WO2002079256A1 - Anticorps et son utilisation - Google Patents

Anticorps et son utilisation Download PDF

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Publication number
WO2002079256A1
WO2002079256A1 PCT/JP2002/002909 JP0202909W WO02079256A1 WO 2002079256 A1 WO2002079256 A1 WO 2002079256A1 JP 0202909 W JP0202909 W JP 0202909W WO 02079256 A1 WO02079256 A1 WO 02079256A1
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Prior art keywords
antibody
ferm
hybridoma
sobm
complex
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PCT/JP2002/002909
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English (en)
Japanese (ja)
Inventor
Naohiro Washida
Toshiko Satake
Kazuki Yano
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Sankyo Company, Limited
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Publication of WO2002079256A1 publication Critical patent/WO2002079256A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/12—Drugs for disorders of the metabolism for electrolyte homeostasis
    • A61P3/14—Drugs for disorders of the metabolism for electrolyte homeostasis for calcium homeostasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • 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).
  • OCIF osteoclastogenesis inhibitory factor
  • sOBM soluble OCIF binding molecule
  • the present invention provides a hybridoma producing the antibody, a method for producing the antibody, a preventive / therapeutic agent for bone metabolism disorders using the antibody as an active ingredient, or a diagnostic agent for bone metabolism disorders using the antibody.
  • the present invention relates to a diagnostic method and a diagnostic kit.
  • Bone metabolism depends on a balance between the activity of osteoblasts, which are responsible for bone formation, and osteoclasts, which are responsible for bone resorption (Chambers, TJ, et al., Vitam. Horm., 46, 41-86).
  • o Bone metabolism disorders are thought to be caused by an imbalance between bone formation and bone resorption, as diseases associated with bone metabolism disorders such as rheumatoid arthritis, osteoarthritis, osteoporosis, Known are hypercalcemia, bone Paget's disease, and renal osteotrophic disease
  • Rheumatoid arthritis is an intractable inflammatory disease with the synovium as the main lesion. If it progresses, it causes cartilage 'bone destruction, leading to joint function deterioration.In addition, various extra-articular symptoms may spread inflammatory diseases to systemic organs, resulting in the patient's QOL ( quality of life).
  • the criteria for early rheumatoid arthritis are being prepared because it is necessary to start treatment for early rheumatoid arthritis and to suppress the progression of bone dystonia.
  • 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 phalangeal joint (PIP), ankle or metatarsophalangeal joint (MTP) swelling lasting more than one week, (4) symmetric swelling lasting more than one 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 p43 (1988)), and rheumatoid arthritis is diagnosed if 4 out of 6 items apply.
  • setting a sensitive standard for such early diagnosis will inevitably sacrifice specificity. For this reason, specific disease markers for early diagnosis of rheumato
  • osteoblasts and osteoclasts are known to interact closely, and this phenomenon is called force-linking.
  • 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 (TNF c, transformer growth factor- ⁇ (TGF- ⁇ ) such as is this and force s SaiwaiushitoraTsuge acting promoted or inhibited Rereru (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., ⁇ ' (Endocr inology, 136, 2367-2376,
  • osteoclast differentiation factor As a factor involved in osteocyte formation, a molecule called osteoclast differentiation factor (ODF) 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).
  • 0BM (0CIF binding molecule; 0BM) was successfully cloned. It became clear that this 0BM was the virtual 0DF.
  • TRANCE W099 / 29865
  • RAKL W098 / 284236
  • This 0BM is a type II membrane-bound protein Yes, along with soluble OBM (sOBM), whose transmembrane region has been deleted, is a factor that supports and promotes osteoclast differentiation and maturation in the osteoclastogenesis system in vitro. (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.
  • 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 OCIF / sOBM complex in plasma of healthy subjects and rheumatoid factor-positive patients using this enzyme immunoassay, more OCIF / sOBM complexes were detected in rheumatoid factor-positive patients.
