+

WO1996009067A1 - Glycoproteine specifique aux osteoclastes pour le diagnostic et le traitement de l'osteoporose - Google Patents

Glycoproteine specifique aux osteoclastes pour le diagnostic et le traitement de l'osteoporose Download PDF

Info

Publication number
WO1996009067A1
WO1996009067A1 PCT/US1994/010700 US9410700W WO9609067A1 WO 1996009067 A1 WO1996009067 A1 WO 1996009067A1 US 9410700 W US9410700 W US 9410700W WO 9609067 A1 WO9609067 A1 WO 9609067A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
antigen
group
radioisotope
contacting
Prior art date
Application number
PCT/US1994/010700
Other languages
English (en)
Inventor
Philip A. Osdoby
Original Assignee
Washington University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Washington University filed Critical Washington University
Priority to PCT/US1994/010700 priority Critical patent/WO1996009067A1/fr
Publication of WO1996009067A1 publication Critical patent/WO1996009067A1/fr

Links

Classifications

    • 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
    • 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
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to immunodiagnostic tests for bone resorption. and to methods for diagnosing and treating osteoporotic disease.
  • Osteoclasts are multinucleated giant cells responsible for bone resorption formed from the fusion of mononuclear hematopoietic precursors belonging to the mononuclear phagocyte system.
  • the cascade of events and signals that guide osteoclast development through precursor proliferation, chemotaxis, and attachment to bone, fusion, and cytodifferentiation are likely dependent, in part, on diffusible osteoblast products and/or matrix components.
  • Osteoclasts are found in areas of active bone erosion in close contact with the bone surface in pits termed resorption bays or lacunae of Howship. Osteoclasts contain numerous mitochondria and vacuoles, many being acid phosphatase-positive lysosomes to facilitate bone resorption. At the site of bone resorption the surface of the cell is highly folded to form a ruffled membrane. Many lysosomes are concentrated at the bases of clefts between irregular cell extensions (lobopodia) and the plasma membrane has a dense, bristly undercoating similar to that of coated vesicles in other cells active i endocytosis.
  • the ruffled membrane Around the perimeter of the ruffled membrane is a zone of acti filaments and here the osteoclast is closely attached to the bone surface, s forming a limit to the resorptive activities of the ruffled membrane.
  • the siz of the ruffled membrane varies with hormonal treatment, increasing whe resorption of bone is stimulated and disappearing in the presence of agent favoring bone deposition, such as calcitonin.
  • osteoclasts are responsible for the removal of bone, althoug exactly how is not known. Demineralization of the bone matrix occurs locall where the ruffled membrane of the osteoclast approaches the bone surface. Osteoclasts may attach bone after its organic lining has been removed b osteoblasts or macrophages to expose the mineralized surface (T . Chambers, et al., Calcif. Tissue Int., 35:566-570, 1984). There is also muc evidence that osteoclasts can readily phagocytose collagen and other organi components of the matrix.
  • Agents responsible for stimulating osteoclasts to resorb bone appear to b multiple, including factors released by osteoblasts and probably various othe cells such as macrophages and lymphocytes. Various humoral factors such as parathyroid hormone are also involved in bone resorption.
  • osteoclasts The cell lineage of osteoclasts has been much studied, and it is no established based upon labelling experiments with grafted cells that they arise by fusion of mononuclear cells that originate in the bone marrow (P. Ash, e al., Nature, 283:669-670. 1980; J.S. Ko, et al., Am. J. Anal, 161:415-425, 1981) rather than from osteoblasts or osteoprogenitor cells, as was previousl believed.
  • Several lines of evidence point to a close similarity betwee osteoclasts and mononuclear phagocytes (macrophages) which can also fuse to form giant multinuclear cells with phagocytic function.
  • Osteoblasts are distinguished from other giant multinucleated cells by the antigens they express in response to the factors that trigger their formation. For instance, it is known that bone- associated giant cells express an antigen called the 121 F osteoclast antigen, and it is believed that development of the giant cell ruffled membrane and the presence of the 121 F osteoclast antigen are related to the formation of the giant cells in response to resorbable materials that are subject to extracellular dissolution or to components of the bone matrix, including osteocalcin, in association with bone mineral (D. Webber, et al., J. Bone and Min. Res., 5:401-410, 1990).
  • osteoclasts In homeostasis, remodelling of the interiors of bone is marked by the balanced activities of osteoclasts and osteoblasts; osteoclasts first excavate a cylindrical tunnel (a 'cutting cone') by concerted action, and then they are followed by osteoblasts which fill in the space created by concentric deposition around a centrally ingrowing blood vessel, forming a "closing cone.” Concentric deposition of lamellae of parallel-fibered bone follows on wall of resorption cavities and around vascular channels, which are thereby narrowed.
  • Osteoporotic diseases are characterized by a loss of homeostasis in bon reso ⁇ tion and bone growth.
  • osteoporosis is a well-define feature of another disease such as Cushing's syndrome.
