be provided in combinations other than those explicitly stated. Still further embodiments and advantages of the present invention will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.

The present invention provides a therapeutic or preventive agent for psoriasis based on the expression of CARD14 in hematopoietic cells, a therapeutic or preventive agent for multiple sclerosis, a therapeutic or preventive agent for reducing side effects caused by TLR-based/IMQ/immunostimulatory adjuvants, and a therapeutic or preventive agent for γδ T cell-mediated diseases and/or complications of immune intervention, and allows easy screening of these.

Both TLR7 and TLR9 are required for IMQ-induced psoriasiform dermatitis, but not for IL-23-induced psoriasiform dermatitis. Both ears from WT and Tlr7 ^−/− Tlr9 ^−/− mice (n=4 mice/group) were treated with IMQ for 6 consecutive days or injected with 500 ng of IL-23 every other day for 5 days. (A) Ear thickness was measured daily before injection. Data are representative of four independent experiments and results are shown as mean ± standard deviation (s.d.), ^** indicates P<0.01; all by Scheffe's test. (B) H&E staining in the skin of control and IMQ mice on the last day of each group. Bars indicate 100 μm. Data are representative of four independent experiments. (C) Ear thickness was monitored daily before injection with IL-23. Data are representative of two independent experiments and results are shown as mean ± s.d. d. All results are Scheffe's assay. (D) H&E staining of the skin of phosphate-buffered saline (PBS)- and IL-23-injected mice on the last day of the experiment. Bars indicate 100 μm. Data are representative of two independent experiments. Generation of Card14 ^−/− mice. Card14 ^−/− mice were generated by replacing exons 2 and 3 of the Card14 gene with the neomycin (neo) resistance gene. (A) Structure of mouse Card14 locus (WT allele), Card14 targeting vector, and predicted mutated Card14 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 and mutant alleles using primers P1, P2, and P3. Genomic DNA was extracted from mouse tails. The WT product was 350 bp, and the knockout product was 250 bp. Lane M represents the base pair marker. (C) Card14 mRNA levels in epidermal and dermal cells from WT and Card14 ^−/− mice. Data were analyzed using real-time qPCR and Card14 expression relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown (n=4 mice/group). CARD14 is essential for the development of skin inflammation in both IMQ-induced and IL-23-induced psoriasis-like models. Ears from WT and Card14 ^−/− mice (n=4 mice/group) were treated with IMQ cream for 6 consecutive days or injected with IL-23 every other day for 5 days. (A) Ear thickness was measured daily before injection. Data are representative of at least 5 independent experiments and results are shown as mean ± s.d., ^* indicates P<0.05, ^** indicates P<0.01; all by Scheffe's test. (B) H&E, keratin 5, and Ki67 staining of skin on the last day of control and mice receiving IMQ. Bars indicate 100 μm. Data are representative of at least 5 independent experiments. (C) Ear thickness was monitored daily before injection with IL-23. Data are representative of 4 independent experiments and results are shown as mean ± s.d. d., ^** indicates P<0.01; all by Scheffe test. (D) H&E, keratin 5, and Ki67 staining of skin in PBS- and IL-23-injected mice on the last day of the experiment. Bars indicate 100 μm. Data are representative of four independent experiments. CARD14 in hematopoietic cells, but not in radioresistant skin-resident cells, plays a key role in psoriasis. (A) Reconstituted bone marrow chimeras (WT BM → WT mice (WT→WT), WT BM → Card14 ^−/− mice (WT→KO), Card14 ^−/− BM → Card14 ^−/− mice (KO→KO) and Card14 ^−/− BM → WT mice (KO→WT), n=4-6 mice/group) were treated with IMQ cream as described in Figure 4. Ear thickness was monitored daily. Data are representative of three independent experiments, and results are shown as mean ± s.d., ^** indicates P<0.01; all by Scheffe test. (B) WT, Rag2 ^−/− , Rag2 ^−/− Il2rg ^−/− , and Tcrd ^−/− mice (n=4 mice/group) were treated with IMQ cream for 6 consecutive days. Ear thickness was measured daily before cream treatment. Data are representative of three independent experiments, and results are shown as mean ± s.d., ^* indicates P<0.05, ^** indicates P<0.01; all by Scheffe test. CARD14 is expressed in γδ T cells and is required for IL-17 and IL-22 production 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 relative to GAPDH. Data are representative of 4 independent experiments. (C) Frozen sections from IL-23-injected WT (left) and Card14 ^−/− mice (right) were stained with DAPI (blue), CARD14 Ab (green), and GL3 (anti-TCRγ) mAb (red) for immunofluorescence staining. Merge indicates overlay. Images were Z-stack deconvolved at 0.5 mm steps. Images were magnified 100x. Data are representative of two independent experiments. (D and E) Epidermal cell suspensions from WT and Card14 ^−/− mice in the IMQ or IL-23 models were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, and intracellular IL-17 and IL-22 expression was analyzed by flow cytometry. Regional flow plots gated on CD45 ⁺ cells are representative of three independent experiments. Data are shown as mean ± s.d., ^* indicates P <0.05; Scheffe test. CARD14 does not interfere with the adjuvant effect of IMQ against co-administered antigens. For ovalbumin (OVA) immunization, WT, Tlr7 ^−/− Tlr9 ^−/− , and Card14 ^−/− mice (n=5-6 mice/group) were injected intradermally in the ear with 100 μg OVA protein (20 μL/ear) on days 0 and 7. Blood was collected on day 14 and antibody titers were measured. For induction of psoriasiform dermatitis, mouse ears were treated with IMQ cream for 6 consecutive days as previously described. (A) Ear thickness was measured daily before cream treatment and injection. Data are representative of two independent experiments, and results are shown as mean ± s.d., ^* indicates P<0.05; all by Scheffe test. (B) OVA-specific _IgG1 and _IgG2c in serum were measured using ELISA. Data are representative of two independent experiments, and results are shown as mean ± s.d., ^** indicates P<0.01; all by Scheffe test. 7 shows the symptom-alleviating effect of CARD14 deficiency in an experimental demyelinating disease model. The results show the 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. Data are shown as mean±SEM. The left panel shows the time-course evaluation (days) based on the EAE score, and the right panel shows the incidence rate. It was shown that CARD14 deficiency had a symptom-alleviating effect in an experimental demyelinating disease model, indicating that CARD14 plays an important role in multiple sclerosis.

The present invention will be described below. Throughout this specification, singular expressions should be understood to include the concept of the plural, unless otherwise specified. Therefore, singular articles (e.g., in the case of English, "a", "an", "the", etc.) should be understood to include the concept of the plural, unless otherwise specified. In addition, it should be understood that the terms used in this specification are used in the sense commonly used in the field, unless otherwise specified. Therefore, unless otherwise defined, all technical terms and scientific and technical terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In the case of conflict, this specification (including definitions) will take precedence.

As used herein, "CARD14" refers to a caspase recruitment domain-containing
It refers to a protein also called CARD protein 14 or CARD-containing MAGUK protein 2 (Carma 2) and the gene encoding it. It may also be indicated as BIMP2; CARMA2; PRP; PSORS2; PSS1, and the accession numbers for its human amino acid sequence are NP_001244899 (human) and NP_570956 (mouse), and the accession numbers for its mRNA sequence are NM_001257970 (human) and NM_130886 (mouse). The amino acid sequence of CARD14 is, for example, SEQ ID NO: 2 (human) and SEQ ID NO: 4 (mouse). The base sequence of CARD14 mRNA is, for example, SEQ ID NO: 1 (human) and SEQ ID NO: 3 (mouse). The amino acid sequence of CARD14 is not limited as long as it has the activity of CARD14. Therefore, it will be understood that not only a protein having an amino acid sequence set forth in a particular SEQ ID NO or accession number (or a nucleic acid encoding the same) can be used in the present invention, but also a functionally active analog or derivative thereof, or a functionally active fragment thereof, or a homolog thereof, or a mutant encoded by a nucleic acid that hybridizes to the nucleic acid encoding the protein under high or low stringency conditions, so long as it meets the specific objectives of the present invention.

As used herein, "derivatives", "analogs" or "variants" preferably include, but are not limited to, molecules that contain a region substantially homologous to the protein of interest (e.g., CARD14), which in various embodiments are at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identical over the same size amino acid sequence or when compared to sequences aligned by computer homology programs known in the art, or the nucleic acid encoding such a molecule is hybridizable to sequences encoding the component protein under (highly) stringent, moderately stringent or non-stringent conditions. This refers to proteins that are the product of modification of naturally occurring proteins by amino acid substitutions, deletions and additions, respectively, and whose derivatives still exhibit the biological functions of the naturally occurring proteins, although not necessarily to the same degree. The biological functions of such proteins can be examined, for example, by suitable and available in vitro assays described herein or known in the art. "Functionally active" as used herein refers to a polypeptide, i.e., a fragment or derivative, that has a structural, regulatory, or biochemical function of a protein, such as biological activity, according to the embodiment to which the polypeptide, i.e., fragment or derivative, of the invention pertains. Although the present invention primarily discusses humans with respect to CARD14, it is understood that many animals other than humans, particularly mammals, are known to express CARD14 and therefore these animals are also within the scope of the present invention, particularly mammals.

Thus, a representative nucleotide sequence for CARD14 is:
(a) a polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 1 or 3, or a fragment thereof;
(b) a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or 4, or a fragment thereof;
(c) a polynucleotide encoding a variant polypeptide or a fragment thereof having one or more amino acid mutations selected from the group consisting of substitutions, additions, and deletions in the amino acid sequence set forth in SEQ ID NO: 2 or 4, and having biological activity;
(d) a polynucleotide which 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 of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or 4, or a fragment thereof;
(f) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides (a) to (e) and encodes a polypeptide having biological activity; or (g) a polynucleotide that has a base sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the polynucleotides (a) to (e) or their complementary sequence, and encodes a polypeptide having biological activity. Here, biological activity typically refers to the activity possessed by CARD14 or the ability to distinguish it from other proteins present in the same organism as a marker.
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) a polypeptide having one or more amino acid mutations selected from the group consisting of substitutions, additions, and deletions in the amino acid sequence set forth in SEQ ID NO: 2 or 4, and having biological activity;
(c) a polypeptide encoded by a splice variant or allelic variant of the nucleotide sequence set forth in SEQ ID NO: 1 or 3;
(d) a polypeptide which is a species homologue of the amino acid sequence set forth in SEQ ID NO: 2 or 4; or (e) a polypeptide which has an amino acid sequence which is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the polypeptides (a) to (d) and has biological activity. Here, biological activity typically refers to the ability to distinguish the activity or marker of CARD14 from other proteins present in the same organism (e.g., the ability to contain a region which can function as a specific epitope when used as an antigen).

In the context of the present invention, a "substance that binds to CARD14", a "binding agent for CARD14" or a "CARD14 interacting molecule" is a molecule or substance that binds to CARD14 at least temporarily. For detection purposes, it is advantageous to be able to indicate the binding (e.g., it is labeled or can be labeled), and for therapeutic purposes, it is advantageous to further bind a therapeutic agent. Substances that bind to CARD14 can be, for example, antibodies, antisense oligonucleotides, siRNAs, low molecular weight molecules (LMWs), binding peptides, aptamers, ribozymes and peptidomimetics. Substances that bind to CARD14 or CARD14 interacting molecules can be inhibitors of CARD14, including, for example, binding proteins or binding peptides directed against CARD14, in particular against the active site of CARD14, as well as nucleic acids directed against the CARD14 gene. Nucleic acids against CARD14 refer to, for example, double-stranded or single-stranded DNA or RNA, or modifications or derivatives thereof, that inhibit expression of the CARD14 gene or activity of CARD14, and include, but are not limited to, antisense nucleic acids, aptamers, siRNAs (small interfering RNAs), and ribozymes. As used herein, "binding protein" or "binding peptide" for CARD14 refers to any protein or peptide that binds to CARD14, and includes, but is not limited to, antibodies (e.g., polyclonal or monoclonal antibodies), antibody fragments, and functional equivalents directed against CARD14.

As used herein, the terms "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 acids may be natural, non-natural, or modified. 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 (e.g., conjugation with a labeling moiety). The definition also includes, for example, polypeptides containing one or more analogs of amino acids (e.g., including non-natural amino acids), peptide-like compounds (e.g., peptoids), and other modifications known in the art. As used herein, "amino acid" is a collective term for an organic compound having an amino group and a carboxyl group. When an antibody according to an embodiment of the present invention includes a "specific amino acid sequence," any amino acid in the amino acid sequence may be chemically modified. Any of the amino acids in the amino acid sequence may form a salt or a solvate. Any of the amino acids in the amino acid sequence may be L-type or D-type. In such cases, the protein according to the embodiment of the present invention can be said to contain the above-mentioned "specific amino acid sequence." Examples of chemical modifications that are known to occur in vivo on amino acids contained in proteins include N-terminal modifications (e.g., acetylation, myristoylation, etc.), C-terminal modifications (e.g., amidation, glycosylphosphatidylinositol addition, etc.), and side chain modifications (e.g., phosphorylation, sugar chain addition, etc.). As long as the object of the present invention is met, the amino acids may be natural or unnatural.

As used herein, "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably to refer to a polymer of nucleotides of any length. The terms also include "oligonucleotide derivatives" or "polynucleotide derivatives." "Oligonucleotide derivatives" or "polynucleotide derivatives" refer to oligonucleotides or polynucleotides that contain derivatives of nucleotides or have unusual bonds between nucleotides, and are used interchangeably. Specific examples of such oligonucleotides include 2'-O-methyl-ribonucleotides, oligonucleotide derivatives in which a phosphodiester bond in an oligonucleotide has been converted to a phosphorothioate bond, oligonucleotide derivatives in which a phosphodiester bond in an oligonucleotide has been converted to an N3'-P5' phosphoramidate bond, oligonucleotide derivatives in which a ribose and a phosphodiester bond in an oligonucleotide have been converted to a peptide nucleic acid bond, oligonucleotide derivatives in which uracil in an oligonucleotide has been replaced with C-5 propynyl uracil, oligonucleotide derivatives in which uracil in an oligonucleotide has been replaced with C-5 thiazole uracil, oligonucleotide derivatives in which cytosine in an oligonucleotide has been replaced with C-5 propynyl cytosine, oligonucleotide derivatives in which cytosine in an oligonucleotide has been replaced with phenoxazine-modified cytosine, oligonucleotide derivatives in which ribose in DNA has been replaced with 2'-O-propyl ribose, and oligonucleotide derivatives in which ribose in an oligonucleotide has been replaced with 2'-methoxyethoxy ribose. Unless otherwise indicated, a particular nucleic acid sequence is also intended to encompass its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by creating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res.19:5081(1991); Ohtsuka et al., J.Biol.Chem.260:2605-2608(1985); Rossolini et al., Mol.Cell.Probes 8:91-98(1994)). As used herein, "nucleic acid" is also used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. As used herein, "nucleotide" may be natural or non-natural.

As used herein, "gene" refers to a factor that determines a genetic trait, and "gene" can refer to "polynucleotide," "oligonucleotide," and "nucleic acid."

As used herein, the term "homology" of a gene refers to the degree of identity between two or more gene sequences, and generally, "homology" refers to a high degree of identity or similarity. Thus, the higher the homology between two genes, the higher the identity or similarity of their sequences. Whether two genes have homology can be determined by direct comparison of the sequences or, in the case of nucleic acids, by hybridization methods under stringent conditions. When two gene sequences are compared directly, the genes are homologous if the DNA sequences between the gene 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% identical. Thus, as used herein, "homolog" or "homologous gene product" refers to a protein in another species, preferably a mammal, that exerts the same biological function as a protein component of a complex as further described herein. Such a homolog may also be referred to as an "orthologous gene product." It is understood that such homologs, homologous gene products, orthologous gene products, etc. can also be used as long as they meet the objectives of the present invention.

Amino acids may be referred to herein by either their commonly known three letter symbols or the one letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may likewise be referred to by their commonly accepted one letter codes. In this specification, comparisons of amino acid and base sequence similarity, identity and homology are calculated using the sequence analysis tool BLAST with default parameters. Identity searches can be performed, for example, using NCBI's BLAST 2.2.28 (published 2 April 2013). The identity value in this specification usually refers to the value obtained when aligned under default conditions using the above BLAST. However, if a higher value is obtained by changing the parameters, the highest value is regarded as the identity value. If identity is evaluated in multiple regions, the highest value among them is regarded as the identity value. Similarity is a value that takes into account similar amino acids in addition to identity.