  • enzyme immunoassay enzyme immunoassay
  • the present invention provides an antibody that binds to the OCIF / sOBM complex, a polyclonal antibody that binds to the OCIF / sOBM complex, a monoclonal antibody that binds to the OCIF / sOBM complex, a humanized monoclonal antibody that binds to the OCIF / sOBM complex,
  • a method for producing these antibodies which comprises collecting these antibodies from a culture of cells producing these antibodies; a pharmaceutical composition containing these antibodies; Prophylactic and therapeutic agents for bone metabolism disorders (rheumatoid arthritis, osteoarthritis, osteoporosis, hypercalcemia, bone jet disease, renal osteodystrophy, etc.), and bone metabolism disorders containing these antibodies
  • the present invention has been made to solve the above problems.
  • the present invention (1) an antibody that binds to a complex of an osteoclastogenesis inhibitory factor (OCIF) and a soluble OCIF binding molecule (sOBM);
  • OCIF osteoclastogenesis inhibitory factor
  • sOBM soluble OCIF binding molecule
  • a cell producing the antibody according to any one of (1) to (9) is cultured, and then the antibody according to any one of (1) to (9) is cultured from the culture.
  • a pharmaceutical composition comprising the antibody according to any one of (1) to (9),
  • a preventive or therapeutic agent for bone metabolism disorder comprising the antibody according to any one of (1) to (9) as an active ingredient;
  • the bone loss tf 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 metabolism disorder according to (15) is one selected from the group consisting of rheumatoid arthritis, osteoarthritis, osteoporosis, hypercalcemia, bone pageet disease, and renal osteodystrophy.
  • a method for diagnosing bone metabolism disorder comprising the following steps [1] and [2]: [1] Step of measuring the amount of the complex of 0CIF and sOBM contained in a sample of a subject or a healthy subject:
  • a diagnostic kit for bone metabolism disorder comprising at least one antibody according to any one of (1) to (9).
  • An antibody produced by hybridoma 01-30 (FERM BP-7872) is contained as an antibody for immobilization, and hybridoma H-0BM1 (FERM BP-6264) or hybrid is used.
  • An osteoclast inhibitor (0CIF) and a soluble 0CIF binding molecule comprising using at least one of the antibodies according to any one of (1) to (9).
  • Antibodies produced by Hypri-Doma 01-30 are contained as antibodies for immobilization, and Hypri-Doma H-0BM1 (FERM BP-6264) or Hybridoma # 20a (FERM)
  • 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, Di syndrome), osteopenia due to reduced gravitational load or fixation or immobilization of limbs, ⁇ single jet disease, osteomyelitis, infectious lesions due to bone loss, solid tumors (breast cancer, lung cancer, kidney cancer,
  • the antibody provided by the present invention is not particularly limited as long as it binds to the OCIF / sOBM complex, and may be either a polyclonal antibody or a monoclonal antibody.
  • Such antibodies High Priestess dormer 01-30 provided by the present invention (FERM BP - 7872) by the: Ri site monoclonal antibodies produced specifically binds (epito pe) to specifically bind to monoclonal antibodies Specific to the site (epitope) that specifically binds to the monoclonal antibody (# 207) produced by hybridoma H-0BM1 (FERM BP-6264) or hybridoma # 207 (FERM BP-7953). Monoclonal antibodies and the like that bind thereto can be exemplified. As a preferred example, hybridoma 01-30
  • FERM BP-7872 monoclonal antibody (# 207) produced by hybridoma H-0BM1 (FERM BP-6264) or hybridoma # 207 (FERM BP-7953).
  • animals immunized when the antibody of the present invention is obtained include humans, mammals other than humans, and birds. Suitable animals are different from the species from which the antigen is derived.
  • the term “antigen” includes the meaning of an immunogen (i-thigh unogen).
  • Antigens used for obtaining the antibodies of the present invention include 0CIF derived from humans or non-human mammals, analogs thereof, mutants thereof, derivatives thereof, and the like (above, TO96 / 26197, WO097 Hereinafter, all of them are simply referred to as “0CIF.”;), Human or non-human mammal-derived 0BM, its analogs, its mutants, its derivatives, etc.
  • the OCIF / sOBM complex is generally 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.
  • 0CIF or sOBM may be recombinant.Recombinant 0CIF or sOBM should be collected from a culture of prokaryotic cells or animal cells such as E.
