MX2008008584A - Anti-periostin antibody and pharmaceutical composition for preventing or treating periostin-related disease containing the same. - Google Patents

Abstract

It is intended to provide an antibody against periostin having an anti-cell adhesion activity, particularly the anti-periostin antibody having an ability to neutralize an anti-cell adhesion action, and a preventive or therapeutic agent for a periostin-related disease using the antibody. It is also intended to provide detection and determination methods for the periostin in a sample using the antibody and a diagnostic method for a periostin-related disease by determining the amount of the periostin by the method.

Description

ANTIBODY AGAINST PERIOSTINE, AND A PHARMACEUTICAL COMPOSITION THAT UNDERSTANDS IT TO PREVENT OR TREAT AN ILLNESS IN WHICH THE PERIOSTINE IS INVOLVED FIELD OF THE INVENTION The present invention relates to antibodies against periostin isoforms having anti-cell adhesive properties, especially anti-periostin antibodies which have the ability to neutralize anti-cell adhesive properties. More specifically, it relates to anti-periostin antibodies that specifically recognize a site responsible for anti-cell adhesion of periostin which have anti-cell adhesive properties specifically expressed in interstitial tissue during tissue restructuring such as cardiac hypertrophy, which are useful for prevention or treatment of diseases related to periostin, such as cardiac deficiency, or are useful for the diagnosis of these diseases.

BACKGROUND OF THE INVENTION Chronic heart failure is a condition in which the heart can not pump enough blood to several organs due to decreased myocardial contractility. Conventionally, it has been treated with cardiotonic drugs that increase myocardial contractility such as digitalis drugs. However, these drugs have been shown to impair vital forecasts during long-term administration, due to excessive energy consumption of the myocardium. Thus, recently predominant therapies are those that use diuretics, β-blockers or angiotensin inhibitors that reduce the excessive load on the heart through the sympathetic nervous system or activated renin-angiotensin-aldosterone system in the condition of cardiac deficiency. However, patients with heart failure have limited activities in their daily lives and can not maintain their quality of life because they are prohibited from doing heavy exercise or the like. In addition, the vital prognosis of patients with cardiac deficiency can not be fully assured. Therefore, it is desirable to develop a new effective drug for the treatment of cardiac deficiency, which allows an improvement in the quality of life and an improvement in the long-term vital prognosis. On the other hand, periostin is an extracellular matrix protein and consists of a polypeptide having a molecular weight of about 90,000. Each polypeptide chain has a signal sequence, a domain rich in cysteine, a domain repeated four times, and a C-terminal domain. Periostin was first called osteoblast-specific factor-2 (OSF-2) and was isolated and identified as a gene specifically expressed in the mouse osteoblast cell line MC3T3-EI (patent document 1, non-patent document 1), and it was later known as periostin and was reported to have adhesion promoter activity in osteoblast cells (non-patent document 2). In early studies, it is thought that periostin is an extracellular matrix specifically expressed in bone tissue. However, it is currently known that it is expressed not only in bone tissue but also very highly in the onset of cardiac deficiency (non-patent document 3, non-patent document 4), aneurysms (non-patent document 5), cancers highly metastatic (non-patent document 6, non-patent document 7, non-patent document 8), pre-eclampsia (non-patent document 9) as well as very slightly in normal tissue. In addition, some splice variants of periostin have been shown to be expressed in osteoblasts (non-patent document 1, non-patent document 2, non-patent document 10, non-patent document 3). As for the functions of periostin, a periostin splice variant consisting of 81 1 amino acids (corresponding to PN-2 in Figure 1) (non-patent document 2) and a splice variant of periostin which consists of 782 amino acids (non-patent document 1 1) are reported to have cellular adhesive properties. On the contrary, it has been reported that a periostin splice variant consisting of 838 amino acids (corresponding to PN-1 in Figure 1) prevents cardiac fibroblasts from adhering to a plate coated with the splice variant. of periostin, that is, has no cell adhesive activity; the gene expression of the periostin splice variant consisting of 838 amino acids (corresponding to PN-1 in Figure 1) is significantly increased in model rats with cardiac deficiency compared to normal rats; this variant is an aggravation factor that induces cardiac dilation; and because the survival rate is significantly increased by inhibiting the expression of this protein (non-patent document 3). In addition, there is a report of a prophylactic or therapeutic agent for cardiac deficiency, in which an anti-sense nucleotide against the splice variant of periostin consisting of 838 amino acids is used to suppress the expression of the splice variant of periostin (patent document 2). As shown above, it has been suggested that the expression of the periostin gene is related to the pathology of cardiac deficiency, but the relationship between the structure of splice variants of periostin and cardiac deficiency is unknown. In this way, we have made an attempt to clarify the structure of periostin related to the pathology of cardiac deficiency when using antibodies. As regards periostin antibodies, there are reports of an antibody related to the inhibition of periostin chemotaxis (non-patent document 12) and an antibody having inhibitory activity against periostin-induced cell growth (non-patent document 13) . However, there is no report of antibodies that show the structure of a region responsible for the periostin cellular adhesive activity nor a report that shows the relationship between the periostin cell adhesive activity and diseases such as cardiac deficiency. Patent Document 1: JPA No. HEI-5-268982 Patent Document 2: Re-publication WO02 / 020055 Patent Document 3: International Publication WO2005 / 019471 Non-patent document 1: Takeshita S. et al., Biochem J (1993) 294, 271-8 Non-patent document 2: Horiuchi K. et al., J. Bone Miner. Res. (1999) 14, 1239-49 Non-patent document 3: Katsuragi N. et al., Circulation (2004) 1 10, 1806-13 Non-patent document 4: Wang D. et al., Hypertension (2003) 42, 88-95 Non-patent document 5: Peters DG. et al., Stroke (2001) 32, 1036-42 Non-patent document 6: Shao R. et al., Mol Cell Biol. (2004) 24, 3992-4003 Non-patent document 7: Gonzalez HE. et al., Arch Otolaryngol Head Neck Surg. (2003) 129, 754-9 Non-patent document 8: Sasaki H. et al., Breast Cancer Res Treat. (2003) 77, 245-52 Non-patent document 9: Sasaki H. et al., Am J Obstet Gynecol. (2002) 186, 103-8 Non-patent document 10: Litvin J. et al., J Cell Biochem. (2004) 92, 1044-61 Non-patent document 1 1: Gillan L. et al., Cancer Res. (2002) 62, 5358-64 Non-patent document 12: Lindner V. et al., Arterioscler Thromb Vasc Biol. (2005) 25, 77-83 Non-patent document 13: Tai IT. et al., Carcinogenesis (2005) 26, 908-15.

BRIEF DESCRIPTION OF THE INVENTION The present invention aims to provide, for example, a novel and effective prophylactic or therapeutic agent for cardiac deficiency, which allows an improvement in the quality of life and an improvement in the long-term vital prognosis. More specifically, the present invention seeks to provide an antibody against a periostin isoform having anti-cellular adhesive activity, specifically the recognition of a site responsible for anti-cell adhesion. The present invention also seeks to provide a hybridoma that produces the antibody, a method for producing the hybridoma, and a method for the production of the antibody through the culture of the hybridoma. The present invention also aims to provide a pharmaceutical composition comprising the antibody to prevent or treat a disease wherein a periostin isoform having anti-cellular adhesive activity is involved. The present invention also has the purpose of providing a method for preventing or treating a disease wherein a periostin isoform having anti-cellular adhesive activity is involved, comprising the administration of the pharmaceutical composition to a patient, as well as a method for the diagnosis of the disease. We clarify that a splice variant of periostin that does not have cellular adhesive activity (PN-1) has anti-cell adhesive activity, that is, the activity of separation of adhered cells. On the other hand, we also confirm that periostin which has cellular adhesive activity (PN-2) does not have anti-cellular adhesive activity, that is, it does not release adherent cells. In addition, we noticed a difference in structure and cellular adhesive activity between periostin cutting and splice variants that have anti-cellular adhesive activity (PN-1) and periostin that does not show anti-cellular adhesive activity (PN-2), and considers that the disease related to periostin isoforms having anticellular adhesive activity can be prevented or treated by inhibiting a region specifically present in periostin splice variants that have anti-cell adhesive activity. In other words, we consider that inhibitors against said region may be useful as prophylactic or therapeutic agents for diseases related to periostin isoforms having anti-cellular adhesive activity.

Analysis of splicing variants of periostin highly expressed during cardiac deficiency reveal that the C-terminal domains where the splice variants are formed consist of exons 15 to 23; specifically rats having the following variants (1) to (4): (1) a variant that retains all exons (called PN-1; which consist of 838 amino acids shown as SEQ ID NO: 1; the cDNA sequence shown as SEQ ID NO: 6), (2) a variant lacking exon-17 (called PN-2; which consists of 81 1 amino acids shown as SEQ ID NO: 5; 27 amino acids (exon 17) shown in SEQ ID NO: 3 are deleted from PN-1; the cDNA sequence shown as SEQ ID NO: 7), (3) a variant lacking exon-21 (called PN-3; consisting of 810 amino acids), (4) a variant lacking exon-17 and exon -21 (called PN-4, which consists of 783 amino acids). In addition to rats, mouse and human PN-1 and PN-2 are also found (mouse PN-1: SEQ ID NO: 8 (amino acid sequence), SEQ ID NO: 9 (cDNA sequence), PN-2 of mouse: SEQ ID NO: 10 (amino acid sequence), SEQ ID NO: 1 1 (cDNA sequence): human PN-1: SEQ ID NO: 12 (cDNA sequence); PN-2 of human; NO: 13 (amino acid sequence), SEQ ID NO: 14 (cDNA sequence)). Among these, PN-1 is highly expressed while PN-2 and PN-3 are expressed to a much lesser degree in the tissue with rat cardiac hypertrophy. Thus, we contemplate the preparation of an antibody that specifically recognizes the residual portion of amino acid encoded by exon-17 as an inhibitor against that site, which site is structurally different in PN-1 and PN-2 and exclusively found in PN- 1 . In order to prepare an antibody, the material used as an immunogen must be hydrophobic, and if an antibody is to be prepared using a part of a large polypeptide such as a protein, the part used as an immunogen must be exposed in the surface of the protein to form a part of the epitope. Thus, for the purpose of examining the possibility of using the peptide chain exon-17 as an antigen, an epitope search is performed initially using the Accelrys software that is widely used in the field of bioinformatics Mac Vector 7.2. It is judged from "hydrophilicity", "surface probability" and "antigenicity" that TTKIITKLVEPKIKVIQGSLQPIIKTE (SEQ ID NO: 3) of the exon-17 region is mainly hydrophobic, suggesting that this region is very unlikely to be exposed on the surface of the protein molecule and has no immunogenicity, so that it can not be used to prepare an antibody, and therefore, it is presumed that it can be difficult to use to prepare practically an antibody. However, we ventured to prepare an antibody against the amino acid sequence encoded by exon-17 by assuming that the use of an antibody against a polypeptide region encoded by exon-17 found specifically in PN-1 may be more suitable to specifically inhibit the PN-1 functions. A peptide consisting of 27 amino acids constituting a peptide encoded by the region of exon-7 is synthesized and used to immunize rabbits, and the resulting serum is purified to give an IgG fraction, whereby a polyclonal anti-peptide antibody is prepared. -expon-17 When the periostin PN-1 protein is then added to a confluent culture to 80% of heart fibroblasts, almost 100% cell detachment (ie, anti-cell adhesive activity) is observed. The administration of the anti-exon-17 peptide antibody to this experimental system inhibits the cellular separation mediated by the periostin protein PN-1, which shows that the anti-exon-17 peptide antibody is an antibody that has a neutralizing activity against PN periostin protein. -1 . Then, the rats of acute myocardial infarction model are prepared and the weekly administration of the anti-exon-17 peptide antibody continues showing that the dilation of the heart is significantly inhibited 4 weeks after the preparation of the models, and that the cardiac function It gets better. It is also shown that these properties are sustained and cardiac fibrosis is inhibited even 8 weeks after the preparation of the models. This indicates that the anti-exon-17 peptide antibody is an antibody that has the activity of inhibiting cardiac dilation and cardiac fibrosis associated with the progress of the cardiac deficiency condition, and the activity of improving cardiac function, leading to the embodiment of the present invention.

