WO2002062990A1 - Antibody and utilization thereof - Google Patents

Abstract

An antibody binding to a complex of osteoclastogenesis inhibitory factor (OCIF) with soluble OCIF-binding molecule (sOBM), i.e., OCIF/sOBM complex occurring in bodily fluids; hybridomas producing this antibody; and preventives or remedies for bone metabolic errors containing this antibody as the active ingredient. A method for diagnosing bone metabolic errors, in particular, rheumatoid arthritis which comprises quantifying the OCIF/sOMB complex; and a kit for diagnosing bone metabolic errors. This antibody is useful in diagnosing, preventing or treating bone metabolic errors, in particular, joint diseases such as rheumatoid arthritis or in analytical reagents for laboratory use, etc.

Description

 Description · Antibodies and their use

[Technical field]

 The present invention relates to an antibody that binds to a complex of an osteoclastogenesis inhibitory factor (OCIF) and a soluble OCIF binding molecule (sOBM).

 Furthermore, the present invention relates to a hybridoma producing the antibody, a preventive / therapeutic agent for bone metabolic disorder comprising the antibody as an active ingredient, or a diagnostic drug, a diagnostic method, and a diagnostic kit for bone metabolic disorder using the antibody. About.

[Background technology]

 Bone metabolism depends on a balance between the activities of osteoblasts, which are responsible for bone formation, and osteoclasts, which are responsible for bone resorption. Bone metabolism disorders are thought to be caused by an imbalance between bone formation and bone resorption. As diseases accompanied by abnormal bone metabolism, rheumatoid arthritis, osteoarthritis, osteoporosis, hypercalcemia, bone pageet disease, and renal osteodystrophy are known. Rheumatoid arthritis is an intractable inflammatory disease in which the synovium is the main lesion. If the lesion progresses, it will cause cartilage and bone destruction, leading to impaired joint function. In addition, various extra-articular manifestations can spread inflammatory diseases to systemic organs, resulting in a significant decrease in the patient's quality of life.

The criteria for early rheumatoid arthritis are being developed because it is necessary to start treatment for early rheumatoid arthritis and suppress the progression of bone fracture. The diagnostic criteria are (1) Morning stiffness lasting for more than 15 minutes for more than one week, (2) Swelling of three or more joint areas lasting for more than one week, (3) Wrist joint, metacarpophalangeal joint (MCP), Proximal finger joint (PIP), ankle or metatarsophalangeal joint (MTP) swelling lasting more than a week, (4) symmetric swelling lasting more than a week, (5) Detection of rheumatoid factor, (6) X-ray changes of the hand or foot, soft tissue fusiform swelling and bone atrophy, or bone erosion (Yamae, Nippon Medical News, 3360, p43 (198 8)), and chronic if 4 out of 6 items apply Diagnosed with rheumatoid arthritis You. However, setting a sensitive standard for such early diagnosis will inevitably sacrifice specificity. For this reason, specific disease markers for early diagnosis of rheumatoid arthritis are currently being sought. '

 As mentioned above, the cells responsible for bone metabolism are osteoblasts and osteoclasts. These cells are known to interact closely, and this phenomenon is called force coupling. In other words, for the differentiation and maturation of osteoclasts, various cytokines secreted by osteoblast-like stromal cells, such as interleukin (IL) -1, IL-6, IL-11, macrophage colony stimulating factor (M -CSF), tumor necrosis factor (TNFc), transforming growth factor β (TGF-J3), etc. act as a promoter or suppressor (Raisz: Disorder of Bone and Mineral Metabolism, 287-311, 1992; Suda et al .: Principle of Bone biology, 87-102, 1996; Suda et al .: Endocrine Reviews, 4, 226-270, 1995; Lacey et al .: Endocrinology, 136, 2367-2376, 1995) Osteoblast-like stromal cells adhere to immature osteoclast progenitor cells and osteoclasts to differentiate osteoclasts, resorb bone by mature and mature osteoclasts, respectively. It is known to play an important role in the function of cells. As a factor involved in osteocyte formation, a molecule called osteoclast differentiation factor (0DF) expressed on the membrane of osteoblast-like stromal cells was assumed (Suda et al.,: Endocrine Rev. 13, 66-80, 1992; Suda et al.,: Bone, 17, 87S-91S, 1995).

Tsuda et al. Found an osteoclastogenesis inhibitory factor (0CIF) in a culture of human fetal lung fibroblasts IMR 90 (ATCC CCL-186), and found that recombinant 0CIF The effect of improving bone metabolism in vivo was successfully confirmed (W096 / 26217). And it was shown that 0CIF promotes osteogenesis by specifically inhibiting osteoclast differentiation and maturation. 0CIF is sometimes called osteoprotegerin (W097 / 23614), and 0CIF used in the present invention includes all substances called by these names. In addition, we successfully cloned an OCIF binding molecule (OCBM) from an osteoblast-like stromal cell line, ST-2 cDNA library. It became clear after the discovery of 0BM that this 0BM was a hypothetical 0DF. Around the same time as the discovery of 0BM, the same protein as 0BM has been published as TRANCE (W099 / 29865) or RANKL (W098 / 28426). 0BM used in the present invention is called by these names. Includes all materials. This 0BM is a type II membrane-bound protein, and together with soluble OBM (sOBM), whose transmembrane region has been deleted, supports the differentiation and maturation of osteoclasts in the osteoclast-forming system in vitro. And clarified that it is a promoting factor (W098 / 46644). 0BM is present in osteoblasts, activated T cells, etc., and 0CIF suppresses osteoclast formation by binding to 0BM and blocking its biological activity.

 Yano et al. Confirmed the presence of 0CIF in human serum and found that it strongly correlated with aging and bone metabolic disorders (Yano et al .; Journal of Bone and Mineral Research, 14 (4) , 518-527 (1999)). It is expected that sOBM is produced with the progression of bone metabolism disorder, and this is expected to bind to 0CIF present in serum to form a complex.However, a measurement system for the complex of 0CIF and sOBM has not yet been developed. Absent.