In one embodiment of the present invention, "several" may be, for example, 10, 8, 6, 5, 4, 3, or 2, or any of these values or less. It is known that a polypeptide that has one or several amino acid residues deleted, added, inserted, or substituted with other amino acids maintains its biological activity (Mark et al., Proc Natl Acad Sci U S A. 1984 Sep; 81 (18): 5662-5666., Zoller et al., Nucleic Acids Res. 1982 Oct 25; 10 (20): 6487-6500., Wang et al., Science. 1984 Jun 29; 224 (4656): 1431-1433.). Antibodies with deletions or the like can be produced, for example, by site-directed mutagenesis, random mutagenesis, or biopanning using an antibody phage library. For example, the KOD-Plus- Mutagenesis Kit (TOYOBO CO., LTD.) can be used for site-specific mutagenesis. From mutant antibodies in which deletions or other modifications have been introduced, it is possible to select antibodies with the same activity as the wild type 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, or may be within a range between any two of these values. The above-mentioned "homology" may be calculated by calculating the ratio of the number of homologous amino acids in two or more amino acid sequences according to a method known in the art. Before calculating the ratio, the amino acid sequences of the amino acid sequences to be compared are aligned, and gaps are introduced into a part of the amino acid sequence if necessary to maximize the ratio of identical amino acids. Methods for alignment, methods for calculating the ratio, methods for comparison, and computer programs related thereto are well known in the art (e.g., BLAST, GENETYX, etc.). In this specification, "homology" can be expressed as a value measured by NCBI's BLAST unless otherwise specified. When comparing amino acid sequences with BLAST, Blastp can be used with default settings as an algorithm. The measurement results are quantified as Positives or Identities.

In this specification, "polynucleotides that hybridize under stringent conditions" refers to well-known conditions commonly used in the art. Such polynucleotides can be obtained by colony hybridization, plaque hybridization, Southern blot hybridization, or the like, using a polynucleotide selected from the polynucleotides of the present invention as a probe. Specifically, the term refers to a polynucleotide that can be identified by hybridizing a filter on which DNA derived from a colony or plaque is immobilized in the presence of 0.7 to 1.0 M NaCl at 65°C, and then washing the filter at 65°C using a 0.1 to 2x SSC (saline-sodium citrate) solution (the composition of a 1x SSC solution is 150 mM sodium chloride and 15 mM sodium citrate). For example, the following conditions can be adopted as "stringent conditions". (1) using low ionic strength and high temperature for washing (e.g., 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate (SDS) at 50°C), (2) using denaturing agents such as formamide during hybridization (e.g., 50% (v/v) formamide and 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM NaCl ... (3) incubate overnight at 37°C in a solution containing 20% formamide, 5x SSC, 50mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20mg/ml denatured sheared salmon sperm DNA, and then wash the filter with 1x SSC at about 37-50°C. The formamide concentration may be 50% or more. The wash time may be 5, 15, 30, 60, or 120 minutes or more. Several factors such as temperature and salt concentration may affect the stringency of the hybridization reaction, and details can be found in Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995). Examples of "highly stringent conditions" are 0.0015 M sodium chloride, 0.0015 M sodium citrate, 65-68°C, or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide, 42°C. Hybridization can be performed according to the method described in experimental textbooks such as Molecular Cloning 2nd ed., Current Protocols in Molecular Biology, Supplement 1-38, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995). Here, sequences that hybridize under stringent conditions preferably exclude sequences that contain only A sequences or only T sequences. Moderately stringent conditions can be easily determined by those skilled in the art based on, for example, the length of DNA, and are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Vol. 1, 7.42-7.45 Cold Spring Harbor Laboratory Press, 2001, and includes the use of a pre-wash solution of 5xSSC, 0.5% SDS, 1.0 mM ethylenediaminetetraacetic acid (EDTA) (pH 8.0) for nitrocellulose filters, hybridization conditions of about 50% formamide, 2xSSC-6xSSC (or other similar hybridization solutions such as Stark's solution in about 50% formamide at about 42°C) at about 40-50°C, and washing conditions of 0.5xSSC, 0.1% SDS at about 60°C. Thus, the polypeptides used in the present invention also include polypeptides encoded by nucleic acid molecules that hybridize under high or moderate stringency conditions to nucleic acid molecules encoding the polypeptides specifically described in the present invention.

As used herein, a "purified" substance or biological factor (e.g., nucleic acid or protein, etc.) refers to a substance or biological factor from which at least a portion of naturally associated factors have been removed. Thus, the purity of the biological factor in a purified biological factor is usually higher (i.e., concentrated) than the state in which the biological factor is normally present. As used herein, the term "purified" means that the same type of biological factor is present in an amount of 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. The substance or biological factor used in the present invention is preferably a "purified" substance. As used herein, an "isolated" substance or biological factor (e.g., nucleic acid or protein, etc.) refers to a substance or biological factor from which naturally associated factors have been substantially removed. The term "isolated" as used herein does not necessarily refer to purity, as this may vary depending on the purpose, but if necessary, preferably means that 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 of the same type of biological factor is present. The material used in the present invention is preferably an "isolated" material or biological factor.

As used herein, a "corresponding" amino acid or nucleic acid or portion refers to an amino acid or nucleotide in a polypeptide or polynucleotide molecule (e.g., CARD14) that has or is predicted to have the same action as a specific amino acid or nucleotide or portion in a polypeptide or polynucleotide that is used as a reference for comparison. In particular, in an enzyme molecule, it refers to an amino acid that is present at a similar position in the active site and contributes similarly to catalytic activity, and in a composite molecule, it refers to a corresponding portion (e.g., a transmembrane domain, etc.). For example, in the case of an antisense molecule, it may be a similar portion in an orthologue that corresponds to a specific portion of the antisense molecule. The corresponding amino acid may be, for example, a specific amino acid that is cysteinized, glutathionylated, S-S bond formed, oxidized (e.g., oxidation of the methionine side chain), formylated, acetylated, phosphorylated, glycosylated, myristylated, or the like. Alternatively, the corresponding amino acid may be an amino acid that is responsible for dimerization. Such a "corresponding" amino acid or nucleic acid may be a region or domain that spans a certain range. In such a case, it is referred to as a "corresponding" region or domain in this specification. Such corresponding regions or domains are useful in designing composite molecules in the present invention.

As used herein, a "corresponding" gene (e.g., a polynucleotide sequence or molecule) refers to a gene (e.g., a polynucleotide sequence or molecule) that has or is expected to have the same function in a certain species as a given gene in a species that is used as a reference for comparison, and when there are multiple genes with such function, it refers to those that have the same evolutionary origin. Thus, a gene that corresponds to a certain gene may be an ortholog of that gene. Thus, for each human CARD14, a corresponding CARD14 can be found in other animals (particularly mammals). Such corresponding genes can be identified using techniques well known in the art. Thus, for example, a corresponding gene in a certain animal (e.g., a mouse) can be found by searching a database containing the sequences of that animal using the sequence of a reference gene (e.g., CARD14, etc. (SEQ ID NO: 1, 3, etc. or SEQ ID NO: 2, 4, etc.) of the corresponding gene as a query sequence.

As used herein, a "fragment" refers to a polypeptide or polynucleotide having a sequence length of 1 to n-1 relative to the full-length polypeptide or polynucleotide (length n). The length of the fragment can be changed as appropriate depending on the purpose. For example, the lower limit of the length for a polypeptide can be 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or more amino acids, and lengths represented by integers not specifically listed here (e.g., 11, etc.) can also be suitable as the lower limit. In addition, for a polynucleotide, the lower limit can be 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, or more nucleotides, and lengths represented by integers not specifically listed here (e.g., 11, etc.) can also be suitable as the lower limit. In the present specification, such fragments are understood to be within the scope of the present invention, for example, if the full-length fragment functions as a marker, therapeutic agent, or target molecule, as long as the fragment itself also functions as a marker, therapeutic agent, or target molecule.

In accordance with the present invention, the term "activity" as used herein refers to the function of a molecule in its broadest sense. Activity generally includes, but is not intended to be limiting, the biological, biochemical, physical or chemical functions of a molecule. Activity includes, for example, enzymatic activity, the ability to interact with other molecules, and the ability to activate, promote, stabilize, inhibit, suppress or destabilize the function of other molecules, stability, ability to localize to a particular subcellular location. Where applicable, the term also relates to the function of protein complexes in their broadest sense.

As used herein, the term "biological function" refers to a specific function that a gene, a nucleic acid molecule, or a polypeptide related thereto may have in a living body, including, but not limited to, the production of specific antibodies, enzymatic activity, and the conferring of resistance. In the present invention, CARD14 belongs to the membrane-associated guanylate kinase (MAGUK) family and functions as a molecular scaffold that specializes in the plasma membrane region to form the assembly of a multiprotein complex, and is also known to belong to the CARD family, have a specific caspase-associated recruitment domain (CARD), and function as a positive regulator of specific interaction with BCL10, phosphorylation of BCL, NF-κB activation, and cell apoptosis, and is therefore related to these functions, but is not limited to these. As used herein, a biological function may be exerted by "biological activity". As used herein, the term "biological activity" refers to the activity that a certain factor (e.g., polynucleotide, protein, etc.) may have in a living body, and includes activities that exert various functions (e.g., transcription promoting activity), and also includes, for example, the activity of activating or inactivating a certain molecule by interacting with another molecule. When two factors interact with each other, the biological activity may be the binding between the two molecules and the biological change that occurs as a result. For example, when one molecule is precipitated with an antibody, the other molecule is also co-precipitated, and the two molecules are considered to be bound. Therefore, observing such co-precipitation is one method of judgment. For example, when a certain factor is an enzyme, its biological activity includes its enzymatic activity. In another example, when a certain factor is a ligand, the ligand includes the binding to the corresponding receptor. Such biological activity can be measured by techniques well known in the art. Thus, "activity" refers to various measurable indicators that indicate or reveal binding (either directly or indirectly); affect a response (i.e., have a measurable effect in response to some exposure or stimulus), such as the affinity of a compound to bind directly to a polypeptide or polynucleotide of the invention, or, for example, a measure of the amount of an upstream or downstream protein or other similar function after some stimulus or event.

As used herein, the "value" of CARD14 may be any direct or indirect value related to CARD14, such as the amount of CARD14 protein, the amount of mRNA expression, enzyme activity, etc. This "value" may be used in the present invention as an indicator of reducing complications of psoriasis, multiple sclerosis, γδ T cell mediated diseases and/or immune interventions, and/or side effects of TLR-based/IMQ/immunostimulatory adjuvants.

As used herein, the term "expression" of a gene, polynucleotide, polypeptide, etc. refers to the gene, etc. undergoing a certain action in vivo to become a different form. Preferably, the term refers to the gene, polynucleotide, etc. being transcribed and translated to become a polypeptide, but the transcription to produce mRNA is also an aspect of expression. Thus, as used herein, the term "expression product" includes such a polypeptide or protein, or mRNA. More preferably, such a polypeptide form may be one that has undergone post-translational processing. For example, the expression level of CARD14 can be determined by any method. Specifically, the expression level of CARD14 can be known by evaluating the amount of CARD14 mRNA, the amount of CARD14 protein, and the biological activity of CARD14 protein. Such measurements can be used in companion diagnostics. The amount of CARD14 mRNA or protein can be determined by methods detailed elsewhere in this specification or other methods known in the art.

As used herein, the term "functional equivalent" refers to any entity that has the same intended function but a different structure compared to the original entity of interest. Therefore, it is understood that the functional equivalent of "CARD14" or its antibody is not CARD14 or its antibody itself, but includes mutants or variants of CARD14 or its antibody (e.g., amino acid sequence variants, etc.) that have the biological action of CARD14, as well as those that can be changed to CARD14 or its antibody itself or mutants or variants of this CARD14 or its antibody at the time of acting (e.g., nucleic acid encoding CARD14 or its antibody itself or mutants or variants of CARD14 or its antibody, and vectors, cells, etc. containing the nucleic acid). In the present invention, it is understood that the functional equivalent of CARD14 or its antibody can be used in the same way as CARD14 or its antibody, even if not specifically mentioned. Functional equivalents can be found by searching databases, etc. As used herein, the term "search" refers to the use of a nucleic acid sequence to find other nucleic acid sequences having a particular function and/or property, either electronically or by biological or other methods. Examples of electronic searches include BLAST (Altschul et al., J. Mol. Biol. 215:403-410(1990)), FASTA (Pearson & Lipman, Proc. Natl. Acad. Sci., USA 85: 2444-2448(1988)), Smith and Waterman method (Smith
and Waterman, J. Mol. Biol. 147:195-197(1981)), and Needleman and Wunsch method (Needleman and Wunsch, J. Mol. Biol. 48:443-453(1970)). Biological searches include, but are not limited to, stringent hybridization, macroarrays in which genomic DNA is attached to a nylon membrane or the like, or microarrays in which genomic DNA is attached to a glass plate (microarray assay), PCR, and in situ hybridization. In this specification, it is intended that the genes used in the present invention should also include corresponding genes identified by such electronic searches and biological searches.

As the functional equivalent of the present invention, an amino acid sequence in which one or more amino acids have been inserted, substituted or deleted, or added to one or both ends thereof can be used. In this specification, "an amino acid sequence in which one or more amino acids have been inserted, substituted or deleted, or added to one or both ends thereof" means that the amino acid sequence has been modified by a well-known technical method such as site-directed mutagenesis, or by natural mutation, by substitution of a number of amino acids to the extent that may occur naturally. The modified amino acid sequence may be, for example, an amino acid sequence in which 1 to 30, preferably 1 to 20, more preferably 1 to 9, even more preferably 1 to 5, and particularly preferably 1 to 2 amino acids have been inserted, substituted or deleted, or added to one or both ends thereof. The modified amino acid sequence may preferably be an amino acid sequence having one or more (preferably one or several, or 1, 2, 3, or 4) conservative substitutions in the amino acid sequence of CARD14. Here, "conservative substitution" means replacing 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, a hydrophobic residue may be replaced with another hydrophobic residue, or a polar residue may be replaced with another polar residue having the same charge. Functionally similar amino acids that can be used for such substitutions are known in the art for each amino acid. Specific examples of non-polar (hydrophobic) amino acids include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Positively charged (basic) amino acids include arginine, histidine, and lysine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

As used herein, "inhibitor" or "suppressant" or "inhibitor" (all of which correspond to the English term inhibitor) refers to a substance or factor that inhibits the biological action of a target entity (e.g., a receptor or a cell) against that receptor or cell. The CARD14 inhibitor of the present invention includes factors that can temporarily or permanently reduce or eliminate the action or function of the target CARD14 or cells expressing CARD14. Such factors include, but are not limited to, antibodies, antigen-binding fragments thereof, derivatives thereof, functional equivalents thereof, antisense, and nucleic acid forms such as RNAi factors such as siRNA.

In this specification, the term "agonist" refers to a substance that exerts or enhances the biological action of a receptor on a target entity (e.g., a receptor). In addition to natural agonists (also called ligands), synthetic or modified agonists can be used. In this specification, the term "antagonist" refers to a substance that suppresses or inhibits the expression of a biological action of a receptor on a target entity (e.g., a receptor). In addition to natural antagonists, synthetic or modified agonists can be used. In addition to those that competitively suppress or inhibit with agonists (or ligands), there are also those that suppress or inhibit noncompetitively. They can also be obtained by modifying agonists. Since antagonists suppress or inhibit physiological phenomena, they can be included in the concept of inhibitors (suppressing agents or inhibitors) or inhibitory or inhibiting factors. Therefore, in this specification, antagonists are essentially used synonymously with "inhibitors."

In the present specification, the term "antibody" broadly includes polyclonal antibodies, monoclonal antibodies, multispecific antibodies, chimeric antibodies, and anti-idiotypic antibodies, as well as fragments thereof, such as Fv fragments, Fab' fragments, F(ab')2 and Fab fragments, and other recombinantly produced conjugates or functional equivalents (e.g., chimeric antibodies, humanized antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single chain antibodies, scFV, diabodies, sc(Fv)2 (single chain (Fv)2), scFv-Fc). Furthermore, such antibodies may be covalently linked or recombinantly fused to enzymes, such as alkaline phosphatase, horseradish peroxidase, α-galactosidase, etc. The anti-CARD14 antibody used in the present invention may be of any origin, type, shape, etc., as long as it binds to the CARD14 protein. Specifically, known antibodies such as non-human animal antibodies (e.g., mouse antibodies, rat antibodies, camel antibodies), human antibodies, chimeric antibodies, and humanized antibodies can be used. In the present invention, monoclonal or polyclonal antibodies can be used as the antibodies, but monoclonal antibodies are preferred. The antibodies preferably bind specifically to the CARD14 protein. The antibodies include modified and unmodified antibodies. The modified antibodies may be antibodies bound to various molecules such as polyethylene glycol. The modified antibodies can be obtained by chemically modifying the antibodies using known techniques.

In one embodiment of the present invention, a "polyclonal antibody" can be produced, for example, by administering an immunogen containing an antigen of interest to a mammal (e.g., rat, mouse, rabbit, cow, monkey, etc.), bird, etc., to induce production of polyclonal antibodies specific to the antigen. The administration of the immunogen may be followed by injection of one or more immunizing agents and, if desired, an adjuvant. Adjuvants may be used to increase the immune response and may include Freund's adjuvant (complete or incomplete), mineral gels (e.g., aluminum hydroxide), or surfactants (e.g., lysolecithin), etc. Immunization protocols are known in the art and may be performed by any method that induces an immune response, tailored to the host organism of choice (Protein Experiment Handbook, Yodosha (2003):86-91.).

