JP5754039B2 - Anti-TRPV2 antibody - Google Patents

Description

The present invention relates to an anti-TRPV2 antibody that recognizes the extracellular domain of TRPV2 (transient receptor potential cation channel, subfamily V, member 2), which is a Ca ²⁺ channel, as an epitope and specifically inhibits the activity of TRPV2. About. Furthermore, it is related with the TRPV2 inhibitor containing the said anti- TRPV2 antibody.

The Trp (transient receptor potential) gene product superfamily is classified into three families: TRPC (7 types), TRPV (6 types), and TRPM (8 types), all of which are ion channels that penetrate the plasma membrane six times. Ca ²⁺ is mainly passed through. Many ion channels present in cell membranes have different channel characteristics, and in order to screen for drugs that react specifically with them, it is necessary to use an assay system adapted to them. This is a significant difference from G protein-coupled receptors (GPCRs), which have relatively limited cellular responses.

TRPV2 is a stretch-activated Ca ²⁺ channel (Non-patent Documents 1-3), which is present in the intracellular membrane system in normal tissues, but is activated by migrating to the cell membrane with muscle degenerative diseases such as muscular dystrophy and cardiomyopathy. This contributes to abnormal Ca ²⁺ influx into cells (Non-patent Documents 2 and 4). A drug that specifically inhibits TRPV2 acts only on denatured muscles and is considered to reduce muscle degeneration, and may have a therapeutic / symptom-relieving effect on muscle degenerative diseases, and is expected to be developed.

Gadolinium, used as a blocker for conventional stretch-sensitive Ca ²⁺ channels, is thought to act on Ca ²⁺ channels in general, and ruthenium red is thought to act on TRPV families (TRPV1 to TRPV6) in general, both of which are nonspecific Drug (Non-Patent Documents 5 and 6). A low molecular compound capable of specifically inhibiting TRPV2 is disclosed in Patent Document 1.

  An anti-rat TRPV2 polyclonal antibody is commercially available as an antibody reactive to TRPV2 (KM019, manufacturer: Transgenic Co., Ltd.). Such an antibody uses a partial peptide at the C-terminus of TRPV2 as an immunogen, and its use is an immunohistochemical analysis, and there is no disclosure about the inhibition of TRPV2 activity.

JP 2009-149534 A

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)

  An object of the present invention is to provide an anti-TRPV2 antibody that recognizes the extracellular domain of TRPV2 as an epitope and has a function of specifically inhibiting the activity of TRPV2.

  The inventors of the present invention have made extensive studies to solve the above-mentioned problems, and have produced an antibody using a specific partial peptide of TRPV2 or a cell expressing TRPV2 as an immunogen, and the antibody specifically inhibits TRPV2. As a result, the present invention was completed.

That is, this invention consists of the following.
1. An anti-TRPV2 antibody that recognizes the extracellular domain of TRPV2 as an epitope and has a function of specifically inhibiting the activity of TRPV2.
2. 2. The TRPV2 extracellular domain, wherein a region comprising at least 5 or more amino acid sequences selected from amino acid sequences between the fifth transmembrane region and the sixth transmembrane region is recognized as an epitope. Anti-TRPV2 antibody.
3. Of the extracellular domain of TRPV2, a region containing at least 5 amino acid sequences selected from the amino acid sequence of TTVTEKPTLGQEEEPVPYGGIL (SEQ ID NO: 1), or in the amino acid sequence of the region, 1 to 2 amino acids are substituted or missing 3. The anti-TRPV2 antibody according to the above item 1 or 2, which recognizes a region consisting of any amino acid sequence deleted, added, inserted or modified as an epitope.
4). 4. The anti-TRPV2 antibody according to any one of the preceding items 1 to 3, wherein a region consisting of GQEEEPVPY (SEQ ID NO: 2) in the extracellular domain of TRPV2 is recognized as an epitope.
5. 5. The anti-TRPV2 antibody according to any one of items 1 to 4, which is a polyclonal antibody.
6). 5. The anti-TRPV2 antibody according to any one of items 1 to 4, which is a monoclonal antibody.
7). A TRPV2 inhibitor comprising the anti-TRPV2 antibody according to any one of 1 to 6 above.
8). A myocyte degeneration inhibitor comprising the anti-TRPV2 antibody according to any one of items 1 to 6.

The anti-TRPV2 antibody of the present invention does not affect the activity of other family proteins of Trp, and can specifically inhibit the Ca2 ⁺ influx activity of TRPV2. Furthermore, since the anti-TRPV2 antibody of the present invention uses the extracellular domain of TRPV2 as an epitope, it can react with TRPV2 present on the cell membrane of living cells. Further, the anti-TRPV2 antibody of the present invention has an action of suppressing muscle cell degeneration. The anti-TRPV2 antibody of the present invention is considered to be capable of inhibiting the activity of TRPV2 on the cell membrane in the pathological state, and can be a promising therapeutic drug candidate for dilated cardiomyopathy, which has only a therapeutic means for transplantation. In addition, the anti-TRPV2 antibody of the present invention is expected to be used as an experimental tool in the functional analysis of TRPV2 and the mechanism analysis of myocyte degeneration.

It is a figure which shows the reactivity of the anti- TRPV2 monoclonal antibody of this invention. Example 1 It is a figure which shows the reactivity of the anti- TRPV2 polyclonal antibody of this invention. (Example 2) It is a figure which shows the TRPV2 activity inhibitory effect of the anti- TRPV2 antibody of this invention. (Experimental example 1) It is a figure which shows the TRPV2 activity inhibitory effect in the myocyte of the anti- TRPV2 antibody of this invention. (Experimental example 2) It is a figure which shows the inhibitory effect with respect to myocyte degeneration of the anti- TRPV2 antibody of this invention. (Experimental example 3) It is a figure which shows the analysis result of the epitope mapping of the anti- TRPV2 antibody of this invention. (Experimental example 4)

  The present invention relates to an antibody having a function of recognizing the extracellular domain of TRPV2 as an epitope and specifically inhibiting the activity of TRPV2.

