JP2023169439A - Anti-human TRPV2 antibody - Google Patents

Abstract

To provide an anti-human TRPV2 antibody or a fragment thereof that recognizes the extracellular domain of human TRPV2 as an epitope.SOLUTION: There is provided an anti-human TRPV2 monoclonal antibody or a fragment thereof that recognizes the extracellular domain of human TRPV2 as an epitope.SELECTED DRAWING: None

Description

The present invention relates to an anti-human TRPV2 antibody or a fragment thereof that recognizes the extracellular domain of the Ca ²⁺ channel TRPV2 (transient receptor potential cation channel, subfamily V, member 2) as an epitope. Furthermore, the present invention relates to human TRPV2 inhibitors containing the anti-human TRPV2 antibody or fragment thereof.

The Trp (transient receptor potential) gene product superfamily includes TRPC (7 species), TRPV (6 species), TRPM (8 species), TRPA (1 species), TRPP (4 species), and TRPML (3 species) in mammals. They are classified into 6 families, all of which are ion channels that penetrate the plasma membrane 6 times, and mainly allow Ca ²⁺ to pass through. Many ion channels that exist in cell membranes have different channel characteristics, and in order to screen for drugs that specifically react with these channels, it is necessary to use an assay system that is compatible with them. This is in sharp contrast to G protein-coupled receptors (GPCRs), which have relatively limited cellular responses.

TRPV2 is a stretch-activated Ca ²⁺ channel (Non-Patent Documents 1-3), and although it exists in the endomembrane system in normal tissues, it migrates to the cell membrane and becomes activated in diseases such as muscular dystrophy and cardiomyopathy. This contributes to abnormal Ca ²⁺ influx into cells (Non-patent Documents 2, 4). Drugs that specifically inhibit TRPV2 are thought to act on degenerated muscles and/or myocardium and reduce degeneration, and are expected to be developed as they may be effective in treating and alleviating the symptoms of muscle and heart diseases. has been done.

Gadolinium, which is conventionally used as a blocker of stretch-sensitive Ca ²⁺ channels, is thought to act on Ca ²⁺ channels in general, and ruthenium red is thought to act on the entire TRPV family (TRPV1 to TRPV6), both of which are non-active. It is a specific drug (Non-patent Documents 5, 6). A low-molecular compound that can specifically inhibit TRPV2 is disclosed in Patent Document 1, but the technology is different from that of an antibody that recognizes human TRPV2 epitope. Patent Document 2 discloses an antibody that recognizes the mouse TRPV2 sequence and has the function of specifically inhibiting TRPV2 activity. There are no known examples of producing antibodies that have the function of specifically inhibiting TRPV2 activity.

Furthermore, an anti-rat TRPV2 polyclonal antibody is commercially available as an antibody having reactivity with TRPV2 (KM019, manufacturer: Transgenic Co., Ltd.). This antibody uses a C-terminal partial peptide of TRPV2 as an immunogen and recognizes the intracellular domain, and its use is for immunohistochemical analysis, and there is no disclosure regarding the inhibition of TRPV2 activity. do not have.

Japanese Patent Application Publication No. 2009-149534 Patent No. 5754039

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

The present invention provides anti-human TRPV2 antibodies and fragments thereof that recognize the extracellular domain of human TRPV2 (hereinafter also referred to as hTRPV2) as an epitope, as well as anti-human TRPV2 antibodies that recognize the extracellular domain of human TRPV2 as an epitope and An object of the present invention is to provide an anti-human TRPV2 antibody or a fragment thereof that has the function of specifically inhibiting TRPV2 activity.

In order to solve the above problems, the present inventors first studied Japanese Patent No. 5754039, which is a prior art document related to the invention of an antibody that recognizes the mouse TRPV2 sequence and has the function of specifically inhibiting TRPV2 activity. We designed an experiment analogous to the content of Examples, produced multiple different immunogens (cells or peptides) that could be assumed from the Examples, and attempted to obtain the desired anti-human TRPV2 antibody.
However, as far as it can be inferred from the contents of prior art documents, etc., anti-human TRPV2 antibodies that recognize the desired extracellular domain of human TRPV2 as an epitope, or that recognize the extracellular domain of human TRPV2 as an epitope and have the activity of human TRPV2 We have not been able to obtain an anti-human TRPV2 antibody that has the ability to specifically inhibit TRPV2.
Based on the above results, the present inventors performed immunization with human TRPV2-expressing cells that were stimulated with a specific substance (cannabidiol) using the phage display method to express human TRPV2 on the cell membrane with high efficiency. He came up with the idea of using a library prepared from animals and conducted extensive research. As a result, we have developed an anti-human TRPV2 antibody that recognizes the desired extracellular domain of human TRPV2 as an epitope, and an anti-human TRPV2 antibody that recognizes the extracellular domain of human TRPV2 as an epitope and has the function of specifically inhibiting the activity of human TRPV2. The present invention was completed by confirming the acquisition of human TRPV2 antibodies. That is, the present invention relates to:
[1] An anti-human TRPV2 monoclonal antibody or a fragment thereof that 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 TRPV2 activity.
[3] A region containing at least five or more amino acid sequences selected from the amino acid sequences between the fifth transmembrane region and the sixth transmembrane region in the extracellular domain of human TRPV2 is recognized as an epitope, and TRPV2 The monoclonal antibody or fragment thereof according to [1] or [2], which specifically inhibits the activity of.
[4] A region containing at least five amino acid sequences selected from the amino acid sequences between the first transmembrane region and the second transmembrane region in the extracellular domain of human TRPV2 is recognized as an epitope, and TRPV2 The monoclonal antibody or fragment thereof according to [1], which specifically inhibits the activity of.
[5] Among the extracellular domains of human TRPV2, the following:
1) Recognizes as an epitope a region containing at least 5 or more consecutive amino acid sequences selected from the amino acid sequence shown in SEQ ID NO: 2, and specifically inhibits TRPV2 activity, or
2) Recognizes as an epitope a region containing at least 5 or more consecutive amino acid sequences selected from the amino acid sequence shown in SEQ ID NO: 2 in which 1 to 2 amino acids have been substituted, added, or deleted. , specifically inhibits TRPV2 activity,
The monoclonal antibody or fragment thereof according to any one of [1] to [3].
[6] Among the extracellular domains of human TRPV2,
1) Recognizes the region containing the amino acid sequence shown in SEQ ID NO: 3 as an epitope and specifically inhibits TRPV2 activity, or
2) recognizes as an epitope a region containing an amino acid sequence in which 1 to 2 amino acids are substituted, added, or deleted from the amino acid sequence shown in SEQ ID NO: 3, and specifically inhibits TRPV2 activity;
The monoclonal antibody or fragment thereof according to any one of [1] to [3] or [5].
[7] Recognizes the amino acid sequence shown in SEQ ID NO: 3 as an epitope in the extracellular domain of human TRPV2, and specifically inhibits TRPV2 activity, [1] to [3], [5], or [ 6] or a fragment thereof.
[8] The monoclonal antibody or fragment thereof according to any one of [1] to [7], which is a humanized antibody.
[9] The heavy chain variable region is as follows:
(i) CDR1 containing the amino acid sequence shown by SEQ ID NO: 4
(ii) CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and (iii) CDR3 containing the amino acid sequence shown in SEQ ID NO: 6.
including;
The light chain variable region is:
(iv) CDR1 containing the amino acid sequence shown by SEQ ID NO: 7
(v) CDR2 containing the amino acid sequence shown in RMS, and (vi) CDR3 containing the amino acid sequence shown in SEQ ID NO: 8.
The monoclonal antibody or fragment thereof according to any one of [1] to [3] and [5] to [8], comprising:
[10] The heavy chain variable region is as follows:
(i) CDR1 containing the amino acid sequence shown by SEQ ID NO: 9
(ii) CDR2 containing the amino acid sequence shown in SEQ ID NO: 10, and (iii) CDR3 containing the amino acid sequence shown in SEQ ID NO: 11.
including;
The light chain variable region is:
(iv) CDR1 containing the amino acid sequence shown by SEQ ID NO: 12
(v) CDR2 containing the amino acid sequence shown in WAS, and (vi) CDR3 containing the amino acid sequence shown in SEQ ID NO: 13.
The monoclonal antibody or fragment thereof according to [1], comprising:
[11] The heavy chain variable region has an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 14, and the light chain variable region has an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 15. The monoclonal antibody or fragment thereof according to any one of [1] to [3] and [5] to [8], which is
[12] The heavy chain variable region has an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 16, and the light chain variable region has an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 17. The monoclonal antibody or fragment thereof according to [1], which is
[13] Treatment of muscle diseases and/or heart diseases, comprising the anti-human TRPV2 monoclonal antibody or fragment thereof according to any one of [1] to [3], [5] to [9], and [11] agent or prophylactic agent.
[14] The muscle disease is a neuromuscular junction and muscle disease in ICD10 G70-G73 or myopathy in M60-M63, and the heart disease is ischemic heart disease in I20-I25 or I30-I52 in ICD10. The therapeutic or preventive agent according to [13], which is another type of heart disease.
[15] Myopathy is primary myopathy of G71 in ICD10 or other myopathy of M62, and heart disease is angina pectoris of I20 in ICD10, acute myocardial infarction in I21, recurrent myocardial infarction in I22, I23 Secondary complications of acute myocardial infarction, I24 Other acute ischemic heart disease, I25 Chronic ischemic heart disease, I30 Acute pericarditis, I31 Other diseases of the pericardium, I32 Other classifications I33 acute and subacute endocarditis, I34 non-rheumatic mitral valve disorders, I35 non-rheumatic aortic valve disorders, I36 non-rheumatic tricuspid valve disorders, I37 pulmonary artery Valve disorders, I38 endocarditis (valvular unknown), endocarditis and valvular disorders in diseases classified elsewhere in I39, acute myocarditis in I40, myocarditis in diseases classified elsewhere in I41, I42 Cardiomyopathy, I43 Cardiomyopathy in other classifications, I44 Atrioventricular block and left bundle branch block, I45 Other conduction disorders, I46 Cardiac arrest, I47 Paroxysmal tachycardia, I48 Atrial fibrillation and flutter in I49, other arrhythmias in I50, heart failure in I50, complications of heart disease in I51 and description of heart disease with unclear diagnosis, or other heart disorders in diseases classified elsewhere in I52 The therapeutic or preventive agent according to [13], which is.
[16] Muscle disease is ICD10 G71.0 muscular dystrophy, G71.1 myotonic disorder, G71.2 congenital myopathy, G71.3 mitochondrial myopathy (not elsewhere classified), G71.8 G71.9 unspecified primary myopathy, M62.0 muscle dissection, M62.1 other muscle rupture (non-traumatic), M62.2 muscle ischemic infarction , M62.3 immobility syndrome (paraplegia), M62.4 muscle contracture, M62.5 muscle wasting and atrophy (not elsewhere classified), M62.6 muscle strain, M62.8 Other manifested myopathies include unspecified myopathies at M62.9, and cardiac diseases include acute transmural myocardial infarction of the anterior wall of I21.0 on ICD10, and acute transmural myocardial infarction of the inferior wall of I21.1 on ICD10. sexual myocardial infarction, I21.2 acute transmural myocardial infarction at other sites, I21.3 acute transmural myocardial infarction (site unknown), I21.4 acute subendocardial myocardial infarction, I21.9 Acute myocardial infarction (details unknown), I25.0 described as atheroma sclerosing cardiovascular disease, I25.1 atheroma sclerosing heart disease, I25.2 old myocardial infarction, I25.3 ventricular aneurysm, I25.4 coronary artery aneurysm, I25.5 ischemic cardiomyopathy, I25.6 painless (asymptomatic) myocardial deficiency blood, I25.8 other types of chronic ischemic heart disease, I25.9 chronic ischemic heart disease (unspecified), I42.0 dilated cardiomyopathy, I42.1 obstructive hypertrophic cardiomyopathy, I42.2 Other hypertrophic cardiomyopathy, I42.3 Endomyocardial (eosinophilic) disease, I42.4 Endocardial fibroelastosis, I42.5 Other restrictive cardiomyopathy, I42 .6 Alcoholic cardiomyopathy, I42.7 Cardiomyopathy due to drugs and other external factors, I42.8 Other cardiomyopathy, I42.9 Cardiomyopathy (unspecified), I50.0 Congestive heart failure , I50.1 left ventricular failure, or I50.9 heart failure (unspecified), the therapeutic or preventive agent according to [13].
[17] The muscle disease is muscular dystrophy with ICD10 G71.0, and the heart disease is acute myocardial infarction (unspecified) with I21.9, dilated cardiomyopathy with I42.0, or left ventricular failure with I50.1 The therapeutic or preventive agent according to [13], which is.
[18] The therapeutic or preventive agent according to [13], wherein the muscle disease and/or heart disease is muscular dystrophy with ICD10 G71.0.
[19] Myopathy and/or heart disease is acute myocardial infarction (details unknown) with ICD10 I21.9, dilated cardiomyopathy I42.0, or left ventricular failure I50.1, described in [13] therapeutic or prophylactic agents.
[20] A diagnostic agent for muscle diseases and/or heart diseases, comprising the anti-human TRPV2 monoclonal antibody or fragment thereof according to any one of [1] to [19].
[21] A diagnostic agent for muscle diseases and/or heart diseases, comprising the anti-human TRPV2 monoclonal antibody or fragment thereof according to [10] or [12].
[22] A diagnostic kit for myopathies and/or heart diseases, comprising the antibody or fragment thereof according to any one of [1] to [12].
[23] The muscle disease is neuromuscular junction and muscle disease in ICD10 G70-G73 or myopathy in M60-M63, and the heart disease is ischemic heart disease in I20-I25 or I30-I52 in ICD10 The diagnostic kit according to [20], which is another type of heart disease.
[24] A method to assist in the diagnosis of muscle diseases and/or heart diseases, which comprises the following:
(i) Quantifying the expression level of TRPV2 in a sample collected from a test animal using the monoclonal antibody or fragment thereof according to any one of [1] to [12];
(ii) Comparing the expression level of TRPV2 quantified in (i) with the expression level of TRPV2 in a sample collected from a healthy animal (control value), and (iii) Based on the results of (ii), ), if the expression level of TRPV2 quantified in ) is greater than the control value, the test animal may be suffering from a muscle disease and/or heart disease, or is currently suffering from a muscle disease and/or heart disease. A method comprising the step of determining that the patient is likely to suffer from a muscle disease and/or a heart disease in the future.
[25] A method to assist in diagnosing the progression of muscle disease and/or heart disease, which includes:
(i) The monoclonal antibody or fragment thereof according to any one of [1] to [12] may be used to treat subjects who are or may be suffering from myopathies and/or heart diseases. Quantifying the expression level of TRPV2 in a sample collected from a test animal;
(ii) Comparing the expression level of TRPV2 quantified in (i) with the expression level of TRPV2 (control value) in a sample collected from an animal suffering from muscle disease and/or heart disease at a specific stage of progression. , and (iii) Based on the results of (ii), if the expression level of TRPV2 quantified in (i) is greater than the control value, the degree of progression of the muscle disease and/or heart disease of the test animal is higher than that of the control. The myopathic disease of the test animal is determined to be higher than the progression level of the animal suffering from the disease, or is judged to be highly likely to be suffering from muscle disease and/or heart disease, and is lower than the control value. and/or determining that there is a high possibility that the heart disease has improved or that the person is not suffering from the muscle disease and/or the heart disease.
[26] A method to assist in diagnosing the progression of muscle disease and/or heart disease, which includes the following:
(i) A test animal suffering from or possibly suffering from muscle disease and/or heart disease using the monoclonal antibody or fragment thereof according to any one of claims 1 to 12. Quantifying the expression level of TRPV2 in the sample collected from
(ii) A step of comparing the expression level of TRPV2 determined in (i) with the expression level of TRPV2 in a sample previously collected from the test animal (control value), and (iii) comparing the result of (ii). Based on this, if the expression level of TRPV2 quantified in (i) is greater than the control value, it is determined that the test animal has advanced muscle disease and/or heart disease, or is suffering from muscle disease and/or heart disease. If it is determined that there is a high possibility that the test animal is suffering from muscle disease and/or heart disease, and if the value is lower than the control value, there is a possibility that the test animal has improved muscle disease and/or heart disease, or is not suffering from muscle disease and/or heart disease. A method comprising the step of determining that

The anti-human TRPV2 antibody or fragment thereof of the present invention does not affect the activities of other family proteins of Trp, and can specifically inhibit the Ca ²⁺ influx activity of TRPV2. Furthermore, since the anti-human TRPV2 antibody or fragment thereof of the present invention uses the extracellular domain of TRPV2 as an epitope, it is capable of reacting with TRPV2 present on the cell membrane of living cells. Furthermore, the anti-human TRPV2 antibody or fragment thereof of the present invention has the effect of suppressing degeneration of muscle cells and cardiomyocytes. The anti-human TRPV2 antibody or fragment thereof of the present invention is thought to be capable of inhibiting the activity of TRPV2 on cell membranes during pathological conditions, and may be a potential therapeutic drug candidate for muscle diseases and/or heart diseases. Furthermore, the anti-human TRPV2 antibody or fragment thereof of the present invention is expected to be used as an experimental tool in functional analysis of TRPV2, mechanism analysis of degeneration of muscle cells and cardiomyocytes, and the like. Furthermore, TRPV2 is known to be expressed on the cell membrane of skeletal muscle cells, cardiomyocytes, and peripheral blood mononuclear cells during pathological conditions of muscle diseases and heart diseases (Non-Patent Documents 2, 7, and 8). Therefore, the anti-human TRPV2 antibody or fragment thereof of the present invention can be used as a drug for diagnosing the occurrence or possibility of suffering from muscle diseases or heart diseases by examining the expression or expression level of TRPV2 on cell membranes using the antibody. It can also be used as In addition, by comparing the past expression level of TRPV2 on cell membranes measured using the antibody with the current expression level, it is possible to diagnose the progress of the pathological condition of muscle diseases and heart diseases, and to treat the disease. It is also possible to evaluate the effectiveness of therapeutic drugs used in clinical trials. Furthermore, the anti-human TRPV2 antibodies of the present invention that do not have the function of specifically inhibiting the activity of human TRPV2 are considered to be highly safe for living organisms, and therefore can be used as companion diagnostic agents.