In one embodiment of the present invention, a "monoclonal antibody" includes a population of antibodies that correspond to a substantially single epitope, except for antibodies with a small amount of naturally occurring mutations. Alternatively, the population of antibodies may be substantially identical, except for antibodies with a small amount of naturally occurring mutations. Monoclonal antibodies are highly specific and differ from conventional polyclonal antibodies, which typically contain different antibodies that correspond to different epitopes. In addition to their specificity, monoclonal antibodies are useful in that they can be synthesized from hybridoma cultures that are not contaminated by other immunoglobulins. The term "monoclonal" may indicate the characteristic of being obtained from a substantially homogeneous antibody population, but does not imply that the antibody must be produced by any particular method. For example, monoclonal antibodies may be produced by methods similar to the hybridoma method described in "Kohler G, Milstein C., Nature. 1975 Aug 7;256(5517):495-497." Alternatively, monoclonal antibodies may be produced by recombinant methods similar to those described in U.S. Pat. No. 4,816,567. Alternatively, monoclonal antibodies may be isolated from phage antibody libraries using techniques similar to those described in "Clackson et al., Nature. 1991 Aug 15;352(6336):624-628," or "Marks et al., J Mol Biol. 1991 Dec 5;222(3):581-597." Alternatively, monoclonal antibodies may be produced by methods described in "Protein Experiment Handbook, Yodosha (2003):92-96."

In one embodiment of the present invention, a "humanized antibody" is an antibody that has, for example, one or more CDRs from a non-human species, a framework region (FR) from a human immunoglobulin, and a constant region from a human immunoglobulin, and that binds to a desired antigen. Antibody humanization can be performed using a variety of techniques known in the art (Almagro et al., FRont Biosci. 2008 Jan 1;13:1619-1633.). For example, CDR grafting (Ozaki et al., Blood. 1999 Jun 1;93(11):3922-3930.), Re-surfacing (roguska et al., Proc Natl Acad Sci U S A. 1994 Feb 1;91(3):969-973.), or FR shuffling (Damschroder et al., Mol Immunol. 2007 Apr;44(11):3049-3060. Epub 2007 Jan 22.) can be used. To alter (preferably improve) antigen binding, amino acid residues in the human FR region may be replaced with corresponding residues from the CDR donor antibody. This FR replacement can be performed by methods well known in the art (Riechmann et al., Nature. 1988 Mar 24;332(6162):323-327.). For example, FR residues important for antigen binding may be identified by modeling the interactions of CDR and FR residues, or unusual FR residues at specific positions may be identified by sequence comparison.

In one embodiment of the present invention, a "human antibody" is an antibody whose heavy chain variable and constant regions and light chain variable and constant regions are derived from genes encoding human immunoglobulin. Major methods for producing human antibodies include the transgenic mouse method for producing human antibodies and the phage display method. In the transgenic mouse method for producing human antibodies, a functional human Ig gene is introduced into a mouse in which endogenous Ig has been knocked out, and human antibodies with diverse antigen-binding abilities are produced instead of mouse antibodies. Furthermore, by immunizing this mouse, it is possible to obtain human monoclonal antibodies using the conventional hybridoma method. For example, the antibody can be produced by the method described in "Lonberg et al., Int Rev Immunol. 1995;13(1):65-93." The phage display method is a system in which a foreign gene is expressed as a fusion protein on the N-terminus of a coat protein (g3p, g10p, etc.) of a filamentous phage, such as M13 or T7, which is typically an E. coli virus, so as not to lose the infectivity of the phage. For example, it can be produced by the method described in "Vaughan et al., Nat Biotechnol. 1996 Mar;14(3):309-314."

In one embodiment of the present invention, an "anti-CARD14 antibody" includes an antibody that has binding ability to CARD14. The method for producing this anti-CARD14 antibody is not particularly limited, but it may be produced, for example, by immunizing a mammal or bird with CARD14.

It is understood that the "functional equivalent" of an "antibody against CARD14 (anti-CARD14 antibody) or a fragment thereof" includes, for example, in the case of an antibody, the antibody itself and a fragment thereof that have the binding activity and, if necessary, the inhibitory activity of CARD14, as well as chimeric antibodies, humanized antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single-chain antibodies, scFVs, diabodies, sc(Fv)2 (single chain (Fv)2), scFv-Fc, and the like.

In one embodiment of the present invention, from the viewpoint of particularly strong suppression of a disease, the anti-CARD14 antibody is preferably an anti-CARD14 antibody that specifically binds to a specific epitope of CARD14.

The anti-CARD14 antibody according to one embodiment of the present invention may be a monoclonal antibody. A monoclonal antibody can act on CARD14 more efficiently than a polyclonal antibody. From the viewpoint of efficiently producing an anti-CARD14 monoclonal antibody, it is preferable to immunize chickens with CARD14.

The antibody class of the anti-CARD14 antibody according to one embodiment of the present invention is not particularly limited, but may be, for example, IgM, IgD, IgG, IgA, IgE, or IgY.

The anti-CARD14 antibody according to one embodiment of the present invention may be an antibody fragment having antigen-binding activity (hereinafter, also 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 one in which a mouse, human, or chicken antibody partial sequence is fused. Such fusion proteins are also included in one form of the anti-CARD14 antibody according to this embodiment.

An anti-CARD14 antibody according to one embodiment of the present invention may be, for example, an antibody obtained through a process of immunizing an organism with purified CARD14, CARD14-expressing cells, or a CARD14-containing lipid membrane. From the viewpoint of enhancing the therapeutic effect against CARD14-positive diseases, it is preferable to use CARD14-expressing cells for immunization.

An anti-CARD14 antibody according to one embodiment of the present invention may be an antibody having a CDR set of an antibody obtained through a process of immunizing an organism with purified CARD14, CARD14-expressing cell cells, or a CARD14-containing lipid membrane. From the viewpoint of enhancing the therapeutic effect against CARD14-positive diseases, it is preferable to use CARD14-expressing cells for immunization. The CDR set is a set of heavy chain CDR1, 2, and 3, and light chain CDR1, 2, and 3.

The anti-CARD14 antibody according to one embodiment of the present invention may have any binding strength as long as the objective is achieved, and examples thereof include, but are not limited to, at least 1.0× ¹⁰ or more, 2.0× ¹⁰ or more, 5.0× ¹⁰ or more, and 1.0× ¹⁰ or more, and typically may have a dissociation constant (KD) value of 1.0× ¹⁰ or more.

An anti-CARD14 antibody according to one embodiment of the present invention may be an antibody that binds to wild-type or mutant CARD14. Mutant types include those resulting from differences in DNA sequences between individuals. The amino acid sequence of wild-type or mutant CARD14 has a homology of preferably 80% or more, more preferably 90% or more, more preferably 95% or more, and particularly preferably 98% or more to the amino acid sequence shown in SEQ ID NO: 2 or 4.

In one embodiment of the present invention, the "CDR (complementarity determining region)" is a region of an antibody that actually contacts an antigen to form a binding site. Generally, the CDR is located on the Fv (variable region: including the heavy chain variable region (VH) and the light chain variable region (VL)) of an antibody. Generally, the CDR includes CDR1, CDR2, and CDR3, each of which is composed of about 5 to 30 amino acid residues. It is known that the CDR of the heavy chain in particular contributes to the binding of the antibody to the antigen. It is also known that, among the CDRs, CDR3 contributes most to the binding of the antibody to the antigen. For example, "Willy et al., Biochemical and Biophysical Research Communications Volume 356, Issue 1, 27 April 2007, Pages 124-128" describes that the binding ability of an antibody was increased by modifying the heavy chain CDR3. The Fv region other than the CDR is called the framework region (FR), which consists of FR1, FR2, FR3, and FR4, and is relatively well conserved among antibodies (Kabat et al., "Sequence of Proteins of Immunological Interest", US Dept. Health and Human Services, 1983.). In other words, the factors that characterize the reactivity of an antibody are the CDRs.
and in particular in the heavy chain CDRs.

Several methods for determining the definition and location of CDRs have been reported. For example, the Kabat definition (Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) or Chothia's definition (Chothia et al., J. Mol. Biol., 1987; 196: 901-917) may be adopted. In one embodiment of the present invention, the Kabat definition is adopted as a preferred example, but is not necessarily limited to this. In some cases, the definition may be determined taking into consideration both the Kabat definition and the Chothia definition. For example, the overlapping portion of CDRs according to each definition, or a portion including both CDRs according to each definition, may be regarded as a CDR. A specific example of such a method is the method of Martin et al. (Proc. Natl. Acad. Sci. USA, 1989;86:9268-9272) using Oxford Molecular's AbM antibody modeling software, which is a compromise between the Kabat and Chothia definitions. Such CDR information can be used to produce mutants that can be used in the present invention. Such antibody mutants can be produced that contain one or several (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) substitutions, additions, or deletions in the framework of the original antibody, but do not contain mutations in the CDRs.

As used herein, "antigen" refers to any substance that can be specifically bound by an antibody molecule. As used herein, "immunogen" refers to an antigen that can initiate lymphocyte activation resulting in an antigen-specific immune response. As used herein, "epitope" or "antigenic determinant" refers to a site in an antigen molecule to which an antibody or lymphocyte receptor binds. Methods for determining epitopes are well known in the art, and such epitopes can be determined by one of skill in the art using such well-known and conventional techniques when provided with the primary sequence of nucleic acid or amino acid. It is understood that the antibodies of the present invention can be similarly utilized with antibodies having other sequences, so long as the epitopes are the same.

It is understood that the antibodies used in this specification may have any specificity as long as undesirable effects such as side reactions are reduced. Therefore, the antibodies used in the present invention may be polyclonal or monoclonal antibodies.

As used herein, the term "subject" refers to an object that is the subject of diagnosis, detection, or treatment of the present invention (e.g., an organism such as a human, or cells, blood, serum, etc. extracted from an organism).

In the present specification, the terms "drug", "agent" or "factor" (each of which corresponds to the English term agent) are used interchangeably in a broad sense and may refer to any substance or other element (e.g., energy such as light, radioactivity, heat, electricity, etc.) as long as it can achieve the intended purpose. Examples of such substances include, but are not limited to, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (e.g., DNA such as cDNA and genomic DNA, and RNA such as mRNA), polysaccharides, oligosaccharides, lipids, organic small molecules (e.g., hormones, ligands, signaling substances, organic small molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (e.g., small molecule ligands, etc.)), and composite molecules thereof. Representative examples of factors specific to a polynucleotide include, but are not limited to, polynucleotides having a certain sequence homology (e.g., 70% or more sequence identity) with respect to the sequence of the polynucleotide and polypeptides such as transcription factors that bind to promoter regions. Agents specific for a polypeptide typically include, but are not limited to, an antibody or a derivative or analog thereof (e.g., a single-chain antibody) specifically directed against the polypeptide, a specific ligand or receptor when the polypeptide is a receptor or ligand, and a substrate when the polypeptide is an enzyme.

As used herein, "treatment" refers to preventing, preferably maintaining, more preferably alleviating, and even more preferably eradicating a disease or disorder (e.g., allergy) from worsening when that condition occurs, and includes exerting a symptom-improving or preventive effect on the patient's disease or one or more symptoms associated with the disease. Preliminary diagnosis followed by appropriate treatment is called "companion treatment," and diagnostic agents for this purpose are sometimes called "companion diagnostic agents."

In this specification, the term "therapeutic agent" refers broadly to any drug capable of treating a target condition (e.g., a disease such as allergy). In one embodiment of the present invention, the "therapeutic agent" may be a pharmaceutical composition containing an active ingredient and one or more pharmacologically acceptable carriers. The pharmaceutical composition may be produced, for example, by mixing the active ingredient with the carrier and using any method known in the technical field of pharmaceutical formulations. The therapeutic agent may be in any form that is used for treatment, and may be the active ingredient alone or a mixture of the active ingredient and any other ingredient. The shape of the carrier is not particularly limited, and may be, for example, a solid or liquid (e.g., a buffer solution). The therapeutic agent for allergies includes drugs used for preventing allergies (prophylactic drugs) or allergy suppressants.

As used herein, "prevention" refers to preventing a certain disease or disorder (e.g., allergy) from occurring before that state occurs. Diagnosis can be performed using the agent of the present invention, and, if necessary, the agent of the present invention can be used to prevent, for example, allergies, or preventive measures can be taken.

In this specification, the term "prophylactic drug (agent)" refers broadly to any drug that can prevent a target condition (e.g., a disease such as an allergy).

Thus, as used herein, an "agent" (or drug, detection agent, etc.) that "specifically" interacts with (or binds to) a biological agent such as a polynucleotide or polypeptide includes one whose affinity for the biological agent, such as the polynucleotide or polypeptide, is typically equal to or higher than, and preferably significantly (e.g., statistically significantly) higher than its affinity for other unrelated polynucleotides or polypeptides (e.g., those with less than 30% identity). Such affinity can be measured, for example, by hybridization assays, binding assays, etc.

As used herein, a first substance or factor "specifically" interacts with (or binds to) a second substance or factor means that the first substance or factor interacts with (or binds to) the second substance or factor with a higher affinity than it does with substances or factors other than the second substance or factor (particularly other substances or factors present in a sample containing the second substance or factor). Examples of specific interactions (or binding) for a substance or factor include, but are not limited to, hybridization in nucleic acids, antigen-antibody reactions in proteins, enzyme-substrate reactions, and other reactions between nucleic acids and proteins, protein-lipid interactions, and nucleic acid-lipid interactions. Thus, when both substances or factors are nucleic acids, "specifically interacting" with a first substance or factor includes the first substance or factor having at least partial complementarity to the second substance or factor. For example, when both substances or factors are proteins, examples of "specific" interaction (or binding) of a first substance or factor with a second substance or factor include, but are not limited to, interactions due to antigen-antibody reactions, interactions due to receptor-ligand reactions, and enzyme-substrate interactions. When two types of substances or factors include proteins and nucleic acids, "specific" interaction (or binding) of a first substance or factor with a second substance or factor includes interactions (or binding) between an antibody and its antigen. By utilizing such specific interactions or binding reactions, it is possible to detect or quantify a target substance in a sample.

As used herein, "detection" or "quantification" of polynucleotide or polypeptide expression may be accomplished using any suitable method, including, for example, measurement of mRNA and immunological measurement methods, including binding or interaction with a detection agent, testing agent, or diagnostic agent. Examples of molecular biological measurement methods include, for example, Northern blot, dot blot, or PCR. Examples of immunological measurement methods include, for example, ELISA using microtiter plates, radioimmunoassay (RIA), fluorescent antibody method, luminescence immunoassay (LIA), immunoprecipitation (IP), immunodiffusion (SRID), immunoturbidimetric (TIA), Western blot, and immunohistochemical staining. Examples of quantification methods include ELISA or RIA. Genetic analysis may also be performed using arrays (e.g., DNA arrays, protein arrays). DNA arrays are broadly reviewed in "DNA Microarrays and the Latest PCR Methods," a special edition of Cell Engineering, edited by Shujunsha. Protein arrays are reviewed in Nat Genet. 2002 Dec;32 Suppl:526-532. In addition to the above, gene expression analysis methods include, but are not limited to, RT-PCR, RACE, SSCP, immunoprecipitation, two-hybrid systems, in vitro translation, and the like. Such further analysis methods are described, for example, in Genome Analysis Experimental Methods: Nakamura Yusuke Lab Manual, edited by Nakamura Yusuke Yodosha (2002), and all descriptions therein are incorporated by reference.

As used herein, "expression level" refers to the amount of a polypeptide or mRNA, etc., expressed in a cell, tissue, etc. of interest. Examples of such expression level include the expression level of the polypeptide of the present invention at the protein level, evaluated by any appropriate method, including immunological measurement methods such as ELISA, RIA, fluorescent antibody method, Western blotting, and immunohistochemical staining, using the antibody of the present invention, or the expression level of the polypeptide used in the present invention at the mRNA level, evaluated by any appropriate method, including molecular biological measurement methods such as Northern blotting, dot blotting, and PCR. "Change in expression level" means an increase or decrease in the expression level of the polypeptide used in the present invention at the protein level or mRNA level, evaluated by any appropriate method, including the immunological measurement method or molecular biological measurement method. By measuring the expression level of a certain marker, various detections or diagnoses based on the marker can be performed.

As used herein, "reduction" or "suppression" of an activity or expression product (e.g., protein, transcript (RNA, etc.)) or synonyms thereof refers to a decrease in the amount, quality, or effect of a particular activity, transcript, or protein, or an activity that is reduced. When "elimination" is used as a reduction, it refers to an activity, expression product, etc. becoming below the detection limit, and is sometimes specifically referred to as "elimination." As used herein, "elimination" is encompassed by "reduction" or "suppression."

As used herein, "increase" or "activation" of an activity or expression product (e.g., protein, transcript (e.g., RNA, etc.)) or synonyms thereof refers to an increase or increasing activity in the amount, quality, or effect of a particular activity, transcript, or protein.

