JP6852926B2 - Disease activity determination / prognosis evaluation of preventive / therapeutic agents for diseases related to cell migration regulation and diseases of lung interstitium - Google Patents

Description

The present invention relates to a regulator of cell migration (including cell infiltration), a preventive / therapeutic agent for a disease related to cell migration, and a screening method for a preventive / therapeutic agent for a disease related to cell migration. More specifically, a prophylactic and / or therapeutic agent for a disease related to cell migration, which contains a substance that inhibits the function of leucine-rich α2 glycoprotein, and a cell migration regulator using the inhibition of the function of leucine-rich α2 glycoprotein as an index. , Related to the method of screening candidate substances for disease prevention / therapeutic agents related to cell migration. Further, the present invention relates to a method for determining disease activity and a method for evaluating prognosis of a disease (for example, interstitial pneumonia) using leucine-rich α2 glycoprotein as a marker of disease activity.

Previously, in diseases related to angiogenesis, the concentration of leucine rich alpha 2 glycoprotein (LRG) (hereinafter, also referred to as LRG) in blood became a disease marker, and conventionally It was reported that LRG can be an excellent marker of disease activity because it correlates more strongly with these disease activities than C-reactive protein (hereinafter, also referred to as CRP), which has been used as an inflammation marker (hereinafter, also referred to as CRP). Patent Document 1, Non-Patent Documents 1 and 2). The present inventors have also reported that quantifying the serum LRG concentration can be a biomarker for tuberculosis testing (Patent Document 2).

However, the function of LRG in the above diseases has not yet been clarified, and it remains completely unclear whether LRG can be a therapeutic target in relation to other than angiogenesis.

Japanese Unexamined Patent Publication No. 2010-286279 Japanese Unexamined Patent Publication No. 2013-213774

Serada S et al., Ann Rheum Dis, Vol. 69, pages 770-774, 2010 Serada S et al., Inflamm Bowel Dis, Vol.18, pages 2169-2179, 2012

As a result of diligent studies, the present inventors conducted experiments on cell infiltration and cell migration, found that LRG is related to regulation of cell invasion and cell migration, and that LRG functions as a cell invasion and cell migration promoter. Therefore, in diseases related to cell infiltration and cell migration, especially in the acute phase (onset stage, acute exacerbation stage, etc.) of diseases such as inflammatory diseases and immune diseases, especially in the acute phase of inflammation (onset stage, acute exacerbation stage, etc.) By finding that it acts on the above, it was found that the LRG inhibitor can be provided as a therapeutic agent or a preventive agent for the acute phase (onset phase, acute exacerbation phase, etc.) of these diseases, and the present invention is completed. I let you.

Bleomycin-induced interstitial pneumonia was induced in LRG-deficient mice and wild-type mice, and the pathophysiology of both was compared. As a result, it was found that in LRG-deficient mice, neutrophil infiltration was significantly reduced and inflammation was milder than in wild-type mice. These results indicate that LRG is involved in the pathophysiology of acute stages (onset stage, acute exacerbation stage, etc.) of diseases such as inflammatory diseases and immune diseases, especially in the acute stage of inflammation (onset stage, acute exacerbation stage, etc.). It shows that.

Therefore, an object of the present invention is to clarify the function of LRG in the acute phase (onset stage, acute exacerbation stage, etc.) of diseases such as inflammatory disease and immune disease, especially in the acute phase of inflammation (onset stage, acute exacerbation stage, etc.). The acute phase (onset stage, acute exacerbation stage, etc.) of these inflammatory diseases, immune diseases, etc. that target LRG based on the function, especially the acute stage of inflammation (onset stage and acute exacerbation). Providing preventive / therapeutic agents for the stage, etc., and using the functional regulation of LRG as an index, the acute stage (onset stage, acute exacerbation stage, etc.) of new inflammatory diseases, immune diseases, etc., especially acute inflammation It is to provide a means for searching for substances having prophylactic and / or therapeutic activity in the stage (onset stage, acute exacerbation stage, etc.).

Furthermore, the present inventors have clarified that LRG is highly expressed in patients with interstitial pneumonia, and found that LRG can be a marker for interstitial pneumonia.

Interstitial pneumonia is a disease in which bilateral diffuse shadows are observed on chest radiographs as a widespread diffuse lung disease, in which the interstitium of the lung is the site of inflammation. Although KL-6 has been put into practical use as a diagnostic marker for interstitial pneumonia, KL-6 is not a universal marker, and further development of a marker has been desired. It has become clear that it can be developed as a marker that can accurately evaluate the disease activity of interstitial pneumonia.

As a result of further studies based on these findings, the present inventors have completed the present invention.

That is, the present invention is as follows.
<Cell migration inhibitor>
(1) A cell migration inhibitor containing an inhibitor (LRG inhibitor) of leucine-rich α2 glycoprotein (LRG).
(2) The agent according to item (1), wherein the LRG inhibitor contains a substance that inhibits the expression or function of leucine-rich α2 glycoprotein (LRG).
(3) The LRG expression inhibitor is
(A) Antisense nucleic acid against transcripts of LRG gene,
(B) Ribozyme nucleic acid for the transcript of the LRG gene, or (c) Nucleic acid or precursor thereof having RNAi activity for the transcript of the LRG gene.
Item (2).
(4) The agent according to item (2) or (3), wherein the LRG function inhibitor is an antibody against LRG.
(5) The agent according to any one of items (1) to (4), wherein the cell migration inhibitor is for treatment and / or prevention in the acute phase of a disease.
(6) The agent according to item (5), wherein the treatment and / or prevention targets inflammation in the disease.
(7) The agent according to item (5) or (6), wherein the disease is an inflammatory disease or an immune disease.
(8) The diseases include inflammatory bowel disease (eg, ulcerative colitis, Crohn's disease, etc.), autoinflammatory disease (eg, Bechette's disease, adult Still's disease, systemic juvenile idiopathic arthritis, cryopyrin-related periodic fever syndrome). , Familial Mediterranean fever, etc.), Collagenosis / rheumatic diseases (eg, vasculitis syndrome, polymyositis, dermatomyositis, systemic erythematosus, scleroderma, rheumatoid arthritis, tonic spondylitis, psoriatic arthritis, etc.), And other inflammatory autoimmune diseases (eg, idiopathic interstitial pneumonia, irritable pneumonia, glomerular nephritis, etc.), which are selected from the group consisting of any of items (5) to (7). The agent according to item 1.
<Acute pharmaceutical composition>
(A1) A pharmaceutical composition containing an inhibitor (LRG inhibitor) of leucine-rich α2 glycoprotein (LRG) for treatment and / or prevention in the acute phase of a disease.
(A2) The pharmaceutical composition according to item (A1), wherein the treatment and / or prevention targets inflammation in the disease.
(A3) The pharmaceutical composition according to item (A1) or (A2), wherein the disease is an inflammatory disease or an immune disease.
(A4) The diseases include inflammatory bowel disease (eg, ulcerative colitis, Crohn's disease, etc.), autoinflammatory disease (eg, Bechette's disease, adult Still's disease, systemic juvenile idiopathic arthritis, cryopyrin-related periodic fever syndrome). , Familial Mediterranean fever, etc.), Collagenosis / rheumatic diseases (eg, vasculitis syndrome, polymyositis, dermatomyositis, systemic erythematosus, scleroderma, rheumatoid arthritis, tonic spondylitis, psoriatic arthritis, etc.), The pharmaceutical composition according to any one of items (A1) to (A3), which is an idiopathic interstitial pneumonia, irritable pneumonia, glomerular nephritis, etc., and other inflammatory autoimmune diseases (eg, idiopathic interstitial pneumonia, irritable pneumonia, glomerular nephritis, etc.). Stuff.
(A5) The pharmaceutical composition according to any one of Items (A1) to (A4), wherein the LRG inhibitor contains a substance that inhibits the expression or function of leucine-rich α2 glycoprotein (LRG).
(A6) The LRG expression inhibitor is
(A) Antisense nucleic acid against transcripts of LRG gene,
The pharmaceutical composition according to item (A5), which is (b) a ribozyme nucleic acid for a transcript of the LRG gene, or (c) a nucleic acid having RNAi activity for a transcript of the LRG gene or a precursor thereof.
(A7) The pharmaceutical composition according to item (A5) or (A6), wherein the function inhibitor of LRG is an antibody against LRG.
<Screening>
(X1) The following steps (A) to (C):
(A) A step of contacting a cell containing a nucleic acid encoding an LRG gene or a reporter protein under the control of the transcriptional regulatory region of the gene with a test substance;
(B) The step of measuring the expression level of the LRG gene or LRG protein or reporter protein in the cells; and (C) the LRG gene or LRG protein or reporter protein as compared with the case of measuring in the absence of the test substance. For treatment and / or prevention of disease in the acute phase, including the step of selecting a test substance with reduced expression as a candidate for a substance used for treatment and / or prevention of the disease in the acute phase of the disease. A screening method for substances used in.
(X2) The LRG is brought into contact with the test substance, and the test substance having the ability to bind to the LRG is selected as a candidate substance used for treatment and / or prevention in the acute phase of the disease. , A method of screening substances used for treatment and / or prevention in the acute phase of a disease.
(X3) The following steps (A) to (C):
(A) A step of contacting LRG with the cell membrane fraction of target cells in the acute phase of inflammatory disease in the presence of a test substance;
(B) A step of measuring the amount of LRG bound to the cell membrane fraction; and (C) A test in which the amount of LRG bound to the cell membrane fraction is reduced as compared with the case of measuring in the absence of the test substance. Item 5. The screening method according to Item (X1) or (X2), which comprises the step of selecting a substance as a candidate for a substance used for treatment and / or prevention in the acute phase of a disease.
(X4) A test substance selected as a candidate for a substance used for treatment and / or prevention in the acute phase of a disease is applied to an inflammatory phase in the acute phase, and the inflammatory response in the acute phase in the model is suppressed. The method according to any one of paragraphs (X1) to (X3), further comprising testing whether or not.
(X5) The following steps (A) to (C):
(A) A step of contacting a target cell in the acute phase of an inflammatory disease with a test substance in the presence and absence of LRG;
(B) The step of measuring the degree of inflammatory reaction in the cells under each condition; and (C) Suppressing the acute inflammatory reaction in the presence of LRG as compared with the case of measuring in the absence of the test substance. However, test substances that did not suppress the acute inflammatory response in the absence of LRG are selected as candidates for substances that inhibit the function of LRG and are used for treatment and / or prevention in the acute phase of the disease. A method of screening for substances used for treatment and / or prevention in the acute phase of a disease, including steps.
(X6) The method according to item (X5), further comprising a step of inducing an inflammatory response in the target cell.
(X7) The method according to item (X5) or (X6), which elicits an inflammatory reaction in the acute phase by stimulation with _{TNFα or H 2} O _2.
(X8) The method according to any one of (X5) to (X7), wherein the target cell is an intestinal epithelial cell, a synovial cell, an alveolar epithelial cell or a bronchial epithelial cell.
<Test for disease activity of lung interstitium>
(Y24) A method for determining the disease activity of a lung interstitial disease in a subject, which includes a step of measuring the concentration of leucine-rich α2 glycoprotein (LRG) in a biological sample derived from the subject. A determination method in which the disease activity of a lung interstitial disease of the subject is evaluated based on the concentration of the LRG.
(Y25) The determination method according to item (Y24), wherein when the concentration of the LRG is higher than that of a healthy person, it is determined that the subject has high disease activity of the lung interstitial disease.
(Y26) The determination method according to item (Y25), wherein the LRG concentration of the healthy person is 3 μg / ml.
(Y27) The determination method according to any one of items (Y24) to (Y26), wherein the disease of the lung interstitium is interstitial pneumonia or pulmonary fibrosis.
(Y28) The pulmonary interstitial disease is idiopathic pulmonary fibrosis (IPF), chronic hypersensitivity pneumonia (CHP), collagen disease-related interstitial pneumonia (CVD-IP), idiopathic non-specific interstitial pneumonia. (NSIP), interstitial pneumonia with dermatitis, exfoliative interstitial pneumonia, lymphocytic interstitial pneumonia, diffuse alveolar disorder, or granulomatous interstitial pneumonia, items (Y24)-( The determination method according to any one of Y27).
(Y29) The determination method according to any one of Items (Y24) to (Y28), wherein the concentration of the LRG is measured by contacting the biological sample with a reagent capable of binding to the LRG. ..
(Y30) The determination method according to item (Y29), wherein the reagent is an anti-LRG antibody or a fragment thereof.
(Y31) The determination method according to any one of Items (Y24) to (Y30), wherein the biological sample is selected from the group consisting of serum and plasma.
(Y32) A determination for determining disease activity of lung interstitial disease, including a reagent capable of binding to leucine-rich α2 glycoprotein (LRG).
(Y33) The determination agent according to item (Y32), wherein the disease of the lung interstitium is interstitial pneumonia or pulmonary fibrosis.
(Y34) The pulmonary interstitial disease is idiopathic pulmonary fibrosis (IPF), chronic hypersensitive pneumonia (CHP), collagen disease-related interstitial pneumonia (CVD-IP), idiopathic non-specific interstitial pneumonia. (NSIP), interstitial pneumonia with dermatitis, exfoliative interstitial pneumonia, lymphocytic interstitial pneumonia, diffuse alveolar disorder, or granulomatous interstitial pneumonia, item (Y32) or ( The determination agent according to Y33).
(Y35) The determination agent according to any one of Items (Y32) to (Y34), wherein the reagent can bind to a labeled or labeled molecule.
(Y36) The determination agent according to any one of Items (Y32) to (Y35), wherein the reagent is an anti-LRG antibody or a fragment thereof.
(Y37) A kit for detecting and / or quantifying leucine-rich α2 glycoprotein (LRG), which comprises the determination agent according to any one of (Y32) to (Y36).
(Y38) The kit according to item (Y37), further comprising a secondary antibody capable of binding to the reagent.
(Y39) A method for assessing the prognosis of lung interstitial disease, derived from a subject who has or is determined to be at risk of having the lung interstitial disease. It includes the step of measuring the concentration of leucine-rich α2 glycoprotein (LRG) in a biological sample, determines the disease activity of the lung interstitium based on the concentration of the LRG, and determines the disease activity of the lung interstitium based on the disease activity. A method by which the prognosis of interstitial disease is determined.
(Y40) The method according to item (Y39), wherein the prognosis of the disease of the lung interstitium is determined based on the disease activity determined by comparing the concentration of the LRG with the concentration of LRG before treatment. ..
(Y41) The method according to item (Y39) or item (Y40), wherein the disease of the lung interstitium is interstitial pneumonia or pulmonary fibrosis.
(Y42) The pulmonary interstitial disease is idiopathic pulmonary fibrosis (IPF), chronic hypersensitivity pneumonia (CHP), collagen disease-related interstitial pneumonia (CVD-IP), idiopathic non-specific interstitial pneumonia. (NSIP), interstitial pneumonia with dermatitis, exfoliative interstitial pneumonia, lymphocytic interstitial pneumonia, diffuse alveolar disorder, or granulomatous interstitial pneumonia, item (Y39) or The method according to (Y40). (Y43) The method according to any one of (Y39) to (Y42), wherein the concentration of the LRG is measured by contacting the biological sample with a reagent capable of binding to the LRG.
(Y44) The method according to item (Y43), wherein the reagent is an anti-LRG antibody or a fragment thereof.
(Y45) The method according to any one of Items (Y39) to (Y44), wherein the biological sample is selected from the group consisting of serum and plasma.
(Y46) A prognostic agent for determining the prognosis of a disease of lung interstitium, which comprises a reagent capable of binding to leucine-rich α2 glycoprotein (LRG).
(Y47) The prognosis-determining agent according to item (Y46), wherein the disease of the lung interstitium is interstitial pneumonia or pulmonary fibrosis.
(Y48) The pulmonary interstitial disease is idiopathic pulmonary fibrosis (IPF), chronic hypersensitive pneumonia (CHP), collagen disease-related interstitial pneumonia (CVD-IP), idiopathic non-specific interstitial pneumonia. (NSIP), interstitial pneumonia with dermatitis, exfoliative interstitial pneumonia, lymphocytic interstitial pneumonia, diffuse alveolar disorder, or granulomatous interstitial pneumonia, item (Y46) or ( Y47). The prognosis determining agent. (Y49) The prognostic agent according to any one of Items (Y46) to (Y48), wherein the reagent can bind to a labeled or labeled molecule.
(Y50) The prognosis-determining agent according to any one of Items (Y46) to (Y49), wherein the reagent is an anti-LRG antibody or a fragment thereof.
(Y51) A kit for detecting and / or quantifying leucine-rich α2 glycoprotein (LRG), which comprises the prognostic agent according to any one of (Y46) to (Y50).
(Y52) The kit according to item (Y51), further comprising a secondary antibody capable of binding to the reagent.