FIG. 1 is a schematic diagram of a vector incorporating VH and VL genes. Figure 2 shows the results of a confirmation assay for anti-human TRPV2 antibodies in mouse antiserum. When anti-His tag antibody was used, bands were confirmed at the 100 kDa and 75 kDa positions (2-a). Also, when immunized mouse antiserum was used as the primary antibody, bands were confirmed at the 100 kDa and 75 kDa positions (2-b). Figure 3 shows the results of evaluating the binding activity of antiserum using hTRPV2-expressing HEK293 cells. Figure 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. Figure 5 shows the results of Cell ELISA using hTRPV2-expressing HEK293 cells using phage colonies recovered by panning. FIG. 6A shows the results of Cell ELISA using the prepared E. coli culture supernatant (01a), which was immobilized on a streptavidin plate after solubilizing biotinylated hTRPV2-expressing HEK293 cells. Although no high binding activity was confirmed for 01a, monoclonal antibodies 01a005, 01a016, 01a018, and 01a033 showed slightly high binding activity in HEK293 cells expressing biotinylated hTRPV2. FIG. 6B shows the results of Cell ELISA using the prepared E. coli culture supernatant (02a), which was immobilized on a streptavidin plate after solubilizing biotinylated hTRPV2-expressing HEK293 cells. All the monoclonal antibodies obtained from 02a showed high specificity for biotinylated hTRPV2-expressing HEK293 cells. Figure 7 shows the results of FACS analysis of the binding of two representative clones, 01a033 (mAb001) and 02a001 (mAb002), to hTRPV2-expressing HEK293 cells using a 10-fold concentrated E. coli culture supernatant. Figure 8 shows an immunoprecipitation experiment using hTRPV2-expressing HEK293 cell lysate using antibody-bound beads prepared from the E. coli culture supernatant of a total of three selected representative clones 01a033 (mAb001), 02a001 (mAb002) and a negative control antibody. This is the result of doing this. FIG. 9 shows the results of concentration estimation and purity confirmation using OD280 nm measurement and SDS-PAGE regarding the purification of anti-human TRPV2 IgG antibody from an anti-human TRPV2 IgG antibody-producing cell line. Figure 10 shows the results of confirming the IgG antibody reaction against hTRPV2-expressing cells. Figure 11 shows the results of a confocal laser microscope examination of the intersection between anti-mouse TRPV2 antibody (mAb88-2) and anti-human TRPV2 antibody (mAb002). Figure 12 shows the 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). This is the result of checking the intersection. Figure 13 shows the results of evaluating the inhibition of Ca ²⁺ 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).

The present invention provides an antibody or a fragment thereof that recognizes the extracellular domain of human TRPV2 as an epitope, and an anti-human antibody that recognizes the extracellular domain of human TRPV2 as an epitope and has the function of specifically inhibiting the activity of human TRPV2. Concerning TRPV2 antibodies or fragments thereof.
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 provides methods for examining TRPV2, which is expressed not only on skeletal muscle cells and cardiac muscle cells but also on the cell membranes of peripheral blood mononuclear cells during pathological conditions of muscle diseases and heart diseases, using the antibody. and/or related to a diagnostic (determination) drug or a diagnostic (determination) method for heart disease.
Furthermore, the present invention provides a therapeutic or preventive agent for myopathies and/or heart diseases, comprising the anti-human TRPV2 antibody of the present invention or a fragment thereof, or a therapeutic or preventive agent for myopathies and/or heart diseases using the antibody. Regarding prevention methods, etc.

1. Antibody of the Invention The human TRPV2 gene has been reported as Genbank accession No. NM_016113. The amino acid sequence of human TRPV2 is shown in SEQ ID NO: 1 (Genbank accession No. NM_016113) in the sequence listing. TRPV2, like other proteins in the Trp family, is structurally characterized by having a six-transmembrane domain.

The anti-human TRPV2 antibody or fragment thereof of the present invention recognizes as an epitope a region consisting of at least five or more amino acid sequences selected from the amino acid sequence of the extracellular domain in SEQ ID NO: 1 above. The extracellular domain of TRPV2 is a portion of TRPV2 that exists on the cell membrane and is substantially outside the cell membrane. The amino acid sequence of the extracellular domain of human TRPV2 is at positions 409-434, 496-500, and/or 554-619 in SEQ ID NO: 1. 5 etc.), preferably two or less amino acids, more preferably one amino acid may be substituted, deleted, added, inserted or modified.

Preferably, the anti-human TRPV2 antibody or fragment thereof of the present invention has at least five amino acid sequences selected from the amino acid sequence between the fifth transmembrane region and the sixth transmembrane region of the extracellular domain of TRPV2. It recognizes a region consisting of an amino acid sequence as an epitope. The 5th transmembrane region and the 6th transmembrane region refer to the 5th and 6th regions counting from the N-terminal side of the region that penetrates the plasma membrane, and the anti-human TRPV2 antibody or fragment thereof of the present invention A region selected from highly hydrophilic amino acid sequences sandwiched between these regions is recognized as an epitope. Specifically, the amino acid sequence between the 5th transmembrane region and the 6th transmembrane region of the extracellular domain of human TRPV2 is from position 554 to 619 of SEQ ID NO: 1. The amino acid sequence may have one to several (2, 3, 4, 5, etc.), preferably 2 or less, and more preferably 1 amino acids substituted, deleted, added, inserted, or modified.

More preferably, the anti-human TRPV2 antibody or fragment thereof of the present invention comprises the amino acid sequence of positions 574 to 595 of human TRPV2 (SEQ ID NO: 1) (SVQPMEGQEDEGNGAQYRGILE: SEQ ID NO: 2) and one to several (2, 3, 4, 5, etc.), preferably 2 or less, more preferably 1 amino acid is substituted, deleted, added, inserted or modified, and consists of at least 5 or more amino acid sequences. It recognizes a region as an epitope.

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

In another embodiment of the anti-human TRPV2 antibody or fragment thereof of the present invention, at least five or more amino acids selected from the amino acid sequence between the first transmembrane region and the second transmembrane region of the extracellular domain of TRPV2. There are also those that recognize a region consisting of the amino acid sequence as an epitope. The first transmembrane region and the second transmembrane region refer to the first and second regions counted from the N-terminal side of the region that penetrates the plasma membrane, and the anti-human TRPV2 antibody or fragment thereof of the present invention A region selected from highly hydrophilic amino acid sequences sandwiched between these regions is recognized as an epitope. Specifically, the amino acid sequence between the first transmembrane region and the second transmembrane region of the extracellular domain of human TRPV2 is from position 412 to 434 of SEQ ID NO: 1. The amino acid sequence may have one to several (2, 3, 4, 5, etc.), preferably 2 or less, and more preferably 1 amino acids substituted, deleted, added, inserted, or modified.

The anti-human TRPV2 antibody or fragment thereof of the present invention has the function of specifically inhibiting TRPV2 activity. TRPV2 is a stretch-activated Ca ²⁺ channel that exists in the endomembrane system of cells in normal tissues, but in diseases such as muscular dystrophy and cardiomyopathy, it migrates to the cell membrane and becomes activated, leading to abnormal infiltration into cells. Contributes to Ca ²⁺ influx. TRPV2 activity means Ca ²⁺ influx into cells, and the anti-human TRPV2 antibody of the present invention or its fragment recognizes the extracellular domain as an epitope, so it is present on the cell membrane of living cells. It is thought that it is possible to recognize TRPV2 and inhibit TRPV2 activity. Furthermore, the anti-human TRPV2 antibody or fragment thereof of the present invention has the function of specifically inhibiting the activity of TRPV2 without substantially affecting the activity of other Trp family proteins.

The monoclonal antibodies or fragments of the present invention include monoclonal antibodies, single chain antibodies, or portions of the above antibodies that have antigen-binding properties, such as Fab fragments or fragments produced by Fab expression libraries. The anti-human TRPV2 monoclonal antibody or fragment thereof of the present invention is derived from mammals including humans, and examples thereof include human, mouse, rat, hamster, rabbit, goat, and horse types. . Furthermore, the antibody or fragment thereof of the present invention is not limited to IgG, but may be IgM or the like.

As used herein, an epitope is a region of an antigen that an antibody binds to. In certain embodiments, it includes any portion of an antigen that can specifically bind to an immunoglobulin or a T-cell receptor or a B-cell receptor. Antigenic determinants include chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in certain embodiments have specific three-dimensional structural characteristics and/or specific May have charged characteristics. In certain embodiments, an antibody can be said to specifically bind an antigen if the antibody preferentially recognizes the target antigen in a complex mixture of proteins and/or macromolecules.

The basic structure of antibody molecules is common to each class and consists of 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 (Immunology Illustrated (I. Roi tt, J. Brostoff, D. Male edition)). Heavy chains usually consist of polypeptide chains containing about 440 amino acids, and each class has a characteristic structure, with γ, μ, α, δ, and ε corresponding to IgG, IgM, IgA, IgD, and IgE. It's called a chain. Furthermore, IgG includes IgG1, IgG2, IgG3, and IgG4, which are called y1, y2, y3, and y4, respectively. Light chains usually consist of polypeptide chains containing about 220 amino acids, and two types are known, L-type and K-type, and are called λ and κ chains, respectively. The peptide composition of the basic structure of an antibody molecule consists of two homologous heavy chains and two light chains linked by disulfide bonds (S-S bonds) and non-covalent bonds, and has a molecular weight of 150,000 to 190,000. The two light chains can pair with any heavy chain. Each antibody molecule is always made up of two identical light chains and two identical heavy chains.

There are four intrachain s-s bonds in the heavy chain (five in the μ and ε chains) and two in the light chain, forming one loop for every 100-110 amino acid residues, and this three-dimensional structure is Each loop is similar and is called a structural unit or domain. The N-terminal domains of both heavy and light chains have variable amino acid sequences, even if they are from the same class (subclass) of the same species, and are called variable regions (V regions). (The heavy chain variable region domain is designated VH and the light chain variable region domain is designated VL). The amino acid sequence on the C-terminal side is almost constant for each class or subclass and is called the constant region (C region) (each domain is expressed as CH1, CH2, CH3, or CL, respectively).

The antigen-determining site of an antibody is composed of VH and VL, and the specificity of binding depends on the amino acid sequence of this site. On the other hand, biological activities such as complement and binding with various cells reflect differences in the structure of the C region of each class Ig. Variability in the light and heavy chain variable regions has been found to be largely confined to three small hypervariable regions present in either chain, which are comprised of complementarity determining regions (CDRs). )It is called.

As the method for producing the anti-human TRPV2 monoclonal antibody or fragment thereof of the present invention, any method known per se or any method to be developed in the future can be employed. Furthermore, various efforts are required to produce human antibodies against specific antigens. For example, reference can be made to Sato, K. et al, Cancer Res., 53, 851-856, 1993 and Japanese Patent Application Publication No. 2008-161198. The type of human antibody is not particularly limited, and may be Fab type or intact type. In order to effectively exhibit antibody activity, intact type antibodies are desirable. Intact type antibodies are not particularly limited, but for example, they may be chimeric antibodies in which the complementary strand 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. can. Since amino acid substitutions occur frequently in the CDR region, from the standpoint of antigenicity, an antibody may contain human antibodies having human-derived CDRs and CDRs derived from the immunized animal species.

The CDRs of the light chain and heavy chain variable regions in the anti-human TRPV2 monoclonal antibodies or fragments thereof of the present invention are delimited according to the Kabat, Chothia, AbM, or IMGT algorithm (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 Reeset 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 one embodiment of the present invention, as the CDRs of the light chain/heavy chain variable regions in an anti-human TRPV2 monoclonal antibody or a fragment thereof, CDR1 in the heavy chain variable region is SEQ ID NO: 4 (GFSLTSFG), and CDR2 is SEQ ID NO: 5. (IWSGGIT), CDR3 contains/consists of the amino acid sequence shown by SEQ ID NO: 6 (LYSHPHAMDY), CDR1 in the light chain variable region is SEQ ID NO: 7 (KSLLHSNGITY), CDR2 is RMS, and CDR3 is SEQ ID NO: 8 (MQHLEYPLT). Included are those comprising/consisting of the indicated amino acid sequence. In the CDR of the light chain/heavy chain variable region of the anti-human TRPV2 monoclonal antibody or fragment thereof of the present invention, 1 to several (2, 3, 4, 5, etc.), preferably 2 nucleotides are present in these base sequences. or more preferably one amino acid may be substituted.
Furthermore, as for the light chain/heavy chain variable regions, the heavy chain variable region has an amino acid sequence that is at least 95% identical to the amino acid sequence shown by SEQ ID NO: 14, and the light chain variable region has an amino acid sequence shown by SEQ ID NO: 15. Preferably, the heavy chain variable region has an amino acid sequence that is at least 97% identical to the amino acid sequence shown in SEQ ID NO: 14, and the light chain variable region has an amino acid sequence that is at least 97% identical to the amino acid sequence shown in SEQ ID NO: 14. The heavy chain variable region may have an amino acid sequence that is at least 97% identical to the amino acid sequence shown in SEQ ID NO: 15, and more preferably, the heavy chain variable region has an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO: 14. , the light chain variable region has an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO: 15.

As another aspect of the present invention, as the CDRs of the light chain/heavy chain variable regions in the anti-human TRPV2 monoclonal antibody or its fragment, CDR1 in the heavy chain variable region is SEQ ID NO: 9 (GFSLTTYG), and CDR2 is SEQ ID NO: 10 (GFSLTTYG). MGWDGKK), CDR3 contains/consists of the amino acid sequence shown by SEQ ID NO: 11 (DGGYTWFAY), CDR1 in the light chain variable region is shown by SEQ ID NO: 12 (QSLLNSRTRKNY), CDR2 is shown by WAS, and CDR3 is shown by SEQ ID NO: 13 (KQSYNLFT). Examples include those comprising/consisting of an amino acid sequence. In the CDR of the light chain/heavy chain variable region of the anti-human TRPV2 monoclonal antibody or fragment thereof of the present invention, 1 to several (2, 3, 4, 5, etc.), preferably 2 nucleotides are present in these base sequences. or more preferably one amino acid may be substituted.
Furthermore, as for the light chain/heavy chain variable regions, the heavy chain variable region has an amino acid sequence that is at least 95% identical to the amino acid sequence shown by SEQ ID NO: 16, and the light chain variable region has an amino acid sequence shown by SEQ ID NO: 17. Examples include anti-human TRPV2 monoclonal antibodies or fragments thereof that recognize as an epitope the extracellular domain of human TRPV2, which has an amino acid sequence that is at least 95% identical to the amino acid sequence, preferably the heavy chain variable region is represented by SEQ ID NO: 16. The light chain variable region is at least 97% identical to the amino acid sequence shown in SEQ ID NO: 17, and more preferably the heavy chain variable region is at least 97% identical to the amino acid sequence shown in SEQ ID NO: 17. The region has an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO: 16, and the light chain variable region has an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO: 17. .

The antigen for producing the anti-human TRPV2 antibody or fragment thereof of the present invention may be a full-length protein or peptide fragment of human TRPV2, or a cell expressing a full-length protein or peptide fragment of human TRPV2, with particular limitations. Not done. For the production of anti-human TRPV2 monoclonal antibodies or fragments thereof, it is preferable to use cells that express human TRPV2 (cells for human TRPV2 expression). The human TRPV2-expressing cell may be any cell that expresses human TRPV2 on its cell membrane, but human TRPV2 may be expressed in mammalian cells (e.g., human, monkey, rat, mouse, hamster, etc.). It was expressed in cells collected from Examples of the mammalian cells include HEK293, COS7, CHO-K1, NIH3T3, Balb3T3, FM3A, L929, SP2/0, P3U1, B16, and P388. Furthermore, TRPV2 expressed in mammalian cells is preferably a full-length protein. In the present invention, it is preferable to use HEK cells for expressing human TRPV2 in which the full-length human TRPV2 protein is expressed in HEK293 cells, and the cells are subjected to specific treatments (stimulation with compounds (such as cannabidiol)). You may also use 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 etc. of the immunized animal. Furthermore, monoclonal antibodies and hybridomas that produce the monoclonal antibodies can be produced by fusing immunized human or animal-derived B lymphocytes with various myeloma cells, specifically by the method described below. can.

In the monoclonal antibody production of the present invention, the antigen dissolved or suspended in a suitable buffer such as phosphate buffer (PBS) can be used as the antigen solution. The antigen solution may usually be 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. Furthermore, when the antigen alone has low antigenicity, such as a peptide antigen, it can be used after being cross-linked to a suitable carrier protein such as albumin or keyhole limpet hemocyanin (KLH). Examples of animals to be immunized with the antigen include warm-blooded animals such as humans, mice, rats, hamsters, horses, goats, and rabbits.