In this specification, the term "label" refers to an entity (e.g., substance, energy, electromagnetic wave, etc.) that distinguishes a target molecule or substance from others. Examples of such labeling methods include RI (radioisotope) method, fluorescence method, biotin method, chemiluminescence method, etc. When a plurality of markers of the present invention or factors or means for capturing them are labeled by a fluorescence method, the labels are made with fluorescent substances having different maximum fluorescence emission wavelengths. The difference in maximum fluorescence emission wavelength is preferably 10 nm or more. When labeling a ligand, any substance that does not affect the function can be used, but Alexa ^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 corresponds to a wide range of fluorescent wavelengths. It is very stable, bright, and has low pH sensitivity compared to other fluorescent dyes of the corresponding wavelengths. Examples of combinations of fluorescent dyes having a maximum fluorescence wavelength of 10 nm or more include a combination of Alexa ^TM 555 and Alexa ^TM 633, and a combination of Alexa ^TM 488 and Alexa ^TM 555. When labeling a nucleic acid, any dye that can bind to the base moiety can be used, but it is preferable to use cyanine dyes (e.g., Cy3 and Cy5 of the CyDyeTM series), rhodamine 6G reagent, 2-acetylaminofluorene (AAF), AAIF (iodine derivative of AAF), etc. Examples of fluorescent substances having a difference in maximum fluorescence wavelength of 10 nm or more include a combination of Cy5 and rhodamine 6G reagent, a combination of Cy3 and fluorescein, and a combination of rhodamine 6G reagent and fluorescein. In the present invention, such labels 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 carry out such methods appropriately depending on the label and the target object.

As used herein, the term "kit" refers to a unit in which the parts to be provided (e.g., test agents, diagnostic agents, therapeutic agents, antibodies, labels, instructions, etc.) are provided, usually in two or more compartments. This kit form is preferred when the purpose is to provide a composition that should not be provided in a mixed state for reasons of stability, etc., but is preferably mixed immediately before use. Such a kit is advantageously provided with instructions or instructions that preferably describe how to use the parts to be provided (e.g., test agents, diagnostic agents, therapeutic agents, etc.) or how to handle the reagents. When the kit is used as a reagent kit in this specification, the kit usually includes instructions, etc., that describe how to use the test agents, diagnostic agents, therapeutic agents, antibodies, etc.

In this specification, "instructions" refers to instructions for a physician or other user on how to use the present invention. The instructions include instructions on how to use the detection method of the present invention, how to use a diagnostic agent, or how to administer a medicine or the like. The instructions may also include instructions on the site of administration, such as oral or esophageal administration (e.g., by injection). The instructions are prepared in accordance with a format prescribed by the regulatory agency of the country in which the present invention is implemented (e.g., the Ministry of Health, Labor and Welfare in Japan, the Food and Drug Administration (FDA) in the United States, etc.), and clearly state that they have been approved by the regulatory agency. The instructions are so-called package inserts, and are usually provided in paper form, but are not limited thereto, and may also be provided in the form of electronic media (e.g., a homepage provided on the Internet, e-mail, etc.).

(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 the function, etc. Therefore, the "CARD14 inhibitor" includes any substance that inhibits the expression of CARD14 and the function of CARD14, as well as any substance that ultimately inhibits the action of CARD14 even if the mechanism of action is unknown, and may be, but is not limited to, an antibody, an antibody fragment, a derivative thereof, a nucleic acid, a small molecule, or other antagonist. Such various antibodies, etc. are described elsewhere in this specification. Preferably, such a CARD14 inhibitor is advantageously one that specifically inhibits CARD14. This is because it can reduce side effects caused by effects on factors other than CARD14.

In the present invention, a "substance that inhibits the expression of CARD14" may be one that acts at any stage, such as the transcription level of a nucleic acid (CARD14 gene) encoding CARD14, the level of post-transcriptional regulation, the level of translation into CARD14 protein, or the level of post-translational modification. Therefore, examples of substances that inhibit the expression of CARD14 include substances that inhibit the transcription of the CARD14 gene (e.g., antigene), substances that inhibit the processing of an initial transcription product into mRNA, substances that inhibit the transport of mRNA into the cytoplasm, substances that inhibit the translation of CARD14 from mRNA (e.g., antisense nucleic acid, miRNA) or substances that degrade mRNA (e.g., siRNA, ribozyme), and substances that inhibit the post-translational modification of an initial translation product. Any substance that acts at any stage can be preferably used.

A preferred example of a transcription product here is mRNA.

Preferably, the substance that specifically inhibits the translation of the CARD14 gene mRNA into CARD14 (or degrades the mRNA) is a nucleic acid that contains a base sequence that is complementary or substantially complementary to the base sequence of these mRNAs, or a part of the base sequence.

A base sequence substantially complementary to the base sequence of the mRNA of the CARD14 gene means a base sequence having such a degree of complementarity that it can bind to a target sequence of the mRNA and inhibit its translation (or cleave the target sequence) under physiological conditions of CARD14-producing cells (e.g., neutrophils) in a mammal to which the compound is administered, and specifically means, for example, a base sequence having a homology of about 90% or more, preferably about 95% or more, more preferably about 97% or more with a base sequence completely complementary to the base sequence of the mRNA (i.e., the base sequence of the complementary strand of the mRNA) in the overlapping region. The "homology of base sequences" in the present invention can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expectation value=10; gaps allowed; filtering=ON; match score=1; mismatch score=-3).

More specifically, examples of base sequences that are complementary or substantially complementary to the base sequence of the mRNA of the CARD14 gene include the following (k) or (l): (k) a base sequence that is complementary or substantially complementary to the base sequence represented by SEQ ID NO: 1 or 3; (l) a base sequence that hybridizes under stringent conditions with the complementary strand sequence of the base sequence represented by SEQ ID NO: 1 or 3 and is complementary or substantially complementary to a sequence that encodes a protein having at least one function of CARD14. The stringent conditions are as described above. The functions of CARD14 include, but are not limited to, the function of membrane-associated guanylate kinase (MAGUK), the function of acting as a molecular scaffold for assembling multiple protein complexes in specific regions of the plasma membrane, the function of interacting with BCL10 and the caspase-associated recruitment function due to the presence of a caspase-associated recruitment domain, the function of acting as a positive regulator of NF-κB activation and cell apoptosis, the function of inducing NF-κB activation and phosphorylation of BCL10, etc.

Preferred examples of the mRNA of the CARD14 gene include the mRNA of human CARD14 containing the base sequence represented by SEQ ID NO:1 (Genbank Accession No. NM_052972), or orthologs thereof in other mammals (e.g., mouse CARD14 (SEQ ID NO:3, Genbank Accession No. NM_029796), etc.), as well as splice variants, allelic mutants, polymorphic variants, etc. thereof.

The "part of a base sequence complementary or substantially complementary" to the base sequence of the mRNA of the CARD14 gene is not particularly limited in length or position, so long as it can specifically bind to the mRNA of the CARD14 gene and inhibit protein translation from the mRNA (or degrade the mRNA), but in terms of sequence specificity, it contains a part that is complementary or substantially complementary to the target sequence, having at least 10 bases, preferably about 15 bases or more.

Specifically, preferred examples of nucleic acids containing a base sequence complementary or substantially complementary to the base sequence of mRNA of CARD14 gene, or a part thereof, include any of the following (1) to (3):
(1) an antisense nucleic acid against the mRNA of the CARD14 gene;
(2) a ribozyme nucleic acid for the mRNA of the CARD14 gene;
(3) A nucleic acid or a precursor thereof having RNAi activity against the mRNA of the CARD14 gene.

(1) Antisense nucleic acid against mRNA of CARD14 gene In the present invention, an "antisense nucleic acid against mRNA of CARD14 gene" is a nucleic acid containing a base sequence complementary or substantially complementary to the base sequence of the mRNA, or a part thereof, and has the function of inhibiting protein synthesis by binding to the target mRNA by forming a specific and stable double strand.

Antisense nucleic acids include polydeoxyribonucleotides containing 2-deoxy-D-ribose, polyribonucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, other polymers with non-nucleotide backbones (e.g., commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special linkages, provided that the polymers contain nucleotides with configurations that allow base pairing or base attachment as found in DNA and RNA. They may be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, DNA:RNA hybrids, and may also be unmodified polynucleotides (or unmodified oligonucleotides), those with known modifications, such as those labeled, capped, or methylated, those with one or more natural nucleotides replaced with analogs, those with intramolecular nucleotide modifications, such as those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), those with charged linkages or sulfur. The nucleic acid may have a linkage (e.g., phosphorothioate, phosphorodithioate, etc.), a side group such as a protein (e.g., nuclease, nuclease inhibitor, toxin, antibody, signal peptide, poly-L-lysine, etc.) or a sugar (e.g., monosaccharide, etc.), an intercurrent compound (e.g., acridine, psoralen, etc.), a chelator (e.g., metal, radioactive metal, boron, oxidizing metal, etc.), an alkylator, or a modified linkage (e.g., α-anomeric nucleic acid, etc.). As used herein, the terms "nucleoside", "nucleotide", and "nucleic acid" include those containing not only purine and pyrimidine bases, but also those containing other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleosides and modified nucleotides may also have sugar moieties modified, for example, one or more hydroxyl groups may be replaced with halogens, aliphatic groups, or converted to functional groups such as ethers or amines.

As described above, the antisense nucleic acid may be DNA or RNA, or may be a DNA/RNA chimera. When the antisense nucleic acid is DNA, the RNA:DNA hybrid formed by the target RNA and the antisense DNA can be recognized by endogenous RNase H to cause selective degradation of the target RNA. Therefore, in the case of antisense DNA that directs degradation by RNase H, the target sequence may be not only a sequence in the mRNA, but also a sequence of an intron region in the initial translation product 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 target region of the antisense nucleic acid of the present invention is not particularly limited in length as long as the translation into the protein CARD14 is inhibited by hybridization of the antisense nucleic acid, and may be the entire sequence or a partial sequence of the mRNA encoding CARD14, and may be as short as about 10 bases or as long as the entire sequence of the mRNA or initial transcription product. Considering the ease of synthesis, antigenicity, intracellular transport problems, etc., an oligonucleotide consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases, is preferred, but is not limited thereto. Specifically, the 5'-end hairpin loop, 5'-end 6-base pair repeat, 5'-end untranslated region, translation initiation codon, protein coding region, open reading frame (ORF) translation termination codon, 3'-end untranslated region, 3'-end palindrome region, or 3'-end hairpin loop of the CARD14 gene may be selected as a preferred target region of the antisense nucleic acid, but is not limited thereto.

Furthermore, the antisense nucleic acid of the present invention may not only hybridize with the mRNA or initial transcription product of the CARD14 gene to inhibit translation into protein, but may also bind to these genes, which are double-stranded DNA, to form a triplex and inhibit transcription into RNA (antigene).

The nucleotide molecules constituting the antisense nucleic acid may be natural DNA or RNA, but may contain various chemical modifications to improve stability (chemical and/or enzymatic) and specific activity (affinity with RNA). For example, in order to prevent degradation by hydrolases such as nucleases, the phosphoric acid residues (phosphates) of each nucleotide constituting the antisense nucleic acid may be replaced with chemically modified phosphoric acid residues such as phosphorothioate (PS), methylphosphonate, and phosphorodithioate. In addition, the hydroxyl group at the 2'-position of the sugar (ribose) of each nucleotide may be replaced with -OR (R=CH ₃ (2'-O-Me), CH ₂ CH ₂ OCH ₃ (2'-O-MOE), CH ₂ CH ₂ NHC(NH)NH ₂ , CH ₂ CONHCH ₃ , CH ₂ CH ₂ CN, etc.). Furthermore, the base portion (pyrimidine, purine) may be chemically modified, for example by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base, or by substituting a thiocarbonyl group for the carbonyl group at the 2-position.

The sugar conformation of RNA is predominantly C2'-endo (S type) and C3'-endo (N type), and in single-stranded RNA, these two conformations exist in equilibrium, but when double-stranded RNA is formed, it is fixed to the N type. Therefore, in order to impart strong binding ability to target RNA, bridged nucleic acid (BNA) (locked nucleic acid (LNA)) (Imanishi, T. et al., Chem. Commun., 1653-9, 2002; Jepsen, JS et al., Oligonucleotides, 14, 130-46, 2004) and ENA (Morita, K. et al., Nucleosides Nucleotides Nucleic Acids, 22, 1619-21, 2003), which are RNA derivatives in which the conformation of the sugar moiety is fixed to the N type by bridging the 2' oxygen and 4' carbon, may also be preferably used.

The antisense oligonucleotide of the present invention can be prepared by determining the target sequence of an mRNA or an initial transcription product based on the cDNA sequence or genomic DNA sequence of the CARD14 gene, and synthesizing a complementary sequence thereto using a commercially available automated DNA/RNA synthesizer (Applied Biosystems, Beckman, etc.). In addition, the antisense nucleic acids containing the various modifications described above can also be chemically synthesized by known methods.

(2) Ribozyme nucleic acid for mRNA of CARD14 gene Other preferred examples of nucleic acids containing a base sequence complementary or substantially complementary to the base sequence of mRNA of CARD14 gene or a part thereof include ribozyme nucleic acids capable of specifically cleaving the mRNA within the coding region. In the narrow sense, "ribozyme" refers to RNA having an enzymatic activity of cleaving nucleic acid, but in this specification, it is used as a concept that includes DNA as long as it has sequence-specific nucleic acid cleavage activity. The most versatile ribozyme nucleic acid is self-splicing RNA found in infectious RNA such as viroids and virusoids, and hammerhead and hairpin types are known. The hammerhead type exerts enzymatic activity with about 40 bases, and by making several bases at both ends adjacent to the part that has a hammerhead structure (total of about 10 bases) complementary to the desired cleavage site of mRNA, it is possible to specifically cleave only the target mRNA. This type of ribozyme nucleic acid has the additional advantage that it does not attack 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 can be made single-stranded by using a hybrid ribozyme linked with an RNA motif derived from a viral nucleic acid capable of specifically binding to an RNA helicase [Proc. Natl. Acad. Sci. USA, 98(10): 5572-5577 (2001)]. Furthermore, when the ribozyme is used in the form of an expression vector containing DNA encoding it, a hybrid ribozyme can be made by further linking a sequence in which a tRNA has been modified in order to promote the transfer of the transcript to the cytoplasm [Nucleic Acids Res., 29(13): 2780-2788 (2001)].

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

siRNA can be designed based on the cDNA sequence information of the target gene, for example, according to the rules proposed by Elbashir et al. (Genes Dev., 15, 188-200 (2001)) and Teramoto et al. (FEBS Lett. 579(13):p2878-82(2005)). The target sequence of siRNA is in principle 15 to 50 bases long, preferably 19 to 49 bases long, more preferably 19 to 27 bases long, and may be, for example, AA+(N)19 (a base sequence of 19 bases following AA), AA+(N)21 (a base sequence of 21 bases following AA), or A+(N)21 (a base sequence of 21 bases following A).

The nucleic acid of the present invention may have additional bases at the 5' or 3' end. The length of the additional bases is usually about 2 to 4 bases, and the total length of the siRNA is 19 bases or more. The additional bases may be DNA or RNA, but using DNA may improve the stability of the nucleic acid. Examples of such additional base sequences include, but are not limited to, ug-3', uu-3', tg-3', tt-3', ggg-3', guuu-3', gttt-3', ttttt-3', and uuuuu-3'.

The siRNA may also have a protruding sequence (overhang) at the 3' end, specifically an overhang with dTdT (dT represents the deoxythymidine residue of deoxyribonucleic acid) added. Alternatively, the siRNA may have a blunt end with no additional terminal addition.

In addition, the sense strand and the antisense strand of siRNA may have different numbers of bases, for example, "aiRNA" in which the antisense strand has protruding sequences (overhangs) at the 3' and 5' ends. A typical aiRNA has an antisense strand of 21 bases, a sense strand of 15 bases, and an overhang structure of 3 bases at each end of the antisense strand (Sun, X. et al., Nature Biotechnology Vol. 26 No. 12 p. 1379, International Publication WO2009/029688 pamphlet). The position of the target sequence is not particularly limited, but it is desirable to select a target sequence from the 5'-UTR and up to about 50 bases from the start codon, as well as from a region other than the 3'-UTR. For the candidate target sequences selected based on the above rules or other factors, a homology search software such as BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) is used to check whether there is any homology in the consecutive 16-17 base sequences in mRNAs other than the target, and the specificity of the selected target sequence is confirmed. For the target sequence whose specificity has been confirmed, a double-stranded RNA consisting of a sense strand having a 3'-end overhang of TT or UU at 19-21 bases after AA (or NA) and an antisense strand having a sequence complementary to the 19-21 bases and a 3'-end overhang of TT or UU may be designed as an siRNA. In addition, a short hairpin RNA (shRNA), which is a precursor of siRNA, can be designed by appropriately selecting any linker sequence (e.g., about 5-25 bases) capable of forming a loop structure and linking the sense strand and antisense strand via the linker sequence.

The sequences of siRNA and/or shRNA can be searched using search software provided free of charge on various websites. Examples of such websites include, but are not limited to, siRNA Target Finder (http://www.ambion.com/jp/techlib/misc/siRNA_finder.html) and pSilencer (registered trademark) Expression Vector Insert Design Tool (http://www.ambion.com/jp/techlib/misc/psilencer_converter.html) provided by Ambion, and GeneSeer (http://codex.cshl.edu/scripts/newsearchhairpin.cgi) provided by RNAi Codex.

The ribonucleoside molecules constituting the siRNA may also be modified in the same manner as in the case of the antisense nucleic acid described above in order to improve stability, specific activity, etc. However, in the case of siRNA, if all ribonucleoside molecules in natural RNA are replaced with modified ones, RNAi activity may be lost, so it is necessary to introduce the minimum number of modified nucleosides that enable the RNA-induced silencing complex (RISC) to function.