  Regarding the gene of TRPV2, Genbank accession No. NM_016113 as human TRPV2, Genbank accession No. NM_011706 as mouse TRPV2, and Genbank accession No. NM_017207 as rat TRPV2 have been reported. The amino acid sequence of human TRPV2 is shown in SEQ ID NO: 3 of the Sequence Listing, the amino acid sequence of mouse TRPV2 is shown in SEQ ID NO: 4 of the Sequence Listing, and the amino acid sequence of rat TRPV2 is shown in SEQ ID NO: 5 of the Sequence Listing. As with other proteins in the Trp family, TRPV2 has a six-transmembrane region as a structural feature.

  The anti-TRPV2 antibody of the present invention recognizes a region comprising at least 5 amino acid sequences selected from the amino acid sequence of the extracellular domain as an epitope. The extracellular domain of TRPV2 is a portion that exists substantially outside the cell membrane of TRPV2 existing on the cell membrane. The amino acid sequence of the cellular domain of TRPV2 is, for example, positions 409-434, 496-500, and / or 554-619 of SEQ ID NO: 3 for human TRPV2, and 406-431, 493-497 of SEQ ID NO: 4 for mouse TRPV2. Or positions 551 to 616, and positions 411 to 436, 498 to 502, and / or 556 to 621 of SEQ ID NO: 5 in rat TRPV2, but in these amino acid sequences, 1 to 2 amino acids are substituted, deleted or added The amino acid sequence may be inserted or modified.

  Preferably, the anti-TRPV2 antibody of the present invention consists of at least 5 amino acid sequences selected from amino acid sequences between the fifth transmembrane region and the sixth transmembrane region in the extracellular domain of TRPV2. A region is recognized as an epitope. The fifth transmembrane region and the sixth transmembrane region mean the fifth and sixth regions counted from the N-terminal side among the regions penetrating the plasma membrane, and the anti-TRPV2 antibody of the present invention contains these regions. A region selected from sandwiched highly hydrophilic amino acid sequences is recognized as an epitope. Among the extracellular domains of TRPV2, the amino acid sequence between the fifth transmembrane region and the sixth transmembrane region is specifically, positions 554 to 619 of SEQ ID NO: 3 in human TRPV2, and SEQ ID NO: 4 in mouse TRPV2. 551 to 616 in rat TRPV2, and positions 556 to 621 in SEQ ID NO: 5. In these amino acid sequences, 1 to 2 amino acids are substituted, deleted, added, inserted or modified. Also good.

  More preferably, in the anti-TRPV2 antibody of the present invention, the amino acid sequence at positions 568 to 589 (TTVTEKPTLGQEEEPVPYGGIL: SEQ ID NO: 1) of mouse TRPV2 (SEQ ID NO: 4) and 1-2 amino acids in the amino acid sequence are substituted or deleted. A region consisting of at least 5 amino acid sequences selected from added, inserted or modified amino acid sequences is recognized as an epitope.

  More preferably, the anti-TRPV2 antibody of the present invention recognizes, as an epitope, a region consisting of the amino acid sequence of the 577-585th amino acid sequence (GQEEEPVPY: SEQ ID NO: 2) of mouse TRPV2 (SEQ ID NO: 4).

The anti-TRPV2 antibody of the present invention has a function of specifically inhibiting the activity of TRPV2. TRPV2 is a stretch-activated Ca ²⁺ channel and exists in the intracellular membrane system in normal tissues. However, TRPV2 moves to the cell membrane and is activated due to muscle degenerative diseases such as muscular dystrophy and cardiomyopathy. Contributes to Ca ²⁺ inflow. The activity of TRPV2 means that Ca ²⁺ is allowed to flow into the cell. Since the anti-TRPV2 antibody of the present invention recognizes the extracellular domain as an epitope, it recognizes TRPV2 present on the cell membrane of living cells. It is considered possible to inhibit the activity of TRPV2. The anti-TRPV2 antibody of the present invention has a function of specifically inhibiting the activity of TRPV2 without substantially affecting the activity of other Trp family proteins.

  The anti-TRPV2 antibody of the present invention may be a monoclonal antibody or a polyclonal antibody. The antibody of the present invention is derived from mammals including humans, and examples thereof include human type, mouse type, rat type, hamster type, rabbit type, goat type, and horse type. The antibody of the present invention is not limited to IgG, and may be IgM.

  As a method for producing the anti-TRPV2 monoclonal antibody of the present invention, a method known per se or any method developed in the future can be adopted. In addition, various devices 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 Laid-Open No. 2008-161198. The type of human antibody is not particularly limited, and may be Fab type or intact type. In order to exert the antibody activity effectively, an intact type antibody is desirable. The intact type antibody is not particularly limited. For example, the antibody complementarity determinig region (CDR) is derived from the original animal species, and the constant region (C region) is a chimeric antibody derived from an appropriate human. It can be. Since CDR regions frequently undergo amino acid substitution, from the viewpoint of antigenicity, a human antibody having a human-derived CDR and a CDR derived from an immunized animal species may be included.