[Disclosure of the Invention]

 The present inventors have conducted intensive searches in view of such a situation, and as a result, have found a monoclonal antibody having extremely high affinity for a complex of OCIF and sOBM (OCIF / sOBM complex). Furthermore, an enzyme immunoassay (enzyme immunoassay; EIA) method capable of measuring the OCIF / sOBM complex using these antibodies has been established. In addition, as a result of measuring the amount of 0CIF / sOBM complex in plasma of healthy individuals and rheumatoid factor-positive patients using this enzyme immunoassay, OCIF / sOBM complex was detected in rheumatoid factor-positive patients, and rheumatoid arthritis was detected. The correlation with the factor was confirmed.

 Thus, the present invention is directed to antibodies that bind to the OCIF / sOBM complex, polyclonal antibodies that bind to the OCIF / sOBM complex, monoclonal antibodies that bind to the OCIF / sOBM complex, and humanized antibodies that bind to the 0C IF / sOBM complex. Monoclonal antibodies, pharmaceutical compositions containing these antibodies, and bone marrow disorders (rheumatoid arthritis, osteoarthritis, osteoporosis, hypercalcemia, bone paget disease, renal bone disorders) It is an object of the present invention to provide a preventive agent and a therapeutic agent for nutrition, etc., a kit for diagnosing bone metabolism disorders containing these antibodies, an OCIF / sOBM complex measurement kit, and the like.

 That is, the present invention

(1) an antibody that binds to a complex of an osteoclastogenesis inhibitory factor (OCIF) and a soluble OCIF binding molecule (sOBM); (2) binding to osteoclast formation inhibitory factor (0CIF), the antibody according to (1),

 (3) binding to a soluble 0CIF binding molecule (sOBM), the antibody according to (1) or (2),

(4) the antibody according to any one of (1) to (3), which is a polyclonal antibody,

(5) a monoclonal antibody, the antibody according to any one of (1) to (3),

(6) the antibody according to (5), which is a humanized antibody;

 (7) The geometry according to (5), which is produced by Hybridoma 01-30 (FERM BP-7872).

 (8) the antibody according to (5), which is produced by hybridoma H-0BM1 (FERM BP-6264);

 (9) Hypri-Dorma 01-30 (FERM BP-7872),

 (10) Hybrid Dorma H-0BM1 (FERM BP-6264),-

(11) The pharmaceutical composition containing the antibody according to any one of (1) to (8),

(12) a prophylactic or therapeutic agent for bone dysfunction tf disorder, comprising the antibody according to any one of (1) to (8) as an active ingredient;

 (13) Bone metabolism disorder is one selected from the group consisting of rheumatoid arthritis, osteoarthritis, osteoporosis, hypercalcemia, bone pageet disease and renal osteodystrophy. The preventive or therapeutic agent for bone metabolic disorder according to (12),

 (14) A method for diagnosing bone metabolism disorder, comprising the following steps [1] and [2]:

 [1] Step of measuring the amount of OCIF / sOBM complex contained in a sample from a subject or a healthy subject:

 [2] When the amount of the complex contained in the sample of the subject is larger than the amount of the complex contained in the sample of a healthy subject, Determining that the subject is suffering from

 (15) The diagnostic method according to (14), wherein the antibody according to any one of (1) to (8) is used.

 (16) the method for diagnosing bone metabolism disorder according to (14) or (15), wherein the sample is plasma or synovial fluid;

 (17) The method for diagnosing bone metabolism disorder according to any one of (14) to (16), wherein the bone metabolism disorder is rheumatoid arthritis or a joint disease.

(18) a kit for diagnosing bone metabolism disorder, comprising at least one antibody according to any one of (1) to (8); (19) The diagnostic kit according to (18), comprising at least two antibodies according to any one of (1) to (8).

About.

 The antibody that binds to the OCIF / sOBM complex provided by the present invention is useful for prevention, treatment and / or diagnosis of bone metabolism disorders.

 In the present invention, bone metabolism disorders include primary osteoporosis (senile osteoporosis, postmenopausal osteoporosis and idiopathic juvenile osteoporosis), endocrine osteoporosis (hyperthyroidism, parathyroidism, Cushing's syndrome and acromegaly) ), Osteoporosis associated with hypogonadism (hypopituitarism, Klinefelter syndrome and Turner syndrome), Hereditary and congenital forms of osteoporosis (osteogenesis imperfecta, homocystinuria, menkes disease and illness) Iddy syndrome), osteopenia due to reduced gravitational load or fixation or immobilization of limbs, pageet's disease, osteomyelitis, infectious lesions due to bone loss, solid tumors (breast cancer, lung cancer, kidney cancer, prostate cancer, etc.) Caused by hypercalcemia, hematological malignancies (multiple myeloma, lymphoma and leukemia), idiopathic hypercalcemia, hyperthyroidism or Hypercalcemia associated with renal insufficiency, osteopenia due to steroid administration, osteopenia due to administration of other drugs (such as methotrexate and immunosuppressants such as cyclosporin A, heparin and antiepileptic drugs), Osteopenia associated with renal insufficiency, surgical procedures, gastrointestinal disorders (small bowel disorders, large bowel disorders, chronic hepatitis, gastrectomy, primary biliary cirrhosis and p-cirrhosis), osteopenia associated with rheumatoid arthritis, etc. Osteopenia due to various rheumatism, bone destruction and joint destruction due to various rheumatism such as rheumatoid arthritis, rheumatic rheumatism, osteoarthritis, periodontal bone loss, bone metastasis of cancer (osteolytic metastasis), traumatic injury, Gaucher disease, sickle cell anemia, bone dystrophy or bone cell death associated with systemic lupus erythematosus or non-traumatic injury, osteodystrophy such as renal osteodystrophy, low alkaline phosphine Turn-Ze hypertriglyceridemia, osteopenia associated with diabetes, osteopenia accompanying nutritional disorder or eating disorders, and other osteopenia and the like.

The antibody provided by the present invention is not particularly limited as long as it binds to the OCIF / sOBM complex, and may be any of a polyclonal antibody and a monoclonal antibody. Examples of such an antibody include a monoclonal antibody specifically binding to a site (epitope) to which a monoclonal antibody produced by Hypridoma 01-30 (FERM BP-7872) provided by the present invention, Hypridoma H-0BM1 Specific to the site (epitope) to which the monoclonal antibody (# 207) produced by (FERM BP-6264) binds Preferred examples thereof include a monoclonal antibody that binds, and preferred examples thereof include a monoclonal antibody produced by Hypri-Doma 01-30 (FERM BP-7872) and a monoclonal antibody produced by Hybridoma H-0BMUFERM BP-6264) ( # 207) and the like.