As a solution to the problems described above, the present invention provides an antibody against a periostin isoform having anti-cellular adhesive activity specifically expressed in the heart during cardiac deficiency or the like, particularly an antibody that specifically recognizes a site responsible for anti-cell adhesion. That is, the present invention includes the following aspects. (1) An antibody against a periostin isoform having anti-cellular adhesive activity, which specifically recognizes a site responsible for periostin anti-cell adhesion, and which has the ability to neutralize the anti-cellular adhesive activity of periostin. (2) The antibody as defined in (1), wherein the site responsible for anti-cellular adhesion of periostin is the amino acid sequence encoded by exon-17 or a portion thereof. (3) The antibody as defined in (2), wherein the amino acid sequence encoded by exon-17 or a part thereof is one of the amino acid sequences selected from the group of SEQ IDS NO: 3, 4, 21 , 22, 23, 24, 26 and 34. (4) The antibody as defined in (3), wherein the amino acid sequence is the amino acid sequence of SEQ IDS NO: 3, 4 or 21. (5) The antibody as defined in any of (1) to (4), which is a monoclonal antibody. (6) The antibody as defined in (5) produced by a hybridoma cell line FERM BP-10718. (7) A hybridoma obtainable by a method comprising the steps of: immunizing a mammal with a peptide having one of the amino acid sequences selected from the group of SEQ IDS NO: 3, 4, and 21 or a peptide that introduces residues of cysteine at its N-terminus; and fusing a mammalian antibody producing cell with a myeloma cell. (8) A FERM BR-10718 hybridoma cell line. (9) A method for producing an antibody as defined in (5), comprising the steps of: immunizing a mammal with a peptide having one of the amino acid sequences selected from the group of SEQ IDS NO: 3, 4, and 21 or a peptide that introduces cysteine residues into its N- terminal; fusing a cell that produces antibody from the mammal with a myeloma cell; and cultivate the obtained hybridoma. (10) The method as defined in (9), wherein the hybridoma is a hybridoma cell line FERM BP-10718. (11) A pharmaceutical composition comprising the antibody as defined in any of (1) to (6). (12) A pharmaceutical composition for preventing or treating a disease in which a periostin isoform having anti-cellular adhesive activity is involved, comprises the antibody as defined in any of (1) to (6). (13) The pharmaceutical composition as defined in (12), wherein the disease is heart failure, myocardial infarction, cardiac dilation, cardiac hypertrophy, cardiac fibrosis, cardiomyopathy, myocarditis, valvular disease, cancer, aneurysm, arteriosclerosis, neurodegenerative disease central, kidney disease, rheumatoid arthritis, osteoporosis, pulmonary emphysema, pulmonary hypertension, chronic obstructive pulmonary disease (COPD), nephritis, pancreatitis, hepatitis, liver fibrosis or pulmonary fibrosis. (14) A method for preventing or treating a disease in which a periostin isoform having anti-cellular adhesive activity is involved, comprises administering the antibody as defined in any of (1) to (6) to a patient. (15) The method as defined in (14), wherein the disease is cardiac deficiency, myocardial infarction, cardiac dilation, cardiac hypertrophy, cardiac fibrosis, cardiomyopathy, myocarditis, valvular disease, cancer, aneurysm, arteriosclerosis, central neurodegenerative disease , kidney disease, rheumatoid arthritis, osteoporosis, pulmonary emphysema, pulmonary hypertension, chronic obstructive pulmonary disease (COPD), nephritis, pancreatitis, hepatitis, liver fibrosis or pulmonary fibrosis. (6) A method for diagnosing a disease in which a periostin isoform having anti-cellular adhesive activity is involved, comprising measuring the amount of the periostin isoform in a biological sample by using the antibody as defined in any of (1) to (6). (17) The method as defined in (16) wherein the antibody is a labeled antibody. (18) The method as defined in (16) or (17), wherein the disease is cardiac deficiency, myocardial infarction, cardiac dilation, cardiac hypertrophy, cardiac fibrosis, cardiomyopathy, myocarditis, valvular disease, cancer, aneurysm, arteriosclerosis , central neurodegenerative disease, kidney disease, rheumatoid arthritis, osteoporosis, pulmonary emphysema, pulmonary hypertension, chronic obstructive pulmonary disease (COPD), nephritis, pancreatitis, hepatitis, liver fibrosis or pulmonary fibrosis. (19) A method for detecting or quantifying a periostin isoform having anti-cell adhesive activity in a sample using the antibody as defined in any of (1) to (6). (20) A diagnostic reagent for a disease in which a periostin isoform having anti-cellular adhesive activity is involved, comprising the antibody as defined in any of (1) to (6). (21) The method as defined in (20), wherein the disease is heart failure, myocardial infarction, cardiac dilation, cardiac hypertrophy, cardiac fibrosis, cardiomyopathy, myocarditis, valvular disease, cancer, aneurysm, arteriosclerosis, central neurodegenerative disease , kidney disease, rheumatoid arthritis, osteoporosis, pulmonary emphysema, pulmonary hypertension, chronic obstructive pulmonary disease (COPD), nephritis, pancreatitis, hepatitis, liver fibrosis or pulmonary fibrosis.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic diagram showing a splice variant of rat periostin. Figure 2 is a diagram showing test results of the anti-cellular adhesive properties of rat PN-1 (Example 2). Figure 3 is a diagram showing results of the inhibition assay of rat PN-1 by anti-rat exon-17 peptide antibody (example 3). Figure 4A is a diagram showing results of the inhibition test of cardiac dilation by exon-17 antirate peptide antibody 4 weeks after the preparation of acute myocardial infarction models (example 4, anterior wall thickness, wall thickness later). Figure 4B is a diagram showing results of the inhibition test of cardiac dilation by anti-rat exon-17 peptide antibody 4 weeks after the preparation of acute myocardial infarction models (Example 4: end-diastolic internal diameter, extreme-systolic internal diameter, cardiac function). Figure 4C is a diagram showing results of the inhibition test of cardiac dilation by exon-17 antirate peptide antibody 4 weeks after the preparation of models of acute myocardial infarction (Example 4: heart rate, infarct area). Figure 5A is a diagram showing results of the inhibition test of cardiac dilation by exon-17 antirate peptide antibody 8 weeks after the preparation of models of acute myocardial infarction (Example 4: anterior wall thickness, wall thickness later). Figure 5B is a diagram showing results of the inhibition test of cardiac dilation by anti-rat exon-17 peptide antibody 8 weeks after the preparation of acute myocardial infarction models (Example 4: end-diastolic internal diameter, extreme-systolic internal diameter, cardiac function). Figure 5C is a diagram showing test results of the inhibition of cardiac dilation by anti-rat exon-17 peptide antibody 8 weeks after the preparation of models of acute myocardial infarction (Example 4: heart rate, infarct area) ). Figure 6A is a diagram showing hemodynamics of model rats treated with anti-rat exon-17 peptide antibody (Example 4: LVP, heart rate). Figure 6B is a diagram showing hemodynamics of model rats treated with anti-rat exon-17 peptide antibody (Example 4: (+) dP / dt, (-) dP / dt). Figure 6C is a diagram showing hemodynamics of model rats treated with anti-rat exon-17 peptide antibody (Example 4: SEP, DEP, LVEDP). Figure 7 is a diagram showing the results of the histological analysis of model rats treated with anti-rat exon-17 peptide antibody (Example 4). Fig. 8 is a diagram showing the diameters of minor axes of myocardial cells of model rats treated with anti-rat peptide exon-17 antibody (Example 4). Figure 9A is a diagram showing the results of gene expression analysis of model rats treated with anti-rat exon-17 peptide antibody (example 4: G3PDH). Figure 9B is a diagram showing the results of gene expression analysis of model rats treated with anti-rat exon-17 peptide antibody (Example 4: ET-1 / G3, Angiotensinogen / G3). Figure 9C is a diagram showing the results of gene expression analysis of model rats treated with anti-rat exon-17 peptide antibody (Example 4: oc-MHC / G3, -MHC / G3). Figure 9D is a diagram showing the results of gene expression analysis of model rats treated with anti-rat exon-17 peptide antibody (Example 4: Col-l / G3, Col-III / G3). Figure 9E is a diagram showing the results of gene expression analysis of model rats treated with anti-rat peptide exon-17 antibody (Example 4: TGF-p / G3, TNF-oc / G3).

The figure 10 is a diagram showing test results of the anti-cellular adhesive properties of human PN-1 (Example 1 5). Figure 11 is a diagram showing results of the inhibition assay of human PN-1 activity by anti-human monoclonal antibody exon-17 (Example 16). Figure 2A is a diagram showing results of the inhibition test of cardiac dilation by anti-human monoclonal exon-17 antibody 4 weeks after the preparation of models of acute myocardial infarction (Example 17: anterior wall thickness, rear wall thickness). Figure 12B is a diagram showing the results of the inhibition test of cardiac dilation by anti-human monoclonal antibody exon-17 4 weeks after the preparation of acute myocardial infarction models (Example 17: end-diastolic internal diameter, extreme-systolic internal diameter, cardiac function). Figure 12C is a diagram showing results of the inhibition test of cardiac dilation by anti-human monoclonal antibody exon-17 4 weeks after the preparation of models of acute myocardial infarction (Example 17: heart rate, infarct area) ).