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

Since the present invention has revealed that LRG is involved in the exacerbation of pathological conditions of diseases related to cell migration, it is related to cell migration by, for example, inhibiting the expression or function of LRG using an LRG inhibitor. Diseases, disorders, conditions, etc. (for example, acute phase (onset phase, acute exacerbation phase, etc.) of diseases such as inflammatory diseases and immune diseases), especially acute phase of inflammation (onset phase, acute exacerbation phase, etc.) Can be treated or prevented. In addition, an inhibitor of LRG, for example, a substance used for treatment and / or prevention in the acute phase of a disease using the expression or function inhibition of LRG as an index, and thus a disease related to cell migration, particularly a disease. Therapeutic or prophylactic agents of substances used for treatment and / or prevention in the acute phase can be screened. In addition, the present invention allows LRG to be used as a marker for interstitial pneumonia.

FIG. 1 shows that LRG is highly expressed in neutrophils and Resident macrophages. (Left) LRG relative expression is seen in each tissue. Spleen cells, spleen-derived TB cells, peripheral blood-derived neutrophils (Gr1-positive), peritoneal macrophages, and liver were collected from wild-type mice, and mRNA was extracted. The expression of LRG was examined by real time PCR. The expression level of LRG was corrected by the expression of HPRT1. From the left, the figure shows splenocytes, splenocyte-derived T cells, B cells, peripheral blood-derived neutrophils (Gr1 positive), peritoneal macrophages, and liver. (Right) The expression of LRG protein was observed by Western blotting. Liver and bone marrow-derived neutrophils were collected from wild-type mice and proteins were extracted. The protein lysate was subjected to sugar chain cleavage treatment (GPF +) with glycopeptidase F (Takara), and then the change in the molecular weight of LRG was compared with the untreated sample by Western blotting. The figure shows the liver in the left two columns and the neutrophils in the right two columns, respectively, from the left, with no GPF and with GPF treatment. It can be seen that there is a difference in sugar chain modification between LRG produced by neutrophils and macrophages and LRG produced by the liver. Highly glycosylated LRG was prominently detected in neutrophils but not in serum, liver and colon. FIG. 2 shows that LRG is abundant in neutrophil granules. LRG in human neutrophils was examined by immunoelectron microscopy. Black particles (gold colloidal particles) such as the tip of the arrow indicate LRG. The lower left figure is a reduced view, and the lower right is an enlarged photograph. In the lower left figure, the circled area indicates neutrophils, and the black particles at the tip of the arrow indicate gold colloidal particles, where LRG is localized. FIG. 3 shows that LRG is expressed on alveolar and bronchial epithelial cells in bleomycin-induced interstitial pneumonia. Wild-type mice (C57BL / 6, female) were anesthetized and bleomycin (1.5 mg / kg) was intratracheally administered to induce interstitial pneumonia and pulmonary fibrosis. Lungs were collected from mice in the number of days shown in the figure after administration, sections were prepared after extension and fixation, and LRG expression was examined by immunohistochemical staining. The upper left shows control, the upper right shows 7 days after bleomycin stimulation, the lower left shows 14 days after bleomycin stimulation, and the lower right shows 21 days after bleomycin stimulation. FIG. 4 shows that in bleomycin-induced interstitial pneumonia, inflammation is mild in LRG knockout mice. This is the result of inducing interstitial pneumonia in mice by intratracheal bleomycin administration. (Left) Changes in body weight over time. ■ (black square) indicates knockout (KO), and ● (black circle) indicates wild type (WT). LRG knockout mice had reduced weight loss after bleomycin administration compared to wild-type mice. (Right) The expression level of TNFα. The expression level of TNFα was examined by extracting mRNA from the lung and examining the expression of TNFα by real-time PCR. In the figure, it is shown in unstimulated (before stimulation) (-) (left), day 3 (center), and day 7 (right). LRG knockout mice had reduced lung expression of the inflammatory cytokine TNFα compared to wild-type mice. FIG. 5 shows that neutrophil infiltration is significantly reduced in LRG knockout mice in bleomycin-induced interstitial pneumonia. Mice treated with bleomycin were analyzed for the number of days indicated. Alveolar lavage fluid (BALF) was collected from each individual mouse using PBS (0.5 mL x 3 times), the number of cells in the fluid was counted, and the cell fraction was analyzed by FACS. (Left) The total number of cells in BALF, the left is the 1st day and the right is the 7th day. Numerical is a cell number (× 10 ^4). (Right) The number of neutrophils (Ly6G +) in BALF, the left is the 1st day and the right is the 7th day. Numerical is a cell number (× 10 ^4). FIG. 6 shows low collagen expression in LRG knockout mice in bleomycin-induced interstitial pneumonia. The results of extracting mRNA from the lungs of mice to which bleomycin was administered and examining the expression of collagen 1α2 by real-time PCR are shown. The y-axis shows the relative value, and the control is shown from the left, and the 3rd day, the 7th day, and the 14th day are shown. FIG. 7 shows that the LRG concentration in the serum of patients with interstitial pneumonia (IP) is higher than that in healthy subjects. The graph on the left shows serum LRG levels in all patients with interstitial pneumonia and in healthy individuals (HC). Statistical analysis was performed using the Mann-Whitney U test. The graph on the right shows chronic hypersensitivity pneumonitis (CHP), collagen disease-related interstitial pneumonitis (CVD-IP), idiopathic pulmonary fibrosis (IPF), idiopathic nonspecific interstitial pneumonitis (NSIP), and healthy subjects. It shows the LRG concentration in serum in (HC). The vertical axis shows the concentration of LRG (μg / ml). Statistical analysis was performed using the Kruskal-Wallis test followed by the Steel test. FIG. 8 shows the LRG concentration and the KL-6 concentration in the serum of IP patients. Each graph shows the serum LRG and KL-6 concentrations of CHP (upper left), CVD-IP (upper right), IPF (lower left) and NSIP (lower right). The vertical axis shows the concentration of LRG (μg / ml), and the horizontal axis shows the concentration of KL-6 (U / ml). LRG levels correlate with KL-6 levels in the serum of IPF patients. FIG. 9 shows the LRG concentration and% DLCO in the serum of IP patients. The upper graph shows serum LRG concentration and% DLCO in CVD-IP (left) and NSIP (right). The lower graph shows serum KL-6 concentration and% DLCO in CVD-IP (left) and NSIP (right). The vertical axis shows LRG concentration (μg / ml) or KL-6 (U / ml), and the horizontal axis shows% DLCO. LRG concentrations in the serum of CVD-IP and NSIP patients correlate with% DLCO. FIG. 10 shows the LRG concentration and% VC in the serum of IP patients. The upper graph shows serum LRG concentration and% VC in NSIP. The lower graph shows serum KL-6 concentration and% VC in NSIP. The vertical axis shows LRG concentration (μg / ml) or KL-6 (U / ml), and the horizontal axis shows% VC. LRG levels correlate with% VC in NSIP patient sera. FIG. 11 shows the LRG concentration and VC in the serum of IP patients. The upper graph shows serum LRG concentration and VC in NSIP. The lower graph shows serum KL-6 concentration and VC in NSIP. The vertical axis shows LRG concentration (μg / ml) or KL-6 (U / ml), and the horizontal axis shows VC (liter). LRG levels correlate with VC in NSIP patient sera. FIG. 12 shows serum LRG concentrations in patients with interstitial pneumonia with dermatomyositis before and during treatment. The vertical axis shows the LRG concentration (μg / ml), and the horizontal axis shows the pre-treatment, 2 weeks, 4 weeks, 8 weeks, 12 weeks, and healthy subjects from the left. It is shown that the serum LRG concentration was high in patients with interstitial pneumonia with dermatomyositis before treatment, but significantly decreased after treatment. Statistical analysis was performed using the Kruskal-Wallis test and the Scheffe posthoc test. FIG. 13 shows an analysis of serum LRG concentrations in each patient with interstitial pneumonia with dermatomyositis. The vertical axis shows the LRG concentration (μg / ml), and the horizontal axis shows from the left before, 2 weeks, 4 weeks, 8 weeks, and 12 weeks after the treatment. FIG. 14 shows an analysis of serum LRG concentrations in each patient with interstitial pneumonia with dermatomyositis. The vertical axis shows the LRG concentration (μg / ml), and the horizontal axis shows from the left before, 2 weeks, 4 weeks, 8 weeks, and 12 weeks after the treatment. FIG. 15 shows an analysis of serum LRG concentrations in each patient with interstitial pneumonia with dermatomyositis. The vertical axis shows the LRG concentration (μg / ml), and the horizontal axis shows from the left before, 2 weeks, 4 weeks, 8 weeks, and 12 weeks after the treatment. FIG. 16 shows an analysis of serum LRG concentrations in each patient with interstitial pneumonia with dermatomyositis. The vertical axis shows the LRG concentration (μg / ml), and the horizontal axis shows from the left before, 2 weeks, 4 weeks, 8 weeks, and 12 weeks after the treatment. FIG. 17 shows an analysis of serum LRG concentrations in each patient with interstitial pneumonia with dermatomyositis. The vertical axis shows the LRG concentration (μg / ml), and the horizontal axis shows from the left before, 2 weeks, 4 weeks, 8 weeks, and 12 weeks after the treatment.

Hereinafter, the present invention will be described. Throughout the specification, it should be understood that the singular representation also includes its plural concept, unless otherwise stated. Therefore, it should be understood that singular articles (eg, "a", "an", "the", etc. in English) also include their plural concept, unless otherwise noted. It should also be understood that the terms used herein are used in the meaning commonly used in the art unless otherwise noted. Thus, unless otherwise defined, all terminology and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, this specification (including definitions) takes precedence.

The present invention is based, at least in part, on the discovery that LRG is involved in the regulation of cell migration, cell infiltration. The findings indicate that LRG is a marker for cell migration-related diseases, such as the acute phase (onset phase, acute exacerbation phase, etc.) of diseases such as inflammatory and immune disorders, especially the acute phase of inflammation (onset phase and acute exacerbation). Not only can it be used as a marker for the state of (stage, etc.), but also in the acute stage (onset stage, acute exacerbation stage, etc.) of diseases such as inflammatory diseases and immune diseases, especially in the acute stage of inflammation (onset stage, acute exacerbation stage, etc.) It shows that it can also be a drug discovery target. That is, LRG inhibitors are used for the prevention and / or treatment of the acute phase (onset stage, acute exacerbation stage, etc.) of diseases such as inflammatory diseases and immune diseases, especially the acute phase of inflammation (onset stage, acute exacerbation stage, etc.). In addition to being useful, using the LRG protein and cells / animals that express it, novel LRG inhibitors, and thus the acute phase (onset phase, acute exacerbation phase, etc.) in diseases such as inflammatory diseases and immune diseases, especially inflammation It is also possible to search for substances that can be used as preventive / therapeutic agents for the acute phase (onset phase, acute exacerbation phase, etc.) of the disease. As used herein, the term "acute phase" is used in the sense commonly used in the art, and typically, the onset of symptoms / symptoms is rapid, and in some cases, the patient is in a life-threatening state and the course is short. It can be said that it is a time when symptoms suddenly appear and medically appropriate management is required, and typically refers to the onset stage of a disease (acute disease, chronic disease, etc.), the acute exacerbation stage, and the like.

I. LRG or a nucleic acid encoding it LRG is a known protein and is represented by SEQ ID NO: 2 or 4 known as Genbank Accession No .: NP_443204 or Genbank Accession No .: NP_084072. A protein containing an amino acid sequence of human or mouse LRG, or an amino acid sequence substantially the same as that of the same. In the present specification, proteins and peptides are described with an N-terminal (amino-terminal) at the left end and a C-terminal (carboxyl-terminal) at the right end according to the convention of peptide notation.

As used herein, LRG refers to cells of humans and other warm-blooded animals (eg, mice, rats, cows, monkeys, dogs, pigs, sheep, rabbits, guinea pigs, hamsters, chickens, etc.) [eg, neutrophils, etc.] Alternatively, it may be isolated and purified from a tissue [for example, blood, etc.] by a known protein separation and purification technique.

Examples of the "amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence substantially the same as this" include the following (a) to (e):
(A) Amino acid sequence shown in SEQ ID NO: 2;
(B) In the amino acid sequence shown in SEQ ID NO: 2, one or more amino acids are deleted, added, inserted or substituted, and an amino acid sequence imparting an activity of promoting cell migration and / or inflammatory reaction;
(C) An amino acid sequence having 90% or more homology with the amino acid sequence shown in SEQ ID NO: 2 and imparting an activity of promoting cell migration and / or inflammatory reaction;
(D) Amino acid sequence encoded by DNA having the base sequence shown in SEQ ID NO: 1;
(E) An amino acid sequence encoded by a DNA having a complementary strand sequence of the nucleotide sequence shown in SEQ ID NO: 1 and hybridizing under stringent conditions and imparting an activity of promoting cell migration and / or an inflammatory reaction. ..

Specifically, the amino acid sequence of the ortholog in another mammal of the human LRG protein consisting of the amino acid sequence represented by SEQ ID NO: 2, or the splice of the human LRG protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or its ortholog. Amino acid sequences in variants, allergen variants or polymorphic variants can be mentioned.

Here, "homologity" means the optimum alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm is used for the optimum alignment of sequences. It means the ratio (%) of the same amino acid and similar amino acid residues to all the overlapping amino acid residues in which the introduction of a gap in one or both can be considered). "Similar amino acids" mean amino acids that are similar in physicochemical properties, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn). ), Basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), amino acids with small side chains (Gly, Ala, Ser, Thr, Met), etc. Amino acids classified into groups can be mentioned. Such substitutions with similar amino acids are expected to result in no change in protein phenotype (ie, conservative amino acid substitutions). Specific examples of conservative amino acid substitutions are well known in the art and are described in various literatures (see, eg, Bowie et al., Science, 247: 1306-1310 (1990)).

For the homology of amino acid sequences in the present specification, the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) is used, and the following conditions (expected value = 10; allow gaps; matrix = BLOSUM62; filtering) = OFF) can be calculated. Other algorithms for determining the homology of amino acid sequences include, for example, the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993) [The algorithm is NBLAST. And incorporated into the XBLAST program (version 2.0) (Altschul et al., Nucleic Acids Res., 25: 3389-3402 (1997))], Needleman et al., J. Mol. Biol., 48: 444-453 (1970). Algorithm described in [The algorithm is incorporated in the GAP program in the GCG software package], Myers and Miller, CABIOS, 4: 11-17 (1988) [The algorithm is included in the CGC sequence alignment software package. Included in some ALIGN programs (version 2.0)], Pearson et al., Proc. Natl. Acad. Sci. USA, 85: 2444-2448 (1988) [The algorithm is in the GCG software package. Is incorporated into the FASTA program], etc., which can also be preferably used.

The stringent condition in (e) above is, for example, the condition described in Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.1-6.3.6, 1999, for example, 6 × SSC (sodium chloride / sodium). Hybridization at citrate) / 45 ° C, followed by one or more washings at 0.2 × SSC / 0.1% SDS / 50-65 ° C, but those skilled in the art will provide equivalent stringency. Hybridization conditions can be appropriately selected.

More preferably, the "amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2" is about 90% or more, preferably about 95% or more, more preferably the amino acid sequence represented by SEQ ID NO: 2. Examples include amino acid sequences having about 96% or more, more preferably about 97% or more, particularly preferably about 98% or more, and most preferably about 99% or more identity.

"A protein containing an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2" contains an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2 and is represented by SEQ ID NO: 2. It is a protein having substantially the same function as the protein consisting of the amino acid sequence to be obtained.

Here, "substantially homogeneous function" means that the properties are qualitatively the same, for example, physiologically or pharmacologically, and the degree of function (eg, about 0.1 to about 10). Quantitative factors such as times, preferably 0.5 to 2 times) and the molecular weight of the protein may be different. In addition, although the detailed role of LRG in vivo is unknown at this time, a protein having an activity of promoting cell migration and / or an inflammatory reaction can be regarded as a "protein having substantially homogeneous function". it can.

Here, the activity of promoting cell migration and / or inflammatory reaction can be measured using, for example, an in vitro or in vivo cell migration and / or inflammatory (sexual disease) model described later.

As the LRG protein in the present invention, for example, (i) 1 to 30, preferably 1 to 10, and more preferably 1 to number (5, 4, 3 or 2) in the amino acid sequence represented by SEQ ID NO: 2. Amino acid sequence lacking one amino acid, (ii) 1 to 30, preferably 1 to 10, and more preferably 1 to number (5, 4, 3 or 2) in the amino acid sequence represented by SEQ ID NO: 2. Amino acid sequence with additional amino acids, (iii) 1 to 30, preferably 1 to 10, and more preferably 1 to number (5, 4, 3 or 2) to the amino acid sequence represented by SEQ ID NO: 2. 1 to 30, preferably 1 to 10, and more preferably 1 to number (5, 4, 3 or 2) in the amino acid sequence in which the amino acid of is inserted, (iv) the amino acid sequence represented by SEQ ID NO: 2. It also includes proteins containing an amino acid sequence in which one amino acid is replaced with another amino acid, or (v) an amino acid sequence in which they are combined.