  The antigen for producing the anti-TRPV2 antibody of the present invention may be a full-length protein or peptide fragment of TRPV2 or a cell expressing the full-length protein or peptide fragment of TRPV2, and is not particularly limited. For the production of an anti-TRPV2 monoclonal antibody, cells expressing TRPV2 (TRPV2 expression cells) are preferably used. The cells expressing TRPV2 may be any cells that express TRPV2 on the cell membrane, but TRPV2 was collected from mammalian cells (eg, human, monkey, rat, mouse, hamster, etc.). Cells). Examples of the mammalian cells include HEK293, COS7, CHO-K1, NIH3T3, Balb3T3, FM3A, L929, SP2 / 0, P3U1, B16, and P388. Moreover, it is preferable that TRPV2 expressed in a mammalian cell is a full-length protein. In the present invention, it is preferable to use an HEK cell for expressing TRPV2 in which a full-length protein of TRPV2 is expressed in HEK293 cells. The antibody of the present invention can be obtained by immunizing a mammal with TRPV2 expression HEK cells as an antigen, preferably together with an adjuvant, and collecting the serum of the immunized animal. In addition, a monoclonal antibody and a hybridoma producing the monoclonal antibody can be prepared by fusing immunized human or animal-derived B lymphocytes with various myeloma cells, specifically by the method described below. it can.

In the production of the monoclonal antibody of the present invention, an antigen solution obtained by dissolving or suspending the antigen in an appropriate buffer such as a phosphate buffer (PBS) can be used. The antigen solution may be usually prepared to a concentration containing about 50 to 500 μg / ml or about 1 × 10 ^{6 to} 1 × 10 ⁹ cells / ml of the antigen substance. In addition, when the antigenicity is low by itself, such as a peptide antigen, it can be used after being crosslinked with an appropriate carrier protein such as albumin or keyhole limpet hemocyanin (KLH). Examples of animals 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. The adjuvants that can be used here are Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), Ribi (MPL), Ribi (TDM), Ribi (MPL + TDM), pertussis vaccine (Boredetella pertussis vaccine), Muramyl Dipeptide (MDP), aluminum adjuvant (ALUM), and combinations thereof are exemplified, but a combination using FCA at the time of primary immunization and FIA or Ribi adjuvant at the time of booster immunization may be used.

The immunization method can appropriately change the injection site, schedule, etc. depending on the type of antigen used and the presence / absence of adjuvant mixing. For example, when a mouse is used as an immunized animal, an immune disease mouse (Balb / c nu -nu mice) were injected with 0.05 to 1 mL of adjuvant mixed antigen solution (antigen substance 10 to 200 μg, or 1x10 ^{6 to} 1x10 ⁷ cell) intraperitoneally, subcutaneously, intramuscularly, or (tail) intravenously. A booster is performed 1 to 4 times every about 4 to 21 days, and a final immunization is further performed after about 1 to 4 weeks. The antigen solution can also be administered without using an adjuvant by injecting intraperitoneally with an increased amount of antigen. The antibody titer is examined by collecting blood about 5 to 10 days after the boost. The antibody titer can be measured by a conventional method according to the antibody titer assay described below. About 3 to 5 days after the final immunization, spleen cells are separated from the immunized animal to obtain antibody-producing cells.

  The anti-TRPV2 monoclonal antibody of the present invention can be prepared, for example, according to the method of Kohler and Milstein (Nature 256, 495-497, 1975). As myeloma cells, those derived from mice, rats, humans, etc. are used, for example, mouse myeloma P3X63-Ag8, P3X63-Ag8-U1, P3NS1-Ag4, SP2 / o-Ag14, P3X63-Ag8.653 etc. Examples are myeloma cells. Some myeloma cells produce an immunoglobulin light chain, and if this is used as a fusion target, the immunoglobulin heavy chain produced by the antibody-producing cell and this light chain may bind randomly. In particular, it is preferable to use myeloma cells that do not produce an immunoglobulin light chain, such as P3X63-Ag8 · 653 and SP2 / o-Ag14. The antibody-producing cells and the myeloma cells are preferably derived from the same species, particularly the same strain. The myeloma cells may be stored according to a method known per se. For example, those subcultured in a general medium supplemented with horse, rabbit or fetal calf serum are stored by freezing. For cell fusion, cells in the logarithmic growth phase are preferably used.

  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, spleen cells and myeloma cells are suspended in a suitable medium or buffer containing about 30 to 60% PEG at a mixing ratio of 1 to 10: 1, preferably 5 to 10: 1, and a temperature of about 25 to The reaction may be performed for about 30 seconds to 3 minutes under conditions of 37 ° C. and pH 6-8. After completion of the reaction, the cells are washed, the PEG solution is removed, the suspension is resuspended in a medium, and seeded in a microtiter plate to continue the culture.

  Cells after the fusion operation are cultured in a selective medium to select hybridomas. The selection medium is a medium in which the parent cell line can be killed and only the fused cells can grow, and a hypoxanthine-aminopterin-thymidine (HAT) medium is usually used. Selection of hybridomas is usually carried out by exchanging a part of the medium, preferably about half of the medium, with the selective medium 1 to 7 days after the fusion operation, and further culturing while repeating the same medium exchange every 2 or 3 days. The well where the hybridoma colony is growing is confirmed by microscopic observation.

  More specifically, the anti-TRPV2 monoclonal antibody of the present invention can be prepared by the following method. Hybridoma producing the antibody of the present invention by inoculating an immune disease mouse with TRPV2 expression HEK cells, and collecting the spleen after 2-4 weeks and fusing the antibody-producing cells contained in the spleen with myeloma cells Can be produced.

  In order to know whether the growing hybridoma is producing the desired antibody, the culture supernatant may be collected and an antibody titer assay may be performed by a method known per se. Specifically, binding activity can be obtained by staining with IC (immunocytochemistry), IF (immunofluorescence), or IHC (immunohistochemistry) staining using TRV2 expression HEK cells or TRPV2 expression HEK cell-derived proteins as antigens. The antibody-producing cells possessed can be selected.

