TW202221036A - Anti-human TRPV2 antibody - Google Patents

Abstract

The present invention provides an anti-human TRPV2 antibody, which recognizes an extracellular domain of human TRPV2 as an epitope, or a fragment thereof.

Description

Anti-human TRPV2 antibody

The present invention relates to an anti-human TRPV2 which recognizes the extracellular domain of TRPV2 (transient receptor potential cation channel, subfamily V, member 2, transient receptor potential cation channel, subclass V, member 2) as an epitope as a Ca ²⁺ channel antibodies or fragments thereof. Furthermore, the present invention relates to a human TRPV2 inhibitor and the like comprising the anti-human TRPV2 antibody or fragment thereof.

The Trp (transient receptor potential, transient receptor potential) gene product superfamily is classified in mammals as TRPC (transient receptor potential canonical, classical transient receptor potential) (7 species), TRPV (transient receptor potential vanilloid, vanillin subtypes) Transient receptor potential) (6 types), TRPM (transient receptor potential melastatin, melastatin transient receptor potential) (8 types), TRPA (transient receptor potential ankyrin, ankyrin transient receptor potential) (1 type), TRPP (transient receptor potential polycystin, polycystic kidney protein transient receptor potential) (4 types), TRPML (transient receptor potential mucolipin, mucolipin transient receptor potential) (3 types) of 6 families, all of which are 6 times permeable Membrane ion channel, mainly through Ca ²⁺ . Most of the ion channels present in the cell membrane have different channel properties, and in order to screen for drugs that specifically react with these, it is necessary to use an analysis system suitable for them. This aspect is quite different from G protein-coupled receptors (GPCRs) where cellular responses are relatively limited.

TRPV2 is a stretch-activated Ca ²⁺ channel (Non-Patent Documents 1 to 3), exists in the endomembrane system of normal tissues, and is transferred to the cell membrane with diseases such as muscular dystrophy and cardiomyopathy, where it is activated and contributes to Ca ² . ⁺ Abnormal influx into cells (Non-Patent Documents 2 and 4). It is considered in the industry that an agent that specifically inhibits TRPV2 acts on degenerated muscles and/or myocardium, thereby reducing degeneration, and may have therapeutic/symptom relieving effects for muscle diseases or cardiac diseases, and development is expected.

The industry believes that gadolinium, which is used as a blocker of previous stretch-sensitive Ca ²⁺ channels, acts on all Ca ²⁺ channels, and that ruthenium red acts on all TRPV families (TRPV1-TRPV6), which are non-specific drugs (Non-patent documents 5 and 6). Patent Document 1 discloses low-molecular-weight compounds capable of specifically inhibiting TRPV2, but the technology is different from antibodies that recognize human TRPV2 epitopes. Patent Document 2 discloses an antibody that recognizes the mouse TRPV2 sequence and has a function of specifically inhibiting the activity of TRPV2, but an antibody that recognizes the sequence of human TRPV2, or an antibody that recognizes the sequence of human TRPV2 and has the activity of specifically inhibiting TRPV2 The production example of the functional antibody is not known.

Further, as an antibody reactive against TRPV2, an anti-rat TRPV2 polyclonal antibody (KM019, manufacturer: TRANS GENIC Co., Ltd.) is commercially available. The antibody recognizes the intracellular domain by using a partial peptide at the C-terminus of TRPV2 as an immunogen, and its use is for the analysis of immunohistochemistry, and no disclosure has been made about the activity inhibition of TRPV2. prior art literature Patent Literature

Patent Document 1: Japanese Patent Laid-Open No. 2009-149534 Patent Document 2: Japanese Patent No. 5754039 Non-patent literature

Non-Patent Document 1: Circulation Research; 93: 829-838 (2003) Non-Patent Document 2: J. Cell Biology; 161(5): 957-967 (2003) Non-Patent Document 3: J. Endocrinology; 191: 515-523 (2006) Non-Patent Document 4: Hum Mol Genet.; 18(5): 824-834 (2009) Non-Patent Document 5: J. Neuroscience; 28(24): 6231-6238 (2008) Non-Patent Document 6: Diabetologia; 51: 2252-2262 (2008) Non-Patent Document 7: Cardiovasc Res; 99: 760-768 (2013) Non-Patent Document 8: Intern Med; 57: 311-318 (2018)

[Problems to be Solved by Invention]

The object of the present invention is to provide an anti-human TRPV2 antibody and a fragment thereof that recognize the extracellular domain of human TRPV2 (hereinafter sometimes referred to as hTRPV2) as an epitope, and an anti-human TRPV2 antibody that recognizes the extracellular domain of human TRPV2 as an epitope and that specifically inhibits human An anti-human TRPV2 antibody or fragment thereof that functions as a function of TRPV2 activity. [Technical means to solve problems]

In order to solve the above-mentioned problems, the present inventors first designed an implementation of Japanese Patent No. 5754039 based on the prior art document related to the invention of an antibody that recognizes the mouse TRPV2 sequence and has a function of specifically inhibiting the activity of TRPV2 By analogy experiments like the example, a plurality of different immunogens (cells or peptides) that can be assumed according to this example were produced, and the desired anti-human TRPV2 antibody was tried to be obtained. However, within the range that can be deduced from the contents of the prior art documents, etc., the desired anti-human TRPV2 antibody that recognizes the extracellular domain of human TRPV2 as an epitope, or that recognizes the extracellular domain of human TRPV2 as an epitope and has specificity has not been obtained. Anti-human TRPV2 antibodies that function to inhibit the activity of human TRPV2. Accepting the above-mentioned results, the present inventors have repeatedly conducted intensive studies focusing on the use of gene banks prepared from animals immunized with cells for human TRPV2 expression, which are expressed by phage display by specifying Stimulation with a substance (cannabidiol) enables the efficient expression of human TRPV2 on the cell membrane. As a result, it was confirmed that a desired anti-human TRPV2 antibody that recognizes the extracellular domain of human TRPV2 as an epitope, or an anti-human TRPV2 antibody that recognizes the extracellular domain of human TRPV2 as an epitope and has a function of specifically inhibiting the activity of human TRPV2 is obtained. antibody, thereby completing the present invention. That is, the present invention relates to the following. [1] An anti-human TRPV2 monoclonal antibody or fragment thereof, which recognizes the extracellular domain of human TRPV2 as an epitope. [2] The monoclonal antibody or fragment thereof according to [1], which recognizes the extracellular domain of human TRPV2 as an epitope and specifically inhibits the activity of TRPV2. [3] The monoclonal antibody or fragment thereof according to [1] or [2], wherein the extracellular domain recognizing human TRPV2 comprises an amino acid sequence selected from the group consisting of the fifth transmembrane region and the sixth transmembrane region The region of at least 5 or more amino acid sequences is used as an epitope to specifically inhibit the activity of TRPV2. [4] The monoclonal antibody or fragment thereof according to [1], wherein the extracellular domain recognizing human TRPV2 comprises at least five amino acid sequences selected from the group consisting of the first transmembrane region and the second transmembrane region The region of the above amino acid sequence acts as an epitope and specifically inhibits the activity of TRPV2. [5] The monoclonal antibody or fragment thereof according to any one of [1] to [3], which is in the extracellular domain of human TRPV2, 1) Recognize a region comprising at least 5 or more consecutive amino acid sequences selected from the amino acid sequence represented by SEQ ID NO: 2 as an epitope to specifically inhibit the activity of TRPV2, or 2) Identify a region comprising at least 5 or more consecutive amino acid sequences selected from the amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2 with substitution, addition, or deletion of 1 to 2 amino acids as a table It specifically inhibits the activity of TRPV2. [6] The monoclonal antibody or fragment thereof according to any one of [1] to [3] or [5], which is in the extracellular domain of human TRPV2, 1) Recognize a region comprising the amino acid sequence represented by SEQ ID NO: 3 as an epitope to specifically inhibit the activity of TRPV2, or 2) Recognize the region including the amino acid sequence in which 1-2 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 3 as an epitope to specifically inhibit the activity of TRPV2. [7] The monoclonal antibody or its fragment according to any one of [1] to [3], [5], or [6], which recognizes SEQ ID NO: 3 in the extracellular region of human TRPV2 The amino acid sequence of TRPV2 is used as an epitope to specifically inhibit the activity of TRPV2. [8] The monoclonal antibody or fragment thereof according to any one of [1] to [7], wherein the antibody is a humanized antibody. [9] The monoclonal antibody or fragment thereof according to any one of [1] to [3], [5] to [8], wherein the heavy chain variable region comprises the following: (i) CDR (complementarity determining region, complementarity determining region) 1 comprising the amino acid sequence represented by SEQ ID NO: 4 (ii) CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5, and (iii) CDR3 comprising the amino acid sequence represented by SEQ ID NO: 6, The light chain variable region includes the following: (iv) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 7 (v) CDR2 comprising the amino acid sequence represented by RMS, and (vi) CDR3 comprising the amino acid sequence represented by SEQ ID NO: 8. [10] The monoclonal antibody or fragment thereof according to [1], wherein the heavy chain variable region comprises the following: (i) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 9, (ii) CDR2 comprising the amino acid sequence represented by SEQ ID NO: 10, and (iii) CDR3 comprising the amino acid sequence represented by SEQ ID NO: 11, The light chain variable region includes the following: (iv) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 12, (v) CDR2 comprising the amino acid sequence represented by WAS, and (vi) CDR3 comprising the amino acid sequence represented by SEQ ID NO: 13. [11] The monoclonal antibody or fragment thereof according to any one of [1] to [3], [5] to [8], wherein the heavy chain variable region is the amino acid represented by SEQ ID NO: 14 The amino acid sequence of which the sequence is at least 95% identical, and the light chain variable region is the amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 15. [12] The monoclonal antibody or fragment thereof according to [1], wherein the heavy chain variable region is an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 16, and the light chain variable region It is an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 17. [13] The monoclonal antibody or fragment thereof according to any one of [1] to [3], [5] to [8], wherein the heavy chain variable region is the same as that of SEQ ID NOs: 22, 24, 25, and 26 Or the amino acid sequence represented by 27 is at least 95% identical, and the light chain variable region is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 23. [14] The monoclonal antibody or fragment thereof according to any one of [1] to [3], [5] to [8], wherein the heavy chain variable region is the same as that represented by SEQ ID NO: 25, 26 or 27 The amino acid sequence is at least 95% identical to the amino acid sequence, and the light chain variable region is the amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 23. [9a] An anti-human TRPV2 monoclonal antibody or fragment thereof, wherein the heavy chain variable region comprises the following: (i) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4, (ii) CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5, and (iii) CDR3 comprising the amino acid sequence represented by SEQ ID NO: 6, The light chain variable region includes the following: (iv) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 7, (v) CDR2 comprising the amino acid sequence represented by RMS, and (vi) CDR3 comprising the amino acid sequence represented by SEQ ID NO: 8. [10a] an anti-human TRPV2 monoclonal antibody or fragment thereof, wherein the heavy chain variable region comprises the following: (i) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 9, (ii) CDR2 comprising the amino acid sequence represented by SEQ ID NO: 10, and (iii) CDR3 comprising the amino acid sequence represented by SEQ ID NO: 11, The light chain variable region includes the following: (iv) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 12, (v) CDR2 comprising the amino acid sequence represented by WAS, and (vi) CDR3 comprising the amino acid sequence represented by SEQ ID NO: 13. [11a] The monoclonal antibody or fragment thereof according to [9a], wherein the heavy chain variable region is an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 14, and the light chain variable region is an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 15. [12a] The monoclonal antibody or fragment thereof according to [10a], wherein the heavy chain variable region is an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 16, and the light chain variable region It is an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 17. [13a] The monoclonal antibody or fragment thereof according to [9a], wherein the heavy chain variable region is an amino group that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 22, 24, 25, 26 or 27 Acid sequence, the light chain variable region is an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 23. [14a] The monoclonal antibody or fragment thereof according to [9a], wherein the heavy chain variable region is an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 25, 26 or 27, and the light The chain variable region is an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 23. [15] A therapeutic or preventive agent for muscle disease and/or heart disease, comprising, for example, [1] to [3], [5] to [9], [9a], [11], [11a], [ The anti-human TRPV2 monoclonal antibody or fragment thereof according to any one of 13], [13a], [14] and [14a]. [16] The therapeutic or preventive agent according to [15], wherein the muscle disease is neuromuscular junction and muscle disease of G70-G73 in ICD10 or the muscle disorder of M60-M63, and the heart disease is I20 in ICD10 - I25 ischemic heart disease or I30-I52 other types of heart disease. [17] The therapeutic or prophylactic agent according to [15], wherein the muscle disease is primary muscle disorder of G71 in ICD10 or other muscle disorder of M62, and the heart disease is angina pectoris of I20 and acute muscle disorder of I21 in ICD10 Myocardial infarction, recurrent myocardial infarction in I22, secondary complications of acute myocardial infarction in I23, other acute ischemic heart disease in I24, chronic ischemic heart disease in I25, acute pericarditis in I30, I31 I32 Other diseases of the pericardium, I32 Pericarditis classified in other diseases, I33 Acute and subacute endocarditis, I34 Nonrheumatic mitral valve disorder, I35 Nonrheumatic main disease Arterial valve disorders, non-rheumatic tricuspid valve disorders in I36, pulmonary valve disorders in I37, endocarditis (valve unknown) in I38, endocarditis and cardiac valve disorders in other diseases classified in I39, I40 Acute myocarditis, I41 Myocarditis among other diseases, I42 Cardiomyopathy, I43 Myocarditis among other diseases, I44 Atrioventricular block and left bundle branch block, I45 Other conduction disorders, I46 cardiac arrest, I47 paroxysmal tachycardia, I48 atrial microfibrillation and atrial flutter, I49 other arrhythmias, I50 heart failure, I51 heart disease Complications and records of cardiac diseases whose diagnosis is unclear, or other cardiac disorders classified as other diseases in I52. [18] The therapeutic or prophylactic agent according to [15], wherein the muscle disease is G71.0 muscular dystrophy, G71.1 myotonic disorder, G71.2 congenital myopathy, G71.3 Mitochondrial myopathy (other unclassified), G71.8 Other primary muscular disorders, G71.9 Primary muscular disorders of unknown detail, M62.0 Myosogenesis, M62. 1. Other muscle rupture (non-traumatic), M62.2, ischemic infarction of muscle, M62.3, immobility syndrome (paraplegia), M62.4, muscle contracture, M62.5, muscle wasting and atrophy ( Others not classified), M62.6 Muscle damage, M62.8 Other express muscle disorder, M62.9 Unspecified muscle disorder, Cardiac disease is acute transmural anterior wall of I21.0 in ICD10 Myocardial infarction, acute transmural myocardial infarction of inferior wall of I21.1, acute transmural myocardial infarction of other parts of I21.2, acute transmural myocardial infarction of I21.3 (unknown location), acute transmural myocardial infarction of I21.4 Acute subendocardial myocardial infarction, acute myocardial infarction in I21.9 (details unknown), atherosclerotic cardiovascular disease in I25.0, atherosclerotic heart disease in I25.1, and atherosclerotic heart disease in I25.1. 2, old myocardial infarction, I25.3, ventricular aneurysm, I25.4, coronary aneurysm, I25.5, ischemic cardiomyopathy, I25.6, painless <asymptomatic> myocardial ischemia, I25.8 Other types of chronic ischemic heart disease, I25.9 Chronic ischemic heart disease (details unknown), I42.0 Dilated cardiomyopathy, I42.1 Obstructive hypertrophic cardiomyopathy, I42 .2 Other hypertrophic cardiomyopathy, I42.3 Endomyocardial (eosinophilic) disease, I42.4 Endomyocardial fibroelastosis, I42.5 Other restrictive cardiomyopathy, I42. 6 Alcoholic cardiomyopathy, I42.7 Cardiomyopathy caused by drugs and other external factors, I42.8 Other cardiomyopathy, I42.9 Cardiomyopathy (details unknown), I50.0 Congestive heart failure , Left ventricular failure at I50.1, or heart failure at I50.9 (details unknown). [19] The therapeutic or preventive agent according to [15], wherein the muscle disease is muscle dystrophy of G71.0 in ICD10, and the heart disease is acute myocardial infarction (details unknown) of I21.9, I42.0 of dilated cardiomyopathy, or left ventricular failure of I50.1. [20] The therapeutic or preventive agent according to [15], wherein the muscle disease and/or the heart disease is muscle dystrophy of G71.0 in ICD10. [21] The therapeutic or prophylactic agent according to [15], wherein the muscle disease and/or heart disease are acute myocardial infarction (details unknown) of I21.9 in ICD10, dilated cardiomyopathy of I42.0, or I50.1 Left ventricular failure. [22] A diagnostic drug for muscle disease and/or heart disease, comprising the anti-human TRPV2 monoclonal antibody or a fragment thereof according to any one of [1] to [14] and [9a] to [14a]. [23] A diagnostic drug for muscle disease and/or heart disease, comprising the anti-human TRPV2 monoclonal antibody or a fragment thereof as described in [10] or [12]. [24] A kit for diagnosis of muscle disease and/or heart disease, comprising the anti-human TRPV2 monoclonal antibody as described in any one of [1] to [14], [9a] to [14a] or the same Fragment. [25] The diagnostic kit according to [24], wherein the muscle disease is G70-G73 neuromuscular junction and muscle disease or M60-M63 muscle disorder in ICD10, and the heart disease is I20 in ICD10 - I25 ischemic heart disease or I30-I52 other types of heart disease. [26] A method for assisting the diagnosis of muscle disease and/or heart disease, comprising the following steps: (i) using the anti-human TRPV2 monoclonal antibody or its fragment as described in any one of [1] to [14], [9a] to [14a], to TRPV2 in the sample collected from the test animal Quantitative performance; (ii) comparing the expression of TRPV2 quantified in (i) with the expression of TRPV2 in samples collected from healthy animals (control values); and (iii) Based on the result of (ii), when the expression level of TRPV2 quantified in (i) is greater than the control value, it is determined that the above-mentioned subject animal suffers from muscle disease and/or heart disease, or currently suffers from muscle disease and The possibility of heart disease, or the possibility of developing muscle disease and/or heart disease in the future. [27] A method for assisting the diagnosis of the degree of progression of muscle disease and/or cardiac disease, comprising the steps of: (i) using the anti-human TRPV2 monoclonal antibody or fragment thereof as described in any one of [1] to [14], [9a] to [14a], for patients suffering from muscle disease and/or heart disease, or the presence of Quantification of the expression of TRPV2 in samples collected from the subject animals of the possibility of having the disease; (ii) comparing the expression of TRPV2 quantified in (i) with the expression of TRPV2 in samples collected from animals suffering from muscle disease and/or cardiac disease at a specified degree of progression (control value); and (iii) Based on the result of (ii), when the expression level of TRPV2 quantified in (i) is greater than the control value, it is judged that the degree of progression of muscle disease and/or cardiac disease of the above-mentioned subject animal is higher than that of the control animal The degree of progression of the animal suffering from the disease, or the possibility of suffering from muscle disease and/or heart disease is high, and when it is less than the control value, it is judged that the muscle disease and/or heart disease of the above-mentioned tested animal is improving, or not suffering from High likelihood of muscle disease and/or heart disease. [28] A method for assisting the diagnosis of the degree of progression of muscle disease and/or heart disease, comprising the steps of: (i) using the anti-human TRPV2 monoclonal antibody or fragment thereof as described in any one of [1] to [14], [9a] to [14a], for patients suffering from muscle disease and/or heart disease, or the presence of Quantification of the expression of TRPV2 in samples collected from the subject animals of the possibility of having the disease; (ii) comparing the expression of TRPV2 quantified in (i) with the expression of TRPV2 in samples collected from the subject animal in the past (control value); and (iii) Based on the results of (ii), when the expression level of TRPV2 quantified in (i) is greater than the control value, it is judged that the muscle disease and/or heart disease of the above-mentioned subject animal is progressing, or suffering from muscle disease and The possibility of heart disease is high, and when it is less than the control value, it is judged that the muscle disease and/or heart disease of the tested animal is improving, or the possibility of not suffering from muscle disease and/or heart disease is high. [Effect of invention]

The anti-human TRPV2 antibody or its fragment of the present invention does not affect the activity of other Trp family proteins, and can specifically inhibit the Ca ²⁺ influx activity of TRPV2. Furthermore, the anti-human TRPV2 antibody or fragment thereof of the present invention uses the extracellular domain of TRPV2 as an epitope, and thus can react with TRPV2 existing on the cell membrane of living cells. In addition, the anti-human TRPV2 antibody or fragment thereof of the present invention has the effect of inhibiting the degeneration of muscle cells and cardiomyocytes. The anti-human TRPV2 antibody or fragment thereof of the present invention is believed to be capable of inhibiting the activity of TRPV2 on cell membranes in diseased state, and can be a powerful candidate therapeutic drug for muscle disease and/or cardiac disease. In addition, the anti-human TRPV2 antibody or fragment thereof of the present invention can be expected to be used as an experimental tool in the functional analysis of TRPV2 or the mechanism analysis of the degeneration of muscle cells or cardiomyocytes. In addition, it is known that TRPV2 is expressed on cell membranes in skeletal muscle cells, cardiomyocytes, and peripheral blood mononuclear cells in the condition of muscle disease or cardiac disease (Non-Patent Documents 2, 7 and 8). Therefore, the anti-human TRPV2 antibody or fragment thereof of the present invention can also be used as an agent for diagnosing the development or possibility of suffering from muscle disease or cardiac disease by investigating the expression or expression level of TRPV2 on the cell membrane using the antibody. Furthermore, by comparing the expression level of TRPV2 on the cell membrane in the past measured using the antibody with the current expression level, it is also possible to diagnose the degree of progression of muscle disease or cardiac disease, or to treat the disease. And the evaluation of the effect of the therapeutic drugs used. Furthermore, the anti-human TRPV2 antibody of the present invention, which does not have the function of specifically inhibiting the activity of human TRPV2, is considered to be highly safe for living organisms, and thus can also be used as a companion diagnostic drug.

The present invention relates to an antibody or a fragment thereof that recognizes the extracellular domain of human TRPV2 as an epitope, and an anti-human TRPV2 antibody or a fragment thereof that recognizes the extracellular domain of human TRPV2 as an epitope and has a function of specifically inhibiting the activity of human TRPV2 . The present invention relates to a human TRPV2 inhibitor or a cardiomyocyte and/or myocyte degeneration inhibitor comprising the anti-human TRPV2 antibody or fragment thereof of the present invention. In addition, the present invention relates to a diagnosis (determination) drug, or a diagnosis (determination) method for a muscle disease and/or heart disease, which comprises using the antibody to study not only skeletal muscle cells or cardiac muscle in the condition of muscle disease or heart disease cells, and TRPV2 expressed on the cell membrane of peripheral blood mononuclear cells. Further, the present invention relates to a therapeutic or preventive agent for muscle disease and/or cardiac disease comprising the anti-human TRPV2 antibody of the present invention or a fragment thereof, or a method for treating or preventing muscle disease and/or cardiac disease using the antibody Wait.

1. Antibodies of the present invention The gene of human TRPV2 is reported with GenBank Accession No. NM_016113. The amino acid sequence of human TRPV2 is shown in SEQ ID NO: 1 of the Sequence Listing (GenBank Accession No. NM_016113). Like other proteins of the Trp family, TRPV2 has, for example, a six-pass transmembrane region as a structural feature.

The anti-human TRPV2 antibody or fragment thereof of the present invention recognizes a region comprising at least 5 or more amino acid sequences selected from the amino acid sequence of the extracellular domain in SEQ ID NO: 1 as an epitope. The extracellular domain of TRPV2 is the portion that exists substantially on the outside of the cell membrane of TRPV2 that is present on the cell membrane. The amino acid sequence of the extracellular domain of human TRPV2 is 409-434, 496-500, and/or 554-619 in SEQ ID NO: 1, and may also be 1 to several (2, 3) of these amino acid sequences. , 4, 5, etc.), preferably within 2, more preferably 1 amino acid sequence by substitution, deletion, addition, insertion or modification.

Preferably, the anti-human TRPV2 antibody or fragment thereof of the present invention recognizes that the extracellular domain of TRPV2 comprises at least 5 or more amino acid sequences selected from the amino acid sequence between the 5th transmembrane region and the 6th transmembrane region. regions as epitopes. The 5th transmembrane region and the 6th transmembrane region mean the regions at the 5th and 6th positions from the N-terminal side in the region penetrating the plasma membrane, and the anti-human TRPV2 antibody or fragment thereof of the present invention recognizes a region selected from these regions The sandwiched region in the amino acid sequence with higher hydrophilicity serves as an epitope. In the extracellular domain of human TRPV2, the amino acid sequence between the 5th transmembrane region and the 6th transmembrane region is specifically 554-619 of SEQ ID NO: 1, and can also be 1 in these amino acid sequences. ~several (2, 3, 4, 5, etc.), preferably within 2, more preferably 1 amino acid sequence in which the amino acid is substituted, deleted, added, inserted or modified.

More preferably, the anti-human TRPV2 antibody or its fragment of the present invention recognizes the amino acid sequence (SVQPMEGQEDEGNGAQYRGILE: SEQ ID NO: 2) comprising the amino acid sequence selected from positions 574 to 595 of human TRPV2 (SEQ ID NO: 1) and 1 to 1 in the amino acid sequence. Several (2, 3, 4, 5, etc.), preferably within 2, more preferably at least 5 or more amino groups in the amino acid sequence in which one amino acid is substituted, deleted, added, inserted or modified Regions of acid sequences serve as epitopes.