  • 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 a protein, and examples thereof include a phosphate buffer.
  • the solvent may contain sodium salt, a surfactant and the like.
  • the pH range of the solvent is 5 to 10, preferably 6 to 8.
  • the range of the heat retention temperature is 0 to 40 ° C, preferably 0 to 37 ° C, more preferably 4 to 25 ° C.
  • the range of this time depends on the reaction temperature, the concentration of 0CIF and sOBM, the mixing ratio thereof, and the like, but is usually 1 hour to 1 week, preferably 6 hours to 2 days.
  • the desired OCIF / sOBM complex can be obtained by electrophoresis (such as PAGE under non-denaturing conditions), EIA using a combination of anti-0CIF antibody and anti-sOBM antibody or anti-0BM antibody, etc.
  • electrophoresis such as PAGE under non-denaturing conditions
  • EIA using a combination of anti-0CIF antibody and anti-sOBM antibody or anti-0BM antibody, etc.
  • the OCIF / sOBM complex thus obtained and confirmed is purified, if necessary, and then purified or selected from an antigen for immunizing an animal when obtaining the antibody of the present invention, and an antibody of the present invention.
  • the method can be used as a standard for measuring the amount of the OCIF / sOBM complex in the test sample.
  • 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, a physiological saline solution), and administered together with an immunological adjuvant (for example, Freund's complete adjuvant) intraperitoneally or intravenously, if necessary. Immunization is generally performed 3 to 4 times at intervals of 1 to 2 weeks. Alternatively, an in vitro sensitization method can also be used.
  • a solvent for example, a physiological saline solution
  • an immunological adjuvant for example, Freund's complete adjuvant
  • spleen cells prepared from the spleen extracted on the third day are fused with a bone marrow tumor cell line (myeloma) to produce a hybridoma by a conventional method.
  • myeloma bone marrow tumor cell line
  • Examples of mouse-derived myeloma include P3X63, Ag8.653 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, and fusion is performed by adding polyethylene glycol to a medium without fetal calf serum (FCS), followed by culturing in HAT selection medium with FCS. And select the fused cells (Hypridoma).
  • FCS fetal calf serum
  • Hypridoma fetal calf serum
  • 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.
  • Antibodies can be purified from the culture supernatant obtained by culturing the hybridoma according to a conventional method, or from ascites obtained by inoculating intraperitoneally into an animal. Antibodies contained in the culture supernatant or ascites are analyzed by salting out, ion exchange and gel chromatography, protein It can be purified by a commonly used method such as affinity chromatography using A or G (Harlow, E. & Lane, D., Antibodies, Cold Spring Harbor Lab. (1988)).
  • the antibody of the present invention thus obtained can be used for OCIF / sOBM immunoassay or assay.
  • measurement or assay include Western blotting, immunoprecipitation, EIA, radioimmunoassay (RIA) and the like.
  • measurements or assays include samples from humans or non-human mammals (blood, plasma, serum, tissue, synovial fluid, urine, lymph, etc.), cells (cultured cells, cell lines, etc.), The culture supernatant, their extracts, their partially purified fractions, and the like can be used as samples, and suitable samples are plasma or synovial fluid.
  • 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];
  • the subject 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 a bone metabolic disorder Determining that the subject is suffering from the disease.
  • 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. If the number is large, it is determined that the subject is suffering from bone metabolism disorder, or the amount of the OCIF / sOBM complex contained in the sample of the subject or a healthy subject is determined three times each. Measuring The average value of the OCIF / sOBM complex contained in the sample of the subject is twice the standard deviation of the average value of the OCIF / sOBM complex contained in the sample of the healthy subject. If the value is equal to or greater than the value obtained by adding the calculated values, it is determined that the subject is suffering from bone loss and haze disorder.
  • the antibody provided by the present invention is used when measuring the amount of the OCIF / sOBM complex in the above step [1].
  • various EIAs such as ELISA, sandwich EIA and the like can be exemplified.
  • sandwich EIA Harlow, E. & Lane, D., Antibodies , Cold Spring Harbor Lab. (1988)
  • sandwich EIA at least two antibodies of the present invention are selected as a primary antibody and a secondary antibody, the primary antibody is immobilized on an insoluble carrier or the like, and the secondary antibody is used as a labeled antibody for OCIF / sOBM detection.