 Examples of animals immunized with the antigen when obtaining the antibody of the present invention include humans, mammals other than humans, and birds. Suitable animals are different from the species from which the antigen is derived.

 Antigens used for obtaining the antibody of the present invention include 0CIF derived from humans or non-human mammals, analogs thereof, mutants thereof, derivatives thereof, etc. (W096 / 26197, W097 / 23614: Hereinafter, all of them are simply referred to as “0CIF.”), 0BM derived from mammals other than human or human, analogs thereof, mutants thereof, derivatives thereof, etc.

(WO ^98/46644 discloses, TO "/ ²⁹⁸⁶⁵ JP, TO ^98/28426 discloses, W098 / 46751 Report:. Below, all of them simply referred to as" 0BM "), s0BM (W098 / 46644 discloses, W099 / 29865, W098 / 28426, and W098 / 46751), and OCIF / sOBM complexes derived from humans or mammals other than humans.

The OCIF / sOBM complex is usually used for the isolation and purification of proteins from biological samples (tissue, blood, etc.) or cells (cultured cells, cell lines, etc.) collected from humans or non-human mammals. [2] 0CIF derived from human or non-human mammal, previously obtained and isolated and purified as necessary, and human or non-human mammal The resulting sOBM can be obtained by dissolving it in a solvent, mixing and then keeping the mixture warm. The species derived from 0CIF and sOBM in [2] is not particularly limited, but the species derived from 0CIF and the species derived from sOBM are preferably the same. The solvent for dissolving 0CIF or sOBM may be any solvent that is usually used for dissolving proteins, and examples thereof include a phosphate buffer. The solvent may contain sodium salt, a surfactant and the like. Further, the pH range of the solvent is 5 to 10, preferably 6 to 8. The range of the mixing ratio between 0CIF and sOBM is 0CIF: sOBM = 1: 0.1 to 1: 100, preferably 0CIF: sOBM = 1: 0.2 to 1: 5. The range of the incubation temperature is 0 to 40 ° C, preferably 0 to 37 ° (more preferably 4 to 25 ° C.) The range of this time is the reaction temperature, the concentration of 0CIF and sOBM, and their mixture. Although it depends on the ratio, it is usually 1 hour to 1 week, preferably 6 hours to 2 days. Confirm that the desired OCIF / sOBM complex was obtained by electrophoresis (PAGE under non-denaturing conditions, etc.), EIA method combining anti-0CIF antibody and anti-sOBM antibody or anti-0BM antibody, etc. Can be. The OCIF / sOBM complex thus obtained and confirmed is purified as necessary, and then used as an antigen for immunizing an animal when obtaining the antibody of the present invention, and used for purification or selection of the antibody of the present invention. Means and the like.

 The polyclonal of the present invention can be obtained by collecting blood from an animal previously immunized with an antigen to obtain a serum fraction, and then obtaining it by affinity chromatography using an OCIF / sOBM complex.

 The monoclonal antibody of the present invention can be obtained by the following method. That is, the above-mentioned antigen is diluted with a solvent (for example, physiological saline) and administered to a mammal intraperitoneally or intravenously together with an immunological adjuvant (for example, Freund's complete adjuvant) as necessary. Immunization is generally performed 3 to 4 times at intervals of 1 to 2 weeks. Alternatively, an in vitro sensitization method can be used. After intravenously administering an antigen diluted with physiological saline or the like to the animal, spleen cells prepared from the spleen removed on the third day are fused with a bone marrow lupus hematoma cell line (myeloma) to produce a hybridoma by a conventional method. I do. Examples of mouse-derived myeloma include P3X63, Ag8.63 and Sp2 / 0-Agl4. Cell fusion between splenocytes and myeloma is generally performed by a known method, for example, the method of Koehler and Milstein (Koehler, G. and Milstein, C. Nature, 256, 495-497, 1975). The sensitized splenocytes and myeloma are mixed in the usual ratio of the number of cells, fused with a fetal calf serum (FCS) -free medium supplemented with polyethylene glycol, and cultured in a HAT selection medium supplemented with FCS. And select the fused cells (Hypridoma). Using the hybridoma culture supernatant, a hybridoma producing the target antibody that specifically recognizes the antigen is selected by a commonly used antibody detection method such as the EIA method. By cloning the obtained cells by the limiting dilution method, stable hybridomas can be established (Harlow, E. & Lane, D., Antibodies, Cold Spring Harbor Lab. (1988)).

Antibodies can be purified from the culture supernatant obtained by culturing the hybridoma in a conventional manner, or from ascites obtained by inoculating the animal intraperitoneally. Antibodies contained in the culture supernatant or ascites can be analyzed by commonly used methods such as salting out, ion exchange and gel chromatography, and affinity chromatography using protein A or G. (Harlow, E. & Lane, D., Antibodies, Cold Spring Harbor Lab. (1988)). [1] After selecting monoclonal antibodies that bind to 0CIF, 0BM, or sOBM from many of the monoclonal antibodies thus obtained, the ability to select antibodies that bind to the OCIF / sOBM complex from among them. Alternatively, [2] by selecting an antibody that directly binds to the OCIF / sOBM complex, a hybridoma producing the desired antibody can be obtained. From the obtained hybridoma culture, an antibody that binds to the OCIF / sOBM complex is obtained by using affinity chromatography using the OCIF / sOBM complex in addition to the above-described purification method. be able to.

 The antibody of the present invention thus obtained can be used for immunoassay or assay of OCIF / sOBM. Examples of such measurement or assay include Western blotting, immunoprecipitation, EIA, radioimmunoassay (RIA) and the like. For such measurement or assay, biological samples (blood, tissue, synovial fluid, urine, lymph, etc.) from mammals other than humans or humans, cells (cultured cells, cell lines, etc.), The culture supernatant, their extracts, their partially purified fractions, etc. can be used as samples.