Further preferably, the anti-human TRPV2 antibody or fragment thereof of the present invention recognizes a region comprising the amino acid sequence of the amino acid sequence of human TRPV2 at positions 579 to 583 (EGQED) (SEQ ID NO: 3) as an epitope.

As another aspect of the anti-human TRPV2 antibody or its fragment of the present invention, the extracellular domain that recognizes TRPV2 includes at least five amino acid sequences selected from the group consisting of the first transmembrane region and the second transmembrane region. Regions of the above amino acid sequences are used as epitopes. The first transmembrane region and the second transmembrane region mean the regions at positions 1 and 2 from the N-terminal side in the region penetrating the plasma membrane, and the anti-human TRPV2 antibody or fragment thereof of the present invention recognizes a region selected from these regions. The region in the amino acid sequence with higher hydrophilicity of the clip acts as an epitope. In the extracellular domain of human TRPV2, the amino acid sequence between the first transmembrane region and the second transmembrane region is specifically 412-434 of SEQ ID NO: 1, and can also be 1 in these amino acid sequences. ~several (2, 3, 4, 5, etc.), preferably within 2, more preferably 1 amino acid sequence in which the amino acid is substituted, deleted, added, inserted or modified.

The anti-human TRPV2 antibody or fragment thereof of the present invention has the function of specifically inhibiting the activity of TRPV2. TRPV2 is a stretch-activated Ca ²⁺ channel, which exists in the endocellular membrane system in normal tissues, and is transferred to the cell membrane with diseases such as muscular dystrophy and cardiomyopathy, and is activated to facilitate the abnormal influx of Ca ²⁺ into the cell. The activity of TRPV2 means the influx of Ca ²⁺ into cells. Since the anti-human TRPV2 antibody of the present invention or its fragment recognizes the extracellular domain as an epitope, it is considered that it can recognize TRPV2 existing on the cell membrane of living cells and inhibit the activity of TRPV2. In addition, the anti-human TRPV2 antibody or its fragment of the present invention does not substantially affect the activity of other Trp family proteins, and has the function of specifically inhibiting the activity of TRPV2.

The monoclonal antibody or fragment of the present invention includes a monoclonal antibody, a single-chain antibody, or a portion of the above-mentioned antibody having antigen-binding properties such as a Fab fragment or a fragment generated from a Fab expression gene library. The anti-human TRPV2 monoclonal antibody or fragment thereof of the present invention is derived from mammals including humans, such as human type, mouse type, rat type, hamster type, rabbit type, goat type, or horse type. In addition, the antibody or its fragment of the present invention is not limited to IgG, but may be IgM or the like.

In this specification, an epitope is a region of an antigen to which an antibody binds. In one embodiment, any portion of an antigen that can specifically bind to an immunoglobulin, T cell receptor, or B cell receptor is included. Epitopes include chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphonium or sulfonate groups, and in certain embodiments, may have specific three-dimensional structural characteristics and/or specificity charge characteristics. In certain embodiments, an antibody, in a complex mixture of proteins and/or macromolecules, is capable of specifically binding to an antigen where the antibody preferentially recognizes the target antigen.

The basic structure of antibody molecules is common to all types, including a heavy chain with a molecular weight of 50,000 to 70,000 and a light chain with a molecular weight of 20,000 to 30,000 (Immunological Diagrams (I. Roitt, J. Brostoff, D. Male)). The heavy chain usually contains a polypeptide chain containing about 440 amino acids, and each type has a characteristic structure, corresponding to IgG, IgM, IgA, IgD, and IgE, which are called γ, μ, α, δ, and ε chains. Furthermore, IgG1, IgG2, IgG3, and IgG4 exist in IgG, and they are referred to as y1, y2, y3, and y4, respectively. The light chain usually includes a polypeptide chain containing about 220 amino acids, and two types of L-type and K-type are known, which are called λ and κ chains respectively. The basic structure of the antibody molecule consists of two heavy chains and two light chains that are homologous to each other through disulfide bonds (S-S bonds) and non-covalent bonds, with molecular weights ranging from 150,000 to 190,000. Two light chains can be paired with either heavy chain. Each antibody molecule is always formed from the same 2 light chains and the same 2 heavy chains.

The intrachain s-s bond forms 4 loops on the heavy chain (5 on the μ and ε chains), and forms 2 loops on the light chain, forming 1 loop for every 100-110 residues relative to the amino acid, This steric structure is similar between rings and is called a building block or region. The region where the heavy chain and the light chain are both located at the N-terminus is called the variable region (V region) (heavy chain variable region) even if it is a sample of the same type (subtype) from the same animal, its amino acid sequence is not necessarily the same. regions are denoted VH and light chain variable regions are denoted VL). In contrast, the amino acid sequence on the C-terminal side is approximately constant in each type or subtype, and is called a constant region (C region) (each region is represented by CH1, CH2, CH3 or CL, respectively).

The epitope of an antibody includes VH and VL, and the specificity of binding depends on the amino acid sequence of the site. On the other hand, biological activities such as binding to complement or various cells reflect differences in the structure of the C region of each type of Ig. It can be seen that the variability of the variable regions of the light chain and the heavy chain is roughly limited to three smaller hypervariable regions present in either chain, and these regions are called complementarity determinig regions (CDRs).

The method for producing the anti-human TRPV2 monoclonal antibody or its fragment of the present invention can be a method known per se, or any method developed in the future. In addition, various efforts are required to produce human-type antibodies against a specific antigen. For example, refer to Sato, K. et al, Cancer Res., 53, 851-856, 1993 or Japanese Patent Laid-Open No. 2008-161198. The type of the human type antibody is not particularly limited, and it may be a Fab type or an intact type. In order to effectively exert the activity of the antibody, an intact antibody is preferable. The intact antibody is not particularly limited. For example, it can be a chimeric antibody in which the complementary chain determining region (CDR) of the antibody is derived from the original animal species, and the constant region (C region) is derived from an appropriate human. Since amino acid substitutions occur frequently in the CDR regions, from an antigenicity point of view, human antibodies having CDRs from humans and CDRs from immunized animal species can be included.

The CDRs of the variable regions of the light chain and heavy chain in the anti-human TRPV2 monoclonal antibodies of the present invention or fragments thereof show boundaries according to the Kabat, Chothia, AbM, or IMGT algorithms (Martinet al. (1989) Proc. Natl Acad. Sci. USA 86: 9268-9272, Martin et al. (1991) Methods Enzymol. 203: 121-153, Pedersen et al. (1992) Immunomethods 1: 126, and Rees et al. (1996) In Sternberg M.J.E. (ed.), Protein Structure Prediction, Oxford University Press, Oxford, pp. 141-172, Lefranc 1999 Nucleic Acid Research 27:209-212).

As an aspect of the present invention, as the CDRs of the variable regions of the light chain and heavy chain in the anti-human TRPV2 monoclonal antibody or its fragment, the following can be exemplified: CDR1 in the variable region of the heavy chain includes SEQ ID NO: 4 (GFSLTSFG) represents or consists of the amino acid sequence, CDR2 contains or consists of the amino acid sequence represented by SEQ ID NO: 5 (IWSGGIT), CDR3 contains the amino acid sequence represented by SEQ ID NO: 6 (LYSHPHAMDY) or consisting of, and CDR1 in the light chain variable region comprises or consists of the amino acid sequence represented by SEQ ID NO: 7 (KSLLHSNGITY), CDR2 comprises or consists of the amino acid sequence represented by RMS, and CDR3 comprises The amino acid sequence represented by SEQ ID NO: 8 (MQHLEYPLT) or composed thereof. In the CDRs of the variable regions of the light chain and heavy chain in the anti-human TRPV2 monoclonal antibody or its fragment of the present invention, in these base sequences, 1 to several (2, 3, 4, 5, etc.) ), preferably within two, more preferably one amino acid can be substituted. In addition, as the variable region of the light chain and the heavy chain, the heavy chain variable region is an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 14, and the light chain variable region is the same as the amino acid sequence of SEQ ID NO: 14. The amino acid sequence represented by No. 15 is at least 95% identical to the amino acid sequence, preferably the heavy chain variable region is an amino acid that is at least 97% identical to the amino acid sequence represented by SEQ ID NO: 14 The light chain variable region is an amino acid sequence that is at least 97% identical to the amino acid sequence represented by SEQ ID NO: 15, and more preferably, the heavy chain variable region is an amine represented by SEQ ID NO: 14 The amino acid sequence of which the amino acid sequence is at least 99% identical, and the light chain variable region is the amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 15. In addition, as another embodiment of the variable region of the light chain and the heavy chain, the variable region of the heavy chain is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 22, 24, 25, 26 or 27. The amino acid sequence of the light chain variable region is an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 23. Preferably, the heavy chain variable region is the same as that of SEQ ID NO: 22 and 24. , the amino acid sequence represented by 25, 26 or 27 is at least 97% identical to the amino acid sequence and the light chain variable region is at least 97% identical to the amino acid sequence represented by SEQ ID NO: 23. , and preferably the heavy chain variable region is an amino acid sequence that is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 22, 24, 25, 26 or 27, and the light chain variable region is the same as the sequence number 22, 24, 25, 26 or 27. The amino acid sequence represented by No. 23 is at least 99% identical to the amino acid sequence. In addition, as another embodiment of the variable region of the light chain and the heavy chain, the variable region of the heavy chain can be an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 25, 26 or 27. And the light chain variable region is an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 23, preferably the heavy chain variable region is represented by SEQ ID NO: 25, 26 or 27. The amino acid sequence is at least 97% identical to the amino acid sequence and the light chain variable region is the amino acid sequence that is at least 97% identical to the amino acid sequence represented by SEQ ID NO: 23, and is preferably a heavyweight The chain variable region is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 25, 26 or 27 and the light chain variable region is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 23 the same amino acid sequence.

As another aspect of the present invention, as the CDRs of the variable regions of the light chain and heavy chain in the anti-human TRPV2 monoclonal antibody or its fragments, the following can be exemplified: CDR1 in the variable region of the heavy chain includes SEQ ID NO: 9 (GFSLTTYG) represents or consists of amino acid sequence, CDR2 contains or consists of amino acid sequence represented by SEQ ID NO: 10 (MGWDGKK), CDR3 contains amino acid sequence represented by SEQ ID NO: 11 (DGGYTWFAY) or consisting of, and CDR1 in the light chain variable region comprises or consists of the amino acid sequence represented by SEQ ID NO: 12 (QSLLNSRTRKNY), CDR2 comprises or consists of the amino acid sequence represented by WAS, and CDR3 comprises The amino acid sequence represented by SEQ ID NO: 13 (KQSYNLFT) or constituted thereof. In the CDRs of the variable regions of the light chain and heavy chain in the anti-human TRPV2 monoclonal antibody or its fragment of the present invention, in these base sequences, 1 to several (2, 3, 4, 5, etc.) ), preferably within two, more preferably one amino acid can be substituted. In addition, as the variable region of the light chain and the heavy chain, an anti-human TRPV2 monoclonal antibody or a fragment thereof that recognizes the extracellular domain of human TRPV2 as an epitope can be exemplified, and the heavy chain variable region in the extracellular domain of the human TRPV2 is the same as that of the human TRPV2. The amino acid sequence represented by SEQ ID NO: 16 is at least 95% identical to the amino acid sequence, and the light chain variable region is the amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 17, preferably For example, the heavy chain variable region is at least 97% identical to the amino acid sequence represented by SEQ ID NO: 16 and the light chain variable region is at least 97% identical to the amino acid sequence represented by SEQ ID NO: 17. The amino acid sequence of SEQ ID NO: 16 is more preferably the amino acid sequence of the heavy chain variable region is at least 99% identical to the amino acid sequence represented by SEQ ID NO: 16, and the light chain variable region is the amino acid sequence of SEQ ID NO: 17. The amino acid sequences represented are at least 99% identical to amino acid sequences.

The antigen used to produce the anti-human TRPV2 antibody or its fragment of the present invention can be the full-length protein or peptide fragment of human TRPV2, or the cell expressing the full-length protein or peptide fragment of human TRPV2, which is not particularly limited. When producing an anti-human TRPV2 monoclonal antibody or a fragment thereof, it is preferable to use cells expressing human TRPV2 (cells for expressing human TRPV2). The above-mentioned cells expressing human TRPV2 can be any as long as they express human TRPV2 on the cell membrane, but the cells that express human TRPV2 in mammalian cells (for example, cells collected from humans, monkeys, rats, mice, hamsters, etc.) medium performers. Examples of the mammalian cells include HEK293, COS7, CHO-K1, NIH3T3, Balb3T3, FM3A, L929, SP2/0, P3U1, B16, P388, and the like. Also, TRPV2 expressed in mammalian cells is preferably a full-length protein. In the present invention, it is preferable to use HEK (Human Embryonic Kidney, human embryonic kidney) cells for expression of human TRPV2 in which the full-length protein of human TRPV2 is expressed in HEK293 cells. Compounds (cannabidiol, etc.) stimulation, etc.). The antibody of the present invention can be obtained by immunizing a mammal with HEK cells expressing human TRPV2 as an antigen, preferably together with an adjuvant, and collecting serum or the like of the immunized animal. In addition, monoclonal antibodies and fusion tumors producing the monoclonal antibodies can be produced by fusing B lymphocytes derived from immunized humans or animals with various myeloma cells, specifically, by the method described below.

In the production of the monoclonal antibody of the present invention, an antigen solution obtained by dissolving or suspending the above-mentioned antigen in a suitable buffer such as phosphate buffered saline (PBS) can be used. The antigen solution is usually prepared at a concentration of about 50 to 500 μg/ml of antigen protein or about 1×10 ⁶ to 1×10 ⁹ cells/ml of antigen-expressing cells. In addition, when the peptide antigen is equal to the low antigenicity at this concentration, it can be used by bridging to an appropriate carrier protein such as albumin or keyhole cyanogen (KLH). Examples of animals immunized and sensitized with this antigen include warm-blooded animals such as humans, mice, rats, hamsters, horses, goats, and rabbits.

In this case, in order to increase the responsiveness of the immunized animal to the antigen, the antigen solution may be mixed with an adjuvant and administered. Examples of adjuvants that can be used here include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), Ribi (MPL (monophosphoryl lipid, monophosphoryl lipid)), Ribi (TDM (trehalose dicorynomycolate, two) Mycolic acid trehalose), Ribi (MPL+TDM), pertussis vaccine (Boredetella pertussis vaccine), muramid dipeptide (MDP), aluminum adjuvant (ALUM), and combinations of these, the following combinations can be used: FCA was used for booster immunization, and FIA or Ribi adjuvant was used for booster immunization.

The immunization method can be appropriately changed according to the type of antigen used, the presence or absence of adjuvant mixture, etc., the injection site, schedule, etc. nu mice), 0.05-1 mL of adjuvant mixed antigen solution (10-200 μg of antigenic protein, or 1×10 ⁶ to 1×10 ⁷ cells of antigen-expressing cells) was injected into the abdominal cavity, subcutaneously, intramuscularly or ( tail) intravenously, 1 to 4 booster immunizations were performed at intervals of about 4 to 21 days from the primary immunization, and the final immunization was performed about 1 to 4 weeks later. The antigen solution can also be administered by increasing the amount of antigen by intraperitoneal injection without the use of an adjuvant. Antibody titers were collected about 5 to 10 days after booster immunization for investigation. The measurement of the antibody titer can be performed according to the antibody titer analysis described later by a commonly performed method. About 3 to 5 days after the final immunization, spleen cells were isolated from the immunized animal to obtain antibody-producing cells.

The anti-human TRPV2 monoclonal antibody of the present invention can be prepared according to, for example, the method of Kohler and Milstein (Kohler and Milstein, Nature 256, 495-497, 1975). As myeloma cells, those derived from mice, rats, humans, etc. can be used, for example, mouse myeloma P3X63-Ag8, P3X63-Ag8-U1, P3NS1-Ag4, SP2/o-Ag14, P3X63-Ag8・653 can be exemplified and other cultured myeloma cell lines. There are myeloma cells that produce immunoglobulin light chains. If they are used as fusion objects, the immunoglobulin heavy chains produced by antibody-producing cells may be randomly bound to the light chains. Myeloma cells of immunoglobulin light chain, such as P3X63-Ag8·653 or SP2/o-Ag14, etc. Antibody-producing cells and myeloma cells are preferably from the same animal, especially the same strain. The preservation method of myeloma cells may be carried out according to a method known per se, for example, in the case of subculture in a normal medium supplemented with fetal horse, fetal rabbit, or fetal bovine serum, preservation is performed by freezing. In addition, it is preferable to use cells in the logarithmic proliferative phase for cell fusion.

As a method for producing a fusion tumor by fusing an antibody-producing cell with a myeloma cell, a method using polyethylene glycol (PEG), a method using Sendai virus, a method using an electrofusion apparatus, and the like can be exemplified. For example, spleen cells and myeloma cells are suspended in a suitable medium or buffer containing about 30-60% PEG at a mixing ratio of 1-10:1, preferably 5-10:1, at a temperature of about 25 The reaction can be carried out for about 30 seconds to 3 minutes under the conditions of ~37°C and pH value of 6 to 8. After the reaction, the cells were washed to remove the PEG solution, resuspended in the medium, and seeded in a microtiter plate to continue the culture.

Cells after fusion operation are cultured in selection medium for selection of fusion tumors. The selective medium is a medium that kills the parent cell line and can only proliferate the fused cells, usually hypoxanthine-aminopterin-thymidine (HAT) medium. The selection of the fusion tumor is usually carried out by replacing a part of the medium, preferably about half the amount, with the selection medium, usually 1 to 7 days after the fusion operation, and then repeating the same medium exchange and culturing every 2 or 3 days. . The wells of the colony growth of the fusion tumor were confirmed by microscopic observation.

More specifically, the anti-human TRPV2 monoclonal antibody of the present invention can be produced by the following method. Human TRPV2 expression HEK cells are inoculated into immune disease mice, spleens are harvested 2 to 4 weeks later, and antibody-producing cells contained in the spleen are fused with myeloma cells, thereby producing a fusion tumor producing the antibody of the present invention.

In order to know whether the growing fusion tumor is producing the desired antibody, the culture supernatant is collected, and the antibody titer analysis can be performed by a method known per se. Specifically, human TRPV2-expressing HEK cells or proteins derived from human TRPV2-expressing HEK cells can be used as antigens and screened by IC (immunocytochemistry), IF (immunofluorescence), and IHC (immunohistochemistry) staining methods. Antibody-producing cells with binding activity.

For example, whether the obtained antibody recognizes the extracellular domain of human TRPV2 can be confirmed by the following method. The above-mentioned fusion tumor culture supernatant was reacted with a population of HEK cells expressing human TRPV2 and a population of HEK cells not expressing human TRPV2, and then reacted with a labeled secondary antibody, and measured by flow cytometry. Human TRPV2 expressing HEK cells that have not reacted with the antibody can be screened for those contained in a fluorescent region having a higher intensity than that of human TRPV2 expressing HEK cells and a population of HEK cells that do not express human TRPV2 as specifically binding to human TRPV2 cells. Antibodies to the outer domain.

In addition, whether the obtained antibody has the function of inhibiting the activity of human TRPV2 can be appropriately confirmed using the TRPV2-specific analysis method described in Japanese Patent Laid-Open No. 2007-259745 (Japanese Patent Application No. 2006-088323).

Furthermore, by referring to the method for measuring the activity of proteins of the Trp family other than TRPV2 described in Japanese Patent Laid-Open No. 2009-149534 (Japanese Patent Application No. 2007-326606 ), it can be appropriately confirmed whether the antibody of the present invention does not substantially inhibit TRPV2 The activity of proteins other than the Trp family is affected.

Furthermore, the pure lines producing the monoclonal antibody of the present invention are isolated by limiting dilution method, soft agar method, method using fluorescence excitation cell sorter, and the like. For example, in the case of the limiting dilution method, the colony of the fusion tumor is serially diluted and cultured with the medium so that it becomes about 1 cell/well, whereby the pure line of the fusion tumor producing the target antibody can be isolated. If the obtained antibody-producing fusion tumor pure line is frozen in the coexistence of about 10% (v/v) dimethyl sulfoxide (DMSO) or glycerol and other cryoprotectants and stored at -70 ~ -196 ℃, it can be stored About half a year to semi-permanent preservation. Cells were rapidly thawed in a thermostat at about 37°C when used. Ideally, it should be used after thorough washing to avoid residual cytotoxicity of cryoprotectant.

The method for obtaining the monoclonal antibody from the fusion tumor can be appropriately selected according to the desired amount, the properties of the fusion tumor, and the like. For example, a method of obtaining ascites from a mouse transplanted with the fusion tumor, a method of obtaining from a culture supernatant by cell culture, and the like can be exemplified. If it is a fusion tumor that can proliferate in the mouse abdominal cavity, a high concentration of several mg/mL monoclonal antibody can be obtained from the ascites fluid. Fusion tumors that do not proliferate in vivo are obtained from culture supernatants of cell cultures. The monoclonal antibody obtained by cell culture, although the amount of antibody produced is lower than that in vivo, it has the advantage of less contamination of immunoglobulins or other impurities contained in the abdominal cavity of mice and easy purification.

When monoclonal antibodies are obtained from the intraperitoneal cavity of mice transplanted with fusion tumors, for example, fusion tumors (about 10 ⁶ or more) are transplanted into pre-administered pristane (2,6,10,14-tetramethyl) Pentadecane) and other substances with immunosuppressive effects in the abdominal cavity of BALB/c mice, the retained ascites was collected after about 1 to 3 weeks. In the case of heterologous fusion tumors (eg, mice and rats), nude mice, radiation-treated mice are preferably used.

On the other hand, when the antibody is obtained from the cell culture supernatant, the fusion tumor is obtained by culturing the fusion tumor using a culture method such as a high-density culture method or a spinner flask culture method in addition to the stationary culture method used for cell maintenance. Antibody-containing culture supernatant. The serum contained in the culture medium contains impurities such as other antibodies and albumin, and in many cases, antibody purification becomes complicated, so it is desirable to reduce the addition to the culture medium. Furthermore, it is preferable to adapt the fusion tumor to a serum-free medium by a conventional method, and to use a serum-free medium for culturing. Antibody purification is facilitated by culturing in serum-free medium.

Purification of monoclonal antibodies from ascites fluid or culture supernatant can be performed by a method known per se. For example, as a purification method of immunoglobulin, by applying the previously known differentiation method using salting out using ammonium sulfate or sodium sulfate, polyethylene glycol (PEG) differentiation method, ethanol differentiation method, DEAE (diethylaminoethyl, diethyl ether) Aminoethyl) ion exchange chromatography, gel filtration, etc. can be easily achieved. Furthermore, when the monoclonal antibody is IgG, it can be purified by affinity chromatography using a protein A-binding carrier, which is convenient.

As another production method, for example, a so-called combinatorial antibody library in which antibody fragments are displayed on the surface of E. coli phage can be constructed, and desired antibodies can be obtained by screening antibodies by biopanning. In this case, the desired antibodies can be screened without immunizing the animals. The human TRPV2 antibody of the present invention is preferably obtained by this production method.

As a screening method for monoclonal antibodies, for example, a display method can be exemplified. As a method for preparing a phage-displayed antibody library, for example, the following ones are mentioned, but not limited thereto.

The phage to be used is not particularly limited, and generally, fibrous phage (Ff phage (bacteriophage)) can be preferably used. As a method of presenting a foreign protein on the surface of a phage, a method of presenting/presenting a foreign protein in the form of a fusion protein with any of the coat proteins of g3p, g6p to g9p can be exemplified, and it is usually used for g3p or g8p The method of fusion of the N-terminal side. As a phage display vector, except 1) the coat protein gene of the phage genome is introduced in the form of fusion of the foreign gene, so that all the coat proteins displayed on the surface of the phage are presented in the form of fusion proteins with the foreign protein, for example, For example: 2) the gene encoding the fusion protein and the wild-type coat protein gene are inserted separately so that the fusion protein and the wild-type coat protein are expressed simultaneously; or 3) the Escherichia coli containing the phagemid vector with the gene encoding the fusion protein is inserted Infect a helper phage with a wild-type coat protein gene to produce phage particles that express both the fusion protein and the wild-type coat protein, etc. However, in the case of 1), if a large exogenous protein is fused, the ability to infect will be lost, Therefore, in order to prepare an antibody library, the type 2) or 3) is used.

As a specific carrier, those described in Holt et al. (Curr. Opin. Biotechnol., 11: 445-449, 2000) can be exemplified. For example, pCES1 (J. Biol. Chem., 274: 18218-18230, 1999 ref.) is a Fab phenotype phagemid vector, which is under the control of a lactose promoter and is configured with a code encoding g3p downstream of the message peptide The DNA (Deoxyribonucleic Acid, deoxyribonucleic acid) of the constant region of the kappa light chain is configured with the DNA encoding CH3 downstream of the g3p message peptide, and g3p is configured with the His-tag, c-myc tag, and amber stop codon (TAG) coding sequence. When introduced into Escherichia coli with the amber mutation, Fab is displayed on the g3p coat protein, but when it is expressed in the HB2151 strain without the amber mutation, a soluble Fab antibody is produced. Further, as a phagemid vector for the scFv expression type, for example, pHEN1 (J. Mol. Biol., 222:581-597, 1991) and the like can be used. On the other hand, as a helper phage, M13-KO7, VCSM13, etc. can be used, for example.