DETAILED DESCRIPTION OF THE INVENTION In one embodiment, the present invention provides an antibody against a periostin isoform having anti-cellular adhesive activity. Periostin here is one of the extracellular matrix proteins and is known to include some splice variants, some of which are specifically expressed in the heart during cardiac deficiency or the like. In the present invention, antibodies can be used as substances (i.e., as inhibitors) for the inhibition of functions of a periostin splice variant specifically expressed in the heart during cardiac deficiency or the like due to its high specificity, security for humans and for other reasons. In the present invention, an antibody can be prepared against an antigen composed of a chemically synthesized peptide consisting of an amino acid sequence encoded by the exon-17 region of the C-terminal domain wherein a splice variant is formed, although said peptide can also be obtained from any source by enzymatic digestion of periostin protein or by genetic engineering techniques. In the present invention, the expression "having anti-cellular adhesive activity" means that it has the action of separating or removing adhered cells. Similarly, the expression "having no anti-cell adhesive activity" means that the adhered cells neither separate nor detach. In this case, the cells maintain their attached state. The presence or absence of anti-cell adhesive activity can be determined by culturing cells (eg, heart fibroblasts) in a culture dish to allow cells to adhere to the culture dish, the addition of a test sample and additional culture of the cells, washing of the plate to remove the separated cells, staining of remaining cells, and confirmation of the state of the adhered cells. In the present invention, periostin isoforms having anti-cell adhesive activity preferably include periostin isoforms consisting of an amino acid sequence of SEQ ID NO: 1 (rat periostin PN-1, 838 amino acids), SEQ ID NO: 2 (human periostin PN-1, 836 amino acids having a shorter N-terminal signal sequence by 2 amino acids than that of rat periostin), or SEQ ID NO: 8 (mouse periostin PN-1). Regions responsible for the anti-cellular adhesive activity of periostin include, for example, the exon-17 site. Specific examples include a residual portion of amino acids shown in SEQ ID NO: 3 of a periostin isoform having an amino acid sequence shown in SEQ ID NO: 1 (672-698 amino acids of SEQ ID NO: 1), a residual part of amino acid shown in SEQ ID NO: 4 of a periostin isoform having an amino acid sequence shown in SEQ ID NO: 2 (670-696 amino acids of SEQ ID NO: 2), and a residual portion of amino acid shown in SEQ ID NO : 21 of a periostin isoform having an amino acid sequence shown in SEQ ID NO: 8 (672-698 amino acids of SEQ ID NO: 8). Additional examples include an amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 26, as well as SEQ ID NO: 34 (an amino acid sequence shown in SEQ ID NO. : 22 or SEQ ID NO: 23, as well as an amino acid sequence consisting of the 1st or 6th N-terminal amino acid residues of SEQ ID NO: 22 or SEQ ID NO: 23). The term "neutralization of the anti-cellular adhesive activity of periostin" means that the action or activity of the "region responsible for the anti-cellular adhesive activity of periostin" above is inhibited, and more specifically means that the action or activity of periostin. it is inhibited using, for example, an antibody capable of specifically recognizing the above responsible site of anti-cell adhesive activity. In a modality, antibodies of the present invention are monoclonal antibodies and polyclonal antibodies obtained by using the antigens as described above. The "monoclonal antibodies" herein refer to any monoclonal antibody that shows reactivity against the antigens described above, and the "monoclonal antibodies" include natural antibodies obtained by immunization of mammals such as mice, rats, hamsters, guinea pigs or rabbits with the antigens, chimeric monoclonal antibodies (chimeric antibodies) and humanized monoclonal antibodies (humanized antibodies; CDR-grafted antibodies) that can be prepared by using genetic recombination techniques, as well as human monoclonal antibodies (human antibodies) that can be prepared by using transgenic animals that produce human antibody or the like. Antibodies of the present invention include monoclonal antibodies having any isotype such as IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD or IgE, preferably IgG (IgG1, IgG2, IgG3, IgG4) or IgM. When the peptides described above are used as antigens, these can be used alone as antigens but can also be used for immunization by adsorption to a macromolecular material such as polyvinyl pyrrolidone, latex or polymethyl methacrylate, or coupling to a carrier protein such as KLH ( Californian limpet hemocyanin) or BSA (bovine serum albumin), and any method can be used. Generally, the peptides can preferably be coupled to a carrier protein by means of known methods (eg, "New series of Development of Drugs, Vol. 14, Hirokawa Publishing Co., 1991"). The peptides are coupled to a carrier protein via cysteine residues introduced at the C- or N-terminus of the peptides so that the peptides have directionality. Interleavers commonly used in the art may be used as long as they are suitable for this purpose. Suitable crosslinkers include succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (subsequently abbreviated as "SMCC") or N-hydroxysuccinamide ester of 3-maleimidobenzoic acid (MBS). Monoclonal antibodies are prepared by culturing hybridomas prepared by the cell fusion method of Kohler and Milstein (G. Kohler et al. Nature (1975) 256, 495-7) to secrete the antibodies and isolate them from the cultures. That is, a mammal is immunized with a peptide having an amino acid sequence encoded by exon-17 or the like and then the cells that produce antibody from this animal are fused to myeloma cells to provide hybridomas. The search for antibodies that produce exon-17 binding hybridomas is performed, for example, with an enzyme immunoassay (later abbreviated as "ELISA") for hybridoma supernatants using a microplate in which it has been immobilized on antigen. Animals to be immunized are not specifically limited, but include several mammals such as mice, rats, guinea pigs, rabbits, sheep, goats, cats, dogs, etc. Among the animals listed above, Balb / c mice are generally used for the preparation of monoclonal antibodies because they are easy to handle or for other reasons, but other strains of mice can also be used. The concentration of the antigen used for immunization here is selected to form sufficient amounts of antigenically stimulated lymphocytes. Preferably, 1 -100 μg of an antigen is diluted to an appropriate concentration with physiological saline or the like and suspended in complete Freund's adjuvant or incomplete Freund's adjuvant or the like, and the suspension is administered to an animal by intraperitoneal injection. or subcutaneous or other means. The administration is performed once to several times every 2-4 weeks. The final immunization is usually performed by administering a solution of 1-100 μg of the antigen in physiological saline by intravenous or subcutaneous injection or other means. Several days after the final immunization, antibody producing cells such as lymphocytes, preferably spleen cells or lymph node cells, are removed from the animal immunized for cell fusion. Cell fusion using spleen cells as antibody-producing cells is explained below, although cells that produce antibody other than spleen cells can also be used for cell fusion. Spleen cells prepared from the spleen aseptically removed 3-4 days after the final immunization are fused to appropriate myeloma cells in the presence of a fusion promoter. Myeloma cells used for fusion can be derived from mammals, but generally those derived from the same species as the animal used for immunization. Several cell lines are already known, for example, SP2 / 0-Ag14 (SP2) (Nature, 276, 269 (1978), NS-1 -Ag4 / 1 (NS-), P3-X63Ag8U.1 (P3U1) [Curr Top.Microbiol.Immunol.81, 1-7 (1978): available from ATCC under ATCC No. CRL-1597], P3-NS1 -1 -Ag4-1, P3-X63Ag8 (P3), FO, X63Ag8.653 (X63.653), 210.RCY3.Ag1 .2.3, S194 / 5XXO.BU1, SKO-007, GM15006TG-A12 and the like are preferably used for mice, and Y3.Ag1 .2.3 and the like are preferably used for rats Preferred fusion promoters include polyethylene glycol (PEG) having a molecular weight of 1000-6000 and Sendai virus (HVJ) Generally, the ratio of spleen cells and myeloma cells during fusion is preferably 10: 1 -2: 1 Hybridomas can be separated from fused cells by culturing a mixture of unfused, splenic cells, unfused myeloma cells and cells fused in a selective medium that inhibits the survival of unfused myeloma cells for an appropriate period until the unfused cells die (approximately 1 week). The selective medium can be, for example, HAT medium (medium containing hypoxanthine, aminopterin and thymidine). In this selective medium, unfused myeloma cells die, and unfused, splenic cells die after a certain period of time (after approximately 1 week) because they are non-tumorous cells, so that hybridomas can be obtained by selection. of viable cells. Hybridomas producing desired antibodies can be obtained by searching for the strains that produce the desired antibodies and cloning the strains to prepare monoclonal antibodies by standard limiting dilution. In this manner hybridomas obtained which produce monoclonal antibodies of the present invention can grow in medium suitable for their growth and can be easily stored in low temperature freezers or liquid nitrogen. In this way hybridomas obtained can produce antibodies by growing in nutrient medium or in the abdominal cavity of a mammal, and the antibodies produced can be purified from culture supernatants or the ascites fluid or serum of the mammal. As an example of the hybridomas of the present invention, a hybridoma can be used which is deposited under FERM BP-10718 on November 1, 2006 with the international depository patent agency, the National Institute of Advanced Industrial Science and Technology. The purification of the antibodies can be carried out by standard isolation / purification methods such as centrifugation, dialysis, salting with ammonium sulfate or the like, ion exchange chromatography using a DEAE column or the like, gel filtration, affinity chromatography, etc. The isotopes and subclasses of monoclonal antibodies thus obtained can be determined using an identification method such as Ouchterlony assay, ELISA or RIA. The Ouchterlony test is convenient but requires a concentration operation if the concentration of monoclonal antibody is low. When ELISA or RIA is used, however, the isotopes and subclasses of the monoclonal antibodies can be identified by direct reaction of the culture supernatant with a solid phase adsorbed by antigen and by the use of antibodies corresponding to various immunoglobulin isotypes and subclasses. as secondary antibodies. Most convenient methods employ commercially available identification equipment (eg, Mouse Typer kit; Bio-Rad) or similar. Protein quantification can be performed by the Folin-Lowry method and the calculation of the absorbance at 280 nm [1.4 (OD280) = 1 mg / ml of immunoglobulin]. Monoclonal antibodies thus obtained of the present invention specifically recognize an amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, an amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4, a isoform of periostin (PN-1) having an amino acid sequence shown in SEQ ID NO: 34, a peptide consisting of an amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4, or a peptide having a sequence of amino acids shown in SEQ ID NO: 34. Preferably, the monoclonal antibodies of the present invention can specifically recognize and bind to a peptide consisting of the amino acid sequence YTTKIITKVV (SEQ ID NO: 26), ie, a peptide consisting of an amino acid sequence that covers the -1 to tyrosine to the 9a valine from the N-terminus of the amino acid sequence of the human periostin peptide exon-17 chain (SEQ ID NO: 4), and a peptide consisting of of a sequence of amino acids covering the 669th tyrosine at 679a valine from the N-terminus of the amino acid sequence of human periostin PN-1 (SEQ ID NO: 2). That is, the monoclonal antibodies of the present invention can specifically recognize an amino acid sequence site (TTKIITKVV; SEQ ID NO: 22), or a portion thereof, which covers the threonine from N-terminal to 9a valine the amino acid sequence of the human periostin peptide exon-17 chain (SEQ ID NO: 4). More preferably, the monoclonal antibodies of the present invention can recognize and bind to a peptide comprising substitutions of alanine at the 1 st and 8-10 nd amino acids of the N-terminus of a peptide consisting of an amino acid sequence (YTTKIITKVV; SEQ ID NO: 26) which covers from -1 to tyrosine to 9a valine from the N-terminus of the human periostin exon-17 peptide chain (SEQ ID NO: 4). That is, the monoclonal antibodies of the present invention can specifically recognize at least one amino acid sequence site (SEQ ID NO: 34), or a portion thereof, which covers the threonine to the threonine 6a from the N-terminus of the amino acid sequence of the human periostin peptide exon-17 chain (SEQ ID NO: 4) or the amino acid sequence of the rat periostin exon-17 peptide chain (SEQ ID NO: 3) . In addition, the polyclonal antibodies of the present invention have the activity of suppressing or inhibiting the anti-cellular adhesive properties of human periostin protein PN-1, ie, neutralizing the anti-cellular adhesive properties of human periostin protein PN-1. In addition, the polyclonal antibodies of the present invention can suppress cardiac dilation, cardiac hypertrophy and cardiac fibrosis that are induced during cardiac deficiency or the like to improve cardiac function. When antibodies are used as antibodies of the present invention, polyclonal antibodies can be obtained by standard methods such as the method described in "New Lecture on Biochemical Experiments, 12, edited by the Japanese Biochemical Society, Tokyo Kagaku Dozin, 1992." Animals to be immunized are not specifically limited, but include horses, goats, sheep, rabbits, guinea pigs, mice, chickens, etc. When a rabbit is immunized, an antigen is diluted to an appropriate concentration with physiological saline or the like and suspended in complete Freund's adjuvant, incomplete Freund's adjuvant or aluminum hydroxide adjuvant or the like, and the suspension is injected into a dose of 10-1000 μg per animal followed by 1 -3 booster injections after 2-4 weeks to provide antisera. Subcutaneous injection at multiple sites is preferred. The preparation of anti-serum polyclonal antibodies can be carried out by the method as described for the purification of monoclonal antibodies.

Polyclonal antibodies obtained in this manner of the present invention specifically recognize an amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and an amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4, a isoform of periostin (PN-1) having an amino acid sequence shown in SEQ ID NO: 34, a peptide consisting of an amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4, or a peptide having an amino acid sequence shown in SEQ ID NO: 34. Preferably, the polyclonal antibodies of the present invention can specifically recognize and bind to a peptide consisting of the amino acid sequence YTTKIITKVV (SEQ ID NO: 26), ie, a peptide which consists of an amino acid sequence that covers from 1 to tyrosine to 9a valine from the N-terminus of the amino acid sequence of the human periostin peptide exon-17 chain (SEQ ID NO: 4), and a peptide which consists of a amino acid sequence covering the 669th tyrosine at 679a valine from the N-terminus of the amino acid sequence of human periostin PN-1 (SEQ ID NO: 2). That is, the polyclonal antibodies of the present invention can specifically recognize an amino acid sequence site (TTKIITKVV; SEQ ID NO: 22), or a portion thereof, which covers from the N-terminal threonine to the 9th valine of the amino acid sequence of the human periostin peptide exon-17 chain (SEQ ID NO: 4). More preferably, the polyclonal antibodies of the present invention can recognize and bind to a peptide comprising substitutions of alanine in the 1 st and 8-10 nd amino acids of the N-terminus of a peptide consisting of an amino acid sequence (YTTKIITKVV; NO: 26) covering from -1 to tyrosine to 9a valine from the N-terminus of the peptide chain exon-17 of human periostin (SEQ ID NO: 4). That is, the polyclonal antibodies of the present invention can specifically recognize at least one amino acid sequence site (SEQ ID NO: 34), or a portion thereof, which covers the threonine to the threonine 6a from the N-terminus of the amino acid sequence of the human periostin peptide exon-17 chain (SEQ ID NO: 4) or the amino acid sequence of the rat periostin exon-17 peptide chain (SEQ ID NO: 3) . In addition, the polyclonal antibodies of the present invention have the activity of suppressing or inhibiting the anti-cellular adhesive properties of human periostin-1 protein, ie, which neutralizes the anti-cellular adhesive properties of human periostin-1 protein. In addition, the polyclonal antibodies of the present invention can suppress cardiac dilation, cardiac hypertrophy and cardiac fibrosis induced during cardiac deficiency or the like to improve cardiac function.

Preparation of humanized antibodies Immunoglobulin G (later simply referred to as "IgG") consists of two light polypeptide chains having a molecular weight of about 23,000 (later referred to as "light chain") and two heavy polypeptide chains having a weight molecular weight of approximately 50000 (later referred to as "heavy chain"). The light and heavy chains have a repeating structure of conserved amino acid sequence regions consisting of approximately 10 residues, which constitutes a basic unit of three-dimensional structure of IgG (later referred to as "domain"). The heavy and light chains consist of 4 and 2 successive domains, respectively. In both heavy and light chains, the amino acid sequence of the amino terminal domain is more variable between antibody molecules than that of other domains, and this domain is called variable domain (later referred to as "domain V"). At the amino terminus of IgG, the V domains of the heavy and light chains are complementary to form a variable region. By contrast, the remaining domains collectively form a constant region. The constant region has a characteristic sequence of each of the animal species, for example, the mouse IgG constant region differs from the human IgG constant region so that mouse IgG is recognized as foreign matter by the human immune system , resulting in a response of human anti-mouse antibody (later referred to as "HAMA") (Schroff RW et al, Cancer Res. (1985) 45, 879-85). In this manner, mouse antibodies can not be administered repeatedly to humans. In order to administer said antibodies to humans, the antibody molecules must be modified to prevent the HAMA response while maintaining the specificity of the antibodies. According to the results of X-ray crystallography, said domain is generally in the form of an elliptical cylindrical structure formed of two antiparallel beta sheets consisting of 3 to 5 beta chains. In the variable region, three loops for each of the V domains of the heavy or light chains are assembled to form an antigen binding site. These loops are called regions of complementary determination (later referred to as "CDR"), which are more variable in amino acid sequence. The remaining parts of the variable region different from the CDR serve to maintain the structures of the CDRs and are called "frames". Kabatt et al. collect a number of primary sequences of heavy and light chain variable regions and prepare a classification table of the primary sequences in CDR and frames on the basis of sequence conservation (Kabatt et al., SEQUENCES OF IMMUNOLOGICAL INTEREST, 5th edition, NIH publication , No. 91 -3242, EA). Frames are further classified into a plurality of subgroups that have common amino acid sequence patterns. The presence of a consensus framework between the mouse and human sequences is also found. Said studies on structural aspects of IgG lead to the development of the procedures for the preparation of humanized antibodies described later. At an early stage of the studies, chimeric antibodies having a variable region of a mouse antibody fused to a constant region of a human antibody are proposed (Morrison SL et al Proc Nati Acad Sci US A. (1984) 81, 6851 -5). However, said chimeric antibodies can induce a HAMA response, especially when administered in the long term, because they still contain many non-human amino acid residues (Begent et al., Br. J. Cancer, (1990) 62, 487 ). A method for the further reduction of amino acid residues derived from a non-human mammal that can induce a HAMA response to humans by transferring only the CDRs into a human antibody is proposed (Peter T et al., Nature, (1986) 321, 522 -5), but the grafting of only the CDRs is normally insufficient to maintain immunoglobulin activity against the antigen. On the other hand, Chothia et al. they use X-ray crystallography data in 1987 to find (a) the amino acid sequences of the CDRs containing a site directly linked to the antigen and a site that maintains the structures of the CDRs, and possible three-dimensional structures of the CDRs are classified into patterns Multiple structures (canonical structures), and (b) classes of canonical structures are determined not only by CDR but also the types of amino acids in specific locations in the frame (Chothia C. et al., J. Mol. Biol. ) 196, 901-17). Based on this finding, a document suggests that when CDR grafting is used, the amino acid residues or a part of the framework can also be grafted onto a human antibody in addition to the CDR sequences (JPA No. HEI-4-502408). Generally, an antibody derived from a non-human mammal that has CDR to be grafted is defined as a "donor", and a human antibody in which the CDRs are grafted is defined as "acceptor"., and considerations in CDR grafting are to preserve the CDR structures to the extent possible to maintain the activity of the immunoglobulin molecule. To achieve this goal, two points must be kept in mind, that is, (a) the acceptor subgroup must be selected, and (b) the amino acid residue can be selected from the donor's frame. Queen et al, propose methods to conceive immunoglobulins wherein an amino acid residue in the framework of a donor is grafted onto an acceptor in addition to the CDR sequences when at least one of the following criteria is satisfied (JPA No. HEI-4 -502408): (a) the amino acid in the framework region of the acceptor is rare for that position and the corresponding amino acid in the donor is common for that position; (b) the amino acid is immediately adjacent to one of the CDR; or (c) the amino acid is predicted to have a side chain atom within about 3 angstroms of the CDRs in a three dimensional immunoglobulin model and to be able to interact with the antigen or the CDRs of the humanized antibody. The DNA encoding the heavy or light chain of an anti-exon-17 monoclonal antibody of the present invention can be obtained by preparing mRNA from hybridoma cells that produce the anti-exon-17 monoclonal antibody, the conversion of MRNA in cDNA upon reverse transcriptase and after isolation of the DNA encoding the heavy or light chain of the antibody.