  Whether the obtained antibody recognizes the extracellular domain of TRPV2 can be confirmed, for example, by the following method. The hybridoma culture supernatant is reacted with an HEK cell population for expressing TRPV2 and an HEK cell population not expressing TRPV2, and then reacted with a labeled secondary antibody, and measured with a flow cytometer. Specifically binds to TRPV2 extracellular domain that is higher in intensity than that of HEV cells for expression of TRPV2 that have not reacted with the antibody and that is contained in a fluorescent region that is higher in intensity than the HEK cell population that does not express TRPV2. Possible antibodies can be selected.

  Whether the obtained antibody has a function of inhibiting the activity of TRPV2 is confirmed by using a TRPV2-specific assay method described in JP-A-2007-259745 (Japanese Patent Application No. 2006-088323). be able to.

  Further, as to whether the antibody of the present invention does not substantially affect the activity of Trp family proteins other than TRPV2, other than TRPV2 described in JP-A-2009-149534 (Japanese Patent Application No. 2007-326606). This can be confirmed by referring to the method for measuring the activity of Trp family proteins.

  Further, 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 target antibody can be isolated by culturing a hybridoma colony by serial dilution with a medium so as to be about 1 cell / well. The resulting antibody-producing hybridoma clone is 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 half a year. It can be stored semipermanently. Cells are used after being rapidly thawed in a thermostatic chamber at around 37 ° C. It is desirable to use after thoroughly washing so that the cytotoxicity of the cryoprotectant does not remain.

  The method for obtaining the monoclonal antibody from the hybridoma can be appropriately selected depending on the required amount and the properties of the hybridoma. For example, a method of obtaining from mouse ascites transplanted with the hybridoma, a method of obtaining from the culture supernatant by cell culture, and the like are exemplified. A hybridoma capable of growing in the mouse abdominal cavity can obtain a high concentration of monoclonal antibody of several mg / mL from ascites. Hybridomas that cannot grow in vivo are obtained from the culture supernatant of the cell culture. Acquiring monoclonal antibodies by cell culture has the advantage that antibody production is lower than in vivo, but there is little contamination with immunoglobulins and other contaminants contained in the mouse abdominal cavity, and purification is easy.

When the monoclonal antibody is obtained from the abdominal cavity of a mouse transplanted with a hybridoma, for example, the intraperitoneal cavity of a BALB / c mouse to which a substance having an immunosuppressive action such as pristane (2, 6, 10, 14-tetramethylpentadecane) has been administered in advance. Hepaticoma (about 10 ⁶ or more) is transplanted, and ascites collected after about 1 to 3 weeks is collected. In the case of heterologous hybridomas (for example, mice and rats), it is preferable to use nude mice or radiation-treated mice.

  On the other hand, when the antibody is obtained from the cell culture supernatant, for example, in addition to the stationary culture method used for cell maintenance, the hybridoma is cultured by using a culture method such as a high-density culture method or a spinner flask culture method. A culture supernatant containing is obtained. The serum contained in the culture solution contains other contaminants such as antibodies and albumin, and antibody purification is often complicated, so it is desirable to reduce the addition to the culture solution. More preferably, the hybridoma is acclimated to a serum-free medium by a conventional method and cultured using the serum-free medium. Antibody purification is facilitated by culturing in a serum-free medium.

  Purification of monoclonal antibodies from ascites and culture supernatant can be performed by a method known per se. For example, as a method for purifying immunoglobulins, a fractionation method by salting out using ammonium sulfate or sodium sulfate, a polyethylene glycol (PEG) fractionation method, an ethanol fractionation method, a DEAE ion exchange chromatography method, a gel filtration method, etc. It is 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 desired antibody can be obtained by constructing a so-called combinatorial antibody library displaying antibody fragments on the surface of Escherichia coli phage and screening the antibody by biopanning. In this case, the desired antibody can be screened without going through the immunization work on the animal.

  The polyclonal antibody of the present invention can be produced according to a method known per se or a method analogous thereto. For example, when the antigen itself or the antigen is a peptide, a complex of the peptide and a carrier protein is prepared, and the warm-blooded animal is immunized in the same manner as the above-described monoclonal antibody production method. It can be produced by collecting the contents and performing separation and purification of the antibody.

  The anti-TRPV2 polyclonal antibody of the present invention is a partial peptide selected from the extracellular domain of TRPV2, preferably a partial peptide selected from the region between the fifth transmembrane region and the sixth transmembrane region, more preferably a mouse. It can be prepared by using a partial peptide consisting of the amino acid sequence (TTVTEKPTLGQEEEPVPYGGIL: SEQ ID NO: 1) at positions 568 to 589 of TRPV2 (SEQ ID NO: 4) as an antigen.

  The polyclonal antibody of the present invention can be collected from blood, ascites, etc., preferably from blood of immunized warm-blooded animals. The polyclonal antibody titer in the antiserum can be measured in the same manner as the antibody titer of the hybridoma culture supernatant described above, for example. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described monoclonal antibody separation and purification.

  The selection of a polyclonal antibody that recognizes the extracellular domain of TRPV2 can also be selected using flow cytometry in the same manner as measured for the hybridoma culture supernatant. In addition, selection of a polyclonal antibody having a function of inhibiting the activity of TRPV2 can also be performed using the technique described in Japanese Patent Application Laid-Open No. 2007-259745 (Japanese Patent Application No. 2006-088323).

  The anti-TRPV2 antibody of the present invention preferably has a function of suppressing muscle cell degeneration. Degeneration of muscle cells is observed in various diseases, and it is considered that the disease state is improved by suppressing / relaxing the degeneration. The ability to suppress muscle cell degeneration is confirmed by subjecting muscle cells cultured on a silicon membrane to stretch stimulation for a predetermined time to induce degeneration, and then measuring the creatinine kinase activity of the culture supernatant. can do.