In addition, as other phage display vectors, a designer (CysDisplayTM technology of Morphosys company), etc. can also be exemplified in the following manner: the 3' end of the antibody gene and the 5' end of the coat protein gene are respectively linked with a cysteine-encoding nucleic acid. The sequence of codons allows the two genes to be expressed simultaneously (not in the form of fusion proteins), and the antibody can be presented on the coat protein on the surface of the phage through the S-S bonds of the introduced cysteine residues to each other.

As the type of antibody library, naive/non-immune gene library, synthetic gene library, immune gene library and the like can be exemplified.

The original/non-immunized gene pool was obtained by RT-PCR (Reverse transcription polymerase chain reaction, reverse transcription polymerase chain reaction) to obtain the normal human retained VH and VL genes, and these were randomly selected to the gene pool obtained in the above-mentioned phage display vector. Usually, mRNA (messenger Ribonucleic acid, messenger ribonucleic acid) of lymphocytes derived from peripheral blood, bone marrow, tonsils, etc. of normal people is used as a template. In order to eliminate the bias of the V gene of the disease course, etc., the amplification of only mRNA from IgM that has not undergone class conversion by antigen sensitization is specifically referred to as the initial gene pool. Representative examples include the gene bank of CAT company (refer to J. Mol. Biol., 222: 581-597, 1991, Nat. Biotechnol., 14: 309-314, 1996), the gene bank of MRC company Library (refer to Annu. Rev. Immunol., 12: 433-455, 1994), Gene Bank of Dyax Corporation (refer to J. Biol. Chem., 1999 (above), Proc. Natl. Acad. Sci. USA, 14: 7969-7974, 2000) and so on.

The synthetic gene library is to select functional specific antibody genes in human B cells, replace the part of the antigen-binding region of the V gene fragment such as CDR3 with DNA encoding a random amino acid sequence of appropriate length, and perform gene libraryization become. Since a gene bank can be constructed from the beginning using a combination of VH and VL genes that produce a functional scFv or Fab, it is considered that the expression efficiency or stability of the antibody is excellent. As a representative one, there can be exemplified: the HuCAL gene bank of Morphosys company (refer to J.Mol. Biol., 296: 57-86, 2000), the gene bank of BioInvent company (refer to Nat. Biotechnol., 18: 852, 2000), the gene bank of Crucell company (refer to Proc. Natl. Acad. Sci. USA, 92: 3938, 1995, J. Immunol. Methods, 272: 219-233, 2003) and the like.

The immunized gene pool is to repeatedly immunize experimental animals such as mice with a specific antigen, and after the antibody response is sufficiently improved, the lymphocytes or auto-cancer, auto-immune gene pools of the animal are treated in the same manner as the above-mentioned initial/non-immunized gene pool. mRNA is prepared from human lymphocytes collected from patients with immune diseases, infectious diseases, etc., or those who have been vaccinated, whose antibody titers have increased in blood against the target antigen, or human lymphocytes that have been artificially immunized with the target antigen by the above-mentioned in vitro immunization method. , Amplify VH and VL genes by RT-PCR method and conduct gene pooling. Since the target antibody gene is initially included in the gene bank, the target antibody can also be obtained from a relatively small-sized gene bank.

The step of screening for antibodies against a target antigen by phage display is called panning. Specifically, for example, the antigen-immobilized carrier is brought into contact with the phage library, the unbound phage is washed and removed, the bound phage is eluted from the carrier, and E. coli is infected to multiply the phage, and the above series of operations are repeated for 3 to About 5 times, the phage showing the antigen-specific antibody is concentrated. As the carrier for immobilizing the antigen, various carriers used for normal antigen-antibody reaction or affinity chromatography can be exemplified, for example, insoluble polysaccharides such as agarose, dextran, and cellulose; Synthetic resins such as amines and silicon; or microdiscs, tubes, films, columns, beads, etc. containing glass, metal, etc., and sensor chips of surface plasmon resonance (SPR) can be exemplified. The immobilization of the antigen may be carried out by physical adsorption, or by a chemical binding method used for insolubilization and immobilization of proteins, enzymes, and the like. For example, a biotin-(streptavidin) system and the like can be preferably used. The non-binding phage can be washed with blocking solution such as BSA (Bovine Serum Albumin, bovine serum albumin) solution (1-2 times), PBS containing surfactant such as Tween (3-5 times), etc. It is also reported that it is better to use a citric acid buffer (pH value of 5) and the like. Acids (eg, 0.1 M hydrochloric acid, etc.) are usually used for elution of specific phages, or cleavage by a specific protease (eg, a gene sequence encoding a trypsin cleavage site can be introduced into the junction of the antibody gene and the coat protein gene. In this case, since the wild-type coat protein is present on the surface of the eluted phage, even if all coat proteins are expressed in the form of fusion proteins, it can infect E. coli and proliferate) or use the competitive dissolution of soluble antigens, or use S-S bonds. Reduction (eg, in CysDisplayTM described above, after panning, the antibody is dissociated from the coat protein using a suitable reducing agent, whereby the antigen-specific phage can be recovered). In the case of elution with an acid, after neutralization with tris(hydroxymethyl)aminomethane or the like, the elutriated phage is infected with Escherichia coli, and after culturing, the phage is recovered by a conventional method.

After the phage presenting antigen-specific antibodies were concentrated by panning, the phage was infected with E. coli, and the E. coli was inoculated on the plate for cell colonization. The phage is recovered again, and the antigen-binding activity is confirmed by the above-mentioned antibody titer assay (eg, ELISA, RIA (radioimmunoassay, radioimmunoassay), etc.) or by FACS or SPR.

The antibody is isolated and purified from the selected phage presenting antigen-specific antibodies. In the case of using, for example, a vector into which an amber stop codon is introduced at the junction of the antibody gene and the coat protein gene as the phage presentation vector, if the phage is infected Escherichia coli without amber mutation (such as HB2151 strain) produces soluble antibody molecules and secretes them into the periplasm or medium, so the extracellular components can be recovered by dissolving the cell wall with lysozyme, and the same purification technology as above can be used conduct. If a His-tag or c-myc tag is introduced in advance, it can be easily purified using Immobilized Metal Affinity Chromatography (IMAC, immobilized metal ion affinity chromatography) method, an anti-c-myc antibody column, or the like. Furthermore, when cleavage by a specific protease is used in panning, if the protease is activated, the antibody molecule is separated from the surface of the phage, so that the target antibody can be purified by performing the same purification operation.

The anti-human TRPV2 monoclonal antibodies or fragments thereof of the present invention include antibodies obtained from mammals (preferably mouse antibodies), chimeric antibodies, and humanized antibodies. The monoclonal antibodies or fragments of the present invention can be any of mouse antibodies, chimeric antibodies, or humanized antibodies.

In this specification, "chimeric antibody" means an antibody in which the sequences of the variable regions (VH and VL) of the heavy and light chains are derived from non-human animal species and the sequences of the constant regions (CH and CL) are derived from humans. The sequence of the variable region is preferably derived from an animal species from which fusion tumors can be easily produced, such as mouse, rat, and rabbit, and the sequence of the constant region is preferably derived from an animal species to be administered.

As a method for producing a chimeric antibody, for example, the method described in the specification of US Pat. No. 6,331,415, or a method in which a part thereof is modified, etc., can be exemplified.

The obtained chimeric heavy chain and chimeric light chain expression vectors are used to transform host cells. As the host cell, animal cells can be exemplified, for example, Chinese hamster ovary (CHO) cells, monkey-derived COS-7 cells, Vero cells, rat-derived GHS cells, etc., in addition to the above-mentioned mouse myeloma cells. For the transformation, any method that can be applied to animal cells can be used, and preferably, electroporation and the like can be used. After culturing for a certain period of time in a medium suitable for the host cells, the culture supernatant is recovered and purified by the same method as above, whereby the chimeric monoclonal antibody can be isolated. Alternatively, it is also possible to use the germline cells of animals that have established gene transfer technology, such as cattle, goats, chickens, etc., and have accumulated technical know-how for the mass reproduction of livestock (poultry) as host cells, and use conventional methods to produce gene transfer animals. The chimeric monoclonal antibodies are easily and abundantly obtained from the milk or eggs of the obtained animals. Furthermore, corn, rice, wheat, soybean, tobacco and other plant cells that have established gene transfer technology and are cultivated in large quantities as main crops can also be used as host cells. Transgenic plants are prepared by particle gun method or Ti vector method, and a large amount of chimeric monoclonal antibody can be obtained from the obtained seeds or leaves.

In this specification, a "humanized antibody" means that the sequences present in all regions other than the complementarity determining regions (CDRs) of the variable regions (ie, the framework regions (FRs) in the constant and variable regions) are derived from humans And only the sequences of the CDRs are derived from antibodies of other mammalian species. As other mammalian species, for example, animal species that can easily produce fusion tumors, such as mice, rats, and rabbits, are preferred.

Examples of methods for producing humanized antibodies include those described in US Pat. Nos. 5,225,539, 5,585,089, 5,693,761, 5,693,762, European Patent Application Publication No. 239400, and International Publication No. 92/19759. method or a method by changing a part of them, etc. Specifically, in the same manner as in the case of the chimeric antibody described above, after isolation of DNA encoding VH and VL from mammalian species other than human (eg, mouse), automated DNA sequencing is used by conventional methods sequenced using a known antibody sequence database [for example, Kabat database (refer to Kabat et al., "Sequences of Proteins of Immunological Interest", US Department of Health and Human Services, Public Health Service, NIH ed., 5th edition, 1991) etc.] analyzed the obtained base sequence or the amino acid sequence deduced from it, and determined the CDRs and FRs of the two strands. A base sequence was designed to replace the CDR coding regions of the base sequences encoding the light and heavy chains of human antibodies having FR sequences similar to the determined FR sequences with the base sequences encoding the determined heterologous CDRs. The base sequence is divided into fragments of about 20 to 40 bases, and the sequence complementary to the base sequence is further divided into fragments of about 20 to 40 bases so as to alternately overlap with the above-mentioned fragments. Fragments are synthesized using a DNA synthesizer, hybridized and ligated according to conventional methods, whereby DNA encoding VH and VL having FRs from humans and CDRs from other mammalian species can be constructed. In order to more rapidly and efficiently transplant CDRs from other mammalian species into VH and VL from humans, site-directed mutagenesis using PCR is preferably used. As such a method, the successive CDR transplantation method etc. which are described in Unexamined-Japanese-Patent No. 5-227970 are mentioned, for example.

Furthermore, in the production of the humanized antibody by the above-mentioned method, the antigen-binding activity of the human antibody FR in which only the amino acid sequences of the CDRs are grafted to the template may be lower than that of the original non-human antibody. . In this case, it is effective to graft together several FR amino acids surrounding the CDR. Examples of the FR amino acids of the grafted non-human antibody include amino acid residues that are important for maintaining the steric structure of each CDR, and such amino acid residues can be estimated by steric structure prediction using a computer. .

Humanized antibody-producing cells or transgenic animals and plants can be obtained by ligating the DNAs encoding VH and VL obtained in the above-described manner with the DNAs encoding CH and CL from humans, respectively, and introducing them into appropriate host cells.

As an alternative method for producing humanized antibodies without using CDR grafting in which mouse CDRs are grafted into the variable regions of human antibodies, for example, methods based on the preserved structure-function correlation between antibodies can be exemplified. , to determine which amino acid residues within the non-human variable region are candidates for replacement. This method can be implemented according to, for example, European Patent No. 0571613, US Patent No. 5,766,886, US Patent No. 5,770,196, US Patent No. 5,821,123, US Patent No. 5,869,619, and the like. In addition, if the amino acid sequence information of VH and VL of the base non-human antibody can be obtained for the production of humanized antibodies using this method, it can be easily performed by, for example, using the contracted antibody production service provided by Xoma Corporation. conduct.

Humanized antibodies can also be modified into scFv, scFv-Fc, minibody, dsFv, Fv, etc., using genetic engineering methods in the same way as chimeric antibodies. By using appropriate promoters, Escherichia coli or yeast can be used. and other microorganisms can also be produced. In addition, humanized antibodies can also be obtained by preparing an antibody library used in the above-mentioned phage display method from animals such as mice and rats, and humanizing the obtained antibodies, but the invention is not limited thereto.

The humanized antibody obtained in the above-described manner can bind to the epitope region of wild-type human TRPV2 can be confirmed by the above-described binding assay using a polypeptide comprising the peptide region.

As an aspect of the anti-human TRPV2 antibody or its fragment of the present invention, it preferably has the function of inhibiting the degeneration of cardiomyocytes and muscle cells. The degeneration of cardiomyocytes and muscle cells can be seen in various diseases, and the industry believes that the disease can be improved by inhibiting and alleviating the degeneration. The function of inhibiting the degeneration of cardiomyocytes and muscle cells can be achieved by applying stretch stimulation to cardiomyocytes and muscle cells cultured on silicon membranes for a predetermined period of time in order to induce degeneration, and measuring the creatine kinase activity of the culture supernatant thereafter. Undergo verification.

Confirmation of antibody binding can be carried out by any well-known analytical method, for example, by direct and indirect sandwich analysis, flow cytometry and immunoprecipitation analysis, etc. (Zola, Monoclonal Antibodies: A Manual of Techniques, (CRC Press, Inc. 1987) pp. 147-158). In the present invention, the binding of the anti-human TRPV2 antibody or its fragment to the antigen can be determined by the methods exemplified above.

The determination of the binding constant (Ka) of the anti-human TRPV2 antibody of the present invention or its fragment, or the identification of other antibodies of the present invention that competitively bind to human TRPV2 with the obtained antibody of the present invention, can use competition assays such as competition ELISA . In the above-described binding assay using an antigen-immobilized solid phase, the competition assay is performed by allowing free antigen or known antibody to coexist in the reaction system of the solid phase and the test antibody. For example, a test antibody solution of known concentration and a mixture of adding antigens of various concentrations to the test antibody solution are brought into contact with the antigen-immobilized solid phase for incubation, and the amount of label on the solid phase is measured. Scatchard analysis can be performed based on the measured values at each free antigen concentration, and the slope of the graph can be calculated as the binding constant. On the other hand, for the antigen-immobilized solid phase, the labeled known antibody (the antibody of the present invention) is reacted with the test antibody of various concentrations, and the test antibody that reduces the labeled amount on the solid phase in a concentration-dependent manner is selected. Antibodies, whereby antibodies that bind to human TRPV2 competitively with the antibodies of the present invention can be identified.

Epitope parsing can be performed by various well-known methods (Epitope Mapping Protocols/Second Edition, Mike Schutkowski, Ulrich Reineke, Ann NY AcadSci. 2010 Jan; 1183:267-87.). More detailed analysis of the epitope of the present antibody can be performed by binding inhibition analysis, homology scanning and/or alanine scanning (eg, Japanese Patent Laid-Open No. 2009-159948, Science, 244: 1081-1085 (1989)) .

Binding affinity can be determined by Berzofsky et al., "Antibody-Antigen Interactions" Fundamal Immunology, Paul, W.E., Ed., Raven Press New York, NY (1984), Kuby, Janis Immunology, W.H. Freeman and Company New York, NY (1992), and the method described in this manual was confirmed. Specifically, as a general method for measuring the affinity of an antibody to an antigen, ELISA, RIA, surface plasmon resonance method, and the like can be exemplified. In addition, the binding affinity of the present invention was determined by ELISA, SPR, immunohistochemical staining, and immunoblotting. Affinity measured by interaction with a specific antibody-antigen may vary, for example, when measured under different conditions such as salt concentration, pH value, and the like. Therefore, determination of parameters such as affinity and other antigen binding parameters such as KD, IC50, etc., are preferably performed in standardized solutions of antibody and antigen, as well as in standardized buffers.

2. Agents, reagents, kits, etc. of the present invention Furthermore, the present invention also relates to a human TRPV2 inhibitor or a cardiomyocyte and/or myocyte degeneration inhibitor comprising the anti-human TRPV2 antibody or fragment thereof of the present invention as an active ingredient. Human TRPV2 inhibitors or cardiomyocytes and/or myocyte degeneration inhibitors can also be used in the form of pharmaceutical compositions, and can also be used as experimental tools (for example: reagents, kits, etc.) to specifically inhibit human TRPV2, or to inhibit and relieve cardiac muscle. Experimental tools (eg, reagents, kits, etc.) for degeneration of cells and/or muscle cells are used in vivo or in vitro. In addition, the anti-human TRPV2 antibody or fragment thereof of the present invention can be used as a diagnostic (determination) drug, a diagnostic (determination) reagent, or a diagnostic (determination) kit for cardiomyocytes and/or myocyte degenerative diseases.

The anti-human TRPV2 antibody or fragment thereof of the present invention can also be used in a state of being contained in a composition as an active ingredient. In addition, when the composition comprising the anti-human TRPV2 antibody of the present invention or its fragment is used for medical purposes, one or more of the anti-human TRPV2 antibody of the present invention or its fragment can be included in an effective amount, and then A pharmaceutically acceptable carrier may be included. In the present invention, pharmaceutically acceptable salts include, for example, alkali metal salts such as sodium salts and potassium salts or basic addition salts such as alkaline earth metal salts; inorganic acid salts such as hydrochloride, sulfate and nitrate; Acid addition salts such as organic acid salts such as acetate and propionate, etc.

The composition can be administered orally or parenterally in the form of a pharmaceutical composition. In the case of oral administration, the anti-human TRPV2 antibody of the present invention can also be used in the form of usual preparations, for example, in the form of any of the following dosage forms: lozenges, powders, granules, capsules and other solids; water preparations; oily suspensions; or liquid preparations such as syrups or elixirs. In the case of parenteral administration, the anti-human TRPV2 antibody of the present invention can be used in the form of aqueous or oily suspension injections and nasal drops. In its preparation, conventional excipients, binders, lubricants, aqueous solvents, oily solvents, emulsifiers, suspending agents, preservatives, stabilizers and the like can be optionally used.

When used in the form of a pharmaceutical composition, it is preferably applied to diseases caused by activation of TRPV2, muscle diseases, and heart diseases. Diseases caused by activation of TRPV2 include allergies, immune system diseases, gastrointestinal disorders, cancer, etc. (Adv Exp Med Biol 2020 1131:505-517, Scientific Reports 2019 9 1554, J Clin Med 2019 8(5): 662, Eur J Neurosci 2016 44(7) 2493-2503, Cancer Res 2010 70(3) 1225-1235), but not necessarily limited thereto.

The anti-human TRPV2 antibody or fragment thereof of the present invention is also effective as a therapeutic or preventive agent for muscle disease and/or heart disease. Examples of muscle diseases include neuromuscular G70-G73 in ICD10 (International Statistical Classification of Diseases and Related Health Problems 10th Revision, hereinafter referred to as ICD10) issued by the World Health Organization (WHO) in 2016. Diseases of myoneural junction and muscle or Disorders of muscles of M60-M63. As a muscle disease, G71 in ICD10 (Primary disorders of muscles) or M62 (Other disorders of muscle) in ICD10 can be preferably exemplified. More preferred examples of muscle diseases include: G71.0 Muscular dystrophy, G71.1 Myotonic disorders, G71.2 Congenital myopathies, G71 .3 Mitochondrial myopathy (not elsewhere classified) (Mitochondrial myopathy, not elsewhere classified), G71.8 Other primary disorders of muscles, G71.9 Unspecified primary Primary disorder of muscle, unspecified, M62.0 Diastasis of muscle, M62.1 Other rupture of muscle (nontraumatic), M62.2 Ischaemic infarction of muscle, Immobility syndrome (paraplegic) of M62.3, Contracture of muscle of M62.4, Contracture of muscle of M62.5 Muscle wasting and atrophy, not elsewhere classified, M62.6 Muscle strain, or M62.8 Other specified disorders of muscle or M62.9 Disorder of muscle, unspecified. As a muscle disease, the muscle dystrophy of G71.0 in ICD10 can be mentioned more preferably. As the disease type of muscle dystrophy G71.0 in the above-mentioned ICD10, specifically, there can be exemplified: autosomal recessive/childhood type/Johnson type-like or Baker-like (autosomal recessive, childhood type, resembling) Duchenne or Becker), benign [Becker], benign scapuloperoneal with early contractures [Emery-Dreifuss], distal distal), facioscapulohumeral, limb-girdle, ocular, oculopharyngeal, scapuloperoneal, or severe [Duchenne]). Examples of heart diseases include I20-I25 in ICD10, ischemic heart diseases, and I30-I52, other forms of heart diseases. Preferred examples of heart diseases include: Angina pectoris (Angina pectoris) of I20 in ICD10, acute myocardial infarction (Acute myocardial infarction) of I21, recurrent myocardial infarction (Subsequent myocardial infarction) of I22, and acute myocardial infarction of I23. Secondary complications (Certain current complications following acute myocardial infarction), I24 other acute ischaemic heart diseases (Other acute ischaemic heart diseases), I25 chronic ischemic heart disease (Chronic ischemic heart disease), I30 acute Acute pericarditis, I31 Other diseases of pericardium, I32 Pericarditis in diseases classified elsewhere, I33 Acute and Subacute endocarditis (Acute and subacute endocarditis), I34 non-rheumatic mitral valve disorders (Nonrheumatic mitral valve disorders), I35 non-rheumatic aortic valve disorders (Nonrheumatic aortic valve disorders), I36 non-rheumatic Tricuspid valve disorders (Nonrheumatic tricuspid valve disorders), I37 Pulmonary valve disorders (Pulmonary valve disorders), I38 Endocarditis (valve unspecified) (Endocarditis, valve unspecified), I39 Heart disease classified as other Endocarditis and heart valve disorders in diseases classified elsewhere, Acute myocarditis in I40, Myocarditis in diseases classified elsewhere in I41, Myocardium in I42 Cardiomyopathy, I43 Cardiomyopathy in other diseases diseases classified elsewhere), I44 Atrioventricular and left bundle-branch block, I45 Other conduction disorders, I46 Cardiac arrest, I47 Paroxysmal tachycardia (Paroxysmal tachycardia), I48 Atrial fibrillation and atrial flutter, I49 Other cardiac arrhythmias, I50 Heart failure failure), I51 Complications and ill-defined descriptions of heart disease, or I52 Other heart disorders in diseases classified elsewhere). More preferred examples of heart diseases include: acute transmural myocardial infarction of anterior wall of I21.0 in ICD10, acute transmural myocardial infarction of inferior wall of I21.1 transmural myocardial infarction of inferior wall), acute transmural myocardial infarction of other sites in I21.2, acute transmural myocardial infarction (unknown location) in I21.3 (Acute transmural myocardial infarction of other sites) of unspecified site), I21.4 Acute subendocardial myocardial infarction, I21.9 Acute myocardial infarction (unspecified), I25.0 described as atherosclerosis Patients with sclerosing cardiovascular disease (Atherosclerotic cardiovascular disease, so described), atherosclerotic heart disease (I25.1), old myocardial infarction (Old myocardial infarction) in I25.2, and I25.3 Aneurysm of heart, Coronary artery aneurysm and dissection of I25.4, Ischaemic cardiomyopathy of I25.5, Painless < Asymptomatic of I25.6 > Myocardial ischemia (Silent myocardial ischaemia), other forms of chronic ischaemic heart disease (Other forms of chronic ischaemic heart disease) of I25.8, chronic ischaemic heart disease (details unknown) of I25.9 (chronic ischaemic heart disease) heart disease, unspecified), dilated cardiomyopathy of I42.0, obstructive hypertrophic cardiomyopathy of I42.1 phic cardiomyopathy), I42.2 Other hypertrophic cardiomyopathy, I42.3 Endomyocardial (eosinophilic) disease, I42.4 Endocardial fiber Endocardial fibroelastosis, I42.5 other restrictive cardiomyopathy, I42.6 alcoholic cardiomyopathy, I42.7 caused by drugs and other external factors Cardiomyopathy due to drugs and other external agents, I42.8 Other cardiomyopathies, I42.9 Cardiomyopathy (unspecified), I50.0 Congestive heart failure heart failure), I50.1 left ventricular failure (Left ventricular failure), or I50.9 heart failure (unspecified) (Heart failure, unspecified). More preferable examples of the heart disease include acute myocardial infarction (details unknown) of I21.9 in ICD10, dilated cardiomyopathy of I42.0, or left ventricular failure of I50.1. Specific examples of the disease type of acute myocardial infarction (details unknown) of I21.9 in the above-mentioned ICD10 include myocardial infarction (acute) NOS (Myocardial infarction (acute) NOS). As the disease type of dilated cardiomyopathy of I42.0 in the above-mentioned ICD10, specifically, congestive cardiomyopathy can be mentioned. Specific examples of the disease type of I50.1 left ventricular failure in the above-mentioned ICD10 include: Cardiac asthma, Left heart failure, Pulmonary edema (heart disease NOS or Heart failure) (Oedema of lung/Pulmonary oedema, with mention of heart disease NOS or heart failure).

The agent of the present invention can be used in combination with other agents. When used in combination, the agent of the present invention and other agents may be provided in the form of a single medicine, or may be provided in the form of a pharmaceutical combination or kit comprising a plurality of preparations prepared separately. The administration period of the agent used in combination with the agent of the present invention is not limited, and the agent of the present invention and the agent used in combination may be administered to the subject at the same time, or may be administered separately (eg, consecutively, at intervals, etc.). The dose of the concomitant drug may be in accordance with the dose used clinically, and can be appropriately selected according to the subject of administration, the route of administration, the disease, the combination, and the like.