When an amino acid sequence is inserted, deleted, added or substituted as described above, the position of the insertion, deletion, addition or substitution is particularly limited as long as the protein can promote cell migration and / or inflammatory response. Not done.

Here, as a method for artificially deleting, adding, inserting or substituting an amino acid, for example, a conventional site-specific mutagenesis is applied to the DNA encoding the amino acid sequence shown in SEQ ID NO: 2. After that, a method of expressing this DNA by a conventional method can be mentioned. Here, as the site-specific mutagenesis method, for example, a method using an amber mutation (gaped duplex method, Nucleic Acids Res., 12,9441-9456 (1984)), or a PCR method using a mutagenesis primer. And so on.

Preferred examples of LRG are, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 2 (Genbank Accession No. NP_443204), or its ortholog in other mammals (eg, mouse LRG protein (SEQ ID NO: 4, Genbank). Accession No. NP_084072), etc.), allergic variants, polymorphic variants [eg, monobasic polymorphisms (SNPs)], etc.

The "nucleic acid encoding LRG" represents a nucleic acid containing the amino acid sequence represented by SEQ ID NO: 2 or a base sequence encoding substantially the same amino acid sequence as shown in (a) to (e) above. .. Specifically, the following (f) to (j):
(F) A base sequence encoding the amino acid sequence shown in SEQ ID NO: 2.
(G) A base encoding an amino acid sequence in which one or more amino acids are deleted, added, inserted or substituted in the amino acid sequence shown in SEQ ID NO: 2 and imparting an activity that promotes cell migration and / or inflammatory reaction. Array,
(H) A nucleotide sequence encoding an amino acid sequence having 90% or more homology with the amino acid sequence shown in SEQ ID NO: 2 and imparting an activity of promoting cell migration and / or inflammatory reaction,
(I) The nucleotide sequence shown in SEQ ID NO: 1.
(J) An amino acid that is a base sequence that hybridizes under stringent conditions to a DNA having a complementary strand sequence of the base sequence shown in SEQ ID NO: 1 and that imparts an activity of promoting cell migration and / or inflammatory reaction. Nucleotide sequence encoding sequence,
Nucleic acid having.

Here, the gene may be either DNA such as cDNA or genomic DNA, or RNA such as mRNA, and is a concept including both a single-stranded nucleic acid sequence and a double-stranded nucleic acid sequence. Further, in the present specification, the nucleic acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3 and the like are DNA sequences for convenience, but when indicating RNA sequences such as mRNA, thymine (T) is referred to as uracil (U). To be understood as.

Alternatively, a typical nucleotide sequence of LRG is
(A) A polynucleotide having the nucleotide sequence shown in SEQ ID NO: 1 or a fragment sequence thereof;
(B) A polynucleotide encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or a fragment thereof;
(C) In the amino acid sequence set forth in SEQ ID NO: 2, one or more amino acids is a modified polypeptide or fragment thereof having one mutation selected from the group consisting of substitutions, additions and deletions, and biology. A polynucleotide encoding a variant polypeptide having a specific activity;
(D) A polynucleotide that is a splice variant or allelic variant of the nucleotide sequence set forth in SEQ ID NO: 1 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 a fragment thereof;
(F) A polynucleotide that hybridizes to any one of the polynucleotides (a) to (e) under stringent conditions and encodes a polypeptide having biological activity; or (g) (a) to At least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one polynucleotide or complementary sequence thereof in (e). It can be a polynucleotide encoding a polypeptide having a biological activity and having a base sequence of. Here, the biological activity typically refers to the activity of LRG, the ability to be distinguished from other proteins existing in the same organism as a marker, the activity of promoting cell migration and / or inflammatory reaction, and the like. Say.

The amino acid sequence of LRG is
(A) A polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or a fragment thereof;
(B) In the amino acid sequence set forth in SEQ ID NO: 2, a polypeptide in which one or more amino acids have one mutation selected from the group consisting of substitutions, additions and deletions, and have biological activity;
(C) A polypeptide encoded by a splice or allelic variant of the nucleotide sequence set forth in SEQ ID NO: 1.
(D) A polypeptide that is a species homologue of the amino acid sequence set forth in SEQ ID NO: 2; or at least 70% and at least 80% identity to any one of the polypeptides (e) (a)-(d). , A polypeptide having an amino acid sequence of at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% and having biological activity. Here, the biological activity can be typically distinguished from other proteins present in the same organism as the activity or marker of LRG (eg, can function as a specific epitope when used as an antigen). Includes regions), activity that promotes cell migration and / or inflammatory response, etc.

Preferred examples of nucleic acids encoding LRG are, for example, human LRG cDNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 (Genbank Accession No. NM_052972), or its ortholog in other mammals (eg, mouse LRG cDNA (eg, mouse LRG cDNA). SEQ ID NO: 3, Genbank Accession No. NM_029796), etc.), allergen variants, polymorphic variants [eg, monobasic polymorphisms (SNPs)] and the like.

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

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

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

The present invention provides an LRG inhibitor, for example, a cell migration inhibitor containing a substance that inhibits the expression of LRG or a substance that inhibits the function. As used herein, the term "cell migration inhibitor" means any agent that inhibits cell migration and / or cell infiltration. Cell migration means that when an inflammatory reaction occurs locally in the body, circulating leukocytes move out of the vasculature and move to the same region by the action of various cytokines and migration factors produced there, especially in acute inflammation. It is supposed to be involved in the period.

The present invention relates to an acute phase (onset phase, acute exacerbation phase, etc.), particularly inflammation, in a disease such as an inflammatory disease or an immune disease, which contains an LRG inhibitor, for example, a substance that inhibits the expression of LRG or a substance that inhibits the function. Provide preventive and / or therapeutic agents for the acute phase (onset phase, acute exacerbation phase, etc.) of the disease.

As used herein, the term "LRG inhibitor" refers to any substance that inhibits the action of LRG, and its mechanism of action includes inhibiting the expression of LRG, inhibiting its function, and the like. Therefore, "LRG inhibitors" include substances that inhibit the expression of LRG, substances that inhibit the function of LRG, and any other substance that ultimately inhibits the action of LRG even if the mechanism of action is unknown. Including.

II. Substances that inhibit LRG expression In the present invention, "substances that inhibit LRG expression" are the transcriptional level of the nucleic acid encoding LRG (LRG gene), the level of post-transcriptional regulation, the level of translation into LRG protein, and the post-translational level. It may act at any stage, such as the level of modification. Therefore, as substances that inhibit the expression of LRG, for example, substances that inhibit transcription of the LRG gene (eg, antigene), substances that inhibit the processing of early transcripts to mRNA, and substances that inhibit the transport of mRNA to the cytoplasm. Includes substances that inhibit the translation of LRG from mRNA (eg, antisense nucleic acid, miRNA) or degrade mRNA (eg, siRNA, ribozyme), or inhibit post-translational modification of the initial translation product. .. Any substance that acts at any stage can be preferably used, but more preferably, a substance selected from the group consisting of the following (1) to (3) is exemplified.
(1) Antisense nucleic acid against transcripts of LRG gene,
(2) Ribozyme nucleic acid for transcripts of LRG gene,
(3) A nucleic acid having RNAi activity against a transcript of the LRG gene or a precursor thereof.

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

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

A base sequence that is substantially complementary to the base sequence of the mRNA of the LRG gene binds to the target sequence of the mRNA under the physiological conditions of LRG-producing cells (eg, neutrophils) in the mammal to be administered. It means a base sequence having a degree of complementarity that can inhibit its translation (or cleave the target sequence), and specifically, for example, a base sequence that is completely complementary to the base sequence of the mRNA (that is, , The base sequence of the complementary strand of mRNA) and the base sequence having about 90% or more, preferably about 95% or more, more preferably about 97% or more homology with respect to the overlapping region.

The "base sequence homology" in the present invention uses the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) and the following conditions (expected value = 10; allow gaps; filtering = ON; It can be calculated with match score = 1; mismatch score = -3).

More specifically, as the base sequence complementary or substantially complementary to the base sequence of the mRNA of the LRG gene, the following (k) or (l):
(K) A base sequence complementary or substantially complementary to the base sequence represented by SEQ ID NO: 1;
(l) Encodes a protein having a base sequence that hybridizes with the complementary strand sequence of the base sequence represented by SEQ ID NO: 1 under stringent conditions and having an activity of promoting cell migration and / or inflammatory reaction. A base sequence that is complementary or substantially complementary to the sequence;
Can be mentioned.

The stringent conditions are as described above.

Preferred examples of LRG gene mRNA are human LRG mRNAs containing the nucleotide sequence represented by SEQ ID NO: 1 (Genbank Accession No. NM_052972), or their orthologs in other mammals (eg, mouse LRG (SEQ ID NO:)). 3. Genbank Accession No. NM_029796), etc.), and splice variants, aller variants, polymorphic variants, etc. of them can be mentioned.

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

Specifically, any of the following (1) to (3) is preferably exemplified as a nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of mRNA of the LRG gene or a part thereof. Be done:
(1) Antisense nucleic acid against mRNA of LRG gene,
(2) Ribozyme nucleic acid for mRNA of LRG gene,
(3) Nucleic acid having RNAi activity against mRNA of LRG gene or a precursor thereof.

(1) Antisense Nucleic Acid Against mRNA of LRG Gene The "antisense nucleic acid against mRNA of LRG gene" in the present invention includes a base sequence complementary to or substantially complementary to the base sequence of the mRNA, or a part thereof. It is a nucleic acid having a function of suppressing protein synthesis by forming a specific and stable double strand and binding to the target mRNA.

Antisense nucleic acids include polydeoxyribonucleotides containing 2-deoxy-D-ribose, polyribonucleotides containing D-ribose, and other types of polymers that are N-glycosides of purine or pyrimidine bases. Other polymers with a non-nucleotide skeleton (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special bindings (provided that the polymer is a base as found in DNA or RNA). (Contains a nucleotide having an arrangement that allows the pairing and attachment of a base). They may be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, DNA: RNA hybrids, as well as unmodified polynucleotides (or unmodified oligonucleotides), known modifications. Additions, such as those with a label known in the art, those with a cap, those that are methylated, those in which one or more natural nucleotides are replaced with relatives, those with intramolecular nucleotide modifications. Those with uncharged bonds (eg, methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.), those with charged or sulfur-containing bonds (eg, phosphorothioate, phosphorodithioate, etc.) Those having side chain groups such as proteins (eg, nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc.) and sugars (eg, monosaccharides, etc.), intercurrent Those with compounds (eg, aclysine, solarene, etc.), those containing chelate compounds (eg, metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, modified It may be one having a bond (for example, an α-anomeric type nucleic acid). Here, the "nucleoside", "nucleotide" and "nucleic acid" may include those containing purine and pyrimidine bases as well as those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleosides and modified nucleotides may also have modified sugar moieties, for example, one or more hydroxyl groups substituted with halogens, aliphatic groups, etc., or functional groups such as ethers, amines, etc. It may have been converted.

As described above, the antisense nucleic acid may be DNA, RNA, or a DNA / RNA chimera. When the antisense nucleic acid is DNA, the RNA: DNA hybrid formed by the target RNA and the antisense DNA can be recognized by endogenous RNase H and cause selective degradation of the target RNA. Therefore, in the case of antisense DNA directed to degradation by RNase H, the target sequence may be not only the sequence in mRNA but also the sequence of the intron region in the initial translation product of the LRG gene. The intron sequence can be determined by comparing the genomic sequence with the cDNA base sequence of the LRG gene using a homology search program such as BLAST or FASTA.

The length of the target region of the antisense nucleic acid of the present invention is not particularly limited as long as the hybridization of the antisense nucleic acid inhibits the translation into protein: LRG, and encodes LRG. The entire sequence or partial sequence of the mRNA to be used may be used, and the short one may be about 10 bases, and the long one may be the full sequence of the mRNA or the initial transcript. Considering the ease of synthesis, antigenicity, intracellular translocation, and the like, oligonucleotides consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases are preferable, but the oligonucleotide is not limited thereto. Specifically, the 5'end hairpin loop of the LRG gene, the 5'end 6-base pair repeat, the 5'end untranslated region, the translation start codon, the protein coding region, the ORF translation stop codon, and the 3'end untranslated region. , 3'end parindrome regions or 3'end hairpin loops can be selected as preferred target regions for antisense nucleic acids, but are not limited thereto.

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

The nucleotide molecules that make up the antisense nucleic acid may be native DNA or RNA, but various chemistries to improve stability (chemical and / or counter-enzyme) and specific activity (affinity with RNA). Modifications can be included. For example, in order to prevent degradation by a hydrolyzing enzyme such as nuclease, the phosphate residue (phosphate) of each nucleotide constituting the antisense nucleic acid is chemically modified with, for example, phosphorothioate (PS), methylphosphonate, phosphorodithionate, etc. It can be replaced with a phosphoric acid residue. In addition, the hydroxyl group at the 2'position of the sugar (ribose) of each nucleotide is changed to -OR (R = CH ₃ (2'-O-Me), CH ₂ CH ₂ OCH ₃ (2'-O-MOE), CH ₂ It _{may be replaced with CH 2} NHC (NH) NH ₂ , CH ₂ CONHCH ₃ , CH ₂ CH ₂ CN, etc.). Further, the base moiety (pyrimidine, purine) may be chemically modified, for example, introduction of a methyl group or a cationic functional group at the 5-position of the pyrimidine base, or substitution of the carbonyl group at the 2-position with thiocarbonyl. Can be mentioned.

The conformation of the sugar portion of RNA is dominated by two, C2'-endo (S type) and C3'-endo (N type), and in single-strand RNA, it exists as an equilibrium between the two, but it is double-stranded. When is formed, it is fixed to N type. Therefore, BNA (LNA) (Imanishi), which is an RNA derivative in which the conformation of the sugar portion is fixed to N type by cross-linking 2'oxygen and 4'carbon in order to impart strong binding ability to the target RNA. , T. et al., Chem. Commun., 1653-9, 2002; Jepsen, JS et al., Oligonucleotides, 14, 130-46, 2004) and ENA (Morita, K. et al., Nucleosides Nucleotides Nucleic Acids) , 22, 1619-21, 2003) can also be preferably used.

The antisense oligonucleotide of the present invention determines the target sequence of mRNA or early transcript based on the cDNA sequence or genomic DNA sequence of the LRG gene, and is a commercially available DNA / RNA automatic synthesizer (Applied Biosystems, Beckman). Etc.), which can be prepared by synthesizing a sequence complementary to this. In addition, all of the antisense nucleic acids containing the above-mentioned modifications can be chemically synthesized by a method known per se.

(2) Ribozyme nucleic acid for mRNA of LRG gene
Another preferred example of a nucleic acid containing a base sequence complementary or substantially complementary to the base sequence of the mRNA of the LRG gene or a part thereof is a ribozyme nucleic acid capable of specifically cleaving the mRNA inside the coding region. Can be mentioned. In a narrow sense, "ribozyme" refers to RNA having an enzymatic activity of cleaving nucleic acid, but in the present specification, it is used as a concept including DNA as long as it has sequence-specific nucleic acid cleaving activity. The most versatile ribozyme nucleic acid includes self-splicing RNA found in infectious RNAs such as viroid and virusoid, and hammerhead type and hairpin type are known. The hammer head type exerts enzymatic activity at about 40 bases, and several bases at both ends adjacent to the part having the hammer head structure (about 10 bases in total) are arranged in a sequence complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA. This type of ribozyme nucleic acid has the additional advantage of not attacking genomic DNA because it uses only RNA as a substrate. When the mRNA of the LRG gene has a double-stranded structure by itself, the target sequence is made single-strand by using a hybrid ribozyme in which an RNA motif derived from a viral nucleic acid capable of specifically binding to RNA helicase is linked. Can be [Proc. Natl. Acad. Sci. USA, 98 (10): 5572-5577 (2001)]. Furthermore, when the ribozyme is used in the form of an expression vector containing the DNA encoding it, it should be a hybrid ribozyme in which tRNA-modified sequences are further linked in order to promote the transfer of the transcript to the cytoplasm. You can also [Nucleic Acids Res., 29 (13): 2780-2788 (2001)].

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

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

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

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

Further, the siRNA may have a different number of bases in the sense strand and the antisense strand. For example, "aiRNA" in which the antisense strand has a protruding sequence (overhang) at the 3'end and the 5'end. Can be mentioned. A typical aiRNA consists of 21 bases in the antisense strand and 15 bases in the sense strand, and has an overhang structure of 3 bases at each end of the antisense strand (Sun, X. et al., Nature Biotechnology Vol26 No. .12 p1379, International Publication No. WO 2009/029688 Pamphlet).