Preparation of human antibodies As used herein, "human antibody" or "human immunoglobulin" means an immunoglobulin in which all regions constituting the immunoglobulin including heavy chain variable regions (VH) and heavy chain constant regions (CH) as well as light chain variable regions (VL) and light chain constant regions (CL) are derived from genes encoding a human immunoglobulin. In other words, it means an antibody in which the heavy chain is derived from a human immunoglobulin heavy chain gene and the light chain is derived from a human immunoglobulin light chain gene. Human antibodies can be prepared by standard methods, for example, by immunizing a transgenic animal prepared by integrating at least one human immunoglobulin gene into the site of a non-human mammal such as a mouse with an antigen, same way as described above for the preparation of monoclonal antibodies. For example, transgenic mice that produce human antibodies can be prepared by methods described in previous documents (Méndez MJ et al., Nature Genetics (1997) 15, 146-56, Green LL et al., Nature Genetics (1994) 7, 13-21. , JPA HEI-4-504365; International Publication No. WO94 / 25585; Nikkei Sciences, June, pp. 40-50, 1995; Nils Lonberg et al., Nature (1994) 368, 856-9, and JPA No. HEI- 6-500233). Antibodies used in the present invention are not limited to whole antibody molecules and can be antibody fragments or derivatives as long as they can neutralize the activity of a periostin isoform having anti-cell adhesive activity. Antibody fragments include, for example, Fab, F (ab ') 2, Fv, single chain antibody (scFV), disulfide-stabilized antibody (dsFV), a peptide containing CDR, etc. Among the antibody fragments of the present invention, Fab, F (ab ') 2 and the like can be obtained by treating an antibody that inhibits the anti-cellular adhesive activity of periostin with a proteolytic enzyme such as papain or pepsin, or alternatively, they can be prepared by constructing a gene encoding the resulting antibody fragment and introducing this construct into an expression vector, followed by expression in an appropriate host cell. Among the antibody fragments of the present invention, scFv can be prepared by linking together an H chain V region and an L chain V region of an antibody that inhibits the anti-cell adhesive activity of periostin by using an appropriate peptide linker or Similary. Alternatively, scFv can be prepared by constructing a DNA segment encoding the entire sequences or desired amino acid sequences of a gene encoding an H chain or V chain H region of the above antibody and a gene encoding an L chain or L chain region of the antibody, and introducing this construct into an expression vector, followed by expression in a suitable host cell. Among the antibody fragments of the present invention, dsFv is an antibody fragment in which polypeptides modified to replace an amino acid residue by a cysteine residue in both V regions of H chains and L of an antibody that inhibits the anti-cellular adhesive activity of periostin are linked together between these cysteine residues via a disulfide bond. An amino acid residue to be replaced by a cysteine residue can be selected by determining the estereostructural estimation of the antibody. dsFv can be prepared by constructing a DNA segment encoding the entire sequence or a desired amino acid sequence of a gene encoding the antibody fragment, and introducing this construct into an expression vector, followed by expression in an appropriate host cell. Among the antibody fragments of the present invention, a CDR-containing peptide comprises at least one or more CDR regions selected from the CDR regions in H or L chains of an antibody that inhibits the anti-cellular adhesive activity of periostin. Also, multiple CDR regions can be linked together by techniques using an appropriate peptide linker or the like. The CDR-containing peptide can also be prepared through the construction of a DNA segment encoding the entire sequence or a desired amino acid sequence of a gene encoding the peptide, and introducing this construct into an expression vector, followed by expression in an appropriate host cell. Alternatively, the CDR-containing peptide can also be prepared by chemical synthesis such as Fmoc or tBoc method. In the present invention, it is also possible to use prior antibody derivatives or antibody fragments, which are modified to have a low molecular weight protein or compound attached thereto. These modifications can be made by known techniques. In one embodiment, the antibodies, antibody fragments or derivatives of the present invention can be used to prevent or treat diseases in which a periostin isoform having anti-cellular adhesive activity is involved. "Diseases in which a periostin isoform having anti-cellular adhesive activity is involved" refers to diseases during which a gene of a periostin isoform having anti-cellular adhesive activity is highly expressed and the production of an encoded protein by the gene it increases. It also refers to diseases whose pathology is exacerbated by an increase in the gene or protein. Said diseases in which a periostin isoform having anti-cellular adhesive activity is involved are not specifically limited, but include heart failure, myocardial infarction, enlargement of the heart (dilation), cardiac hypertrophy, cardiac fibrosis, cardiomyopathy, myocarditis, disease valvular, cancers, aneurysm, arteriesclerosis, central neurodegenerative disease, renal diseases, rheumatoid arthritis, osteoporosis, pulmonary emphysema, pulmonary hypertension, chronic obstructive pulmonary disease (COPD), nephritis (acute and chronic), pancreatitis (acute and chronic), hepatitis ( acute and chronic), hepatic fibrosis, or pulmonary fibrosis. Cancers for which the antibodies of the present invention can be applied include, but are not limited to, cancers of the breast, large intestine, lung, malignant melanoma, bone, pancreas, stomach, skin, uterus, ovary, rectum, colon, uterus , Fallopian tubes, esophagus, small intestine, thyroid, parathyroid, adrenal gland, prostate, bladder and kidney, especially cancers of breast, large intestine, lung and malignant melanoma. The present invention also provides a diagnostic reagent for a disease (eg, cardiac deficiency) in which a periostin isoform having anti-cell adhesive activity is involved, prepared by labeling an antibody as described above with a label. Markers that can be used here include enzymes, radioisotopes, fluorescent inks, etc. The enzymes used here are not specifically limited as long as they meet the criteria such as high change number, stability even after conjugation, and the ability to react specifically with their substrates to develop color, etc., and enzymes used in immunoassays. Standard enzyme (ETA) can be used. Examples of preferred enzymes include peroxidases, β-galactosidases, alkaline phosphatases, glucose oxidase, acetylcholine esterase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, etc. Enzyme inhibitors and coenzymes and the like can also be used. The conjugation of these enzymes and antibodies can be carried out by known methods using crosslinkers such as maleimide compounds. Substrates that can be used are known materials, selected depending on the enzyme used. For example, when the enzyme used is peroxidase, 3,3 ', 5,5'-tetramethylbenzidine can be used, or when the enzyme used is an alkaline phosphatase, parinitrophenol, or the like, can be used. Radioisotopes that can be used as markers include those used in the standard radioimmunoassay (RIA) such as 1251 and 3H. Fluorescent inks that can be used are those used in the standard fluoroimmunoassay such as fluorescent isothiocyanate (FITC) and tetramethyl rhodamine isothiocyanate (TRITC). The present diagnostic reagent can also be used as immunohistological staining capable of specifically staining affected interstitial tissue of the heart. When labeled with a radioisotope, it can also be used to imagine the injury during cardiac deficiency through internal administration. The present invention also provides a method for the detection or quantification of a periostin isoform having anti-cellular adhesive activity in a biological sample obtained by the preparation of human or animal blood serum, i.e. in serum, comprising the use of the antibody, antibody fragment or derivative of the present invention. The present invention further provides a method for diagnosing a disease (eg, cardiac deficiency) wherein a periostin isoform having anti-cellular adhesive activity is involved, comprising detection or quantification of the periostin isoform. In the present method, a periostin sofma having anti-cell adhesive activity can be detected by the so-called double-layer ELISA (enzyme-linked immunosorbent assay). When the diagnostic equipment of the present invention is used, a sample is initially contacted with a plate in which a primary anti-periostin antibody has been immobilized to form a complex, and a secondary anti-periostin antibody labeled with a marker it binds to this complex, and then the intensity of the marker signal in this ternary complex is measured, whereby a periostin isoform having anti-cell adhesive activity can be detected or quantified. In particular, since a periostin isoform having anti-cellular adhesive activity is a splice variant that is specifically expressed during the pathology of cardiac deficiency or the like, the pathology in cardiac deficiency or the like can be diagnosed at monitor your production In this manner, an antibody of the present invention can be used here as the secondary antibody by labeling the antibody. Pharmaceutical compositions comprising the antibodies, fragments or antibody derivatives of the present invention as active ingredients can be prepared using carriers and / or excipients or other additives, which are used in standard formulation techniques. The active ingredients of the pharmaceutical compositions according to the present invention are preferably administered in admixture with carriers, excipients, known pharmacologically acceptable diluents or the like by any mode of administration normally used for pharmaceutical preparations, for example, by oral or parenteral route ( for example, intravenous, intramuscular or subcutaneous). For example, the pharmaceutical compositions of the present invention can be prepared by properly mixing the active ingredients with carriers, flavors, stabilizing excipients, diluents, emulsifiers, solutions, suspensions, physiologically acceptable syrups or the like, and can be used in the form of tablets, powders, granules, solutions or similar. Additives which can be incorporated into tablets or the like include, for example, binders such as gelatin and lubricants such as corn starch. The tablets can be coated with a sugar coating or a gastric or enteric film. In the capsule dosage form, the above compositions may further comprise liquid carriers. Sterile injectable compositions can also be prepared by applying standard formulas. Injectable aqueous vehicles include isotonic solutions containing glucose and the like, which can be used in combination with appropriate solubilizers such as polyethylene glycol, etc. The compositions can also be incorporated with pH regulators, stabilizers, preservatives, antioxidants, sedative agents and the like. For oral administration, when the active ingredients are likely to be broken down in the digestive tract, the compositions can be administered orally as formulations that are resistant to decomposition in the digestive tract, for example, as microcapsules that encapsulate the active ingredients within liposomes. . It is also possible to use other modes of administration with the intention of absorption through the different mucous membranes of the digestive tract, including rectal, intranasal, sublingual and transpulmonary routes. In this case, the compositions may be administered in the form of suppositories, nose drops, sublingual tablets, transpulmonary agents or the like. When the pharmaceutical compositions of the present invention are used for therapeutic purposes, their dosage is established in a therapeutically effective dosage, which varies depending on, for example, age and body weight of a subject to which the composition will be administered, the severity of the symptoms and a route of administration, and in this way the administration is determined on an individual basis. In general, the daily adult dosage for oral administration is approximately 0.1 to 1000 mg, provided as a single dose or in divided doses. For continuous intravenous administration, the compositions can be administered in the range of 0.01 μg / kg / min to 1.0 μg / kg / min, desirably 0.025 μg / kg / min to 0.1 μg / kg / min. The following examples further illustrate the present invention in detail and specifically, however, in this way limiting the invention.

EXAMPLES EXAMPLE OF PREPARATION 1 Search for periostin by subtraction 1 - . 1 - . 1 - . 1 - . 1 - . 1 -1 Preparation of rats of pathological model of cardiac deficiency and collection of left ventricular samples Rats sensitive to salt Dah1 male (Dahl-S) (Shimizu Laboratory Supplies) are raised on a diet high in salt at 8% of 6 weeks of age , and the left ventricle of three animals each is collected in stage of cardiac hypertrophy (11 weeks of age) and stage of cardiac deficiency (14 weeks of age). 1 -2 Preparation of total RNA mRNA is prepared from approximately 500 mg of the left ventricle using ISOGEN (Nippon Gene) as instructed by the manufacturer. Next, mRNA of about 40 μg of the combined total RNA of three animals each is purified in the stage of cardiac hypertrophy and cardiac deficiency using Fast Track 2.0 equipment (Invitrogen) as instructed by the manufacturer to recover approximately 3 μg of mRNA in each step . 1 -3 Subtraction of cDNA The cDNA subtraction is performed using PCR-select cDNA subtraction equipment (Clontech) as instructed by the manufacturer. That is, cDNA is synthesized from 2 μ? of each mRNA obtained in previous 1-2 and is digested with restriction enzyme Rsal. Then, subtractive hybridization is performed using synthesized mRNA from the 14-week-old animals as the analyzing cDNA and the cDNA synthesized from the animals at 1 1-week-old as the driving cDNA after 2 adapters included in the kit have been linked separately to the analyzing cDNA. Then, a cDNA fragment with altered expression level is amplified specifically by PCR using primers complementary to the adapters to provide the amplification product 1. A similar subtraction operation is performed using the cDNA synthesized from the 1 1-week-old animals as the analyzing cDNA and the cDNA synthesized from the 14-week-old animals as the driving cDNA to provide the amplification product 2. 1 -4 Classification dot blot A. Preparation of dot biots The amplification product 1 is TA cloned in a PCR II vector (Invitrogen) and the clones having the insert fragment are selected. The insert fragment of each clone is amplified by the PCR reaction, and then every 1 μ? of the reaction solution is treated with heat and then dot blot analysis is performed on 2 nylon membrane filters (Boehringer) and fixed with a UV interlayer (Stratagene).

B. Preparation of cDNA probes Amplification product 1 is digested with restriction enzymes Rsal and Eael, Smal to remove the adapters and subjected to random priming with DIG-dUTP using DIG upper DNA labeling / detection equipment II ( Boehringer) as instructed by the manufacturer to prepare cDNA probe 1. Similarly, cDNA probe 2 is prepared from amplification product 2.