As another drug, especially in the case of a therapeutic or preventive drug for muscle disease and/or heart disease, specifically, a steroid preparation (deflazacort) can be used as a therapeutic drug for muscle disease. , prednisone, prednisolone and other glucocorticoids), exon skipping (exon skipping), gene therapy, cell medicine, etc., and further examples of recovery can be exemplified, but not limited thereto. In addition, if it is a therapeutic drug for heart disease, ACE (Angiotensin-converting enzyme, angiotensin-converting enzyme) inhibitors, angiotensin II receptor antagonists, beta blockers, diuretics, antialdosterone drugs, h-ANP (human atrial natriuretic peptide, human atrial natriuretic peptide), foxglove preparations, cardiotonic agents, and various cell medicines, etc., and implantable devices such as pacemakers/defibrillators, or including the use of cardiac catheters The medical device for ablation may include, but is not limited to, surgical therapy including heart transplantation, left ventricular reconstruction, and mitral valve reconstruction. Furthermore, there is a case in which the above-mentioned therapeutic drug for heart disease is used in combination with the muscle disease in which the symptoms of cardiac function are reduced. In this specification, "therapeutic drugs for muscle diseases and/or heart diseases" include not only drugs for the purpose of radical treatment of muscle diseases and/or heart diseases, but also drugs for inhibiting tauopathies and dementia-related diseases, for example. The medicine for the purpose of development, the medicine for the purpose of inhibiting the development can also be used as a "preventive medicine for muscle disease and/or heart disease".

The diagnostic drug, diagnostic kit, or diagnostic reagent of the present invention is one for diagnosing (determining) muscle disease and/or cardiac disease. The above-mentioned diagnostic drugs and the like of the present invention are used to confirm the expression of TRPV2 on the cell membrane of peripheral blood monocytes or cardiomyocytes or muscle cells, or to measure the cytoplasm and on the cell membrane of peripheral blood mononuclear cells or cardiomyocytes or muscle cells, Or a reagent for the expression level of TRPV2 on the cell membrane (eg, anti-TRPV2 of the present invention or a fragment thereof), and muscle disease and/or heart disease can be diagnosed by this confirmation or measurement.

The anti-human TRPV2 antibody or fragment thereof can be a fluorescently labeled antibody, an enzyme-labeled antibody, a streptavidin-labeled antibody, a biotin-labeled antibody, or a radiolabeled antibody. In addition, the anti-human TRPV2 antibody can usually be dissolved in water or an appropriate buffer (eg: TE (Tris-Ethylenediaminetetraacetic acid, Tris-Ethylenediaminetetraacetic acid) buffer, PBS at an appropriate concentration. etc.) in the form of an aqueous solution, or a form of a freeze-dried product is contained in the diagnostic drug, kit, or reagent of the present invention. In addition, when measuring the expression level of TRPV2, it can be measured using one type of antibody, or multiple types, preferably two types of antibodies can be used for measurement (eg, immunostaining, flow cytometry, sandwich ELISA, etc.).

The diagnostic drug, kit, or reagent of the present invention may further contain other components necessary for carrying out the method according to the method for confirming or measuring TRPV2. For example, in the case of measuring by the Western blotting method, the kit of the present invention may further contain detection reagents such as blotting buffer, labeling reagent, blotting membrane, standard solution, and the like. Here, as the "standard solution", a purified sample of TRPV2 can be exemplified. For example, the peptide of the present invention can be dissolved in water or an appropriate buffer solution (for example: TE buffer, fetal bovine-containing solution) so as to obtain a specific concentration. Serum, PBS, etc.) in an aqueous solution.

When the measurement is performed by sandwich ELISA, the diagnostic drug, kit, or reagent of the present invention may further include a solid-phase antibody measurement plate, a cleaning solution, and the like in addition to the above. When the measurement is performed by an agglutination method including a latex agglutination method, an antibody-coated latex, gelatin, etc. may be included. In the case of measuring by chemical fluorescence method or chemical fluorescence electron method, antibody-bound magnetic particles and a suitable buffer may be included. In the detection of TRPV2 using LC/MS, LC-MS/MS (Liquid chromatography-mass spectrometry/Mass Spectrometry, liquid chromatography-mass spectrometry/mass spectrometry) or immunochromatography, it may include antibody-coated TRPV2. Column or micro-column, micro-chip as part of the inspection machine. Furthermore, in the case of a time-resolved fluorescence assay or a similar fluorescence assay, a plurality of labeled anti-human TRPV2 antibodies and other required components may be included as a composition.

3. The diagnosis (determination) method of the present invention, etc. In the case of using the anti-human TRPV2 antibody or fragment thereof of the present invention as a diagnostic drug for muscle disease and/or heart disease, for example, peripheral blood mononuclear cells can be isolated from the blood of a patient, and peripheral blood mononuclear cells (eg, peripheral blood mononuclear cells) can be isolated. : The expression level of TRPV2 in T cells, B cells, etc.) is measured, and the classification of the disease, the diagnosis (judgment) of whether or not the disease is suffered, the diagnosis (judgment) of whether the possibility of suffering the disease is high at present (judgment), Severity (eg: severe, moderate, mild, etc.) or the diagnosis (judgment) of the degree of progression. Furthermore, for example, by diagnosing the severity or progression of the disease, it is also possible to evaluate the effect of the therapeutic drug used for the treatment of the disease. In addition, companion diagnostics (determination) can be performed on whether or not the anti-human TRPV2 antibody or fragment thereof of the present invention is applicable. The measured (expressed) amount of TRPV2 in peripheral blood mononuclear cells can be the amount of TRPV2 present in the cytoplasm of peripheral blood mononuclear cells and on the cell membrane (plasma membrane), or the amount of TRPV2 present in peripheral blood mononuclear cells. The amount of TRPV2 present on the cell membrane is preferably the amount of TRPV2 present on the cell membrane. The cells to be measured (expressed) of TRPV2 in the diagnostic (determination) method of the present invention or the like may be muscle cells or cardiomyocytes in addition to peripheral blood mononuclear cells. The expression of TRPV2 can be confirmed by a generally known method such as flow cytometry or immunostaining, and the measurement or quantification of the expression level of TRPV2 can be carried out by flow cytometry (FACS) or immunostaining.

The diagnostic (determination) method of the present invention is characterized by detecting TRPV2 in a sample collected from a subject animal. In addition, the diagnostic method of the present invention may, for example, include the following steps as specific steps: (i) quantifying TRPV2 in a sample collected from a subject animal; and (ii) quantifying the amount of TRPV2 quantified in (i) Comparisons were made with the amount of TRPV2 in samples collected from healthy animals (hereinafter referred to as "control values").

As a result of the comparison in (ii), when the amount of TRPV2 quantified in (i) is greater than the control value, it means that the above-mentioned subject animal suffers from muscle disease and/or heart disease, or currently suffers from muscle disease and/or Likelihood of heart disease or future muscle disease and/or heart disease.

Furthermore, the diagnostic method of the present invention may include the following steps in addition to the above-mentioned steps: (iii) based on the result of (ii), when the amount of TRPV2 quantified in (i) is greater than the control value, determine that the above-mentioned tested animal suffers from the disease Muscle disease and/or heart disease, or steps where there is a current likelihood of developing muscle disease and/or heart disease, or a likelihood of developing muscle disease and/or heart disease in the future.

Furthermore, the present invention also relates to a method for assisting in the diagnosis (determination) of the degree of progression of muscle disease and/or cardiac disease. The method for assisting diagnosis of the present invention is also characterized in that TRPV2 is detected in a sample collected from a subject animal. In addition, the method for auxiliary diagnosis of the present invention may, for example, include the following steps as a specific step: (i) carrying out TRPV2 in a sample collected from a subject animal suffering from muscle disease and/or heart disease or having the possibility of suffering from it. quantify; and (ii) compare the amount of TRPV2 quantified in (i) with the amount of TRPV2 in a sample collected from the subject animal in the past (hereinafter "control value").

As a result of the comparison in (ii), when the amount of TRPV2 quantified in (i) is greater than the control value, it means that the muscle disease and/or heart disease of the above-mentioned subject animal is progressing, or suffering from muscle disease and/or The possibility of heart disease is high, and when it is less than the control value, it means that the muscle disease and/or heart disease of the tested animal is improving, or the possibility of not suffering from muscle disease and/or heart disease is high. The method for auxiliary diagnosis of the present invention may include the following steps in addition to the above steps: (iii) based on the result of (ii), when the amount of TRPV2 quantified in (i) is greater than the control value, determine the above-mentioned test animal Muscle disease and/or heart disease is progressing, or the possibility of suffering from muscle disease and/or heart disease is high. When it is less than the control value, it is judged that the muscle disease and/or heart disease of the tested animal is improving, or not suffering from it. High likelihood of muscle disease and/or heart disease.

In addition, the method for assisting the diagnosis (determination) of the degree of progression of muscle disease and/or heart disease of the present invention, for example, may also include the following steps as other specific steps: (i) and (ii) correlate the amount of TRPV2 quantified in (i) with the amount of TRPV2 since suffering from muscle disease and/or cardiac disease at a specified degree of progression; The amount of TRPV2 in the samples collected from the animals (hereinafter referred to as "control values") was compared.

As a result of the comparison in (ii), when the amount of TRPV2 quantified in (i) is greater than the control value, it means that the muscle disease and/or cardiac disease of the above-mentioned subject animal is more advanced than that of the control animal suffering from the disease. The degree of progression of the diseased animal, or the possibility of suffering from muscle disease and/or heart disease is high. When it is less than the control value, it means that the muscle disease and/or heart disease of the above-mentioned tested animal is improving, or does not suffer from muscle disease. and/or high likelihood of heart disease. The method for auxiliary diagnosis of the present invention may include the following steps in addition to the above-mentioned steps: (iii) based on the result of (ii), when the amount of TRPV2 quantified in (i) is greater than the control value, judge the above-mentioned animal to be tested. The degree of progression of muscle disease and/or heart disease is higher than that of the control animals suffering from the disease, or the possibility of suffering from muscle disease and/or heart disease is high, and when it is less than the control value, it is judged that the above-mentioned tested The animal's muscle disease and/or heart disease is improving, or the possibility of not suffering from muscle disease and/or heart disease is high.

The determination and testing (results) of the above-mentioned possibilities are useful at least in assisting the definitive diagnosis of muscle disease and/or cardiac disease by a doctor.

In addition, as mentioned above, since the amount of TRPV2 on the cell membrane of peripheral blood mononuclear cells is increased in muscle disease and/or heart disease, it is useful for patients undergoing treatment for muscle disease and/or heart disease or for prevention The present invention also relates to a method for evaluating the therapeutic effect of muscle disease and/or heart disease reserve group in the treatment of onset of muscle disease and/or heart disease.

The method for evaluating the therapeutic effect of the present invention is also characterized in that TRPV2 is detected in a sample collected from a subject animal. In addition, the method for evaluating the therapeutic effect of the present invention may, for example, include the following steps as specific steps: (i) administering a drug for the treatment of muscle disease and/or heart disease since the beginning or for preventing the onset of muscle disease and/or heart disease and (ii) compare the amount of TRPV2 quantified in (i) with a sample collected from the test animal in the past (e.g., before starting the drug treatment). The amount of TRPV2 (hereinafter referred to as the "control value") in the sample collected after the start of the administration treatment and before the collection time point of (i) was compared. As a result of the comparison in (ii), when the amount of TRPV2 quantified in (i) is greater than the control value, it means that the current administration (or the selected therapeutic drug) is not effective, and when it is less than the control value When , it means that the current administration treatment (or the selected treatment drug) is effective. The method for evaluating the therapeutic effect of the present invention may include the following steps in addition to the above steps: (iii) based on the result of (ii), when the amount of TRPV2 quantified in (i) is greater than the control value, the evaluation is the current administration The treatment (or the selected therapeutic drug) is not effective, and the current administered treatment (or the selected therapeutic drug) is evaluated as effective when it is less than the control value.

In this specification, the therapeutic drugs used in the administration therapy include not only the approved and marketed therapeutic drugs, but also the concept of new drugs under development in clinical trials. That is, the method for evaluating the therapeutic effect of the present invention can also be used in the monitoring of the drug effect in clinical trials and the like.

Animals that can be tested by the method of the present invention are not particularly limited as long as they express TRPV2, and examples thereof include mammals (e.g., humans, monkeys, cows, pigs, horses, dogs, cats, sheep, goats, rabbits, hamsters, guinea pigs, mice, rats, etc.), birds (eg: chickens, etc.), etc. Mammals are preferred, and humans are more preferred. The biological sample derived from the test animal to be the sample is not particularly limited, and examples thereof include blood, muscle tissue (biopsy sample), saliva, urine, and the like. More preferably blood, muscle tissue (biopsy sample).

As the "control value" used in the method of the present invention, the amount of TRPV2 in the control sample, or the amount of TRPV2 obtained by measuring the control in advance, or the amount of TRPV2 that is set, does not need to be measured simultaneously with the method of the present invention. Here, in order to set a control value, a plurality of individuals may be used as a control group, and an average value of the measured values of the plurality of individuals may be used as a control value. That is, as a control value, the above-mentioned determination and the like also include the use of the amount of TRPV2 in a control sample obtained from a control group (healthy animals, animals suffering from muscle disease and/or heart disease at a specific degree of progression, etc.) within the scope of the method of the present invention.

When the "TRPV2 in the sample collected from the tested animal" is greater than "the amount of TRPV2 in the sample collected from the healthy animal (control value)", it can be diagnosed (determined) that the tested animal currently suffers from muscle disease and/or possibility of heart disease or future muscle disease and/or heart disease. As the amount of "TRPV2 in a sample collected from a test animal", 1.1 times to 10 times the control value can be preferably exemplified. More preferably, 1.1 times to 8 times can be mentioned. More preferably, it is 1.2 to 5 times. The optimum can be 1.2 times to 3 times.

When the "TRPV2 in the sample collected from the tested animal" is greater than "the amount of TRPV2 in the sample collected from the tested animal in the past (control value)", the muscle disease of the tested animal and/or can be determined Heart disease is progressing. As the amount of "TRPV2 in the sample collected from the test animal", 1.1 times to 10 times the control value can be preferably exemplified. More preferably, 1.1 times to 8 times can be mentioned. More preferably, it is 1.2 to 5 times. The optimum can be 1.2 times to 3 times.

When the "TRPV2 in the sample collected from the tested animal" is less than "the amount of TRPV2 in the sample collected from the tested animal in the past (control value)", it can be determined that the muscle disease and/or the heart disease is improving . As the amount of "TRPV2 in the sample collected from the test animal", 0.1 times to 0.9 times the control value can be preferably exemplified. More preferably, it is 0.2 to 0.9 times. More preferably, it is 0.3 to 0.9 times. The optimum is 0.4 to 0.9 times.

When the "TRPV2 in the sample collected from the tested animal" is greater than "the amount of TRPV2 in the sample collected from the animal suffering from muscle disease and/or cardiac disease at a specific degree of progression (control value)" , it can be determined that the degree of progression of muscle disease and/or heart disease in the tested animal is higher than that in the control animal suffering from the disease. As the amount of "TRPV2 in a sample collected from a test animal", 1.1 times to 10 times the control value can be preferably exemplified. More preferably, 1.1 times to 8 times can be mentioned. More preferably, it is 1.2 to 5 times. The optimum can be 1.2 times to 3 times.

When "TRPV2 in the sample collected from the tested animal" is less than "the amount of TRPV2 in the sample collected from the animal suffering from muscle disease and/or cardiac disease at a specific degree of progression (control value)" , it can be determined that muscle disease and/or heart disease is improving. As the amount of "TRPV2 in the sample collected from the test animal", 0.1 times to 0.9 times the control value can be preferably exemplified. More preferably, it is 0.2 to 0.9 times. More preferably, it is 0.3 to 0.9 times. The optimum is 0.4 to 0.9 times.

Quantitative analysis of TRPV2 can be performed by normalizing the amount of TRPV2 in a sample with the amount of a standard protein (internal standard protein). That is, using the above method, after quantifying the amount of TRPV2 in the sample and the amount of the standard protein, the ratio of the signals between the two (TRPV2/standard protein) is calculated, and the amount of TRPV2 in the sample is compared with the amount of the standard protein. The ratio can be expressed. The standard protein only needs to be a protein that stably expresses a certain amount, and is preferably a protein that is commonly expressed in most tissues or cells. For example, proteins necessary for cell survival, such as RNA synthase, energy-generating enzymes, ribosomal proteins, and cytoskeletal proteins, are coded by genes (housekeeping genes). Specifically, it is not particularly limited, and examples thereof include proteins such as β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-tubulin. In particular, beta actin can be exemplified.

In addition, instead of the above-mentioned control value, the threshold value of the TRPV2 amount of peripheral blood mononuclear cells or cardiomyocytes or muscle cells related to muscle disease and/or heart disease can be preset, and the TRPV2 amount of the tested animal is related to the threshold value. value to compare. The (expressed) amount of TRPV2 in peripheral blood mononuclear cells or cardiomyocytes or muscle cells determined in the setting of the above cut-off value can be the amount of TRPV2 present in the cytoplasm of each cell and on the cell membrane (plasma membrane), or It may be the amount of TRPV2 present on the cell membrane of each cell, preferably the amount of TRPV2 present on the cell membrane. For example, when the amount of TRPV2 on the cell membrane of peripheral blood mononuclear cells of the test animal or on the cell membrane (plasma membrane) of cardiomyocytes or muscle cells is above the threshold value, the test animal can be diagnosed as suffering from muscle disease and/or or heart disease, or have a current or future likelihood of developing muscle disease and/or heart disease.

The "critical value" is a value that can satisfy both a higher diagnostic sensitivity (diagnosis rate with disease) and a higher diagnostic specificity (diagnosis rate without disease) when a disease is determined based on this value. For example, a value that shows a higher positive rate in individuals with muscle disease and/or heart disease and a higher negative rate in individuals without muscle disease and/or heart disease can be set as a critical value.

The method of calculating the critical value is well known in the art. For example, the amount of TRPV2 on the cell membrane of peripheral blood mononuclear cells in individuals with muscle disease and/or heart disease and in individuals without muscle disease and/or heart disease is calculated, and the diagnostic sensitivity and As for the diagnostic specificity, an ROC (Receiver Operating Characteristic) curve was created using commercially available analysis software based on these equivalent values. Then, a value at which the diagnostic sensitivity and diagnostic specificity are as close to 100% as possible is obtained, and this value can be used as a critical value. In addition, for example, it is also preferable to use the "mean value + 2 standard deviation" of the amount of TRPV2 on the cell membrane of peripheral blood mononuclear cells of many healthy animals as the critical value. If this value is used, the muscle can be judged with good sensitivity and specificity. Disease and/or heart disease onset. Further, for example, a value at which the similarity ratio between the diagnostic sensitivity and the diagnostic specificity becomes the maximum is obtained from the ROC curve, and this value is used as a critical value, whereby a muscle disease and/or a heart disease can be determined with high sensitivity. Or it is also better to set the point with the lowest diagnostic ability in the ROC curve, that is, the point farthest from the line where the area under the ROC curve becomes 0.5, as the critical value, that is, calculate "sensitivity + specificity - 1", and set this value to be the largest. The point of value is set as the critical value.

As a threshold value for the amount of TRPV2 on the cell membrane of peripheral blood mononuclear cells, for example, the ratio of TRPV2 present on the cell membrane of peripheral blood mononuclear cells in a sample collected from a subject animal is 15% to 40% (for example, , 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%).

When the method of the present invention is used for the diagnosis of muscle diseases and/or cardiac diseases, in addition to TRPV2, changes in other diagnostic markers of muscle diseases and/or cardiac diseases can also be studied. As diagnostic markers for muscle diseases and/or heart diseases, for example, troponin, BNP (Brain Natriuretic Peptide, B-type natriuretic peptide), ANP, CK (creatine kinase), myoglobin, AST (aspartate aminotransferase, aspartate aminotransferase), ALT (alanine aminotransferase, alanine aminotransferase), LDH (lactate dehydrogenase, lactate dehydrogenase), aldolase, etc., which can be detected according to well-known and commonly used detection methods . Moreover, in the diagnosis of muscle disease and/or heart disease, etc., in addition to TRPV2, a machine or the like can be used for diagnosis. As a diagnostic method using a machine or the like, in the case of muscle diseases, for example, needle electromyography, MRI (Magnetic Resonance Imaging, magnetic resonance imaging), etc. are mentioned, and in the case of cardiac diseases, for example, electrocardiogram, cardiac ultrasound, etc. are mentioned.

4. The preventive and therapeutic methods of the present invention The anti-human TRPV2 antibody or its fragment of the present invention, as described above, does not affect the activity of other Trp family proteins, and can specifically inhibit the Ca ²⁺ influx activity of TRPV2. Therefore, the antibody is suitable for the prevention and treatment of muscle diseases such as muscle dystrophy and cardiomyopathy or cardiac diseases related to the Ca ²⁺ influx.

In the prevention and treatment method of the present invention, for example, in the above-mentioned method for diagnosing (determining) muscle disease and/or heart disease of the present invention, etc., it is determined that the subject animal suffers from muscle disease and/or heart disease, and currently suffers from muscle disease. When the possibility of disease and/or heart disease, or the possibility of developing muscle disease and/or heart disease in the future, by administering the anti-human TRPV2 antibody or fragment thereof of the present invention, the prevention and treatment of the disease can be carried out . In addition, in the prevention and treatment method of the present invention, the anti-human TRPV2 antibody or fragment thereof of the present invention may be used in combination with the above-mentioned therapeutic or preventive drugs for muscle diseases and/or cardiac diseases.

The form of administration of the pharmaceutical composition to the subject, the route of administration, and the like can be appropriately determined according to the purpose and the subject to be administered. For example, intravenous, intraarterial, subcutaneous, intramuscular, transdermal, nasal, or oral administration can be performed in appropriate administration forms such as injection, transdermal, inhalation/nasal, and oral. Usually, the antibody is administered intravenously and subcutaneously as a general injection, and the administration form and route of administration can also be adapted to the pharmaceutical composition of the present invention.

The dose and schedule of administration of the active ingredient in various formulations can be appropriately adjusted according to the purpose of administration, the age and body weight of the subject, and the like. For example, the dose of the monoclonal antibody of the present invention can be selected within the range of 0.0001 mg to 1,000 mg/kg body weight in one dose. Or it can also be selected within the range of 0.001 to 100,000 mg per patient. However, the dosage of the therapeutic agent of the present invention is not limited to these.

The administration schedule of the pharmaceutical composition, or the therapeutic or preventive agent of the present invention can be appropriately adjusted according to the purpose of administration, the age and body weight of the subject, and the like. For example, the frequency of administration is usually in the range of about once a week to about once every 3 months, more preferably in the range of about once every 2 weeks to about once every 10 weeks, for example, every 4-8 weeks 1 time. The antibody of the present invention is preferably administered parenterally, intramuscularly or subcutaneously, intravenously, such as in the cubital fossa or other peripheral veins. As a preventive treatment, it is generally preferred to administer the antibody of the present invention once a month to once every 2 to 3 months, or less frequently. The number of times of administration may be one time, or a plurality of times (for example, 2 to 5 times) may be administered at intervals of several days or weeks. Also, development can be monitored by periodic evaluation. In the case of repeated administration for at least several days, the treatment can be repeated according to the symptoms until the symptoms of the disease are improved to a certain extent. The desired dose can be delivered to the patient by single or multiple bolus administration, or continuous infusion of the antibody, depending on the pattern of pharmacokinetic decay of the antibody. Example

Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited by these examples.

Comparative Example 1-1. Failed Examples 1) and 2) of hTRPV2 Antibody Production 1) Synthesis of a partial peptide CATESVQPMEGQEDEGNGAQYRGIL, which is a region with higher hydrophilicity between the 5th transmembrane region and the 6th transmembrane region of human TRPV2 (hTRPV2), that is, the amino acid sequence at positions 571 to 594. The KLH of the carrier was prepared as an immunogen. 200 μL of immunogen at 2 mg/mL was mixed with 200 μL of adjuvant (complete adjuvant (FREUND)) to make an emulsion, and the resultant was used to immunize 4-week-old female mice (C3H) (day 1). ). The 2nd (4th day) and 3rd (7th day) adjuvant was changed to incomplete adjuvant (FREUND), and the same amount of immunogen was used for immunization. The final immunization was performed 3 days later. Cell fusion was performed. 2) Or similarly, 3 partial peptides (p571-594/p558-575/p579-595; CATESVQPMEGQEDEGNGAQYRGIL/CQEAWRPEAPTGPNATESV) were synthesized in the region with higher hydrophilicity between the 5th transmembrane region and the 6th transmembrane region of hTRPV2 /EGQEDEGNGAQYRGILEC), respectively combined with KLH as a carrier. Thereafter, the peptides were mixed to prepare an immunogen of 2 mg/mL, immunized with the same immunization schedule as above, and cell fusion was performed.

Comparative Example 1-2. Cell fusion by PEG method Hypertrophic groin lymph nodes were removed from immunized mice, and cells were recovered therefrom. The recovered lymph node-derived cells were mixed with myeloma cells (P3X63Ag8U.1, P3U1 ATCC [CRL-1597] from mouse myeloma), and PEG4000 (MERCK) (with RPMI (Roswell Park Memorial) was added to the pellets after centrifugation. Institute, Roswell Park Memorial Institute) medium for equal dilution) for cell fusion. After washing with RPMI medium, the cells were suspended in FBS (Fetal Bovine Serum, fetal bovine serum)-HAT medium, and inoculated into a 96-well plate. After the inoculation, the medium was changed, and the culture supernatant was sampled from the 96-well plate at the stage of confirming the formation of a fusion tumor colony for screening.