The position of the target sequence is not particularly limited, but it is desirable to select the target sequence from 5'-UTR and about 50 bases from the start codon, and from regions other than 3'-UTR. For the target sequence candidate group selected based on the above rules and others, BLAST (http://www.ncbi.nlm) is checked to see if there is homology in the consecutive sequences of 16-17 bases in the non-target mRNA. Check using homology search software such as .nih.gov / BLAST /) to confirm the specificity of the selected target sequence. For the target sequence with confirmed specificity, the sense strand having the 3'end overhang of TT or UU at 19-21 bases after AA (or NA), and the sequence complementary to the 19-21 bases and TT or A double-stranded RNA consisting of an antisense strand with a 3'end overhang of UU may be designed as a siRNA. For the short hairpin RNA (shRNA), which is a precursor of siRNA, an arbitrary linker sequence (for example, about 5-25 bases) capable of forming a loop structure is appropriately selected, and the sense strand and the antisense strand are selected. It can be designed by linking via a linker sequence.

The siRNA and / or shRNA sequences can be searched using the search software provided free of charge on various websites. Such sites include, for example, the siRNA Target Finder (http://www.ambion.com/jp/techlib/misc/siRNA_finder.html) provided by Ambion and the insert design tool for pSilencer® Expression Vector (registered trademark). http://www.ambion.com/jp/techlib/misc/psilencer_converter.html), GeneSeer provided by RNAi Codex (http://codex.cshl.edu/scripts/newsearchhairpin.cgi), but limited to these Not done.

The ribonucleoside molecule constituting siRNA may also undergo the same modifications as in the case of the antisense nucleic acid described above in order to improve stability, specific activity and the like. However, in the case of siRNA, the RNAi activity may be lost if all ribonucleoside molecules in the native RNA are replaced with the modified form, so it is necessary to introduce the minimum modified nucleoside that allows the RISC complex to function. ..

Specifically, as the modification, in order to improve the stability (chemical and / or counter-enzyme) and specific activity (affinity with RNA) of a part of the nucleotide molecules constituting siRNA, as well as the natural DNA. Can be replaced with RNA that has undergone various chemical modifications (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 phosphate residues (phosphates) of each nucleotide constituting siRNA are chemically modified phosphates such as phosphorothioate (PS), methylphosphonate, and phosphorodithionate. Can be replaced with a residue. In addition, the hydroxyl group at the 2'position of the sugar (ribose) of each nucleotide can be changed to -OR (R = CH ₃ (2'-O-Me), CH ₂ CH ₂ OCH ₃ (2'-O-MOE), CH ₂ CH ₂ NHC (NH) NH ₂ , CH ₂ CONHCH ₃ , CH ₂ CH ₂ CN, etc.), may be replaced with a fluorine atom (-F). Further, the base moiety (pyrimidine, purine) may be chemically modified, for example, introduction of a methyl group or a cationic functional group at the 5-position of the pyrimidine base, or substitution of the carbonyl group at the 2-position with thiocarbonyl. Can be mentioned. In addition, the modification method for antisense nucleic acid described in (1) above can be used. Alternatively, chemical modification (2'-deoxylation, 2'-H) that replaces a part of RNA in siRNA with DNA may be performed. Further, an artificial nucleic acid (LNA: Locked Nucleic Acid) in which the 2'position and the 4'position of sugar (ribose) are _{crosslinked with -O-CH 2- and the conformation is fixed to N type may be used.}

In addition, the sense strand and antisense strand that make up siRNA are ligands, peptides, sugar chains, antibodies, lipids, positive charges, and cell membranes that specifically recognize receptors present on the cell surface via a linker. It may be chemically bonded to oligoarginine, Tat peptide, Rev peptide, Ant peptide, etc. that adsorb and penetrate the surface layer.

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

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

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

Based on the nucleotide sequence information, the siRNA can be chemically synthesized according to a conventional method using, for example, an automatic DNA / RNA synthesizer such as 394 Applied Biosystems, Inc. synthesizer. For example, Caruthers et al., 1992, Methods in Enzymology 211,3-19, Thompson et al., International Publication WO 99/54459, 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 Examples include the methods described in et al., 1990 Nucleic Acids Res., 18,5433, and US Pat. No. 6,01311. Specifically, it can be synthesized using a nucleic acid protecting group known to those skilled in the art (for example, a dimethoxytrityl group at the 5'end) and a coupling group (for example, phosphoramidite at the 3'end). That is, the protecting group at the 5'end is deprotected with an acid such as TCA (trichloroacetic acid) to carry out a coupling reaction. Then, after capping with an acetyl group, the next nucleic acid condensation reaction is carried out. In the case of siRNA containing modified RNA or DNA, modified RNA (for example, 2'-O-methylnucleotide, 2'-deoxy-2'-fluoronucleotide) may be used as a raw material, and a coupling reaction may be used. Conditions can be adjusted as appropriate. In addition, when introducing a phosphorothioate bond in which the phosphate bond moiety is modified, a vocabulary reagent (3H-1,2-benzodithiol-3-one 1,1-dioxide) can be used.

Alternatively, the oligonucleotides may be synthesized separately and joined together after synthesis, for example by hybridization (Moore et al., 1992, Science 256,9923; Draper et al. International Publication WO 93/23569; Shabarova et al. , 1991, Nucleic Acids Research 19,4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16,951; Bellon et al., 1997, Nucleosides & Nucleotides, Bellon et al., 1997, Bioconjugate Chem.8,204), or synthetic and / or deprotection After that, they may be connected together by hybridization. siRNA molecules can also be synthesized by tandem synthesis. That is, both siRNA strands are synthesized as a single contiguous oligonucleotide separated by a cleaving linker, which is then cleaved to produce separate siRNA fragments, which are hybridized and purified. .. The linker may be a polynucleotide linker or a non-nucleotide linker.

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

Another preferred example of a nucleic acid containing a base sequence complementary or substantially complementary to the base sequence of the mRNA of the LRG gene or a part thereof is a microRNA (miRNA) that targets the mRNA. Examples of human miRNAs that target human LRG mRNA include has-miR-220b, has-miR-93, has-miR-95 and the like. The miRNA can also be prepared according to the method described for siRNA described above.

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

The LRG expression inhibitory activity of these nucleic acids should be investigated using a transformant into which a nucleic acid encoding LRG has been introduced, an in vivo or in vitro LRG gene expression system, or an in vivo or in vitro LRG protein translation system. Can be done.

The substance that inhibits the expression of LRG in the present invention is not limited to nucleic acids containing a base sequence complementary to or substantially complementary to the base sequence of mRNA of the LRG gene as described above or a part thereof, and production of LRG. It may be another substance such as a low molecular weight compound as long as it directly or indirectly inhibits. Such a substance can be obtained, for example, by the screening method of the present invention described later.

III. Substances that inhibit the function of LRG In the present invention, the “substance that inhibits the function of LRG” may be any substance as long as it inhibits the function of LRG once functionally produced.

Specifically, as a substance that suppresses the function of LRG, for example, an antibody against LRG can be mentioned. The antibody may be either a polyclonal antibody or a monoclonal antibody. These antibodies can be produced according to a method for producing an antibody or antiserum known per se. The isotype of the antibody is not particularly limited, but IgG, IgM or IgA is preferable, and IgG is particularly preferable. Further, the antibody is not particularly limited as long as it has at least a complementarity determining region (CDR) for specifically recognizing and binding to a target antigen, and is not particularly limited as long as it has a complete antibody molecule, for example, Fab, Fab', F. Fragments such as (ab') ₂ , scFv, scFv-Fc, minibodies, diabodies and other genetically engineered conjugate molecules, or polyethylene glycol (PEG) and other molecules with protein stabilizing activity. It may be a modified derivative thereof or the like.

In a preferred embodiment, an antibody against LRG is used as a human-targeted drug, so that the antibody (preferably a monoclonal antibody) is an antibody that reduces the risk of exhibiting antigenicity when administered to humans. Specifically, a fully human antibody, a humanized antibody, a mouse-human chimeric antibody and the like, and particularly preferably a fully human antibody. Humanized antibodies and chimeric antibodies can be genetically engineered according to conventional methods. Although a fully human antibody can be produced from a human-human (or mouse) hybridoma, in order to provide a large amount of antibody stably and at low cost, a human antibody-producing mouse or a phage display method is used. It is desirable to use and manufacture.

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

According to the present invention, an LRG inhibitor such as a substance that inhibits the expression or function of LRG is useful as an agent that suppresses cell migration and cell infiltration (also referred to as "cell migration inhibitor" in the present specification).

LRG inhibitors such as substances that inhibit the expression or function of LRG show the activity of suppressing cell migration and cell infiltration promoted by LRG, and thus are acute in diseases (for example, inflammatory diseases, autoimmune diseases, etc.). Useful for prevention and / or treatment of the phase.

Therefore, drugs containing substances that inhibit the expression or function of LRG are used in the acute phase (onset stage, acute exacerbation stage, etc.) of diseases such as inflammatory diseases and immune diseases, especially in the acute phase of inflammation (onset stage and acute exacerbation). It can be used as a prophylactic and / or therapeutic agent. In the present specification, the prophylactic and / or therapeutic agents for the acute phase (onset stage, acute exacerbation stage, etc.) of diseases such as inflammatory diseases and immune diseases, particularly the acute phase of inflammation (onset stage, acute exacerbation stage, etc.) are referred to. It may be called "anti-acute disease substance", "anti-acute disease drug", etc. Preventive and / or therapeutic agents for the acute phase (onset phase, acute exacerbation phase, etc.) of inflammatory and immune diseases, especially the acute phase of inflammation (onset phase, acute exacerbation phase, etc.) are "anti-acute phase inflammatory substances (drugs). ) ”,“ Anti-acute autoimmune disease substance (drug) ”, etc.

IV. Drugs containing antisense nucleic acids, ribozyme nucleic acids, siRNAs and their precursors
It is homologous (or complementary) to the antisense nucleic acid of the present invention that can complementarily bind to the transcript of the LRG gene and suppress the translation of the protein from the transcript, and the transcript (mRNA) of the LRG gene. ) SiRNA (or ribozyme) that has a base sequence and can cleave the transcript by targeting the transcript, shRNA that is a precursor of the siRNA, etc. (hereinafter, collectively referred to as "nucleic acid of the present invention"). Since it suppresses the expression of LRG in vivo and suppresses cell migration and cell infiltration promoted by LRG, it suppresses the acute phase (onset phase and acute exacerbation phase) in diseases such as inflammatory diseases and immune diseases. Etc.), especially useful for the prevention and / or treatment of the acute phase of inflammation (onset phase, acute exacerbation phase, etc.).

The medicament containing the nucleic acid of the present invention is low in toxicity, and human or non-human mammals (eg, rats, rabbits, sheep, pigs, cows, cats, etc.) can be used as a liquid preparation as it is or as a pharmaceutical composition having an appropriate dosage form. It can be administered orally or parenterally (eg, intravascularly, subcutaneously, etc.) to dogs, monkeys, etc.

When the nucleic acid of the present invention is used as a therapeutic or prophylactic agent for the above-mentioned diseases in the acute phase, it can be formulated and administered according to a method known per se. That is, the nucleic acid of the present invention is inserted alone or into a suitable expression vector for mammalian cells such as a retroviral vector, an adenovirus vector, an adenovirus-associated virus (adeno-associated virus) vector, and then inserted in a functional manner. It can be formulated according to conventional means. The nucleic acid can be administered as is, or with an adjunct to promote ingestion, by a catheter such as a gene gun or hydrogel catheter. Alternatively, it can be aerosolized and locally administered intratracheally as an inhalant.

Further, for the purpose of improving pharmacokinetics, prolonging the half-life, and improving intracellular uptake efficiency, the nucleic acid may be prepared (injection) alone or together with a carrier such as liposome and administered intravenously, subcutaneously, or the like. ..

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

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

Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets, film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups. Agents, emulsions, suspending agents and the like can be mentioned. Such compositions may be prepared by known methods and may contain carriers, diluents or excipients commonly used in the pharmaceutical field. As the carrier and excipient for tablets, for example, lactose, starch, sucrose, and magnesium stearate are used.

It is convenient that the parenteral or oral pharmaceutical compositions described above are prepared in dosage forms of dosage units that are compatible with the dosage of the active ingredient. Dosage forms of such dosage units include, for example, tablets, pills, capsules, injections (ampoules), and suppositories. The nucleic acid of the present invention is preferably contained, for example, usually 5 to 500 mg per dosage unit dosage form, particularly 5 to 100 mg for injections and 10 to 250 mg for other dosage forms.

The dose of the above-mentioned drug (or pharmaceutical composition) containing the nucleic acid of the present invention varies depending on the administration target, target disease, symptom, administration route, etc., and is, for example, inflammatory bowel inflammation (UC) or Crohn's disease (CD). ), Etc., inflammatory bowel disease (IBD), interstitial pneumonia, collagen disease / rheumatoid disease (for example, systemic erythematosus (SLE), rheumatoid arthritis, etc.) The nucleic acid of the present invention is usually about 0.01 to 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight, about 1 to 5 times a day, preferably about 1 to 5 times a day. It is convenient to administer by intravenous injection about 1 to 3 times a day. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If the symptoms are particularly severe, the dose may be increased according to the symptoms.

In addition, each composition described above may contain other active ingredients as appropriate as long as it does not cause an unfavorable interaction by blending with the nucleic acid of the present invention.

V. Drugs containing antibodies against LRG, low molecular weight compounds that inhibit the expression or function of LRG, etc.
Antibodies to LRG and low molecular weight compounds that inhibit the expression or function of LRG can inhibit the production or function of LRG. Therefore, these substances inhibit the expression or function of LRG in the living body and suppress the cell migration and cell infiltration promoted by LRG. Therefore, these substances suppress the acute phase (onset phase) in diseases such as inflammatory diseases and immune diseases. It is useful for the prevention and / or treatment of the acute phase (onset phase, acute exacerbation phase, etc.) of inflammation.

Drugs containing the above antibodies and low molecular weight compounds are low toxicity and can be used as is as a solution or as a pharmaceutical composition of an appropriate dosage form in humans or mammals (eg, rats, rabbits, sheep, pigs, cows, cats). , Dogs, monkeys, etc.) can be administered orally or parenterally (eg, intravascular administration, subcutaneous administration, etc.).

The above antibody or low molecular weight compound may be administered by itself or as a suitable pharmaceutical composition. The pharmaceutical composition used for administration may include the above-mentioned antibody or low molecular weight compound or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such pharmaceutical compositions are provided in dosage forms suitable for oral or parenteral administration.

As the composition for parenteral administration, for example, injections, suppositories and the like are used, and the injections are dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections and drip injections. May be included. Such injections can be prepared according to known methods. As a method for preparing an injection, for example, the antibody or low molecular weight compound of the present invention or a salt thereof can be prepared by dissolving, suspending or emulsifying it in a sterile aqueous solution or oily solution usually used for injections. As the aqueous solution for injection, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents are used, and suitable solubilizing agents such as alcohol (eg, ethanol) and polyalcohol (eg, eg) are used. Propylene glycol (polyethylene glycol), nonionic surfactant [eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)] and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection solution is preferably filled in a suitable ampoule. The suppository used for rectal administration may be prepared by mixing the above antibody or a salt thereof with a usual suppository base.

Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets, film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups. Agents, emulsions, suspending agents and the like can be mentioned. Such compositions may be prepared by known methods and may contain carriers, diluents or excipients commonly used in the pharmaceutical field. As the carrier and excipient for tablets, for example, lactose, starch, sucrose, and magnesium stearate are used.

It is convenient that the parenteral or oral pharmaceutical compositions described above are prepared in dosage forms of dosage units that are compatible with the dosage of the active ingredient. Dosage forms of such dosage units include, for example, tablets, pills, capsules, injections (ampoules), and suppositories. Antibodies and low molecular weight compounds are usually contained in an amount of 5 to 500 mg per dosage form, particularly preferably 5 to 100 mg for injections and 10 to 250 mg for other dosage forms.

The dose of the above-mentioned drug containing the above-mentioned antibody or low-molecular-weight compound or a salt thereof varies depending on the administration target, target disease, symptom, administration route, etc., but for example, when used for the treatment / prevention of IBD. Is usually about 0.01 to 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight, 1 to 5 times a day, with an antibody or low molecular weight compound as a single dose. It is convenient to administer by intravenous injection, preferably about 1 to 3 times a day. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If the symptoms are particularly severe, the dose may be increased according to the symptoms.

Each of the above compositions may contain other active ingredients as long as the combination with the above antibody or low molecular weight compound does not cause an unfavorable interaction.