  • the preferred antibodies of the present invention are monoclonal antibodies produced by hybridoma 01-30 (FERM BP-7872) and hybridoma H-0BM1 (FE RM BP-6264) or hybridoma # 207 (FERM BP -When combining the monoclonal antibody (# 207) produced by 7953), it is preferable to use the former as the primary antibody and the latter as the secondary antibody.
  • the phrase “j is subject having bone metabolism disorder” includes, in addition to the fact that the subject already has bone metabolism disorder,
  • the subject and the healthy subject in the present invention are mammals other than humans or humans.
  • the above step [1] in the diagnostic method of the present invention is included in the present invention as a method for measuring an OCIF / sOBM complex.
  • the present invention provides a kit for diagnosing a bone metabolism disorder in a human or a non-human mammal.
  • the kit is not particularly limited as long as it contains at least one antibody of the present invention, but preferably contains at least two antibodies of the present invention when used for measurement by sandwich EIA. One of them is an antibody immobilized on an insoluble carrier, and the other is a labeled antibody.
  • the kit of the present invention appropriately contains a lysing agent, a diluent for a sample, a diluent for a labeled antibody, an OCIF / sOBM complex standard, a detergent, a substrate for a labeled enzyme, a reaction stopping solution for a labeled enzyme, and the like. Is also good.
  • the insoluble carrier for example, polymers such as polystyrene, polyethylene, polypropylene, polyester, polyacryl nitrile, latex, polymers such as magnetic fine particles in which latex is coated with metal, and combinations thereof can be specified. Wear.
  • the shape of the insoluble carrier can be various shapes such as tray shape, spherical shape, container shape, test tube, porous filter, and microtiter plate.
  • 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 (hereinafter referred to as “P0D”), alkaline phosphatase,] 3-galactosidase, glucose oxidase, and the like. Fluorescent substances such as fluorescein isothiosinate, and Fi co pyridinium protein, as the light-emitting substance, Isorushinoru, etc. Cie genin, and as the radioactive material as possible out to the like 125 1, 131 1, 14 c ,. 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 for biotin and the like.
  • the labeling substance is an enzyme
  • a substrate and, if necessary, a color former are used to measure its activity.
  • the POD As enzymes, the 0 2 as substrate, 2 a color former, 2 '- Ajinoji - (3-E chill benz thiazoline sulfonic acid) Anmoniumu salt (ABTS), 5-Aminosarichiru acid. -Phenylenediamine, 3,3 ', 5,5'-tetramethylbenzine, etc., as substrate when using enzyme phosphatase as enzyme.
  • fluorescein-di- ( ⁇ -D-galatatoviranoside) fluorescein-di- ( ⁇ -D-galatatoviranoside)
  • 4 -nitrophenylphosphate 4 -nitrophenylphosphate
  • 4-methylumbelliferyl phosphate etc.
  • -methylumberiferi / le / 3-D-galactopyranoside can be used.
  • any lysing agent may be used as long as it is commonly used for immunological measurement.
  • the pH range of phosphate buffer, Tris-HCl buffer, acetate buffer, etc. ranges from 6.0 to 8.0.
  • 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.
  • kit for diagnosing bone metabolism disorders provided by the present invention is also encompassed in the present invention as a kit for measuring an OCIF / sOBM complex.
  • the present invention provides a pharmaceutical composition containing the antibody of the present invention, and an agent for preventing or treating bone metabolism disorders.
  • the monoclonal antibody is contained in the pharmaceutical composition of the present invention and administered to a human, and the monoclonal antibody is derived from a mammal other than a human, the monoclonal antibody is preferably humanized.
  • HAMA human antimouse antibody response
  • an antibody is composed of two immunoglobulin heavy chains each having a molecular weight of about 50,000 (hereinafter referred to as “heavy chains”) and two immunoglobulin light chains each having a molecular weight of about 23,000 (hereinafter referred to as “light chains”). It is configured.