 The present invention provides a method for diagnosing a bone metabolism disorder in a human or a non-human mammal. The diagnostic method is not particularly limited as long as it includes a step of measuring the amount or concentration of the OCIF / sOBM complex, and includes, for example, the following steps [1] and [2];

[1] Step of measuring the amount of OCIF / sOBM complex contained in a sample of a subject or a healthy subject:

[2] When the amount of the complex contained in the sample of the subject is larger than the amount of the complex contained in the sample of a healthy subject, the subject has abnormal bone metabolism. The process of determining that you have a disease. In a preferred diagnostic method, in the above step [2], the amount of the complex contained in the sample of the subject is twice as large as the amount of the complex contained in the sample of a healthy subject. In the case of more than the above, it is determined that the subject suffers from bone metabolism disorder. Further, in a preferred diagnostic method, in the step [1], when the amount of the OCIF / sOBM complex is measured, Use the antibodies provided by Ming. As a measurement method using such an antibody, various EIAs such as ELISA, sandwich EIA and the like can be exemplified. Preferably, sandwich EIA (Harlow, E. & Lane, D., Antibodies, Cold Spring Harbor Lab. (1988)). In the case of sandwich EIA, select at least two antibodies of the present invention as the primary antibody and the secondary antibody, immobilize the primary antibody on an insoluble carrier, and use the secondary antibody as a labeled antibody for OCIF / sOBM detection. I do. A preferred antibody of the present invention, a monoclonal antibody produced by Halibri-Doma 01-30 (FERM BP-7787) and a monoclonal antibody (# 207) produced by Hypri-Doma H-0BM1 (FERM BP-6264) When combined, it is desirable to use the former as the primary antibody and the latter as the secondary antibody.

 The present invention provides a kit for diagnosing anomalous bone loss in a human or non-human mammal. The kit is not particularly limited as long as it contains at least one antibody of the present invention, but when used for measurement by sandwich EIA, it contains at least two antibodies of the present invention, preferably One is an antibody immobilized on an insoluble carrier, and the other is a labeled antibody. The kit of the present invention may also contain a lysing agent, a detergent, a substrate for a labeling enzyme, and the like.

 As the insoluble 1 "raw carrier, for example, polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, latex, polymers such as magnetic fine particles in which latex is coated with metal, etc., and combinations thereof can be specified. The shape of the insoluble carrier can be various shapes such as tray shape, spherical shape, container shape, test tube, porous filter and the like.

As the labeling substance of the labeled antibody, it is advantageous to use enzymes, fluorescent substances, luminescent substances, radioactive substances and the like. Enzymes include peroxidase, alkaline phosphatase,] 3-galactosidase, glucose oxidase, etc., fluorescent substances include fluorescein isothiosinate, phycopyriprotein, etc., and luminescent substances include isorcinol, lucigenin, etc. And ¹²⁵ I, ¹³¹ I, ¹⁴ C, ¾, etc. can be used as the radioactive material. Further, the present invention is not limited to those exemplified above, and is not particularly limited as long as it can be used for an immunological assay.

When the labeling substance is an enzyme, a substrate and, if necessary, a color former are used to measure its activity. If peroxidase is used as the enzyme, the substrate H ₂ O ₂ as a color former, 2,2′-azinodi- [3-ethylbenzthiazoline sulfonic acid] ammonium salt (ABTS), 5-aminosalicylic acid, 0-phenylenediamine, 3, 3 ', 5,5'-Tetramethylbenzidine and the like. When alkaline phosphatase is used as the enzyme, 0-ditrophenyl phosphate and 4-methyl'umbelliferyl phosphate are used as substrates. In the case of using 3-D-galactosidase, fluorescein-di-(-D-galactovyranoside), 4-methylbenrefrylinoleno-β-ϋ-galactopyranoside and the like can be used as substrates.

 Any lysing agent may be used as long as it is commonly used for immunological measurement.For example, ρΗ containing phosphate buffer, Tris ^: acid buffer, acetate buffer, etc. is 6.0 to 8.0. Are shown as preferred examples. Further, as the detergent, those commonly used for immunological measurement are also used as they are. Examples include physiological saline, phosphate buffer containing saline, Tris buffer containing saline, and mixtures thereof. These detergents may further contain a nonionic surfactant such as Triton X-100, Tween20 or Brij35, and an ion surfactant such as sodium dodecyl sulfate or CHAPS.

 The present invention provides a pharmaceutical composition containing the antibody of the present invention, and an agent for preventing or treating bone metabolism disorders.

 In the case where a monoclonal antibody is contained in a pharmaceutical composition, and when the monoclonal antibody is derived from a mammal other than a human, the monoclonal antibody is preferably humanized.

 In order to humanize a monoclonal antibody derived from a mammal other than human, the complementarity determining region (hereinafter referred to as “CDR”) has been determined. The entire sequence and the framework (region work region) It is necessary to set the amino acid sequence of the variable region so that a part of the amino acid residue of the sequence is transplanted to a human antibody. This design follows the following method.

 When designing for humanization, the guidelines for selecting subgroups of receptor are:

 (1) A natural combination of a known human antibody immunoglobulin chain (hereinafter referred to as “heavy chain”) and an immunoglobulin light chain (hereinafter referred to as “light chain”) having a natural amino acid sequence is prepared and used as it is. ,

(2) The combination as a subgroup to which the heavy and light chains belong is preserved, but the heavy and light chains are Using a reamino acid sequence or consensus sequence that is derived from different human antibodies and has high identity to the amino acid sequences of the donor heavy and light chains,

Either is selected. In the present invention, the above guidelines can be followed.

 (3) It is also possible to adopt a method of selecting the heavy chain and light chain FR having the highest identity with the donor FR from a library of primary sequences of human antibodies without considering the combination of subgroups. It is possible. By these selection methods, the identity of the amino acid in the FR portion between the donor and the acceptor can be at least 70% or more. By adopting this method, it becomes possible to reduce the number of amino acid residues to be transplanted from a donor, and to induce a human anti-mouse antibody (Human Anti Mouse Antibody) response (Schloff et al., Cancer Res., 45, 879-85 (1985)).

 In addition, the operation of predicting the tertiary structure from the primary sequence of an antibody molecule has limited prediction accuracy, and the role of an amino acid residue that rarely appears in the subgroup to which the donor belongs cannot be sufficiently specified. In accordance with the method of Queen et al. (See Japanese Patent Application Laid-Open No. 4-502408), it is generally difficult to determine which amino acid residue, donor or acceptor, should be selected at such a position. According to the selection method of (3), the chance of making such a judgment can be significantly reduced.