Pharmaceutical compositions containing the above-mentioned antisense nucleic acid against LRG, ribozyme nucleic acid, siRNA and its precursors, antibodies against LRG, low molecular weight compounds that suppress the expression or function of LRG, etc. are inflammatory diseases. Diseases such as inflammatory diseases and immune diseases as prophylactic and / or therapeutic agents for the acute phase (onset stage, acute exacerbation stage, etc.), especially the acute phase of inflammation (onset stage, acute exacerbation stage, etc.) in diseases such as immune diseases. It can be used for the treatment, prevention, or prevention of progression of the acute phase (onset phase, acute exacerbation phase, etc.), especially the acute phase of inflammation (onset phase, acute exacerbation phase, etc.) Specific examples of such diseases include inflammatory bowel diseases (IBD) (eg, ulcerative colitis (UC), Crohn's disease (CD), etc.) and autoinflammatory diseases (eg, Bechet's disease, adult Still's disease). , Systemic juvenile idiopathic arthritis, cryopyrin-related periodic fever syndrome, familial Mediterranean fever, Castleman's disease, interstitial pneumonia, etc.), collagen disease / rheumatic disease (eg, vasculitis syndrome, polymyositis, dermatitis) , Systemic erythematosus (SLE), scleroderma, rheumatoid arthritis (RA), tonic spondylitis, psoriatic arthritis, etc.), and other inflammatory autoimmune disorders (eg, idiopathic interstitial pneumonia, hypersensitivity) (Pneumonia, glomerulonephritis, etc.), etc.), but are not limited to these, and any disorder in which LRG is involved in exacerbation of the pathological condition is included in the target disease of the present invention. For example, rheumatoid arthritis has been said to be a chronic disease in the past, but in recent years early detection and early treatment have become more important, and the term "chronic" rheumatoid arthritis has been judged to be inappropriate (https: //). (See www.ryumachi-jp.com/info/yogo.html), the term "chronic" has been removed and used. Therefore, it is understood that the present invention is also effective for the acute phase of rheumatoid arthritis. It is understood that other diseases are generally useful because cell migration and cell infiltration are often involved in the symptoms occurring in the acute phase.

Inflammatory diseases / immunity with pharmaceuticals containing the above-mentioned antisense nucleic acid against LRG, ribozyme nucleic acid, siRNA and its precursors, antibodies against LRG, low molecular weight compounds that suppress the expression or function of LRG, etc. When used for the treatment or prevention of the acute phase (onset phase, acute exacerbation phase, etc.) of a disease such as a disease, especially the acute phase of inflammation (onset phase, acute exacerbation phase, etc.), it may be used alone. It may be used in combination with an agent (for example, another anti-inflammatory agent) having an action of preventing and / or treating one or more kinds of diseases such as inflammatory diseases and immune diseases.

The concomitant drug is not particularly limited, but is, for example, mesalazine, corticosteroids (eg, betamethasone, prednisolone, hydrocortisone, dexamethasone, etc.), non-steroidal anti-inflammatory drugs (NSAIDs; eg, salicylic acid, anthranic acid, etc.). System, arylic acid system, propionic acid system, oxycam system, pyrin system), anti-TNFα antibody (influximab, adalimumab), anti-rheumatic drug (eg, immunomodulatory drug such as Actalit, immunosuppressive drug such as methotrexate, anti-TNFα antibody And biologics such as etanercept).

VI. Screening of drug candidate compounds for diseases As described above, inhibition of LRG expression and / or function suppresses cell migration and cell infiltration promoted by LRG, and thus in diseases such as inflammatory diseases and immune diseases. It is useful for the prevention and / or treatment of the acute phase (onset phase, acute exacerbation phase, etc.), especially the acute phase of inflammation (onset phase, acute exacerbation phase, etc.). Therefore, compounds that inhibit the expression and / or function of LRG are used in the acute phase (onset stage, acute exacerbation stage, etc.) of diseases such as inflammatory diseases and immune diseases, especially in the acute phase of inflammation (onset stage, acute exacerbation stage, etc.). ) Can be used as a prophylactic and / or therapeutic agent.

Therefore, LRG-producing cells can be used in the acute phase (onset phase, acute exacerbation phase, etc.) of diseases such as inflammatory diseases and immune diseases by using the expression level and / or function of LRG (or LRG gene) as an index. In particular, it can be used as a tool for screening substances that can be used as prophylactic and / or therapeutic agents in the acute phase of inflammation (onset phase, acute exacerbation phase, etc.).

When screening for a compound that inhibits the expression or function of LRG, the screening method involves culturing cells capable of producing LRG in the presence and absence of a test substance, and expressing LRG under both conditions. Includes comparing the amount or degree of function. Compounds that inhibit LRG function can also be screened by testing their ability to bind to purified LRG proteins and their inhibitory activity to LRG proteins and their binding proteins (eg, LRG receptors). ..

The cells capable of producing LRG used in the above screening method may be human or other mammalian cells that naturally express them or biological samples containing them (eg, blood, tissues, organs, etc.). There are no particular restrictions. In the case of blood, tissues, organs, etc. derived from non-human animals, they may be isolated from the living body and cultured, or the test substance may be administered to the living body itself, and the biological samples are isolated after a certain period of time. You may.

In addition, examples of cells having the ability to produce LRG include various transformants prepared by known and commonly used genetic engineering techniques. As the host, for example, animal cells such as H4IIE-C3 cells, HepG2 cells, HEK293 cells, COS7 cells, and CHO cells are preferably used.

Specifically, it hybridizes with the DNA encoding LRG (that is, the base sequence represented by SEQ ID NO: 1 or the base sequence having complementarity to the base sequence under stringent conditions, and in SEQ ID NO: 2. A DNA containing a nucleotide sequence encoding a polypeptide having the same function as a protein consisting of the represented amino acid sequence) is ligated downstream of a promoter in an appropriate expression vector and introduced into a host animal cell. be able to.

The method for preparing the gene encoding LRG will be described below.

The genes encoding LRG are the usual genetic engineering methods (eg, Sambrook J., Frisch EF, Maniatis T., Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory). It can be obtained according to the method described in press) etc. That is, the DNA encoding LRG is, for example, a cDNA or cDNA derived from the cell / tissue that produces LRG by synthesizing an appropriate oligonucleotide as a probe or primer based on the nucleotide sequence represented by SEQ ID NO: 1. It can be cloned from the library using the hybridization method or PCR method. Hybridization can be performed, for example, according to the method described in Molecular Cloning 2nd Edition (above). In addition, when a commercially available library is used, hybridization can be performed according to the method described in the instruction manual attached to the library.

The DNA base sequence can be determined by using a known kit, for example, Mutan ^TM- super Express Km (Takara Shuzo Co., Ltd.), Mutan ^TM- K (Takara Shuzo Co., Ltd.), ODA-LA PCR method, Gapped duplex method, etc. The conversion can be performed according to a method known per se, such as the Kunkel method, or a method similar thereto.

The cloned DNA can be used as is for the purpose, digested with a restriction enzyme if desired, or after adding a linker. The DNA may have ATG as a translation initiation codon on its 5'end and TAA, TGA or TAG as a translation termination codon on its 3'end. These translation start codons and translation stop codons can be added using a suitable synthetic DNA adapter.

Then, using the obtained LRG gene, an LRG protein can be produced and obtained according to a usual genetic engineering method.

For example, a culture obtained by preparing a plasmid capable of expressing the LRG gene in a host cell, introducing the plasmid into the host cell, transforming the plasmid, and culturing the transformed host cell (transformant). All you have to do is get the LRG from the thing. The plasmid contains, for example, genetic information that can be replicated in a host cell, can replicate autonomously, is easily isolated and purified from the host cell, and can function in the host cell. A gene in which a gene encoding LRG has been introduced into an expression vector having the above and having a detectable marker can be preferably mentioned. Various expression vectors are commercially available.

For example, the expression vector used for expression in Escherichia coli is an expression vector containing promoters such as lac, trp, and tac, which are commercially available from Pharmacia, Takara Bio, and the like. A restriction enzyme used for introducing a gene encoding LRG into the expression vector is also commercially available from Takara Bio Inc. If it is necessary to induce higher expression, the ribosome binding region may be linked upstream of the DNA encoding LRG. Examples of the ribosome binding region used include those described in reports by Guarente L. et al. (Cell 20, p543) and Taniguchi et al. (Genetics of Industrial Microorganisms, p202, Kodansha).

In addition, animal cell expression plasmids (eg, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo); bacteriophage such as λ phage; animal viral vectors such as retrovirus, vaccinia virus, adenovirus, etc. It can also be used. The promoter may be any promoter as long as it is suitable for the host used for gene expression. For example, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (laus sarcoma virus) promoter, MoMuLV (Molony mouse leukemia virus) LTR, HSV-TK (simple herpesvirus thymidine kinase) promoter, β-actin. A gene promoter, aP2 gene promoter, etc. are used. Of these, the EF-α promoter, CAG promoter, CMV promoter, SRα promoter and the like are preferable.

As the expression vector, in addition to the above, a vector containing an enhancer, a splicing signal, a poly A addition signal, a selectable marker, an SV40 origin of replication (hereinafter, may be abbreviated as SV40ori) and the like may be used. it can.

Examples of the selectable marker include a dihydrofolate reductase gene (hereinafter, sometimes abbreviated as dhfr, methotrexate (MTX) resistance), an ampicillin resistance gene (hereinafter, ^{sometimes abbreviated as amp r} ), and a neomycin resistance gene (hereinafter, may be abbreviated as amp r). Hereinafter, G418 resistance) and the like, which may be abbreviated ^{as neo r, can be mentioned.} In particular, when dhfr gene-deficient Chinese hamster cells are used and the dhfr gene is used as a selectable marker, the target gene can also be selected using a medium containing no thymidine.

LRG-expressing cells can be produced by transforming a host with an expression vector containing the above-mentioned LRG-encoding DNA.

Examples of host cells include prokaryotic or eukaryotic microbial cells, insect cells, mammalian cells and the like. Examples of mammalian cells include HepG2 cells, HEK293 cells, HeLa cells, human FL cells, monkey COS-7 cells, monkey Vero cells, Chinese hamster ovary cells (hereinafter abbreviated as CHO cells), and dhfr gene-deficient CHO cells (hereinafter abbreviated as CHO cells). hereinafter, CHO (dhfr ^-) cell), mouse L cells, mouse AtT-20, mouse myeloma cells, mouse myeloma cells, rat H4IIE-C3 cells, rat GH3 cells can be used. For example, Escherichia coli and the like can be preferably mentioned from the viewpoint of facilitating mass preparation of LRG.

The plasmid obtained as described above can be introduced into the host cell by a conventional genetic engineering method. The transformant can be cultured by the usual method used for culturing microorganisms, insect cells or mammalian cells. For example, in the case of Escherichia coli, culture is carried out in a medium containing an appropriate carbon source, nitrogen source and micronutrients such as vitamins. As the culturing method, either solid culture or liquid culturing may be used, and liquid culturing such as aeration stirring culture method is preferable.

Transformation can be carried out by a calcium phosphate coprecipitation method, a PEG method, an electroporation method, a microinjection method, a lipofection method or the like. For example, the method described in Cell Engineering Supplement 8 New Cell Engineering Experiment Protocol, 263-267 (1995) (published by Shujunsha), Virology, Vol. 52, 456 (1973) can be used.

The transformed cells obtained as described above, mammalian cells capable of producing native LRG, or tissues / organs containing the cells are, for example, the minimum essential medium (MEM) containing about 5 to 20% of fetal calf serum. ) [Science, Vol. 122, 501 (1952)], Dalbeco Modified Eagle's Medium (DMEM) [Virology, Vol. 8, 396 (1959)], RPMI 1640 Medium [The Journal of the American Medical Association, Vol. 199, 519 (1967)] ], 199 medium [Proceeding of the Society for the Biological Medicine, Vol. 73, 1 (1950)]. The pH of the medium is preferably about 6-8. Culturing is usually carried out at about 30-40 ° C, with aeration and stirring as needed.

The LRG protein may be obtained in combination with the methods usually used for isolation and purification of general proteins. For example, the transformant obtained by the above culture may be removed by centrifugation or the like, and LRG may be purified from the culture supernatant in the same manner as described above. When the LRG protein accumulates in the cells of the transformant obtained by the above culture, for example, after collecting the transformant by centrifugation or the like, the cells are crushed or lysed, and it is necessary. For example, the protein may be solubilized and purified by performing various chromatographic steps such as ion exchange, hydrophobicity, and gel filtration alone or in combination. Further operations may be performed to restore the higher-order structure of the purified protein.

In carrying out the screening of the present invention, examples of the test substance include proteins, peptides, non-peptidic compounds, synthetic compounds, fermented products, cell extracts, plant extracts, animal tissue extracts and the like. The substance may be a novel substance or a known substance.

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

The contact of the test substance with the cells is, for example, the above-mentioned medium and various buffers (for example, HEPES buffer, phosphate buffer, phosphate buffered saline, Tris-hydrochloric acid buffer, borate buffer, acetic acid. It can be carried out by adding the test substance in (buffer solution, 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 in the range of, for example, about 0.1 nM to about 100 μM. The incubation time includes, for example, about 10 minutes to about 24 hours.

When the cells producing LRG are provided in the form of a non-human mammalian individual, the condition of the individual animal is not particularly limited, but for example, an inflammatory disease model animal in which inflammation is induced by a drug or genetic modification (for example, IBD model animals such as mice in which colitis was induced by drugs such as sodium dextran sulfate (DSS) and 2.4.6-trinitrobenzene sulfonic acid (TNBS), and genetically modified mice such as IL-10 knockout mice and IL-2 knockout mice. , Collagen-induced arthritis (CIA) mice, SKG mice, PD-1 knockout mice, K / BxN mice, RA model animals such as synobiolin Tg mice, etc.). There are no particular restrictions on the breeding conditions of the animals used, but it is preferable that the animals are bred in an environment of SPF grade or higher. Contact of the test substance with the cells is performed by administration of the test substance to the individual animal. The administration route is not particularly limited, and examples thereof include intravenous administration, intraarterial administration, subcutaneous administration, intradermal administration, intraperitoneal administration, oral administration, intratracheal administration, and rectal administration. The dose is not particularly limited, but for example, about 0.5 to 20 mg / kg can be administered as a single dose 1 to 5 times a day, preferably 1 to 3 times a day for 1 to 14 days.

Alternatively, the above screening method can be performed by contacting the test substance with an extract of the cells or LRG isolated and purified from the cells instead of the cells having the ability to produce LRG.

(Measurement of LRG gene or LRG expression level)
The present invention is characterized by comparing the expression of the protein (gene) in cells capable of producing LRG with and without the presence of a test substance, such as inflammatory diseases and immune diseases. Provided is a method for screening a substance that can be used for prevention and / or treatment of an acute phase (onset phase, acute exacerbation phase, etc.), particularly an acute phase of inflammation (onset phase, acute exacerbation phase, etc.). The cells used in this method, the type of the test substance, the mode of contact between the test substance and the cells, and the like are the same as described above.

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

Therefore, more specifically, the present invention
(A) Cells capable of producing LRG were cultured in the presence and absence of the test substance, and the amount of mRNA encoding the protein under both conditions was measured using the detection nucleic acid of the present invention. , Prevention and / or prevention of acute phase (onset phase, acute exacerbation phase, etc.), especially acute phase of inflammation (onset phase, acute exacerbation phase, etc.) in diseases such as inflammatory diseases and immune disorders, which are characterized by comparison. Methods for screening for substances that can be used therapeutically, and (b) cells capable of producing LRG were cultured in the presence and absence of the test substance, and the amount of the protein under both conditions was presented in the book. The acute phase (onset phase, acute exacerbation phase, etc.) of diseases such as inflammatory diseases and immune diseases, which is characterized by measurement and comparison using the detection antibody of the present invention, particularly the acute phase of inflammation (onset phase and acute phase). Provided is a method for screening substances that can be used for prevention and / or treatment of (exacerbation period, etc.).

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

(I) Tested on normal or disease models (eg DSS or TNBS-induced colitis model) non-human mammals (eg mice, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.) After a certain period of time (30 minutes to 3 days, preferably 1 to 2 days, more preferably 1 to 24 hours) after administration of the substance, blood or a specific organ (eg, brain) Etc.), or obtain a tissue or cell isolated from an organ.

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

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

The detection and quantification of the expression level of the LRG gene (mRNA) can be carried out by a known method such as Northern blot method or RT-PCR method using RNA prepared from the cells or complementary polynucleotide transcribed from the RNA. Specifically, the presence or absence of expression of the LRG gene in RNA and its expression by using a polynucleotide having at least 15 consecutive bases in the base sequence of the LRG gene and / or a polynucleotide complementary thereto as a primer or a probe. Levels can be detected and measured. Such probes or primers can be designed based on the base sequence of the LRG gene, for example, using primer 3 (http://primer3.sourceforge.net/) or vector NTI (manufactured by Infomax). ..