At this time, in order to increase the responsiveness of the immunized animal to the antigen, the antigen solution can be mixed with an adjuvant and administered. Adjuvants that can be used here are Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), Ribi (MPL), Ribi (TDM), Ribi (MPL+TDM), Boredetella pertussis vaccine, and muramyl. Dipeptide (MDP), aluminum adjuvant (ALUM), and combinations thereof are exemplified, but a combination using FCA for the initial immunization and FIA or Ribi adjuvant for the booster immunization may also be used.

The immunization method can be adjusted depending on the type of antigen used and the presence or absence of adjuvant mixture, etc., and the injection site, schedule, etc. can be changed as appropriate. Inject 0.05 to 1 mL of adjuvant mixed antigen solution (10 to 200 μg of antigen protein, or 1 × 10 ⁶ to 1 × 10 ⁷ cells) into adjuvant-mixed antigen solution intraperitoneally, subcutaneously, intramuscularly, or (tail) vein (nu mice). After the initial immunization, booster immunizations are given 1 to 4 times approximately every 4 to 21 days, followed by a final immunization approximately 1 to 4 weeks later. The antigen solution can also be administered without using an adjuvant by intraperitoneally injecting a larger amount of antigen. Antibody titers are determined by collecting blood approximately 5 to 10 days after the booster immunization. The antibody titer can be measured by a commonly used method according to the antibody titer assay described below. Approximately 3 to 5 days after the final immunization, splenocytes are isolated from the immunized animal to obtain antibody-producing cells.

The anti-human TRPV2 monoclonal antibody of the present invention can be produced, for example, according to the method of Kohler and Milstein (Kohler and Milstein, Nature 256, 495-497, 1975). Myeloma cells derived from mice, rats, humans, etc. are used, such as mouse myeloma P3X63-Ag8, P3X63-Ag8-U1, P3NS1-Ag4, SP2/o-Ag14, P3X63-Ag8・653, etc. Myeloma cells are exemplified. Some myeloma cells produce immunoglobulin light chains, and when these are used as a fusion target, the light chains may randomly bind to the immunoglobulin heavy chains produced by antibody-producing cells. In particular, it is preferable to use myeloma cells that do not produce immunoglobulin light chains, such as P3X63-Ag8.653 and SP2/o-Ag14. The antibody-producing cells and myeloma cells are preferably derived from animals of the same species, especially animals of the same strain. Myeloma cells may be preserved according to methods known per se; for example, cells subcultured in a general medium supplemented with horse, rabbit, or fetal bovine serum are preserved by freezing. Furthermore, it is preferable to use cells in the logarithmic growth phase for cell fusion.

Examples of methods for producing hybridomas by fusing antibody-producing cells and myeloma cells include methods using polyethylene glycol (PEG), methods using Sendai virus, and methods using an electrofusion device. For example, splenocytes 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, and at a temperature of about 25-25%. The reaction may be carried out at 37°C and pH 6 to 8 for about 30 seconds to 3 minutes. After the reaction is completed, the cells are washed, the PEG solution is removed, and the cells are resuspended in the medium, seeded in a microtiter plate, and cultured.

The cells after the fusion operation are cultured in a selective medium to select hybridomas. The selective medium is a medium that kills the parent cell line and allows only the fused cells to proliferate, and is usually a hypoxanthine-aminopterin-thymidine (HAT) medium. Selection of hybridomas is usually performed by replacing a portion of the medium, preferably about half, with a selection medium 1 to 7 days after the fusion operation, and culturing while repeating the same medium exchange every 2 to 3 days. Confirm wells in which hybridoma colonies are growing by microscopic observation.

More specifically, the anti-human TRPV2 monoclonal antibody of the present invention can be produced by the following method. HEK cells for expressing human TRPV2 are inoculated into immunologically diseased mice, the spleen is collected 2 to 4 weeks later, and the antibody-producing cells contained in the spleen are fused with myeloma cells to produce the antibody of the present invention. Hybridomas can be produced.

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

Whether the obtained antibody recognizes the extracellular domain of human TRPV2 can be confirmed, for example, by the following method. The above hybridoma culture supernatant is reacted with a HEK cell population for expressing human TRPV2 and a HEK cell population that does not express human TRPV2, and then reacted with a labeled secondary antibody and measured using a flow cytometer. Specific to the human TRPV2 extracellular domain, the fluorescence range is higher than that of HEK cells expressing human TRPV2 that have not been reacted with antibodies and is higher than the HEK cell population that does not express human TRPV2. It is possible to select antibodies that can bind to specific antibodies.

In addition, whether the obtained antibody has the function of inhibiting the activity of human TRPV2 can be determined using the TRPV2-specific assay method described in JP-A-2007-259745 (Japanese Patent Application No. 2006-088323). It can be confirmed.

Furthermore, as to whether the antibody of the present invention does not substantially affect the activity of proteins of the Trp family other than TRPV2, please refer to This can be confirmed as appropriate by referring to the method for measuring the activity of Trp family proteins.

Furthermore, a single clone producing the monoclonal antibody of the present invention is isolated by a limiting dilution method, a soft agar method, a method using a fluorescence excitation cell sorter, or the like. For example, in the case of the limiting dilution method, a hybridoma clone that produces the antibody of interest can be isolated by serially diluting a hybridoma colony in a medium to approximately 1 cell/well and culturing it. The obtained antibody-producing hybridoma clones can be frozen in the presence of a cryoprotectant such as about 10% (v/v) dimethyl sulfoxide (DMSO) or glycerin and stored at -70 to -196°C for about six months to Can be stored semi-permanently. Before use, cells should be rapidly thawed in a constant temperature bath at around 37°C. It is desirable to thoroughly wash the cryoprotectant before use to avoid residual cytotoxicity.

The method for obtaining monoclonal antibodies from hybridomas can be appropriately selected depending on the required amount, properties of the hybridoma, etc. Examples include a method of obtaining the hybridoma from the ascites of a mouse transplanted with it, a method of obtaining the hybridoma from the culture supernatant by cell culture, and the like. If a hybridoma can grow in the mouse peritoneal cavity, monoclonal antibodies at a high concentration of several mg/mL can be obtained from ascites fluid. Hybridomas that cannot be grown in vivo are obtained from the culture supernatant of cell culture. Obtaining monoclonal antibodies by cell culture has the advantage that, although the amount of antibody produced is lower than in vivo, there is less contamination by immunoglobulins and other contaminants contained in the mouse peritoneal cavity, and purification is easy.

When monoclonal antibodies are obtained from the intraperitoneal cavity of a mouse transplanted with a hybridoma, for example, the peritoneal cavity of a BALB/c mouse to which a substance with an immunosuppressive effect such as pristane (2,6,10,14-tetramethylpentadecane) has been administered is used. Hybridomas (approximately 10 ⁶ or more) are transplanted into the body, and collected ascites is collected approximately 1 to 3 weeks later. In the case of heterologous hybridomas (eg, mouse and rat), it is preferable to use nude mice or radiation-treated mice.

On the other hand, when obtaining antibodies from cell culture supernatants, for example, in addition to the static culture method used for cell maintenance, the hybridoma is cultured using a culture method such as a high-density culture method or a spinner flask culture method. Obtain a culture supernatant containing . Since the serum contained in the culture solution contains impurities such as other antibodies and albumin, which often complicates antibody purification, it is desirable to add less serum to the culture solution. More preferably, the hybridoma is adapted to a serum-free medium by a conventional method and then cultured using the serum-free medium. Antibody purification is facilitated by culturing in serum-free medium.

Monoclonal antibodies can be purified from ascites fluid or culture supernatant by methods known per se. For example, conventional immunoglobulin purification methods include salting-out fractionation using ammonium sulfate or sodium sulfate, polyethylene glycol (PEG) fractionation, ethanol fractionation, DEAE ion exchange chromatography, gel filtration, etc. This can be easily achieved by applying. Furthermore, when the monoclonal antibody is IgG, it can be easily purified by affinity chromatography using a protein A-binding carrier.

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

Examples of methods for screening monoclonal antibodies include display methods. Examples of methods for producing phage display antibody libraries include, but are not limited to, the following.

The phage used is not particularly limited, but filamentous phage (Ff bacteriophage) is usually preferably used. Examples of methods for displaying foreign proteins on the phage surface include expressing and displaying them as a fusion protein on coat proteins g3p, g6p to g9p, but g3p or g8p is usually used. This method involves fusing it to the N-terminus of Phage display vectors include 1) those in which a foreign gene is introduced in the form of a fusion with the coat protein gene of the phage genome, so that all of the coat protein displayed on the phage surface is displayed as a fusion protein with the foreign protein; ) A gene encoding the fusion protein is inserted separately from the wild-type coat protein gene to simultaneously express the fusion protein and the wild-type coat protein, and 3) E. coli carrying a phagemid vector carrying the gene encoding the fusion protein. In the case of 1), phage particles that simultaneously express the fusion protein and the wild-type coat protein are produced by infecting a helper phage carrying the wild-type coat protein gene. Since the ability to infect is lost, types 2) or 3) are used to create antibody libraries.

Specific vectors include those described by Holt et al. (Curr. Opin. Biotechnol., 11: 445-449, 2000). For example, pCES1 (see J. Biol. Chem., 274: 18218-18230, 1999) contains DNA encoding the kappa light chain constant region and the g3p signal peptide downstream of the g3p signal peptide under the control of a single lactose promoter. This is a Fab expression type phagemid vector in which the g3p coding sequence is placed downstream of CH3-encoding DNA, His-tag, c-myc tag, and amber stop codon (TAG). When introduced into E. coli that has the amber mutation, Fab is displayed on the g3p coat protein, but when expressed in strains such as HB2151 that do not have the amber mutation, soluble Fab antibodies are produced. Furthermore, as a scFv expression type phagemid vector, pHEN1 (J. Mol. Biol., 222:581-597, 1991) and the like can be used, for example. On the other hand, examples of helper phages include M13-KO7 and VCSM13.

In addition, as another phage display vector, sequences containing cysteine-encoding codons are linked to the 3' end of the antibody gene and the 5' end of the coat protein gene, respectively, and both genes are simultaneously and separately (rather than as a fusion protein). Examples include those designed so that the antibody can be expressed and displayed on the coat protein on the phage surface via S-S bonds between the introduced cysteine residues (CysDisplayTM technology of Morphosys).

Types of antibody libraries include naive/non-immune libraries, synthetic libraries, immune libraries, and the like.

A naive/non-immunized library is a library obtained by obtaining VH and VL genes possessed by normal humans by RT-PCR and randomly cloning them into the above-mentioned phage display vectors. . Usually, mRNA etc. derived from normal human peripheral blood, bone marrow, lymphocytes such as tonsils are used as a template. In order to eliminate V gene bias such as disease history, we specifically call a library that amplifies only IgM-derived mRNA that has not undergone class switching due to antigen sensitization a naive library. Representative examples include the CAT library (see J. Mol. Biol., 222: 581-597, 1991; Nat. Biotechnol., 14: 309-314, 1996), the MRC library (Annu Rev. Immunol., 12: 433-455, 1994), Dyax library (J. Biol. Chem., 1999 (supra); Proc. Natl. Acad. Sci. USA, 14: 7969-7974, 2000).

The synthetic library selects a functional specific antibody gene in human B cells and replaces the antigen-binding region of the V gene fragment, such as CDR3, with DNA encoding a random amino acid sequence of appropriate length. It has been made into a library. Since libraries can be constructed from a combination of VH and VL genes that produce functional scFv and Fab from the beginning, it is said to have excellent antibody expression efficiency and stability. Representative examples include Morphosys' HuCAL library (see J. Mol. Biol., 296: 57-86, 2000), BioInvent's library (see Nat. Biotechnol., 18: 852, 2000), Crucell's library (see Proc. Natl. Acad. Sci. USA, 92:3938, 1995; J. Immunol. Methods, 272: 219-233, 2003), and the like.

Immunized libraries are created by repeatedly immunizing experimental animals such as mice with a specific antigen to sufficiently increase the antibody response, and then using the animal's lymphocytes, patients with cancer, autoimmune diseases, infectious diseases, etc. The above-mentioned naive/non-immune live lymphocytes are collected from lymphocytes collected from people with increased blood antibody titers against the target antigen, such as those who have received vaccinations, or from human lymphocytes that have been artificially immunized with the target antigen by the above-mentioned in vitro immunization method. mRNA was prepared in the same manner as for the library, and the VH and VL genes were amplified by RT-PCR to create a library. Since the antibody gene of interest is contained in the library from the beginning, the antibody of interest can be obtained even from a library of relatively small size.

The process of selecting antibodies against a target antigen using phage display is called panning. Specifically, for example, a carrier on which an antigen is immobilized is brought into contact with a phage library, unbound phages are washed away, bound phages are eluted from the carrier, and E. coli is infected to multiply the phages. By repeating this series of operations about 3 to 5 times, phage that present antigen-specific antibodies are concentrated. As carriers for immobilizing antigens, there are various carriers used in normal antigen-antibody reactions and affinity chromatography, such as insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicone, and glass. , microplates, tubes, membranes, columns, beads, etc. made of metal, etc., and surface plasmon resonance (SPR) sensor chips. Physical adsorption may be used to immobilize the antigen, or chemical bonding, which is used to insolubilize and immobilize proteins, enzymes, etc., may be used. For example, a biotin-(strept)avidin system or the like is preferably used. To wash unbound phages, a blocking solution such as a BSA solution (1-2 times), PBS containing a surfactant such as Tween (3-5 times), etc. can be sequentially used. There are also reports that it is preferable to use a citrate buffer (pH 5). For elution of specific phages, acids (e.g., 0.1 M hydrochloric acid, etc.) are usually used, but cleavage with specific proteases (e.g., a gene sequence encoding a trypsin cleavage site at the junction between the antibody gene and coat protein gene) is used. In this case, the wild-type coat protein is displayed on the surface of the eluted phage, making it possible to infect and propagate E. coli even if all of the coat protein is expressed as a fusion protein). Competitive elution with soluble antigen or reduction of S-S bonds (for example, in the aforementioned CysDisplayTM, after panning, antigen-specific phages can be recovered by dissociating antibodies and coat proteins using an appropriate reducing agent). elution is also possible. When eluted with acid, the eluted phage is neutralized with Tris or the like, then infected with E. coli, and after culturing, the phage is recovered by a conventional method.

After phages presenting antigen-specific antibodies are concentrated by panning, E. coli is infected with the phages, and the E. coli is seeded onto a plate to perform cell cloning. The phages are collected again, and the antigen-binding activity is confirmed by measurement using the antibody titer measurement method described above (eg, ELISA, RIA, etc.), FACS, or SPR.

Isolation and purification of antibodies from phage clones that display selected antigen-specific antibodies can be carried out, for example, when using a vector in which an amber stop codon has been introduced at the junction between the antibody gene and coat protein gene as a phage display vector. When this phage is used to infect Escherichia coli that does not have the amber mutation (e.g. HB2151 strain), soluble antibody molecules are produced and secreted into the periplasm or the medium, so the cell wall is lysed with lysozyme etc. and the extracellular fraction is fraction can be collected and performed using purification techniques similar to those described above. If a His-tag or c-myc tag is introduced, it can be easily purified using immobilized metal affinity chromatography (IMAC) or an anti-c-myc antibody column. In addition, when using cleavage with a specific protease during panning, since the antibody molecules are separated from the phage surface when the protease acts, the target antibody can be purified by performing the same purification procedure. be able to.

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. Monoclonal antibodies or fragments of the invention may be murine, chimeric, or humanized antibodies.

As used herein, "chimeric antibody" refers to an antibody in which the heavy chain and light chain variable region (VH and VL) sequences are derived from a non-human animal species, and the constant region (CH and CL) sequences are derived from a human. means. The sequence of the variable region is preferably derived from an animal species in which hybridomas can be easily produced, such as mouse, rat, rabbit, etc., and the sequence of the constant region is preferably derived from the animal species to be administered.

Examples of methods for producing chimeric antibodies include the method described in US Pat. No. 6,331,415 or a partially modified method thereof.

Host cells are transformed with the obtained chimeric heavy chain and chimeric light chain expression vectors. Examples of host cells include animal cells, such as the mouse myeloma cells described above, Chinese hamster ovary (CHO) cells, monkey-derived COS-7 cells, Vero cells, and rat-derived GHS cells. For transformation, any method applicable to animal cells may be used, but electroporation is preferred. After culturing for a certain period of time in a medium suitable for the host cell, a chimeric monoclonal antibody can be isolated by collecting the culture supernatant and purifying it in the same manner as above. Alternatively, transgenic animals can be produced by conventional methods using germline cells of animals for which transgenic technology has been established, such as cows, goats, and chickens, and for which mass breeding know-how has been accumulated as livestock (poultry). By doing so, chimeric monoclonal antibodies can be easily obtained in large quantities from the resulting animal's milk or eggs. Furthermore, transgenic technology has been established for corn, rice, wheat, soybean, tobacco, etc., and plant cells that are cultivated in large quantities as major crops are used as host cells, and microinjection into protoplasts, electroporation, and intact cells can be performed. It is also possible to produce transgenic plants using the particle gun method or Ti vector method, and obtain large amounts of chimeric monoclonal antibodies from the resulting seeds, leaves, etc.

As used herein, a "humanized antibody" means that the sequences of all regions other than the complementarity determining regions (CDRs) present in the variable region (i.e., the framework regions (FR) in the constant region and variable region) are human. refers to an antibody in which only the CDR sequences are derived from another mammalian species. Preferred examples of other mammalian species include animal species in which hybridomas can be easily produced, such as mice, rats, and rabbits.