Specifically, the modification can be a replacement of a part of the nucleotide molecules constituting the siRNA with natural DNA or with RNA that has been subjected to various chemical modifications in order to improve stability (chemical and/or enzymatic) and specific activity (affinity with RNA) (see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163). For example, in order to prevent degradation by hydrolases such as nucleases, the phosphoric acid residues (phosphates) of each nucleotide constituting the siRNA can be replaced with chemically modified phosphoric acid residues such as phosphorothioate (PS), methylphosphonate, and phosphorodithioate. In addition, the hydroxyl group at the 2'-position of the sugar (ribose) of each nucleotide may be _replaced with _{-OR (R = CH3 (2'-O-Me), CH2CH2OCH3 ( 2' -} O-MOE), _CH2CH2NHC (NH _{) NH2} , _{CH2CONHCH3 , CH2CH2CN} , etc.) or a fluorine atom (-F). Furthermore, the base portion (pyrimidine, purine) may be chemically modified, for example, by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base, or by replacing the carbonyl group at the 2-position with a thiocarbonyl. In addition, the modification method for antisense nucleic acid described in (1) above can be used. Alternatively, chemical modification (2'-deoxylation, 2'-H) may be performed to replace a part of the RNA in the siRNA with DNA. Alternatively, 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.sub.2-- to fix the conformation to N-type may be used.

The sense and antisense strands constituting the siRNA may also be chemically bonded via a linker to a ligand, peptide, sugar chain, antibody, lipid, or oligoarginine, Tat peptide, Rev peptide, or Ant peptide, which specifically recognizes a receptor present on the cell surface, or which has a positive charge or molecular structure and adsorbs to and penetrates the cell membrane surface.

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

In the present specification, a nucleic acid designed to generate an siRNA for the mRNA of the CARD14 gene in vivo is also defined as being included in the nucleic acid containing a base sequence complementary or substantially complementary to the base sequence of the mRNA of the CARD14 gene, or a part thereof. Examples of such a nucleic acid include the above-mentioned shRNA and an expression vector constructed to express siRNA. shRNA can be prepared by designing an oligo-RNA containing a base sequence in which the sense strand and the antisense strand of a target sequence on an mRNA are linked by inserting a spacer sequence of a length (e.g., about 5 to 25 bases) that can form an appropriate loop structure, and synthesizing this with a DNA/RNA automatic synthesizer. There are two types of vectors that express shRNA: tandem type and stem loop (hairpin) type. The former is a vector in which an expression cassette for the sense strand of siRNA and an expression cassette for the antisense strand are linked in tandem, and each strand is expressed in a cell and annealed to form a double-stranded siRNA (dsRNA). On the other hand, the latter is a vector in which an expression cassette for shRNA is inserted, and the shRNA is expressed in the cell and processed by dicer to form dsRNA. Although a pol II promoter (e.g., CMV immediate early promoter) can also be used as the promoter, a pol III promoter is generally used to ensure accurate transcription of short RNA. Examples of pol III promoters include mouse and human U6-snRNA promoters, human H1-RNase PRNA promoter, and human valine-tRNA promoter. In addition, a sequence of four or more consecutive Ts is used as a transcription termination signal.

The siRNA or shRNA expression cassette constructed in this manner is then inserted into a plasmid vector or a viral vector. Examples of such vectors include viral vectors such as retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes virus, and Sendai virus, as well as animal cell expression plasmids.

The above siRNA can be chemically synthesized according to standard methods using an automated DNA/RNA synthesizer such as a 394 Applied Biosystems, Inc. synthesizer based on the nucleotide sequence information. For example, the methods described in Caruthers et al., 1992, Methods in Enzymology 211, 3-19, Thompson et al., International Publication WO99/54459, Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol. Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, Usman et al., 1987 J. Am. Chem. Soc., 109, 7845, Scaringe et al., 1990 Nucleic Acids Res., 18, 5433, and U.S. Pat. No. 6,001,311 can be mentioned. Specifically, it can be synthesized using a nucleic acid protecting group (e.g., a dimethoxytrityl group at the 5' end) and a coupling group (e.g., a phosphoramidite at the 3' end) known to those skilled in the art. That is, the protecting group at the 5' end is deprotected with an acid such as TCA (trichloroacetic acid) and a coupling reaction is carried out. Then, after capping with an acetyl group, the next nucleic acid condensation reaction is carried out. In the case of siRNA containing modified RNA or DNA, modified RNA (e.g., 2'-O-methyl nucleotide, 2'-deoxy-2'-fluoro nucleotide) can be used as the raw material, and the coupling reaction conditions can be adjusted appropriately. In addition, when introducing a phosphorothioate bond with a modified phosphate bond, a beaucage reagent (3H-1,2-benzodithiol-3-one 1,1-dioxide) can be used.

Alternatively, the oligonucleotides may be synthesized separately and linked together after synthesis, e.g., by ligation (Moore et al., 1992, Science 256, 9923; Draper et al. WO 93/23569; Shabarova et al., 1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al., 1997, Nucleosides & Nucleotides, Bellon et al., 1997, Bioconjugate Chem. 8, 204), or linked together after synthesis and/or deprotection by hybridization. siRNA molecules can also be synthesized by tandem synthesis, i.e., both siRNA strands are synthesized as a single contiguous oligonucleotide separated by a cleavable linker, which is then cleaved to generate separate siRNA fragments, which are hybridized and purified. The linker may be a polynucleotide linker or a non-nucleotide linker.

The synthesized siRNA molecules can be purified using methods known to those of skill in the art, such as by gel electrophoresis or by high performance liquid chromatography (HPLC).

Another preferred example of a nucleic acid containing a base sequence complementary or substantially complementary to the base sequence of the mRNA of the CARD14 gene, or a part thereof, is a microRNA (miRNA) that targets the mRNA. The miRNA can also be prepared according to the method described above for the siRNA.

Nucleic acids containing a base sequence complementary or substantially complementary to the base sequence of the mRNA of the CARD14 gene or a part thereof can be provided in a special form such as liposomes or microspheres, or in a form to which other molecules are added. Examples of the added form include polycations such as polylysine, which act to neutralize the charge of the phosphate group backbone, and hydrophobic lipids (e.g., phospholipids, cholesterol, etc.) that enhance interaction with cell membranes and increase uptake of nucleic acids. Preferred lipids for addition include cholesterol and its derivatives (e.g., cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3' or 5' end of the nucleic acid, and can be attached via bases, sugars, or intramolecular nucleoside bonds. Other groups include capping groups specifically located at the 3' or 5' end of the nucleic acid to prevent degradation by nucleases such as exonucleases and RNases. Such capping groups include, but are not limited to, hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.

The CARD14 expression inhibitory activity of these nucleic acids can be examined using 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 CARD14 protein translation system.

The substance that inhibits the expression of CARD14 in the present invention is not limited to a nucleic acid that contains a base sequence that is complementary or substantially complementary to the base sequence of the mRNA of the CARD14 gene as described above, or a part thereof, and may be other substances such as low molecular weight compounds as long as they directly or indirectly inhibit the production of CARD14. Such substances can be obtained, for example, by the screening method of the present invention described below.

In the present invention, a "substance that inhibits the function of CARD14" may be any substance as long as it inhibits CARD14, once functionally produced, from exerting its function.

Specifically, examples of substances that suppress the function of CARD14 include antibodies against CARD14. The antibodies may be either polyclonal or monoclonal antibodies. These antibodies can be produced according to known methods for producing antibodies or antisera. The isotype of the antibody is not particularly limited, but is preferably IgG, IgM, or IgA, and more preferably IgG. The antibody is not particularly limited as long as it has at least a complementarity determining region (CDR) for specifically recognizing and binding to a target antigen, and may be a complete antibody molecule, or a fragment such as Fab, Fab', or F(ab') ₂ , a genetically engineered conjugate molecule such as scFv, scFv-Fc, a minibody, or a diabody, or a derivative thereof modified with a molecule having a protein stabilizing effect such as polyethylene glycol (PEG).

In a preferred embodiment, the antibody against CARD14 is used as a pharmaceutical intended for administration to humans, and therefore 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, etc., and is particularly preferably a fully human antibody. Humanized antibodies and chimeric antibodies can be produced by genetic engineering according to standard methods. Furthermore, fully human antibodies can also be produced from human-human (or mouse) hybridomas, but in order to provide large amounts of antibodies stably and at low cost, it is desirable to produce human antibody-producing mice or phage disks.

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

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

Preferred Embodiments
The preferred embodiments of the present invention are described below. The embodiments provided below are provided for a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is clear that a person skilled in the art can make appropriate modifications within the scope of the present invention in consideration of the description in this specification. It is also understood that the following embodiments of the present invention can be used alone or in combination.

<Therapeutic or preventive agent for psoriasis and screening method>
In one aspect, the present invention provides a method for screening a therapeutic or preventive agent for psoriasis based on the expression of CARD14 in hematopoietic cells. Although CARD14 is a conventionally known molecule, it has not been found that a therapeutic or preventive agent for psoriasis can be screened by observing its expression in hematopoietic cells, particularly γδ T cells, among which epidermal γδ T cells, and this has been provided for the first time in the present invention. Without wishing to be bound by theory, it is understood that a CARD inhibitor, such as an agent that suppresses or eliminates the expression of CARD14 in hematopoietic cells, particularly γδ T cells, among which preferably epidermal γδ T cells, can be used as a therapeutic or preventive agent for psoriasis. Thus, the method of the present invention includes the steps of contacting a candidate substance with hematopoietic cells and determining the expression of CARD14 in hematopoietic cells, where it is determined that the candidate substance can be used as a therapeutic or preventive agent for psoriasis if the candidate substance suppresses or eliminates the expression of CARD14.

CARD inhibitors, such as agents that suppress or eliminate the expression of CARD14, include, for example, inhibitors of the expression product of CARD14, such as antibodies, antibody fragments, functional equivalents, etc., or antisense nucleic acids of CARD14, molecules with RNAi function, etc.

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

In the screening method of the present invention, in order to provide a better therapeutic or preventive agent, it is preferable to confirm that CARD14 is not expressed in cells (also called "non-hematopoietic cells"; for example, skin resident cells) that are not hematopoietic cells (particularly γδ T cells, and among these, preferably epidermal γδ T cells). Here, in a preferred embodiment, it is desirable that the hematopoietic cells and the non-hematopoietic cells are of allogeneic origin, and more advantageously, that they are of the same individual. In one preferred embodiment, the method of the present invention further includes a step of contacting the candidate substance with the non-hematopoietic cells, and selecting the candidate substance as the therapeutic or preventive agent if CARD14 has no substantial effect on expression. In this specification, "has no substantial effect" means that CARD14 can exert its function even if there is an effect.

The psoriasis that is the subject of the present invention may include, for example, imoquimide (IMQ)-induced psoriasis and IL-23-induced psoriasis.

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

In the present invention, specific delivery to hematopoietic cells is achieved by a delivery system guided by a specific molecule, such as an antibody specific to the hematopoietic cells (e.g., γδ T cells).

<Therapeutic or preventive agents for multiple sclerosis and screening methods thereof>
In one aspect, the present invention provides a method for screening a therapeutic or preventive agent for multiple sclerosis based on the expression of CARD14 in hematopoietic cells. Although CARD14 is a conventionally known molecule, it has not been found that a therapeutic or preventive agent for multiple sclerosis can be screened by observing its expression in hematopoietic cells, particularly γδ T cells, among which epidermal γδ T cells, and this has been provided for the first time in the present invention. Without wishing to be bound by theory, it is understood that a CARD inhibitor, such as an agent that suppresses or eliminates the expression of CARD14 in hematopoietic cells, particularly γδ T cells, among which preferably epidermal γδ T cells, can be used as a therapeutic or preventive agent for multiple sclerosis. In the present invention, since the role of CARD14 was clearly demonstrated in an experimental autoimmune encephalomyelitis (EAE) model, which is a model of multiple sclerosis (autoimmune disease model), it is believed that multiple sclerosis can be prevented or treated using a mechanism based on CARD14. Thus, the method of the present invention includes the steps of contacting a candidate substance with hematopoietic cells and determining the expression of CARD14 in the hematopoietic cells, where, 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 preventive agent for psoriasis.

CARD inhibitors, such as agents that suppress or eliminate the expression of CARD14, include, for example, inhibitors of the expression product of CARD14, such as antibodies, antibody fragments, functional equivalents, etc., or antisense nucleic acids of CARD14, molecules with RNAi function, etc.

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

In the screening method of the present invention, in order to provide a better therapeutic or preventive agent, it is preferable to confirm that CARD14 is not expressed in cells ("non-hematopoietic cells"; for example, skin resident cells) that are not hematopoietic cells (particularly γδ T cells, and among these, preferably epidermal γδ T cells). Here, in a preferred embodiment, it is desirable that the hematopoietic cells and the non-hematopoietic cells are of allogeneic origin, and more advantageously, are of the same individual origin. In one preferred embodiment, the method of the present invention further includes a step of contacting the candidate substance with the non-hematopoietic cells, and selecting the candidate substance as the therapeutic or preventive agent if CARD14 has no substantial effect on expression.

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

In the present invention, specific delivery to hematopoietic cells is achieved by a delivery system guided by a specific molecule, such as an antibody specific to the hematopoietic cells (e.g., γδ T cells).

<Therapeutic or preventive agents for reducing side effects caused by TLR-based/IMQ/immunostimulatory adjuvants and screening methods thereof>
In one aspect, the present invention provides a method for screening a therapeutic or preventive agent for multiple sclerosis based on the expression of CARD14 in hematopoietic cells. Although CARD14 is a conventionally known molecule, it has not been found that a therapeutic or preventive agent for reducing side effects caused by TLR-based/IMQ/immunostimulatory adjuvants can be screened by observing its expression in hematopoietic cells, particularly γδ T cells, among which epidermal γδ T cells, and this has not been found to be the first to be provided in the present invention. Without wishing to be bound by theory, it is understood that a CARD inhibitor, such as an agent that suppresses or eliminates the expression of CARD14 in hematopoietic cells, particularly γδ T cells, among which preferably epidermal γδ T cells, can be used as a therapeutic or preventive agent for reducing side effects caused by TLR-based/IMQ/immunostimulatory adjuvants. As demonstrated herein, it has been demonstrated that both TLR7 and TLR9 signaling in combination is necessary for the full development of IMQ-induced psoriasiform dermatitis, while the IL-23-induced psoriasiform dermatitis model directly activates downstream of TLR7 and TLR9-mediated 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 demonstrate a clear difference in innate immune signaling between the IMQ-induced psoriasiform model and the IL-23-induced psoriasiform model. Through such findings, it is possible to screen therapeutic or preventive agents for reducing side effects caused by TLR-based/IMQ/immunostimulatory adjuvants. This can be an excellent target for reducing side effects caused by IMQ-based adjuvants or other TLR-based adjuvants or immune stimulatory adjuvants. This will inhibit innate immune activation without suppressing adaptive immune responses specific to the co-administered antigen. Thus, the method of the present invention includes the steps of contacting a candidate substance with hematopoietic cells and determining the expression of CARD14 in the hematopoietic cells, wherein if the candidate substance inhibits or eliminates the expression of CARD14, it is determined that the candidate substance can be used as a therapeutic or preventive agent for psoriasis.

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

CARD inhibitors, such as agents that suppress or eliminate the expression of CARD14, include, for example, inhibitors of the expression product of CARD14, such as antibodies, antibody fragments, functional equivalents, etc., or antisense nucleic acids of CARD14, molecules with RNAi function, etc.

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

In the screening method of the present invention, in order to provide a better therapeutic or preventive agent, it is preferable to confirm that CARD14 is not expressed in cells ("non-hematopoietic cells", for example, skin-resident cells) that are not hematopoietic cells (particularly γδ T cells, and among these, preferably epidermal γδ T cells). Here, in a preferred embodiment, it is desirable that the hematopoietic cells and the non-hematopoietic cells are of allogeneic origin, and more advantageously, are of the same individual origin. In one preferred embodiment, the method of the present invention further includes a step of contacting the candidate substance with the non-hematopoietic cells, and selecting the candidate substance as the therapeutic or preventive agent if CARD14 has no substantial effect on expression.

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

In the present invention, specific delivery to hematopoietic cells is achieved by a delivery system guided by a specific molecule, such as an antibody specific to the hematopoietic cells (e.g., γδ T cells).

<Therapeutic or preventive agents for γδ T cell-mediated diseases and/or complications of immune intervention and screening methods thereof>
In one aspect, the present invention provides a method for screening a therapeutic or preventive agent for multiple sclerosis based on the expression of CARD14 in hematopoietic cells. Although CARD14 is a conventionally known molecule, it has not been found that a therapeutic or preventive agent for γδ T cell-mediated diseases and/or complications of immune intervention can be screened by observing its expression in hematopoietic cells, particularly γδ T cells, among which epidermal γδ T cells, and this has been provided for the first time in the present invention. Without wishing to be bound by theory, it is understood that a CARD inhibitor, such as an agent that suppresses or eliminates the expression of CARD14 in hematopoietic cells, particularly γδ T cells, among which preferably epidermal γδ T cells, can be used as a therapeutic or preventive agent for γδ T cell-mediated diseases and/or complications of immune intervention. Thus, the method of the present invention includes the steps of contacting a candidate substance with hematopoietic cells and determining the expression of CARD14 in the hematopoietic cells, wherein if the candidate substance inhibits or eliminates the expression of CARD14, it is determined that the candidate substance can be used as a therapeutic or preventive agent for psoriasis.