When the Northern blot method is used, the primer or probe is ^{labeled with a radioisotope (32} P, ³³ P, etc .: RI) or a fluorescent substance, and the RNA is transferred to a nylon membrane or the like according to a conventional method. After hybridizing with, the double strand of the formed primer or probe (DNA or RNA) and RNA is used as a signal derived from the label (RI or fluorescent substance) of the primer or probe as a radiation detector ( BAS-1800II, manufactured by Fuji Film Co., Ltd.) or a method of detecting and measuring with a fluorescence detector can be exemplified. In addition, the probe is labeled according to the protocol using the AlkPhos Direct Labeling and Detection System (manufactured by Amersham Pharamcia Biotech), hybridized with RNA derived from cells, and then the signal derived from the labeled substance of the probe is multi-buy. You can also use the method of detection and measurement with the Oimager STORM860 (manufactured by Amersham Pharmacia Biotech).

When using the RT-PCR method, a pair of cDNAs prepared from cell-derived RNA according to a conventional method and prepared based on the sequence of the LRG gene so that the target LRG gene region can be amplified using this as a template. A method of hybridizing a primer (a normal strand that binds to the above-mentioned cDNA (-strand) and a reverse strand that binds to the + strand) and performing a PCR method according to a conventional method to detect the obtained amplified double-stranded DNA. Can be exemplified. The amplified double-stranded DNA is detected by performing the above PCR using a primer labeled with RI or a fluorescent substance in advance, thereby producing the labeled double-stranded DNA.
A method of detecting the above, a method of transferring the produced double-stranded DNA to a nylon membrane or the like according to a conventional method, and a method of hybridizing with the labeled primer as a probe for detection can be used. The generated labeled double-stranded DNA product can be measured with an Agilent 2100 bioanalyzer (manufactured by Yokogawa Analytical Systems) or the like. In addition, SYBR Green RT-PCR Reagents (manufactured by Applied Biosystems) prepares an RT-PCR reaction solution according to the protocol, and the reaction is reacted with ABI PRIME 7900 Sequence Detection System (manufactured by Applied Biosystems) to detect the reaction product. You can also do it.

The expression of the LRG gene in the cells to which the test substance was added was 2/3 times or less, preferably 1/2 times or less, and more preferably 1/3 times the expression level in the control cells to which the test substance was not added. If the following, the test substance can be selected as a substance that suppresses the expression of the LRG gene.

Screening for substances that change the expression level of LRG can also be performed by a reporter gene assay using the transcriptional regulatory region of the LRG gene. Here, the "transcriptional regulatory region" usually refers to a range of several kb to several tens of kb upstream of the chromosomal gene, for example, (i) 5'-race method (5'-RACE method) (for example, 5). Steps to determine the 5'end by conventional methods such as'-fullRaceCoreKit (manufactured by Takara Bio), oligocap method, S1 primer mapping, etc .; (ii) Genome Walker Kit (Clonetech) The 5'-upstream region can be obtained using (manufactured by) or the like, and the obtained upstream region can be identified by a method including the step of measuring the promoter activity;

A nucleic acid encoding a reporter protein (hereinafter referred to as "reporter gene") is ligated downstream of the transcriptional regulatory region of the LRG gene in a functional manner to construct a reporter protein expression vector. The vector may be prepared by a method known to those skilled in the art. That is, ordinary genetic engineering described in "Molecular Cloning: A Laboratory Manual 2nd edition" (1989), Cold Spring Harbor Laboratory Press, "Current Protocols In Molecular Biology" (1987), John Wiley & Sons, Inc., etc. The transcriptional regulatory region of the LRG gene excised according to the procedure can be integrated on a plasmid containing the reporter gene.

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

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

Further, as a method for measuring the expression level of the reporter gene, a method corresponding to each reporter gene may be used. For example, when a luciferase gene is used as a reporter gene, the transformed cell is cultured for several days to obtain an extract of the cell, and then the extract is reacted with luciferin and ATP to chemiluminescent, and the luminescence intensity thereof is obtained. The promoter activity can be detected by measuring. At this time, a commercially available luciferase reaction detection kit such as Pickagene Dual Kit (registered trademark; manufactured by Toyo Ink Mfg. Co., Ltd.) can be used.

Specifically, as a method for measuring the amount of protein in LRG, for example,
(I) The detection antibody of the present invention is competitively reacted with the sample solution and the labeled LRG, and the labeled protein bound to the antibody is detected to quantify the LRG in the sample solution. Method and
(Ii) After reacting the sample solution with the detection antibody of the present invention insolubilized on the carrier and another labeled detection antibody of the present invention simultaneously or continuously, the labeling agent on the insolubilized carrier. A method of quantifying LRG in a sample solution by measuring the amount (activity) of the sample solution can be mentioned.

The detection and quantification of the protein expression level of LRG can be quantified according to a known method such as Western blotting using an antibody that recognizes LRG. Western blotting uses an antibody that recognizes LRG as the ^{primary antibody, and then binds to the primary antibody labeled with a radioactive isotope such as 125} I, a fluorescent substance, or an enzyme such as horseradish peroxidase (HRP) as the secondary antibody. It can be carried out by labeling with the antibody to be used and measuring the signals derived from these labeling substances with a radiation measuring device (BAI-1800II: manufactured by Fuji Film Co., Ltd., etc.), a fluorescence detector, or the like. In addition, after using an antibody that recognizes LRG as the primary antibody, it is detected according to the protocol using the ECL Plus Western Blotting Detection System (manufactured by Amersham Pharmacia Biotech), and then detected by the multi-biomager STORM860 (manufactured by Amersham Pharmacia Biotech). It can also be measured.

The above-mentioned antibody is not particularly limited in its form, and may be a polyclonal antibody using LRG as an immunogen, a monoclonal antibody, and further, at least continuous among the amino acid sequences constituting LRG, usually. Antibodies having antigen-binding property to a polypeptide consisting of 8 amino acids, preferably 15 amino acids, and more preferably 20 amino acids can also be used.

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

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

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

The method for quantifying LRG using the detection antibody of the present invention is not particularly limited, and the amount of antibody, antigen or antibody-antigen complex corresponding to the amount of antigen in the sample solution is chemically or physically determined. Any measuring method may be used as long as it is detected by means and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competitive method, immunometric method and sandwich method are preferably used. In terms of sensitivity and specificity, for example, it is preferable to use the sandwich method described later.

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

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

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

In the competitive method, the LRG in the sample solution and the labeled LRG are competitively reacted with the antibody, and then the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation), B, F The LRG in the sample solution is quantified by measuring the labeled amount. In this reaction method, a soluble antibody is used as an antibody, and a liquid phase method in which B / F separation is performed using polyethylene glycol or a secondary antibody against the antibody (primary antibody), and immobilization as a primary antibody. An antibody is used (direct method), or a soluble primary antibody is used, and a immobilized antibody is used as the secondary antibody (indirect method).

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

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

In applying these individual immunological measurement methods to the quantification method of the present invention, no special conditions, operations, or the like are required to be set. The LRG measurement system may be constructed by adding the usual technical considerations of those skilled in the art to the usual conditions and operation methods in each method. For details of these general technical means, review articles, books, etc. can be referred to.

For example, "Radioimmunoassay" edited by Hiroshi Irie (Kodansha, published in 1974), "Continued Radioimmunoassay" edited by Hiroshi Irie (Kodansha, published in 1979), "Enzyme Immunoassay" edited by Eiji Ishikawa et al. (Published in 1993), Eiji Ishikawa et al., "Enzyme Immunoassay" (2nd edition) (Medical Shoin, published in 1982), Eiji Ishikawa et al., "Enzyme Immunoassay" (3rd edition) (Medical Shoin, Showa) Published in 1987), "Methods in ENZYMOLOGY" Vol.70 (Immunochemical Techniques (Part A)), Immunochemical Techniques (Part B), Immunochemical Techniques (Part C), Immunochemical Techniques (Part C)) (Immunochemical Techniques (Part D: Selected Immunoassays)), Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies)) (The above is published by Academic Press) and so on.

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

For example, in the above screening method, the expression level (mRNA amount or protein amount) of LRG in the presence of the test substance is about 20% or more, preferably about 30%, as compared with the case in the absence of the test substance. As described above, more preferably, when about 50% or more is inhibited, the test substance is used as an LRG expression inhibitor, and therefore, in the acute phase (onset phase, acute exacerbation phase, etc.) in diseases such as inflammatory diseases and immune diseases. In particular, it can be selected as a candidate for a substance that can be used for the prevention and / or treatment of the acute phase of inflammation (onset phase, acute exacerbation phase, etc.).

Alternatively, in the above screening method, instead of the cell expressing the LRG gene, a cell containing a reporter gene under the control of the transcriptional regulatory region inherent in the LRG gene can be used. Such cells may be cells, tissues, organs or individuals of transgenic animals into which a reporter gene (eg, luciferase, GFP, etc.) under the control of the transcriptional regulatory region of the LRG gene has been introduced. When such cells are used, the expression level of LRG can be evaluated by measuring the expression level of the reporter gene using a conventional method.

(Measurement of LRG function)
The screening method of the present invention can also be performed using whether or not the test substance inhibits the function of LRG as an index.

Since LRG is a protein secreted mainly from neutrophils into the blood, substances capable of binding to LRG protein are LRG protein and its binding partner protein (for example, LRG receptor on the surface of target cells). It is thought that the function of LRG can be inhibited by blocking the interaction with. Therefore, candidates for LRG function inhibitors can be screened using the binding ability to LRG as an index.

For example, the test substance is adsorbed on each well of the well plate, an LRG solution labeled with an appropriate labeling agent is added to each well and incubated, and then the liquid phase is removed, and the amount of labeling bound to the solid phase is measured after washing. By doing so, it is possible to detect a test substance having the ability to bind to LRG. Instead of directly labeling LRG, labeled anti-LRG antibodies can also be used to detect LRG bound to the solid phase. Alternatively, a solution of the test substance is passed through a carrier on which LRG is immobilized (eg, an affinity column), and the test substance retained on the carrier is passed through a substance having the ability to bind to LRG, that is, an inflammatory disease / immune disease. It can also be selected as a candidate for substances that can be used for the prevention and / or treatment of the acute phase (onset phase, acute exacerbation phase, etc.) of diseases such as, especially the acute phase of inflammation (onset phase, acute exacerbation phase, etc.). it can.

The candidate substance thus obtained actually prevents the acute phase (onset stage, acute exacerbation stage, etc.) of diseases such as inflammatory diseases and immune diseases, especially the acute phase of inflammation (onset stage, acute exacerbation stage, etc.). And / or whether or not it has a therapeutic effect, the candidate substance is applied to an appropriate disease model (for example, bleomycin-induced interstitial pneumonia), and whether or not the disease response in the model is suppressed is tested. It can be confirmed by. In vivo and in vitro models can be used as such models. As an in vivo model, for example, a bleomycin-induced interstitial pneumonia model (wild-type mouse (C57BL / 6, female) is anesthetized, and bleomycin (1.5 mg / kg) is administered intratracheally to interstitial pneumonia / lung. Can induce fibrosis). In addition to these, a DSS-induced colitis model (can be prepared by allowing non-human animals to drink water containing 3 to 5% (w / v) of DSS with a molecular weight of 5,000 to 10,000 for 5 to 10 days). , IBD model, CIA model (complete Freund), such as TNBS-induced colitis model (which can be prepared by dissolving TNBS in 50% ethanol and intrarectally administering to non-human animals, for example, in an amount of 50 μg / g body weight). By injecting a non-human animal with a monoclonal antibody cocktail that recognizes an epitope in CB11 of type II collagen, a CAIA model (which can be prepared by immunizing a non-human animal with an adjuvant and emulsion of type II collagen). RA models and the like (which can be prepared) can also be used, but are not limited thereto. On the other hand, as an in vitro model, target cells in diseases (for example, inflammatory diseases, autoimmune diseases, etc.) (for example, intestinal epithelial cells in IBD, synovial cells in RA, alveolar epithelial cells or bronchial epithelial cells in pneumonia, etc.) ) (For example, Caco-2 cell culture system, synovial fibroblast culture system derived from synovial tissue of RA patients, etc.), but is not limited thereto. These in vitro models can be stimulated with acute cytokines such as TNFα _{, active oxygen such as H 2} O ₂ , LPS, etc., or acute cytokine-producing cells such as monocytes, macrophages, and neutrophils, if necessary. Complex culture with (eg, ^{using a Transwell TM} culture system, etc., target cells (eg, Caco-2 cells) in the upper compartment, inflammatory cytokine-producing cells (macrophage-like THP-1 cells, RAW264.7) in the lower compartment A culture system in which cells) are individually cultured in a single layer) can elicit a reaction in the acute phase.

Whether or not the candidate substance has an inhibitory effect on the acute phase of the disease can be determined by whether or not the reaction in the above model is suppressed by the addition of the candidate substance. For example, in the above drug-induced bleomycin-induced interstitial pneumonia model animal, changes in body weight (suppression of weight loss due to onset, degree of recovery from weight loss, etc.), lung condition, epithelium at the site of lung disease, etc. The presence or absence and / or the degree of the effect on the acute phase of the disease can be determined by using the degree of damage and cell infiltration as an index. On the other hand, when a monolayer culture system of intestinal epithelial cells, which is an in vitro model, is used, the reaction in the acute phase of the disease is based on the decrease in transepithelial electrical resistance (TER) value, LDH production, and increase in IL-8 expression. The degree can be evaluated.

In another preferred embodiment of the invention, LRG function inhibitors can be screened by testing the binding inhibitory activity of the LRG protein and its binding protein (eg, LRG receptor). Regarding LRG receptors and biological substances that bind to LRG, since LRG is a protein that is mainly secreted from neutrophils into the blood, the acute phase (onset phase and onset phase) of diseases such as inflammatory diseases and immune diseases It is strongly suggested that there is a physiological binding partner of LRG among the proteins expressed on the surface of target cells in the acute phase (such as acute exacerbation), especially in the acute phase of inflammation (such as onset and acute exacerbation). .. Therefore, the membrane fraction of the target cell (for example, in the case of intestinal epithelial cell, alveolar epithelial cell or bronchial epithelial cell, the membrane fraction on the mucosal proper layer side is isolated according to a conventional method and solidified on a suitable carrier. Tested by synchronizing, contacting the solid phase labeled LRG protein in the presence and absence of the test substance, and comparing to the amount of LRG bound to the cell membrane fraction in the absence of the test substance. When the amount of LRG bound to the cell membrane fraction is significantly low in the presence of the substance, the test substance is used in the acute phase (onset phase, acute exacerbation phase, etc.) of diseases such as inflammatory diseases and immune diseases, especially inflammation. Can be selected as a candidate for a therapeutic or preventive substance for the acute phase (onset phase, acute exacerbation phase, etc.) of the disease. The candidate substance thus selected is the acute phase (onset phase) in a disease such as an inflammatory disease or an immune disease. And acute exacerbation period), especially whether or not it has a preventive or therapeutic effect in the acute stage of inflammation (onset stage, acute exacerbation stage, etc.) can be confirmed by the same method as described above.

In yet another embodiment of the present invention, the above-mentioned in vitro inflammation model is used to inhibit the function of LRG to prevent an acute phase (onset phase, acute exacerbation phase, etc.) in a disease such as an inflammatory disease or an immune disease, particularly. Substances that have an action to treat or prevent the acute phase of inflammation (onset phase, acute exacerbation phase, etc.) can also be screened in one step. The method is the following steps (1) to (3):
(1) A step of contacting a target cell of a disease (for example, an inflammatory disease, an autoimmune disease, etc.) with a test substance in the presence or absence of LRG (2) A disease in the cell under each condition (for example) For example, a step of measuring the degree of reaction (for example, inflammatory reaction, autoimmune reaction) of inflammatory disease, autoimmune disease, etc. (3) Compared with the case of measuring in the absence of a test substance, LRG The test substance that suppresses the reaction in the presence and does not suppress the reaction in the absence of LRG inhibits the function of LRG to treat diseases (for example, inflammatory diseases, autoimmune diseases, etc.) or Includes the step of selecting as a candidate for a prophylactic agent.

The method may further include, if necessary, a step of inducing a reaction associated with the acute phase of the disease at or before or after the step (1) above. Methods for inducing reactions related to the acute phase of the disease include, for example, stimulation with inflammatory cytokines such as TNFα _{, active oxygen such as H 2} O ₂ , LPS, etc., or acute monocytes, macrophages, neutrophils, etc. Examples include complex culture with cytokine-producing cells in the stage. In one preferred embodiment, for example, ^{using a Transwell TM} culture system or the like, target cells (eg, Caco-2 cells) in the upper compartment and inflammatory cytokine-producing cells (macrophage-like THP-1 cells, RAW264) in the lower compartment. A method of culturing each of .7 cells) in a single layer can be mentioned. In this case, add LRG to the medium in the lower compartment. LPS or the like may be further added to the medium to induce inflammation. The test substance is usually added to the medium in the lower compartment, but for example, screening for components contained in foods that can be absorbed in the intestine and inhibit LRG function, or orally administrable LRG function inhibitors. The test substance can be added to the medium in the upper compartment.