Examples of methods for producing humanized antibodies include the methods described in U.S. Patent Nos. 5,225,539, 5,585,089, 5,693,761, 5,693,762, European Patent Application No. 239400, and International Publication No. 92/19759; Examples include a method in which the method is partially modified. Specifically, in the same manner as in the case of the chimeric antibody described above, DNA encoding VH and VL derived from a mammalian species other than humans (e.g., mouse) is isolated, and then DNA encoding VH and VL is isolated using an automatic DNA sequencer (e.g., Applied Biosystems, etc.), the resulting nucleotide sequence or amino acid sequence deduced from it is sequenced using a known antibody sequence database [e.g., Kabat database (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.] to determine the CDRs and FRs of both chains. designing a nucleotide sequence in which the CDR coding region of the nucleotide sequence encoding the light chain and heavy chain of a human antibody having an FR sequence similar to the determined FR sequence is replaced with the nucleotide sequence encoding the determined heterologous CDR; The base sequence is divided into fragments of about 20 to 40 bases, and sequences complementary to the base sequence are further divided into fragments of about 20 to 40 bases so as to alternately overlap with the fragments. Each fragment is synthesized using a DNA synthesizer, and by hybridizing and ligating them according to a conventional method, DNA encoding VH and VL having human-derived FRs and other mammalian species-derived CDRs is constructed. be able to. To more quickly and efficiently transplant CDRs from other mammalian species into human VHs and VLs, it is preferable to use site-directed mutagenesis by PCR. Such a method includes, for example, the sequential CDR transplantation method described in JP-A-5-227970.

In addition, in producing a humanized antibody by the method described above, if only the CDR amino acid sequence is grafted onto the template human antibody FR, the antigen-binding activity may be lower than that of the original non-human antibody. In such cases, it is effective to simultaneously transplant some of the FR amino acids around the CDR. The non-human antibody FR amino acids to be transplanted include amino acid residues that are important for maintaining the 3D structure of each CDR, and such amino acid residues can be estimated by 3D structure prediction using a computer. .

By ligating the DNA encoding VH and VL obtained in this way with DNA encoding human-derived CH and CL, respectively, and introducing them into appropriate host cells, humanized antibody-producing cells or transgenic cells can be created. You can get plants and animals.

As an alternative method to create humanized antibodies without using CDR grafting, in which murine CDRs are grafted onto the variable regions of human antibodies, e.g. Examples include methods for determining which amino acid residues within the amino acid residues are candidates for substitution. This method can be carried out as described in, 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, etc. In addition, humanized antibodies can be produced using this method, if the VH and VL amino acid sequence information of the non-human antibody is obtained, for example, by using the contract antibody production service provided by It can be done easily.

Like chimeric antibodies, humanized antibodies can be modified into scFv, scFv-Fc, minibody, dsFv, Fv, etc. using genetic engineering methods, and can also be produced in microorganisms such as Escherichia coli and yeast by using an appropriate promoter. can be done. Furthermore, humanized antibodies can also be obtained by producing an antibody library used in the above-mentioned phage display method from an animal such as a mouse or rat, and then humanizing the obtained antibody, but is not limited thereto.

The ability of the humanized antibody thus obtained to bind to the epitope region of wild-type human TRPV2 can be confirmed by the above-mentioned binding assay using a polypeptide containing the peptide region.

One embodiment of the anti-human TRPV2 antibody or fragment thereof of the present invention preferably has the function of suppressing degeneration of cardiomyocytes and muscle cells. Degeneration of cardiomyocytes and muscle cells is observed in various diseases, and it is thought that the pathological conditions can be improved by suppressing or alleviating the degeneration. The function of suppressing degeneration of cardiomyocytes and muscle cells is achieved by subjecting cardiomyocytes and muscle cells cultured on silicone membranes to a stretching stimulus for a predetermined period of time to induce degeneration, and then increasing the creatinine kinase activity of the culture supernatant. This can be confirmed by measuring.

Confirmation of antibody binding can be performed using any well-known assay method, such as direct and indirect sandwich assays, flow cytometry, and immunoprecipitation assays (Zola, Monoclonal Antibodies: A Manual of Techniques, (CRC Press, Inc.) .1987) pp. 147-158). In the present invention, the binding between an anti-human TRPV2 antibody or a fragment thereof and an antigen can be measured by the methods listed above.

To measure the binding constant (Ka) of the anti-human TRPV2 antibody of the present invention or a fragment thereof or to identify other antibodies of the present invention that bind to human TRPV2 competitively with the already obtained antibody of the present invention, competitive ELISA is used. Competitive assays such as can be used. A competitive assay is carried out in a binding assay using the above-mentioned antigen-immobilized solid phase by allowing free antigen or a known antibody to coexist in the reaction system between the solid phase and the test antibody. For example, a test antibody solution with a known concentration and a mixture of antigens with various concentrations added to the test antibody solution are incubated in contact with an antigen-immobilized solid phase, and the amount of each label on the solid phase is measured. . Scatchard analysis is performed from the measured values at each free antigen concentration, and the slope of the graph can be calculated as a binding constant. On the other hand, a labeled known antibody (the antibody of the present invention) and a test antibody at various concentrations were reacted with the antigen-immobilized solid phase to reduce the amount of label on the solid phase in a concentration-dependent manner. By selecting a test antibody, it is possible to identify an antibody that binds to human TRPV2 competitively with the antibody of the present invention.

Epitope analysis can be performed by various known methods (EpitopeMapping Protocols/Second Edition, Mike Schutkowski, Ulrich Reineke, Ann NY AcadSci. 2010 Jan; 1183:267-87.). More detailed analysis of the epitope of this antibody can be performed by binding inhibition assay, homolog scan and/or alanine scan (for example, JP 2009-159948A, Science, 244: 1081-1085 (1989)). .

Binding affinities are 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 methods described herein. Specifically, common methods for measuring the affinity of antibodies to antigens include ELISA, RIA, and surface plasmon resonance. Furthermore, the binding affinity of the present invention has been determined by ELISA, SPR, immunohistochemical staining, and immunoblot. Affinity, measured by interaction of a particular antibody with an antigen, can vary when measured under different conditions, eg, salinity, pH, etc. Therefore, measurements of affinity and other parameters of antigen binding, such as KD, IC50, etc., are preferably performed with standardized solutions of antibody and antigen, and with standardized buffers.

2. Agents, reagents, kits, etc. of the present invention Furthermore, the present invention extends to human TRPV2 inhibitors or cardiomyocyte and/or myocyte degeneration inhibitors containing the anti-human TRPV2 antibody or fragment thereof of the present invention as an active ingredient. Human TRPV2 inhibitors or myocardial cell and/or myocyte degeneration inhibitors can be used as pharmaceutical compositions, but also as experimental tools (e.g., reagents, kits, etc.) to specifically inhibit human TRPV2, or as myocardial cell degeneration inhibitors. It can also be used in vivo or in vitro as an experimental tool (eg, reagent, kit, etc.) to inhibit or alleviate degeneration of cells and/or muscle cells. Furthermore, the anti-human TRPV2 antibody of the present invention or a fragment thereof can be used as a diagnostic (determination) agent, a diagnostic (determination) reagent, or a diagnostic (determination) kit for cardiomyocyte and/or myocyte degenerative diseases.

The anti-human TRPV2 antibody or fragment thereof of the present invention can also be used as an active ingredient in a composition. Furthermore, when a composition containing the anti-human TRPV2 antibody of the present invention or a fragment thereof is used for pharmaceutical purposes, it contains an effective amount of one or more of the anti-human TRPV2 antibodies of the present invention or a fragment thereof, and It may also contain a biologically acceptable carrier. In the present invention, pharmaceutically acceptable salts include basic addition salts such as alkali metal salts and alkaline earth metal salts such as sodium salts and potassium salts, and inorganic acid salts such as hydrochlorides, sulfates, and nitrates. Examples include acid addition salts such as salts and organic acid salts such as acetates and propionates.

Such compositions can be administered orally or parenterally as pharmaceutical compositions. For oral administration, the anti-human TRPV2 antibody of the present invention can be administered in conventional formulations, such as solid formulations such as tablets, powders, granules, and capsules; solutions; oily suspensions; or liquid formulations such as syrups or elixirs. It can also be used in any of the following dosage forms. For parenteral administration, the anti-human TRPV2 antibody of the present invention can be used as an aqueous or oily suspension injection, or a nasal solution. In its preparation, conventional excipients, binders, lubricants, aqueous solvents, oily solvents, emulsifiers, suspending agents, preservatives, stabilizers, etc. can be used as desired.

When used as a pharmaceutical composition, it is preferably applied to diseases caused by activation of TRPV2, muscle diseases, and heart diseases. Diseases caused by TRPV2 activation 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 are not necessarily limited to these.

The anti-human TRPV2 antibody or fragment thereof of the present invention is also effective as a therapeutic or preventive agent for muscle diseases and/or heart diseases.
Examples of muscle diseases include diseases of the neuromuscular junction and muscles (G70-G73) in the 2016 edition of ICD10 (International Statistical Classification of Diseases and Related Health Problems 10th Revision; hereinafter referred to as ICD10) published by WHO. junction and muscle) or M60-M63 myopathies (Disorders of muscles).
The muscle disease preferably includes G71 Primary disorders of muscles or M62 Other disorders of muscle in ICD10.
More preferred muscle diseases include G71.0 Muscular dystrophy, G71.1 Myotonic disorders, G71.2 Congenital myopathies, and G71.3 Mitochondrial myopathy ( Mitochondrial myopathy, not elsewhere classified; G71.8 Other primary disorders of muscles; G71.9 Primary disorder of muscle, M62.0 Diastasis of muscle, M62.1 Other rupture of muscle (nontraumatic), M62.2 Ischaemic infarction M62.3 Immobility syndrome (paraplegic), M62.4 Contracture of muscle, M62.5 Muscle wasting and atrophy (classified elsewhere) (Muscle wasting and atrophy, not elsewhere classified), M62.6 Muscle strain, or M62.8 Other specified disorders of muscle or M62.9 Muscle strain. Disorder of muscle, unspecified.
Even more preferred muscle diseases include muscular dystrophy of ICD10 G71.0.
Specifically, the types of muscular dystrophy G71.0 according to ICD10 are autosomal recessive, childhood type, resembling Duchenne or Becker, and benign [Becker]. benign [Becker], benign scapuloperoneal with early contractures [Emery-Dreifuss], distal, facioscapulohumeral facioscapulohumeral, limb-girdle, ocular, oculopharyngeal, scapuloperoneal, or severe [Duchenne] Can be mentioned.
Heart diseases include, for example, Ischemic heart diseases (Ischemic heart diseases) of I20-I25 in ICD10 or Other forms of heart disease (Ischemic heart diseases) of I30-I52.
The cardiac disease is preferably I20 angina pectoris, I21 acute myocardial infarction, I22 subsequent myocardial infarction, I23 secondary complications of acute myocardial infarction. Certain current complications following acute myocardial infarction, I24 Other acute ischaemic heart diseases, I25 Chronic ischemic heart disease, I30 Acute pericarditis. pericarditis), I31 Other diseases of pericardium, I32 Pericarditis in diseases classified elsewhere, I33 Acute and subacute endocarditis I34 Nonrheumatic mitral valve disorders, I35 Nonrheumatic aortic valve disorders, I36 Nonrheumatic tricuspid valve disorders ), I37 Pulmonary valve disorders, I38 Endocarditis, valve unspecified, I39 Endocarditis and valvular disorders in diseases classified elsewhere. heart valve disorders in diseases classified elsewhere), I40 Acute myocarditis, I41 Myocarditis in diseases classified elsewhere, I42 Cardiomyopathy, I43 Cardiomyopathy in diseases classified elsewhere, I44 Atrioventricular and left bundle branch block, I45 Other conduction disorders, I46 Cardiac arrest (Cardiac arrest), I47 Paroxysmal tachycardia, I48 Atrial fibrillation and 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 preferably, the heart disease is acute transmural myocardial infarction of the anterior wall at I21.0 in ICD10, acute transmural myocardial infarction of the inferior wall at I21.1. infarction of inferior wall), I21.2 Acute transmural myocardial infarction of other sites, I21.3 Acute transmural myocardial infarction of unknown site. I21.4 acute subendocardial myocardial infarction, I21.9 acute myocardial infarction (unspecified site), I25.0 atheroma Atherosclerotic cardiovascular disease, so described, I25.1 atherosclerotic heart disease, I25.2 Old myocardial infarction, I25.3 Aneurysm of heart, I25.4 Coronary artery aneurysm and dissection, I25.5 Ischaemic I25.6 Silent myocardial ischaemia, I25.8 Other forms of chronic ischaemic heart disease, I25.9 Chronic ischaemic heart disease, unspecified, I42.0 Dilated cardiomyopathy, I42.1 Obstructive hypertrophic cardiomyopathy, I42.2 Other hypertrophic cardiomyopathy in I42.3, Endocardial (eosinophilic) disease in I42.4, Endocardial fibroelastosis in I42.4 , I42.5 Other restrictive cardiomyopathy, I42.6 Alcoholic cardiomyopathy, I42.7 Cardiomyopathy due to drugs and other external factors. other external agents), I42.8 Other cardiomyopathies, I42.9 Cardiomyopathy, unspecified, I50.0 Congestive heart failure, I50.1 Examples include left ventricular failure or heart failure, unspecified at I50.9.
Even more preferred examples of the heart disease include acute myocardial infarction (details unknown) at ICD10 of I21.9, dilated cardiomyopathy of I42.0, or left ventricular failure of I50.1.
Specifically, the pathological type of acute myocardial infarction (details unknown) at I21.9 in ICD10 includes myocardial infarction (acute) NOS.
Specific examples of the dilated cardiomyopathy type I42.0 in ICD10 include congestive cardiomyopathy.
Specifically, the pathological types of I50.1 left ventricular failure in ICD10 are cardiac asthma, left heart failure, and pulmonary edema (those with a description of 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 drugs. When used in combination, the agent of the present invention and another drug may be provided in the form of a single pharmaceutical, or may be provided in the form of a pharmaceutical combination or kit containing multiple separately formulated preparations. good. The timing of administration of the agent of the present invention and the drug to be used in combination is not limited, and the agent of the present invention and the drug to be used in combination may be administered to the subject at the same time or separately (e.g., consecutively, at intervals of time). etc.) may be administered. The dosage of the drug to be used in combination may be in accordance with the dosage used clinically, and can be appropriately selected depending on the subject of administration, route of administration, disease, combination, etc.

Other drugs include steroid preparations (deflazacort, prednisone, prednisolone, etc. These include, but are not limited to, corticoids), exon skipping, gene therapy, cell medicine, and even rehabilitation. In addition, therapeutic drugs for heart disease include ACE inhibitors, angiotensin II receptor antagonists, β-blockers, diuretics, anti-aldosterone drugs, h-ANP, digitalis preparations, cardiotonic drugs, and various cell medicines. In addition, medical devices include implantable devices such as pacemakers and defibrillators, ablation using cardiac catheters, and surgical treatments including heart transplantation, left ventricular plasty, and mitral valve plasty. but is not limited to this. Furthermore, in cases of myopathies that exhibit symptoms of decreased cardiac function, they may be used in combination with the above-mentioned cardiac disease therapeutics. As used herein, "therapeutic agents for muscle diseases and/or heart diseases" include not only medicines aimed at the radical treatment of muscle diseases and/or heart diseases, but also, for example, suppressing the progression of tauopathy and dementia-related diseases. Also included are drugs aimed at inhibiting the progression of the disease, and drugs aimed at inhibiting the progression of the disease may be used as "prophylactic drugs for muscle diseases and/or heart diseases."

The diagnostic agent, diagnostic kit, or diagnostic reagent of the present invention is for diagnosing (determining) muscle disease and/or heart disease. The above-mentioned diagnostic agents of the present invention may be used to confirm the expression of TRPV2 on the cell membrane of peripheral blood mononuclear cells, cardiomyocytes, or muscle cells, or to confirm the expression of TRPV2 in the cytoplasm or cell membrane of peripheral blood mononuclear cells, cardiomyocytes, or muscle cells. , or a reagent for measuring the expression level of TRPV2 on the cell membrane (eg, the anti-TRPV2 of the present invention or a fragment thereof), and muscle disease and/or heart disease can be diagnosed by the confirmation or measurement.

The anti-human TRPV2 antibody or a fragment thereof may be a fluorescently labeled antibody, an enzyme-labeled antibody, a streptavidin-labeled antibody, a biotin-labeled antibody, or a radioactively labeled antibody. In addition, anti-human TRPV2 antibodies are usually prepared in the form of an aqueous solution dissolved in water or an appropriate buffer (e.g., TE buffer, PBS, etc.) to an appropriate concentration, or in the form of a lyophilized product. It may be included in the diagnostic agent, kit, or reagent of the invention. In addition, when measuring the expression level of TRPV2, it may be measured using one type of antibody, or it may be measured using multiple types, preferably two types of antibodies (for example, immunostaining, flow cytometry, sandwich ELISA method, etc.) may be used.

Depending on the method for confirming or measuring TRPV2, the diagnostic agent, kit, or reagent of the present invention may further contain other components necessary for carrying out the method. For example, when measuring by Western blotting, the kit of the present invention can further contain a blotting buffer, a labeling reagent, a blotting membrane, a detection reagent, a standard solution, and the like. Here, the "standard solution" refers to a purified sample of TRPV2, such as the peptide of the present invention, prepared by adding it to a specific concentration in water or an appropriate buffer (e.g., TE buffer, PBS containing fetal bovine serum, etc.). Examples include dissolved aqueous solutions.