  Furthermore, the present invention extends to a TRPV2 inhibitor or a myocyte degeneration inhibitor comprising the anti-TRPV2 antibody of the present invention as an active ingredient. A TRPV2 inhibitor or a myocyte degeneration inhibitor can also be used as the above-mentioned pharmaceutical composition, but as an experimental tool specifically inhibiting TRPV2 or as an experimental tool suppressing or mitigating myocyte degeneration in vivo. Alternatively, it can be used in vitro.

  The anti-TRPV2 antibody of the present invention can also be used in a state where it is contained in the composition as an active ingredient. When the composition containing the anti-TRPV2 antibody of the present invention is used for pharmaceutical use, the composition contains an effective amount of one or more of the anti-TRPV2 antibodies of the present invention, and further includes a pharmaceutically acceptable carrier. You can leave. 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, inorganic acid salts such as hydrochlorides, sulfates and nitrates, and the like. Examples include acid addition salts such as organic acid salts such as acetates and propionates.

  Such a composition can be administered orally or parenterally as a pharmaceutical composition. In the case of oral administration, the anti-TRPV2 antibody of the present invention is a usual preparation, for example, a solid agent such as a tablet, powder, granule or capsule; a liquid agent; an oily suspension; or a liquid agent such as a syrup or elixir. It can be used as any dosage form. In the case of parenteral administration, the anti-TRPV2 antibody of the present invention can be used as an aqueous or oily suspension injection or nasal solution. In the preparation, conventional excipients, binders, lubricants, aqueous solvents, oily solvents, emulsifiers, suspending agents, preservatives, stabilizers and the like can be arbitrarily used.

  When used as a pharmaceutical composition, it is preferably applied to diseases caused by activation of TRPV2 and muscle degenerative diseases. Examples of diseases caused by activation of TRPV2 include, but are not necessarily limited to, hypertension, allergy, immune system diseases, gastrointestinal diseases, cancer and the like. Examples of the muscle degenerative disease include muscular dystrophy, cardiomyopathy, heart failure, and myocardial infarction.

  Hereinafter, in order to deepen the understanding of the present invention, the present invention will be specifically described with reference to examples and experimental examples, but it goes without saying that these do not limit the scope of the present invention.

(Example 1) Preparation of anti-TRPV2 monoclonal antibody (1) Preparation of HEK cells for mouse TRPV2 expression In this example, full-length mouse TRPV2 having the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing was prepared as disclosed in JP2007-259745A. In accordance with the method described in the publication (Japanese Patent Application No. 2006-088323), HEK cells for mouse TRPV2 expression in which mouse TRPV2 was expressed on the cell membrane were prepared by introducing and expressing in HEK293 cells.

(2) Production of hybridoma Using mouse TRPV2 expression HEK cells prepared in (1) above as an antigen, prepared in PBS to 1 × 10 ^{6 to} 1 × 10 ⁷ cells / mL, and the same amount of Freund's complete adjuvant (freund's comlete adjubant: manufactured by Difco) and emulsified. This emulsified antigen was administered to the abdominal cavity of 4 female BALB / c nu-nu mice at 4 weeks of age, 100 μL per mouse. Further, every two weeks, the above-mentioned antigen prepared at 500 μg / mL with an adjuvant was administered twice at 50 μg per mouse and boosted, and then the antibody titer of the mouse was measured. One week later, mice with high antibody titers were subjected to final immunization by preparing HEK cells for expression of mouse TRPV2 as an antigen to 100 μg / mL with PBS and injecting them from the mouse tail vein.

Three days after the final immunization, BALB / c mice were excised and splenocytes were suspended in the EMEM culture solution to prepare a suspension of splenocytes. Subsequently, the spleen cells were washed 3 times with EMEM culture solution, and the number of cells was calculated to obtain 5 × 10 ⁷ spleen cells. For cell fusion, the P3U1 myeloma cultured cell line was used as the parent cell line. The parent cell line was subcultured in RPMI-1640 culture medium containing 10% of inactivated fetal calf serum (FCS). The cells were further cultured in 10% FCS-containing RPMI-1640 culture medium 3 days before cell fusion, and cells in the logarithmic growth phase were used. The parent cell line was washed three times with RPMI-1640 culture solution, and the number of cells was calculated to obtain 5 × 10 ⁶ to 10 ⁷ viable cells.

  In the RPMI-1640 culture solution, polyethylene glycol-4000 is dissolved to a concentration of 50 (W / V), and mixed so that the ratio of the above spleen cells to the parent cell line is 2: 1. And Milstein: Cell fusion according to the method of Nature (Nature Vol. 256, 495-497 (1975)) and European Journal of Immunology (Eur. J. Immunol. Vol. 6, 511-519, (1976)) Went.

Thereafter, 1 to 3 × 10 ⁶ splenocytes were added to a HAT selection medium containing 100 μM hypoxanthine, 0.4 μM aminopterin and 16 μM thymidine (HAT) in RPMI-1640 medium supplemented with 10% FCS. It was suspended so that it might become / mL. Next, 100 μL of this cell suspension was dispensed into each well of a 96-well microtiter plate, and then cultured in a CO ₂ sterile incubator in a 37 ° C., 95% humidity, 8% CO ₂ atmosphere. Add 1 drop of HAT selection medium to each well on day 1 and 2 after the start of culture, and add 2 drops of HAT selection medium to each well on days 7 and 9 after the start of culture. Culture was performed. Thereafter, the cells were grown in a culture solution containing no HAT, and after about 10 days to 2 weeks, the growing hybridomas were confirmed on a selective medium.