 Generally, an antibody derived from a non-human mammal having a CDR to be transplanted is defined as a “donor”, and a human antibody to which a CDR is transplanted is defined as an “acceptor”. The present invention also complies with this definition.

 When an antibody that binds to the OCIF / sOBM complex is used for the prevention or treatment of various bone metabolic disorders, it can be administered in various forms, but the type of disease, the degree of the disease, the age of the patient, the sex of the patient, etc. Can be appropriately selected according to the conditions. For example, tablets, capsules, powders, granules, and syrups are orally administered, injections are administered alone or mixed with normal replacement fluids such as glucose and amino acids, and intravenously administered or intramuscularly administered alone It is administered subcutaneously, intradermally, intraperitoneally, and suppositories are rectally administered. According to a conventional method, these preparations include known excipients, binders, disintegrants, lubricants, flavoring agents, solubilizing agents, suspending agents, coating agents, and the like, which can be generally used in the pharmaceutical preparation field. It can be formulated using adjuvants.

For molding into tablets, a wide variety of carriers known in the art can be used. You. Examples of such a carrier include excipients such as lactose, sucrose, sodium salt, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, and cayic acid; water, ethanol, 'propanol, simple syrup, Binders such as glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone; dried starch, sodium alginate, agar powder, laminarane powder, sodium hydrogen carbonate, calcium carbonate, Polyoxyethylene sorbitan fatty acid esternoles, sodium laurinole sulfate, monoglyceride stearate, starch, lactose, etc., crushing agents such as sucrose, stearin, cocoa butter, hydrogenated oil; quaternary ammonium base, lauryl sulfate Na Absorption promoters such as lium; humectants such as glycerin and starch; adsorbents such as starch, lactose, dextrin, bentonite, and colloidal keic acid; abundance such as purified talc, stearates, powdered boric acid, polyethylene glycol Agents and the like. In addition, the preparation can be made into tablets coated with a usual coating, if necessary, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, double tablets, and multilayer tablets.

 For molding into a pill form, a wide variety of carriers known in the art can be used. Examples of such carriers include excipients such as glucose, lactose, cocoa butter, starch, hydrogenated vegetable oil, kaolin, and talc; binders such as gum arabic, tragacanth, gelatin, and ethanol; laminaran, agar, and the like. Disintegrant and the like.

 For shaping in the form of suppositories, a wide variety of carriers known in the art can be used. Such carriers include, for example, polyethylene glycol, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides and the like.

When prepared as an injection, the liquid preparation and the suspension are preferably sterilized and isotonic with blood. In order to form these solutions, emulsions and suspensions, any diluent known in the art can be widely used, for example, water, ethanol, propylene glycol, ethoxylated isostearinoleanorecol. And polyoxylated isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. In this case, a sufficient amount of salt, glucose, glycerin, etc. may be included in the pharmaceutical preparation to maintain isotonicity with blood, and ordinary solubilizing agents, buffers, soothing agents, etc. May be added. In addition, if necessary, coloring agents, preservatives, flavors, flavors, sweeteners, and other medicines may be included.

 Although the amount of the antibody that binds to the OCIF / sOBM complex contained in these pharmaceutical preparations is not particularly limited, it is usually 0.1 to 70% by weight, preferably 1 to 30% by weight. %.

 The dosage of the antibody that binds to the OCIF / sOBM complex depends on the symptoms, age, body weight, dosage form, dosage form, etc., but is generally 500 to l, 000 mg / day and 10 to 100 mg / day for adults. The preferred range is 50 to 500 mg.

 The frequency of administration of a drug containing an antibody that binds to the OCIF / sOBM complex as an active ingredient depends on the dosage form, dosage form, etc., but is once every few days, once a day, or several times a day. The present invention also provides a hybridoma that produces the monoclonal antibody of the present invention. Such a hybridoma is not limited as long as it is a cell that produces an antibody that binds to OCIF / sOBM.A site where a monoclonal antibody produced by hybridoma 01-30 (FERM BP-7872) binds ( specifically binds to the site (epitope) that binds to the monoclonal antibody (# 207) produced by hybridoma H-0BM1 (FERM BP-6264) that produces a monoclonal antibody that specifically binds to Examples thereof include a hybridoma producing a monoclonal antibody, and preferred examples thereof include hybridoma 01-30 (FERM BP-7787) and hybridoma H-0BM1 (FERM BP-6264). it can.

[Brief description of drawings]

 FIG. 1 shows the results of analysis of the OCIF / sOBM complex of the present invention (Example 3) by electrophoresis (Native PAGE) under non-denaturing conditions.

 Explanation of reference numerals

 Lane 1: OCIF / sOBM complex

 Lane 2: monomer type 0CIF

 Lane 3: human sOBM

FIG. 2 shows the results of OCIF / sOBM complex measurement by sandwich EIA constructed using the anti-sOBM monoclonal antibody of the present invention (Example 4) as a solid-phased antibody and a rabbit ego anti-OCIF polyclonal antibody as a labeled antibody. FIG. 3 shows the results of measurement of the OCIF / sOBM complex by Sandwich EIΆ constructed using the anti-sOBM monoclonal antibody # 207 of the present invention (Example 4) as a solid-phased antibody and the anti-OCIF monoclonal antibody as a labeled antibody. Show _p

 FIG. 4 shows OCIF / sOBM complex by sandwich EIA constructed with anti-sOBM monoclonal antibody # 20 and anti-OCIF monoclonal antibody 01-30 of the present invention (Example 4) as immobilized antibody or labeled antibody, respectively. 2 shows the measurement results.

 FIG. 5 shows a calibration curve prepared using the OCIF / sOBM complex standard prepared in Example 3 of the present invention (Example 5).

 FIG. 6 shows a healthy subject and a patient with rheumatoid factor positive by Sandwich EIA constructed using the anti-sOBM monoclonal antibody # 207 of the present invention (Example 6) as a labeling antibody and the anti-OCIF monoclonal antibody 01-30 as a solid-phased antibody. 3 shows the measurement results of the OCIF / sOBM complex in plasma of the present invention.