  • the heavy chain and the light chain each have a region having a different amino acid sequence called a variable region at about 110 residues from the amino terminal. Furthermore, among these variable regions, the frequency of amino acid sequence mutation is particularly high, and the region is called a hypervariable region, which is located near the 30th, 50th, and 95th positions from the amino terminus, respectively.
  • these hypervariable regions are assembled and folded so as to form a surface structure to which an antigen can bind.
  • the hypervariable region is referred to as a complementarity determining region (CDR).
  • CDR complementarity determining region
  • the part sandwiched between the two CDRs is called a framework (frame work region: hereinafter referred to as “FR”).
  • FR framework
  • the humanized antibody of the present invention includes a chimeric antibody (Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (Chem.), In which a variable region in a monoclonal antibody derived from a non-human animal is conjugated to a human-derived constant region.
  • CDR-grafted antibody PT Jones et al., Nature 321, 522 (1986)
  • CDR-grafted anti- Riechmann, L., et al., Nature 332, 323-327 (1988); Isaacs, JD. Et al., Lancet 340, 748-752 (1992)
  • 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 the CDR is transplanted is defined as an “acceptor”.
  • the present invention also complies with this definition. .
  • the identity of the amino acid in the FR moiety between the donor and the acceptor can be at least 70% or more.
  • the number of amino acid residues to be transplanted from the donor can be reduced, and the induction of the HAMA response can be reduced.
  • an antibody that binds to the OCIF / sOBM complex When 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, syrups are orally administered, injections are administered intravenously, alone or mixed with normal replenishers such as glucose and amino acids, or intramuscularly administered alone It is administered subcutaneously, intradermally, intraperitoneally, and suppositories are rectally.
  • compositions can be used in the pharmaceutical preparation field, such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspensions, coatings, etc., according to the usual methods. It can be formulated using an auxiliary agent.
  • a wide variety of carriers known in the art can be used.
  • carriers include excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, and caicic acid; water, ethanol, propanol, simple syrup, dextrose, Binders such as starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone; etc .; —dry starch, sodium alginate, agar powder, laminaran powder, sodium hydrogencarbonate, calcium carbonate, polyoxyethylene Disintegrators for sorbitan fatty acid esters, sodium lauryl sulfate, monoglyceride stearate, starch, lactose; disintegrators for sucrose, stearin, cocoa butter, hydrogenated oil, etc .; quaternary ammonium base, sodium lauryl sulfate Ab
  • a wide variety of carriers known in the art can be used.
  • 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. And the like.
  • Such carriers include, for example, polyethylene glycol, cocoa butter, Higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides and the like can be mentioned.
  • the liquid preparation and the suspension are preferably sterilized and isotonic with blood.
  • any diluent known in the art can be widely used, such as water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, Polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters and the like can be mentioned.
  • 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.
  • coloring agents may be included.
  • 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 10 days / 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.
  • the site where the monoclonal antibody produced by hybridoma 01-30 (FERM BP-7872) binds hybridoma that produces a monoclonal antibody that specifically binds to an epitope
  • a monoclonal antibody (# 207) produced by hybridoma H-0BM1 (FERM BP-6264) or hybridoma # 207 (FERM BP-7953)
  • hybridomas that produce a monoclonal antibody that specifically binds to an epitope epitope
  • epitope epitope
  • FIG. 1 shows the results when the OCIF / sOBM complex of the present invention (Example 3) was subjected to electrophoresis (Native PAGE) under non-denaturing conditions.
  • Fig. 2 shows the results of measurement of the OCIF / sOBM complex by sandwich EIA using the anti-sOBM monoclonal antibody of the present invention (Example 4) as a solid-phased antibody and a perch anti-OCIF polyclonal antibody as a labeled antibody. .
  • Fig. 3 shows the results of OCIF / sOBM complex measurement by Sandwich EIA constructed using anti-sOBM monoclonal antibody # 207 as the immobilized antibody and anti-OCIF monoclonal antibody as the labeled antibody in this effort (Example 4). Is shown.
  • FIG. 4 shows the measurement of OCIF / sOBM complex by sandwich EIA in which anti-sOBM monoclonal antibody # 207 and anti-OCIF monoclonal antibody 01-30 of the present invention (Example 4) were respectively immobilized or labeled. The results are shown.