C. Classification One of the dot blot membranes prepared in A above is hybridized with cDNA probe 1 and the other with cDNA probe 2. Specifically, hybridization is performed in Easy Hyb DIG solution at 42 ° C overnight using DIG (Boehringer) upper DNA labeling / detection equipment II as instructed by the manufacturer. The membranes are washed twice with 2 x SSC, 0.1% SDS at room temperature for 5 minutes and twice with 0.1 x SSC, 0.1% SDS at 68 ° C for 15 minutes, and then reacted with labeled DIG antibodies. with alkaline phosphatase in the blocking pH regulator included in the kit, and then ready-to-use CSPD is added to advance chemiluminescence and the X-ray film is exposed. The clones that show a stronger signal in the cDNA probe 1 that in the cDNA probe 2 are selected as positive and sequenced clones. 1 -5 Sequencing Nucleotide sequences are determined by analysis in an automatic DNA sequencer model 3734 (PE Applied Biosystems) using THERMO Sequenase ™ II ink terminator cycle sequencing equipment (Amersham Pharmacia). The genetic sequences obtained in this way are compared to sequences in the GenBank data bank to reveal that one of the clones (SF014) is a gene that has an 86% homology for mouse periostin (GenBank Accession No. D13664).

EXAMPLE OF PREPARATION 2 Cloning of rat periostin cDNA Rat periostin cDNA was isolated by sorting 10 phage sub-clusters of approximately 4000 clones (a total of approximately 40,000 clones) prepared from a rat aortic cDNA library (Clontech) inserted into gt1 1 vector by PCR using primers (1) 5'-GTTCATTGAAGGTGGCGATGGTC-3 '(SEQ ID NO: 15), and (2) 5'-GAGATAAAATCCCTGCATGGTCCT-3' (SEQ ID NO: 16) designated at the base of the nucleotide sequence of SF014 to provide 3 positive sub-groupings. One of the sub-clusters is sorted by hybridization using the PCR-amplified fragment as an alkaline phosphatase-labeled probe using AlkPhos Direct ™ (Amersham Pharmacia) to provide a positive clone rat # 1 periostin. Its insert fragment is subcloned into the EcoRI site of pBluescript II (Stratagene) and the total nucleotide sequence is determined according to the method of Preparation Example 1-5. The resulting clone has a length of about 3 kb corresponding to nucleotide 292 for the 3 'end of mouse periostin (GenBank Accession No. D 3664), which suggests that it is a 5'-truncated clone. In this manner, SMART ™ RACE cDNA amplification kit (Clonthech) is used as instructed by the manufacturer to perform the 5'-RACE reaction using rat aortic cDNA as a template and the primer (2) 5'-GAGATAAAATCCCTGCATGGTCCT -3 '(SEQ ID NO: 16) and an initiator (3) 5'-CACGGTCGATGACATGGACAACACC-3' (SEQ ID NO: 17) designated in the bases of the nucleotide sequence of rat periostin # 1. The resulting PCR product is TA cloned in Invitrogen PCR II vector to provide a clone designated as rat periostin 5'RACE # 1. The nucleotide sequence is determined according to the method of Preparation Example 1-5. The results show that rat periostin 5'RACE # 1 is a longer clone by about 300bp than rat periostin # 1 obtained initially in the 5 'direction with the 5' end being longer by 15bp than the 5 'end of mouse periostin (GenBank Accession No. D13664). Ten subgroups of phage from approximately 40,000 clones (a total of approximately 400,000 clones) prepared from the rat aortic cDNA library are sorted by PCR using an initiator (4) 5'-ACGGAGCTCAGGGCTGAAGATG-3 '(SEQ ID NO: 18) designated at the base of the nucleotide sequence of rat periostin 5'RACE # 1 and the primer (3) 5'-CACGGTCGATGACATGGACAACACC-3 '(SEQ ID NO: 17) to provide 2 positive sub-clusters. One of the sub-clusters is classified by hybridization using the fragment amplified by PCR as a probe to provide a positive clone designated as rat # 2 periostin. The insert fragment is subcloned into the EcoRI site of pBluescript II (Stratagene) and the nucleotide sequence is determined according to the method of Preparation Example 1-5. The resulting clone has a length of approximately 2.6 kb with the 5 'end which is the same as that of the clone obtained with 5'-RACE and the corresponding 3' end up to nucleotide 2410 of mouse periostin (GenBank Accession No. D13664 ). The nucleotide sequence of rat periostin 5'RACE # 1 previously obtained is exactly the same as the nucleotide sequence of the relevant region of rat periostin # 2. The full length of rat periostin cDNA is completed by periostin # 1 rat and periostin # 2 rat. The nucleotide sequence of this full-length cDNA and the translated amino acid sequence of this nucleotide sequence is shown as SEQ ID NO: 6 and 1.

EXAMPLE OF PREPARATION 3 Construction of a myc-His-rat periostin protein fusion expression vector An expression vector having an Myc epitope and 6 histidine tags at the carboxyl terminal of the translated protein of the rat periostin gene coding region obtained in the preparation example 2, and having a CMV promoter, is prepared. Initially, a fragment of approximately 500 bp obtained by the digestion of a rat periostin 5'RACE # 1 obtained in Preparation Example 2 with restriction enzymes EcoRI and Hindlll and a fragment of approximately 2780 bp obtained by periostin digestion # 1 of rat obtained from Preparation Example 2 with restriction enzymes Hindlll and Hpal are ligated to a vector fragment obtained by digestion of the vector pTracer-CMV2 (Invitrogen) with restriction enzymes EcoRI and EcoRV using a ligation kit (Takara Bio Inc.) to provide a plasmid designated as periostin pTracer-C V2 / rat. Perrostin pTracer-CMV2 / rat prepared in this manner is digested with restriction enzymes EcoRI and Smal to provide a fragment of approximately 2330 bp containing the coding region of the rat periostin gene, and PCR is performed using periostin # 1 of rat obtained in Preparation Example 2 as a template and primer (5) 5'-GACCCGGGAAGAACGCATCATC-3 '(SEQ ID NO: 19) designated at the base of the template and initiator sequence (6) 5'-TGGGTGACCCTGAGAACGGCCTTCTCTTGATC- 3 '(SEQ ID NO: 20) designed to insert a BstEII site immediately before the rat periostin stop codon and the amplification product is purified and then digested with Smal and BstEII restriction enzymes to provide a fragment of approximately 270 bp. These two fragments are ligated to a vector fragment obtained by the digestion of an expression vector constructing pcDNA4 / Myc-His / plasmid type C (Invitrogen) with restriction enzymes EcoRI and BstEII using a ligation kit (Takara Bio Inc. .) to provide a plasmid designated pADNc4 / Myc-His / rat periostin. The total nucleotide sequence of the insert is confirmed by the method described in Preparation Example 1-5.

EXAMPLE OF PREPARATION 4 Construction of a baculovirus expression vector The rat plasmid pADNc4 / Myc-His / periostin obtained in the preparation example 3 is digested with restriction enzymes SaCI and Pmel to cut a fragment of rat PN-1 peptide / Myc-His. This fragment is ligated to a vector fragment obtained by digestion of pFastBacHTc (Invitrogen) with restriction enzymes SacI and Kpnl (reduction) using a ligation kit (Takara Bio Inc.) to provide an expression vector designated as pFastBac / periostin. -1 rat / Myc-His. The nucleotide sequence of the insert is confirmed by the method described in Preparation Example 1-5.

EXAMPLE OF PREPARATION 5 Preparation and cultivation of a recombinant baculovirus DH10BAC cells of Escherichia coli are transformed with pFastBac / rat periostin-1 / Myc-His obtained in the preparation example 5 to prepare a recombinant baculovirus. Electrophoresis and PCR confirm that the resulting baculovirus contains the desired insert. Insect Sf9 cells (2 x 106 cells / ml) infected with this recombinant baculovirus in MOI = 0.1 are cultured in a serum-free medium (containing 50 g / ml gentamicin in 2000 ml of Sf-900IISFM (Invitrogen)) a 28 ° C for 4-5 days and then the culture supernatant is collected.

EXAMPLE OF PREPARATION 6 Purification of rat periostin protein To a SP Sepharose rapid flow column (10 ml of bed volume) equilibrated with an equilibrium pH regulator (50 mM sodium acetate pH buffer, pH 6.0, 0.1 M sodium chloride) are applied 2000 ml of the culture supernatant obtained in the preparation example 5, and the fraction with resulting flow passage is grouped as a fraction with flow step of sepharose SP. The column is washed with equilibrium pH regulator until the absorbance at 280 nm approaches 0 (approximately 100 ml) to provide a fraction washed with SP sepharose. The column is eluted with 100 ml of an elution pH buffer (50 mM sodium diacid phosphate, (pH 8.0), 5 mM sodium chloride, 5 mM imidazole) to provide an SP sepharose eluate fraction. Then, 100 ml of SP sepharose eluate fraction is applied to a Ni-NTA agarose column (5 ml bed volume) equilibrated with 50 mM sodium phosphate pH regulator, pH 8.0, 0.5 M sodium chloride. 5 mM imidazole, and the resulting flow-through fraction is pooled as a fraction with Ni-NTA agarose flow passage. The column is washed with approximately 50 ml of a wash pH regulator (50 ml of sodium diacid phosphate, pH 8.0, 0.5 M sodium chloride, 5mM imidazole) to provide a Ni-NTA agarose wash fraction. The column is eluted with approximately 25 ml of each of the elution pH regulators (1) 50 mM sodium diacid phosphate, 0.5 M sodium chloride, 20 mM imidazole, followed by similar compositions except that the imidazole concentrations are (2) 30 mM, (3) 40 mM, (4) 50 mM and (5) 60 mM to provide fractions of Ni-NTA agarose eluate (1) - (6). The fractions shown to contain the desired protein by Western blotting are concentrated at 1 ml or less. Then, the concentrated samples are applied to a gel filtration column (Sephacryl S-200HR < j) 11 mm x 95 cm; bed volume 90) equilibrated with degassed PBS (-) (137 mM NaCl, 8.1 mM Na2HP04, 2.68 mM KCI, 1.47 mM KH2P04) and eluted with PBS (-) and the eluate is lyophilized to provide a periostin protein of purified rat.

EXAMPLE 1 Synthesis of rat exon-17 peptide chain and preparation of its polyclonal antibody against this A specific structure for rat PN-1 is identified as an exon-17 sequence by sequence comparison because cardiac dilation is induced by increased expression of the PN-1 gene in the heart of normal SD rats and the survival rate it is improved by the administration of an antisense oligonucleotide against rat periostin in the heart of model rats with cardiac deficiency DahI and rat PN-1 shows that it does not have cellular adhesive properties in contrast to previously reported PN-2. A peptide having a Cys residue added to the N-terminus of the amino acid sequence constituting this exon-17 is chemically synthesized in 10 mg of yield with a purity of 80% or more. Rabbits (kbl); JW) are immunized with the polypeptide coupled to 6 mg of a KLH carrier protein. FCA (Freund's complete adjuvant) is used in secondary and subsequent immunization. Administration is carried out in 20 dorsal subcutaneous sites at weeks 0, 2, 4 and 6 using a peptide dose of 800 μg / animal in the primary immunization, and 400 g / animal in the secondary and subsequent immunizations. The antibody titrator is determined by ELISA and total sera are collected at week 7. Then, an affinity column is prepared by using a synthetic peptide, and only the antibody that reacts specifically to the exon-17 peptide is harvested. The polyclonal antibody against the peptide encoded by rat periostin exon-17 is hereinafter referred to as a peptide antibody exon-17 antirate.

EXAMPLE 2 In vitro study of the presence or absence of anti-cellular adhesive activity of rat periostin protein (rat PN-1) Rat heart fibroblasts are obtained by a method similar to those described in the literature (Ruwhof C. van Wamel AE, Egas JM, van der Laarse A. Mol Cell Biochem, 2000 May; 208 (1-2): 89-98 , Ashizawa N, Graf K, Do YS, Nunohiro T, Giachelli CM, Meehan WP, Tuan TL, Hsueh WA, J Clin Invest, 1996 Nov. 15: 98 (10): 2218-27). Specifically, 20 SD rats on days 1-2 are anesthetized with ether and the chest is disinfected with ethanol. The heart is isolated and placed in a dish containing PBS (-), where the heart is cut transversely to bleed it and washed additionally with PBS (-) three times, and then PBS (-) is discarded at the lowest possible level and the heart It is crumbled with scissors. Then, the comminuted tissue is stirred in a 1: 1 mixture of PBS (-): collagenase / trypsin at 37 ° C for 15 minutes, and then the cells are lysed by pipetting as completely as possible. Then, the cell lysate is filtered through a platinum mesh in a centrifuge tube, and the platinum mesh is washed with 10 ml of M199 medium containing 10% serum and 10 ml of PBS (-). Then, the centrifuge tube is rotated at 1500 rpm for 10 minutes, and the cells obtained as a pellet fraction are shaken in 20 ml of M199 medium containing 10% serum and placed on a plate. This is allowed to stand at 37 ° C for 1 hour, and then the cells adhered to the dish are collected as rat heart fibroblasts. Rat heart fibroblasts are placed in a 96-well plate at a density of 6.4 x 104 cells / 100 μ? and they are grown overnight, and the culture is then incubated in fresh DMEM medium with 10% FBS containing 10 μg / ml cycloheximide at 37 ° C for 1 hour. Then, the cells are washed twice with DMEM medium (serum free) pre-heated at 37 ° C, and rat periostin protein (rat PN-1) prepared according to the preparation examples is added to DMEM medium. (free of serum) at a final concentration of 10 g / ml. Fibronectin that has cell adhesion promoting properties is used as a positive control and BSA (bovine serum albumin) is used as a negative control. After incubation at 37 ° C for 1 hour, microscopy shows that all cells are separated in the group treated with rat periostin protein (rat PN-1), and the cells are washed twice with PBS (- ) and then fixed in formalin with 10% neutral pH regulated for 30 minutes. The cells are then washed with PBS (-) three times and then stained with crystal violet for 30 minutes. Then, the degree of staining is measured using a plate reader at 550 nm (BIO-RAD, Microplate Reader Model 680) (Figure 2). As a result, the control groups treated with fibronectin and BSA and the untreated group do not show anti-cellular adhesive properties because the adhered cells do not separate, on the contrary to the group treated with rat periostin protein (PN-1). rat) where the cells separate, showing that rat periostin protein (rat PN-1) has a separation effect on adhered cells, ie, anti-cell adhesive properties.