Comparative Example 1-3. Cell ELISA The hTRPV2 expressing HEK293 cells and the negative control HEK293 cells (ATCC CRL-3216 from human embryonic kidney) were inoculated in a 96-well plate (NUNC) for cell ELISA (the number of cells was 1-3×10). ⁴ /well), in order to promote the cytoplasmic membrane transfer of hTRPV2, it was appropriately treated with cannabidiol. The fusion tumor culture supernatant was added to the immobilized plate on which the cells were immobilized at 50 μL/well, and the reaction was carried out at room temperature for 30 minutes. After washing twice (0.5% BSA/2 mM EDTA (ethylenediamine tetraacetic acid, ethylenediamine tetraacetic acid, ethylenediamine tetraacetic acid)/PBS), the peroxidase-labeled goat anti-microbe was diluted with diluent (0.5% BSA/2 mM EDTA/PBS). The mouse IgG (MBL) was diluted 10,000-fold, and the resultant was reacted at room temperature for 30 minutes. After washing 3 times, add a chromogenic substrate for color development for 10 to 15 minutes, and measure the absorbance at 450 to 620 nm. However, positive clones could not be confirmed.

Comparative Example 1-4. Antigen solid-phase ELISA Since no positive clones were found in the above cell ELISA, the binding to the immunogen was evaluated by ELISA, and 200 clones bound to the immunogen were selected. Furthermore, flow cytometric analysis was performed on the 200 clones using hTRPV2-expressing HEK293 cells and HEK293 cells. As a result, 10 clones bound to hTRPV2-expressing HEK293 cells, but also bound to hTRPV2-non-expressing HEK293 cells. As a precaution, 10 clones of culture supernatants were used for hTRPV2 functional analysis, none of which inhibited hTRPV2-induced Ca ²⁺ influx. Therefore, these 10 clones were considered not to have undergone hTRPV2-specific binding. Also, the remaining 190 clones did not bind to either of hTRPV2 expressing HEK293 cells and HEK293 cells. As the reason for not being able to confirm the pure line considered to be hTRPV2-specific, the possibility that HEK293 cells themselves also express hTRPV2 without any difference can also be considered. Therefore, reusing Chinese hamster-derived CHO cells (CHO-K1 ATCC CCL-61) instead of human-derived HEK293 cells, the above 10 clones were evaluated for binding of hTRPV2 expressing CHO cells to CHO cells by flow cytometry analysis. As a result, 8 clones bound to hTRPV2 expressing HEK293 cells but not to hTRPV2 expressing CHO cells. In addition, the other two clones bound to hTRPV2-expressing HEK293 cells and hTRPV2-expressing CHO cells. However, the above two pure lines also bind to HEK293 cells and CHO cells. Therefore, no clones that specifically bound hTRPV2 were obtained.

Comparative Example 1-5. Failure of hTRPV2 antibody production 3) According to the method described in Japanese Patent Laid-Open No. 2007-259745 (Japanese Patent Application No. 2006-088323), full-length human TRPV2 was introduced into HEK293 cells and allowed to Expression, human TRPV2 expressing HEK293 cells were produced by this, treated with cannabidiol, and used as an immunogen. 4-week-old female mice (C3H) were immunized with 200 μL of adjuvant (complete adjuvant (FREUND)), followed by the second cryopreservation described above, with human TRPV2 expressing HEK293 cells (2×10 ⁷ in 400 μL of PBS). ) as an immunogen for each immunization. In adjuvant immunization (day 0), 50 μL of adjuvant was administered to each animal, followed by human TRPV2 expressing HEK293 cells (5 in 100 μL of PBS) on days 1, 4, 7, 10, 13, and 16. ×10 ⁶ ) After a total of 7 immunizations with adjuvant, cell fusion was performed 3 days after the final immunization.

Comparative Example 1-6. Cell fusion by PEG method Hypertrophic groin lymph nodes were removed from immunized mice, and cells were recovered therefrom. The recovered lymph node-derived cells were mixed with myeloma cells (P3X63Ag8U.1, P3U1 ATCC [CRL-1580] from mouse myeloma), and after centrifugation, PEG4000 (MERCK) was added to the particles (equivalent in RPMI medium). dilution) for cell fusion. After washing with RPMI medium, the cells were suspended in FBS-HAT medium and inoculated into a 96-well plate. After the inoculation, the medium was changed, and the culture supernatant was sampled from the 96-well plate at the stage of confirming the formation of a fusion tumor colony for screening.

Comparative Example 1-7. Cell ELISA The hTRPV2 expressing HEK293 cells and the negative control HEK293 cells were inoculated in a 96-well plate (NUNC) for cell ELISA (the number of cells was 1-3×10 ⁴ cells/well), and the cells were treated with cannabis bisulfite. Phenol treatment. The fusion tumor culture supernatant was added to the immobilized plate of the above-mentioned cells in the form of 50 μL/well, and the reaction was carried out at room temperature for 30 minutes. After washing twice (0.5% BSA/2 mM EDTA/PBS), peroxidase-labeled goat anti-mouse IgG (MBL) was diluted 10,000-fold with diluent (0.5% BSA/2 mM EDTA/PBS), and the resulting React at room temperature for 30 minutes. After washing 3 times, add a chromogenic substrate for color development for 10 to 15 minutes, and measure the absorbance at 450 to 620 nm. As a result, more than 76 positive clones were obtained. However, hTRPV2 was not specific for HEK293 cells and was also positive for HEK293 cells as a negative control.

Comparative Example 1-8. Flow Cytometry Analysis Binding to hTRPV2-expressing HEK293 cells and HEK293 cells was confirmed by flow cytometric analysis of the culture supernatants of a total of 76 samples that reacted with hTRPV2-expressing HEK293 cells and had fusion tumors in the cell ELISA. Detection uses PE (phycoerythrin, phycoerythrin)-labeled anti-mouse IgG antibody. As a result, it was suggested in the cell ELISA that the samples that bound to the HEK293 cells expressing hTRPV2 also bound to the HEK293 cells to the same extent, and the clones of the samples expressing the specificity of hTRPV2 to HEK293 cells could not be confirmed. As a reason for not being able to confirm a pure line considered to be hTRPV2-specific, it is also considered that hTRPV2 is also expressed in HEK293 cells themselves, but no difference was seen. Therefore, CHO cells from Chinese hamster ovary (CHO-K1 ATCC CCL-61) were reused instead of HEK293 cells from humans, and flow cytometric analysis was performed using hTRPV2 expressing CHO cells and CHO cells to evaluate the above 76 clones Binding to hTRPV2 expresses CHO cells and CHO cells. As a result, there were 5 clones that bound to both hTRPV2-expressing HEK293 cells and hTRPV2-expressing CHO cells, but these clones also reacted with HEK293 cells and CHO cells. Therefore, it is considered that an antibody that specifically binds to hTRPV2 has not been obtained.

Comparative Example 1-9. Failed Example 4) of hTRPV2 Antibody Production Synthesis of recombinant protein using wheat germ extract (+liposome) as antigen: the amino acid region of the membrane region of hTRPV2 (removing 380 amino acids of the N-terminal and 113 amino acids of the C-terminal of the intracellular domain) 381-650, CellFree Sciences) (1.32 mg/mL, 0.65 mL). The above recombinant protein was mixed with Freund's comlete adjubant (Difco) in a volume ratio of about 1:2 and emulsified. The emulsified antigen was administered to the tail of 8-week-old female mice (B6D2F1) at 60 μL each. After 14 days, the antibody titer test using serum was first performed, and 17 days later, booster immunization was performed with the same amount as the first. After booster immunization, lymphocytes were collected from the iliac lymph nodes on the 4th day, and fused with myeloma SP2 cells (SP2/0-Ag14 from mouse myeloma, ECACC 85072401) by the polyethylene glycol (PEG) method.

Comparative Example 1-10. Antibody titer test using serum Antibody titer examination using serum was performed by flow cytometric analysis using hTRPV2-expressing HEK293 cells treated with cannabidiol and hTRPV2-expressing HEK293 cells not treated with cannabidiol, but neither case was seen with hTRPV2-unexpressed differences in HEK293 cells. Therefore, serum antibody titers were checked by ELISA using recombinant proteins of the amino acid region (positions 381-650) of the membrane fraction of hTRPV2 used for immunization. At 1000-fold dilution of serum, the mice were divided into groups of positive mice and definite non-positive mice. After booster immunization was performed in each group in the manner described above, lymphocytes were collected from the iliac lymph nodes on the 4th day. In RPMI medium, PEG-4000 was dissolved so that the concentration of PEG-4000 was 50(W/V)%, and the above lymphocytes and SP2 cells were mixed so that the ratio of the above-mentioned lymphocytes and SP2 cells was 2:1 to perform cell fusion. The culture medium of the fusion tumor was replaced with the HAT selection medium, and after further culture, the culture supernatant was used for screening by ELISA, and the result was positive in the fusion tumor using the lymphocytes of the positive group in the serum antibody titer test. 54 (out of 300 attempts) and 36 (out of 300 attempts) in the group with ambiguous positives. Each positive supernatant was used for staining of hTRPV2 expressing HEK293 cells, but all the positive fusion tumor supernatants did not react with hTRPV2 expressing HEK293 cells. The fusion tumor supernatant to which hTRPV2 expresses cell-specific binding could not be confirmed.

Comparative example 1-11. Production of anti-hTRPV2 antibody; failure examples 5) and 6) 5) Synthesize a partial peptide (p571-594 CATESVQPMEGQEDEGNGAQYRGIL) with a higher hydrophilicity region between the 5th transmembrane region and the 6th transmembrane region of hTRPV2, that is, the amino acid sequence at positions 571-594 and bind to KLH . This peptide was mixed 1:1 with an adjuvant (Complete Freund's Adjuvant, Difco Laboratories) to immunize New Zealand white rabbits with 0.5 mg of the peptide once a week for 4 weeks. Two rabbits (slc: Nzw Japan SLC) were used to obtain antiserum. 6) Or 3 kinds of synthetic peptides (3 kinds of peptide mixture p571-594/p558-575/p579-595; CATESVQPMEGQEDEGNGAQYRGIL) were synthesized in the region with higher hydrophilicity between the 5th transmembrane region and the 6th transmembrane region of hTRPV2 /CQEAWRPEAPTGPNATESV/EGQEDEGNGAQYRGILEC), bound to KLH, respectively. Each peptide was mixed and used as an immunogen. Specifically, three kinds of partial peptides as antigens were mixed with adjuvant (complete Freund's adjuvant, Difco Laboratories) at 1:1, and 0.6 mg per animal was administered once a week for 4 weeks. (0.2 mg×3) peptide was immunized with 2 New Zealand white rabbits to obtain antiserum.

Comparative Example 1-12. Immunostaining and Immunoblotting Evaluation The obtained antiserum was an antiserum that reacted with each antigenic peptide. However, in order to confirm whether the obtained antiserum recognizes the extracellular domain of TRPV2, using the antiserum, immunoblotting (500-fold dilution) and immunostaining (50-100-fold dilution) of hTRPV2 expressing HEK293 cells were performed. In immunostaining, hTRPV2 expressing HEK293 cells were incubated with each antiserum (50-100-fold dilution) at room temperature for 30 minutes, and then labeled with FITC (Fluorescein Isothiocyanate, fluorescent isothiocyanate) anti-rabbit secondary antibody. The reaction was carried out, and the fluorescence of FITC was observed using a confocal laser microscope. As a result of immunostaining and immunoblotting, all the obtained antisera did not react with hTRPV2 expressing HEK293 cells.

In the above-described manner, experiments were designed based on the analogy of the examples of the prior art document Japanese Patent No. 5754039 related to the invention of an antibody that recognizes the TRPV2 sequence of mice and has a function of specifically inhibiting the activity of TRPV2, and the result is a An attempt was made to obtain the desired anti-human TRPV2 antibodies based on the various immunogens (cells or peptides) assumed in this example. However, within the range that can be deduced from the contents of the prior art documents, etc., the desired anti-human TRPV2 antibody that recognizes the extracellular domain of human TRPV2 as an epitope, or an anti-human TRPV2 antibody that recognizes the extracellular domain of human TRPV2 as an epitope and has specificity has not been obtained. Anti-human TRPV2 antibodies that function to inhibit the activity of human TRPV2.

Example 1. Production of mouse anti-human TRPV2 antibody 1-1. Production and immunization of immunogens Using two kinds of autoimmune disease mice (SLE model mice MRL/lpr and C3H/lpr), two mice each (four mice in total) were immunized by the footpad method at 2-week intervals. The following peptides were immunized as immunogens in the region with higher hydrophilicity between the 5th transmembrane region and the 6th transmembrane region of human TRPV2 (hTRPV2) at 1 week and 3 weeks, and the following peptides were immunized at 5 weeks and 7 weeks. The following full-length hTRPV2 is expressed in HEK293 cells (TRPV2 is expressed at the cytoplasmic membrane by cannabidiol stimulation). One week after the final immunization, blood and lymph nodes were collected, and lymph nodes of mice confirmed to contain the target antibody (stained from extracellular and exhibited inhibitory activity) were used for evaluation with antiserum to prepare a phage library. Immunogen; peptide-KLH 579-595 (Ac)EGQEDEGNGAQYRGILEC-KLH and full-length hTRPV2 expressing HEK293 cells (in accordance with the method described in Japanese Patent Laid-Open No. 2007-259745 (Japanese Patent Application No. 2006-088323), full-length hTRPV2 The amino acid sequence was introduced into HEK293 cells and expressed, thereby producing hTRPV2 expressing HEK293 cells, and using 10 μM cannabidiol (10% FBS/DMEM (Dulbecco's Modified Eagle Medium, Dulbecco's Modified Eagle Medium)) After treatment at 37°C for 15 minutes to increase the expression of TRPV2 in the plasma membrane, the cells were recovered and used as an immunogen.

1-2.2 Production of Antibody Phage Library of 2 species RNA was extracted from each lymph node (mouse number 01, 03, 04) of 3 mice selected by serum evaluation of 4 mice that had been immunized by ISOGEN (NIPPON GENE). cDNA was synthesized from the extracted RNA using SuperScript III Reverse Transcriptase (Invitrogen). Using primers for mouse antibodies, PCR was performed using the synthesized cDNA as a template to amplify the mouse antibody genes VH and Vκ. Mouse No. 01 (Gene Bank No. 01) was amplified alone, and mouse No. 03 and 04 (Gene Bank No. 02) were mixed and amplified. The PCR product of Vκ was inserted into the scFv-cp3 vector using restriction sites. Escherichia coli was transformed with the L chain-inserted vector, and plastids were recovered from the cultured Escherichia coli. The VH was inserted into the plastid (scFv-cp3 vector incorporating the L chain) using other restriction sites. Escherichia coli DH12S (Invitrogen) was transformed with a vector incorporating VH and VL genes. As a result, No.01 antibody library obtained 6.9×10 ⁸ transformants, and No.02 antibody library obtained 5.9×10 ⁸ transformants. The pattern diagram is shown in FIG. 1 . Glycerol feedstock for the production of transformed E. coli. Also, a phage library solution was prepared by infecting the transformed E. coli with the helper phage M13KO7 for panning.

1-3. Confirmation analysis of anti-human TRPV2 antibody in mouse antiserum (1) Immunoblotting using 1% n-Dodecyl-β-D-maltoside (Dojindo ) 1.6×10 ⁷ biotinylated hTRPV2 expressing HEK293 cells (which express hTRPV2 full-length expression vector (Myc-biotinylated-His tag) in HEK293 cells) were solubilized and mixed with Dynabeads MyOne Streptavidin T1 at room temperature. (VERITAS) was spun for 20 minutes to bind to the magnetic beads. After washing, SDS sample buffer was added and boiled at 95°C for 10 minutes. The SDSylated samples were detected by Western blot using antiserum and anti-His-tag antibody. As a result, when the anti-His-tag antibody was used under the condition of exposure time of 10 seconds, bands were confirmed at the positions of 100 kDa and 75 kDa ( FIG. 2 a ). Furthermore, when the exposure time was 3 minutes, when the immunized mouse antiserum was used for the primary antibody, bands were also observed at the positions of 100 kDa and 75 kDa (Fig. 2b). Since bands were observed at the same positions as in the case of using the anti-His tag antibody, it was judged that hTRPV2 was specifically detected by solubilization. The shades of the bands are in the order of mouse number 01>03>04.

(2) Cell ELISA The binding activity of the antisera was assessed using hTRPV2 expressing HEK293 cells. Each antiserum was evaluated at two concentrations (2,000-fold dilution and 10,000-fold dilution), and the results showed that any antiserum showed higher binding activity to hTRPV2-expressing HEK293 cells than the negative control HEK293 cells (Fig. 3).

(3) ELISA of solubilized membrane components (solubilized ELISA) The activity of each antiserum was confirmed using streptavidin plates (Thermo Scientific) in which biotinylated hTRPV2 expressing HEK293 cells were solubilized and immobilized. As a result, it did not react with the TRPV2 non-expressing HEK293 cell solubles as a negative control (those obtained by adding the same tag to a protein X completely unrelated to TRPV2), while the biotinylated hTRPV2 expressing HEK293 cells did not react. The solubilized material reacted specifically, and it was confirmed that the solubilized membrane fraction could be prepared ( FIG. 4 ). The strength of the binding activity to the biotinylated peptide was in the order of mouse number 04>01>03, while the solubilized membrane fraction was in the order of mouse number 01>03>04.

The activity of hTRPV2-expressing 293 cells was confirmed by cell ELISA and solubilization ELISA, and all the methods confirmed specificity in mouse serum. However, in the cell ELISA, the antiserum also reacted with HEK2923 cells that did not express hTRPV2. It is considered that since the individuals used for gene bank production were immunized with HEK293 cells expressing hTRPV2, they reacted with common molecules on the HEK293 cell membrane. Therefore, if cells are used as the antigen for the first panning, it is difficult to concentrate the antibody specific for the target antigen, so peptides are used for the first to third panning antigens. If peptides are used for panning antigens, there is also concern about the possibility of not being able to obtain antibodies that recognize the three-dimensional structure. However, first, we aim to obtain antibodies that exhibit specificity in cell ELISA, and then use peptide antigens to start panning.

1-4. Panning To obtain antibodies against hTRPV2, antigens for panning were prepared. Specifically, after solubilization by attaching a biotinylated tag, it is recovered by streptavidin beads, etc., and the antigen is presented at a high density, thereby improving the concentration efficiency of the target antibody. Biotinylated peptide: 579-595 (Ac)EGQEDEGNGAQYRGILEK-biotin Biotinylated hTRPV2 expressing HEK293 cells: The hTRPV2 full-length expression vector (Myc-biotinylated-His tag) was genetically transfected into HEK293 cells using Transficient (MBL: WU1001). Cells were suspended in DMEM (low glucose) containing 10% FCS (Fetal Calf Serum, fetal calf serum). After 24 hours of culture, the cells were recovered.

implemented panning 1st run: Biotinylated peptide-bound magnetic particle panning 2nd run: Biotinylated peptide-bound magnetic particle panning 3rd time: KLH-modified peptide-binding magnetic particle panning, or Biotinylated peptide + anti-biotin antibody combined with magnetic particle panning After the 4th time, the panning method was carried out by two methods. Step 4-A: Panning using biotinylated hTRPV2-expressing HEK293 cells solubilized and then presented on magnetic particles Step 5-A: panning using biotinylated hTRPV2-expressing HEK293 cells that are solubilized and then presented on magnetic particles via an anti-His-tag antibody, or 4th-B: Cell Panning Using Biotinylated hTRPV2 Expressing HEK293 Cells 5th-B: Cell Panning Using Biotinylated hTRPV2 Expressing HEK293 Cells At each panning, streptavidin-labeled magnetic beads were used for recovery. Unbound phages were removed by washing, and bound phages were eluted by trypsin treatment, and the above operation was repeated.

Binding of the panned recovered phage to hTRPV2 was confirmed by cellular ELISA using hTRPV2 expressing HEK293 cells. As a result, the phage colony (02a 5th) recovered by the 5th panning using the No. 02 phage library showed slightly higher binding activity to hTRPV2 expressing HEK293 cells relative to the negative control ( FIG. 5 ). However, compared with HEK293 cells that did not express TRPV2 (those obtained by adding the same tag to protein X, which is completely unrelated to TRPV2), which was placed as a negative control, it was confirmed that the amount of expression on the membrane was very low, and mouse serum The binding activity of hTRPV2 was lower in HEK293 cells. From this, it was judged that the hTRPV2-specific antibody was contained in the phage colony recovered by the fifth panning, the reactivity of HEK293 cells expressing hTRPV2 exceeding the reactivity against hTRPV2, and 48 clones were isolated from this plate. Also, 48 clones were similarly isolated from the phage colony (01a 5th) recovered by the fifth panning using the No. 01 phage library subjected to the same panning method. That is, in the phage libraries No. 02 and No. 01, the phages recovered by the fifth panning, respectively, contained a clone that bound to hTRPV2-expressing HEK293 cells. These are simply linked, and 002a and 001a are isolated.

1-5. Preparation of monoclonal antibody fragments In cell ELISA evaluation using the phage colony, phage (02a) recovered from phage library No. 02, which had been panned 5 times, showing specificity for hTRPV2-expressing HEK293 cells, and 5 times the same. The phage (01a) recovered from the panned No. 01 phage library was an isolated clone. 48 colonies were picked from each E. coli plate of antibody phage recovered from each panning and cultured by applying IPTG (isopropyl β-D-1-thiogalactopyranoside, isopropyl β-D-1-thiogalactopyranoside Glycoside) was induced to express and secrete the antibody in E. coli culture supernatant, and the obtained E. coli particles were removed as scFv cp3 E. coli culture supernatant and used for cell ELISA evaluation.

1-6. Evaluation of binding properties by ELISA The binding activity of the phages prepared above (02a and 01a above) was evaluated by ELISA in which biotinylated hTRPV2 expressing HEK293 cells were solubilized and then immobilized on a streptavidin plate. As a result, 01a005, 01a016, 01a018, and 01a033 in the 01a phage colony exhibited slightly higher binding activity to biotinylated hTRPV2-expressing HEK293 cells ( FIG. 6A ). These 4 clones exhibited higher binding activity in biotinylated hTRPV2 expressing HEK293 cells compared to the negative control hTRPV2 non-expressing cells (which express the biotinylated protein X, which is completely unrelated to TRPV2), therefore Set as positive pure line. Furthermore, all clones in the 02a phage colony exhibited higher binding activity to biotinylated hTRPV2-expressing HEK293 cells (Fig. 6B).

1-7. VH analysis of monoclonal antibodies Plasmid DNA was prepared from the culture medium picked up and cultured from the E. coli plate of the phage recovered after each panning, and the VH analysis of the ELISA-positive clone described above (1-6) was performed. The results of the analysis showed that the ELISA-positive clones (01a005, 01a016, 01a018, and 01a033) of the 01a gene bank all had the same VH sequence (VH of the 01a gene bank: SEQ ID NO: 16). Then, 02a001-009 in the ELISA-positive clones of the 02a gene bank were analyzed, and the result was that 7 clones were identical (there was one amino acid difference in the frame part), including two clones with one amino acid difference in the CDR2 part. (02a003, 02a005), but the CDR3 considered important for binding activity is the same in all clones (VH of 02a GenBank: SEQ ID NO: 14 (02a001), 18 (02a003 and 02a005), 19 (02a006) (3 types) )). Therefore, in the evaluation of the clones after analysis, 01a033 (hereinafter sometimes referred to as mAb001) with the best specificity in ELISA was selected for the 01a gene pool, and 02a001 (hereinafter sometimes referred to as mAb001) was selected because there was no difference in the binding activity of the 02a gene pool. Also expressed as mAb002). The 02a gene pool is a mixed gene pool of immunized individuals No. 3 and No. 4, but since the pure line with the same VH sequence as 02a001 has higher binding activity, it is presumed that the pure line with lower binding activity such as 01a033 has not been obtained.

1-8. Confirmation of binding to hTRPV2 by cellular ELISA and flow cytometry As a result of the VH analysis of the ELISA-positive clones of the 01a and 02a gene banks, one specific clone was confirmed for each gene bank. Therefore, for the representative clones 01a033 (mAb001) (heavy chain CDR1 (SEQ ID NO: 9) and heavy chain CDR2 (SEQ ID NO: 10) ), heavy chain CDR3 (SEQ ID NO: 11); light chain CDR1 (SEQ ID NO: 12), light chain CDR2 (WAS), light chain CDR3 (SEQ ID NO: 13); heavy chain variable region: SEQ ID NO: 16; light chain variable region Region: SEQ ID NO: 17), 02a001 (mAb002) (heavy chain CDR1 (SEQ ID NO: 4), heavy chain CDR2 (SEQ ID NO: 5), heavy chain CDR3 (SEQ ID NO: 6); light chain CDR1 (SEQ ID NO: 7), light chain The binding activity of CDR2 (RMS), light chain CDR3 (SEQ ID NO: 8) heavy chain variable region: SEQ ID NO: 14; light chain variable region: SEQ ID NO: 15) was evaluated by cellular ELISA using hTRPV2 expressing HEK293 cells. In the above evaluation, each sample was picked from each E. coli plate and cultured, and the expression and secretion of the antibody protein in the E. coli culture supernatant was induced by application of IPTG. Thereafter, the pellet obtained by removing the E. coli particles by centrifugation was concentrated by ammonium sulfate. As a result, both clones showed higher fluorescence intensity to hTRPV2 expressing cells.