The substance that inhibits the expression or function of LRG obtained by using any of the above screening methods of the present invention is an acute phase (onset phase, acute exacerbation phase, etc.), particularly inflammation in diseases such as inflammatory diseases and immune diseases. It is useful as a drug for the prevention and / or treatment of the acute phase (onset phase, acute exacerbation phase, etc.) of.

When the compound obtained by using the screening method of the present invention is used as the above-mentioned preventive / therapeutic agent, it can be formulated in the same manner as the above-mentioned low-molecular-weight compound that inhibits the expression or function of LRG, and has the same administration route and the same administration route. At the dosage, it can be administered orally or parenterally to humans or mammals (eg, mice, rats, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, chimpanzees, etc.) it can.

VII. Method for determining disease activity of lung interstitial disease such as interstitial pneumonia The present invention is a method for determining disease activity of lung interstitial disease in a subject, and a living body derived from the subject. Including the step of measuring the concentration of leucine-rich α2 glycoprotein (LRG) in the sample, the disease activity (ie, the risk of further aggravation) of the subject's lung interstitial disease based on the concentration of the LRG. Provide a judgment method for evaluating. In the present invention, it is highly reliable as a marker for pulmonary interstitial disease that has been conventionally established only in KL-6, and also has disease activity of a type of pulmonary interstitial disease that cannot be examined by KL-6. Markers that can be comprehensively determined are provided. Such a marker has not been provided in the past, and provides a new disease activity determination tool for lung interstitial diseases for which it is difficult to determine disease activity. In addition, lung stromal disease is different from inflammation and other diseases of the lung, and normal pneumonia and interstitial pneumonia have different causes and pathological conditions (different treatment methods). Is considered irrelevant in the field. In addition, although pulmonary interstitial diseases are complications observed in autoimmune diseases, autoimmune diseases may be complicated not only with interstitial pneumonia but also with many diseases such as hypertension and diabetes. It is well known in the field that interstitial pneumonia is not always associated with autoimmune disease, and that even if LRG becomes a biomarker of an autoimmune disease, it does not indicate that it is a marker of interstitial pneumonia. In view of the above, it can be said that the findings of the present invention are truly unexpected. In addition, since inflammation is a common disease in the lung interstitial disease, it is considered that it is related to normal inflammation markers, but it is found that some inflammation is present in a specific inflammation marker such as CRP. However, it cannot be derived that it is a marker of other pathological conditions (interstitial pneumonia in this case), and the function of "interstitial pneumonia" as a disease activity marker can be derived from the findings of other inflammations. It is understood by those skilled in the art. It provides functions not found in KL-6, which has been conventionally used as a marker for lung interstitial diseases, particularly interstitial pneumonia, and in that sense, the ability of the present invention as a marker for lung interstitial diseases. Was sought after in the field and deserves special mention.

Disease activity is judged by the degree of disease symptoms, disease progression rate, organ dysfunction, and the like. High disease activity refers to a condition in which the disease tends to worsen and becomes more severe. Evaluating disease activity is important in determining the therapeutic effect of a disease, predicting the prognosis of treatment, and determining future treatment policies. In addition, it is possible to minimize the aggravation by changing the treatment method to an appropriate one before the aggravation based on the evaluation of the disease activity. "Disease activity" of pulmonary interstitial diseases (eg, interstitial pneumonia) refers to symptoms, results of respiratory function tests such as% DLCO or% VC, imaging tests such as chest CT, arterial blood gas or blood tests, and It is determined by comprehensively evaluating the transition. % DLCO and% VC are particularly important in assessing the disease activity of interstitial pneumonia because they are factors that are directly involved in the exacerbation of the condition. Since serum LRG levels in patients with all types of interstitial pneumonia correlate with the important factors% DLCO and% VC, blood tests for LRG can easily assess activity. The present invention can evaluate the disease activity of a type of lung interstitium that could not be evaluated by conventional activity markers.

% DLCO is the carbon monoxide lung diffusion capacity expressed as a percentage, and the normal range is 80% or more. For VC, vital capacity means the amount of gas from maximum inspiration to maximum expiration. % VC is the vital capacity (VC) expressed as a percentage of the normal predicted value. The normal range of% VC is 80% or more, and those less than 80% are called restrictive disorders. Restrictive disorders include interstitial pneumonia (pulmonary fibrosis), destruction or disappearance of lung parenchyma due to pulmonary tuberculosis or pulmonary resection, pleural effusion, and sometimes neuromuscular diseases such as childhood paralysis and myasthenia gravis.

As used herein, the term "severity" refers to the severity of a symptom of a certain disease, and can be divided into mild, moderate, severe and the like. When the disease is not severe, that is, when the severity is mild to moderate, or even if it is severe, the disease activity is measured to prevent further disease. It can be done to prevent aggravation. In the past, it was difficult to select an appropriate treatment because there was no simple index of disease activity, but according to the present invention, it is possible to prevent the severity from becoming severe by preventing the aggravation. When used in Japan, interstitial pneumonia can be classified into I, II, III, and IV according to the values of _{arterial blood gas at rest and SpO 2 when walking for 6 minutes.}

Even for the same disease, the clinical significance of evaluating the disease activity is great because the course and prognosis differ greatly depending on the disease activity. In the determination method of the present invention, the disease activity of a type of lung interstitial disease that could not be evaluated by the conventional marker can be easily evaluated, which is superior to the conventional marker (for example, KL-6). .. In addition, for example, interstitial pneumonia is one of the intractable diseases in which restrictive ventilatory impairment accompanied by hypoxemia progresses, and there is a strong tendency to suffer from respiratory failure and circulatory failure, which is likely to lead to death. Accurate assessment of disease activity and corresponding therapeutic intervention is essential for pneumonia.

The "subject" targeted by the determination method of the present invention is a human or mammal (for example, mouse, rat, rabbit, sheep, pig, cow, horse, cat, dog, monkey, chimpanzee, human, etc.). , Preferably humans.

As used herein, the term "lung interstitium" refers to, for example, the interlobular wall, the supporting tissue near the pleura, and more specifically, the alveolar septum. "Pulmonary interstitial disease" includes interstitial pneumonia and any lung interstitial disease associated with% DLCO and / or% VC disorders (eg, showing values outside the normal range). included.

In the present specification, "interstitial pneumonia" refers to a group of diseases showing inflammation of the lung interstitium, and some of them progress to post-inflammatory fibrosis (pulmonary fibrosis). Interstitial pneumonia is classified into several diseases, typically idiopathic pulmonary fibrosis (IPF) and chronic hypersensitivity pneumonitis: secondary interstitial pneumonia. CHP), interstitial pneumonia associated with collagen vascular diseases (CVD-IP), non-specific interstitial pneumonia (NSIP), interstitial with dermatitis Diffuse interstitial pneumonia (DIP), lymphoid interstitial pneumonia, diffuse alveolar damage (DAD), granulomatous interstitial pneumonia Etc., but are not limited to these.

The biological sample used in the above determination method is preferably serum, but may be plasma. The biological sample can be of human or mammalian origin.

Typically, the normal value of the LRG concentration is about 3.07 μg / ml, and when the LRG concentration is 5.91 μg / ml or more, it can be judged that the disease activity of the lung interstitial disease is high.

In the above determination method, the LRG concentration in a biological sample can be measured by contacting the biological sample with a reagent capable of binding to LRG (for example, an anti-LRG antibody or a fragment thereof). For example, the various quantitative methods described herein can be used, including nephrometry, competitive methods, immunometric methods, sandwich methods and the like.

The present invention further provides determinations for determining disease activity of the lung interstitium. The determination comprises a reagent capable of binding to LRG, which reagent may be labeled or may be capable of binding to the labeled molecule. As described above, various labeling agents can be used for labeling, and those skilled in the art can select a suitable targeting agent according to the measurement method. Examples of the labeling agent include radioactive isotopes, enzymes, fluorescent substances, luminescent substances and the like.

The present invention also provides a kit for detecting and / or quantifying LRG, which comprises the above-mentioned determination agent. The kit may further include a secondary antibody capable of binding to the above reagents. The secondary antibody can be a labeled antibody that binds to the primary antibody.

VIII. Method for Evaluating Prognosis of Interstitial Pneumonia The present invention is a method for evaluating the prognosis of a disease of lung interstitial disease, and there is a risk of having the disease of the lung interstitial disease or having the disease of the lung interstitial disease. Including the step of measuring the concentration of leucine-rich α2 glycoprotein (LRG) in the biological sample derived from the determined subject, the disease activity of the lung interstitial disease is determined based on the concentration of the LRG. Provided is a method for determining the prognosis of the lung interstitial disease based on the disease activity. In certain embodiments, the prognosis of lung interstitial disease is determined based on disease activity as determined by comparing LRG concentrations to pretreatment LRG concentrations.

As used herein, the term "prognosis" refers to the predicted course and outcome of a patient with a disease. Outcomes include healing, remission and immutability. Prognosis can be assessed by comparison with pre-treatment LRG levels. The measurement of LRG concentration is performed as described above.

The biological sample used in the above prognosis evaluation method is preferably serum, but may be plasma. The biological sample can be of human or mammalian origin.

In the above prognosis evaluation method, the LRG concentration in a biological sample can be measured by contacting the biological sample with a reagent capable of binding to LRG (for example, an anti-LRG antibody or a fragment thereof). For example, the various quantitative methods described herein can be used, including nephrometry, competitive methods, immunometric methods, sandwich methods and the like.

The present invention further provides a prognosis-determining agent for determining the prognosis of pulmonary interstitial diseases such as interstitial pneumonia. The prognostic agent comprises a reagent capable of binding to LRG, which reagent may be labeled or may be capable of binding to the labeled molecule. As described above, various labeling agents can be used for labeling, and those skilled in the art can select a suitable targeting agent according to the measurement method. Examples of the labeling agent include radioactive isotopes, enzymes, fluorescent substances, luminescent substances and the like.

The present invention also provides a kit for detecting and / or quantifying LRG, which comprises the prognostic agent. The kit may further include a secondary antibody capable of binding to the above reagents. The secondary antibody can be a labeled antibody that binds to the primary antibody.

(General technology)
The molecular biological, biochemical, and microbiological methods used herein are well known and commonly used in the art, including those already cited, eg, Sambrook J. et. al. (1989). Molecular Cloning: A Laboratory Manual, 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 ofMethods from Current Protocols in Mo lecular Biology, Wiley, and annual updates; Sninsky, JJ et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press These have been incorporated herein by reference in their relevant parts (which may be all).

For DNA synthesis techniques and nucleic acid chemistry for the production of artificially synthesized genes, see, for example, Gait, MJ (1985). Oligonucleotide Synthesis: A Practical Approach, 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, GMet al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, GT (I996). These have been incorporated herein by reference in the relevant parts.

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

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

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

The present invention has been described above by showing preferred embodiments for ease of understanding. Hereinafter, the present invention will be described based on examples, but the above description and the following examples are provided for purposes of illustration only and not for the purpose of limiting the present invention. Therefore, the scope of the present invention is not limited to the embodiments and examples specifically described in the present specification, but is limited only by the claims.

Hereinafter, the present invention will be further described with reference to Examples, but the present invention is not limited thereto.
(Reference Example 1) Preparation of LRG-deficient mouse An LRG homo-deficient mouse was prepared by the following method.

The mouse LRG gene (Lrg1) was obtained by PCR, inserted into a vector having a diphtheria toxin sequence, and modified as follows to prepare a targeting vector. A neomycin resistance gene (Neo) sandwiched between FRT sequences was inserted downstream of the coding region of the Lrg1 gene (exon 2), and Loxp sequences were further inserted upstream of exon 2 and downstream of Neo, respectively. This targeting vector was introduced into mouse ES cells by electroporation to select neomycin-resistant ES clones. Homologous recombination clones were identified by PCR and used to generate chimeric mice. The offspring of chimeric mice were screened by PCR to obtain the desired heterozygous mice, and then the Neo and Lrg sequences were sequentially deleted by mating with FLP-expressing transgenic mice and Cre-expressing transgenic mice. Lrg homozygous mice were obtained by mating the obtained Lrg1 heterozygous mice with each other.

(Example 1: LRG expression distribution)
In this example, various tissues were taken out from mice, and the diversity of LRG expression in various tissues was observed.

(Materials and methods)
Spleen cells, splenocyte-derived TB cells, blood-derived neutrophils (Gr1-positive), peritoneal macrophages, and liver were collected from wild-type mice (C57BL / 6J, 9-11-week-old female, purchased from Charles River). , MRNA was extracted. Various cells were collected by the MACS® cell separation system (Miltenyi Biotec), and Thy1.2 and CD19-positive cells in splenocytes were used as T cells and B cells, respectively, and Gr1 in blood cells (peripheral blood). Positive cells were collected as neutrophils. In addition, among the cells in the abdominal cavity lavage fluid, the cells adhered to the culture dish were collected as peritoneal macrophages. The mRNA was extracted using the RNeasy Mini Kit (QIAGEN Cat. No. 74106) according to the product manual. The expression of LRG was examined by real time PCR. Real-time PCR was ^{performed according to the product manual of SYBR® Premix Ex Taq TM} (TAKARA RR420A) and analyzed using the 7900HT Fast Real-Time PCR System (applied biosystems). The sequence of primers is as follows. Fw 5'ATCAAGGAAGCCTCCAGGAT (SEQ ID NO: 5), Rv 5'CAGCTGCGTCAGGTTGG (SEQ ID NO: 6). The expression level of LRG was corrected by the expression of HPRT1. In addition, liver and blood-derived neutrophils were collected from wild-type mice to extract proteins. For protein extraction, RIPA buffer (10 mM Tris-HCl pH 7.5, 0.1% sodium deoxycholate, 1% NP-40, 0.1%) supplemented with a proteolytic enzyme inhibitor and a dephosphorylation inhibitor (Nakarai) SDS, 150 mM NaCl) was used. For liver tissue, the supernatant was collected by homogenizing in RIPA buffer, and for neutrophils, cell pellet was dissolved in RIPA buffer. The protein lysate was subjected to sugar chain cleavage treatment (GPF +) with glycopeptidase F (Takara), and then the change in the molecular weight of LRG was compared with the untreated sample by Western blotting. The Western blotting method is as follows. The above protein lysates are separated by SDS-PAGE, transferred to a PVDF membrane, treated with a rabbit anti-mouse LRG antibody (IBL, clone R322), and chemiluminescent using an enzyme-labeled secondary antibody (Western Lightning ECL, Perkin). Detected by Elmer).

(result)
The results are shown in FIG. As is clear from the graph on the left of FIG. 1, it was observed that LRG was highly expressed especially in neutrophils and Resident macrophages. It was also found that LRG produced by neutrophils and macrophages and LRG produced by the liver differ in sugar chain modification.

(Example 2: Observation of LRG with an immunoelectron microscope)
In this example, the expression of LRG was observed using an immunoelectron microscope to examine its distribution.

(Materials and methods)
LRG in human neutrophils was examined by immunoelectron microscopy. The specimen was prepared by the Post-embedding method, and the electron microscope used was HT7700 (Hitachi High-Technologies Corporation). The observation was carried out at Kamakura Techno Science Co., Ltd. under standard shooting conditions. The antibodies used were LRG1 polyclonal antibody (Proteintech, 13224-1-AP) and Goat Anti-Mouse IgG H & L (Abcam, 5nm Gold).

(result)
The results are shown in FIG. In the figure, black particles (gold colloidal particles) such as the tip of the arrow indicate LRG. As shown, LRG was shown to be abundant in neutrophil granules.

(Example 3: Expression of LRG in a bleomycin-induced interstitial pneumonia mouse model)
In this example, the expression of LRG in a bleomycin-induced interstitial pneumonia mouse model was confirmed.

(Materials and methods)
(Preparation of mouse model of bleomycin-induced interstitial pneumonia)
Anesthetize wild-type mice (C57BL / 6, female) and administer bleomycin (1.5 mg / kg) (Bleomycin (registered trademark) injection 5 mg (Nippon Kayaku Co., Ltd.)) into the trachea for interstitial pneumonia. -Induced pulmonary fibrosis, which was used as a mouse model for bleomycin-induced interstitial pneumonia.

(LRG expression)
Lungs were collected from mice 7 days, 14 days, and 21 days after administration, sections were prepared after extension and fixation, and LRG expression was examined by immunohistochemical staining. Immunohistochemical staining was performed as follows. That is, after deparaffinization, a rabbit anti-mouse LRG antibody (IBL, clone R322) was reacted, and the color was developed by DAB using the DAKO REAL Envision Detection System (Dako K5007).