EXAMPLE 3 In vitro study of the neutralizing activity of anti-rat exon-17 peptide antibody SD rat heart fibroblasts obtained by a method similar to that of Example 2 are placed in a 96-well plate at a density of 6.4 x 10 4 cells / 100 μ? and they are grown overnight, and then the culture is incubated in fresh DMEM medium with 10% FBS containing 10 μg / ml of cyclohexamide at 37 ° C for 1 hour. The cells are then washed twice with DMEM (serum free) medium pre-warmed at 37 ° C, and rat periostin protein (rat PN-1) and anti-rat exon-17 peptide antibody are added to DMEM medium. (serum free) at final concentrations of 10 μg / ml and 100 μ9 / G ??, respectively. Rat periostin protein alone is used as a positive control and BSA is used as a negative control. After incubation at 37 ° C for 1 hour, microscopy shows that all cells are separated in the group treated with rat periostin protein alone, and the cells are washed twice with PBS (-) and then fixed in formalin with pH regulated 10% neutral for 30 minutes. Then, the cells are washed with PBS (-) three times and then stained with crystal violet for 30 minutes. Then, the degree of staining is measured using a plate reader at 550 nm (BIO-RAD, Microplate Reader Model 680) (Figure 3). The results show that the anti-rat peptide exon-17 antibody is an antibody that has the activity of inhibiting the separation induced by rat periostin protein (PN-1) from adhered cells, ie, inhibition of the anti-cell adhesive properties of PN-1, that is, neutralization of the anti-cellular adhesive properties of rat periostin protein (rat PN-1).

EXAMPLE 4 Effect of anti-rat peptide exon-17 antibody in rats of acute myocardial infarction model A male Lewis rat weighing 250-300 g is fixed on a surgical table for rats after the animal is completely anesthetized by peritoneal administration of pentobarbital (0.1 ml / 100 g). A tube is inserted orally into the trachea and connected to a rat ventilator (tidal volume of respiration 3 ml, 80 breaths / minute), and the skin is cut laterally from the third left intercostal space of the sternum and the underlying pectoralis major muscle it is also cut laterally, and the intercostal space is opened using a rat rib spacer to expose the heart. Then, the near left coronary artery under the left atrium is ligated with silk 1 .0 using a curved needle that has a diameter of 5 mm. After visual confirmation that the anterior and lateral walls along which the left coronary artery runs have changed from red to white they show sufficient blockage of the coronary bloodstream and the disappearance of movement of the walls at these sites (in the substitute operation group, the needle is passed through the coronary artery and then removed without ligation), the third and fourth ribs are fixed by ligation with silk 3.0 (after the lung expands to remove air that exists outside the lung in the rib cage so that the lung can easily expand). The incision site in the skin is sutured with silk 3.0 in the same way and then observed for some time, and the tube is removed after confirmation of regaining consciousness and resumption of spontaneous breathing. Models of acute myocardial infarction are prepared consecutively by the above procedure. On the next day, percutaneous echocardiography is performed under intranasal anesthesia with isoflurane, and small models of infarction that have infarct size smaller than 20% of the entire periphery of the left ventricle are excluded. The remaining infarct models are classified in order to increase cardiac function, and are alternatively classified into a group treated with anti-rat peptide exon-17 antibody and a group treated with a control antibody (rabbit IgG) every 200 via the vein from the tail. The antibodies are administered to each group on the day after the preparation of the models and at intervals of 6 days after the initial administration, a total of 4 times. The heart is evaluated by echocardiography through the chest wall at intervals of one week until the end of 8 weeks. At the end of 8 weeks, a tube is inserted into the trachea and connected to a ventilator under anesthesia with pentobarbital, and the skin is cut from the left neck and the neck muscles are retracted with forceps to expose the left common carotid artery, and after bleeding is interrupted by ligation at the origin of the left carotid artery, the artery is drilled at a distal site with the use of small scissors and a rat mirror catheter is inserted from that site in such a way that the tip of the catheter with a pressure sensor that reaches inside the left ventricle while the catheter is connected to a computer to measure cardiac function and blood pressure and the like. The results of echocardiography 4 weeks after the preparation of the models show that the reduction of the thickness of the anterior wall and thickness of the posterior wall of the heart is inhibited, the increase of the extreme-diastolic internal diameter and the extreme-systolic internal diameter is inhibits, and that the EF value indicative of the contractile function of the heart is increased in the group treated with anti-rat peptide exon-17 antibody significantly compared to the control group treated with rabbit IgG. In short, cardiac dilation is inhibited, showing that the cardiac function is improved (figure 4A to figure 4C). The results of echocardiography 8 weeks after the preparation of the models also show inhibition of cardiac dilation and improvement of cardiac function in the same way as the results after 4 weeks (Figure 5A to Figure 5C). These results suggest that the anti-rat exon-17 peptide antibody effect that inhibits cardiac dilation and improves cardiac function is still maintained approximately 4 weeks after administration of the antibody. So, hemodynamics show significant differences in maximum derivative of left ventricular pressure ((+) dP / dt), minimum derivative of left ventricular pressure ((-) dP / dt) and left ventricular end-diastolic pressure (LVEDP) in group treated with anti-rat exon-17 peptide antibody compared to the control group treated with IgG, suggesting that cardiac function is improved (Figure 6A to Figure 6C). In sections of the heart stained with Masson trichrome, blue sites decreased in the group treated with anti-rat peptide exon-17 antibody compared to the control group treated with IgG, showing that fibrosis is inhibited (figure 7). An analysis of the diameters of the minor axis of the myocardial cells shows that the reduction of the diameters of the minor axis of myocardial cells is significantly inhibited in the group treated with anti-rat peptide exon-17 antibody compared to the control group treated with IgG (figure 8). This result correlates with the result of echocardiography. In addition, the results of gene expression analysis in infarct sites and non-infarct sites show that the levels of expression of endothelin-1 (ET-1), collagen type I, III and TGF-beta in non-infarct sites decrease significantly in the group treated with the neutralizing antibody compared to the group treated with the control antibody at levels comparable to those of the substitute group, indicating that the condition is improved (Figure 9A to Figure 9E).

EXAMPLE 5 Cloning of full-length human periostin-1 cDNA CDNA prepared from 1 μg of total RNA derived from the human heart (Clontech, catalog No. 64100-1, batch No. 4120493) is used as a template to carry out PCR with KOD plus DNA polymerase (Toyobo Co., Ltd.) using the following primers for full-length cloning: sense chain 5'-AAGCTAGCCACCATGATTCCCTTTTTACCCAT-3 '(SEQ ID NO: 27) and antisense strand 5'-AATCCACAATTTCCCTCAT-3' (SEQ ID NO: 28). The resulting PCR products are cloned using a Zero Blunt TOPO PCR cloning kit (Invitrogen). A 5'-sense chain TAACCAAAGTTGTGGAACCAA-3 '(SEQ ID NO: 29) is prepared from a region corresponding to human periostin exon-17, whereas an antisense chain 5'-TGTGTCTCCCTGAAGCAGTC-3' (SEQ ID NO: 30) ) is prepared from a region corresponding to exon-21, followed by the selection of clones detected with these initiators of this clone group. Then, the nucleotide sequences of the selected clones are determined to select a non-cut and spliced clone, whereby the full-length cloning of human periostin-1 cDNA is completed. The resulting clone is designated as PCR4 / human periostin-1.

EXAMPLE 6 Construction of human periostin-1 expression vector for in vitro translation Plasmid PCR4 / human periostin-1 obtained in Example 5 is digested with restriction enzymes Pme I and Not I to cut a human periostin-1 DNA fragment, which is then mitigated. An expression vector pTNT (Promega), within which CATCACCATCACCATCACTAA (6 x His + stop codon) (SEQ ID NO: 31) has been inserted, is digested enzymatically in the Mlu I site at its multicloning site and then mitigated . The DNA fragment obtained above is ligated to this vector using a ligation kit (TaKaRa Bio Inc.). Then, for the purpose of creating fusion in frame with the His tag, synthetic linkers (sense chain 5'-CTAGAAGACGATTAAGGGAAGGTCGTTCTCAGCTGGAAGTTCTGTTCCAG GGGCCC-3 '(SEQ ID NO: 32) and antisense chain 5'-GGGCCCCTGGAACAGAACTTCCAGCTGAGAACGACCTTCCCTTAATCGTC TT-3' (SEQ ID NO. : 33)) are prepared and ligated to the fragment of the vector digested with restriction enzymes Xba I and Sma I using a ligation kit. The nucleotide sequence of the ligated part is confirmed, and the resulting expression vector is designated pTNT / human periostin-1 / His.

EXAMPLE 7 Protein synthesis by in vitro translation The expression vector obtained in Example 6 is provided for protein synthesis in vitro with coupled transcription / translation systems TNT SP6 Quick (Promega). More specifically, in relation to 2 μg of the expression vector pTNT / periostin-1 / His, 40 μ? of SP6 Quick Master Mix and 1 μ? of 1 mM methionine are added and diluted with water treated with DEPC to give a total volume of 50 μ ?, followed by the reaction at 30 ° C for 90 minutes. The reaction product is stored at -80 ° C until purification.

EXAMPLE 8 Purification of human periostin protein (PN-1) The synthetic protein obtained in example 7 is purified using a MagZ protein purification system (Promega). More specifically, to the synthetic protein obtained in Example 7, 2 volumes of MagZ binding / washing buffer are added and mixed well. The sample prepared in this way is added to MagZ binding particles. After stirring this mixture at 4 ° C for 1 hour, the supernatant is removed and the MagZ binding particles are washed four times with MagZ binding / washing buffer, followed by elution of the synthetic protein with elution pH buffer MagZ. The protein purified in this way is stored at -80 ° C until it is used.

EXAMPLE 9 Preparation of monoclonal antibody against exon-17 peptide chain of human periostin (1) Antigenic preparation A peptide (SEQ ID NO: 25) having a Cys residue added to the N-terminus of the amino acid sequence constituting exon-17 human periostin (SEQ ID NO: 4) is chemically synthesized by the Fmoc method to obtain the peptide with a yield of 10 mg with a purity of 90% or more. As a carrier protein, KLH (5 mg, CALBIOCHEM) is coupled to this peptide to provide an antigenic solution. That is, KLH is dissolved in PBS (0.01 M) and adjusted to 3.3 mg / ml, to which a solution of 0.2524 mg / ml MBS (GE Healthcare Bio-Sciences KK) is added dropwise and reacts shaking to room temperature for 60 minutes. Dichloromethane is used to remove free MBS, to thereby obtain KLH-MB. This KLH-MB (5 mg) is mixed the antigenic peptide (5 mg) dissolved in the pH buffer of 0.01 M sodium phosphate (pH 7.2) and reacts agitation at 4 ° C for 12 hours to obtain the antigenic solution. (2) Immunization Three 6-week-old BALB / c female mice are each injected subcutaneously on both legs with the full volume of a mixed emulsion of the antigen solution (50 μm) containing 100 g of coupled antigenic peptide to KLH obtained in (1) and FCA (Freund's complete adjuvant, 50 μ?). The mice are then injected twice on both legs with a mixed emulsion prepared in situ of the above antigenic solution and FIA (incomplete Freund's adjuvant) in a 2 week interval. The mice are then sacrificed by cervical dislocation and aseptic lymph nodes are collected on their legs. Although RPMI medium (kohjinbio Co., Ltd.) is supplied, the above lymph nodes are crushed and pass through a mesh of approximately 10 μ? T? of pore size to obtain lymph node cells suspended in RPMI medium. This suspension is centrifuged at 1000 rpm for 10 minutes to obtain lymph node cells as a pellet fraction. This pellet fraction is then hemolyzed to remove the red blood cells in a solution (1 ml) prepared by adding a 20 mM HEPES pH regulator (pH 7.4) to a 0.84% ammonium chloride solution, the centrifugation is repeated at 1, 000 rpm for 5 minutes. The resulting pellet fraction (cell fraction) is washed several times with RPMI medium and then used for cell fusion. (3) Preparation of myeloma cells The mouse myeloma cell line P3X63Ag8U.1 (P3U1) which is resistant to 8-azaguanine and does not secrete immunoglobulin is cultured in RPMI medium containing 20% fetal calf serum (FCS) in an incubator with 10% C02, 37 ° C. Cells in the logarithmic growth phase are harvested and centrifuged at 1,000 rpm for 5 minutes to obtain the cells alone as a pellet fraction, which is then suspended in RPMI medium. (4) Cell fusion RPMI medium obtained in (2) containing 108 to 3 x 10 8 immunized lymph node cells and the RPMI medium obtained in (3) containing 108 myeloma cells are mixed and then centrifuged at 1,000 rpm for 10 minutes. The supernatant is gently removed to obtain the cells as a fraction of the pellet, followed by the addition of 1 ml of polyethylene glycol 1500 25% (w / v) (PEG 1500, Boehringer). The cells are further diluted to a total volume of 10 ml by slow addition of RPMI medium. To this suspension, RPMI medium containing 20% FCS (10 ml) is added and allowed to stand for a time, followed by centrifugation at 1,000 rpm for 5 minutes. The resulting pellet fraction (cell fraction) is adjusted to a cell density of 10 6 cells / ml by adding RPMI containing 20% FCS, and this cell suspension is dispensed at 200 μl /? in 96-well culture plates (Corning). After culturing in an incubator with 5% C02 at 37 ° C for 24 hours, HAT solution (Invitrogen) is added and the culture is continued for an additional 2 weeks.