[Best Mode for Carrying Out the Invention]

 Next, the present invention will be described in more detail with reference to examples. However, these are merely examples, and the present invention is not limited thereto. Example 1 Preparation of anti-OCIF monoclonal antibody

Anti-OCIF monoclonal antibody was obtained according to the method described in W096 / 26217. Specifically, spleen cells of BALB / c mice immunized with purified 0CIF as an immunogen were fused with mouse myeloma cells (P3X63.Ag8.653) to produce hybridomas, and 0CIF was used as a solid phase from these cells. Cells producing anti-0CIF-specific antibodies were selected by the activated EIA. Hybridomas producing an anti-0CIF-specific antibody were repeatedly clawed three to five times by the limiting dilution method to select clones with high antibody production. The obtained production strain was administered intraperitoneally to BALB / c mice to which pristane (Aldrich Chemical Co., Ltd.) had been administered in a proportion of 1 to 10 × 10 ⁶ cells, and about 2 weeks later The ascites was collected. From this ascites fluid, affinity chromatography was carried out using Affigurupu Tin A Sepharose (Bio-Rad) according to the protocol attached to the kit to purify 40 types of antibodies. When the purity of the obtained purified antibody was determined by SDS-PAGE, a uniform band was recognized at a molecular weight of about 150,000. Example 2 Preparation of Anti-sOBM Monoclonal Antibody-Anti-sOBM monoclonal antibody was obtained according to the method described in W098 / 46644. That is, spleen cells of a BALB / c mouse immunized with purified sOBM as an immunogen were fused with mouse myeloma cells (P3X63.Ag8.653) to produce a hybridoma, and sOBM was isolated from these cells. Cells producing antibodies specifically recognizing sOBM were selected by immobilized EIA. Cloning of hybridomas in which production of anti-sOBM-specific antibodies was observed was repeated 3 to 5 times by the limiting dilution method, and clones with high antibody production were selected. The obtained strain was pre-administered with pristane (Aldrich Chemical Co., Ltd.). Intraperitoneally 1 to 10 × 10 ⁶ cells / animal of BALB / c mice, and about 2 weeks later The ascites was collected. From this ascites fluid, affinity chromatography was performed using a protein Tin A column (Pharmacia) according to the protocol attached to the kit to purify 40 types of antibodies. When the purity of the obtained purified antibody was analyzed by SDS-PAGE, a uniform band was observed at a molecular weight of about 150,000. Example 3 Preparation of Standard OCIF / sOBM Complex

60 μg of human (monomer) type 0CIF obtained according to the method described in WO 96/26217 in lOraM phosphate buffer (pH 7.4) containing 0.1% Tween20_0.15M NaCl (PBST) / ml, and human sOBM obtained according to the method described in W098 / 46644 was added to 40 μg / ml, and incubated at 4 ° C for 18 hours to prepare a 0CIF / human sOBM complex. (100 / zg / ml as complex). The formation of a complex by this method was partially modified from the method of Davis et al. (Ann. NY Acad. Sci. 121, 404 (1964)) and examined by Native PAGE. That is, the above-mentioned 0CIF / human sOBM complex, monomeric 0CIF, and human sOBM were mixed with an equal amount of sample buffer (0.0625M Tris-HCl (pH 6.8), 15% Glycerol, 0.001% BPB). Then, 10 μl of the sample mixture was analyzed by native PAGE. The electrophoresis gel used was Multigel 4/20 (Daiichi Kagaku), the electrophoresis buffer was 0.025M Tris-0.192M Glycine (ρΗ8.4), and the gel was stained using Silverstin KANT0 (Kanto Chemical). Fig. 1 shows the results. As a result, monomeric 0CIF (lane 2) and human sOBM (lane 3) showed single bands at different positions on Native PAGE. The complex (Lane In 1), a band was detected at a position different from that of monomeric OCIF and human sOBM. Further, in lane 1, no protein was detected at a position corresponding to monomeric 0CIF and human sOBM. From the above results, it was clarified that the monomer type 0CIF and the human sOBM all reacted under these conditions to form a complex. Example 4 Measurement of OCIF / sOBM complex by EIA

(1) Selection of anti-sOBM monoclonal antibody

 Sandwich EIA was constructed using 40 kinds of the anti-sOBM monoclonal antibodies obtained in Example 2 as solid-phased antibodies, and a rabbit ego anti-0CIF polyclonal antibody described in W096 / 26217 as a labeled antibody.標識 Labeling of a heron anti-0CIF polyclonal antibody was performed using a maleimide-activated peroxidase kit (Pierce). Forty anti-sOBM monoclonal antibodies were each dissolved in 0.1 M sodium bicarbonate solution (pH 9.6) at a concentration of 10 μg / ml, and 100 μl aliquots were added to a 96-ml Erymno plate (Nunc). In addition to each well, the mixture was allowed to stand at 4 ° C. for immobilization. The solution of each gel was discarded, 25% (V / V) Block Ace (Snow Brand Milk Products Co., Ltd.) 300 1 was added, and the mixture was allowed to stand at room temperature for 2 hours to block. After blocking, the plate was washed with PBST.

Next, each of the standard OCIF / sOBM complexes obtained in Example 3 was dissolved and diluted in PBST containing 40% blockage, and added to each well in a volume of ΙΟΟμΙ, followed by reaction at room temperature for 2 hours. After 2 hours, the plate was washed with PBST, and peroxidase (P0D) -labeled rabbit herb anti-0CIF polyclonal antibody diluted 1000-fold with PBST containing 25% Block Ace was added to each well at 100 / zl, and reacted at room temperature for 2 hours. . After washing the plate with PBS T, add ΙΟΟμΙ of enzyme substrate solution (ΤΜΒ: ScyTec) to each well to develop color, then add 停止 μΙ of reaction stop solution (ScyTec) to each well. The enzymatic reaction was stopped. The absorbance at 450 nm of each well was measured using a microplate reader (Imnoreader NJ2000: Nippon Intermed). As a result, it was confirmed that out of 40 kinds of anti-sOBM monoclonal antibodies, 8 kinds bound to the OCIF / sOBM complex. Also, as shown in FIG. 2, it was confirmed that # 207 had the highest reactivity with the OCIF / sOBM complex. The Hypri-Doma, which produces # 207, was released on November 5, 1997 at 1-1-3 Higashi, Tsukuba City, Ibaraki Prefecture, Japan, by the Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology, Research Institute Patent Student Product Depositary Center), deposited internationally as H-0BM1 and given accession number FERM BP-6264. ·