(3) Screening Among the hybridomas obtained above, a strain producing an antibody that recognizes the extracellular domain of TRPV2 was selected by the following method.

  The hybridoma culture supernatant was reacted with a population of HEV cells for expressing TRPV2 or a population of HEK cells not expressing TRPV2, and then reacted with a labeled secondary antibody (FITC-labeled goat anti-mouse IgG Zymed). After the reaction, each cell population was measured with a flow cytometer (FACScan, Becton Dickinson), and the background intensity measured without using the hybridoma culture supernatant (a TRK2 expression HEK cell population that did not react with the antibody) The hybridoma contained in the fluorescence region with higher intensity and higher intensity than the HEK cell population not expressing TRPV2 was obtained as an antibody that can specifically bind to the TRPV2 extracellular domain. .

(4) Purification of antibody Each hybridoma strain was reduced in the fetal bovine serum content in the culture solution from 10% to 2% and finally cultured in a serum-free culture solution. 100 ml of the culture supernatant of each hybridoma cultured in serum-free medium for 7 to 7 days is centrifuged at 2,000 rpm for 10 minutes, and the resulting supernatant is filtered through a 0.45 μm filter to remove solid components. Was purified with 1 mL of 6% agarose gel (HiTrapProtein G HP Columns-GE Healthcare Japan) immobilized.

  In order to confirm that the obtained monoclonal antibody specifically binds to the TRPV2 extracellular domain, immunoblotting and immunostaining using live cells (TRK2 expression HEK cells used as an antigen) were performed. In the immunoblot, mouse IgG was used as a control. In immunostaining, TRPV2-expressing HEK293 cells and each antibody were incubated at room temperature for 30 minutes, and then FITC-labeled secondary antibody was reacted, and FITC fluorescence was observed with a confocal laser microscope (FLUOVIEW FV1000, Olympus). The control is mouse IgG (normal mouse IgG, Santa Cruz Biotechnology).

  FIG. 1a shows the result of immunoblotting, and FIG. 1b shows the result of immunostaining. In FIG. 1a, lane 1 was electrophoresed with the protein fraction of HEK293 cells, and lane 2 was electrophoresed with the protein fraction of HEK cells for mouse TRPV2 expression, 1 μg / ml of mouse IgG and monoclonal antibodies 11-6, 88- 2 was immunoblotted using 2. The upper part of FIG. 1b is a photograph of each antibody reacted. Monoclonal antibodies 11-6 and 88-2 were found to be reactive with the extracellular domain of TRPV2.

(Example 2) Preparation of anti-TRPV2 polyclonal antibody The amino acid sequence (SEQ ID NO: 1) at positions 568 to 589, which is a highly hydrophilic region between the fifth transmembrane region and the sixth transmembrane region of mouse TRPV2. The partial peptide possessed was synthesized by the solid phase method, and a polyclonal antibody was prepared using this partial peptide as an antigen. Specifically, a partial peptide as an antigen was immunized to a New Zealand white rabbit together with an adjuvant (complete Freund's adjuvant, Difco Laboratories) to obtain an antiserum. The obtained antiserum was purified as usual to obtain a polyclonal antibody.

  In order to confirm that the obtained antiserum polyclonal antibody is an antibody that recognizes the extracellular domain of TRPV2, immunostaining was performed using antiserum, immunoblot, and live cells (TRK2 expression HEK cells). Went. In the immunoblot, antiserum collected before immunization with the antigen was used as a control. In the immunostaining, as in the method of Example 1, after culturing HEV293 cells for TRPV2 expression and each antiserum for 30 minutes at room temperature, the FITC-labeled secondary antibody was reacted, and the fluorescence of FITC was observed with a confocal laser microscope. did. Antiserum (pre-immune serum) collected before immunization with antigen was used as a control, and as a positive control, a Flag tag was inserted into the extracellular domain of TRPV2, and each Flag tag was anti-Flag antibody and rhodamine labeled secondary Observations were made with antibodies.

  FIG. 2a shows the result of immunoblotting, and FIG. 2b shows the result of immunostaining. In FIG. 2a, lane 1 was electrophoresed with the protein fraction of HEK293 cells, and lane 2 was electrolyzed with the protein fraction of mouse TRPV2 expression HEK cells, and the preimmune serum, serum 591, and serum 592 were each diluted 1000-fold. The sample was immunoblotted. The upper part of FIG. 2b is a photograph obtained by reacting each serum, and the lower part is a positive control. Polyclonal antibodies of serum 591 and serum 592 were found to be reactive against the extracellular domain of TRPV2.

(Example 3) Selection of antibody having activity of inhibiting TRPV2 activity According to a high-throughput screening method using a mouse TRPV2 expression cell (the method described in Japanese Patent Application Laid-Open No. 2007-259745 (Japanese Patent Application No. 2006-088323)). The obtained serum and monoclonal antibody were screened using the inhibitory action of TRPV2 activity as an index.

HEK293 cell line (mouse TRPV2 expression cell) into which mouse TRPV2 specified by accession number FERM P-20837 or FERM P-20836 was introduced was cultured on a 96-well microplate, and a confluent one was used. The fluorescence intensity at a measurement wavelength of 510 nm at excitation wavelengths of 340 nm and 380 nm was measured for cells washed twice with 100 μl of BSS (Balanced Salt solution).
As a control, HEK293 cell line into which mouse TRPV2 was not introduced was also cultured in the same manner, and the fluorescence intensity was measured in the same manner.