Selection by means of ELISA A selection is made to determine the positive wells that show a reaction between the culture supernatant and the antigenic peptide. To be used as an antigenic solution for assay, the antigenic peptide (2 mg) obtained in (1) is coupled to ovalbumin (OVA) as a carrier protein to prepare a conjugate. Each well of a 96-well microvalier plate (Falcon 353912) is covered with the above conjugate (1 μg / ml) when resting overnight at 4 ° C. After washing this plate, the culture supernatant of (4) (50 μ ?, containing monoclonal antibodies) is added dropwise to each well and allowed to stand in an incubator at 37 ° C for 2 hours, followed by washing with PBS (-) (saline with regulated pH of phosphate). After the addition of sheep anti-mouse IgG antibody conjugated with alkaline phosphatase (Zymed), the plate is left to stand in an incubator at 37 ° C for 1 hour, washed with PBS (-) and then develops color for 20 minutes by adding a substrate that develops color (ALP). The absorbance (antibody titrator) at 490 nm OD is measured for each well with a plate reader (BIO-RAD, Model 680 Micro Píate Reader) to confirm its reactivity with the antigenic peptide, to thereby determine the positive wells that show a reaction between the culture supernatant and the antigenic peptide. (6) Cloning of antibody-producing cells Cells in positive wells whose reactivity with the antigenic peptide is confirmed by ELISA in (5) are provided for cloning of antibody-producing cell lines by limiting dilution. That is, cells in the positive wells are placed in each well of a 96 well culture plate and are cultured in an incubator with 5% C02 at 37 ° C for 2 weeks. In the same way as it is used in (5), the reactivity with the antigenic peptide is confirmed by ELISA for the culture supernatant in each well, and cloning by limiting the dilution is repeated again for each positive well to obtain 30 cells that they have a high reactivity with the antigenic peptide and show good growth of the colony. These cells are transferred to 24-well culture plates and cultured in an incubator with 5% C02 at 37 ° C for 2 weeks. In the same manner as used in (5), the reactivity with the antigenic peptide (antibody titrator) is again confirmed by ELISA for each culture supernatant. Cells in 10 wells that show a high absorbance at 490 nm OD, i.e., 10 hybridoma cell lines are determined to be useful as antibody producing cells and are selected.

Hybridoma cell lines Since the antibody-producing cells thus obtained always produce the antibodies of the present invention, ie, anti-human monoclonal antibodies exon-17, the culture supernatant wherein these antibody-producing cells are cultured can be used directly as the antibody solution of the present invention. It should be noted that the anterior antibody producing cell line (hybridoma) No. 1 (SBM337), which produces anti-human monoclonal antibody exon-17, is deposited under FERM BP-10718 on November 1, 2006 with the patent agency International Depository, the National Institute of Advanced Industrial Science and Technology. (7) Confirmation of human periostin protein binding capacity (PN-1) Antibodies produced by the 10 antibody-producing cells obtained in (6) are confirmed to observe their ability to bind to the human periostin protein (PN-1) by means of a dot blotting test. That is, the synthetic protein obtained in Example 8 (300 μ? / ???) is speckled in volumes of 5 μ? in a Hybond-ECL nitrocellulose membrane (GE Healthcare Bio-Science KK) and washed once with TBS solution (10 mM Tris-HCl (pH 8.0), 150 mM NaCl). The blocking pH regulator (Block Ace, Show Brand Milk Products Co., Ltd.) is added and stirred at room temperature for 1 hour. Then a solution of 1 μg / ml of each monoclonal antibody (primary antibody) obtained in (6) is added to the membrane and stirred for 3 hours, the membrane is washed four times with TBS solution which is under stirring for 10 minutes . Then a 0.4 μg / ml solution of a HRP-labeled anti-mouse IgG antibody (Promega) (secondary antibody) is added to the membrane and stirred at room temperature for 1 hour, the membrane is washed four times with TBS solution under stirring for 10 minutes. The detection reagents (ECL plus Western blotting detection system, GE Healthcare Bio-Sciences KK) are added and reacted for 1 minute to detect chemiluminescence. As a result, it is confirmed that all of the 10 antibody-producing cells cloned in (6) bind to human periostin PN-1. (8) Mass production and purification of monoclonal antibody BALB / c mice are administered intra-peritoneally with pristane [2,6,10,14-tetramethyl pentadecane (0.5 ml, Wako Puré Chemical industries, Ltd.)] and is maintained for 2 or 3 weeks. The production hybridomas of monoclonal antibody No, 1 and 3 that have been maintained in the logarithmic growth phase are collected and centrifuged to remove the culture supernatant. To the cells in each pellet fraction, FCS free RPMI medium is added to prepare a cell suspension at a cell density of 1 x 10 7 cells / ml. This cell suspension is injected intraperitoneally in BALB / c mice pre-treated with pristane and, after approximately three weeks, the exudate ascites fluid is collected from the abdominal region by means of a syringe. After each collected ascites fluid is filtered using a filter with a pore size of f 0.22 μ, the filtrates are purified in a routine manner by affinity chromatography on a G-sepharose protein column (Millipore, 1 151 1324) to prepare two anti-human monoclonal exon-17 antibodies.

EXAMPLE 10 Analysis of the site of recognition of monoclonal antibody anti-human exon-17 in peptide chain exon-17 periostin of human The two resulting monoclonal antibodies (No. 1 and No. 3) are analyzed for their recognition sites in the human periostin exon-17 peptide chain (epitope identification). That is, based on an amino acid sequence consisting of 45 amino acids in total between the 9-phenylalanine of the N-terminus and the 9a isoleucine of the C-terminus of the peptide chain exon-17 periostin (SEQ ID NO: 4; 1 to threonine to 27th glutamic acid), the following 36 peptides composed of 10 amino acids are synthesized in a cellulose membrane to prepare a membrane-bound peptide array (SPOT client services from Sigma-Aldrich Japan KK). 1 FKEIPVTVYT 2 KEIPVTVYTT 3 EIPVTVYTTK 4 IPVTVYTTKI 5 PVTVYTTKII 6 VTVYTTKIIT 7 TVYTTKIITK 8 VYTTKIITKY 9 YTTKIITKVV 10 TTKIITKVVE 1 1 TKIITKVVEP 12 KIITKVVEPK 13 IITKVVEPKI 14 ITKVVEPKIK 15 TKVVEPKIKV 16 KVVEPKIKVI 17 VVEPKIKVIE 18 VEPKIKVIEG 19 EPKIKVIEGS 20 PKIKVIEGSL 21 KIKVIEGSLQ 22 IKVIEGSLQP 23 KVIEGSLQPI 24 VIEGSLQPII 25 IEGSLQPIIK 26 EGSLQPIIKT 27 GSLQPIIKTE 28 SLQPIIKTEG 29 LQPIIKTEGP 30 QPIIKTEGPT 31 PIIKTEGPTL 32 IIKTEGPTLT 33 IKTEGPTLTK 34 KTEGPTLTKV 35 TEGPTLTKVK 36 EGPTLTKVKI This membrane is allowed to stand in a small volume of methanol for 5 minutes and then washed three times with TBS solution. Blocking pH regulator (casein, included in SPOT) is added and stirred at room temperature for 2 hours. Then a 1 μg / ml solution of each monoclonal antibody (primary antibody) obtained in example 9 (8) is added to the membrane and stirred for 3 hours, the membrane is washed three times in TBS solution under stirring for 10 minutes. Then a solution 0.4 μ? / ??? of anti-mouse IgG antibody labeled with HRP (Promega) (secondary antibody) is added to the membrane and incubated for 2 hours, the membrane is washed three times with TBS solution under stirring for 5 minutes. Detection reagents (SuperSignal West Peak, Pierce) are added and reacted for 1 minute to detect chemiluminescence. As a result, monoclonal antibodies No. 1 and No. 3 are found to react and bind only to synthetic peptide No. 9 consisting of the amino acid sequence YTTKIITKVV (SEQ ID NO: 26), ie, a peptide comprising of an amino acid sequence that covers from -1 to tyrosine to 9a valine of the N-terminus of the amino acid sequence of the peptide chain exon-17 periostin of human (SEQ ID NO: 4) or that covers from the 669th tyrosine at 679a valine from the N-terminal amino acid sequence of human periostin PN-1 (SEQ ID NO: 2).

EXAMPLE 1 Analysis of the site of recognition of polyclonal antibody exon-17 anti-rat in peptide chain exon-17 periostin of human In the same manner as shown in example 10, the polyclonal antibody prepared in example 1 is analyzed for its recognition site in the human periostin exon-17 peptide chain (identification of the epitope). As a result, as in the case of the monoclonal antibodies in example 10, the polyclonal antibody is found to react with only the synthetic peptide No. 9, indicating that the polyclonal antibody specifically recognizes the same site as the monoclonal antibodies. This suggests that antibodies having the same specificity can be obtained in both cases where an exon-17 rat periostin peptide is used as an antigen to prepare a polyclonal antibody and where an exon-17 human periostin peptide is used as an antigen to prepare a monoclonal antibody.

EXAMPLE 12 Confirmation of rat periostin protein binding capacity (PN-11) The two resulting monoclonal antibodies (No. 1 and No. 3) are confirmed for their ability to bind to rat periostin protein (PN-1) by dot blotting analysis. That is to say, the purified protein obtained in the preparation example 6 (30 μg ml) is mottled in volumes of 5 μ? in a Hybond-ECL nitrocellulose membrane (GE Healthcare Bio-Sciences KK) and washed once with TBS solution (10 mM Tris-HCl (pH 8.0), 150 mM NaCl). Blocking pH regulator (Block Ace, Snow Brand Milk Products Co., Ltd.) is added and stirred at room temperature for 1 hour. Then a 1 g / ml solution of each monoclonal antibody (primary antibody) is added to the membrane and stirred for 3 hours, the membrane is washed four times with TBS solution under stirring for 10 minutes. Then a solution 0.4 μg-g / ml of anti-mouse IgG antibody labeled with HRP (Promega) (secondary antibody) is added to the membrane and stirred at room temperature for 1 hour, the membrane is washed four times with TBS solution under stirring for 10 minutes. The detection reagents (ECL plus Western blotting detection system, GE Healthcare Bio-Sciences KK) are added and reacted for 1 minute to detect chemiluminescence. As a result, it is confirmed that the two resulting monoclonal antibodies also bind to rat periostin PN-1.

EXAMPLE 13 Anti-Human Monoclonal Antibody Epitope Analysis The results of example 10 indicate that the epitope part of each anti-human monoclonal antibody exon-17 recognizes an amino acid sequence (TTKIITKVV; SEQ ID NO: 22) that covers from the N-terminal threonine to the chain 9a valine peptide exon-17 human periostin (SEQ ID NO: 4). Also, the results of example 12 confirm that each anti-human monoclonal antibody exon-17 also binds to rat periostin protein (PN-1). These results suggest that the epitope part of each anti-human monoclonal antibody exon-17 recognizes a region, whose amino acids do not differ between humans and rats, in the amino acid sequence (TTKIITKVV; SEQ ID NO: 22) that covers from the threonine N-terminal to the 9th valine of the peptide chain exon-17 human periostin (SEQ ID NO: 4), that is, the complete amino acid sequence, or a part of it, that covers from the N-terminal threonine to the 7th lysine of the peptide chain exon-17 human periostin (SEQ ID NO: 4), or the peptide chain exon-17 rat periostin (SEQ ID NO: 3). In this way, for example further analysis of the epitope part, the alanine scan is carried out. Based on the amino acid sequence (YTTKIITKVV; SEQ ID NO: 26) which covers the -1 to tyrosine to the 9a valine of the N-terminal chain of the human exon-17 periostin peptide chain (SEQ ID NO: 4), the following 10 peptides modified to replace some amino acids by alanines are synthesized with a purity of 80% or more. # 1 YTTKIITKVV # 2 ATTKIITKAA # 3 AATKIITKAA # 4 AAAKIITKAA # 5 AAAAIITKAA # 6 AAAAAITKAA # 7 ATTKIITAAA # 8 ATTKIIAAAA # 9 ATTKIAAAAA # 10 ATTKAAAAAA Synthetic peptides (1 mg each) are solubilized with 50 μ? PBS (-), are mottled in volumes of 1.5 μ? in a Hybond-ECL nitrocellulose membrane (GE Healthcare Bio-Sciences KK) and washed once with TBS solution. Blocking pH regulator (Block Ace, Snow Brand Milk Products Co., Ltd.) is added and stirred at room temperature for 1 hour. Then a solution of 1 μg / ml of each monoclonal antibody (primary antibody) obtained in example 9 (8) is added to the membrane and stirred for 3 hours, the membrane is washed four times with TBS solution under stirring for 10 minutes . Then a 0.4 μg / ml solution of HRP-labeled anti-mouse IgG antibody (Promega) (secondary antibody) is added to the membrane and stirred at room temperature for 1 hour, the membrane is washed four times with TBS solution under stirring. 10 minutes. Detection reagents (SuperSignal West Peak, Pierce) are added and react for 1 minute to detect chemiluminescence. As a result, the monoclonal antibodies are found to react strongly with the synthetic peptide # 7 shown above (a peptide comprising alanine substitutions in the 1st and 8-1 Oo amino acids of the N-terminus of a peptide consisting of a sequence of amino acids (YTTKIITKVV; SEQ ID NO: 26) that covers from -1 to tyrosine to 9a valine of the N-terminal peptide chain exon-17 human periostin (SEQ ID NO: 4)), reacts weakly with synthetic peptides # 1 (a peptide consisting of an amino acid sequence (YTTKIITKVV; SEQ ID NO: 26) which covers from -1 to tyrosine to 9a valine of the N-terminal chain of the human exon-17 peptide chain periostin (SEQ ID NO: 4)) and # 2 (a peptide comprising alanine substitutions in the 1st and 9th amino acids of the N-term of a peptide consisting of an amino acid sequence (YTTKIITKVV; SEQ ID NO: 26) covering from the - 1 to tyrosine at the 9th valine of the N-terminus of the peptide chain exon-17 human periostin (SEQ ID NO: 4)), and react more weakly with synthetic peptides # 3 (a peptide comprising alanine substitutions at the 1, 2, and 9-1 O-amino acids of the N-terminus of a peptide consisting of an amino acid sequence (YTTKIITKVV; SEQ ID NO: 26) which covers from -1 to tyrosine to 9a valine of the N-terminus of the peptide chain exon-17 human periostin (SEQ ID NO: 4)) and # 8 (a peptide comprising alanine substitutions in the 1st and 7-10th amino acids of the N-terminus of a peptide consisting of an amino acid sequence (YTTKIITKVV; SEQ ID NO: 26) that covers from -1 to tyrosine to 9a valine of the N-terminus of the chain peptide exon-17 human periostin (SEQ ID NO: 4)).