  • 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 the results obtained by using a sandwich EIA constructed with anti-sOBM monoclonal antibody # 207 of the present invention (Example 6) as a labeled antibody and anti-OCIF monoclonal antibody 01-30 as a solid-phased antibody in serum or rheumatism of a healthy subject.
  • 4 shows the results of measuring the amount of OCIF / sOBM complex in plasma of factor-positive patients.
  • Anti-OCIF monoclonal antibody was prepared according to the method of Yano et al. (J. Bone & Mineral Res., 14, 518-527 (1999)). That is, BALB / c mice were immunized with a mixture of human 0CIF monomer and dimer purified according to the method of Tomoyasu et al. (Biochem. Biophys. Res. Co Rat, 245, 382-387 (1998)). Next, the spleen was collected from the mouse. A hybridoma was prepared by cell fusion of the obtained spleen cells and mouse myeoma cells (P3 X63. Ag8.63), and 0CIF was converted to 0CIF using EIA immobilized with 0CIF.
  • the anti-sOBM monoclonal antibody was prepared basically according to the method of Yano et al. (J. Bone & Mineral Res., 14, 518-527 (1999)).
  • BALB mice were immunized with human sOBM purified according to the method described in W098 / 46644, and then spleens were collected from the mice.
  • a hybridoma was prepared by fusing the obtained spleen cells and mouse myeloma cells (P3X63.Ag8.653), and sOBM-specific EIA was performed by immobilizing sOBM from these cells. Cells producing antibodies that bind to were selected. Cloning of hybridomas, in which production of an antibody specifically binding to sOBM was observed, by limiting dilution was performed 3 to 5 times, and clones with high antibody production were selected.
  • the obtained production strain was administered intraperitoneally to BALB / c mice to which pristane (Aldrich Chemical Co., Ltd.) had been administered at a concentration of 1 to 10 ⁇ 10 6 cells / animal.
  • pristane Aldrich Chemical Co., Ltd.
  • affinity chromatography was performed using a protein A column (Pharmacia) according to the protocol attached to the kit to purify 40 types of antibodies.
  • SDS-PAGE SDS-PAGE
  • Fig. 1 shows the results.
  • human 0CIF monomer (lane 2) and human sOBM (lane 3) showed single bands at different positions on Native PAGE.
  • the complex (lane 1) prepared by mixing them, a band was detected at a position different from that of human 0CIF monomer or human sOBM.
  • no protein was detected at the position corresponding to human 0CIF monomer or human sOBM.
  • J Example 4 Measurement of OCIF / sOBM complex by EIA
  • Sandwich EIA was constructed by using 40 kinds of anti-sOBM monoclonal antibodies obtained in Example 2 as solid-phased antibodies, and using a rabbit ego anti-0CIF polyclonal antibody described in W096 / 26217 as a labeled antibody.
  • ⁇ Labeling of a heron anti-0CIF polyclonal antibody was performed using a maleimide-activated peroxidase kit (Pierce). Dissolve each of the 40 anti-sOBM monoclonal antibodies in 0.1 M sodium bicarbonate solution (pH 9.6) to a concentration of 10 ⁇ g / ml, and add 100 ⁇ l aliquots to each well of a 96-well Elymno plate (Nunc). In addition, the mixture was allowed to stand at 4 ° C for immobilization. Discard each well and add 25% (V / V) Solution (manufactured by Dainippon Pharmaceutical Co., Ltd.)
  • each of the OCIF / sOBM complexes obtained in Example 3 was dissolved and diluted in PBST containing 40% block ace, added to each well in a volume of ⁇ , and allowed to react at room temperature for 2 hours. Two hours later, the plate was washed with PBST, and POD-labeled 'Egret anti-0CIF polyclonal antibody, diluted 1000-fold with PBST containing 25% Block Ace, was added to each well, and allowed to react at room temperature for 2 hours. After washing the plate with PBST, add 100 1 enzyme-substrate solution (TMB, ScyTec) to each well to develop color, then add ⁇ ⁇ of reaction stop solution (ScyTec) to each well to perform enzyme reaction. Stopped.