100 μl of 4 μM Fura-2-AM ^(TM) (Dojindo ⁾ dissolved in 10 mM HEPES-BSS was added to the cells and contacted at 37 ° C. for 30 minutes to load the cells with a calcium indicator. Next, the plate was washed twice with 100 μl of 1 mM HEPES-BSS, 50 μl of BSS containing the antibody obtained in Example 1 or 2 was added, and reacted at 37 ° C. for 30 minutes. The same procedure was performed for a 1 mM HEPES-BSS (50 μl) system containing no calcium inhibitor. As a positive control, a 10 mM HEPES-BSS (50 μl) system supplemented with Ca ²⁺ calcium ionophore (ionomycin 10 μM) was similarly performed.

50 μl of 2-APB (2-aminoethoxydiphenyl borate) solution was added to each well and allowed to react at room temperature for 1 minute. The reaction solution had a final concentration of 2-APB of 0.5 mM, a final concentration of CaCl ₂ of 5.25 mM, and a pH of 6.3.

Ca ²⁺ was measured using a fluorescence / luminescence / absorption multiplate reader (Genios Plus (TECAN)). The measurement was performed by measuring the fluorescence intensity at a measurement wavelength of 510 nm at excitation wavelengths of 340 nm and 380 nm. When the strong Ca ²⁺ increase observed in the 2-APB addition system is suppressed in the presence of the antibody, the antibody has the action of inhibiting TRPV2, and serum 591, serum 592, monoclonal antibodies 11-6, 88 I selected -2.

(Experimental Example 1) Confirmation of TRPV2 activity inhibitory effect of antibody

(1) About the antibody obtained in Example 3, using the method for measuring the activity of Trp family proteins other than TRPV2 described in JP-A-2009-149534 (Japanese Patent Application No. 2007-326606), The effect on Trp family proteins was confirmed.

HEK293 cells into which mouse TRPV1 was introduced were used as TRPV1 expression cells, and HEK cells into which mouse TRPC1 was introduced were used as TRPC1 expression cells. Similarly to the measurement of TRPV2 activity, a fluorescent dye was introduced into the cells, TPPV1 was stimulated with 2-APB, TRPC1 was stimulated with thapsigargin, and intracellular Ca ²⁺ increase was measured.

  As a result, it was found that serum 591 and 592 and mouse monoclonal antibodies 11-6 and 88-2 did not substantially affect the activity of TRPV1 and TRPC1, and specifically suppressed TRPV2 activity.

(2) In mouse TRPV2 expression cells, the inhibitory effect on the TRPV2 activity of the antibody was confirmed by monitoring with an intracellular calcium probe using a plate reader. The monoclonal antibody was reacted at 10 ng / ml and the serum (rabbit polyclonal antibody) was diluted 1000 times at room temperature 2 hours before agonist stimulation. Cells loaded with intracellular Ca ²⁺ concentration indicator fura-2 were stimulated with 2APB (2-aminoethoxydiphenyl borate) solution (0.3 mM, 0.5 mM) in the presence of BSS according to a conventional method.

In an experiment under a microscope, cells loaded with intracellular Ca ²⁺ concentration indicator fura-2 were stimulated with 2APB solution (0.3 mM, 0.5 mM, and 1 mM) in the presence of BSS according to a conventional method, and then washed with BSS. Then, the solution was exchanged with a solution containing 2 mM Ca ²⁺ , thermally stimulated (52 ° C. or more for 30 seconds), and finally stimulated with 10 μM ionomycin.

The results are shown in FIG. FIG. 3a shows the results of monitoring the increase in intracellular Ca ²⁺ concentration after addition of TRPV2 agonist 2-APB in the presence of various antibodies. FIG. 3b shows the result of measuring the increase in intracellular Ca ²⁺ concentration under the microscope using the intracellular Ca ²⁺ fluorescent dye fura-2, and is the average and standard deviation of 10 or more cells for TRPV2 expression. The black line is the result of the pre-immune serum as the control, and the gray line is the result of the 591 serum, and each shows the intracellular Ca ²⁺ response to 2APB which is a TRPV2 agonist after reacting at 1000-fold dilution for 2 hours at room temperature. As a result, the increase in intracellular Ca ²⁺ by 2APB stimulation was suppressed by 591 serum. FIG. 3c shows the result of HEK293 cells not expressing TRPV2 (no antibody added). It was found that the intracellular Ca ²⁺ increase by TRPV2 (FIG. 3b black) was suppressed by the antibody to the same level as the level without TRPV2 expression (FIG. 3b gray, FIG. 3c).

(Experimental example 2) Confirmation of inhibitory effect of myocardial TRPV2 activity of antibody Using cultured skeletal muscle cells of cardiomyopathy hamster (J2N-k), a model animal of human cardiomyopathy, antibody suppresses intracellular calcium metabolism abnormality It was confirmed. In the same manner as in Experimental Example 1, an increase in intracellular Ca ²⁺ concentration was measured under a microscope using intracellular Ca ²⁺ fluorescent dye fura-2.

The results are shown in FIG. The average and standard deviation of 10 or more cells for expressing TRPV2 are shown. The black line is the result of the control serum, and the gray line is the result of the 591 serum, and each shows the intracellular Ca ²⁺ response to 2APB, which is a TRPV2 agonist after reacting at 1000-fold dilution for 2 hours at room temperature. As a result, the increase in intracellular Ca ²⁺ by 2APB stimulation was suppressed by 591 serum.

(Experimental example 3) Confirmation of inhibitory effect of antibody on muscle cell degeneration Using cultured skeletal cells of cardiomyopathy hamster (J2N-k), a model animal of human cardiomyopathy, it was confirmed that the antibody inhibits muscle degeneration. . The muscle cells cultured on the silicon membrane were subjected to 20% stretch stimulation for 1 hour, and then the creatinine kinase activity of the culture supernatant was measured by a conventional method. Control sera before immunization was diluted 500-fold, and 591 serum was diluted 500-fold or 250-fold. After reacting at room temperature for 1 hour, stretch stimulation was given.