In addition, in this experiment, the objective was to obtain an antibody that binds to an antigen on the cell membrane. Therefore, flow cytometric analysis was performed by hTRPV2 expressing HEK293 cells expressing hTRPV2 in native form without immobilization or solubilization. As a result, only a small amount of binding was confirmed in 01a033 (mAb001), but hTRPV2-specific binding was confirmed in 02a001 (mAb002). Regarding 01a033 (mAb001), the possibility of recognizing only the pure line in the solubilized state (mAb001) cannot be denied. However, since this experiment was evaluated by scFv, it is considered that the binding activity increases after the IgG conversion. The possibility of binding to hTRPV2 expressing HEK293 cells was confirmed by flow cytometry analysis ( FIG. 7 ).

1-9. Confirmation of binding to hTRPV2 by immunoprecipitation experiments The results of the VH analysis of the ELISA positive clones were that the selected representative clones 01a033 (mAb001), 02a001 (mAb002) and a total of 3 clones of the negative control antibody The culture supernatant was used to prepare beads bound with scFv antibody, and immunoprecipitation experiments using hTRPV2 expressing HEK293 cell solubles were performed. As a result, binding to hTRPV2 (the band at the 100 kDa position) was confirmed only in the beads bound with 01a033 (mAb001) and 02a001 (mAb002) ( FIG. 8 ). Bands were confirmed at the same positions as in the case of using the anti-His-tag antibody, so it was confirmed that either scF _V could immunoprecipitate hTRPV2.

Construction of antibody gene vector ・Introduction of variable regions into expression vectors to construct expression systems of H chain and L chain For the VH and VL fragments of the two pure lines 01a033 (mAb001) and 02a001 (mAb002), using the scFv expression vector as a template, the signal sequence and restriction enzyme sites were added by PCR and amplified. VL continues to make VL-CK fragments by fusion PCR with CK appended (fusion-PCR). These gene fragments were treated with 2 kinds of restriction enzymes, respectively, and incorporated into the H chain vector (pEHX1.1) and L chain vector (pELX2.2) pre-assembled with mouse CH1, 2, and 3 genes. . Regarding the secretion signal sequence, VH4-34 was used for the H chain and VK2-A23 was used for the L chain. VH4-34: MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 20) VK2-A23: MRLLAQLLGLLMLWVPGSSG (SEQ ID NO: 21) Sequence the H chain vector and L chain vector to confirm that the exact gene fragment can be inserted. The H chain vector and the L chain vector were decomposed with two kinds of restriction enzymes, and the H chain vector and the L chain vector were ligated to prepare an IgG antibody expression vector. After the ligated IgG antibody expression vector was introduced into Escherichia coli, plastids were recovered from the cultured transformants using QIAGEN Plasmid Midi Kit (QIAGEN).

・The expression vectors of H chain and L chain were introduced into CHO cells, and it was confirmed that one of the expression vectors of IgG antibody produced by digestion with restriction enzyme (AseI (NEB)) was expressed, and the single-chain plastid was separated by electroporation. Introduced into CHO-K1 (suitable for suspension culture). Suspended in 20 mL of 10% FBS Ham's F12 medium (Wako Pure Chemical Industries, Ltd.), and cultured at 37°C, 5%, and CO ₂ . The next day, puromycin (Sigma) was added so that the final concentration would be 10 μg/mL, and one day later, a part of the culture supernatant was collected and analyzed by ELISA. As a result, IgG expression was confirmed in both 01a033 (mAb001) and 02a001 (mAb002).

Production of mouse antibody gene expression vector into cells and antibody production ・Preparation of anti-human TRPV2 IgG antibody-producing cell line Continue to culture the anti-human TRPV2 IgG antibody-producing cells that have confirmed IgG expression, strip them with 0.05% trypsin-EDTA (Wako Pure Chemical Industries, Ltd.) on the 14th day after gene transfer, and adapt them to cells containing 4 mM L-glutamine (L -Glutamine) serum-free medium (EX-CELL (registered trademark) CD-CHO Fusion (SAFC). During adaptation, 10 μg/mL of puromycin was continuously added to continue selection.

・Purification of anti-human TRPV2 IgG antibody from an anti-human TRPV2 IgG antibody-producing cell line Suspend the acclimated cells in 100 mL of serum-free medium at a concentration of 2.0 to 3.0 × 10 ⁵ cells/mL at 37°C in a Shake culture in 500 mL flasks (Nunc) for 10 days. The culture supernatant was recovered and purified by an affinity column using nProtein A Sepharose 4 Fast Flow (GE Healthcare). After washing with PBS, the antibody was eluted from the column with 0.2 M glycine (Glycin)-HCl (pH 3.0), dialyzed with PBS, and the buffer solution was filtered by ultrafiltration (Amicon Ultra (Millipore)). Replaced with PBS(-) and concentrated. After filtration through a 0.22 μm filter, the concentration and the Purity confirmation (Figure 9).

1-12. hTRPV2-binding activity of anti-human TRPV2 IgG antibody (flow cytometry) ・The HEK293 cells were genetically transfected with biotinylated hTRPV2 expression vector and biotinylated hTRPV2 non-expressing vector (to make it completely irrelevant to TRPV2) The protein X biotinylation obtained from the expression).・Preparation of biotinylated hTRPV2 expressing HEK293 cells The culture was started with D-MEM (10% FBS) medium and subcultured in a subconfluent state. The experiments used cells that proliferated to subconfluence. The biotinylated hTRPV2 expression vector (MBL) was transgenic using Transficient (MBL) according to the manual and cultured for about 24 hours. At the same time, as a negative control, the hTRPV2 non-expression vector was also introduced in the same manner (the protein X, which is completely unrelated to TRPV2, was biotinylated to be expressed). After the cells were detached by pipetting, they were washed with PBS, and the number of cells was adjusted to 2×10 ⁵ per specimen in 1% BSA/PBS.

・Analysis of combined reaction and flow cytometry Antibody binding reaction and washing operations were performed with 1% BSA/PBS. The anti-human TRPV2 IgG antibody prepared above at 5 μg/mL was allowed to bind to hTRPV2 expressing HEK293 cells for 1 hour on ice. After washing once with 1% BSA/PBS, it was reacted with a secondary antibody (Alexa488-labeled anti-mouse IgG (H+L) antibody, 5 μg/mL (Invitrogen)) for 1 hour on ice. After washing twice with 1% BSA/PBS, analysis was performed by a flow cytometer (Beckman, FC500). The MFI (mean fluorescence intensity) value obtained by comparing the X-mean of each pure line with the negative control antibody was obtained. As a result, 01a033 (mAb001) was specific for hTRPV2 expressing cells, but a very small amount was confirmed. degree of integration. 02a001 (mAb002) was also shifted in negative control cells (hTRPV2-non-expressing HEK293 cells), but strongly bound to hTRPV2-expressing HEK293 cells, and two peaks were observed. The large shifted peak on the right is considered to be the response to the forced expression of hTRPV2, and the peak on the left is the response to the very small amount of hTRPV2 present inside the HEK293 cells. It is presumed that in the scFv-cp3 E. coli culture supernatant before IgG conversion, the titer was not sufficient and the shift confirmed only in the forced expression antigen was also compared with the HEK293 cells expressing a small amount by increasing the titer by IgG conversion. hTRPV2 response (Figure 10).

1-13. Confirmation of cross-reactivity between anti-mouse TRPV2 antibody and anti-human TRPV2 antibody ・Confirmation test of crossover using western ink dot method HEK293 cells, mouse TRPV2 (mTRPV2) and hTRPV2 expressing HEK293 cells were solubilized by Lysis buffer. After centrifugation at 16,000×g for 10 minutes at 4° C., the supernatant was separated by 7.5% SDS-PAGE and transcribed into PVDF (Polyvinylidene Difluoride, polyvinylidene fluoride). After blocking the membrane with Blocking One (Nacalai Tesque), anti-mouse TRPV2 antibody (mAb88-2), anti-human TRPV2 antibody (mAb002), and TRPV2 polyclonal antibody pAb (TRPV2) ( A polyclonal antibody prepared using the C-terminal region of TRPV2 as an antigen and an antibody recognizing mTRPV2 and hTRPV2: Non-Patent Document 2), and incubated at 4°C overnight. Wash buffer (Wash buffer) (0.1% Tween-20/TBS) for 3 times in 5 minutes, then with HRP (horseradish peroxidase, horseradish peroxidase)-labeled secondary antibody (goat) diluted 1000 times by Blocking One Anti-mouse IgG (H+L) secondary antibody (Goat anti-Mouse IgG (H+L) Secondary Antibody), HRP or Goat anti-Rabbit IgG (H+L)) was incubated at room temperature for 1 hour. Then, after washing three times with washing buffer (0.1% Tween-20/TBS) for 5 minutes, washing with PBS was further two times, ECL (enhanced chemiluminescence substrate, enhanced chemiluminescence substrate) was added, and chemiluminescence was detected. In pAb(TRPV2), both mTRPV2 and hTRPV2 were stained by immunoblotting, but mAb88-2 only stained mTRPV2 expressing HEK293 cell solubles and mAb002 only hTRPV2 expressing HEK293 cell solubles.

・Confirmation test of crossover by immunostaining mTRPV2-expressing HEK293 cells (upper), hTRPV2-expressing HEK293 cells and HEK293 cells (lower) were fixed (5 minutes) with neutral formaldehyde, and then washed with PBS. Then, it was incubated with anti-mouse TRPV2 antibody (mAb88-2) (0.013 mg/ml) or anti-human TRPV2 antibody (mAb002) (0.021 mg/ml) for 12 hours at 4°C. After being washed with PBS, incubated with Alexa488 anti-mouse IgG diluted 500 times at room temperature for 1 hour, washed with PBS, and examined by confocal laser microscope. As a result, the anti-mouse TRPV2 antibody (mAb88-2) stained only the mTRPV2-expressing HEK293 cells, and the anti-human TRPV2 antibody (mAb002) only stained the hTRPV2-expressing HEK293 cells ( FIG. 11 ).

・Confirmation test of crossover by cell ELISA in 96-well plates were seeded with hTRPV2 expressing HEK293 cells (Fig. 12a) or ^mTRPV2 expressing HEK293 cells (Fig. 12b) at 1-3 x 104 cells/well. The next day, after fixing with PFA (paraformaldehyde) for 5 minutes, washing with PBS, anti-mouse TRPV2 antibody (mAb88-2), anti-human TRPV2 antibody (mAb002, mAb001), and isotype controls. After incubating at room temperature for 30 minutes, wash with PBS. Furthermore, Alexa488-labeled anti-mouse secondary antibody (life technology) was added at 50 μl/well, and the reaction was carried out at room temperature for 30 minutes. After washing with PBS, the fluorescence value was measured by POLARstar Omega (BMG Labtech, Germany) (Fig. 12a and Fig. 12b).

1-14. Determination of KD value of anti-hTRPV2 antibody The measurement of the dissociation constant between the anti-hTRPV2 antibodies (mAb001, mAb002) obtained in the above 1-11. and the ectodomain of hTRPV2 was carried out in the following manner. Synthesis of hTRPV2 extracellular domain-biotin (572-609) (addition of lysine to the C-terminus of the amino acid sequence represented by the amino acid sequence of SEQ ID NO: 1 in the sequence listing from positions 572 to 609 acid residues and biotin peptides). The antibody was captured (captured) by an immobilized anti-mouse IgG (Fc) antibody using a Biacore T200 (GE Healthcare Bioscience), and the assay was performed by a capture method using the antigen as the analyte. Anti-mouse IgG (Fc) antibody (Mouse Antibody Capture Kit, GE Healthcare Bioscience Co., Ltd.) was covalently bound to the sensor chip CM5 (GE Healthcare Bioscience Co., Ltd.) at ~15,000 RU by amine coupling method. Healthcare Bioscience). Immobilization was performed in the same manner for the reference pool. HBS-EP (10 mM HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% Surfactant P20) was used as running buffer. About 10 μg/ml of antibody solution was added to the chip immobilized with anti-mouse IgG (Fc) antibody at a flow rate of 10 μl/min for 180 seconds, and then a solution of hTRPV2 extracellular domain (572-609) was added at a flow rate of 30 μl/min. The serial solution was diluted (10-1000 nM) for 120 seconds and the dissociation phase was monitored for 900 seconds. As a regeneration solution, 10 mM Glycine-HCl (pH 1.7) was added for 180 seconds at a flow rate of 20 μl/min. Data Analysis Using the one-to-one binding model of Biacore Insight Evaluation Software (version 2.0), the binding rate constant ka, the dissociation rate constant kd and the dissociation constant (KD, KD=kd/ka) were calculated. As a result, the KD value of the anti-hTRPV2 antibody (mAb002) was 19.2 nM, and the KD value of the anti-hTRPV2 antibody (mAb001) was 188 nM.

1-15. Assessment of intracellular Ca ²⁺ influx inhibition in HEK293 cells using anti-human TRPV2 antibody. hTRPV2 expressing HEK293 cells were cultured to confluence by poly-D-Lysine-coated 96-well plates. state. The culture medium was removed, and 50 μl of 5 mM Cal520 (Cal520 (registered trademark), AM, AAT Bioquest) dissolved in BSS (Balanced Salt Solution) 0.1% BSA containing 0.5 mM Ca ²⁺ was added at 37°C. The cells were loaded with calcium indicator (Cal520) after exposure for 2.5 hours. After that, the Cal520 loading solution was removed, and 50 μl of a 100 mM cannabidiol (CBD) solution dissolved in BSS containing 0.5 mM Ca ²⁺ was added, and the solution was allowed to stand at 37° C. for 30 minutes. Thereafter, the CBD solution was removed, and anti-hTRPV2 antibodies ( ^mAb001 , mAb002), anti-mTRPV2 antibodies (88-2, 11-6), anti-mTRPV2 antibodies (anti-TRPV2 ( VRL1) (extracellular) antibody (#ACC-039) Alomon lab) and isotype control (IgG) diluted liquid 50 μl were contacted at 4°C for 1 hour. Then, the temperature was returned to 37°C, and after 2 minutes, 50 μl of 2 mM CaCl ₂ and BSS containing 10 mM probenecid were added to start the measurement. The preparations were made so that the final concentrations of _CaCl2 and probenecid were 1.25 mM and 5 mM, respectively. Fluorescence changes reflecting changes in intracellular Ca ²⁺ concentration over time were measured by a fluorescence luminescence kinetic analyzer FDSS7000. The inhibition of the anti-TRPV2 antibody was evaluated by calculating the reaction rate of the well to which the antibody was added, taking the reaction of the well to which only the antibody diluent was added as 100%. The _IC50 of the antibody (mAb002) was 8.9 nM (Figure 13a and Figure 13b). From the above results, it was found that mAb002 has Ca ²⁺ influx inhibitory activity via hTRPV2. In addition, although mAb001 did not exhibit Ca ²⁺ influx inhibitory activity, it observed the activity of binding to hTRPV2, and therefore it is considered useful when an anti-TRPV2 antibody is used as a diagnostic drug. BSS: Balanced Salt Solution (146 mM NaCl, 4 mM KCl, 2 mM MgCl ₂ , 0.5 mM CaCl ₂ , 10 mM glucose, and 10 mM HEPES/Tris, pH 7.2)

1-16. Epitope identification of anti-hTRPV2 antibody Plasmids (Flag-labeled at the C-terminus of hTRPV2) in which the amino acids of the hTRPV2 candidate peptides that became the epitopes of the anti-hTRPV2 antibody, respectively, were replaced by alanine were transfected into HEK293 cells using lipofectamine 3000. Cells were solubilized 24-48 hours after transfection with solubilization buffer (10 mM Tris HCl pH 7.4, 145 mM NaCl, 1 mM EDTA, 1% Np40). Proteins were separated by SDS-PAGE and transcribed to a hydrophobic membrane (PVDF). After blocking with 5% skim milk, wash with tris-buffered saline (TTBS) containing 0.1% Tween 20, and make it with anti-hTRPV2 antibody (mAb002) or anti-flag (anti- flag) antibody (MBL M2) for 12 hours. After washing with TTBS, it was reacted with a 1000-fold diluted HRP-labeled secondary antibody (Goat anti-Mouse IgG (H+L) secondary antibody, HRP Invitrogen) at room temperature for 2 hours. TTBS for cleaning. The chemiluminescence detection agent was prepared according to the manual, and the hydrophobic film was set in the chemiluminescence detection device for exposure, and then introduced into the camera. The expression of hTRPV2 was quantified by the chemiluminescent detection band formed by the labeling with the anti-flag antibody, and analyzed by image J together with the quantification of the detection band formed by the anti-hTRPV2 antibody (mAb002). The amino acid (EGQED, SEQ ID NO: 3) in which the band formed by the binding of the antibody to the peptide disappeared by alanine substitution was determined to be the epitope (Fig. 14a and Fig. 14b).

1-17. Expression of TRPV2 in muscle cells from healthy people and patients with muscular dystrophy and internalization using anti-human TRPV2 antibodies The immortalized human myoblast cell line Hu5/KD3 from healthy people and the immortalized myoblast cell line D4P4 from a patient with Josien's muscular dystrophy (DMD) were used (Biochem Biophys Res Commun 2006; 348: 1383-1388, Mech Dev 2008 ; 125: 257-269, Gene Ther 2011; 18: 857-866) evaluated the internalization of hTRPV2 using anti-hTRPV2 antibodies. In a dish coated with collagen type I, DMEM medium (in high-glucose (4.5 g/ml) DMEM (Sigma) supplemented with 20% FBS, 2% Ultroser G () containing 20% FBS was used. Biosepra, PALL) cultured Hu5/KD3 and D4P4 to a subconfluent state. Thereafter, the medium was changed to DMEM medium containing 2% horse serum and insulin-transferrin-selenium-A (Thermo) to induce differentiation. It was used after confirming the formation of myotubes 10 days after the initiation of differentiation. The expression of hTRPV2 in Hu5/KD3 and D4P4 was investigated. After the cells were fixed (5 minutes) with 2% PFA, they were washed with PBS. After treatment with 0.1% triton-X for 1 minute, blocking (Blocking-One, Nakaraitesque) was performed at 4°C with anti-hTRPV2 antibody (self-made rabbit purified antibody against the C-terminus of hTRPV2 (0.01 mg/ml) and diluted 50-fold. Anti-human Dystrophine antibody (NCL-DYS2, Nococastra) was incubated for 12 hours. After washing with PBS, secondary antibody was added and incubated for 1 hour at room temperature. After washing with PBS, the nuclei were denatured by DAPI. After staining, microscopy was performed using a confocal laser microscope (Olympus Fluoview FV1000). As a result, Dystrophine was expressed in Hu5/KD3, a cell line derived from healthy people, but not in D4P4, a cell line derived from DMD patients. In addition, hTRPV2 was expressed in any cell, but was present on the cell membrane in D4P4, a cell line derived from a DMD patient. In contrast, hTRPV2 was dispersed in the cytoplasm in Hu5/KD3, a cell line derived from a healthy human, and hTRPV2 expressed (Figure 15).

Next, the internalization of hTRPV2 using anti-hTRPV2 antibodies was investigated. Hu5/KD3 and D4P4 were differentiated into myotubes by the methods described above. Then, anti-hTRPV2 antibody (mAb002) (0.01 mg/ml) was added and incubated at 37°C for 1 hour. Then, the cells were fixed with 2% PFA (5 minutes), washed with PBS, and treated with Triton-X. Anti-hTRPV2 antibody (self-made rabbit purified antibody against the C-terminus of hTRPV2 (0.01 mg/ml) was incubated at 4°C for 12 hours. Further, a secondary antibody containing WGA (Thermo), a reagent for staining cell membranes, was added to the chamber. The cells were incubated at room temperature for 1 hour, washed with PBS, stained with DAPI, and examined by a confocal laser microscope (Olympus Fluoview FV1000). As a result, in Hu5/KD3 cells derived from a healthy human cell line, hTRPV2 was hardly observed in WGA-stained cell membranes. However, in D4P4, a cell line derived from a DMD patient, coexistence of WGA and hTRPV2 was observed. Furthermore, the coexistence with WGA was reduced by treating D4P4 from a DMD patient cell line with an anti-hTRPV2 antibody (mAb002) ( FIG. 16 ). Based on the above, the anti-hTRPV2 antibody (mAb002) exhibited hTRPV2 internalization in DMD-derived muscle cell lines.

1-18. Evaluation of intracellular Ca ²⁺ influx inhibition from patients with muscular dystrophy using anti-human TRPV2 antibody Immortal human myoblast cell line Hu5/KD3 from healthy people and immortal myoblast cell line D4P4 from DMD patients were used (Biochem Biophys Res Commun 2006; 348: 1383-1388, Mech Dev 2008; 125: 257-269, Gene Ther 2011; 18: 857-866), inhibition of intracellular ^{Ca influx} by anti-hTRPV2 antibody Evaluation. Hu5/KD3 and D4P4 were cultured and isodifferentiated into myotubes by the method described above. The culture medium was removed, and 5 μM Cal520 (Cal520 (registered trademark), AM, AAT Bioquest) dissolved in BSS 0.1% BSA containing 0.5 mM CaCl ₂ was added, and the cells were contacted at 37°C for 1.5 hours to load the cells with a calcium indicator ( Cal520). Then, the Cal520 loading solution was removed to prepare BSS containing 0.5 mM CaCl ₂ , and after 1 hour at room temperature, intracellular Ca ²⁺ elevation was measured at room temperature using a confocal laser microscope (Olympus Fluoview FV1000). At 1 minute, 3 minutes, and 5 minutes after the start of the measurement, the final concentration of the external solution was 5 mM CaCl ₂ , 100 μM cannabidiol (CBD), and 5 mM probenecid were added to measure the intracellular Ca ²⁺ reaction. . To confirm the effect of the antibody, BSS containing 0.5 mM CaCl ₂ loaded with Cal520 was contacted with anti-hTRPV2 antibody (mAb002) for 1 hour at room temperature, and intracellular Ca ²⁺ elevation was measured at room temperature using a confocal laser microscope. At 1 minute, 3 minutes, and 5 minutes after the start of the measurement, the final concentration of the external solution was 5 mM CaCl ₂ , 100 μM cannabidiol (CBD), and 5 mM probenecid were added to measure the intracellular Ca ²⁺ reaction. . BSS: Balanced Salt Solution (146 mM NaCl, 4 mM KCl, 2 mM MgCl ₂ , 0.5 mM CaCl ₂ , 10 mM glucose, 0.1% BSA, and 10 mM HEPES/Tris, pH 7.4) which As a result, an increase in intracellular Ca ²⁺ was observed by addition of 5 mM CaCl ₂ , CBD, probenecid in D4P4 derived from DMD patient cell line, but not in Hu5/ This phenomenon was not observed in KD3. Furthermore, when an anti-hTRPV2 antibody (mAb002) was added to D4P4 of a DMD patient-derived cell line, the effect of increasing intracellular Ca ²⁺ by 5 mM CaCl ₂ , CBD, and probenecid was not observed ( FIGS. 17 a and 17 b ). ). From the above, the anti-hTRPV2 antibody (mAb002) inhibited intracellular Ca ²⁺ influx in myocytes from muscular dystrophy patients.

Example 2. Production of anti-human TRPV2 humanized antibody 2-0. Selection and production of monoclonal antibodies against human TRPV2 The selection, production and expression of monoclonal antibodies against human TRPV2 are described. The present inventors designed humanized mAb002 antibody, and produced VH12+VL1, VH13+VL1, VH14+VL1, VH15+VL1 and VH16+VL1 by CDR grafting. Humanized mAb002 antibody (VH12+VL1): heavy chain CDR1 (SEQ ID NO: 4), heavy chain CDR2 (SEQ ID NO: 5), heavy chain CDR3 (SEQ ID NO: 6); light chain CDR1 (SEQ ID NO: 7), light chain CDR2 (RMS ), light chain CDR3 (SEQ ID NO: 8); heavy chain variable region (SEQ ID NO: 22); light chain variable region (SEQ ID NO: 23) Humanized mAb002 antibody (VH13+VL1): heavy chain CDR1 (SEQ ID NO: 4), heavy chain CDR2 (SEQ ID NO: 5), heavy chain CDR3 (SEQ ID NO: 6); light chain CDR1 (SEQ ID NO: 7), light chain CDR2 (RMS ), light chain CDR3 (SEQ ID NO: 8); heavy chain variable region (SEQ ID NO: 24); light chain variable region (SEQ ID NO: 23) Humanized mAb002 antibody (VH14+VL1): heavy chain CDR1 (SEQ ID NO: 4), heavy chain CDR2 (SEQ ID NO: 5), heavy chain CDR3 (SEQ ID NO: 6); light chain CDR1 (SEQ ID NO: 7), light chain CDR2 (RMS ), light chain CDR3 (SEQ ID NO: 8); heavy chain variable region (SEQ ID NO: 25); light chain variable region (SEQ ID NO: 23) Humanized mAb002 antibody (VH15+VL1): heavy chain CDR1 (SEQ ID NO: 4), heavy chain CDR2 (SEQ ID NO: 5), heavy chain CDR3 (SEQ ID NO: 6); light chain CDR1 (SEQ ID NO: 7), light chain CDR2 (RMS ), light chain CDR3 (SEQ ID NO: 8); heavy chain variable region (SEQ ID NO: 26); light chain variable region (SEQ ID NO: 23) Humanized mAb002 antibody (VH16+VL1): heavy chain CDR1 (SEQ ID NO: 4), heavy chain CDR2 (SEQ ID NO: 5), heavy chain CDR3 (SEQ ID NO: 6); light chain CDR1 (SEQ ID NO: 7), light chain CDR2 (RMS ), light chain CDR3 (SEQ ID NO: 8); heavy chain variable region (SEQ ID NO: 27); light chain variable region (SEQ ID NO: 23)

2-1. Construction and production of anti-human TRPV2 chimeric antibody cDNA was prepared from mouse fusions secreting mAb002, a functional inhibitory antibody against human TRPV2. The heavy and light chains encoding the cDNA fragments were isolated and sequenced. Next, a chimeric gene was produced by linking the mouse V region cDNA fragment to the human immunoglobulin constant region. The DNA sequences encoding the heavy and light chains of the chimeric antibody were inserted into the pCDNA3.4 vector to construct a full-length IgG expressing plasmid. The expression plasmid was introduced into Expi293F cells to express the chimeric antibody. The antibody was purified, and the protein concentration and purity were determined by OD280 and SDS-PAGE. Purified IgG was confirmed by SDS-PAGE under non-reducing conditions as a band of approximately 150 kDa. Furthermore, it was evaluated from the results of SDS-PAGE that the purity of IgG was considered to be 90% or more.