 (2) Selection of anti-0CIF monoclonal antibody

 Sandwich EIA was constructed using the anti-sOBM monoclonal antibody # 207 obtained in (1) above as a solid-phased antibody and the 40 anti-OCIF monoclonal antibodies obtained in Example 1 as labeled antibodies. Labeling of the anti-0CIF monoclonal antibody was performed using a maleimide-activated peroxidase kit (Pierce). Dissolve the anti-sOBM monoclonal antibody (# 207) in 0.1 M sodium bicarbonate solution (pH 9.6) to a concentration of 10 μg / ml, and add 100 μl aliquots of each solution to a 96-well Elymno plate (Nunc). In addition to the well, the mixture was allowed to stand at 4 ° C for solidification. The solution in each well was discarded, 300 μl of 25% Block Ace was added, and the mixture was allowed to stand at room temperature for 2 hours for blocking. After blocking, the plate was washed with PBST.

 Next, each of the standard OCIF / sOBM complexes obtained in Example 3 was dissolved and diluted in PBST containing 40% blockage, and added in a volume of ΙΟΟμΙ to each well, followed by reaction at room temperature for 2 hours. Two hours later, the plate was washed with PBST, and 40 kinds of P0D-labeled anti-0CIF monoclonal antibodies diluted 1000-fold with ST containing 25% Block Ace were added at 100 // I to each well and reacted at room temperature for 2 hours. . After washing the plate with PBST, add 1 μl of enzyme substrate solution (TMB, ScyTec) to each well to develop color, and add 100 μl of a stop solution (ScyTec) to each well. The enzyme reaction was stopped. The absorbance at 450 nm of each well was measured with a microplate reader. As a result, it was confirmed that out of the 40 anti-0CIF monoclonal antibodies, 6 types bind to the OCIF / sOBM complex. In addition, as shown in FIG. 3, it was revealed that the reactivity with the 01-30 OCIF / sOBM complex was the highest. This hybridoma was established on April 14, 1999 by the National Institute of Advanced Industrial Science and Technology at the National Institute of Advanced Industrial Science and Technology of the Ministry of International Trade and Industry of 1-1-3 Tsukuba-Higashi, Ibaraki, Japan. Deposit No. FERM P-17366, deposited under the Accession No. FERM P-17366, transferred to the International Depositary on February 1, 2002, and deposited under Accession No. FERM BP-7872.

 (3) Selection of immobilization and labeling

The anti-human sOBM monoclonal antibody (# 207) obtained in the above (1) and the anti-OCIF monoclonal antibody (01-30) obtained in the above (2) were each adjusted to 0.1 μg / ml with 0.1M bicarbonate. Dissolve in sodium solution (pH 9.6), 100 μl each 96 ゥ: The plate was added to each well of a rate (Nunc), and allowed to stand at 4 ° C for immobilization. The solution in each gel was discarded, 300/1 25% Block Ace was added, and the mixture was allowed to stand at room temperature for 2 hours for blocking. After blocking, the plate was washed with PBST. 5 ng / ml of the standard OCIF / sOBM complex obtained in Example 3 was dissolved and diluted in PBST containing 40% Block Ace, added to each pellet in 100 μl portions, and reacted at room temperature for 2 hours. Two hours later, la. Wash the rate with PBST and contain 25% of POD-labeled # 1-30 when immobilized antibody is # 207 and 25% POD-labeled # 207 when 01-30 is immobilized antibody After dilution with PBST, 100 / il was added to each well and reacted at room temperature for 2 hours. After washing the plate with PBST, add 100 μl of enzyme substrate solution (TMB, ScyTec) to each well to develop color. Then add 反 応 μΙ of reaction stop solution (ScyTec) to each well. The enzyme reaction was stopped. The absorbance at 450 nm of each gel was measured with a microplate reader. The results are shown in FIG. As a result, the OCIF / sOBM complex was more sensitive when 01-30 was used as the immobilized antibody (primary antibody) and # 207 was used as the labeled antibody (secondary antibody), compared to the reverse combination. It was confirmed that it could be detected. Example 5 Preparation of calibration curve

 Using the OCIF / sOBM complex standard prepared in Example 3 in a concentration range of 0 to 100 ng / ml, the method of Example 4 (3), that is, 01-30 was immobilized antibody, # 207 Using EIA as a labeled antibody, a standard curve was prepared. Fig. 5 shows a typical result. By this EIA method, an increase in absorbance was confirmed with an increase in the concentration of the complex, and it became clear from this calibration curve that the OCIF / sOBM complex could be quantified in a concentration range of 1 to 25 ng / ml. Example 6 Measurement of OCIF / sOBM Complex in Healthy Subjects and Rheumatoid Factor Positive Patient Specimens

The OCIF / sOBM complex in the plasma of healthy subjects and rheumatoid factor-positive patients was measured by sandwich EIA using 01-30 as the immobilized antibody and # 207 as the labeled antibody. 0 Dissolve 1-30 to a concentration of 10 μg / ml in 0.1 M sodium bicarbonate solution (pH 9.6), add ΙΟΟμΙ to each well of a 96 ゥ Elymno plate (Nunc), The mixture was allowed to stand at 4 ° C for solid phase immobilization. Solution was discarded each Uweru, 25% Block Ace (Snow Brand Milk Products Co.) 300 _mu 1 was added and allowed to stand at room temperature for 2 hr for blocking. After blocking, the plate was washed with PBST. Normal pool serum (BioWhitaker) and rheumatic factors Each of the three positive plasmas was dissolved in PBST containing 40% block ace, diluted 2-fold, added in a volume of {θμΐ} in triplicate to each well, and reacted at room temperature for 2 hours. Two hours later, the plate was washed with PBST, and POD-labeled # 207 was diluted with PBST containing 25% Block Ace, and ΙΟΟμΙ was added to each well, followed by reaction at room temperature for 2 hours. After washing the plate with PBST, add 100 / l enzyme substrate solution (T-book, ScyTec) to each well to develop color, and add 停止 μΙ of reaction stop solution (ScyTec) to each well. The enzyme reaction was stopped. The absorbance at 450 nm of each well was measured with a microplate reader. The results are shown in FIG. As a result, the amount of OCIFZsOBM complex was statistically significantly higher in all three rheumatoid factor-positive patients than in healthy subjects (t-test). Therefore, the OCIFZsOBM complex was shown to be useful as a clinical marker for rheumatic diseases. Example 7