(result)
The results are shown in FIG. From the photographic diagram of FIG. 3, it is interpreted that there are strongly stained cells at least in the alveolar epithelium and the bronchial epithelium from an anatomical point of view. Therefore, it was shown that LRG is expressed in alveolar epithelial cells and bronchial epithelial cells in bleomycin-induced interstitial pneumonia.

(Example 4: Bleomycin-induced interstitial pneumonia in LRG knockout mice)
In this example, the degree of inflammation in bleomycin-induced interstitial pneumonia in LRG knockout mice was confirmed.

(Materials and methods)
Bleomycin was administered intratracheally to various mice to induce interstitial pneumonia in the mice. The same reagents as in Example 3 were used. In this example, wild-type mice (C57BL / 6J, 9-11 week old females, purchased from Charles River) and LRG-deficient mice produced in Reference Examples (in this specification, "deficient" mice are "knockout" mice or "knockout" mice. Breomycin was administered to each of the "KO" mice, which are also used interchangeably.) To induce interstitial pneumonia in the mice.

Then, in various mice, the body weights after 3, 7, 10, 12, 14, 17, and 21 days were measured and recorded. In addition, the expression level of TNFα was measured. MRNA was extracted from the lung and the expression of TNFα was examined by real-time PCR. The mRNA extraction and real-time PCR methods were in accordance with Example 1, and primers Fw 5'CCTCCCTCTCATCAGTTCTATGG (SEQ ID NO: 7) and Rv 5'CGTGGGCTACAGGCTTGTC (SEQ ID NO: 8) were used for the detection of TNFα.

(result)
The results are shown in FIG. FIG. 4 (left) shows the results of changes in body weight over time. LRG knockout mice had reduced weight loss after bleomycin administration compared to wild-type mice. FIG. 4 (right) shows the expression level of TNFα. LRG knockout mice had reduced lung expression of the inflammatory cytokine TNFα compared to wild-type mice. From the above, it was found that in bleomycin-induced interstitial pneumonia, inflammation was mild in LRG knockout mice.

(Example 5: Infiltration of neutrophils in LRG knockout mice in bleomycin-induced interstitial pneumonia)
In this example, neutrophil infiltration of LRG knockout mice was observed in bleomycin-induced interstitial pneumonia.

(Materials and methods)
Mice treated with bleomycin were analyzed on days 1 and 7. Alveolar lavage fluid (BALF) was collected from each individual mouse using Dulbecco PBS (-) (0.5 mL x 3 times), the number of cells in the fluid was counted, and the cell fraction was analyzed by FACS. FACS was performed under the following conditions. Cells, FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, Pacific Blue, Horizon V500 labeled antibody against CD45, Gr1, CD19, CD86, CD11c, F4 / 80, Ly6G, CD11b (CD11b) Only Becton Dickinson, others purchased from BioLegend) were stained and analyzed using FACS CantoII (Becton Dickinson) as instructed by the manufacturer.

(result)
The results are shown in FIG. FIG. 5 shows the total number of cells in (left) BALF and FIG. 6 shows the number of neutrophils (Ly6G +) in (right) BALF. In Breo-induced interstitial pneumonia, LRG knockout mice were found to have markedly reduced neutrophil infiltration. Therefore, it is understood that LRG plays a role in enhancing cell infiltration of neutrophils.

(Example 6: In bleomycin-induced interstitial pneumonia, collagen expression is low in LRG knockout mice)
In this example, collagen expression was observed in LRG knockout mice in bleomycin-induced interstitial pneumonia.

(Materials and methods)
MRNA was extracted from the lungs of mice to which bleomycin was administered, and the expression of collagen 1α2 was examined by real-time PCR. The mRNA extraction and PCR methods were the same as those described in Example 1, and Fw 5'TGTTGGCCCATCTGGTAAAGA (SEQ ID NO: 9) and Rv 5'CAGGGAATCCGATGTTGCC (SEQ ID NO: 10) were used as PCR primers.

(result)
The results are shown in FIG. Control It can be seen that in LRG knockout mice, the induction of collagen expression related to fibrosis is low in bleomycin-induced interstitial pneumonia on days 3, 7, and 14. Therefore, it is understood that LRG plays an important role in the progression of pulmonary fibrosis. LRG is closely associated with fibrosis associated with interstitial pneumonia (ie, pulmonary fibrosis), revealing that LRG can be an active marker of pulmonary fibrosis.

(Example 7: Quantification of serum LRG concentration in patients with interstitial pneumonia)
In this example, the serum LRG concentration in the serum of patients with interstitial pneumonia was quantified.

(Method)
Serum LRG levels were quantified in patients with interstitial pneumonia (CHP (n = 11), CDV-IP (n = 12), IPF (n = 10), NSIP (n = 10)). The correlation between serum LRG concentration and serum KL-6 concentration, and the correlation between serum LRG concentration or serum KL-6 concentration and% DLCO, VC,% VC were analyzed. The sandwich method was used to measure the serum LRG concentration.

(result)
The results are shown in FIGS. 7 to 11. The serum LRG concentration of patients with interstitial pneumonia was significantly higher than that of healthy subjects. In addition, the serum LRG concentration was significantly higher than that of healthy subjects in any of CHP, CVD-IP, IPF, and NSIP diseases (Fig. 7). Serum LRG levels were correlated with serum KL-6 levels in IPF patients, but not in other diseases (Fig. 8). Serum LRG concentrations showed a significant correlation with% DLCO in CVD-IP and NSIP patients (Fig. 9). Serum LRG levels were significantly correlated with VC and% VC in NSIP patients (FIGS. 10 and 11). That is, a correlation was found between the degree of pulmonary dysfunction and serum LRG concentration, but no significant correlation was found for KL-6. These results indicate that KL-6 cannot function as a marker for all interstitial pneumonia, while LRG can be a comprehensive marker.

We have demonstrated in the present invention that LRG is involved in the regulation of cell infiltration and / or cell migration. Then, in this example, in NSIP, which is one of the interstitial pneumonia caused by inflammation due to cell infiltration, the serum LRG concentration is closely related to% DLCO and% VC, which are important factors in the evaluation of disease activity. It became clear that it correlates with. Therefore, it can be said that the result of this example or the LRG concentration proved that it can be used as a disease activity marker of NSIP. In addition to this, interstitial pneumonia including CHP, CVD-IP, IPF and NSIP is a disease whose onset mechanism is cell infiltration in common, so LRG is not only NSIP but also CHP, CVD-. It was predicted to be useful for assessing general disease activity of interstitial pneumonia such as IP and IPF. As expected, similar experiments were performed on patients with interstitial pneumonia (CHP, CVD-IP and IPF) other than NSIP, and as with NSIP patients, the levels were significantly higher than in healthy individuals. In addition, a tendency similar to NSIP was observed in relation to% DLCO and% VC. Therefore, those skilled in the art will understand that LRG can be a disease activity marker for various interstitial pneumonia with cell infiltration as a common pathogenic mechanism.
(Relationship with disease activity)

Usually, the disease activity of interstitial pneumonia is to comprehensively evaluate the results of symptoms, respiratory function tests such as% DLCO or% VC, imaging tests such as chest CT, arterial blood gas or blood tests, and their transition. Determined by. % DLCO and% VC are particularly important in assessing disease activity in lung interstitial disease, especially interstitial pneumonia, as they are factors directly involved in the exacerbation of the condition.

In addition, the results of this example revealed that serum LRG correlates with% DLCO and% VC, which are important factors in assessing the disease activity of interstitial pneumonia. That is, it is shown that the activity can be easily evaluated by the blood test of LRG. Therefore, it is understood that the markers of the present invention can be used as an index of disease activity of diseases of pulmonary interstitium such as interstitial pneumonia.

(Example 8: Analysis of serum LRG concentration in patients with interstitial pneumonia with dermatomyositis)
In this example, the serum LRG concentration of patients with interstitial pneumonia with dermatomyositis before and after treatment was analyzed.

(Method)
Serum LRG levels of patients with interstitial pneumonia with dermatomyositis (N = 49) were measured before, 2 weeks, 4 weeks, 8 weeks, and 12 weeks after treatment. Treatment of interstitial pneumonia with dermatomyositis was performed by using a steroid (oral prednisolone / methylprednisolone pulse therapy) in combination with an immunosuppressant (cyclosporine or tacrolimus). Depending on the case, additional pulse therapy with cyclophosphamide was also given. The sandwich method was used to measure the serum LRG concentration.

(result)
The results are shown in FIGS. 12 and 13-17. In patients with interstitial pneumonia with dermatomyositis, the LRG concentration decreased with the course of treatment, and the serum LRG concentration decreased significantly after the treatment as compared with before the treatment (Fig. 12).

The results of Examples 7 and 8 showed that measuring serum LRG levels was highly useful in assessing activity in patients with interstitial pneumonia.

The present invention has been described above based on examples. It is understood by those skilled in the art that this embodiment is merely an example, and that various modifications are possible and that such modifications are also within the scope of the present invention.

As described above, the present invention has been illustrated using the preferred embodiments of the present invention, but it is understood that the scope of the present invention should be interpreted only by the scope of claims. The patents, patent applications and documents cited herein are to be incorporated by reference in their content as they are specifically described herein. Understood.

INDUSTRIAL APPLICABILITY The present invention provides a prophylactic and / or therapeutic agent for a disease related to cell migration, which targets LRG, which is a factor different from the target of conventional therapeutic agents. By providing therapeutic agents with a new mechanism of action, diseases related to cell migration, inflammatory diseases, immune diseases, etc. Acute phase (onset phase, acute exacerbation phase, etc.) of the disease, especially acute phase of inflammation (onset phase, acute exacerbation phase, etc.), and inflammation that has acquired resistance to the therapeutic agent by continuous use of the existing therapeutic agent. Better treatment can be provided to patients in the acute phase (onset stage, acute exacerbation stage, etc.) of diseases such as sexual diseases and immune diseases, especially in the acute phase of inflammation (onset stage, acute exacerbation stage, etc.). Furthermore, the present invention has a preventive purpose of preventing an acute phase (onset phase, acute exacerbation phase, etc.), particularly an acute phase of inflammation (onset phase, acute exacerbation phase, etc.) in diseases such as inflammatory diseases and immune diseases, and inflammation. For the purpose of preventing recurrence of diseases in patients who have a temporary remission of the acute phase (onset phase, acute exacerbation phase, etc.) of diseases such as sexual and immune diseases, especially the acute phase of inflammation (onset phase, acute exacerbation phase, etc.) Can be used. Further, according to the present invention, the acute phase (onset phase, acute exacerbation phase, etc.), particularly inflammation, in diseases such as inflammatory diseases and immune diseases, which exert a therapeutic or preventive effect by inhibiting the expression or function of LRG. It is possible to screen for new prophylactic / therapeutic drug candidates in the acute phase (onset phase, acute exacerbation phase, etc.). In addition, it is possible to judge the disease activity of interstitial pneumonia and evaluate the prognosis using LRG as an index.

SEQ ID NO: 1: Human LRG protein nucleic acid sequence SEQ ID NO: 2: Human LRG protein amino acid sequence SEQ ID NO: 3: Mouse LRG protein nucleic acid sequence SEQ ID NO: 4: Mouse LRG protein amino acid sequence SEQ ID NO: 5: LRG detection primer (Forward) )
SEQ ID NO: 6: LRG detection primer (Reverse)
SEQ ID NO: 7: TNFα detection primer (Forward)
SEQ ID NO: 8: TNFα detection primer (Reverse)
SEQ ID NO: 9: Primer for detecting collagen 1α2 (Forward)
SEQ ID NO: 10: Primer for detecting collagen 1α2 (Reverse)

Claims (29)

A determination agent for determining the disease activity of lung interstitial diseases, which comprises a reagent capable of binding to leucine-rich α2 glycoprotein (LRG). The determination agent according to claim 1, wherein the disease of the lung interstitium is interstitial pneumonia or pulmonary fibrosis. The diseases of the lung interstitial disease are idiopathic pulmonary fibrosis (IPF), chronic hypersensitive pneumonia (CHP), interstitial lung disease-related pneumonia (CVD-IP), and idiopathic non-specific interstitial pneumonia (NSIP). The determination agent according to claim 1, wherein the interstitial pneumonia with dermatitis, exfoliative interstitial pneumonia, lymphocytic interstitial pneumonia, diffuse alveolar disorder, or granulomatous interstitial pneumonia. The determination agent according to any one of claims 1 to 3, wherein the reagent can bind to a labeled or labeled molecule. The determination agent according to any one of claims 1 to 4, wherein the reagent is an anti-LRG antibody or a fragment thereof. A kit for detecting and / or quantifying leucine-rich α2 glycoprotein (LRG), which comprises the determination agent according to any one of claims 1 to 5. The kit of claim 6, further comprising a secondary antibody capable of binding to the reagent. A prognostic agent for determining the prognosis of lung interstitial disease, which comprises a reagent capable of binding to leucine-rich α2 glycoprotein (LRG). The prognosis-determining agent according to claim 8, wherein the disease of the lung interstitium is interstitial pneumonia or pulmonary fibrosis. The interstitial lung diseases are idiopathic pulmonary fibrosis (IPF), chronic hypersensitivity pneumonia (CHP), interstitial lung disease-related pneumonia (CVD-IP), and idiopathic nonspecific interstitial pneumonia (NSIP). The prognosis agent according to claim 8, which is interstitial pneumonia with dermatomyitis, exfoliative interstitial pneumonia, lymphocytic interstitial pneumonia, diffuse alveolar disorder, or granulomatous interstitial pneumonia. .. The prognostic agent according to any one of claims 8 to 10, wherein the reagent can bind to a labeled or labeled molecule. The prognosis-determining agent according to any one of claims 8 to 11, wherein the reagent is an anti-LRG antibody or a fragment thereof. A kit for detecting and / or quantifying leucine-rich α2 glycoprotein (LRG), which comprises the prognostic agent according to any one of claims 8 to 12. 13. The kit of claim 13, further comprising a secondary antibody capable of binding to the reagent. A method of using leucine-rich α2 glycoprotein (LRG) as an index of disease activity of lung interstitial disease in a subject, which is a method of using leucine-rich α2 glycoprotein (LRG) in a biological sample derived from the subject. A method comprising the step of measuring a concentration. The method according to claim 15, wherein when the concentration of LRG shows a high value as compared with a healthy person, it is shown that the subject has high disease activity of a disease of lung interstitium. The method according to claim 16, wherein the concentration of LRG in the healthy person is 3 μg / ml. The method according to any one of claims 15 to 18, wherein the disease of the lung interstitium is interstitial pneumonia or pulmonary fibrosis. The diseases of the lung interstitial disease are idiopathic pulmonary fibrosis (IPF), chronic hypersensitive pneumonia (CHP), interstitial lung disease-related pneumonia (CVD-IP), and idiopathic non-specific interstitial pneumonia (NSIP). , Interstitial pneumonia with dermatitis, exfoliative interstitial pneumonia, lymphocytic interstitial pneumonia, diffuse alveolar disorder, or granulomatous interstitial pneumonia, any one of claims 15-18. The method described in the section. The method according to any one of claims 15 to 19, wherein the concentration of the LRG is measured by contacting the biological sample with a reagent capable of binding to the LRG. The method of claim 20, wherein the reagent is an anti-LRG antibody or fragment thereof. The determination method according to any one of claims 15 to 21, wherein the biological sample is selected from the group consisting of serum and plasma. Leucine-rich α2 glycoprotein (LRG) is a method for evaluating the prognosis of lung interstitial disease, and there is a risk of having the lung interstitial disease or having the lung interstitial disease. A method comprising the step of measuring the concentration of leucine-rich α2 glycoprotein (LRG) in a biological sample derived from a subject determined to be present. The method according to claim 23, wherein the disease activity determined by comparing the concentration of LRG with the concentration of LRG before treatment is used as an index for evaluating the prognosis of the disease of the lung interstitium. The method according to claim 23 or 24, wherein the disease of the lung interstitium is interstitial pneumonia or pulmonary fibrosis. The diseases of the lung interstitial disease are idiopathic pulmonary fibrosis (IPF), chronic hypersensitive pneumonia (CHP), interstitial lung disease-related pneumonia (CVD-IP), and idiopathic non-specific interstitial pneumonia (NSIP). 23 or 24 of claim 23 or 24, which is interstitial pneumonia with dermatitis, exfoliative interstitial pneumonia, lymphocytic interstitial pneumonia, diffuse alveolar disorder, or granulomatous interstitial pneumonia. Method. The method according to any one of claims 23 to 26, wherein the concentration of the LRG is measured by contacting the biological sample with a reagent capable of binding to the LRG. 27. The method of claim 27, wherein the reagent is an anti-LRG antibody or fragment thereof. The method according to any one of claims 23 to 28, wherein the biological sample is selected from the group consisting of serum and plasma.

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