2-2. Confirmation of the binding of the anti-human TRPV2 chimeric antibody to hTRPV2 Using the hTRPV2 expressing 293 cells described in Example 1-12. and the negative control HEK293 cells, the hTRPV2 anti-human TRPV2 chimeric antibody was evaluated by the following method binding activity. Serially diluted chimeric antibody or isotype control (human IgG, Biolegend) was added to approximately ¹ x 105 hTRPV2 forced expressing HEK cells or HEK cells and incubated at 4°C for 40 minutes. After washing with PBS, secondary antibody (1 μg/ml, Alexa Fluor647-labeled affinity-purified goat anti-human (Affinipure Goat Anti-Human) IgG (H+L), Thermo) was added to the cells, followed by incubation at 4°C for 30 minutes . After washing with PBS, the binding of cells was analyzed by flow cytometry (FACS Canto, BD Bioscience). As a result, it was confirmed that the chimeric antibody binds to hTRPV2 expressing 293 cells.

2-3. Humanization Design of Antibody Using Back Mutation The structure of the parent antibody that became the template was modeled by computer-aided homology modeling programs. Humanized antibody systems are designed by CDR grafting from the parental antibody followed by mutation introduction using back mutations. Specifically, the CDRs of the parental antibodies were grafted into human recipients (human IgG) to obtain humanized light chains and humanized heavy chains of each parental antibody. Residues believed to be important for binding activity were selected from the CDR regions, framework regions, and amino acids at the VH-VL interface of the humanized antibody for substitution with that residue in the parent antibody. Affinity was studied by combining 16 humanized heavy chains with 3 humanized light chains that had been back-mutated.

2-4. Construction and production of anti-human TRPV2 humanized antibody The 16 humanized heavy chains and 3 humanized light chains that have been back mutated were combined to synthesize the DNA sequences encoding humanized IgG heavy and light chains, and inserted into the pCDNA3.4 vector to construct a full-length IgG. performance body. Expression plasmids were introduced into Expi293F cells to express the humanized antibody. The binding activity was evaluated using the humanized antibody contained in the culture supernatant.

2-5. Evaluation of the binding of the anti-human TRPV2 humanized antibody in the culture supernatant to hTRPV2 Using the hTRPV2 expressing 293 cells described in Example 1-12. and the negative control HEK293 cells, the hTRPV2-binding activity of the anti-human TRPV2 humanized antibody was evaluated by the method described in Example 2-2. Humanized antibodies were ranked by EC50 for binding to hTRPV2 expressing 293 cells. The results are shown in Tables 1 to 5.

[Table 1] dilution VH1+VL1 VH1+VL2 VH1+VL3 VH2+VL1 VH2+VL2 VH2+VL3 VH3+VL1 VH3+VL2 1 118 193 135 859 567 1119 1077 864 1/3 119 141 117 1022 790 1229 1202 881 1/9 106 115 108 920 684 918 956 684 1/27 97.7 113 99.1 751 542 718 783 609 1/81 90.9 94.2 89.4 600 463 579 672 488 1/243 110 92.2 105 382 401 423 493 437 1/729 87.2 101 96.7 272 359 298 328 313 1/2187 91 102 92.6 195 222 220 249 232 1/6561 102 92.9 98.3 157 165 160 190 168 1/19683 114 94.7 93.3 121 134 144 165 158 1/59049 99.6 95.9 97.8 108 133 115 151 144 1/177147 97.2 101 96.7 98.5 101 138 132 134 EC50 ~7.984 ~1425 28.89 0.9342 0.7138 5.132 1.944 2.638

[Table 2] dilution VH3+VL3 VH4+VL1 VH4+VL2 VH4+VL3 VH5+VL1 VH5+VL2 VH5+VL3 VH6+VL1 1 1090 196 233 201 221 315 227 167 1/3 1199 184 236 206 169 236 193 156 1/9 882 162 172 149 152 166 175 137 1/27 783 138 143 134 134 141 142 132 1/81 613 131 138 130 128 135 132 131 1/243 481 120 120 126 104 129 133 129 1/729 333 110 133 112 137 139 135 129 1/2187 244 134 135 143 110 136 139 135 1/6561 191 138 145 139 124 129 138 137 1/19683 160 121 154 144 136 110 135 128 1/59049 145 138 126 128 140 138 138 138 1/177147 147 126 153 121 139 139 137 134 EC50 1.306 7.474 ~9.062 ~14.08 107.5 31.17 32.51 12.94

[table 3] dilution VH6+VL2 VH6+VL3 Chimera human IgG NC PBS 1 157 199 881 146 110 138 1/3 166 211 1165 118 101 102 1/9 129 166 934 109 98.3 103 1/27 104 113 772 103 99.5 104 1/81 101 100 688 98.4 96.2 96.9 1/243 108 96.1 645 97.1 96.6 104 1/729 100 103 449 98.3 102 91.4 1/2187 105 97.2 320 102 100 105 1/6561 113 102 222 107 101 101 1/19683 104 105 169 103 102 101 1/59049 112 105 140 103 112 107 1/177147 140 108 126 110 125 106 EC50 ~13.22 8.911 0.385 0.000661 NA NA

[Table 4] dilution VH7+VL1 VH8+VL1 VH9+VL1 VH10＋VL1 VH11＋＋VL1 VH7+VL2 VH8+VL2 VH9+VL2 1 142 145 118 115 117 110 111 126 1/3 148 149 130 108 141 115 137 116 1/9 131 116 117 111 120 138 130 126 1/27 111 136 112 113 128 119 119 115 1/81 105 107 108 113 110 122 114 128 1/243 111 111 124 106 111 120 152 148 1/729 128 96.2 107 146 131 150 146 155 1/2187 143 103 144 148 154 135 142 145 1/6561 99 148 117 120 138 154 162 157 1/19683 94.7 125 154 114 146 152 136 166 1/59049 108 120 148 145 168 134 109 163 1/177147 135 116 149 141 152 173 144 128 EC50 ~5.166 ~3.678 0.01183 ~0.1173 ~0.07161 ~0.0 ~0.2504 1.092

[table 5] dilution VH10＋VL2 VH11＋＋VL2 VH7+VL3 VH8+VL3 VH9+VL3 VH10＋VL3 VH11＋＋VL3 Chimera human IgG 1 147 146 146 149 128 150 140 411 166 1/3 149 154 167 169 168 154 163 471 176 1/9 163 149 159 153 178 149 171 557 173 1/27 148 144 152 152 170 169 153 403 157 1/81 119 137 158 137 136 142 142 423 159 1/243 137 144 137 166 142 144 140 368 153 1/729 136 162 137 156 122 147 134 284 163 1/2187 137 130 140 138 158 139 159 225 164 1/6561 143 161 161 139 162 136 169 152 191 1/19683 136 157 124 156 147 143 146 136 202 1/59049 140 156 165 152 158 153 123 145 167 1/177147 151 148 126 139 127 141 120 126 164 EC50 NA ~0.01736 ~0.4182 0.2144 NA ~0.3055 0.002028 0.1012 NA

2-6. Generation of anti-human TRPV2 humanized antibody and confirmation of its binding to human TRPV2 In addition to the 3 humanized antibodies (VH2+VL1, VH3+VL1, VH3+VL3) that showed high affinity among the previously designed humanized antibodies, 7 humanized antibodies (VH12+VL1, VH13+VL1, VH12+VL3, VH13+VL3, VH14+VL1, VH15+VL1, VH16+VL1). For these 10 humanized antibodies, DNA sequences were synthesized and inserted into pCDNA3.4 vector to construct full-length IgG expressing plasmids. Expression plasmids were introduced into Expi293F cells to express the humanized antibody. The antibody was purified, and the protein concentration and purity were determined by OD280 and SDS-PAGE. Purified IgG was confirmed by SDS-PAGE under non-reducing conditions as a band of approximately 150 kDa. Furthermore, according to the result of SDS-PAGE, the purity of IgG was considered to be 90% or more (Fig. 18a, Fig. 18b, Fig. 19).

2-7. hTRPV2 binding activity of anti-human TRPV2 humanized antibody Using the hTRPV2 expressing 293 cells described in Example 1-12. and the negative control HEK293 cells, the hTRPV2-binding activity of the anti-human TRPV2 humanized antibody was evaluated by the method described in Example 2-2. As a result, VH12+VL1, VH13+VL1, VH14+VL1, VH15+VL1, and VH16+VL1 exhibited binding properties equivalent to those of chimeric antibodies. The results are shown in Tables 6 to 8. In Table 8, the values marked with * were not subjected to EC50 calculation.

[Table 6] μg/ml Log(μg/ml) VH12+VL1 VH13＋VL1 VH12+VL3 VH13＋VL3 VH2+VL1 45 1.653213 5251 5010 5039 5335 5089 15 1.176091 5072 5444 4908 5191 4387 5 0.69897 4507 5091 4652 4687 3474 1.666667 0.221849 3570 4582 4291 4498 2159 0.555556 -0.25527 2198 3371 3358 3722 1238 0.185185 -0.73239 1145 1918 1958 2589 659 0.061728 -1.20952 635 1132 1035 1535 375 0.020576 -1.68664 389 711 650 935 265 0.006859 -2.16376 273 511 407 565 212 0.002286 -2.64088 213 399 283 412 181 0.000762 -3.118 183 303 223 274 150 0.000254 -3.59512 153 250 183 249 138 EC50 0.8955 0.3575 0.3119 0.2247 3.143

[Table 7] μg/ml Log(μg/ml) VH3+VL1 VH3+VL3 Chimeric antibody human IgG PBS 45 1.653213 5465 4684 6321 216 137 15 1.176091 5025 4426 6454 172 151 5 0.69897 4547 4079 6173 154 136 1.666667 0.221849 3339 3247 5666 131 145 0.555556 -0.25527 1930 2234 4858 131 151 0.185185 -0.73239 1018 1131 3906 136 149 0.061728 -1.20952 577 654 2692 129 177 0.020576 -1.68664 401 424 1695 136 148 0.006859 -2.16376 298 300 1089 151 181 0.002286 -2.64088 238 240 746 182 146 0.000762 -3.118 192 178 503 163 173 0.000254 -3.59512 167 176 412 161 151 EC50 1.195 0.7525 0.1261 NA NA

[Table 8] μg/ml Log(μg/ml) VH14+VL1 VH15+VL1 VH16+VL1 Chimeric antibody human IgG PBS 45 1.653213 2059.0* 1693.0* 1741.0* 1817.0* 170.0* 177 15 1.176091 3131.0* 2953.0* 3146.0* 2404.0* 163.0* 156 5 0.69897 3523 4516 3718 5667 148 165 1.666667 0.221849 3443 3954 3611 6155 147 156 0.555556 -0.25527 1921 2052 2001 4790 153 157 0.185185 -0.73239 944 975 996 2949 163 161 0.061728 -1.20952 502 520 518 1605 158 152 0.020576 -1.68664 319 396 312 857 161 164 0.006859 -2.16376 236 326 220 516 162 158 0.002286 -2.64088 209 241 206 385 161 166 0.000762 -3.118 195 235 187 316 229 165 0.000254 -3.59512 183 286 185 269 220 234 EC50 0.5256 0.7041 0.5313 0.2 NA NA

2-8. Evaluation of Ca ²⁺ Influx Inhibition in Human TRPV2 Forced Expressing Cells of Anti-human TRPV2 Humanized Antibody The intracellular Ca ²⁺ of anti-human TRPV2 humanized antibody human TRPV2 forced expression cells was evaluated by the method described below Influx inhibitory activity. hTRPV2 expressing HEK293 cells were cultured to a confluent state by poly-D-lysine-coated 96-well plates. The culture medium was removed, and 50 μl of 5 μM Cal520 (Cal520 (registered trademark), AM, AAT Bioquest) dissolved in BSS 0.1% BSA containing 1.8 mM Ca ²⁺ was added, and the cells were exposed to calcium for 2.5 hours at 37°C to load intracellular calcium. indicator (Cal520). Then, anti-hTRPV2 humanized antibodies (VH14+VL1, VH15+VL1, VH16+VL1) and isotype control (IgG, Thermo) 5 μg/mL were contacted at room temperature for 1 hour, and intracellular Ca was measured by confocal laser microscope at room temperature. ²⁺ rise. 100 and 300 seconds after the start of the measurement, the external solution was added to the final concentration of 100 μM cannabidiol (CBD) and 5 mM probenecid, respectively, and the intracellular Ca ²⁺ reaction was measured. BSS: Balanced Salt Solution (146 mM NaCl, 4 mM KCl, 2 mM MgCl ₂ , 1.8 mM CaCl ₂ , 10 mM glucose, 0.1% BSA, and 10 mM HEPES/Tris, pH 7.4) which As a result, in the anti-human TRPV2 humanized antibody (VH14+VL1, VH15+VL1, VH16+VL1) group, the increase in intracellular Ca ²⁺ induced by CBD and probenecid was suppressed compared with the isotype control (IgG) addition group (Fig. 20). Based on the above, the anti-human TRPV2 humanized antibody, like mAb002, inhibited intracellular Ca ²⁺ influx in human TRPV2-expressing cells.

2-9. Evaluation of intracellular Ca ²⁺ influx inhibition from patients with muscular dystrophy by anti-human TRPV2 humanized antibody The immortal myoblast cell line D4P4 from DMD patients used in Example 1-17. was used to evaluate Inhibition of intracellular Ca ²⁺ influx by anti-human TRPV2 humanized antibody. D4P4 was cultured and differentiated into myotubes by the method described in Examples 1-17. The culture medium was removed, and 5 μM Cal520 (Cal520 (registered trademark), AM, AAT Bioquest) dissolved in BSS 0.1% BSA containing 0.5 mM CaCl ₂ was added, and the cells were contacted at 37°C for 1.5 hours to load the cells with a calcium indicator ( Cal520). After that, the anti-human TRPV2 humanized antibodies (VH14+VL1, VH15+VL1, VH16+VL1) and isotype control (IgG, Thermo) 5 μg/mL were contacted at room temperature for 1 hour, and the cells were measured by confocal laser microscope at room temperature. Ca ²⁺ rises. After 100 seconds from the start of the measurement, the outer solution was added to the final concentration of 5 mM CaCl ₂ and 100 μM cannabidiol (CBD), respectively, and the intracellular Ca ²⁺ reaction was measured. BSS: Balanced Salt Solution (146 mM NaCl, 4 mM KCl, 2 mM MgCl ₂ , 0.5 mM CaCl ₂ , 10 mM glucose, 0.1% BSA, and 10 mM HEPES/Tris, pH 7.4) which As a result, in D4P4 supplemented with an isotype control (IgG), intracellular Ca ²⁺ increased by the addition of 5 mM CaCl ₂ and CBD, but when anti-human TRPV2 humanized antibodies (VH14+VL1, VH15+VL1, VH16+VL1), the increase in intracellular Ca ²⁺ produced by 5 mM CaCl ₂ and CBD was inhibited (Fig. 21a, Fig. 21b). From the above, the anti-hTRPV2 humanized antibody, like mAb002, inhibited intracellular Ca ²⁺ influx in muscle cells from patients with muscular dystrophy. [Industrial Availability]

The anti-human TRPV2 antibody or fragment thereof of the present invention is useful for the treatment or prevention of muscle disease and/or cardiac disease. Furthermore, the anti-human TRPV2 antibody or fragment thereof of the present invention is useful as a diagnostic drug for cardiomyocytes and/or myocyte degenerative diseases. This application is based on Japanese Patent Application No. 2020-136837 (filing date: August 13, 2020 ) for which it applied in Japan, the entire contents of which are incorporated in this specification.

Figure 1 is a schematic diagram of a vector incorporating VH and VL genes. Figure 2 shows the results of a confirmatory analysis of anti-human TRPV2 antibodies in mouse antisera. In the case of using an anti-His-tag antibody, bands (2-a) were confirmed at the positions of 100 kDa and 75 kDa. In addition, when the immunized mouse antiserum was used for the primary antibody, bands (2-b) were also confirmed at the positions of 100 kDa and 75 kDa. Figure 3 shows the results of evaluating the binding activity of antiserum using hTRPV2 expressing HEK293 cells. Fig. 4 shows the results of confirming the activity of each antiserum using a streptavidin plate (Thermo Scientific) in which biotinylated hTRPV2 expressing HEK293 cells were solubilized and immobilized. Fig. 5 shows the results of cell ELISA (Cell enzyme linked immunosorbent assay) using hTRPV2 expressing HEK293 cells using phage colonies recovered by panning. Fig. 6A shows the results of cell ELISA performed on the prepared Escherichia coli culture supernatant (01a) using biotinylated hTRPV2 expressing HEK293 cells solubilized and then immobilized on a streptavidin plate. No one with higher binding activity was confirmed from 01a, but monoclonal antibodies 01a005, 01a016, 01a018, and 01a033 showed slightly higher binding activity in biotinylated hTRPV2 expressing HEK293 cells. Fig. 6B shows the results of cell ELISA performed on the prepared E. coli culture supernatant (02a) using biotinylated hTRPV2 expressing HEK293 cells solubilized and then immobilized on a streptavidin plate. All of the monoclonal antibodies obtained from 02a showed high specificity to biotinylated hTRPV2 expressing HEK293 cells. Figure 7 shows the representative clones 01a033 (mAb001) and 02a001 (mAb002), using a 10-fold concentrate of E. coli culture supernatant, by FACS (Fluorescence-Activated Cell Sorting, Fluorescence-Activated Cell Sorting) Results of the study of binding to hTRPV2 expressing HEK293 cells. Figure 8 is the preparation of antibody-binding beads (beads) from E. coli culture supernatants of 3 clones of selected representative clones 01a033 (mAb001), 02a001 (mAb002) and negative control antibodies, and expressed HEK293 cell soluble using hTRPV2 results of immunoprecipitation experiments. Figure 9 is a graph using OD (optical density) 280 nm measurement and SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, ten The results of concentration estimation and purity confirmation of sodium dialkyl sulfate-polyacrylamide gel electrophoresis). Fig. 10 shows the results of confirmation of IgG antibody response to hTRPV2 expressing cells. Figure 11 shows the results of microscopic examination using a confocal laser microscope for the crossover of the anti-mouse TRPV2 antibody (mAb88-2) and the anti-human TRPV2 antibody (mAb002). Figure 12 shows the comparison of anti-human TRPV2 antibody (mAb002), anti-human TRPV2 antibody (mAb001), anti-mouse TRPV2 antibody (88-2) and isotype control against hTRPV2-expressing HEK293 cells (12a) and mTRPV2-expressing HEK293 cells (12b) The result of cross-confirmation. Figure 13 shows the results of evaluating the inhibition of Ca2 ^{+ influx} into HEK293 cells by anti-TRPV2 antibodies (13a and 13b). Figure 14 shows the results of epitope identification of anti-human TRPV2 antibodies (14a and 14b). Fig. 15 is a graph showing the expression of TRPV2 and dystrophin in muscle cell lines derived from healthy people and patients with Crushing's muscular dystrophy. In Figure 15, nuclei within myotubes were stained by DAPI (4',6-diamidino-2-phenylindole, 4',6-diamidino-2-phenylindole). Figure 16 is a graph showing the performance of TRPV2 on muscle cell lines derived from healthy people and patients with Crushing's muscular dystrophy, and muscle cell lines from patients with Crushing's muscular dystrophy treated with mAb002. In Fig. 16, nuclei in myotubes were stained by DAPI, and cell membranes were stained by WGA (wheat germ agglutinin). Figure 17a is a graph of the evaluation of intracellular Ca ²⁺ influx using CBD (Cannabidiol)-probenecid in muscle cell lines derived from healthy individuals and patients with Crohn's muscular dystrophy. Figure 17b is a graph evaluating the effect of mAb002 on intracellular Ca ²⁺ influx with CBD-probenecid in muscle cell line D4P4 from DMD patients. Figure 18a shows the results of SDS-PAGE in reduced and non-reduced states of anti-human TRPV2 humanized antibodies (VH2+VL1, VH3+VL1, VH3+VL3). M represents a marker (Takara, 3452), 1 to 3 represent reduced states (1: VH2+VL1, 2: VH3+VL1, 3: VH3+VL3), 4 to 7 represent non-reduced states (4: VH2+VL1, 5: VH3+VL1, 6 : VH3+VL3, 7: Human IgG). Figure 18b shows the results of SDS-PAGE in reduced and non-reduced states of anti-human TRPV2 humanized antibodies (VH12+VL1, VH13+VL1, VH12+VL3, VH13+VL3). M represents the marker (Takara, 3452), 1 to 4 represent reduced states (1: VH12+VL1, 2: VH12+VL1, 3: VH12+VL3, 4: VH13+VL3), 5 to 9 represent non-reduced states (5: VH12+VL1, 6: VH12+VL1, 7: VH12+VL3, 8: VH13+VL3, 7: human IgG). Figure 19 shows the results of SDS-PAGE of anti-human TRPV2 humanized antibodies (VH14+VL1, VH15+VL1, VH16+VL1) under reduced and non-reduced states. M represents the marker (Takara, 3452), 1 to 3 represent reduced states (1: VH14+VL1, 2: VH15+VL1, 3: VH16+VL1), 4 to 7 represent non-reduced states (4: VH14+VL1, 5: VH15+VL1, 6: VH16+VL1, 7: Human IgG). Fig. 20 is a graph evaluating the effect of anti-human TRPV2 humanized antibody on intracellular Ca ²⁺ influx using CBD and probenecid in human TRPV2-enforced cell lines. Figure 21a is a graph evaluating the effect of human TRPV2 humanized antibody on intracellular Ca ²⁺ influx with 5 mM CaCl ₂ , CBD in muscle cell lines from a patient with Crouching's muscular dystrophy. Fig. 21b is a graph showing the values of IgG, VH14, VH15, VH16 for ΔFmax-F0 of Fig. 21a.