CARD inhibitors, such as agents that suppress or eliminate the expression of CARD14, include, for example, inhibitors of the expression product of CARD14, such as antibodies, antibody fragments, functional equivalents, etc., or antisense nucleic acids of CARD14, molecules with RNAi function, etc.

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

In the screening method of the present invention, in order to provide a better therapeutic or preventive agent, it is preferable to confirm that CARD14 is not expressed in cells ("non-hematopoietic cells"; for example, skin resident cells) that are not hematopoietic cells (particularly γδ T cells, and among these, preferably epidermal γδ T cells). Here, in a preferred embodiment, it is desirable that the hematopoietic cells and the non-hematopoietic cells are of allogeneic origin, and more advantageously, are of the same individual origin. In one preferred embodiment, the method of the present invention further includes a step of contacting the candidate substance with the non-hematopoietic cells, and selecting the candidate substance as the therapeutic or preventive agent if CARD14 has no substantial effect on expression.

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

In the present invention, specific delivery to hematopoietic cells is achieved by a delivery system guided by a specific molecule, such as an antibody specific to the hematopoietic cells (e.g., γδ T cells).

As used herein, the term "γδ T cell-mediated disease" refers to any disease mediated by γδ T cells, and examples of "γδ T cell-mediated disease" include, but are not limited to, psoriasis, multiple sclerosis, inflammation, and cytokineemia.

As used herein, "complications of immune intervention" refers to any complication that occurs when immune intervention is performed, and examples of "complications of immune intervention" include, but are not limited to, inflammation, cytokineemia, etc.

<Screening method details>
When screening for a compound that inhibits the expression or function of CARD14, the screening method includes culturing a cell capable of producing CARD14 in the presence and absence of a test substance, and comparing the expression level or the degree of function of CARD14 under both conditions. Compounds that inhibit the function of CARD14 can also be screened by testing the binding ability to purified CARD14 protein and the binding inhibitory activity between CARD14 protein and its binding protein (e.g., CARD14 receptor). In particular, hematopoietic cells, preferably γδ T cells, are advantageous as such cells, and epidermal γδ T cells are even more advantageous. Alternatively, genetically modified cells may be used. Specifically, such cells can be prepared by linking DNA encoding CARD14 (i.e., DNA containing a base sequence that hybridizes under stringent conditions with the base sequence represented by SEQ ID NO: 1 or 3, preferably SEQ ID NO: 1, or a base sequence complementary to said base sequence, and that encodes a polypeptide having the same function as a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or 4, preferably SEQ ID NO: 2) downstream of a promoter in an appropriate expression vector and introducing the vector into a host animal cell.

In one embodiment, the screening method includes the steps of: (A) contacting a cell containing a nucleic acid encoding a reporter protein under the control of the CARD14 gene or the transcriptional regulatory region of the gene with a test substance; (B) measuring the expression level of the CARD14 gene or CARD14 protein or reporter protein in the cell; and (C) selecting a test substance that reduces the expression level of the CARD14 gene or CARD14 protein or reporter protein compared to when measured in the absence of the test substance as a candidate substance to be used for reducing side effects of psoriasis, multiple sclerosis, TLR-based/IMQ/immunostimulatory adjuvants, or for treating and/or preventing complications of γδ T cell-mediated diseases and/or immune interventions. In one embodiment, CARD14 is contacted with the test substance, and a test substance capable of binding to CARD14 can be selected as a candidate substance to be used for treating and/or preventing psoriasis.

In another embodiment, the present invention includes the steps of (A) contacting CARD14 with a cell membrane fraction of hematopoietic cells (particularly γδ T cells, and preferably epidermal γδ T cells) in the presence of a test substance; (B) measuring the amount of CARD14 bound to the cell membrane fraction; and (C) selecting a test substance that reduces the amount of CARD14 bound to the cell membrane fraction compared to when measured in the absence of the test substance as a candidate substance to be used for reducing side effects of psoriasis, multiple sclerosis, TLR-based/IMQ/immunostimulatory adjuvants, or for treating and/or preventing complications of γδ T cell-mediated diseases and/or immune interventions.

In a further embodiment, the present invention further comprises applying a test substance selected as a candidate substance for use in reducing side effects of psoriasis, multiple sclerosis, TLR-based/IMQ/immunostimulatory adjuvants, or for treating and/or preventing complications of γδ T cell-mediated diseases and/or immune interventions to a model of psoriasis, multiple sclerosis, TLR-based/IMQ/immunostimulatory adjuvants, or a complication of γδ T cell-mediated diseases and/or immune interventions (including, for example, the model animals described in the Examples) and testing whether or not the test substance suppresses a reaction to reduce side effects of psoriasis, multiple sclerosis, TLR-based/IMQ/immunostimulatory adjuvants, or a complication of γδ T cell-mediated diseases and/or immune interventions in the model.

In another embodiment, the present invention provides a method for the treatment of hematopoietic cells (particularly γδ T cells, and preferably epidermal γδ T cells) with a test substance in the presence and absence of CARD14; (B) measuring the degree of complications of γδ T cell-mediated diseases and/or immune interventions or reducing side effects of psoriasis, multiple sclerosis, TLR-based/IMQ/immunostimulatory adjuvants in the cells under each condition; and (C) measuring the degree of complications of psoriasis, multiple sclerosis, TLR-based/IMQ/immunostimulatory adjuvants in the presence of CARD14 compared to those measured in the absence of the test substance. or suppressing the reaction of γδ T cell-mediated disease and/or complications of immune intervention and reducing side effects caused by psoriasis, multiple sclerosis, TLR-based/IMQ/immunostimulatory adjuvants in the absence of CARD14, or selecting a test substance that did not suppress the reaction of γδ T cell-mediated disease and/or complications of immune intervention as a candidate substance to be used for inhibiting the function of CARD14 to reduce side effects caused by psoriasis, multiple sclerosis, TLR-based/IMQ/immunostimulatory adjuvants, or for treating and/or preventing complications of γδ T cell-mediated disease and/or immune intervention.

When carrying out the screening of the present invention, test substances include, for example, proteins, peptides, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc., and these substances may be novel or known.

In addition, when selecting a substance that reduces the expression level of CARD14 or the CARD14 gene, or a substance that reduces the function of CARD14, control cells that are not contacted with the test substance can also be used as a comparison control. Here, "not contacting with the test substance" also includes the case of adding a solvent (blank) in the same amount as the test substance instead of the test substance, or the case of adding a negative control substance that does not affect the expression level of CARD14 or the CARD14 gene, or the function of CARD14.

Contact of the test substance with the cells can be carried out, for example, by adding the test substance to the above-mentioned culture medium or various buffer solutions (e.g., HEPES buffer, phosphate buffer, phosphate-buffered saline, Tris-HCl buffer, borate buffer, acetate buffer, etc.) and incubating the cells for a certain period of time. The concentration of the test substance to be added varies depending on the type of compound (solubility, toxicity, etc.), but is appropriately selected, for example, from within the range of about 0.1 nM to about 100 μM. The incubation time can be, for example, from about 10 minutes to about 24 hours.

When the cells producing CARD14 are provided in the form of a non-human mammalian individual, the state of the individual animal is not particularly limited, but may be, for example, a psoriasis disease model animal in which inflammation is induced by a drug or genetic modification (e.g., IMQ-induced psoriasiform dermatitis, IL-23-induced psoriasiform dermatitis, etc., as exemplified in Example 1), a multiple sclerosis model animal (e.g., the experimental autoimmune encephalomyelitis (EAE) model used in Example 2, etc.), a model animal that develops side effects due to TLR-based/IMQ/immunostimulatory adjuvants (IMQ-induced psoriasiform dermatitis, IL-23-induced psoriasiform dermatitis, etc., in Example 1), or a model animal of a γδ T cell-mediated disease and/or a complication of immune intervention (IMQ-induced psoriasiform dermatitis, IL-23-induced psoriasiform dermatitis, etc., in Example 1). There are no particular limitations on the breeding conditions of the animals used, but it is preferable that they are bred in an environment of SPF grade or higher. The contact of the test substance with the cells is achieved by administering the test substance to the animal. There are no particular limitations on the route of administration, but examples include intravenous administration, intraarterial administration, subcutaneous administration, intradermal administration, intraperitoneal administration, oral administration, intratracheal administration, and rectal administration. There are also no particular limitations on the dosage, but for example, a single dose of about 0.5 to 20 mg/kg can be administered 1 to 5 times a day, preferably 1 to 3 times a day, for 1 to 14 days.

Alternatively, the above screening method can be performed by contacting the test substance with an extract of cells capable of producing CARD14, or with CARD14 isolated and purified from the cells, instead of with cells capable of producing CARD14.

The present invention provides a method for screening for a substance that can be used to prevent and/or treat psoriasis, which is characterized by comparing the expression of the protein (gene) in cells capable of producing CARD14 in the presence and absence of a test substance. The cells, type of test substance, and manner of contact between the test substance and the cells used in this method are the same as those described above.

The expression level of CARD14 can be measured at the RNA level by detecting the mRNA of the CARD14 gene using a nucleic acid capable of hybridizing under stringent conditions with the DNA encoding CARD14 described above, i.e., a nucleic acid (DNA) capable of hybridizing under stringent conditions with the base sequence represented by SEQ ID NO:1 or a base sequence complementary thereto (hereinafter sometimes referred to as the "detection nucleic acid of the present invention"). Alternatively, the expression level can also be measured at the protein level by detecting these proteins using the antibody against CARD14 described above (hereinafter sometimes referred to as the "detection antibody of the present invention").

More specifically, therefore, the present invention provides (a) a method for screening a substance that can be used to reduce side effects caused by psoriasis, multiple sclerosis, TLR-based/IMQ/immunostimulatory adjuvants, or to prevent and/or treat complications of γδ T cell-mediated diseases and/or immune interventions, characterized by culturing cells capable of producing CARD14 in the presence and absence of a test substance, and measuring and comparing the amount of mRNA encoding the protein under both conditions using the detection nucleic acid of the present invention, and (b) a method for screening a substance that can be used to reduce side effects caused by psoriasis, multiple sclerosis, TLR-based/IMQ/immunostimulatory adjuvants, or to prevent and/or treat complications of γδ T cell-mediated diseases and/or immune interventions, characterized by culturing cells capable of producing CARD14 in the presence and absence of a test substance, and measuring and comparing the amount of the protein under both conditions using the detection antibody of the present invention.

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

(i) A test substance is administered to a normal or disease model (e.g., psoriasis, multiple sclerosis, to reduce side effects of TLR-based/IMQ/immunostimulatory adjuvants, or complications of γδ T cell-mediated diseases and/or immune interventions, etc.) non-human mammal (e.g., mouse, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc.), and after a certain period of time (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), blood or a specific organ (e.g., brain, etc.), or tissue or cells isolated from the organ are obtained.

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

(ii) Cells expressing the CARD14 gene (e.g., transformants into which CARD14 has been introduced) are prepared according to the above method, and when the cells are cultured according to a conventional method, a test substance is added to the medium or buffer, and after a certain period of incubation (after 1 to 7 days, preferably after 1 to 3 days, more preferably after 2 to 3 days), CARD14 or the mRNA encoding it contained in the cell culture can be quantified and analyzed in the same manner as in (i) above.

The expression level of the CARD14 gene (mRNA) can be detected and quantified by known methods such as Northern blotting and RT-PCR using RNA prepared from the cells or a complementary polynucleotide transcribed therefrom. Specifically, the presence or absence of expression of the CARD14 gene in RNA and its expression level can be detected and measured by using a polynucleotide having at least 15 consecutive bases in the base sequence of the CARD14 gene and/or its complementary polynucleotide as a primer or probe. Such a probe or primer can be designed based on the base sequence of the CARD14 gene using, for example, primer 3 (http://primer3.sourceforge.net/) or Vector NTI (Infomax).

In the case of using the Northern blot method, the primer or probe is labeled with a radioisotope ( ³² P, ³³ P, etc.: RI) or a fluorescent substance, and then hybridized with RNA derived from a cell that has been transferred to a nylon membrane or the like in a conventional manner, and the formed double strand of the primer or probe (DNA or RNA) and RNA is detected and measured as a signal derived from the label (RI or fluorescent substance) of the primer or probe using a radiation detector (BAS-1800II, Fujifilm Corporation) or a fluorescence detector. Alternatively, the probe may be labeled according to the protocol using AlkPhos Direct Labelling and Detection System (GE Healthcare), hybridized with RNA derived from a cell, and then the signal derived from the label of the probe is detected and measured using a multi-bioimager STORM860 (GE Healthcare).

When using RT-PCR, a method can be exemplified in which cDNA is prepared from cell-derived RNA in a conventional manner, and a pair of primers (positive strand that binds to the above cDNA (-strand), and reverse strand that binds to the + strand) prepared based on the sequence of the CARD14 gene are hybridized with the template so that the target CARD14 gene region can be amplified using the template, and PCR is performed in a conventional manner to detect the resulting amplified double-stranded DNA. The amplified double-stranded DNA can be detected by a method of detecting labeled double-stranded DNA produced by performing the above PCR using primers that have been labeled with RI or a fluorescent substance in advance, or by a method of transferring the produced double-stranded DNA to a nylon membrane or the like in a conventional manner, and using the labeled primer as a probe to hybridize with the membrane and detect the product. The produced labeled double-stranded DNA product can be measured using an Agilent 2100 Bioanalyzer (manufactured by Yokogawa Analytical Systems, Inc.) or the like. Alternatively, an RT-PCR reaction solution can be prepared using SYBR Green RT-PCR Reagents (Applied Biosystems) according to the protocol, and the reaction can be performed using an ABI PRIME 7900 Sequence Detection System (Applied Biosystems) to detect the reaction product.

If the expression of the CARD14 gene in cells to which the test substance has been added is 2/3 or less, preferably 1/2 or less, and more preferably 1/3 or less, of the expression level in control cells to which the test substance has not been added, the test substance can be selected as a substance that inhibits the expression of the CARD14 gene.

Screening for a substance that changes the expression level of CARD14 can also be performed by a reporter gene assay using the transcriptional regulatory region of the CARD14 gene. Here, the "transcriptional regulatory region" generally refers to a range of several kb to several tens of kb upstream of the chromosomal gene, and can be performed, for example, by (i) determining the 5' end by a conventional method such as the 5'-RACE method (which can be performed using, for example, 5'-full RaceCoreKit (manufactured by Takara Bio Inc.)), the oligocapping method, or S1 primer mapping; (ii) determining the 5' end by a conventional method such as the Genome Walker method;
The promoter activity of the upstream region obtained by the method can be determined by a method including the steps of obtaining the 5'-upstream region using a 5'-upstream region detection kit (Clontech) or the like, and measuring the promoter activity of the obtained upstream region.

A reporter protein expression vector is constructed by linking a nucleic acid (hereinafter referred to as a "reporter gene") that functionally encodes a reporter protein downstream of the transcriptional regulatory region of the CARD14 gene. The vector may be prepared by a method known to those skilled in the art. For example, the transcriptional regulatory region of the CARD14 gene excised according to the usual genetic engineering techniques described in Molecular Cloning: A Laboratory Manual 2nd edition (1989), Cold Spring Harbor Laboratory Press, "Current Protocols In Molecular Biology" (1987), John Wiley & Sons, Inc., etc., can be incorporated into a plasmid containing a reporter gene.

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

The reporter gene prepared by operably linking the transcriptional regulatory region of the CARD14 gene can be inserted into a vector that can be used in the cells into which the reporter gene is to be introduced, using standard genetic engineering techniques, to prepare a plasmid, which can then be introduced into an appropriate host cell. Stable transformed cells can be obtained by culturing the vector in a medium with selection conditions according to the selection marker gene carried by the vector. Alternatively, the reporter gene operably linked to the transcriptional regulatory region of the CARD14 gene can be expressed transiently in the host cell.

The expression level of the reporter gene can be measured by a method appropriate for each reporter gene. For example, when a luciferase gene is used as the reporter gene, the transformed cells are cultured for several days, and then an extract of the cells is obtained. The extract is then reacted with luciferin and ATP to cause chemiluminescence, and the luminescence intensity is measured to detect promoter activity. In this case, a commercially available luciferase reaction detection kit such as Picagene Dual Kit (registered trademark; manufactured by Toyo Ink) can be used.

Specific examples of methods for measuring the amount of CARD14 protein include (i) a method in which the detection antibody of the present invention is competitively reacted with a sample liquid and labeled CARD14, and the labeled protein bound to the antibody is detected to quantify CARD14 in the sample liquid, and (ii) a method in which the sample liquid is simultaneously or successively reacted with the detection antibody of the present invention insolubilized on a carrier and another labeled detection antibody of the present invention, and then the amount (activity) of the labeling agent on the insolubilized carrier is measured to quantify CARD14 in the sample liquid.