EXAMPLE 14 Analysis of the recognition site of polyclonal antibody exon-17 anti-rat in peptide chain exon-17 periostin of human In the same manner as that shown in example 13, the polyclonal antibody prepared in example 1 is analyzed for its recognition site in the human periostin exon-17 peptide chain (epitope identification). As a result, as in the case of the monoclonal antibodies in example 13, the polyclonal antibody is found to react strongly with synthetic peptide # 7, reacts weakly with synthetic peptides # 1 and # 2, and reacts weaker still with peptides Synthetic # 3 and # 8, indicating that the polyclonal antibody specifically recognizes the same site as the monoclonal antibodies. This suggests that antibodies having the same specificity can be obtained in both cases where an exon-17 rat periostin peptide is used as an antigen to prepare a polyclonal antibody and where an exon-17 human periostin peptide is used as an antigen to prepare a monoclonal antibody.

EXAMPLE 15 In vitro study of the presence or absence of anti-cellular adhesive activity of human periostin protein (PN-1 of human) In the same manner as shown in example 2, human heart fibroblasts (Dainippon Pharmaceutical Co., Ltd., catalog No. CS-ABI-51 18) are placed in a 96-well plate at a density of 6.4 x 104 cells / 100 μ? and are grown overnight, and the culture is then incubated in fresh CSC medium (Cell System Corporation) with 10% FBS containing 10 μ? /? t? of cycloheximide at 37 ° C for 1 hour. Then, the cells are washed twice with CSC medium (free of serum) pre-heated at 37 ° C, and human periostin protein (PN-1 of human) prepared according to the examples is added to medium CSC (free of charge). serum) at a final concentration of 1 9 / G ?? Fibronectin that has cell adhesion promoting properties is used as a positive control and BSA (bovine serum albumin) that does not have cellular adhesive properties is used as a negative control. After incubation at 37 ° C for 3.5 hours, microscopy shows that all cells are separated in the group treated with human periostin protein, and the cells are washed twice with PBS (-) and then fixed in formalin with Regulated pH 10% neutral for 30 minutes. Then, the cells are washed with PBS (-) three times and then stained with crystal violet for 30 minutes. Then, the degree of staining is measured using a plate reader at 550 nm (BIO-RAD, Microplate Reader Model 680) (Figure 10). As a result, the control groups treated with fibronectin and BSA and the untreated group do not show anti-cellular adhesive properties, on the contrary to the group treated with human periostin protein (human PN-1) where the cells are separated , showing that the human periostin protein (human PN-1) has anti-cell adhesive properties.

EXAMPLE 16 In vitro study of the neutralizing activity of anti-human monoclonal antibody exon-17 In the same manner as shown in Example 3, human heart fibroblasts are placed in a 96-well plate at a density of 6.4 x 10 4 cells / 100 μ? and they are grown overnight, and the culture is then incubated in fresh CSC medium with 10% FBS containing 10 μg / ml cycloheximide at 37 ° C for 1 hour. The cells are then washed twice with CSC medium (serum free) pre-heated at 37 ° C, and human periostin protein (human PN-1) and anti-human exon-17 monoclonal antibody (No. No. 3) are added to CSC medium (serum free) at final concentrations of 1 μ? /? T? and 200 μg / vn \, respectively. Human periostin protein (PN-1 from human) is used individually as a positive control and BSA is used as a negative control. After incubation at 37 ° C for 3.5 hours, microscopy shows that all cells are separated in the group treated with human periostin protein (human PN-1) only, and the cells are washed twice with PBS (-) and then fixed in formalin with 10% neutral pH regulated for 30 minutes. Then, the cells are washed with PBS (-) three times and then stained with crystal violet for 30 minutes. Then, the degree of staining is measured using a plate reader at 550 nm (BIO-RAD, Microplate Reader Model 680) (Figure 11). The results show that anti-human monoclonal antibodies exon-17 are antibodies that have the activity of inhibiting the anti-cellular adhesive properties of human periostin protein (human PN-1), that is, the neutralization of anti-adhesive properties. Human periostin protein cell phones (human PN-1). As shown above, the anti-cellular adhesive properties of human periostin protein (human PN-1) are inhibited by antibodies against exon-17 of human periostin protein (human PN-1) that specifically recognizes a sequence or a part thereof consisting of the 1st or 6th N-terminal amino acids of exon-17, suggesting that exon-17, at least one segment of peptide or a part thereof consisting of 1 or 6 N-terminal amino acids of exon-17 constitutes a region related to the anti-cellular adhesive properties of human periostin protein (human PN-1).

EXAMPLE 17 Effect of anti-human monoclonal antibody exon-17 in rats model of acute myocardial infarction In the same manner as that shown in example 4, a male Lewis rat weighing 250-300 g is fixed on a surgical table for rats after the animal is completely anesthetized by peritoneal administration of pentobarbital (0.1 ml / 100 g). A tube is inserted orally into the trachea and connected to a rat ventilator (tidal volume 3 ml, 80 breaths / minute), and the skin is cut laterally from the third left intercostal space of the sternum and the underlying pectoralis major muscle is also cut laterally, and the intercostal space is opened using a rat rib retractor to expose the heart. Then, the near left coronary artery under the left atrium is ligated with silk 1 .0 using a curved needle that has a diameter of 5 mm. After visual confirmation that the anterior and lateral walls along which the left coronary artery runs have been changed from red to white to show sufficient blockage of the coronary bloodstream and the disappearance of wall movement at these sites ( in the substitute operation group, the needle is passed through the coronary artery and then removed without ligation), the third and fourth ribs are fixed by ligation with silk 3.0 (after the lung expands to remove the air that exists outside of the lung in the rib cage so that the lung can easily expand). The incision site in the skin is sutured with silk 3.0 in the same manner and then observed for a time, and the tube is removed after confirmation of regaining consciousness and resumption of spontaneous breathing. Models of acute myocardial infarction are prepared consecutively by the previous procedure. On the next day, percutaneous echocardiography is performed under intranasal anesthesia with isoflurane, and small infarct models that have an infarct size of less than 20% of the entire periphery of the left ventricle are excluded. The remaining infarction models are classified in order to increase cardiac function, and are classified alternatively in a group treated with monoclonal anti-human monoclonal antibody (No. 3) and a group treated with a control antibody (IgG). of rabbit) every 200 μg via the vein of the tail.

The antibodies are administered to each group on the day following the preparation of the models and at intervals of 6 days after the initial administration, a total of 4 times. The heart is evaluated by echocardiography through the chest wall at intervals of one week until the end of 4 weeks. The results of the echocardiography 4 weeks after the preparation of the models shows that the reduction of the thickness of the anterior wall and the thickness of the posterior wall of the heart are inhibited, the increase of the extreme-diastolic internal diameter and extreme-systolic inner diameter are inhibited, and that the FS value or EF value indicative of the contractile function of the heart is increased in the group treated with anti-human monoclonal antibody exon-17 significantly as compared to the control group treated with rabbit IgG. In short, cardiac dilation is inhibited, showing that cardiac function is improved (Figure 12A to Figure 12C). As shown above, in rats model of acute myocardial infarction, the effects of inhibition of cardiac dilation and improvement of cardiac function are caused by antibodies against human periostin protein exon-17 (human PN-1) having an epitope composed of at least one sequence consisting of the 1-6th N-terminal amino acid of exon-17, suggesting that exon-17 of human periostin protein (human PN-1), especially a region comprising at least one segment of peptide consisting of the 1-6th amino acid N-terminal of exon-17 is a region related to cardiac dilation and reduced cardiac function after myocardial infarction.

Industrial Applicability Diseases in which periostin is involved can be prevented and treated by suppressing the function of a periostin isoform that has highly expressed anti-cellular adhesive activity in a disease such as cardiac deficiency, inhibition of aggravation of the condition and Improvement of tissue function through the use of an antibody against the periostin isoform having anti-cellular adhesive activity. In addition, the presence of the diseases and the degree of symptom progression can be known through the measurement of the amount of periostin isoform in a sample from a patient.

Claims (1)

1. NOVELTY OF THE INVENTION CLAIMS 1 .- An antibody against a periostin soforma having anti-cell adhesive activity, which specifically recognizes a site responsible for periostin cell adhesion, and which has the ability to neutralize the anti-cellular adhesive activity of periostin. 2. The antibody according to claim 1, further characterized in that the site responsible for anti-cellular adhesion of periostin is the amino acid sequence encoded by exon-17 or by means of a part thereof. 3. The antibody according to claim 2, further characterized in that the amino acid sequence encoded by exon-17 or through a part thereof is one of the amino acid sequences selected from the group of SEQ IDS NO: 3, 4, 21, 22, 23, 24, 26 and 34. 4. The antibody according to claim 3, further characterized in that the amino acid sequence is the amino acid sequence of SEQ ID NO: 3, 4 or 21. 5. The antibody according to any of claims 1 to 4, further characterized in that it is a monoclonal antibody. 6. The antibody according to claim 5, further characterized in that it is produced by means of a hybridoma cell line FERM BP-10718. 7. - A hybridoma obtainable through a method comprising the steps of: immunizing a mammal with a peptide having one of the amino acid sequences selected from the group of SEQ IDS NO: 3, 4, and 21 or a peptide that introduces cysteine residues in the N-terminal thereof; and the fusion of a mammalian antibody producing cell with a myeloma cell. 8.- A hybridoma cell line FERM BP-10718. 9. - A method for the production of an antibody of claim 5, comprising the steps of: immunizing a mammal with a peptide having one of the amino acid sequences selected from the group of SEQ IDS NO: 3, 4, and 21 or a peptide that introduces cysteine residues into the N-terminus thereof; the fusion of a cell that produces antibody from the mammal with a myeloma cell; and the culture of the obtained hybridoma. 10. - The method according to claim 9, further characterized the hybridoma is a hybridoma cell line FERM BP-10718. eleven . - A pharmaceutical composition comprising the antibody of any of claims 1-6. 12. - A pharmaceutical composition for preventing or treating a disease wherein is involved a periostin isoform having anti-cellular adhesive activity, comprising the antibody of any of claims 1-6. 13. - The pharmaceutical composition according to claim 12, further characterized by the disease is heart failure, myocardial infarction, cardiac dilation, cardiac hypertrophy, cardiac fibrosis, cardiomyopathy, myocarditis, valvular disease, cancer, aneurysm, arteriosclerosis, neurodegenerative disease central, kidney disease, rheumatoid arthritis, osteoporosis, pulmonary emphysema, pulmonary hypertension, chronic obstructive pulmonary disease (COPD), nephritis, pancreatitis, hepatitis, liver fibrosis or pulmonary fibrosis. 14. A method for preventing or treating a disease wherein a periostin isoform having anticellular adhesive activity is involved, which comprises administering the antibody according to any of claims 1-6 to a patient. 15. - The method according to claim 14, further characterized in that the disease is heart failure, myocardial infarction, cardiac dilation, cardiac hypertrophy, cardiac fibrosis, cardiomyopathy, myocarditis, valvular disease, cancer, aneurysm, arteriesclerosis, central neurodegenerative disease , kidney disease, rheumatoid arthritis, osteoporosis, pulmonary emphysema, pulmonary hypertension, chronic obstructive pulmonary disease (COPD), nephritis, pancreatitis, hepatitis, liver fibrosis or pulmonary fibrosis. 16. A method of diagnosing a disease wherein a periostin isoform having anti-cellular adhesive activity is involved, comprising measuring the amount of the periostin isoform in a biological sample by using the antibody in accordance with any of the claims 1-6. 17. - The method according to claim 16, further characterized in that the antibody is a labeled antibody. 18. - The method according to claim 16 or 17, further characterized in that the disease is heart failure, myocardial infarction, cardiac dilation, cardiac hypertrophy, cardiac fibrosis, cardiomyopathy, myocarditis, valvular disease, cancer, aneurysm, arteriosclerosis, disease central neurodegenerative, kidney disease, rheumatoid arthritis, osteoporosis, pulmonary emphysema, pulmonary hypertension, chronic obstructive pulmonary disease (COPD), nephritis, pancreatitis, hepatitis, liver fibrosis or pulmonary fibrosis. 19. A method for detecting or quantifying a periostin isoform having anti-cell adhesive activity in a sample using the antibody of any of claims 1-6. 20. - A diagnostic reagent for a disease wherein a periostin isoform having anti-cellular adhesive activity is involved, comprising the antibody of any of claims 1-6. twenty-one . - The diagnostic reagent according to claim 20, further characterized by the disease is heart failure, myocardial infarction, cardiac dilation, cardiac hypertrophy, cardiac fibrosis, cardiomyopathy, myocarditis, valvular disease, cancer, aneurysm, arteriosclerosis, central neurodegenerative disease , kidney disease, rheumatoid arthritis, osteoporosis, pulmonary emphysema, pulmonary hypertension, chronic obstructive pulmonary disease (COPD), nephritis, pancreatitis, hepatitis, liver fibrosis or pulmonary fibrosis.