Furthermore, when measuring by sandwich ELISA, the diagnostic agent, kit, or reagent of the present invention can further contain a solid-phase antibody measurement plate, a washing liquid, etc. in addition to the above. When measuring by an agglutination method including a latex agglutination method, antibody-coated latex, gelatin, etc. can be included. When measuring by chemifluorescence method or chemifluorescence electron method, antibody-bound magnetic particles and a suitable buffer solution can be included. Detection of TRPV2 using LC/MS, LC-MS/MS, or immunochromatography methods can include an antibody-coated column or microcolumn or microchip as part of the detection equipment. Furthermore, a time-resolved fluorescence measurement method or a fluorescence measurement method similar thereto may include a plurality of labeled anti-human TRPV2 antibodies and other necessary components.

3. Diagnosis (determination) method of the present invention, etc. When using the anti-human TRPV2 antibody of the present invention or a fragment thereof as a diagnostic agent for muscle diseases and/or heart diseases, for example, peripheral blood mononuclear cells are isolated from the patient's blood. By measuring the expression level of TRPV2 in peripheral blood mononuclear cells (e.g. T cells, B cells, etc.), it is possible to classify the disease, diagnose (determine) whether or not you are suffering from the disease, and determine whether or not you are suffering from the disease. It is possible to diagnose (determine) whether there is a high possibility that the patient is currently suffering from the disease, and to diagnose (determine) the severity (e.g., severe, moderate, mild, etc.) or degree of progression. Furthermore, for example, by diagnosing the severity and degree of progression of the disease, it is also possible to evaluate the effectiveness of therapeutic agents used to treat the disease. Furthermore, companion diagnosis (determination) can be made as to whether the anti-human TRPV2 antibody or fragment thereof of the present invention is applicable. The (expression) amount of TRPV2 in peripheral blood mononuclear cells to be measured may be the amount of TRPV2 present in the cytoplasm and on the cell membrane (plasma membrane) of peripheral blood mononuclear cells, or the amount of TRPV2 present on the cell membrane of peripheral blood mononuclear cells. Although it may be the amount of TRPV2 present on the cell membrane, it is preferably the amount of TRPV2 present on the cell membrane. In addition to peripheral blood mononuclear cells, cells whose TRPV2 (expression) amount is measured in the diagnostic (determination) method of the present invention may be myocytes or cardiomyocytes. The expression of TRPV2 can be confirmed by commonly known methods such as flow cytometry and immunostaining, and the expression level of TRPV2 can be measured and quantified by flow cytometry (FACS) and immunostaining.

The diagnosis (determination) method of the present invention is characterized by detecting TRPV2 in a sample collected from a test animal. In addition, the diagnostic method of the present invention includes specific steps such as (i) quantifying TRPV2 in a sample collected from a test animal, and (ii) quantifying the amount of TRPV2 determined in (i). It may also include a step of comparing the amount of TRPV2 in a sample collected from an animal (hereinafter referred to as a "control value").

As a result of the comparison in (ii), if the amount of TRPV2 quantified in (i) is greater than the control value, the test animal is suffering from or currently suffering from muscle disease and/or heart disease. It indicates that the person may be suffering from a disease and/or heart disease or may suffer from a muscle disease and/or heart disease in the future.

Furthermore, in addition to the above steps, the diagnostic method of the present invention provides that, based on the results of (iii) and (ii), if the amount of TRPV2 quantified in (i) is greater than the control value, the test animal Determining that one is suffering from a disease and/or a heart disease, or is currently suffering from a muscle disease and/or a heart disease, or is likely to suffer from a muscle disease and/or a heart disease in the future. May include.

Furthermore, the present invention also extends to methods for assisting in diagnosing (determining) the degree of progression of muscle diseases and/or heart diseases. The diagnostic aiding method of the present invention is also characterized by detecting TRPV2 in a sample collected from a test animal. In addition, the method of assisting diagnosis of the present invention includes, as a specific step, (i) TRPV2 in a sample collected from a test animal that is or may be suffering from muscle disease and/or heart disease. and (ii) comparing the amount of TRPV2 quantified in (i) with the amount of TRPV2 in a sample previously collected from the test animal (hereinafter referred to as "control value"). But that's fine.

As a result of the comparison in (ii), if the amount of TRPV2 quantified in (i) is greater than the control value, the myopathies and/or heart diseases of the test animal are progressing, or the myopathies and/or heart diseases are progressing. or indicates that the test animal is likely to be suffering from a heart disease, and if the value is lower than the control value, the test animal's muscle disease and/or heart disease is improved, or the test animal is suffering from a muscle disease and/or heart disease. This indicates that there is a high possibility that the patient is not infected.
In addition to the above steps, the method for assisting diagnosis of the present invention provides that, based on the results of (iii) and (ii), if the amount of TRPV2 quantified in (i) is greater than the control value, The test animal is judged to have progressive muscle disease and/or heart disease or is likely to be suffering from muscle disease and/or heart disease, and is lower than the control value. Alternatively, it may include a step of determining that the heart disease has improved or that there is a high possibility that the patient is not suffering from muscle disease and/or heart disease.

In addition, the method of assisting in diagnosing (determining) the degree of progression of muscle disease and/or heart disease of the present invention includes, as another specific step, for example, (i) (ii) quantifying TRPV2 in a sample collected from a test animal that may have a specific stage of myocardial disease and/or heart disease; It may also include a step of comparing the amount of TRPV2 in a sample taken from an affected animal (hereinafter referred to as a "control value").

As a result of the comparison in (ii), if the amount of TRPV2 quantified in (i) is greater than the control value, the degree of progression of the muscle disease and/or heart disease in the test animal is determined to be higher than the control value. The test animal is considered to have a myopathic disease and/or cardiac disease if it is lower than the control value, indicating that the test animal is more advanced than the animal tested or is likely to be suffering from myocardial and/or cardiac disease. This indicates that the person is improving or is likely to be free of muscle and/or heart disease.
The method of assisting diagnosis of the present invention includes, in addition to the above steps, based on the results of (iii) and (ii),
If the amount of TRPV2 quantified in (i) is greater than the control value, the degree of progression of the muscle disease and/or heart disease in the test animal is higher than the degree of progression of the control animal suffering from the disease. , or it is determined that the test animal is likely to be suffering from muscle disease and/or heart disease, and if the value is lower than the control value, the muscle disease and/or heart disease of the test animal is improved, or the muscle disease is determined to be lower than the control value. and/or may include a step of determining that there is a high possibility that the patient does not suffer from heart disease.

The above-mentioned possibility determination and test (result) are useful for at least assisting a doctor in making a definitive diagnosis regarding muscle disease and/or heart disease.

Moreover, as mentioned above, the amount of TRPV2 on the cell membrane of peripheral blood mononuclear cells increases in muscle diseases and/or heart diseases, and therefore, the present invention is useful for patients undergoing treatment for muscle diseases and/or heart diseases, or for patients undergoing treatment for muscle diseases and/or heart diseases. The present invention also extends to methods for evaluating the therapeutic effects of myopathies and/or cardiac disease predispositions that are being treated to prevent the onset of heart diseases.

The method for evaluating therapeutic efficacy of the present invention is also characterized by detecting TRPV2 in a sample collected from a test animal. Further, the method for evaluating the therapeutic effect of the present invention includes, as a specific step, (i) drug treatment for muscle diseases and/or heart diseases or drug therapy for preventing the onset of muscle diseases and/or heart diseases; (ii) quantifying TRPV2 in a sample collected from a test animal that has started treatment; and (ii) quantifying the amount of TRPV2 determined in It may also include a step of comparing the amount of TRPV2 (hereinafter referred to as "control value") in a sample collected before the start of drug treatment, a sample collected after the start of drug treatment and before the time of collection in (i).
As a result of the comparison in (ii), if the amount of TRPV2 quantified in (i) is larger than the control value, it indicates that the current drug treatment (or the selected therapeutic agent) is not effective, and it is smaller than the control value. This indicates that the current medication (or selected treatment) is effective.
The method for evaluating the therapeutic effect of the present invention includes, in addition to the above steps, based on the results of (iii) and (ii), if the amount of TRPV2 quantified in (i) is greater than the control value, The method may include the step of evaluating the treatment (or the selected therapeutic agent) as not being effective, and evaluating the current medication treatment (or the selected therapeutic agent) as being effective if the value is less than a control value.

As used herein, the term "therapeutic drugs used for drug therapy" includes not only therapeutic drugs that have already been approved and are on the market, but also investigational drugs that are currently undergoing clinical trials. That is, the method for evaluating therapeutic efficacy of the present invention can also be used for monitoring drug efficacy in clinical trials.

Animals that can be tested in the method of the present invention are not particularly limited as long as they express TRPV2, and examples include mammals (e.g., humans, monkeys, cows, pigs, horses, dogs, cats, sheep, and goats). , rabbits, hamsters, guinea pigs, mice, rats, etc.), and birds (e.g., chickens, etc.). Preferably it is a mammal, more preferably a human.
The biological sample derived from the test animal that serves as the sample is not particularly limited, and examples thereof include blood, muscle tissue (biopsy sample), saliva, urine, and the like. More preferred are blood and 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 previously measured or set for the control etc. may be used, and the amount of TRPV2 used in the method of the present invention may be used at the same time as the method of the present invention. No need to measure.
In order to set the control value here, it is also possible to use a plurality of individuals as a control group and the average value of the measured values of the plurality of individuals as the control value. That is, the amount of TRPV2 in a control sample derived from an already obtained control group (healthy animals, animals suffering from muscle disease and/or heart disease at a certain stage of progression, etc.) is used as a control value, and the above Making determinations and the like is also included within the scope of the method of the present invention.

If "TRPV2 in the sample collected from the test animal" is greater than "the amount of TRPV2 in the sample collected from a healthy animal (control value)", the test animal is currently suffering from muscle disease and/or heart disease. It can be diagnosed (determined) that the patient may be suffering from muscle disease and/or heart disease in the future.
Preferably, the amount of "TRPV2 in the sample collected from the test animal" is 1.1 to 10 times the control value. More preferably, it is 1.1 times to 8 times. More preferably, it is 1.2 to 5 times. Most preferably, it is 1.2 times to 3 times.

If "TRPV2 in the sample collected from the test animal" is greater than "the amount of TRPV2 in the sample collected in the past from the test animal (control value)", the test animal has muscle disease and/or heart disease. It can be determined that this is progressing.
Preferably, the amount of "TRPV2 in the sample collected from the test animal" is 1.1 to 10 times the control value. More preferably, it is 1.1 times to 8 times. More preferably, it is 1.2 to 5 times. Most preferably, it is 1.2 times to 3 times.

If “TRPV2 in the sample collected from the test animal” is smaller than “the amount of TRPV2 in the sample collected in the past from the test animal (control value)”, muscle disease and/or heart disease has improved. It can be determined that there is.
The amount of "TRPV2 in the sample collected from the test animal" is preferably 0.1 to 0.9 times the control value. More preferred is 0.2 times to 0.9 times. More preferably, it is 0.3 to 0.9 times. Most preferably, it is 0.4 times to 0.9 times.

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

When "TRPV2 in the sample collected from the test animal" is smaller than "the amount of TRPV2 in the sample collected from the animal suffering from muscle disease and/or heart disease at a specific stage (control value)" , it can be determined that muscle disease and/or heart disease are improved.
The amount of "TRPV2 in the sample collected from the test animal" is preferably 0.1 to 0.9 times the control value. More preferred is 0.2 times to 0.9 times. More preferably, it is 0.3 to 0.9 times. Most preferably, it is 0.4 times to 0.9 times.

Quantitative analysis of TRPV2 may be performed by standardizing the amount of TRPV2 in the sample with the amount of standard protein (internal standard protein). That is, after quantifying the amount of TRPV2 in the sample and the amount of standard protein using the above method, the ratio of the two signals (TRPV2/standard protein) is calculated, and the amount of TRPV2 in the sample is calculated based on the presence of the standard protein. It can be expressed as a ratio to the amount.
The standard protein may be any protein that is constantly expressed in a certain amount, and preferably is a protein that is commonly expressed in many tissues and cells. Examples include proteins essential for cell survival, such as proteins encoded by genes (housekeeping genes) such as RNA synthase, energy generation enzymes, ribosomal proteins, and cytoskeletal proteins. Specific examples thereof include, but are not limited to, proteins such as β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-tubulin. Particularly preferred is β-actin.

In addition, instead of the above-mentioned control value, a cut-off value for the amount of TRPV2 in peripheral blood mononuclear cells, cardiomyocytes, or muscle cells related to myopathies and/or heart disease is set in advance, and the amount of TRPV2 in the test animal is You may also compare it with a cutoff value. The (expression) amount of TRPV2 in peripheral blood mononuclear cells, cardiomyocytes, or muscle cells measured in setting the above cutoff value may be the amount of TRPV2 present in the cytoplasm and on the cell membrane (plasma membrane) of each cell, Although it may be the amount of TRPV2 present on the cell membrane of each cell, it is preferably the amount of TRPV2 present on the cell membrane.
For example, if the amount of TRPV2 on the cell membrane of peripheral blood mononuclear cells or the cell membrane (plasma membrane) of cardiomyocytes or muscle cells of a test animal is above the cutoff value, the test animal may be diagnosed with muscle disease or It can be diagnosed that the person is suffering from a heart disease, is currently suffering from a muscle disease and/or a heart disease, or has a possibility of suffering from a muscle disease and/or a heart disease in the future.

The "cutoff value" is a value that satisfies both high diagnostic sensitivity (prevalence rate) and high diagnostic specificity (predictability rate) when a disease is determined based on the value. For example, the cutoff value may be set to a value that shows a high positive rate in individuals who have developed muscle disease and/or heart disease, and a high negative rate in individuals who have not developed muscle disease and/or heart disease. I can do it.

Methods for calculating cutoff values are well known in the art. For example, the amount of TRPV2 on the cell membrane of peripheral blood mononuclear cells in individuals who have developed muscle disease and/or heart disease and individuals who have not developed muscle disease and/or heart disease is calculated, and the diagnostic sensitivity of the calculated value is and diagnostic specificity, and based on these values, create an ROC (Receiver Operating Characteristic) curve using commercially available analysis software. Then, the value when the diagnostic sensitivity and diagnostic specificity are as close to 100% as possible can be determined, and this value can be used as the cutoff value. For example, it is also preferable to set the "average value + 2 standard deviations" of the amount of TRPV2 on the cell membrane of peripheral blood mononuclear cells in a large number of healthy animals as the cutoff value, and using this value will provide good sensitivity and specificity. It becomes possible to determine that a muscle disease and/or a heart disease has developed. Also, for example, by finding the value that maximizes the likelihood ratio between diagnostic sensitivity and diagnostic specificity from the ROC curve and using that value as the cutoff value, it is possible to determine muscle disease and/or heart disease with high sensitivity. It becomes possible to do so. Alternatively, set the cutoff value at the point on the ROC curve with the lowest diagnostic ability, that is, the point farthest from the line where the area under the ROC curve is 0.5, that is, calculate "sensitivity + specificity - 1" and It is also preferable to set the point at which the value reaches the maximum value as the cutoff value.

As a cutoff value for the amount of TRPV2 on the cell membrane of peripheral blood mononuclear cells, for example, the percentage of peripheral blood mononuclear cells in which TRPV2 is present on the cell membrane in samples collected from test animals 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%).

In the method of the present invention, when diagnosing muscle diseases and/or heart diseases, etc., in addition to TRPV2, changes in other diagnostic markers for muscle diseases and/or heart diseases may be investigated. Diagnostic markers for muscle diseases and/or heart diseases include, for example, troponin, BNP, ANP, CK (creatine kinase), myoglobin, AST, ALT, LDH, aldolase, etc., and these can be detected according to well-known and commonly used detection methods. can do. Further, when diagnosing muscle diseases and/or heart diseases, etc., in addition to TRPV2, devices etc. may be used for diagnosis. Diagnostic methods using equipment include, for example, needle electromyography, MRI, etc. for muscle diseases, and electrocardiograms, echocardiography, etc. for heart diseases.

Four. Prevention and treatment methods of the present invention As described above, the anti-human TRPV2 antibody or fragment thereof of the present invention does not affect the activities of other family proteins of Trp, and specifically inhibits the Ca ²⁺ influx activity of TRPV2. It is possible to inhibit Therefore, the antibody is suitable for the prevention and treatment of muscle diseases such as muscular dystrophy and cardiomyopathy, or cardiac diseases that are related to the Ca ²⁺ influx.

In the prevention and treatment method of the present invention, for example, in the method for diagnosing (determining) muscle disease and/or heart disease of the present invention, etc., the test animal is currently suffering from muscle disease and/or heart disease. If it is determined that the person is suffering from a disease and/or heart disease, or has the possibility of suffering from muscle disease and/or heart disease in the future, the anti-human TRPV2 antibody of the present invention or a fragment thereof may be administered. The disease can be prevented and treated. Furthermore, in the prophylactic and therapeutic methods of the present invention, the anti-human TRPV2 antibody of the present invention or a fragment thereof may be used in combination with a therapeutic or prophylactic agent for muscle diseases and/or heart diseases as described above.

The administration form, administration route, etc. of the pharmaceutical composition to a subject can be determined as appropriate depending on the purpose and subject. For example, it can be administered intravenously, arterially, subcutaneously, intramuscularly, transdermally, nasally, or orally in an appropriate dosage form such as an injection, a transdermal solution, an inhalation/nasal solution, or an oral solution. Antibodies are generally administered intravenously or subcutaneously by injection, and such dosage forms and routes can also be applied to the pharmaceutical composition of the present invention.