The protein expression level of CARD14 can be detected and quantified according to known methods such as Western blotting using an antibody that recognizes CARD14. Western blotting can be performed by using an antibody that recognizes CARD14 as a primary antibody, labeling the secondary antibody with an antibody that binds to the primary antibody labeled with a radioisotope such as ¹²⁵ I, a fluorescent substance, an enzyme such as horseradish peroxidase (HRP), or the like, and measuring signals derived from these labeled substances using a radiation meter (BAI-1800II: Fujifilm, etc.), a fluorescence detector, or the like. Alternatively, an antibody that recognizes CARD14 can be used as a primary antibody, followed by detection using ECL Plus Western Blotting Detection System (GE Healthcare) according to the protocol, and measurement using a multi-biometer STORM860 (GE Healthcare).

The above-mentioned antibody is not particularly limited in its form, and may be a polyclonal antibody or a monoclonal antibody using CARD14 as an immunogen. Furthermore, an antibody having antigen-binding ability to a polypeptide consisting of at least consecutive amino acids, usually 8 amino acids, preferably 15 amino acids, and more preferably 20 amino acids, from the amino acid sequence constituting CARD14 can also be used.

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

In the above quantitative method (ii), it is desirable that the two types of antibodies recognize different parts of CARD14. For example, if one antibody recognizes the N-terminus of CARD14, the other antibody can be one that reacts with the C-terminus of the protein.

The labeling agent used in the measurement method using a labeling substance may be, for example, a radioisotope, an enzyme, a fluorescent substance, or a luminescent substance. The radioisotope may be, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], or [ ¹⁴ C]. The enzyme is preferably stable and has a high specific activity, for example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, or malate dehydrogenase. The fluorescent substance may be, for example, fluorescamine or fluorescein isothiocyanate. The luminescent substance may be, for example, luminol, a luminol derivative, luciferin, or lucigenin. Furthermore, a biotin-(strept)avidin system may be used to bind an antibody or an antigen to a labeling agent.

The method for quantifying CARD14 using the detection antibody of the present invention should not be particularly limited, and any method may be used as long as it detects the amount of antibody, antigen, or antibody-antigen complex corresponding to the amount of antigen in a sample solution by chemical or physical means and calculates this from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephelometry, competitive method, immunometric method, and sandwich method are preferably used. In terms of sensitivity and specificity, it is preferable to use, for example, the sandwich method described below.

To insolubilize an antigen or antibody, physical adsorption may be used, or chemical bonding, which is usually used to insolubilize or immobilize proteins or enzymes, may be used. Carriers include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicone, and glass.

In the sandwich method, the sample solution is reacted with the insolubilized detection antibody of the present invention (primary reaction), and then another labeled detection antibody of the present invention is reacted with the sample solution (secondary reaction), after which the amount or activity of the labeling agent on the insolubilized carrier is measured to quantify CARD14 in the sample solution. The primary and secondary reactions may be performed in the reverse order, simultaneously, or at different times. The labeling agent and the method of insolubilization may be similar to those described above. In addition, in the sandwich immunoassay, the antibody used for the immobilized antibody or labeled antibody does not necessarily have to be of one type, and a mixture of two or more types of antibodies may be used for the purpose of improving the measurement sensitivity, etc.

The detection antibody of the present invention can also be used in measurement systems other than the sandwich method, such as the competitive method, immunometric method, or nephelometry.

In the competitive method, CARD14 in the sample solution is competitively reacted with the labeled CARD14 and the antibody, and then the unreacted labeled antigen (F) is separated from the labeled antigen (B) bound to the antibody (B/F separation), and the amount of labeling of either B or F is measured to quantify the amount of CARD14 in the sample solution. This reaction method uses a soluble antibody as the antibody, and performs B/F separation using polyethylene glycol or a secondary antibody against the antibody (primary antibody), and a solid-phase method using a solid-phase antibody as the primary antibody (direct method), or a soluble primary antibody and a solid-phase antibody as the secondary antibody (indirect method).

In the immunometric method, CARD14 in the sample solution and immobilized CARD14 are subjected to a competitive reaction with a fixed amount of labeled antibody, and then the solid and liquid phases are separated; alternatively, CARD14 in the sample solution is reacted with an excess amount of labeled antibody, and then immobilized CARD14 is added to bind unreacted labeled antibody to the solid phase, after which the solid and liquid phases are separated. The amount of label in either phase is then measured to quantify the amount of antigen in the sample solution.

In addition, nephelometry measures the amount of insoluble precipitates that form in a gel or solution as a result of an antigen-antibody reaction. When the amount of CARD14 in the sample solution is small and only a small amount of precipitate is obtained, laser nephelometry, which uses laser scattering, is preferably used.

When applying these individual immunological measurement methods to the quantitative method of the present invention, no special conditions, procedures, etc. are required. A measurement system for CARD14 can be constructed by adding the usual technical considerations of a person skilled in the art to the usual conditions and procedures for each method. For details of these general technical means, please refer to reviews, publications, etc.

For example, see "Radioimmunoassay" edited by Irie Hiroshi (Kodansha, published in 1974), "Radioimmunoassay 2" edited by Irie Hiroshi (Kodansha, published in 1979), "Enzyme Immunoassay" edited by Ishikawa Eiji et al. (Igaku Shoin, published in 1978), "Enzyme Immunoassay" edited by Ishikawa Eiji et al. (2nd edition) (Igaku Shoin, published in 1982), "Enzyme Immunoassay" edited by Ishikawa Eiji et al. (3rd edition) (Igaku Shoin, published in 1987), and "Methods in ENZYMOLOGY" Vol. 70 (Immunochemical Techniques (Part A)), Vol. 73 (Immunochemical Techniques (Part B)), Vol. 74 (Immunochemical Techniques (Part C)), Vol. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), Vol. 92 (Immunochemical Techniques (Part E: For reference, see Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies)) (both published by Academic Press).

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, if the expression level (mRNA amount or protein amount) of CARD14 in the presence of the test substance is inhibited by about 20% or more, preferably about 30% or more, more preferably about 50% or more compared to that in the absence of the test substance, the test substance can be selected as a candidate for a substance that inhibits the expression of CARD14 and can therefore be used to reduce side effects of psoriasis, multiple sclerosis, TLR-based/IMQ/immunostimulatory adjuvants, or to prevent and/or treat complications of γδ T cell-mediated diseases and/or immune interventions.

Alternatively, in the above screening method, cells containing a reporter gene under the control of the endogenous transcriptional regulatory region of the CARD14 gene can be used instead of cells expressing the CARD14 gene. Such cells may be cells, tissues, organs, or individuals of a transgenic animal into which a reporter gene (e.g., 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 standard method.

The screening method of the present invention can also be performed using as an index whether or not a test substance inhibits the function of CARD14. CARD14 belongs to the membrane-associated guanylate kinase (MAGUK) family and functions as a molecular scaffold that forms the assembly of a multiprotein complex specialized in the region of the plasma membrane, and therefore the relationship with these functions and the fact that this protein belongs to the CARD family, has a specific caspase-associated recruitment domain (CARD), and is also known to have specific interaction with BCL10 and to function as a positive regulator of BCL phosphorylation, NF-κB activation, and cell apoptosis, can be used as an index to screen inhibitors.

In yet another embodiment of the present invention, a substance that inhibits the function of CARD14 to reduce side effects caused by psoriasis, multiple sclerosis, TLR-based/IMQ/immunostimulatory adjuvants, or to treat or prevent complications of γδ T cell-mediated diseases and/or immune interventions can be screened in one step using the in vitro model for reducing side effects caused by psoriasis, multiple sclerosis, TLR-based/IMQ/immunostimulatory adjuvants, or to treat or prevent complications of γδ T cell-mediated diseases and/or immune interventions. The method includes the following steps (1) to (3):
(1) contacting target cells of a disease (e.g., psoriasis, multiple sclerosis, for reducing side effects of TLR-based/IMQ/immunostimulatory adjuvants, or complications of γδ T cell-mediated diseases and/or immune interventions, etc.) with a test substance in the presence and absence of CARD14; (2) measuring the response of the disease (e.g., psoriasis, multiple sclerosis, for reducing side effects of TLR-based/IMQ/immunostimulatory adjuvants, or complications of γδ T cell-mediated diseases and/or immune interventions) in the cells under each condition (e.g., a response specific to psoriasis, multiple sclerosis, for reducing side effects of TLR-based/IMQ/immunostimulatory adjuvants, or complications of γδ T cell-mediated diseases and/or immune interventions, etc.); (3) measuring the degree of the reaction (sclerosis-specific reaction, TLR-related reaction, complication-specific symptom) in the presence of CARD14 compared to the reaction measured in the absence of the test substance, and selecting a test substance that inhibits the function of CARD14 and does not inhibit the reaction in the absence of CARD14 as a candidate substance that inhibits the function of CARD14 and exhibits a therapeutic or preventive effect on a disease (e.g., psoriasis, multiple sclerosis, for reducing side effects of TLR-based/IMQ/immunostimulatory adjuvants, or for γδ T cell-mediated diseases and/or complications of immune intervention, etc.).

<Medicine>
The medicament of the present invention may be administered as it is, or may be administered as a suitable pharmaceutical composition. The pharmaceutical composition used for administration may contain the medicament of the present invention and a pharmacologically acceptable carrier, diluent or excipient. Such a pharmaceutical composition is provided in a dosage form suitable for oral or parenteral administration.

For compositions for parenteral administration, for example, injections, suppositories, etc. are used, and injections may include dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, and drip injections. Such injections can be prepared according to known methods. For example, the injections can be prepared by dissolving, suspending, or emulsifying the nucleic acid of the present invention in a sterile aqueous or oily liquid that is usually used for injections. As aqueous solutions for injection, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, etc. are used, and may be used in combination with appropriate dissolving aids, such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castoroil)), etc. As the oily liquid, for example, sesame oil, soybean oil, etc. are used, and benzyl benzoate, benzyl alcohol, etc. may be used in combination as a solubilizing agent. The prepared injection solution is preferably filled into a suitable ampule. Suppositories for rectal administration may be prepared by mixing the above nucleic acid with a normal suppository base.

Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, emulsions, suspensions, etc. Such compositions are produced by known methods and may contain carriers, diluents, or excipients commonly used in the pharmaceutical field. Examples of carriers and excipients for tablets include lactose, starch, sucrose, and magnesium stearate.

The above-mentioned parenteral or oral pharmaceutical compositions are conveniently prepared in a dosage unit form that matches the dosage of the active ingredient. Examples of such dosage unit forms include tablets, pills, capsules, injections (ampoules), and suppositories.

The pharmaceutical of the present invention is low-toxicity and can be administered orally or parenterally (e.g., intravascular administration, subcutaneous administration, etc.) to humans or mammals (e.g., rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.) as a liquid preparation or as a pharmaceutical composition in an appropriate dosage form.

(General technology)
The molecular biological, biochemical and microbiological techniques used herein are well known and commonly used in the art, and include those already cited, for example, Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, FM (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, FM (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, MA (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, FM (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, FM (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. 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, Academic Press, Special Edition of Experimental Medicine "Gene Introduction & Expression Analysis Experimental Methods" Yodosha, 1997, etc., and the relevant parts (which may be the entirety) of these are incorporated by reference in this specification.

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

For example, the oligonucleotides of the present invention can be synthesized by standard methods known in the art, such as by using an automated DNA synthesizer (such as those commercially available from Biosearch, Applied Biosystems, etc.). For example, phosphorothioate oligonucleotides can be synthesized by the method of Stein et al. (Stein et al., 1988, Nucl. Acids Res. 16:3209), and methylphosphonate oligonucleotides can be prepared by using controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. USA 85:7448-7451), etc.

In this specification, "or" is used when "at least one or more" of the items listed in the text can be adopted. The same applies to "alternative." In this specification, when it is stated that "within a range of two values," the range includes the two values themselves.

All references cited herein, including scientific literature, patents, patent applications, and the like, are hereby incorporated by reference in their entirety to the same extent as if each were specifically set forth herein.

The present invention has been described above by showing preferred embodiments for ease of understanding. The present invention will be described below based on examples, but the above description and the following examples are provided for illustrative purposes only and are not provided 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 this specification, but is limited only by the scope of the claims.

Example 1: Evaluation of immunological function in CARD14-deficient mice
In this example, CARD14-deficient mice were generated and their immunological functions were evaluated using two mouse psoriasis-like models, one induced by IMQ cream and the other by injection of recombinant IL-23 protein into the mouse ear.

Because little is known about the immunological role of CARD14 in psoriasis-like dermatitis in vivo, the inventors hypothesized that CARD14 may be involved in TLR7- and/or other innate immune receptor-mediated intracellular signaling pathways that result in activation of NF-κB in the cutaneous innate immune system.

Materials and Methods
(Mouse strain)
Card14 ^−/− (details described below and in Figure 2), Tlr7 ^−/− , Tlr9 ^−/− and Tlr7 ^−/− Tlr9 ^−/− double-deficient mice (Onishi et al., 2015) were bred in our animal facility and maintained under specific pathogen-free conditions. ^{Rag2 −/−} Il2rg ^−/− mice were obtained from Taconic Biosciences. ^{Tcrd −/−} mice were obtained from Jackson Laboratories. C57BL/6 (WT) mice were purchased from CLEA Japan, Inc. (Tokyo, Japan). All experiments were performed in accordance with the Institutional Ethical Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Biomedical Innovation, Health and Nutrition.

(Generation and genotyping of knockout mice)
Card14 ^−/− mice were generated by Lexicon Pharmaceuticals (The Woodlands, TX, USA) using a targeting vector designed to remove exon 2 and part of exon 3 by homologous recombination. PCR was used for routine mouse genotyping. The primers used were:
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 eight times onto the C57BL/6 background, as determined using a marker-assisted speed congenic method, resulting in >99% replacement of the B6 background genome in the N8 generation (Markel et al., 1997, Nature Genetics. 17:280-284).

(Psoriasis-like dermatitis model)
For induction of psoriasiform dermatitis, mice were treated with topical administration of Beselna (sold as Aldara ^™ outside Japan; 3M Pharmaceuticals, St. Paul, Minn.) cream (purchased from Mochida Pharmaceutical Co., Ltd.) containing 62.5 mg of 5% IMQ for six consecutive days as previously described (van der Fits et al., 2009).

Control mice were not treated with cream. Intradermal injections of 20 μl PBS, either alone or containing 500 ng of recombinant mouse IL-23 (BioLegend), were performed in both ears of anesthetized mice every other day for 5 days using a 30-gauge needle as previously described (Zheng et al., 2007). Ear measurements were taken at the center of the ear using a constant pressure pachymeter (PG-16J, Teclock, Japan). Mice were sacrificed at various time points and tissues were harvested.

(Generation of bone marrow chimeras)
BM cells ( ^5x106 ) were obtained by flushing the femurs and tibias of donor WT and Card14 ^-/- mice. Mice (WT and Card14 ^-/- ) received a lethal dose of X-rays (9 Gy) and were then reconstituted by intravenous injection of BM cells via the orbital plexus. Treatment with IMQ cream was performed 6 weeks after reconstitution.

(Histological examination of ear skin)
On the last day of the experiment, mice were sacrificed and ears were collected and fixed in 10N formalin for subsequent embedding in paraffin. Paraffin tissue sections (4 μm in diameter) were deparaffinized and stained with 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 rabbit anti-mouse monoclonal antibody against Ki67 (Thermo Fisher Scientific). Sections were treated in 0.01 M citric acid (adjusted to pH 6.0) for 10 min at 80°C before adding the primary antibody. The secondary antibody used was a peroxidase-labeled polymer conjugated to goat anti-rabbit immunoglobulin (Dako). 3,3'-Diaminobenzidine (Dako) was used as the chromogen, resulting in brown staining (Uchio et al., 2002, Lab Invest. 82:619-628). All slides were scanned at 20x absolute magnification using an Aperio ScanScope (Aperio Technologies).

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

(Preparation of ear cells)
Epidermal cell suspensions were prepared by incubating epidermis with trypsin-EDTA for 45 min at 37°C. A dermal suspension was obtained using a buffer containing 1.6 mg/mL collagenase IV (Worthington) and 0.1 mg/mL Dnase-I (Sigma) in complete RPMI medium (Nacalai Tesque). The cells were then filtered through a 70 μm pore size cell strainer (BD Falcon) to obtain a single cell suspension (Nakashima et al. The Journal of Allergy and Clinical Immunology. 134:100-107, 2014; Tokura et al., 1994, The Journal of Investigative Dermatology. 102:31-38).

RNA Extraction and Real-Time Quantitative PCR
The preserved cells were harvested by centrifugation at 400×g for 5 min. RNA was isolated using the Qiagen RNeasy kit (Qiagen). RNA was then reverse transcribed into cDNA using reverse transcriptase (ReverTra Ace, Toyobo, Osaka, Japan) and oligo-dT primers. The real-time PCR mixture consisted of 10 μL of SYBR Green Master mix (Roche), forward and reverse primers (200 nM for CARD14 and glyceraldehyde-3-phosphate dehydrogenase; GAPDH) and 2 μL of cDNA sample in a total volume of 20 μL. The primers for amplification were 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 reactions were performed for 40 cycles (95°C for 15 min, 60°C for 1 min) in a MicroAmp optical 384-well reaction plate of a LightCycler480 System II (Roche Life Science). The fluorescent signal detected during the threshold cycle (ΔCt) was recorded by the software installed in the instrument. To normalize the target gene levels for variations in RNA and cDNA quality, GAPDH was amplified under the same conditions as the internal control.