 (1) Production of kit for OCIF / sOBM complex measurement (for 80 samples)

 (1) One 96-well plate in which the 01-30 antibody was immobilized by the method of Example 4 and blocked with Block Ace in advance:

 (2) P207-labeled # 207 antibody by the method of Example 4: 10 / Z 1

 (3) OCIF / sOBM complex standard: 1, 2.5, 5, 10, 25 ng / ml at 5 concentrations of 400 each

(4) Sample dilution (PBS solution containing 0.01% Tween20 and 40% Block Ace): 10 ml

(5) Labeled antibody dilution (PBS solution containing 0.01% Tween20 and 25% Block Ace): 10 ml

 (6) 96-well plate washing solution (PBS solution containing 0.1% Tween20): 1 liter

(7) Substrate solution for measuring the activity of the labeled enzyme (here, TMB solution): 10 ml

(8) Reaction stop solution (TMB stopping reagent): 10ml

(2) Quantitation method using kit

To each well of the plate (1), add the sample diluted with the diluent (4), normal pooled serum, and 0 CIF / sOBM complex standard (3) in triplicate 100 // 1 each. After leaving at room temperature for about 2 hours, wash each well of the plate 5 to 6 times with the washing solution (300 µl). An automatic plate washer may be used for this washing operation. 100 μl of POD-labeled # 207 antibody (2) diluted 500 times with diluent (5) on each well of the washed L plate And leave at room temperature for about 2 hours. Wash each well of the plate 5 or 6 times with the washing solution (6). This washing operation may be performed using an automatic plate washer. Add the enzyme substrate solution (7) to each well in a volume of ΙΟΟμΙ, and leave at room temperature for 20-30 minutes. Stop the enzyme reaction by adding ΙΟΟμΙ of Reaction Stop Solution (8) to each well. Measure the absorbance at 450 nm of each well with a microplate reader. A calibration curve for the OCIF / sOBM complex is prepared from the absorbance at 450 nm of each module to which the OCIF / sOBM complex standard (3) has been added, and the OCIF / sOBM complex concentration of each sample is calculated from the calibration curve.

[Possibility of industrial use]

 According to the present invention, an antibody that binds to the OCIF / sOBM complex and a hybridoma that produces the antibody can be obtained.

 The antibody of the present invention is useful for prevention, treatment or diagnosis of bone metabolism disorders. Further, the present invention provides a method for diagnosing bone sickness I abnormality by quantifying the amount of the OCIF / sOBM complex. Further, the present invention provides a kit for diagnosing bone metabolism disorder containing the antibody. Further, the antibody of the present invention can also be used as an analytical reagent for research.

[Reference to deposited biological material]

 (1) Name and address of the depositary institution that deposited the biological material

 Name: National Institute of Advanced Industrial Science and Technology

 Patent Organism Depositary

 Address: Tsukuba East 1-chome, Ibaraki Prefecture, Japan 1 Date of deposit with Central 6th Agency

 November 5, 1997 (Original deposit date)

 February 20, 1998 (Transfer date to deposit based on the Budapest Treaty) Deposit number assigned to deposit by HA institution

 FERM BP-6264

(2) b. Name and address of the depositary institution that deposited the biological material

 Name: National Institute of Advanced Industrial Science and Technology

Patent Organism Depositary Address: Tsukuba East 1-chome, Ibaraki Prefecture, Japan 1 Chuo Exit No. 6

 April 14, 1999 (date of original deposit)

 February 1, 2002 (Transfer date to deposits based on the Budapest Treaty) Deposit number assigned to the deposit by the Thai institution

 FERM BP—7872

Claims

The scope of the claims

1. An antibody that binds to a complex of osteoclastogenesis inhibitory factor (OCIF) and a soluble OCIF binding molecule (sOBM).

 2. The antibody according to claim 1, which binds to osteoclast formation inhibitory factor (0CIF).

3. The antibody according to claim 1 or 2, which binds to a soluble OCIF binding molecule (sOBM).

 4. The antibody according to any one of claims 1 to 3, which is a polyclonal antibody.

 5. The antibody according to any one of claims 1 to 3, which is a monoclonal antibody.

 6. The antibody according to claim 5, which is a humanized antibody.

 7. The antibody according to claim 5, which is produced by hybridoma 01-30 (FERM BP-7872).

 8. The antibody according to claim 5, which is produced by hybridoma H-0BM1 (FERM BP-6264).

 9. Hybrid Dorma 01-30 (FERM BP-7872).

 10. Hybridoma H-0BM1 (FERM BP-6264).

 1 1. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 8.

 12. A preventive or therapeutic agent for bone metabolism disorders, comprising the antibody according to any one of claims 1 to 8 as an active ingredient.

 13. The bone disorder is one selected from the group consisting of rheumatoid arthritis, osteoarthritis, osteoporosis, hypertensive lucidemia, bone pageet disease and renal osteodystrophy. 13. The preventive or therapeutic agent for bone loss f abnormality according to range 12.

 14. A method for diagnosing bone metabolism disorders, including the following steps [1] and [2]:

 [1] Step of measuring the amount of OCIF / sOBM complex contained in a sample from a subject or a healthy subject:

[2] If the amount of the complex contained in the sample of the subject is significantly larger than the amount of the complex contained in the sample of a healthy subject, Abnormalities Determining that the subject is suffering from.

 15. The diagnostic method according to claim 14, wherein the antibody according to any one of claims 1 to 8 is used.

 16. The method for diagnosing abnormal bone metabolism according to claim 14 or 15, wherein the sample is plasma or synovial fluid.

 17. The method for diagnosing bone metabolism disorder according to any one of claims 14 to 16, wherein the bone loss Hi disorder is rheumatoid arthritis or joint disease.

 18. A kit for diagnosing bone metabolism disorder, comprising at least the antibody according to any one of claims 1 to 8.

 19. The diagnostic kit according to claim 18, comprising at least two antibodies according to any one of claims 1 to 8.