  • TMB enzyme-substrate solution
  • ScyTec reaction stop solution
  • the absorbance at 450 nm of each well was measured using a microplate reader (Imnoleader I NJ2000: Intermed Japan).
  • 8 were confirmed to bind to the OCIF / sOBM complex.
  • # 207 had the highest reactivity with the OCIF / sOBM complex.
  • Hypri-Doma which produces # 207
  • Tsukuba East Co., Ltd. 1-1-1 was deposited internationally as H-0BM1 with the 6th Independent Administrative Agency, National Institute of Advanced Industrial Science and Technology (AIST) under the accession number FERM BP-6264.
  • Hypri-Dorma which produces # 207, was established on February 19, 2001 at 1-1-3 Tsukuba-Higashi, Ibaraki, Japan, by the Ministry of International Trade and Industry, Ministry of International Trade and Industry. Tsukuba East Co., Ltd.
  • a 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-OCIF monoclonal antibody was performed using a maleimide-activated peroxidase kit (Pierce). 0. 1M bicarbonate Natoriumu solution to anti-sOBM monoclonal antibody ( ⁇ 1 ⁇ 20 7) becomes 10 ⁇ g / ml (pH9. 6) To ⁇ , each 100 ⁇ ⁇ 96 ⁇ El I Takeno plates (Nunc ) Then, the mixture was allowed to stand still at 4 ° C. to solidify. The solution in each well was discarded, 300 ⁇ l of 25% Block Ace (Dainippon Pharmaceutical Co., Ltd.) was added, and the mixture was left standing at room temperature for 2 hours for blocking. Thereafter, the plate was washed with PBST.
  • each of the OCIF / sOBM complexes obtained in Example 3 was dissolved and diluted in PBST containing 40% Block Ace, and 100 ⁇ l of each was added to each well, followed by reaction at room temperature for 2 hours. After the reaction, wash the plate with PBST, add 100 1 of each of 40 POD-labeled anti-OCIF monoclonal antibodies diluted 1000-fold with PBST containing 25% Block Ace to each well, and react at room temperature for 2 hours. I let it. After washing the plate with PBST, add ⁇ of enzyme substrate solution (TMB, ScyTec) to each well to develop color, and add 100 ⁇ l of reaction stop solution (ScyTec) to each well. The reaction was stopped.
  • TMB enzyme substrate solution
  • ScyTec enzyme substrate solution
  • the anti-human sOM monoclonal antibody # 207 obtained in (1) above or the anti-0CIF monoclonal antibody 01-30 obtained in (2) above was added to a 0.1 M sodium bicarbonate solution at 10 ⁇ g / ml. (pH 9.6), 100 ⁇ l of each solution was added to each plate of 96 Ueno Reim Nobrate (Nunc), and the mixture was allowed to stand at 4 ° C. for solidification. The solution in each well was discarded, 25% Block Ace (manufactured by Dainippon Pharmaceutical Co., Ltd.) (300 ⁇ ) was added, the mixture was allowed to stand at room temperature for 2 hours, and blocking was performed. Thereafter, the plate was washed with PBST.
  • Example 3 5 ng / ml of the OCIF / sOBM complex obtained in Example 3 was dissolved in PBST containing 40% block ace, diluted, added to each well in 100 ⁇ l portions, and reacted at room temperature for 2 hours. . Two hours later, the plate was washed with PBST, and when # 207 was immobilized, POD-labeled 01-30 was used.When 01-30 was immobilized, POD-labeled # 207 was used for 25 times each. Dilute with PBST containing Block Ace and add ⁇ to each well. The reaction was allowed to proceed at room temperature for 2 hours.
  • Example 4 Using the OCIF / sOBM complex standard prepared in Example 3 in the concentration range of 0 to 100 ng / ml, the method of Example 4 (3), that is, using 01-30 as the immobilized antibody, and # The measurement was performed by sandwich EIA using 207 as a labeled antibody, and a calibration curve was prepared. Figure 5 shows a typical result. This EIA method confirmed that the absorbance increased with increasing complex concentration, and this calibration curve revealed that the OCIF / sOBM complex could be quantified in the concentration range of 1 to 25 ng / ml.