Flow cytometry analysis and cell sorting
The following antibodies were purchased from BDPharMingen: anti-CD8a (Ly-2, APC); anti-CD45 (30-F11, PerCP/Cy5.5); anti-mouse γδTCR (GL3, PE); anti-IL-17A (TC11-18H10, PE); and anti-NK1.1 (PK136, PE). The following antibodies from BioLegend were used: anti-CD3e (134-2C11, PerCP/Cy5.5); anti-CD4 (RM4-5, PE/Cy7); anti-CD11b (M1/70, FITC); anti-CD45 (30-F11, PE/Cy7); and anti-TCRβ (H57-597, PerCP/Cy5.5). The following antibodies were purchased from eBioscience: anti-CD19 (1D3, PacificBlue); anti-IL-22 (IL22JOP, APC); and anti-TCRγδ (GL3, APC). For FACS analysis, fixed areas of the ear were hollowed out using Trephine (8-mm, Kai Industries Co., Ltd.). Cells were preincubated with Fc-block (BioLegend) for 15 min and then labeled with appropriate cell surface antibodies for 30 min at 4°C. For intracellular cytokine staining, cells were stimulated with 50 ng/ml PMA (Sigma) and 500 ng/ml ionomycin (Sigma) and treated with GolgiStop (BD Bioscience) for 5 min at 37°C. For intracellular staining, cells were fixed and permeabilized with BD Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences) and then stained with antibodies for 30 min at 4°C. 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 Responses)
Intradermal injections of 20 μl PBS, either alone or containing 100 μg LPS-free OVA (Seikagaku), were performed on days 0 and 7. After blood collection, antibody titers were measured on day 14. Mice's ears were treated for 6 consecutive days with IMQ to induce psoriasiform dermatitis as previously described. OVA-specific antibodies were quantified using ELISA. Briefly, flat-bottom 96-well MaxiSorp plates (Nunc) were coated with 50 μg/mL OVA in 0.5 M NaHCO _3. Plates were incubated overnight at 4° C., washed with PBS containing 0.05% Tween® 20, and blocked for 1 h at room temperature. After washing, 50 μL of diluted serum was incubated for 2 h at room temperature. Plates were then washed and incubated with alkaline phosphate-conjugated goat anti-mouse _IgG1 and _IgG2C antibodies (1:4000, Southern Biotech) for 2 hours at room temperature. Plates were washed and then bound antibodies were visualized using TMB substrate solution (Kirkegaard & Perry Laboratories). The reaction was stopped by adding 2N _H2SO4 and absorbance at ₄₅₀ nm was read on a BioStack ^™ (BioTek Instruments Inc.) microplate reader using the Gen5 ELISA (Biotek®) program.

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

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

Next, we investigated the potential involvement of TLR7 and/or TLR9 using another established psoriasiform dermatitis model induced by administration of IL-23 (Zheng et al., 2007). Consistent with previous reports, injection of recombinant IL-23 on days 0, 2, and 4 resulted in a marked increase in ear thickness on day 5, accompanied by epidermal thickening and dermal inflammatory cell infiltration. All these changes were independent of TLR7 or TLR9 (Figures 1C and 1D). These results demonstrated that both TLR7 and TLR9 signaling in combination was necessary to fully develop IMQ-induced psoriasiform dermatitis, while the IL-23-induced psoriasiform dermatitis model directly activated downstream of TLR7- and TLR9-mediated innate immune activation. While it remains 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 demonstrated clear differences in innate immune signals between the IMQ-induced and IL-23-induced psoriasis-like models.

CARD14 is essential for the development of skin inflammation in both IMQ- and IL-23-induced psoriasis-like models.
To better characterize the role of CARD14 in vivo, we generated CARD14-deficient (Card14 ^−/− ) mice ( FIG. 2 ) and evaluated the role of CARD14 in both IMQ-induced and IL-23-induced psoriasis-like models (van der Fits et al., 2009 ; Zheng et al., 2007 ). We applied IMQ cream for 6 consecutive days or injected IL-23 every other day for 5 days. WT mice had a worsened skin condition and increased skin thickness after application of IMQ, whereas Card14 ^−/− mice were significantly protected from skin inflammation ( FIG. 3A ). Histochemical experiments revealed that hyperproliferative cells stained for keratin 5 and Ki67 in the ear skin of Card14 ^−/− mice were similar to those in control mice, whereas hyperproliferative cells in WT mice receiving IMQ application were strongly increased (Fig. 3B). Furthermore, IL-23-induced ear swelling was significantly suppressed in Card14 ^−/− mice (Fig. 3C). Epidermal hyperkeratinization (seen as hyperproliferative cells stained for 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 indicate that CARD14 is essential for the formation of psoriasis-like dermatitis induced by the application of IMQ cream and the injection of IL-23. Thus, we conclude that CARD14 is required for downstream signaling of TLR7 and TLR9, as well as downstream signaling of IL-23, which does not require TLR7 or TLR9, and is therefore relevant to signaling pathways in adaptive rather than innate immune cells.

CARD14 in hematopoietic cells, but not in radioresistant skin-resident cells, plays a key role in psoriasis.
Although CARD14 has been shown to be important for innate immunity in keratinocytes (Fuchs-Telem et al., 2012; Jordan et al., 2012), our results above 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 generated reciprocal bone marrow (BM) chimeras and further evaluated the role of CARD14 using an IMQ-induced psoriasis-like model. BM cells from WT and Card14 ^−/− mice were transplanted into X-ray irradiated Card14 ^−/− and WT mice, respectively. After 6 weeks, the mice were treated bilaterally with IMQ cream for 6 consecutive days. Psoriasis-like inflammation was induced in Card14 ^−/− and WT mice transplanted with BM from WT mice, but not in irradiated WT or Card14 ^−/− mice transplanted with Card14 − ^/− BM (FIG. 4A). This indicates that CARD14 expression in hematopoietic BM-derived cells was essential for the pathogenesis of psoriasis in the IMQ-induced model, but not in radioresistant skin-resident cells. In addition to RAG genes, we uncovered the potential contribution of T and B cells regulated by natural killer (NK) cells, natural killer T (NKT) cells, innate lymphoid cells (ILCs), and γδT cells. We compared the severity of psoriasis-like models in T cell receptor delta-deficient (Tcrd ^-/- ) mice, which lack gamma delta T cells, and Rag2 ^-/- Il2rg ^-/- mice, which lack T cells, NK cells, NKT cells, and ILCs. Consistent with previous reports (Pantelyushin et al., 2012, The Journal of Clinical Investigation.122:2252-2256), Tcrd ^-/- and Rag2 ^-/- Il2rg ^-/- mice showed a marked reduction in skin swelling after application of IMQ cream compared to WT mice (Figure 4B). Although we cannot exclude the possibility that CARD14 in non-hematopoietic cells, including keratinocytes, plays a role in psoriasis-like inflammation, the above results strongly suggest that CARD14 in BM-derived cells, potentially γδ T cells, is required for the development of IMQ-induced psoriasis-like dermatitis.

CARD14 is expressed in γδ T cells and is required for IL-17 and IL-22 production by γδ T cells.
To test CARD14 expression in several different cell types, cells from ear skin were isolated and mRNA was measured using real-time quantitative polymerase chain reaction (qPCR). Consistent with previous reports (Jordan et al., 2012), CARD14 mRNA was expressed at high levels in keratinocytes (Figure 5A). Among immune cells, CARD14 mRNA in γδ T cells of ear skin was as high as in keratinocytes and higher than other immune cell types (Figures 5A and 5B). To further define the cell type expressing CARD14, we performed confocal microscopy analysis of ear skin using antibodies against CARD14 and T cell receptor γδ (TCRγδ) 5 days after administration of IL-23. CARD14-positive signals were detected in the epidermal region of WT mice receiving IL-23, whereas no CARD14-positive signals were detected in Card14 ^-/- mice receiving IL-23. In addition, TCRγδ-positive signals were detected in the peripheral region between the epidermis and dermis in both WT and Card14 ^-/- mice, and specific TCRγδ-positive signals overlapped with CARD14-positive signals expressed in the cytoplasm, whereas no overlapping regions were detected in Card14 ^-/- mice (FIG. 5C). These data indicate that CARD14-positive γδ T cells are present in psoriasiform skin.

It has been shown that γδ T cells, which produce IL-17 and IL-22, play an important role in the pathogenesis of psoriasis (Cai et al., 2011). It has also been reported that the majority of IL-23 receptor (IL-23R) expressing cells are γδ T cells, and that IL-23R mutations have been found in certain psoriasis patients (Cai et al., 2011; Tsoi et al., 2012). Since IMQ-induced dermatitis and IL-23-induced dermatitis are closely related to the production of IL-17 and IL-23 (Cai et al., 2011; Pantelyushin et al., 2012, The Journal of Clinical Investigation.122:2252-2256; Zheng et al., 2007), we examined IL-17- and IL-22-producing cells recovered from epidermal sheets of WT and Card14 ^−/− mice receiving IMQ or IL-23 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 and TCRγδ ⁺ IL-22 cells than the control group. In contrast, both IMQ- and IL-23-treated Card14 ^{−/ −} mice had significantly fewer epidermal γδ T cells producing IL-17 and IL-22 (Figures 5D and 5E). These data suggest that CARD14 may play an important role in γδ T cells secreting IL-17 and IL-22 in epidermal lesions of psoriatic skin.

CARD14 does not interfere with the adjuvant effect of IMQ on co-administered antigens
Since IMQ is an agonist of TLR7, a commonly used vaccine adjuvant in many other applications, we were interested in whether CARD14 would affect the adjuvant effect of IMQ on co-administered antigens. To address this question, we modified the experimental protocol of a psoriasis-like dermatitis model using IMQ cream, immunizing WT, Tlr7 ^−/− Tlr9 ^−/− and Card14 ^−/− mice with ovalbumin (OVA) via intradermal injection in both ears, while applying IMQ cream topically to the same ears. An increase in ear thickness was observed in WT mice, while Tlr7 ^−/− Tlr9 ^−/− and Card14 ^−/− mice consistently showed significantly thinner ear thickness at day 7, even when the mice were injected with OVA protein (Figure 6A). WT mice were treated with IMQ cream along with OVA immunization and showed significantly higher OVA-specific immunoglobulin (IgG) ₁ and IgG _2c antibody responses compared to the untreated OVA-immunized group, as expected (Fig. 6B). OVA-specific IgG _2c antibody titers were significantly reduced in Tlr7 ^−/− Tlr9 ^−/− mice, whereas Card14 ^−/− mice showed serum anti-OVA IgG _2c levels comparable to those of WT mice (Fig. 6B). These results indicate that IMQ cream has an adjuvant effect on the co-administered protein antigen and that CARD14 does not affect the adjuvant effect. Since IMQ-induced psoriasis is a complication of IMQ cream, developing a CARD14 inhibitor may suppress the pathogenesis of psoriasiform dermatitis without affecting IMQ-induced innate immune function and its adjuvant effect.

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

CARD14 and CARD11 have been shown to be novel MAGUK family members that function as upstream activators of BCL10 and NF-κB signaling (Bertin et al., 2001). CARD14 is expressed in epithelial cells of skin lesions in psoriasis patients (Harden et al., 2014, PloS One. 9:e111255), and we initially hypothesized that CARD14 is a downstream signaling molecule for TLR7-mediated NF-κB activation in epithelial stromal cells such as keratinocytes. However, our results above demonstrate that TLR7 and TLR9 are required for IMQ-induced psoriasiform dermatitis, but not for IL-23-induced psoriasiform dermatitis (Figure 1), while CARD14 is important for both IMQ-induced and IL-23-induced psoriasiform dermatitis (Figure 3). These data suggest that our initial hypothesis was questionable, and rather, CARD14 is downstream of IL-23, meaning that it seems 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 the CARD14-mediated pathogenesis of psoriasiform dermatitis. Although it is not clear which cells express IL-23R, it has been shown that most cells expressing IL-23R are γδ T cells (Awasthi et al., 2009, Journal of Immunology.182:5904-5908.). Our results demonstrate that CARD14 is required for IMQ- and IL-23-mediated production of IL-17 and IL-22 in TCRγδ T cells (Figures 5D and 5E), but further studies are needed to clarify how CARD14 is involved in these intracellular signaling pathways. We have shown that CARD14 is required for psoriatic pathogenesis through suppression of IL-17- and IL-22-producing γδ T cells in two established psoriasis-like dermatitis models.

We found that CARD14 is expressed in γδ T cells in psoriasis-like skin rashes in addition to its expression in epidermal keratinocytes (Figures 5A and 5C). It is known that experimental conditions differ between human patient samples and experimental mouse models, and the amount of TCRγδ cells differs in humans and mice (Cai et al., 2011; Pasparakis et al., 2014, Nature reviews. Immunology. 14:289-301). Thus, the role of CARD14 in humans and mice (including animal models) may differ with respect to either CARD14-expressing cell types or experimental conditions. Further studies are needed to clarify the role of CARD14 in human γδ T cells in psoriasis.

CARD14 may be a good target to reduce the pathogenesis of psoriasiform dermatitis and side effects caused by IMQ-based adjuvants or other TLR-based or immunostimulatory adjuvants. It inhibits innate immune activation without suppressing adaptive immune responses specific to the co-administered antigen. The immunological significance of CARD14-mediated innate and adaptive immune responses should be studied. γδ T cells play an important role in various immune disorders such as multiple sclerosis (Sutton et al., 2009, Immunity. 31:331-341) and other infectious diseases (Carding and Egan, 2002, Nature reviews. Immunology. 2:336-345.; Sutton et al.,2012, European Journal of Immunology. 42:2221-2231).

Example 2: Multiple Sclerosis
Next, in this example, we investigated whether CARD14 deficiency reduces symptoms in an experimental demyelinating disease model of multiple sclerosis. This example was basically performed based on the description in the literature by Rui et al. (Yuxiang Rui, Tasuku Honjo, and Shunsuke Chikuma. Programmed cell death 1 inhibits inflammatory helper T-cell development through controlling the innate immune response. PNAS 2013 110 (40) 16073-16078).

The experimental autoimmune encephalomyelitis (EAE) used in this example is an autoimmune disease model induced by immunization with protein antigens or peptides derived from central nervous tissue, and shares many pathological conditions with multiple sclerosis (MS). Therefore, EAE is commonly used in research on the pathology of MS and in the development of treatments and therapeutic agents.

(mouse)
CARD14 ^−/− mice were generated by gene knockout according to the method of Rui et al.

(EAE mice)
Mice were immunized subcutaneously (sc) with 0.2 mL of an emulsion containing 200 μg of MOG _35-55 peptide (peptide corresponding to MOG35-55 residues), complete Freund's adjuvant (CFA), and 250 μg of heat-killed mycobacteria (HKMTB) according to Rui et al. In addition, mice received two intraperitoneal (ip) injections of 200 ng of pertussis toxin (PTX) at the time of immunization and 48 hours later. In some experiments, suboptimal immunization conditions were used, omitting HKMTB, PTX, or both. Mice were observed daily for clinical signs of disease and EAE scores were assessed as follows:
0, asymptomatic (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 presented as the mean clinical score for each experimental group.

For restimulation assays, spleens were harvested 8 or 30 days after immunization and CD4 ⁺ T cell enriched samples were obtained using anti-CD4 microbeads and the autoMACS system (Miltenyi). In a volume of 1 mL, 2×10 ⁶ isolated CD4 ⁺ T cells were cultured with 2×10 ⁶ mitomycin C-treated splenocytes from CARD-14+/+ mice in the presence of MOG _35-55 peptide (30 μg/mL). Supernatants were harvested 3 days later for cytokine analysis.

Figure 7 shows the 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 (left). The right side shows the incidence (incidence) of CARD14 deficiency, which reduced symptoms in an experimental demyelinating disease model, indicating that CARD14 plays an important role in multiple sclerosis.

Example 3: Screening
In this example, screening is performed for therapeutic or preventive agents for psoriasis, therapeutic or preventive agents for multiple sclerosis, therapeutic or preventive agents for reducing side effects caused by TLR-based/IMQ/immunostimulatory adjuvants, and therapeutic or preventive agents for γδ T cell-mediated diseases and/or complications of immune intervention.

The inhibitory activity against CARD14 is measured by any method known in the art, the inhibitory activity of the candidate substance is measured, and those exhibiting inhibitory activity are identified as inhibitors.

These inhibitors can be administered in models of psoriasis, multiple sclerosis, side effects of TLR-based/IMQ/immunostimulatory adjuvants, or complications of γδ T cell-mediated diseases and/or immune interventions described in Examples 1 or 2, and symptoms, etc., can be monitored over time, and those that can prevent or treat symptoms can be identified as prophylactic or therapeutic agents for these diseases, disorders, or symptoms.

(References: The documents listed are not included as an admission that they are prior art to the present invention.)
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As described above, the present invention has been illustrated using preferred embodiments of the present invention, but it is understood that the scope of the present invention should be interpreted only by the claims. It is understood that the patents, patent applications, and literature cited in this specification should be incorporated by reference to this specification in the same manner as if the contents themselves were specifically set forth in this specification. This application claims priority to Japanese Patent Application No. 2015-200948 filed in Japan on October 9, 2015, the contents of which are incorporated by reference in their entirety in this specification.

This invention has industrial applicability in the biopharmaceutical and pharmaceutical industries.

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 examples.