The dosage and administration schedule of the active ingredients in various preparations can be adjusted as appropriate depending on the purpose of administration, age, body weight, etc. of the subject. For example, the dosage 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 as a single dose. Alternatively, it can 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 therapeutic or prophylactic agent of the present invention can be adjusted as appropriate depending on the purpose of administration, age, body weight, etc. of the subject. For example, the frequency of administration typically ranges from about once per week to about once every three months, more preferably from about once every two weeks to about once every ten weeks, For example, once every 4 to 8 weeks. Preferably, the antibodies of the invention are administered parenterally, intravenously, eg, into the cubital fossa or other peripheral vein, intramuscularly or subcutaneously. For prophylactic treatment, it is generally preferred to administer the antibodies of the invention from once per month to once every two to three months, or less. The number of administrations may be once, or multiple times (for example, 2 to 5 times) at intervals of several days or weeks. Progress may also be monitored by periodic evaluation. When repeatedly administered over at least several days, the treatment is repeated depending on the symptoms until a certain amount of improvement in the symptoms of the disease is achieved. Depending on the pattern of pharmacokinetic decay of the antibody, the desired dose can be delivered to the patient by administering the antibody in single or multiple boluses, or by continuous infusion.

The present invention will be described in detail below with reference to Examples, but the present invention is not limited thereto.

Comparative example 1-1. Examples of failed hTRPV2 antibody production 1) and 2)
1) After synthesizing the partial peptide CATESVQPMEGQEDEGNGAQYRGIL, which has the amino acid sequence of positions 571 to 594, which is a highly hydrophilic region between the 5th and 6th transmembrane regions of human TRPV2 (hTRPV2), it was used as a carrier. It was prepared as an immunogen by binding KLH. 200 μL of immunogen (2 mg/mL) and 200 μL of adjuvant (complete adjuvant (FREUND)) were mixed to form an emulsion, which was used to immunize 4-week-old female mice (C3H) (day 1). For the second (4th day) and third (7th day), the adjuvant was changed to incomplete adjuvant (FREUND) and the same amount of immunogen as the first time was used for immunization, and cell fusion was performed 3 days after the final immunization.
2) Alternatively, three types of partial peptides (p571-594/ p558-575/p579-595; CATESVQPMEGQEDEGNGAQYRGIL/CQEAWRPEAPTGPNATESV /EGQEDEGNGAQYRGILEC) was synthesized, and then KLH was individually conjugated as a carrier. Thereafter, each peptide was mixed, an immunogen of 2 mg/mL was prepared, and the cells were immunized with the same immunization schedule as above for cell fusion.

Comparative example 1-2. Enlarged inguinal lymph nodes were removed from mice immunized with cell fusion using the PEG method, and cells were collected from them. The collected lymph node-derived cells and myeloma cells (mouse myeloma-derived P3X63Ag8U.1, P3U1 ATCC [CRL-1597]) were mixed, centrifuged, and PEG4000 (MERCK) (diluted in equal volume with RPMI medium) was added to the pellet. Cell fusion was performed. After washing with RPMI medium, the cells were suspended in FBS-HAT medium and seeded in a 96-well plate. After seeding, the medium was replaced, and when hybridoma colony formation was confirmed, the culture supernatant was sampled from the 96-well plate and screened.

Comparative example 1-3. Cell ELISA
hTRPV2-expressing HEK293 cells and negative control HEK293 cells (human embryonic kidney-derived ATCC CRL-3216) were seeded in a 96-well plate for Cell ELISA (NUNC) (1 to ³ × 10 cells/well), and hTRPV2 cells were Cannabidiol was treated appropriately to promote plasma membrane translocation. 50 μL/well of hybridoma culture supernatant was added to the immobilized plate on which the above cells were immobilized, and the reaction was allowed to proceed 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 times with diluent (0.5% BSA/2 mM EDTA/PBS) for 30 minutes at room temperature. Made it react. After washing three times, a chromogenic substrate was added to develop color for 10 to 15 minutes, and absorbance was measured at 450 to 620 nm. However, no positive clones were confirmed.

Comparative example 1-4. antigen solid phase ELISA
Since no positive clones were observed in the above Cell ELISA, binding to the immunogen was evaluated by ELISA, and the top 200 clones that bound to the immunogen were selected. Furthermore, when flow cytometry was performed on the 200 clones using hTRPV2-expressing HEK293 cells and HEK293 cells, 10 clones bound to hTRPV2-expressing HEK293 cells, but also bound to HEK293 cells that do not express hTRPV2. As a precaution, we performed an hTRPV2 functional assay using the culture supernatants of 10 clones, but none of the clones inhibited Ca ²⁺ influx by hTRPV2. Therefore, it was considered that these 10 clones did not specifically bind to hTRPV2. Furthermore, the remaining 190 clones did not bind to either hTRPV2-expressing HEK293 cells or HEK293 cells.
One possible reason why clones that seemed to be hTRPV2-specific could not be confirmed was that hTRPV2 was also expressed in HEK293 cells themselves, and there was a possibility that no difference was observed. Therefore, instead of human-derived HEK293 cells, we newly used Chinese hamster-derived CHO cells (CHO-K1 ATCC CCL-61) and evaluated the binding of the above 10 clones to hTRPV2-expressing CHO cells and CHO cells by flow cytometry. did.
As a result, 8 clones bound to hTRPV2-expressing HEK293 cells but not to hTRPV2-expressing CHO cells. In addition, the remaining two clones bound to hTRPV2-expressing HEK293 cells and hTRPV2-expressing CHO cells. However, the above two clones also bound to HEK293 cells and CHO cells. Therefore, no clone specifically binding to hTRPV2 was obtained.

Comparative example 1-5. Example of failure in hTRPV2 antibody production 3)
Human TRPV2-expressing HEK293 cells were created by introducing and expressing full-length human TRPV2 into HEK293 cells according to the method described in Japanese Patent Application Laid-Open No. 2007-259745 (Japanese Patent Application No. 2006-088323), and treated with cannabidiol. was conducted and used as an immunogen. A 4-week-old female mouse (C3H) was immunized with 200 μL of adjuvant (complete adjuvant (FREUND)), and from the second time onward, the above frozen stock and human TRPV2-expressing HEK293 cells (2 × 10 ⁷ cells in 400 μL of PBS) were used as the immunogen. It was used for each immunization.
For adjuvant immunization (day 0), 50 μL of adjuvant was administered per animal, and then on days 1, 4, 7, 10, 13, and 16, human TRPV2-expressing HEK293 cells (5 × ¹⁰ cells in 100 μL of PBS) were administered. ) was immunized a total of 7 times with an adjuvant, and then cell fusion was performed 3 days after the final immunization.

Comparative example 1-6. Enlarged inguinal lymph nodes were removed from mice immunized with cell fusion using the PEG method , and cells were collected from within. The collected lymph node-derived cells and myeloma cells (mouse myeloma-derived P3X63Ag8U.1, P3U1 ATCC [CRL-1580]) were mixed, centrifuged, and PEG4000 (MERCK) (diluted in equal volume with RPMI medium) was added to the pellet. Cell fusion was performed. After washing with RPMI medium, the cells were suspended in FBS-HAT medium and seeded in a 96-well plate. After seeding, the medium was replaced, and when hybridoma colony formation was confirmed, the culture supernatant was sampled from the 96-well plate and screened.

Comparative example 1-7. Cell ELISA
hTRPV2-expressing HEK293 cells and negative control HEK293 cells were seeded in a 96-well plate for Cell ELISA (NUNC) (cell number 1 to 3×10 ⁴ cells/well) and treated with cannabidiol. 50 μL/well of hybridoma culture supernatant was added to the above-described cell immobilized plate, and the reaction was allowed to proceed at room temperature for 30 minutes. After washing twice (0.5% BSA/2mM EDTA/PBS), peroxidase-labeled goat anti-mouse IgG (MBL) was diluted 10,000 times with diluent (0.5% BSA/2mM EDTA/PBS) and reacted for 30 minutes at room temperature. I let it happen. After washing three times, a chromogenic substrate was added to develop color for 10 to 15 minutes, and absorbance was measured at 450 to 620 nm. As a result, more than 76 positive clones were obtained. However, it was not specific to hTRPV2-expressing HEK293 cells, but was also positive for negative control HEK293 cells.

Comparative example 1-8. flow cytometry
For a total of 76 culture supernatant samples that reacted with hTRPV2-expressing HEK293 cells by Cell ELISA and in which hybridomas were present, binding to hTRPV2-expressing HEK293 cells and HEK293 cells was confirmed by flow cytometry. A PE-labeled anti-mouse IgG antibody was used for detection. As a result, as suggested by Cell ELISA, samples that bound to hTRPV2-expressing HEK293 cells also bound to HEK293 cells to the same degree or higher, and no sample clones specific to hTRPV2-expressing HEK293 cells could be confirmed. . The reason why clones that seemed to be hTRPV2-specific could not be confirmed was that hTRPV2 was also expressed in HEK293 cells themselves, and it was thought that no difference could be observed. Therefore, instead of human-derived HEK293 cells, we used new Chinese hamster ovary-derived CHO cells (CHO-K1 ATCC CCL-61) and performed flow cytometry using hTRPV2-expressing CHO cells and CHO cells. and binding to CHO cells was evaluated for the above 76 clones. As a result, there were 5 clones that bound both hTRPV2-expressing HEK293 cells and hTRPV2-expressing CHO cells, but these clones also reacted with HEK293 cells and CHO cells. Therefore, it was considered that an antibody that specifically binds to hTRPV2 had not been obtained.

Comparative example 1-9. Example of failure in hTRPV2 antibody production 4)
Recombinant protein using wheat germ extract (+ liposome) as an antigen; amino acid region of the membrane region of hTRPV2 (380 amino acids at the N-terminus and 113 amino acids at the C-terminus of the intracellular region are removed, positions 381-650, cell-free) Science Inc.) was synthesized (1.32 mg/mL, 0.65 mL). The above recombinant protein and Freund's complete adjuvant (Difco) were mixed at a volume ratio of about 1:2 and emulsified. 60 μL of this emulsified antigen was administered to the tails of 8-week-old female mice (B6D2F1) per mouse. After 14 days, the antibody titer was first checked using serum, and after 17 days, a booster immunization was given in the same amount as the initial dose. On the fourth day after the booster immunization, lymphocytes were collected from the iliac lymph nodes and fused with myeloma SP2 cells (mouse myeloma-derived SP2/0-Ag14, ECACC 85072401) using the polyethylene glycol (PEG) method.

Comparative example 1-10. Antibody titer check using serum Antibody titer checking using serum was performed by flow cytometry using hTRPV2-expressing HEK293 cells treated with cannabidiol and hTRPV2-expressing HEK293 cells without cannabidiol treatment, but in both cases hTRPV2 No difference was observed between non-expressing HEK293 cells. Therefore, we checked the serum antibody titer by ELISA using a recombinant protein of the amino acid region (positions 381-650) of the membrane portion of hTRPV2 used for immunization. The mice were divided into groups of mice that were positive and mice that were clearly not positive at serum dilution of 1:1000, and lymphocytes were collected from the iliac lymph nodes of each group on the 4th day after the booster immunization as described above. PEG-4000 was dissolved in RPMI medium to a concentration of 50 (W/V)%, and the lymphocytes and SP2 cells were mixed at a ratio of 2:1 to perform cell fusion.
The hybridoma culture medium was replaced with HAT selection medium, and after further culturing, the culture supernatant was used for screening by ELISA, and 54 (300 trials), and 36 (of 300 trials) in the group with unclear positive results. Although each positive supernatant was used to stain hTRPV2-expressing HEK293 cells, none of the positive hybridoma supernatants reacted with hTRPV2-expressing HEK293 cells. No hybridoma supernatant binding specifically to hTRPV2-expressing cells could be confirmed.

Comparative example 1-11. Production of anti-hTRPV2 antibody; failure cases 5) and 6)
5) Synthesize a partial peptide (p571-594 CATESVQPMEGQEDEGNGAQYRGIL) with the amino acid sequence of positions 571-594, which is a highly hydrophilic region between the 5th and 6th transmembrane regions of hTRPV2, and bind it to KLH I let it happen. The peptide was mixed 1:1 with an adjuvant (complete Freund's adjuvant, Difco Laboratories) and administered to two New Zealand White rabbits (slc:Nzw Japan SLC) once a week, once per rabbit for four weeks. Antiserum was obtained by immunization with 0.5 mg of peptide.
6) Alternatively, three types of synthetic peptides (three types of peptide cocktail p571-594/p558-575/p579-595; CATESVQPMEGQEDEGNGAQYRGIL/ CQEAWRPEAPTGPNATESV/ EGQEDEGNGAQYRGILEC) were synthesized and conjugated to KLH, respectively. Each peptide was mixed and used as an immunogen. Specifically, three partial peptides, which are antigens, were mixed 1:1 with an adjuvant (complete Freund's adjuvant, Difco Laboratories) and administered to two New Zealand White rabbits once a week for 4 weeks. Antiserum was obtained by immunizing with 0.6 mg (0.2 mg x 3) of the peptide once.

Comparative example 1-12. Immunostaining and immunoblot evaluation The obtained antisera were antisera that reacted with each antigenic peptide. However, in order to confirm whether the obtained antiserum recognizes the extracellular domain of TRPV2, we used the antiserum to perform immunoblotting (500-fold dilution) and immunostaining using hTRPV2-expressing HEK293 cells. 50-100 times dilution). For immunostaining, hTRPV2-expressing HEK293 cells and each antiserum (50-100 times diluted) were incubated at room temperature for 30 minutes, then reacted with FITC-labeled anti-rabbit secondary antibody, and FITC fluorescence was observed using a confocal laser microscope. did. As a result of immunostaining and immunoblotting, all of the obtained antisera did not react with hTRPV2-expressing HEK293 cells.

As mentioned above, we designed an experiment analogous to the content of Examples of Patent No. 5754039, which is a prior art document related to the invention of an antibody that recognizes the mouse TRPV2 sequence and has the function of specifically inhibiting TRPV2 activity. We prepared a plurality of different immunogens (cells or peptides) that could be assumed from the above examples, and attempted to obtain the desired anti-human TRPV2 antibody. However, as far as it can be inferred from the contents of prior art documents, etc., anti-human TRPV2 antibodies that recognize the desired extracellular domain of human TRPV2 as an epitope, or that recognize the extracellular domain of human TRPV2 as an epitope and have the activity of human TRPV2 We have not been able to obtain an anti-human TRPV2 antibody that has the ability to specifically inhibit TRPV2.

Example 1. Mouse anti-human TRPV2 antibody production
1-1. Immunogen production and immunization
Using two types of autoimmune disease mice (SLE model mice MRL/lpr and C3H/lpr), two mice each (four mice in total) were immunized with the foot pad method at two-week intervals. For weeks 1 and 3, the following peptide from the highly hydrophilic region between the 5th and 6th transmembrane regions of human TRPV2 (hTRPV2) was used as an immunogen, and for weeks 5 and 7, the following full-length peptide was used as an immunogen. hTRPV2-expressing HEK293 cells (TRPV2 was expressed in the cell plasma membrane by stimulation with cannabidiol) were immunized. One week after the final immunization, blood and lymph nodes were collected, and lymph nodes from mice that were confirmed to contain the desired antibody (which can be stained from outside the cells and exhibits inhibitory activity) were evaluated using antiserum. , and created a phage library.
Immunogen: Peptide-KLH 579-595 (Ac)EGQEDEGNGAQYRGILEC-KLH and full-length hTRPV2-expressing HEK293 cells (full-length hTRPV2 amino acid sequence was obtained according to the method described in JP-A No. 2007-259745 (Japanese Patent Application No. 2006-088323)). hTRPV2-expressing HEK293 cells were created by introducing and expressing hTRPV2 into HEK293 cells, and the cells were treated with 10 μM cannabidiol (10% FBS/DMEM) at 37°C for 15 minutes to increase TRPV2 expression in the plasma membrane. It was collected and used as an immunogen.

1-2. Preparation of two types of antibody phage libraries Using ISOGEN (Nippon Gene) from each lymph node of 3 mice (mouse numbers 01, 03, 04) selected for serum evaluation among 4 immunized mice. RNA was extracted. cDNA was synthesized from the extracted RNA using SuperScript III Reverse Transcriptase (Invitrogen). PCR was performed using mouse antibody primers and the synthesized cDNA as a template to amplify mouse antibody genes VH and Vκ. Mouse number 01 (No.01 library) was amplified alone, and mouse numbers 03 and 04 (No.02 library) were amplified as a mixture.
The Vκ PCR product was inserted into the scFv-cp3 vector using restriction enzyme sites. E. coli was transformed with the vector into which the L chain had been inserted, and plasmids were recovered from the cultured E. coli. VH was inserted into this plasmid (scFv-cp3 vector into which the L chain had been integrated) using another restriction enzyme site. When E. coli DH12S (Invitrogen) was transformed with the vector incorporating the VH and VL genes, 6.9 x 10 ⁸ transformants were obtained for the No. 01 antibody library and 5.9 x 10 ⁸ transformants were obtained for the No. 02 antibody library. Ta. A schematic diagram is shown in Figure 1.
A glycerol stock of transformed E. coli was prepared. In addition, a phage library solution was prepared by infecting the transformed E. coli with helper phage M13KO7 and used for panning.