  The results are shown in FIG. When reacted with preimmune serum, creatinine kinase activity (ck efflux) is high and degeneration of myocytes is induced, whereas when reacted with 591 serum, at any dilution ratio Degeneration of muscle cells due to stretch stimulation was suppressed.

(Experimental example 4) Epitope mapping In this experimental example, the epitope analysis was performed about the polyclonal antibody 591, the polyclonal antibody 592, and the monoclonal antibody 88-2.
Of the 22 residues of the amino acid sequence at positions 568 to 589 (SEQ ID NO: 1), 15 peptides consisting of 8 amino acid residues were synthesized, shifted by 1 residue, and the reactivity of the antibody against each peptide was confirmed. . Each peptide 1 mg / ml was dot-blotted 1 μl on a nitrocellulose membrane, the membrane was blocked according to a conventional method, reacted with each antibody, and detected by an immunoantibody method.

The results are shown in FIG.
FIG. 6a shows partial peptides 568 to 589 (upper) and 551 to 566 (lower) selected from the region between the fifth transmembrane region and the sixth transmembrane region in the extracellular domain of TRPV2. FIG. 5 shows the results of examining the reactivity to each peptide using a mouse monoclonal antibody 88-2 (10 ng / ml) diluted with 1000 times of 591 serum, 592 serum and control IgG after blocking by doting on a nitrocellulose membrane. All antibodies were found to be highly reactive with peptides 568-589.
The photograph in FIG. 6b shows the actual dot blot results using 591 serum or control IgG. The graph of FIG. 6b is obtained by quantifying the staining intensity of the dot blot using an imaging analyzer. The graph shows the results of 591 serum, 592 serum and monoclonal antibody 88-2 together, and represents the mean and standard deviation of three experiments. All the antibodies showed reactivity with GQEEEPVP (SEQ ID NO: 6) and QEEEPVPY (SEQ ID NO: 7). In addition, 591 serum and 592 serum showed higher reactivity to QEEEPVPY (SEQ ID NO: 7). Monoclonal antibody 88-2 showed higher reactivity to GQEEEPVP (SEQ ID NO: 6). Therefore, these antibodies were considered to recognize around GQEEEPVPY (SEQ ID NO: 2).

  As described above, the anti-TRPV2 antibody of the present invention can recognize the extracellular domain of TRPV2 and can react with TRPV2 expressed in living cells. Further, the anti-TRPV2 antibody of the present invention has a function of inhibiting the activity of TRPV2, and can exert an inhibitory action on TRPV2 expressed in living muscle cells. Activation of TRPV2 is known to occur in a wide range of diseases involving degeneration and cell death of muscle cells, regardless of whether they are cardiac muscle or skeletal muscle. Therefore, it is considered that the anti-TRPV2 antibody of the present invention can be applied not only to dilated cardiomyopathy but also to a therapeutic agent for muscular dystrophy or heart failure in general, or a myocardial protective agent. Furthermore, the antibody of the present invention is considered to be able to suppress the activity of TRPV2 on the cell membrane during a disease state, and can be a promising therapeutic drug candidate for dilated cardiomyopathy, which only has a therapeutic means conventionally. In addition, since the anti-TRPV2 antibody of the present invention is an experimental tool that specifically inhibits TRPV2, it can be used for elucidating myocardial pathology caused by TRPV2 activation and also as a means for confirming the involvement of TRPV2 in various myocardial pathologies it can.

Claims (8)

Anti-TRPV2 antibody having a function of recognizing a region consisting of an amino acid sequence corresponding to GQEEEPVPY (SEQ ID NO: 2) of mouse TRPV2 as an epitope among the extracellular domain of mammalian TRPV2 and specifically inhibiting the activity of TRPV2 . The anti-TRPV2 antibody according to claim 1, wherein the region consisting of an amino acid sequence corresponding to GQEEEPVPY (SEQ ID NO: 2) of mouse TRPV2 is any of the following regions:
1) A region consisting of an amino acid sequence of GQEEEPVPY (SEQ ID NO: 2) in the extracellular domain of mouse TRPV2;
2) A region consisting of an amino acid sequence corresponding to GQEEEPVPY (SEQ ID NO: 2) of mouse TRPV2 in the extracellular domain of human TRPV2;
3) A region consisting of an amino acid sequence corresponding to GQEEEPVPY (SEQ ID NO: 2) of mouse TRPV2 in the extracellular domain of rat TRPV2;
4) A region consisting of any one of the amino acid sequences in which 1 to 2 amino acids are substituted, deleted, added, inserted or modified in the regions 1) to 3) above. The anti-TRPV2 antibody according to claim 1 or 2, wherein a region consisting of GQEEEPVPY (SEQ ID NO: 2) in the extracellular domain of TRPV2 is recognized as an epitope. The extracellular domain of mammalian TRPV2, which was prepared by immunizing a non-human animal with a peptide consisting of an amino acid sequence corresponding to TTVTEKPTLGQEEEPVPYGGIL (SEQ ID NO: 1) of mouse TRPV2 as an antigen. The anti-TRPV2 antibody according to any one of the above. The anti-tumor according to any one of claims 1 to 4, which was prepared by immunizing a non-human animal using a peptide consisting of the amino acid sequence of TTVTEKPTLGQEEEPVPYGGIL (SEQ ID NO: 1) among the extracellular domain of mouse TRPV2 as an antigen. TRPV2 antibody. The anti-TRPV2 antibody according to any one of claims 1 to 5, which is a polyclonal antibody or a monoclonal antibody. A TRPV2 inhibitor comprising the anti-TRPV2 antibody according to any one of claims 1 to 6. A myocyte degeneration inhibitor comprising the anti-TRPV2 antibody according to any one of claims 1 to 6.