  • Example 6 Measurement of the amount of OCIF / sOBM complex contained in a sample
  • the amount of the OCIF / sOBM complex in the serum of healthy subjects or the plasma of rheumatoid factor-positive patients was measured by sandwich EIA using 01-30 as the immobilized antibody and # 207 as the labeled antibody.
  • the results are shown in FIG.
  • the average amount of OCIF OBM complex in plasma of rheumatoid factor-positive patients is larger than twice the average amount of OCIF / sOBM complex in serum of healthy subjects, and It was larger than the average value of the amount of the OCIF / sOBM complex plus twice its standard deviation.
  • the amount of OCIF / sOBM complex contained in the plasma of rheumatoid factor-positive patients was statistically significantly higher than that in the serum of healthy subjects.
  • Substrate solution for measuring the activity of the labeled enzyme (here, TMB solution): 10 ml
  • 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 metabolism disorder 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 a research analysis reagent.
  • FERM BP-7872 B Name and address of the depositary institution that deposited the biological material

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Abstract

L'invention porte sur un anticorps se liant à un complexe d'un facteur inhibiteur d'ostéoclastogénèse (OCIF) auquel est liée une molécule soluble (complexe facteur inhibiteur d'ostéoclastogénèse/molécule soluble), cette liaison se produisant dans les fluides biologiques. L'invention porte sur un hybridome produisant cet anticorps, sur un procédé de production de cet anticorps au moyen de l'hybridome et sur des agents préventifs ou thérapeutiques utilisés ans le dysfonctionnement métabolique osseux contenant cet anticorps comme ingrédient actif. L'invention porte sur un procédé de diagnostic des dysfonctionnements métaboliques osseux (tels que l'arthrite rhumatoïde) visant à quantifier le complexe facteur inhibiteur d'ostéoclastogénèse/molécule soluble. Un kit de diagnostic est également décrit. L'anticorps précité est utile dans le diagnostic, la prévention et le traitement des dysfonctionnements métaboliques osseux (tel que l'arthrite rhumatoïde) ou comme réactif analytique utilisé en laboratoire.
PCT/JP2002/002909 2001-03-26 2002-03-26 Anticorps et son utilisation WO2002079256A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6958350B2 (en) 2001-02-19 2005-10-25 Astrazeneca Ab Chemical compounds
US6960602B2 (en) 2001-03-22 2005-11-01 Astrazeneca Ab Piperidine derivatives as modulators of chemokine receptors
WO2012050154A1 (fr) * 2010-10-13 2012-04-19 学校法人 東京女子医科大学 Inhibiteur de formation d'ostéoclastes contenant un anti-vdac anticorps

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998046644A1 (fr) * 1997-04-15 1998-10-22 Snow Brand Milk Products Co., Ltd. Nouvelle proteine et methode de production associee
WO1999015691A1 (fr) * 1997-09-24 1999-04-01 Snow Brand Milk Products Co., Ltd. Procede permettant de diagnostiquer un dysfonctionnement du metabolisme osseux

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998046644A1 (fr) * 1997-04-15 1998-10-22 Snow Brand Milk Products Co., Ltd. Nouvelle proteine et methode de production associee
WO1999015691A1 (fr) * 1997-09-24 1999-04-01 Snow Brand Milk Products Co., Ltd. Procede permettant de diagnostiquer un dysfonctionnement du metabolisme osseux

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YANO K. ET AL.: "Immunological characterization of circulating osteoprotegerin/osteoclastogenesis inhibitory factor: increased serum concentrations in postmenopausal women wtih osteoporosis", J. BONE MINER. RES., vol. 14, no. 4, 1999, pages 518 - 527, XP002953801 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6958350B2 (en) 2001-02-19 2005-10-25 Astrazeneca Ab Chemical compounds
US6960602B2 (en) 2001-03-22 2005-11-01 Astrazeneca Ab Piperidine derivatives as modulators of chemokine receptors
WO2012050154A1 (fr) * 2010-10-13 2012-04-19 学校法人 東京女子医科大学 Inhibiteur de formation d'ostéoclastes contenant un anti-vdac anticorps

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