1-3． Anti-human TRPV2 antibody confirmation assay in mouse antiserum (1) Immunoblot
1.6×10 ⁷ cells of biotinylated hTRPV2-expressing HEK293 cells (hTRPV2 full-length expression vector (Myc-Biotinylation-His tag) expressed in HEK293 cells) were treated with 1% n-Dodecyl-β-D-maltoside (Dojindo). Solubilized with Dynabeads MyOne Streptavidin T1 (Veritas) and rotated for 20 minutes at room temperature to bind to magnetic beads. After washing, SDS sample buffer was added and boiled at 95°C for 10 minutes. The SDS converted sample was detected using antiserum and anti-His tag antibody using Western blot method. As a result, when the anti-His tag antibody was used under conditions of an exposure time of 10 seconds, bands were confirmed at the positions of 100 kDa and 75 kDa (Fig. 2a). Furthermore, when the exposure time was 3 minutes, bands were observed at the 100 kDa and 75 kDa positions even when immunized mouse antiserum was used as the primary antibody (Figure 2b). In both cases, a band was confirmed at the same position as when an anti-His tag antibody was used, so it was concluded that hTRPV2 was solubilized and specifically detected. The density of the bands was in the order of mouse numbers 01>03>04.

(2) Cell ELISA
The binding activity of the antiserum was evaluated using hTRPV2-expressing HEK293 cells. As a result of evaluating each antiserum at two concentrations (2000-fold dilution and 10000-fold dilution), all antisera showed higher binding activity to hTRPV2-expressing HEK293 cells than to negative control HEK293 cells (Figure 3 ).

(3) ELISA using solubilized membrane fraction (Solubilized ELISA)
The activity of each antiserum was confirmed using a streptavidin plate (Thermo Scientific) in which biotinylated hTRPV2-expressing HEK293 cells were solubilized and immobilized. As a result, the lysate of HEK293 cells expressing biotinylated hTRPV2 did not react with the negative control lysate of HEK293 cells that did not express TRPV2 (expressed with the same tag attached to protein It was confirmed that the solubilized membrane fraction had been prepared because it specifically reacted with the target (Figure 4). The strength of binding activity to biotinylated peptide was in the order of mouse numbers 04>01>03, whereas in the solubilized membrane fraction, the order was mouse numbers 01>03>04.

The activity of hTRPV2-expressing 293 cells was confirmed using Cell ELISA and solubilization ELISA, and both methods confirmed specificity using mouse serum. However, in Cell ELISA, the antiserum also reacted with HEK2923 cells that do not express hTRPV2, and since all the individuals used for library construction were immunized with HEK293 cells that express hTRPV2, common molecules on the HEK293 cell membrane were detected. It was thought that it was a reaction to. For this reason, it was determined that it would be difficult to concentrate antibodies specific to the target antigen if cells were used as the antigen for the first panning, so peptides were used as the antigen for the first to third pannings. There was a concern that if peptides were used as panning antigens, it might not be possible to obtain antibodies that recognize the 3D structure, but we started panning using peptide antigens with the goal of obtaining antibodies that showed specificity in Cell ELISA.

1-4. panning
We created an antigen to be used in panning to obtain antibodies against hTRPV2. Specifically, by adding a biotinylated tag, the target antibody was collected after solubilization using streptavidin beads, etc., and the antigen was presented at high density, thereby increasing the concentration efficiency of the target antibody.
Biotinylated peptide; 579-595 (Ac)EGQEDEGNGAQYRGILEK-biotin
HEK293 cells expressing biotinylated hTRPV2; hTRPV2 full-length expression vector (Myc-Biotinylation-His tag) was transfected into HEK293 cells using Transficient (MBL: WU1001). Cells were suspended in DMEM (low glucose) containing 10% FCS. After 24 hours of culture, cells were collected.

Panning performed
1st round: Biotinylated peptide-bound magnetic particle panning
2nd round: Biotinylated peptide-bound magnetic particle panning
3rd round: KLH-modified peptide-bound magnetic particle panning, or biotinylated peptide + anti-biotin antibody-bound magnetic particle panning
From the fourth time onwards, two different panning methods were used.
4th round-A: After solubilizing biotinylated hTRPV2-expressing HEK293 cells, panning with those presented on magnetic particles
5th round-A: After solubilizing HEK293 cells expressing biotinylated hTRPV2, panning with anti-His tag antibody presented on magnetic particles, or
4th round-B: Cell panning using HEK293 cells expressing biotinylated hTRPV2
5th round-B: Cell panning using biotinylated hTRPV2-expressing HEK293 cells During each panning, cells were collected using streptavidin-labeled magnetic beads. The procedure of removing unbound phages by washing and eluating bound phage by trypsin treatment was repeated.

The binding of the phages recovered by panning to hTRPV2 was confirmed by Cell ELISA using hTRPV2-expressing HEK293 cells. As a result, the phage colony (02a 5th) recovered in the 5th round of panning using the No. 02 phage library showed slightly higher binding activity to hTRPV2-expressing HEK293 cells than the negative control (Figure 5). However, it was confirmed that the expression level on the membrane was considerably lower than in TRPV2 non-expressing HEK293 cells (expressed with the same tag attached to protein Mouse serum binding activity was lower in hTRPV2-expressing HEK293 cells. Therefore, it was determined that the phage colonies recovered in the fifth round of panning that were even slightly more reactive to hTRPV2-expressing HEK293 cells contained hTRPV2-specific antibodies, and 48 clones were isolated from this plate. In addition, 48 clones were similarly isolated from the phage colonies (01a 5th) recovered in the 5th panning using the No. 01 phage library that had been subjected to the same panning method. That is, in the No. 02 and No. 01 phage libraries, clones that bind to hTRPV2-expressing HEK293 cells were included among the phages recovered in the fifth round of panning. They were monocloned and 002a and 001a were isolated.

1-5. Preparation of monoclonal antibody fragment Phage (02a) recovered from phage library No. 02 that was panned five times and showed specificity for hTRPV2-expressing HEK293 cells in Cell ELISA evaluation using phage colonies, and the same A clone was isolated from the phage (01a) recovered from the No. 01 phage library that had been subjected to panning five times. 48 colonies were picked up and cultured from the E. coli plate containing the antibody phage recovered from each panning, and IPTG induction was applied to express and secrete the antibody into the E. coli culture supernatant. After removing the E. coli pellet, the scFv cp3 E. coli It was used as a culture supernatant for Cell ELISA evaluation.

1-6. Evaluation of binding by ELISA The binding activity of the phage prepared above (02a and 01a above) was evaluated by ELISA after solubilizing biotinylated hTRPV2-expressing HEK293 cells and immobilized on a streptavidin plate. As a result, 01a005, 01a016, 01a018, and 01a033 from the 01a phage colony showed slightly high binding activity in HEK293 cells expressing biotinylated hTRPV2 (Fig. 6A). These 4 clones showed higher binding activity in biotinylated hTRPV2-expressing HEK293 cells than in negative control cells that did not express hTRPV2 (expressing biotinylated protein And so. Furthermore, all clones from the 02a phage colony showed high binding activity to HEK293 cells expressing biotinylated hTRPV2 (Fig. 6B).

1-7. VH analysis of monoclonal antibodies Plasmid DNA was prepared from the culture medium in which phages collected after each panning were picked up from the E. coli plate and cultured, and VH analysis of the ELISA-positive clones described above (1-6) was performed. As a result of the analysis, the ELISA positive clones (01a005, 01a016, 01a018, 01a033) of the 01a library all had the same VH sequence (VH of the 01a library: SEQ ID NO: 16). Next, we analyzed 02a001 to 009 from the ELISA-positive clones of the 02a library, and found that 7 clones were identical (with a 1 amino acid difference in the frame part), and 2 clones had a 1 amino acid difference in the CDR2 part (02a003 , 02a005), but CDR3, which is thought to be important for binding activity, was the same in all clones (VH of 02a library: SEQ ID NO: 14 (02a001), 18 (02a003 and 02a005), 19 (02a006 )(3 types)). Therefore, when evaluating the clones after analysis, the 01a library had the best specificity in ELISA, 01a033 (hereinafter sometimes referred to as mAb001), and the 02a library showed differences in binding activity. 02a001 (hereinafter also referred to as mAb002) was selected. The 02a library is a mixed library of immunized individuals No. 3 and No. 4, but because the clone with the same VH sequence as 02a001 has high binding activity, clones with low binding activity such as 01a033 cannot be obtained. It is assumed that there was no such thing.

1-8． Confirmation of binding to hTRPV2 by Cell ELISA and flow cytometry
As a result of VH analysis of ELISA-positive clones from 01a and 02a libraries, one type of specific clone was confirmed in each library. Representative clone 01a033 (mAb001) (heavy chain CDR1 (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 : 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 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) binding activity was evaluated by Cell ELISA using hTRPV2-expressing HEK293 cells. In the above evaluation, each sample was picked up from each E. coli plate, cultured, and subjected to IPTG induction to express and secrete the antibody protein into the E. coli culture supernatant. Thereafter, the E. coli pellet was removed by centrifugation, and the resultant was precipitated and concentrated using ammonium sulfate. As a result, both clones showed high fluorescence intensity against hTRPV2-expressing cells.

In addition, this experiment aims to obtain antibodies that bind to antigens on cell membranes. Therefore, flow cytometry was performed using hTRPV2-expressing HEK293 cells that expressed the native form of hTRPV2 without being immobilized or solubilized. As a result, although only slight binding was confirmed with 01a033 (mAb001), hTRPV2-specific binding was confirmed with 02a001 (mAb002). Regarding 01a033 (mAb001), we cannot deny the possibility that it is a clone that recognizes only the solubilized form, but since this experiment was evaluated using scFv, the binding activity increased after IgG conversion, and the binding activity against hTRPV2-expressing HEK293 cells increased. We considered that there was a good possibility that binding could be confirmed by flow cytometry (Figure 7).

1-9. Confirmation of binding to hTRPV2 by immunoprecipitation experiment
As a result of VH analysis of ELISA-positive clones, beads bound with scFv antibodies were prepared from the culture supernatants of a total of three clones, representative clones 01a033 (mAb001) and 02a001 (mAb002), and a negative control antibody, and hTRPV2-expressing HEK293 cells were prepared. Immunoprecipitation experiments with solubles were performed. As a result, binding to hTRPV2 (band at 100 kDa) was confirmed only with beads to which 01a033 (mAb001) and 02a001 (mAb002) were bound (Figure 8). In both cases, a band was confirmed at the same position as when anti-His tag antibody was used, confirming that all scF _Vs were able to immunoprecipitate hTRPV2.

1-10． Construction of antibody gene vector and introduction of variable region into expression vector to construct H chain and L chain expression system
The VH and VL fragments of two clones 01a033 (mAb001) and 02a001 (mAb002) were amplified by PCR using the scFv expression vector as a template while adding a signal sequence and a restriction enzyme site, respectively. For VL, a VL-CK fragment was prepared by fusion-PCR in which CK was subsequently added.
These gene fragments were each treated with two types of restriction enzymes and incorporated into an H chain vector (pEHX1.1) and a L chain vector (pELX2.2) into which the mouse CH1, 2, 3 genes had been previously incorporated. As secretion signal sequences, 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)
The H chain vector and L chain vector were sequenced and it was confirmed that the correct gene fragment had been inserted. The H chain vector and L chain vector were digested with two types of restriction enzymes, and the H chain vector and L chain vector were ligated to produce an IgG antibody expression vector. After introducing the linked IgG antibody expression vector into E. coli, plasmids were recovered from the cultured transformants using the QIAGEN Plasmid Midi Kit (QIAGEN).

・Introduce H chain and L chain expression vectors into CHO cells and confirm expression Digest one site of the prepared IgG antibody expression vector with restriction enzyme (AseI (NEB)) and electroporate the single-stranded plasmid. The cells were introduced into CHO-K1 (adapted for suspension culture) by ration. The cells were suspended in 20 mL of 10% FBS Ham's F12 medium (Wako Pure Chemical Industries, Ltd.) and cultured at 37°C, 5%, and CO2. The next day, puromycin (Sigma) was added to a final concentration of 10 μg/mL, and one day later, a portion of the culture supernatant was collected and analyzed by ELISA. As a result, IgG expression was confirmed in both 01a033 (mAb001) and 02a001 (mAb002).

1-11. Preparation of mouse antibody gene expression vector-introduced cells and antibody production/preparation of anti-human TRPV2 IgG antibody-producing cell lines
Continue to culture the anti-human TRPV2 IgG antibody-producing cells that have confirmed IgG expression, and detach them with 0.05% trypsin-EDTA (Wako Pure Chemical Industries, Ltd.) on the 14th day after gene transfer, and transfer to serum-free medium containing 4 mM L-Glutamine. (Aclimated to EX-CELL (registered trademark) CD-CHO Fusion (SAFC). During acclimatization, puromycin was added at 10 μg/mL and selection was continued.

・Purification of anti-human TRPV2 IgG antibody from anti-human TRPV2 IgG antibody-producing cell line Suspend the cells that have completed conditioning in 100 mL of serum-free medium at a concentration of 2.0 to 3.0 x ¹⁰ cells/mL for 10 days. , shaking culture was performed in a 500 mL flask (Nunc) at 37°C. The culture supernatant was collected and purified using 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 Glycin-HCl (pH 3.0), and after dialysis with PBS, it was transferred to PBS(-) by ultrafiltration (Amicon Ultra (Millipore)). Buffer replacement and concentration were performed. After filtration with a 0.22 μm filter, concentration and purity were confirmed by OD280 nm measurement and CBB staining of the gel subjected to SDS-PAGE (Figure 9).

1-12. hTRPV2 binding activity of anti-human TRPV2 IgG antibody (flow cytometry)
・Cells used
HEK293 cells transfected with a biotinylated hTRPV2 expression vector and a biotinylated hTRPV2 non-expression vector (expressing biotinylated protein X, which is completely unrelated to TRPV2) were used.
・Preparation of HEK293 cells expressing biotinylated hTRPV2
Culture was started in D-MEM (10% FBS) medium and subconfluent was subcultured. Cells grown to subconfluence were used in the experiment.
Biotinylated hTRPV2 expression vector (MBL) was transfected using Transficient (MBL) according to the manual, and cultured for about 24 hours. At the same time, as a negative control, an hTRPV2 non-expression vector (expressing a biotinylated protein X, which is completely unrelated to TRPV2) was introduced in the same manner. The cells were detached by pipetting, washed with PBS, and the number of cells was adjusted to 2×10 ⁵ per sample with 1% BSA/PBS.

・Binding reaction and flow cytometry analysis Antibody binding reaction and washing operation were performed using 1% BSA/PBS. 5 μg/mL of the anti-human TRPV2 IgG antibody prepared above 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)) on ice for 1 hour. After washing twice with 1% BSA/PBS, analysis was performed using a flow cytometer (Beckman, FC500).
The MFI value was determined by comparing the X-mean of each clone with that of a negative control antibody. Although 01a033 (mAb001) was specific to hTRPV2-expressing cells, it was found to have minimal binding. Although a shift was observed in 02a001 (mAb002) in negative control cells (hTRPV2 non-expressing HEK293 cells), it strongly bound to hTRPV2-expressing HEK293 cells, and two peaks were confirmed. The significantly shifted peak on the right is considered to be a response to forcedly expressed hTRPV2, and the peak on the left is thought to be a response to hTRPV2 that is extremely slightly endogenous to HEK293 cells. In the scFv-cp3 E. coli culture supernatant before IgG conversion, the titer was insufficient and the shift was only confirmed with the forced expression antigen. It is presumed that this reaction also occurred (Fig. 10).

1-13. Confirmation of species cross-reactivity between anti-mouse TRPV2 antibody and anti-human TRPV2 antibody / Cross-reactivity confirmation test using Western blot
HEK293 cells, mouse TRPV2 (mTRPV2) and hTRPV2-expressing HEK293 cells were solubilized with Lysis buffer. After centrifugation at 16,000 × g for 10 min at 4°C, the supernatant was separated on 7.5% SDS-PAGE and then transferred to PVDF. After blocking the membrane with Blocking One (Nacalai Tesque), anti-mouse TRPV2 antibody (mAb88-2) diluted 500 times with Blocking One, anti-human TRPV2 antibody (mAb002), TRPV2 polyclonal antibody pAb (TRPV2) (C-terminus of TRPV2) A polyclonal antibody prepared using the region as an antigen and capable of recognizing both mTRPV2 and hTRPV2 (Non-Patent Document 2) was added and incubated at 4°C overnight. After washing 3 times for 5 minutes with Wash buffer (0.1% Tween-20/TBS), add HRP-labeled secondary antibody (Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP or Goat anti-Mouse IgG (H+L) diluted 1000 times with Blocking One). -Incubated with Rabbit IgG (H+L) for 1 hour at room temperature. After that, washed 3 times for 5 minutes with Wash buffer (0.1% Tween-20/TBS), further washed 2 times with PBS, and ECL (enhanced chemiluminescence substrate) was added to detect chemiluminescence. pAb (TRPV2) stains both mTRPV2 and hTRPV2 by immunoblotting, mAb88-2 stains only mTRPV2-expressing HEK293 cell lysates, and mAb002 stains only mTRPV2-expressing HEK293 cell lysates. Only hTRPV2-expressing HEK293 cell lysates were stained.