           <![CDATA[<110> NATIONAL CEREBRAL AND CARDIOVASCULAR CENTER]]> SUMITOMO DAINIPPON PHARMA CO., LTD.<! [CDATA[<120> Anti-Human TRPV2 Antibody]]> <![CDATA[<130> 093169]]> <![CDATA[<150> JP 2020-136837]]> <![CDATA[<151> 2020- 08-13]]> <![CDATA[<160> 27 ]]> <![CDATA[<170> PatentIn v3.5]]> <![CDATA[<210> 1]]> <![CDATA[ <211> 764]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<220>]]> <![CDATA[<221 > MISC_FEATURE]]> <![CDATA[<222> (574)..(595)]]> <![CDATA[<223> epitope (20 amino acids)]]> <![CDATA[< 220>]]> <![CDATA[<221> MISC_FEATURE]]> <![CDATA[<222> (579)..(583)]]> <![CDATA[<223> Epitope (5 amines base acid)]]> <![CDATA[<400> 1]]> Met Thr Ser Pro Ser Ser Ser Pro Val Phe Arg Leu Glu Thr Leu Asp 1 5 10 15 Gly Gly Gln Glu Asp Gly Ser Glu Ala Asp Arg Gly Lys Leu Asp Phe 20 25 30 Gly Ser Gly Leu Pro Pro Met Glu Ser Gln Phe Gln Gly Glu Asp Arg 35 40 45 Lys Phe Ala Pro Gln Ile Arg Val Asn Leu Asn Tyr Arg Lys Gly Thr 50 55 60 Gly Ala Ser Gln Pro Asp Pro Asn Arg Phe Asp Arg Asp Arg Leu Phe 65 70 75 80 Asn Ala Val Ser Arg Gly Val Pro Glu Asp Leu Ala Gly Leu Pro Glu 85 90 95 Tyr Leu Ser Lys Thr Ser Lys Tyr Leu Thr Asp Ser Glu Tyr Thr Glu 100 105 110 Gly Ser Thr Gly Lys Thr Cys Leu Met Lys Ala Val Leu Asn Leu Lys 115 120 125 Asp Gly Val Asn Ala Cys Ile Leu Pro Leu Leu Gln Ile Asp Arg Asp 130 135 140 Ser Gly Asn Pro Gln Pro Leu Val Asn Ala Gln Cys Thr Asp Asp Tyr 145 150 155 160 Tyr Arg Gly His Ser Ala Leu His Ile Ala Ile Glu Lys Arg Ser Leu 165 170 175 Gln Cys Val Lys Leu Leu Val Glu Asn Gly Ala Asn Val His Ala Arg 180 185 190 Ala Cys Gly Arg Phe Phe Gln Lys Gly Gln Gly Thr Cys Phe Tyr Phe 195 200 205 Gly Glu Leu Pro Leu Ser Leu Ala Ala Cys Thr Lys Gln Trp Asp Val 210 215 220 Val Ser Tyr Leu Leu Glu Asn Pro His Gln Pro Al a Ser Leu Gln Ala 225 230 235 240 Thr Asp Ser Gln Gly Asn Thr Val Leu His Ala Leu Val Met Ile Ser 245 250 255 Asp Asn Ser Ala Glu Asn Ile Ala Leu Val Thr Ser Met Tyr Asp Gly 260 265 270 Leu Leu Gln Ala Gly Ala Arg Leu Cys Pro Thr Val Gln Leu Glu Asp 275 280 285 Ile Arg Asn Leu Gln Asp Leu Thr Pro Leu Lys Leu Ala Ala Lys Glu 290 295 300 Gly Lys Ile Glu Ile Phe Arg His Ile Leu Gln Arg Glu Phe Ser Gly 305 310 315 320 Leu Ser His Leu Ser Arg Lys Phe Thr Glu Trp Cys Tyr Gly Pro Val 325 330 335 Arg Val Ser Leu Tyr Asp Leu Ala Ser Val Asp Ser Cys Glu Glu Asn 340 345 350 Ser Val Leu Glu Ile Ile Ala Phe His Cys Lys Ser Pro His Arg His 355 360 365 Arg Met Val Val Leu Glu Pro Leu As n Lys Leu Leu Gln Ala Lys Trp 370 375 380 Asp Leu Leu Ile Pro Lys Phe Phe Leu Asn Phe Leu Cys Asn Leu Ile 385 390 395 400 Tyr Met Phe Ile Phe Thr Ala Val Ala Tyr His Gln Pro Thr Leu Lys 405 410 415 Lys Gln Ala Ala Pro His Leu Lys Ala Glu Val Gly Asn Ser Met Leu 420 425 430 Leu Thr Gly His Ile Leu Ile Leu Leu Gly Gly Ile Tyr Leu Leu Val 435 440 445 Gly Gln Leu Trp Tyr Phe Trp Arg Arg His Val Phe Ile Trp Ile Ser 450 455 460 Phe Ile Asp Ser Tyr Phe Glu Ile Leu Phe Leu Phe Gln Ala Leu Leu 465 470 475 480 Thr Val Val Ser Gln Val Leu Cys Phe Leu Ala Ile Glu Trp Tyr Leu 485 490 495 Pro Leu Leu Val Ser Ala Leu Val Leu Gly Trp Leu Asn Leu Leu Tyr 500 505 510 Tyr Thr Arg Gly Phe Gl n His Thr Gly Ile Tyr Ser Val Met Ile Gln 515 520 525 Lys Val Ile Leu Arg Asp Leu Leu Arg Phe Leu Leu Ile Tyr Leu Val 530 535 540 Phe Leu Phe Gly Phe Ala Val Ala Leu Val Ser Leu Ser Gln Glu Ala 545 550 555 560 Trp Arg Pro Glu Ala Pro Thr Gly Pro Asn Ala Thr Glu Ser Val Gln 565 570 575 Pro Met Glu Gly Gln Glu Asp Glu Gly Asn Gly Ala Gln Tyr Arg Gly 580 585 590 Ile Leu Glu Ala Ser Leu Glu Leu Phe Lys Phe Thr Ile Gly Met Gly 595 600 605 Glu Leu Ala Phe Gln Glu Gln Leu His Phe Arg Gly Met Val Leu Leu 610 615 620 Leu Leu Leu Ala Tyr Val Leu Leu Thr Tyr Ile Leu Leu Leu Asn Met 625 630 635 640 Leu Ile Ala Leu Met Ser Glu Thr Val Asn Ser Val Ala Thr Asp Ser 645 650 655 Trp Ser Il e Trp Lys Leu Gln Lys Ala Ile Ser Val Leu Glu Met Glu 660 665 670 Asn Gly Tyr Trp Trp Cys Arg Lys Lys Gln Arg Ala Gly Val Met Leu 675 680 685 Thr Val Gly Thr Lys Pro Asp Gly Ser Pro Asp Glu Arg Trp Cys Phe 690 695 700 Arg Val Glu Glu Val Asn Trp Ala Ser Trp Glu Gln Thr Leu Pro Thr 705 710 715 720 Leu Cys Glu Asp Pro Ser Gly Ala Gly Val Pro Arg Thr Leu Glu Asn 725 730 735 Pro Val Leu Ala Ser Pro Pro Lys Glu Asp Glu Asp Gly Ala Ser Glu 740 745 750 Glu Asn Tyr Val Pro Val Gln Leu Leu Gln Ser Asn 755 760 <![CDATA[<210> 2]]> <![CDATA[<211> 22]] > <![CDATA[<212> PRT]]> <![CDATA[<213> Homo sapiens]]> <![CDATA[<400> 2]]> Ser Val Gln Pro Met Glu Gly Gln Glu Asp Glu Gly Asn Gly Ala Gln 1 5 10 15 Tyr Arg Gly Ile Leu Glu 20 <![CDATA[<210> 3]]> <![CDATA[<211> 5]]> <![CDATA[<212> PRT]] > <![ CDATA[<213> Homo sapiens]]> <![CDATA[<400> 3]]> Glu Gly Gln Glu Asp 1 5 <![CDATA[<210> 4]]> <![CDATA[<211> 8 ]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> 02a001 Antibody ( 002 antibody) heavy chain CDR1]]> <![CDATA[<400> 4]]> Gly Phe Ser Leu Thr Ser Phe Gly 1 5 <![CDATA[<210> 5]]> <![CDATA[< 211> 7]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Heavy chain CDR2 of 02a001 antibody (002 antibody)]]> <![CDATA[<400> 5]]> Ile Trp Ser Gly Gly Ile Thr 1 5 <![CDATA[<210> 6]]> <![CDATA [<211> 10]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[< 223> Heavy chain CDR3 of 02a001 antibody (002 antibody)]]> <![CDATA[<400> 6]]> Leu Tyr Ser His Pro His Ala Met Asp Tyr 1 5 10 <![CDATA[<210> 7] ]> <![CDATA[<211> 11]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> 02a001 antibody (002 antibody) light chain CDR1]]> <![CDATA[<400> 7]]> Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr 1 5 10 <![ CDATA[<210> 8]]> <![CDATA[<211> 9]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA [<220>]]> <![CDATA[<2 23> Light chain CDR3 of 02a001 antibody (002 antibody)]]> <![CDATA[<400> 8]]> Met Gln His Leu Glu Tyr Pro Leu Thr 1 5 <![CDATA[<210> 9]]> <![CDATA[<211> 8]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220>]]> <! [CDATA[<223> Heavy chain CDR1 of 01a033 antibody (001 antibody)]]> <![CDATA[<400> 9]]> Gly Phe Ser Leu Thr Thr Tyr Gly 1 5 <![CDATA[<210> 10 ]]> <![CDATA[<211> 7]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220>]] > <![CDATA[<223> Heavy chain CDR2 of 01a033 antibody (001 antibody)]]> <![CDATA[<400> 10]]> Met Gly Trp Asp Gly Lys Lys 1 5 <![CDATA[<210 > 11]]> <![CDATA[<211> 9]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220> ]]> <![CDATA[<223> 01a033 antibody (001 antibody) heavy chain CDR3]]> <![CDATA[<400> 11]]> Asp Gly Gly Tyr Thr Trp Phe Ala Tyr 1 5 <![ CDATA[<210> 12]]> <![CDATA[<211> 12]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA [<220>]]> <![CDATA[<223> 01a033 antibody (001 antibody) light chain CDR1]]> <![CDATA[<400> 12]]> Gln Ser Leu Leu Asn Ser Arg Thr Arg Lys Asn Tyr 1 5 10 <![CDATA[<210> 13]]> <![CDATA[<211> 8]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual sequence]] > <![CDATA[<220>]]> <![CDATA[<223> 01a033 antibody (001 antibody) light chain CDR3]]> <![CDATA[<400> 13]]> Lys Gln Ser Tyr Asn Leu Phe Thr 1 5 <![CDATA[<210> 14]]> <![CDATA[<211> 118]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> 02a001 antibody (002 antibody) heavy chain variable region]]> <![CDATA[<400> 14]]> Gln Val Gln Leu Lys Gln Ser Gly Pro Val Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Phe 20 25 30 Gly Ile His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Ser Gly Gly Ile Thr His Tyr Asn Ser Ala Leu Met 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Lys Leu Tyr Ser His Pro His Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <![CDATA[<210> 15]]> <![CDATA[<211> 112]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>] ]> <![CDATA[<223> 02a001 antibody (002 antibody) Light chain variable region]]> <![CDATA[<400> 15]]> Asp Ile Val Met Thr Gln Ala Ala Pro Ser Val Pro Val Thr Pro Gly 1 5 10 15 Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Ile Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Ala Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His 85 90 95 Leu Glu Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <![CDATA[<210> 16]]> <![CDATA[<211> 117]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> 01a033 antibody (001 antibody) heavy chain variable region]]> <![CDATA[<400> 16]]> Gln Val Gln Leu Lys Glu Ser Gly Pro Asp Leu Val Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Thr Tyr 20 25 30 Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Thr Met Gly Trp Asp Gly Lys L ys Tyr Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Asn Gln Val Phe Leu 65 70 75 80 Lys Leu Ser Ser Leu Gln Thr Glu Asp Thr Ala Met Tyr Tyr Cys Thr 85 90 95 Arg Asp Gly Gly Tyr Thr Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ala 115 <![CDATA[<210> 17]]> <![CDATA[<211> 112]]> < ![CDATA[<212> PRT]]> <![CDATA[<213> artificial sequence]]> <![CDATA[<220>]]> <![CDATA[<223> 01a033 antibody (001 antibody) Light chain variable region]]> <![CDATA[<400> 17]]> Asn Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr Asn Leu Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <![CDATA[<210> 18]]> <![CDATA[<211> 118]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]] > <![CDATA[<220>]]> <![CDATA[<223> 02a003 or 02a005 heavy chain variable region]]> <![CDATA[<400> 18]]> Gln Val Gln Leu Lys Gln Ser Gly Pro Val Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Phe 20 25 30 Gly Ile His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Ser Gly Gly Ile Thr His Tyr Asn Ser Asp Leu Met 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Lys Leu Tyr Ser His Pro His Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <![CDATA[<210> 19]]> <![CDATA[<211> 118]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220>]]> <! [CDATA[<223> 02a006 antibody heavy chain variable region]]> <![CDATA[<400> 19]]> Gln Val Gln Leu Lys Gln Ser Gly Pro Val Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Phe 20 25 30 Gly Ile His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Ser Gly Gly Ile Thr His Tyr Asn Ser Ala Leu Met 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Asp Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Lys Leu Tyr Ser His Pro His Ala Met Asp Tyr Trp Gly Thr Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <![CDATA[<210> 20]]> <![CDATA[<211> 19] ]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Secret Message Sequence] ]> <![CDATA[<400> 20]]> Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser <![CDATA[<210> 21]]> < ![CDATA[<211> 20]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220>]]> <![ CDATA[<223> Secretory Message Sequence]]> <![CDATA[<400> 21]]> Met Arg Leu Leu Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Val Pro 1 5 10 15 Gly Ser Ser Gly 20 <![CDATA[<210> 22]]> <![CDATA[<211> 118]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Heavy chain variable region]]> <![CDATA[<400> 22]]> Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Phe 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Trp Ser Gly Gly Ile Thr His Tyr Asn Ser Ala Leu Met 50 55 60 Ser Arg Val Ser Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Val Thr Ala Ala Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Lys Leu Tyr Ser His Pro His Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <![CDATA[<210> 23]]> <![CDATA[<211 > 112]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Light Chain Variable Region]]> <![CDATA[<400> 23]]> Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Ser Ly s Ser Leu Leu His Ser 20 25 30 Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His 85 90 95 Leu Glu Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <![CDATA[<210> 24]]> <![CDATA[<211> 118]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Heavy chain variable region]]> <![CDATA[<400> 24]]> Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Phe 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Trp Ser Gly Gly Ile Thr His Tyr Asn Ser Ala Leu Met 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Phe Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Me t Tyr Tyr Cys Ala 85 90 95 Lys Leu Tyr Ser His Pro His Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <![CDATA[<210> 25]]> <![CDATA [<211> 118]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[< 223> heavy chain variable region]]> <![ CDATA[<400> 25]]> Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Phe 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Trp Ser Gly Gly Ile Thr His Tyr Asn Ser Ala Leu Met 50 55 60 Ser Arg Val Ser Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Phe Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Lys Leu Tyr Ser His Pro His Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <![CDATA[<210> 26]]> <![CDATA[<211> 118]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Manual Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Heavy chain variable region]]> <![CDATA[<400> 26]]> Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Phe 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Trp Ser Gly Gly Ile T hr His Tyr Asn Ser Ala Leu Met 50 55 60 Ser Arg Val Ser Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Lys Leu Tyr Ser His Pro His Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <![CDATA[<210> 27]]> <![CDATA[<211> 118]]> <![CDATA[<212> PRT]]> <![CDATA[<213> Artificial Sequence]]> <![CDATA[<220>]]> <![CDATA[<223> Heavy Chain Variable Region] ]> <![CDATA[<400> 27]]> Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Phe 20 25 30 Gly Ile His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Trp Ser Gly Gly Ile Thr His Tyr Asn Ser Ala Leu Met 50 55 60 Ser Arg Val Ser Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Phe Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Lys Leu Tyr Ser His Pro His Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115
      

Claims (28)

An anti-human TRPV2 monoclonal antibody or fragment thereof, which recognizes the extracellular domain of human TRPV2 as an epitope. The monoclonal antibody or fragment thereof according to claim 1, which recognizes the extracellular domain of human TRPV2 as an epitope, and specifically inhibits the activity of TRPV2. The monoclonal antibody or fragment thereof according to claim 1 or 2, wherein the extracellular domain recognizing human TRPV2 comprises at least 5 or more amino groups selected from the amino acid sequence between the 5th transmembrane region and the 6th transmembrane region The region of the acid sequence acts as an epitope that specifically inhibits the activity of TRPV2. The monoclonal antibody or its fragment according to claim 1, wherein the extracellular domain recognizing human TRPV2 comprises at least 5 or more amino acid sequences selected from the amino acid sequence between the first transmembrane region and the second transmembrane region This region acts as an epitope and specifically inhibits the activity of TRPV2. The monoclonal antibody or fragment thereof of any one of claims 1 to 3, which is in the extracellular domain of human TRPV2, 1) Recognize a region comprising at least 5 or more consecutive amino acid sequences selected from the amino acid sequence represented by SEQ ID NO: 2 as an epitope to specifically inhibit the activity of TRPV2, or 2) Identify a region comprising at least 5 or more consecutive amino acid sequences selected from the amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2 with substitution, addition, or deletion of 1 to 2 amino acids as a table It specifically inhibits the activity of TRPV2. The monoclonal antibody or fragment thereof of any one of claims 1 to 3 or 5, which is in the extracellular domain of human TRPV2, 1) Recognize a region comprising the amino acid sequence represented by SEQ ID NO: 3 as an epitope to specifically inhibit the activity of TRPV2, or 2) Recognize the region including the amino acid sequence in which 1-2 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO: 3 as an epitope to specifically inhibit the activity of TRPV2. The monoclonal antibody or its fragment according to any one of claims 1 to 3, 5, or 6, which recognizes the amino acid sequence represented by SEQ ID NO: 3 as an epitope in the extracellular region of human TRPV2, and has a specific inhibited the activity of TRPV2. The monoclonal antibody or fragment thereof of any one of claims 1 to 7, wherein the antibody is a humanized antibody. The monoclonal antibody or fragment thereof of any one of claims 1 to 3, 5 to 8, wherein the heavy chain variable region comprises the following: (i) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 4, (ii) CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5, and (iii) CDR3 comprising the amino acid sequence represented by SEQ ID NO: 6, The light chain variable region includes the following: (iv) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 7, (v) CDR2 comprising the amino acid sequence represented by RMS, and (vi) CDR3 comprising the amino acid sequence represented by SEQ ID NO: 8. The monoclonal antibody or fragment thereof of claim 1, wherein the heavy chain variable region comprises the following: (i) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 9, (ii) CDR2 comprising the amino acid sequence represented by SEQ ID NO: 10, and (iii) CDR3 comprising the amino acid sequence represented by SEQ ID NO: 11, The light chain variable region includes the following: (iv) CDR1 comprising the amino acid sequence represented by SEQ ID NO: 12, (v) CDR2 comprising the amino acid sequence represented by WAS, and (vi) CDR3 comprising the amino acid sequence represented by SEQ ID NO: 13. The monoclonal antibody or its fragment according to any one of claims 1 to 3, 5 to 8, wherein the heavy chain variable region is an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 14, The light chain variable region is an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 15. The monoclonal antibody or fragment thereof according to claim 1, wherein the variable region of the heavy chain is an amino acid sequence that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 16, and the variable region of the light chain is the amino acid sequence represented by SEQ ID NO: 17 The indicated amino acid sequence is at least 95% identical to the amino acid sequence. The monoclonal antibody or fragment thereof of any one of claims 1 to 3, 5 to 8, wherein the heavy chain variable region is at least 95 times the amino acid sequence represented by SEQ ID NO: 22, 24, 25, 26 or 27 % identical amino acid sequence, the light chain variable region is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 23. The monoclonal antibody or fragment thereof of any one of claims 1 to 3, 5 to 8, wherein the heavy chain variable region is an amine that is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 25, 26 or 27 The amino acid sequence of the light chain variable region is at least 95% identical to the amino acid sequence represented by SEQ ID NO: 23. A therapeutic or preventive agent for muscle disease and/or heart disease, comprising the anti-human TRPV2 monoclonal antibody or a fragment thereof according to any one of claims 1 to 3, 5 to 9, 11, 13 and 14. The therapeutic or prophylactic agent of claim 15, wherein muscle disease is neuromuscular junction and muscle disease of G70-G73 in ICD10 or muscle disorder of M60-M63, and heart disease is ischemia of I20-I25 in ICD10 Sexual heart disease or other types of heart disease I30-I52. If the therapeutic or preventive agent of claim 15, the muscle disease is primary muscle disorder of G71 in ICD10 or other muscle disorder of M62, and the heart disease is angina pectoris of I20, acute myocardial infarction of I21, and acute myocardial infarction of I22 of ICD10. Recurrent myocardial infarction, secondary complications of acute myocardial infarction in I23, other acute ischemic heart disease in I24, chronic ischemic heart disease in I25, acute pericarditis in I30, pericarditis in I31 Other diseases, I32 Pericarditis classified in other diseases, I33 Acute and subacute endocarditis, I34 Nonrheumatic mitral valve disorder, I35 Nonrheumatic aortic valve disorder, I36 Non-rheumatic tricuspid valve disorder, I37, pulmonary valve disorder, I38, endocarditis (valve unknown), I39, endocarditis and heart valve disorders classified as other diseases, I40, acute myocarditis, I41 Myocarditis among other diseases, I42 cardiomyopathy, I43 Myocardium among other diseases, I44 Atrioventricular block and left bundle branch block, I45 Other conduction disorders , I46 sudden cardiac arrest, I47 paroxysmal tachycardia (symptom), I48 atrial microfibrillation and atrial flutter, I49 other arrhythmias, I50 heart failure, I51 heart disease complications and diagnosis Records of unspecified cardiac disease, or other cardiac disorders classified as other diseases in I52. The therapeutic or prophylactic agent of claim 15, wherein the muscle disease is muscular dystrophy in G71.0 of ICD10, myotonic disorder in G71.1, congenital myopathy in G71.2, and mitochondria in G71.3 Myopathy (other unclassified), G71.8 Other primary muscle disorders, G71.9 Primary muscle disorders of unknown detail, M62.0 Muscle dissociation, M62.1 Other muscle ruptures (non-traumatic), ischemic infarction of muscle in M62.2, immobility syndrome in M62.3 (paraplegia), muscle contracture in M62.4, muscle wasting and atrophy in M62.5 (other unclassified) ), Muscle damage in M62.6, Muscle disorder in M62.8, Muscle disorder with unknown details in M62.9, Heart disease is acute transmural myocardial infarction in the anterior wall of I21.0 in ICD10, I21. 1 Acute transmural myocardial infarction in inferior wall, acute transmural myocardial infarction in other parts of I21.2, acute transmural myocardial infarction (location unknown) in I21.3, acute subendocardial infarction in I21.4 Myocardial infarction, acute myocardial infarction (details unknown) in I21.9, atherosclerotic cardiovascular disease in I25.0, atherosclerotic heart disease in I25.1, old myocardium in I25.2 Infarction, ventricular aneurysm in I25.3, coronary (symptomatic) aneurysm in I25.4, ischemic cardiomyopathy in I25.5, painless <asymptomatic> myocardial ischemia in I25.6, and others in I25.8 Type of chronic ischemic heart disease, chronic ischemic heart disease of I25.9 (details unknown), dilated cardiomyopathy of I42.0, obstructive hypertrophic cardiomyopathy of I42.1, other hypertrophy of I42.2 Cardiomyopathy, I42.3 Endomyocardial (eosinophilic) disease, I42.4 Endomyocardial fibroelastosis, I42.5 Other restrictive cardiomyopathy, I42.6 Alcoholic myocardium I42.7 Cardiomyopathy caused by drugs and other external factors, I42.8 Other cardiomyopathy, I42.9 Cardiomyopathy (details unknown), I50.0 Congestive heart failure, I50.1 Left ventricular failure, or heart failure at I50.9 (details unknown). The therapeutic or preventive agent of claim 15, wherein the muscle disease is muscle dystrophy of G71.0 in ICD10, the heart disease is acute myocardial infarction (details unknown) of I21.9, and dilated cardiomyopathy of I42.0 , or left ventricular failure of I50.1. The therapeutic or preventive agent according to claim 15, wherein the muscle disease and/or heart disease is muscle dystrophy of G71.0 in ICD10. The therapeutic or prophylactic agent of claim 15, wherein the muscle disease and/or heart disease is acute myocardial infarction (details unknown) of I21.9 in ICD10, dilated cardiomyopathy of I42.0, or left ventricle of I50.1 exhaustion. A diagnostic drug for muscle disease and/or heart disease, comprising the anti-human TRPV2 monoclonal antibody or a fragment thereof according to any one of claims 1 to 14. A diagnostic medicine for muscle disease and/or heart disease, comprising the anti-human TRPV2 monoclonal antibody or its fragment according to claim 10 or 14. A diagnostic kit for muscle disease and/or heart disease, comprising the anti-human TRPV2 monoclonal antibody or a fragment thereof according to any one of claims 1 to 14. The diagnostic kit of claim 24, wherein the muscle disease is neuromuscular junction and muscle disease of G70-G73 in ICD10 or muscle disorder of M60-M63, and the heart disease is ischemia of I20-I25 in ICD10 Sexual heart disease or other types of heart disease I30-I52. A method for assisting the diagnosis of muscle disease and/or heart disease, comprising the following steps: (i) using the anti-human TRPV2 monoclonal antibody or fragment thereof as claimed in any one of claims 1 to 14, to quantify the amount of expression of TRPV2 in a sample collected from a test animal; (ii) comparing the expression of TRPV2 quantified in (i) with the expression of TRPV2 in samples collected from healthy animals (control values); and (iii) Based on the result of (ii), when the expression level of TRPV2 quantified in (i) is greater than the control value, it is determined that the above-mentioned subject animal suffers from muscle disease and/or heart disease, or currently suffers from muscle disease and The possibility of heart disease, or the possibility of developing muscle disease and/or heart disease in the future. A method for assisting the diagnosis of the degree of progression of muscle disease and/or heart disease, comprising the following steps: (i) Using the anti-human TRPV2 monoclonal antibody or fragment thereof according to any one of claims 1 to 14, on a sample collected from a test animal suffering from muscle disease and/or heart disease, or at the possibility of suffering from it The expression of TRPV2 in the TRPV2 was quantified; (ii) comparing the expression of TRPV2 quantified in (i) with the expression of TRPV2 in samples collected from animals suffering from muscle disease and/or cardiac disease at a specified degree of progression (control value); and (iii) Based on the result of (ii), when the expression level of TRPV2 quantified in (i) is greater than the control value, it is judged that the degree of progression of muscle disease and/or cardiac disease of the above-mentioned subject animal is higher than that of the control animal The degree of progression of the animal suffering from the disease, or the possibility of suffering from muscle disease and/or heart disease is high, and when it is less than the control value, it is judged that the muscle disease and/or heart disease of the above-mentioned tested animal is improving, or not suffering from High likelihood of muscle disease and/or heart disease. A method for assisting the diagnosis of the degree of progression of muscle disease and/or heart disease, comprising the following steps: (i) Using the anti-human TRPV2 monoclonal antibody or fragment thereof according to any one of claims 1 to 14, on a sample collected from a test animal suffering from muscle disease and/or heart disease, or at the possibility of suffering from it The expression of TRPV2 in the TRPV2 was quantified; (ii) comparing the expression of TRPV2 quantified in (i) with the expression of TRPV2 in samples collected from the subject animal in the past (control value); and (iii) Based on the results of (ii), when the expression level of TRPV2 quantified in (i) is greater than the control value, it is judged that the muscle disease and/or heart disease of the above-mentioned subject animal is progressing, or suffering from muscle disease and The possibility of heart disease is high, and when it is less than the control value, it is judged that the muscle disease and/or heart disease of the tested animal is improving, or the possibility of not suffering from muscle disease and/or heart disease is high.

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