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

・Cross-reactivity confirmation test using Cell ELISA
HEK293 cells expressing hTRPV2 (FIG. 12a) or HEK293 cells expressing mTRPV2 (FIG. 12b) were seeded at 1-3×10 ⁴ cells/well in a 96 well plate. The next day, after fixing with PFA for 5 minutes, washing was performed with PBS, and anti-mouse TRPV2 antibody (mAb88-2), anti-human TRPV2 antibody (mAb002, mAb001), and isotype control were added to each cell. After incubation for 30 minutes at room temperature, it was washed with PBS. Furthermore, Alexa488-labeled anti-mouse secondary antibody (life technology) was added at 50 μl/well and allowed to react at room temperature for 30 minutes. After washing with PBS, fluorescence values were measured using POLARstar Omega (BMG Labtech, Germany). (Figure 12a, and Figure 12b)

1-14. Measurement of KD value of anti-hTRPV2 antibody 1-11 above. The dissociation constant between the obtained anti-hTRPV2 antibodies (mAb001, mAb002) and the hTRPV2 extracellular domain was measured as follows.
hTRPV2 extracellular domain-biotin (572-609) (a polypeptide with a lysine residue and biotin added to the C-terminus of the amino acid sequence shown from positions 572 to 609 of the amino acid sequence of SEQ ID NO: 1 in the sequence listing) was synthesized. Using Biacore T200 (GE Healthcare Biosciences), the measurement was performed using a capture method in which the antibody was captured on an immobilized anti-mouse IgG (Fc) antibody and the antigen was measured as an analyte. ~15,000 RU of anti-mouse IgG (Fc) antibody (Mouse Antibody Capture Kit, GE Healthcare Biosciences, Inc.) was covalently bound to the sensor chip CM5 (GE Healthcare Biosciences) using the amine coupling method. A reference cell was also immobilized in the same manner. HBS-EP (10 mM HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% Surfactant P20) was used as a running buffer. After adding an antibody solution of approximately 10 μg/ml at a flow rate of 10 μl/min for 180 seconds onto a chip immobilized with an anti-mouse IgG (Fc) antibody, a diluted serial solution of hTRPV2 extracellular domain (572-609) ( 10-1000 nM) was added for 120 seconds at a flow rate of 30 μl/min and the dissociation phase was monitored for 900 seconds. As a regeneration solution, 10 mM Glycine-HCl (pH 1.7) was added at a flow rate of 20 μl/min for 180 seconds. For data analysis, the association rate constant ka, dissociation rate constant kd, and dissociation constant (KD; KD=kd/ka) were calculated using the one-to-one binding model of the analysis software (Biacore Insight Evaluation Software, version 2.0). . As a result, the KD value of anti-hTRPV2 antibody (mAb002) was 19.2 nM, and the KD value of anti-hTRPV2 antibody (mAb001) was 188 nM.

1-15. Evaluation of HEK293 intracellular Ca ²⁺ influx inhibition using anti-human TRPV2 antibody
HEK293 cells expressing hTRPV2 were cultured in a poly-D-Lysine coated 96 well plate until confluent. After removing the culture medium, 50 μl of 5 mM Cal520 (Cal520 (registered trademark), AM, AAT Bioquest) dissolved in 0.5 mM Ca ²⁺ -containing BSS 0.1% BSA was added, and the cells were left in contact for 2.5 hours at 37°C. was loaded with a calcium indicator (Cal520). Thereafter, the Cal520 loading solution was removed, 50 μl of 100 mM cannabidiol (CBD) solution dissolved in BSS containing 0.5 mM Ca ²⁺ was added, and the mixture was allowed to stand at 37° C. for 30 minutes. After that, remove the CBD liquid and use anti-hTRPV2 antibodies (mAb001, mAb002), anti-mTRPV2 antibodies (88-2, 11-6), anti-mTRPV2 antibodies (Anti-TRPV2 (VRL1) (extracellular) Antibody (#ACC-039)Alomon lab). , isotype control (IgG) was contacted with 50 μl of BSS containing 0.5 mM Ca ²⁺ diluted in 0.01% BSA for 1 hour at 4°C. Thereafter, the temperature was returned to 37° C., and after 2 minutes, 50 μl of BSS containing 2 mM CaCl ₂ and 10 mM probenecid was added to start measurement. Final concentrations of CaCl ₂ and probenecid were adjusted to 1.25 mM and 5 mM, respectively. Fluorescence changes reflecting changes in intracellular Ca ²⁺ concentration over time were measured using a fluorescence kinetics analyzer FDSS7000. Inhibition by anti-TRPV2 antibody was evaluated by calculating the reaction rate of wells containing antibody, with the response of wells containing only antibody dilution taken as 100%. The IC ₅₀ of the antibody (mAb002) was 8.9 nM (Figure 13a and Figure 13b).
BSS; Balanced Salt Solution (146mM NaCl, 4mM KCl, 2mM _MgCl2 , 0.5mM CaCl2 _, 10mM glucose, and 10mM HEPES/Tris, pH7.2)

1-16. Identification of epitope of anti-hTRPV2 antibody A plasmid in which the amino acids of the hTRPV2 candidate peptide, which serve as the epitope of the anti-hTRPV2 antibody, were each replaced with alanine (Flag labeled at the C-terminus of hTRPV2) was transfected into HEK293 cells using Lipofectamine 3000. Cells 24-48 hours after transfection were solubilized 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 transferred to a hydrophobic membrane (PVDF). After blocking with 5% skim milk, it was washed with Tris-buffered saline (TTBS) containing 0.1% Tween 20, and reacted with anti-hTRPV2 antibody (mAb002) or anti-flag antibody (MBL M2) at 4°C 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, and then washed again with TTBS. A chemiluminescence detection agent was prepared according to the manual, and the hydrophobic membrane was set in a chemiluminescence detection device, exposed to light, and captured into a camera. The expression of hTRPV2 was determined by quantifying the chemiluminescence detection band labeled with an anti-flag antibody, and analyzed using image J along with the quantification of the detection band using an anti-hTRPV2 antibody (mAb002). It was determined that the amino acid (EGQED, SEQ ID NO: 3) that caused the disappearance of the band due to antibody-peptide binding by alanine substitution was an epitope (FIGS. 14a and 14b).

The anti-human TRPV2 antibody or fragment thereof of the present invention is useful for treating or preventing muscle diseases and/or heart diseases. Furthermore, the anti-human TRPV2 antibody or fragment thereof of the present invention is useful as a diagnostic agent for cardiomyocyte and/or myocyte degenerative diseases.

Claims (26)

An anti-human TRPV2 monoclonal antibody or fragment thereof that recognizes the extracellular domain of human TRPV2 as an epitope. 2. The monoclonal antibody or fragment thereof according to claim 1, which recognizes the extracellular domain of human TRPV2 as an epitope and specifically inhibits TRPV2 activity. A region containing at least five amino acid sequences selected from the amino acid sequences between the fifth and sixth transmembrane regions in the extracellular domain of human TRPV2 is recognized as an epitope, and the activity of TRPV2 is activated. 3. The monoclonal antibody or fragment thereof according to claim 1 or 2, which specifically inhibits. A region containing at least five or more amino acid sequences selected from the amino acid sequences between the first transmembrane region and the second transmembrane region in the extracellular domain of human TRPV2 is recognized as an epitope, and the activity of TRPV2 is detected. 2. The monoclonal antibody or fragment thereof according to claim 1, which specifically inhibits. Among the extracellular domains of human TRPV2, the following:
1) Recognizes as an epitope a region containing at least 5 or more consecutive amino acid sequences selected from the amino acid sequence shown in SEQ ID NO: 2, and specifically inhibits TRPV2 activity, or
2) Recognizes as an epitope a region containing at least 5 or more consecutive amino acid sequences selected from the amino acid sequence shown in SEQ ID NO: 2 in which 1 to 2 amino acids have been substituted, added, or deleted. , specifically inhibits TRPV2 activity,
The monoclonal antibody or fragment thereof according to any one of claims 1 to 3. Among the extracellular domains of human TRPV2,
1) Recognizes the region containing the amino acid sequence shown in SEQ ID NO: 3 as an epitope and specifically inhibits TRPV2 activity, or
2) recognizes as an epitope a region containing an amino acid sequence in which 1 to 2 amino acids are substituted, added, or deleted from the amino acid sequence shown in SEQ ID NO: 3, and specifically inhibits TRPV2 activity;
The monoclonal antibody or fragment thereof according to any one of claims 1 to 3 or 5. According to any one of claims 1 to 3, 5, or 6, which recognizes the amino acid sequence shown by SEQ ID NO: 3 as an epitope in the extracellular domain of human TRPV2 and specifically inhibits TRPV2 activity. monoclonal antibodies or fragments thereof. The monoclonal antibody or fragment thereof according to any one of claims 1 to 7, which is a humanized antibody. The heavy chain variable region is:
(i) CDR1 containing the amino acid sequence shown by SEQ ID NO: 4
(ii) CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and (iii) CDR3 containing the amino acid sequence shown in SEQ ID NO: 6.
including;
The light chain variable region is:
(iv) CDR1 containing the amino acid sequence shown by SEQ ID NO: 7
(v) CDR2 containing the amino acid sequence shown in RMS, and (vi) CDR3 containing the amino acid sequence shown in SEQ ID NO: 8.
The monoclonal antibody or fragment thereof according to any one of claims 1 to 3 and 5 to 8, comprising: The heavy chain variable region is:
(i) CDR1 containing the amino acid sequence shown by SEQ ID NO: 9
(ii) CDR2 containing the amino acid sequence shown in SEQ ID NO: 10, and (iii) CDR3 containing the amino acid sequence shown in SEQ ID NO: 11.
including;
The light chain variable region is:
(iv) CDR1 containing the amino acid sequence shown by SEQ ID NO: 12
(v) CDR2 containing the amino acid sequence shown in WAS, and (vi) CDR3 containing the amino acid sequence shown in SEQ ID NO: 13.
The monoclonal antibody or fragment thereof according to claim 1, comprising: The heavy chain variable region has an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 14, and the light chain variable region has an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 15. The monoclonal antibody or fragment thereof according to any one of claims 1 to 3 and 5 to 8. The heavy chain variable region has an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 16, and the light chain variable region has an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 17. The monoclonal antibody or fragment thereof according to claim 1. A therapeutic or preventive agent for muscle diseases and/or heart diseases, comprising the anti-human TRPV2 monoclonal antibody or fragment thereof according to any one of claims 1 to 3, 5 to 9, and 11. The muscle disease is neuromuscular junction and muscle disease in ICD10 G70-G73 or myopathy in M60-M63, and the heart disease is ischemic heart disease in I20-I25 or other types of I30-I52 in ICD10. 14. The therapeutic or preventive agent according to claim 13, which is a heart disease. Myopathy is primary myopathy of G71 in ICD10 or other myopathy of M62, and cardiac disease is angina pectoris in I20, acute myocardial infarction in I21, recurrent myocardial infarction in I22, acute myocardial infarction in I23 in ICD10. Secondary complications of myocardial infarction, I24 other acute ischemic heart disease, I25 chronic ischemic heart disease, I30 acute pericarditis, I31 other pericardial diseases, I32 diseases classified elsewhere Pericarditis, I33 acute and subacute endocarditis, I34 non-rheumatic mitral valve disorders, I35 non-rheumatic aortic valve disorders, I36 non-rheumatic tricuspid valve disorders, I37 pulmonic valve disorders, I38 endocarditis (valvular unknown), I39 endocarditis and valvular disorders in diseases classified elsewhere, I40 acute myocarditis, I41 myocarditis in diseases classified elsewhere, I42 myocardium cardiomyopathy in diseases classified other than I43, I44 atrioventricular block and left bundle branch block, I45 other conduction disorders, I46 cardiac arrest, I47 paroxysmal tachycardia, and I48 atrial fibrillation. other heart arrhythmias in I49, heart failure in I50, complications of heart disease in I51 and description of heart disease with unclear diagnosis, or other heart disorders in diseases classified other than I52, The therapeutic or prophylactic agent according to claim 13. If the muscle disease is ICD10 G71.0 muscular dystrophy, G71.1 myotonic disorder, G71.2 congenital myopathy, G71.3 mitochondrial myopathy (not elsewhere classified), G71.8 other Primary myopathy, G71.9 unspecified primary myopathy, M62.0 muscle tear, M62.1 other muscle tear (non-traumatic), M62.2 muscle ischemic infarction, M62. Immobility syndrome (paraplegia) in 3, Muscle contracture in M62.4, Muscle wasting and atrophy (not elsewhere classified) in M62.5, Muscle strain in M62.6, Other manifestations in M62.8 Myopathy was diagnosed as a myopathy, M62.9 was an unspecified myopathy, and the cardiac disease was an acute transmural myocardial infarction in the anterior wall of I21.0 in ICD10, and an acute transmural myocardial infarction in the inferior wall of I21.1 in ICD10. , I21.2 acute transmural myocardial infarction at other sites, I21.3 acute transmural myocardial infarction (site unknown), I21.4 acute subendocardial myocardial infarction, I21.9 acute myocardial infarction (Details unknown), I25.0 described as atheroma sclerosing cardiovascular disease, I25.1 atheroma sclerosing heart disease, I25.2 old myocardial infarction, I25.3 ventricular aneurysm, I25.4 coronary artery aneurysm, I25.5 ischemic cardiomyopathy, I25.6 painless myocardial ischemia, I25 .8 other types of chronic ischemic heart disease, I25.9 chronic ischemic heart disease (unspecified), I42.0 dilated cardiomyopathy, I42.1 obstructive hypertrophic cardiomyopathy, I42.2 other hypertrophic cardiomyopathy in I42.3, endocardial (eosinophilic) disease in I42.4, endocardial fibroelastosis in I42.5, other restrictive cardiomyopathy in I42.6 Alcoholic cardiomyopathy, I42.7 Cardiomyopathy due to drugs and other external factors, I42.8 Other cardiomyopathy, I42.9 Cardiomyopathy (unspecified), I50.0 Congestive heart failure, I50. 14. The therapeutic or prophylactic agent according to claim 13, which is left ventricular failure of I50.9 or heart failure of I50.9 (details unknown). The muscle disease is muscular dystrophy with ICD10 G71.0, and the heart disease is acute myocardial infarction (unspecified) with I21.9, dilated cardiomyopathy with I42.0, or left ventricular failure with I50.1. The therapeutic or prophylactic agent according to claim 13. 14. The therapeutic or preventive agent according to claim 13, wherein the muscle disease and/or heart disease is muscular dystrophy with ICD10 G71.0. The therapeutic agent according to claim 13, wherein the myocardial disease and/or heart disease is acute myocardial infarction (details unknown) of I21.9 in ICD10, dilated cardiomyopathy of I42.0, or left ventricular failure of I50.1. or prophylactic agents. A diagnostic agent for myopathies and/or heart diseases, comprising the anti-human TRPV2 monoclonal antibody or fragment thereof according to any one of claims 1 to 19. A diagnostic agent for muscle diseases and/or heart diseases, comprising the anti-human TRPV2 monoclonal antibody or fragment thereof according to claim 10 or 12. A kit for diagnosing muscle diseases and/or heart diseases, comprising the antibody or fragment thereof according to any one of claims 1 to 12. Myopathy is G70-G73 Neuromuscular Junction and Muscle Disease or M60-M63 Myopathy in ICD10, and Cardiac Disease is Ischemic Heart Disease I20-I25 or Other I30-I52 in ICD10. 21. The diagnostic kit according to claim 20, which is a type of heart disease. A method to aid in the diagnosis of muscular and/or cardiac diseases, comprising:
(i) quantifying the expression level of TRPV2 in a sample collected from a test animal using the monoclonal antibody or fragment thereof according to any one of claims 1 to 12;
(ii) Comparing the expression level of TRPV2 quantified in (i) with the expression level of TRPV2 in a sample collected from a healthy animal (control value), and (iii) Based on the results of (ii), ), if the expression level of TRPV2 quantified in ) is greater than the control value, the test animal may be suffering from a muscle disease and/or heart disease, or is currently suffering from a muscle disease and/or heart disease. A method comprising the step of determining that the patient is likely to suffer from a muscle disease and/or a heart disease in the future. A method to aid in diagnosing the progression of muscle and/or heart disease, the method comprising:
(i) A test animal suffering from or possibly suffering from muscle disease and/or heart disease using the monoclonal antibody or fragment thereof according to any one of claims 1 to 12. Quantifying the expression level of TRPV2 in the sample collected from
(ii) Comparing the expression level of TRPV2 quantified in (i) with the expression level of TRPV2 (control value) in a sample collected from an animal suffering from muscle disease and/or heart disease at a specific stage of progression. , and (iii) Based on the results of (ii), if the expression level of TRPV2 quantified in (i) is greater than the control value, the degree of progression of the muscle disease and/or heart disease of the test animal is higher than that of the control. The myopathic disease of the test animal is determined to be higher than the progression level of the animal suffering from the disease, or is judged to be highly likely to be suffering from muscle disease and/or heart disease, and is lower than the control value. and/or determining that there is a high possibility that the heart disease has improved or that the person is not suffering from the muscle disease and/or the heart disease. A method to aid in diagnosing the progression of muscle and/or heart disease, the method comprising:
(i) A test animal suffering from or possibly suffering from muscle disease and/or heart disease using the monoclonal antibody or fragment thereof according to any one of claims 1 to 12. Quantifying the expression level of TRPV2 in the sample collected from
(ii) A step of comparing the expression level of TRPV2 determined in (i) with the expression level of TRPV2 in a sample previously collected from the test animal (control value), and (iii) comparing the result of (ii). Based on this, if the expression level of TRPV2 quantified in (i) is greater than the control value, it is determined that the test animal has advanced muscle disease and/or heart disease, or is suffering from muscle disease and/or heart disease. If it is determined that there is a high possibility that the test animal is suffering from muscle disease and/or heart disease, and if the value is lower than the control value, there is a possibility that the test animal has improved muscle disease and/or heart disease, or is not suffering from muscle disease and/or heart disease. A method comprising the step of determining that