  • a skeletal disorde that could also be considered osteoporosis is the major characteristic of certain heritable diseases o connective tissue, such as forms of osteogenesis imperfecta. In mos instances of osteoporosis, however, no other disease is apparent.
  • Thi category of osteoporosis can be conveniently considered to comprise severa forms.
  • osteoporotic is periodontal disease in which bon reso ⁇ tion is increased over the normal homeostatic level by metaboli byproducts of bacterial gum infection, resulting in loss of teeth. Another for occurs in children or young adults of both sexes and with normal gonada function. This form is frequently termed idiopathic osteoporosis, although most of the other forms are in fact also of unknown pathogenesis. So-called type I osteoporosis is found in a relatively small subset of postmenopausal women between 51 and 65 years of age and is characterized by an accelerated and disproportionate loss of trabecular bone as contrasted with cortical bone.
  • Decreased parathyroid gland function in this group of individuals may be compensatory to increased bone reso ⁇ tion.
  • So-called type II osteoporosis is found in a large proportion of women and men over the age of 75. In these individuals circulating levels of parathyroid hormone tends to be higher than normal. Both groups may have decreased mean circulating levels of 1,25(OH 2 )D 3 compared to age-matched controls, but levels are often in the normal range (Harrison's Principles of Internal Medicine, ed. E. Braunwald, et al., 11th Edition, McGraw-Hill Book Company: New York, 1987, page 1884- 1885). All of these disease states are referred to as osteoporosis or osteoporotic diseases.
  • osteoporosis The causes of osteoporosis are many, ranging from hypogonadism to calcium malabsorption. For instance, osteoclast development is governed by the interactions of humoral, local, and matrix-derived signals. Further, osteoclast reso ⁇ tive activity requires the expression of functional cell surface receptors, enzymes, and ion channels. Specific plasma membrane changes that occur during osteoclast maturation may therefore play a pivotal role in normal and pathological bone remodeling processes. Although many modulators such as, calcitonin, GM-CSF, and prostaglandins modulate osteoclast activity and development, it is not clear which factors act early or late in osteoclast ontogeny. Therefore diagnosis of osteoporotic disease depends upon development of tests that can be used to monitor changes that occur during osteoclast maturation and the rates of bone resorption and bone growth to determine when an imbalance in normal homeostatic processes indicates decreasing bone mass that can lead to fragile bones.
  • modulators such as, calcitonin, GM-CSF, and prostaglandins
  • a substantially purified osteoclast-specific plasma membrane-associated glycoprotein 47J expressed by giant multinucleated osteoclast cells associated with bone resorption processes is disclosed as well as monoclonal antibodies that bind to antigenic epitopes on the 47J glycoprotein.
  • a substantially similar human osteoclast-specific plasma membrane-associated glycoprotein expressed by human giant multinucleated osteoclast cells is also disclosed.
  • this invention provides both in vitro and in vivo methods for monitoring the progress of osteoporotic disease by repeated contact of patient samples with an antibody specific for a 47J antigenic epitope.
  • this invention provides a method for ameliorating osteoporotic disease in an animal by administering to the patient a therapeutically effective amount of a monoclonal antibody or fragment thereof that specifically binds to 47J antigen.
  • FIGURE 1A is a phase micrograph of avian frozen bone section reacted with 47M Mab. Arrow indicates a multinucleated osteoclast reactive with 47J.
  • FIGURE 1B is an immunohistograph of the bone section shown in FIGURE IA showing immunohistochemical staining of frozen avian bone section with 47M
  • FIGURE 2 is an bar graph showing the results of the ELISA of CHAPS detergent of dead and live osteoclasts (OC), marrow derived giant cells (MNGC), macrophages (MNC), fibroblasts (Fib), and osteoblasts (OB).
  • OC dead and live osteoclasts
  • MNGC marrow derived giant cells
  • MNC macrophages
  • Fib fibroblasts
  • OB osteoblasts
  • FIGURE 3 is a bar graph comparing the absorbance of marrow mononuclear cell cultures exposed to osteoblast conditioned media (OBCM) or phorbol-12- myristate-13-acetate (PMA) to induce formation of 47J-specific antigen in the giant cells which subsequently formed.
  • the control is marrow mononuclear cell cultures not exposed to OBCM or PMA.
  • FIGURE 4 is a photograph of an SDS-PAGE analysis of immunopurified 47J antigen under reduced (lane 1) and non-reduced (lane 3) 14 C-labeled standards are shown in lane 2. 35 S labeled immunoaffinity purified antigen obtained from PMA induced giant cells is shown in lane 4.
  • FIGURE 6 is a photograph of a gel showing deglycosyiation analysis by reducing SDS-PAGE of immunoaffinity purified 47J antigen labeled with 125 l and treated either with (lane 1) or without (lane 2) n-glycanase.
  • the present invention provides a substantially purified osteoclast-specifi plasma membrane-associated glycoprotein 47J expressed by gian multinucleated osteoclast cells associated with bone reso ⁇ tion processes an monoclonal antibodies that bind to antigenic epitopes on the glycoprotein.
  • Th antigenic glycoprotein has an apparent molecular weight as determined b SDS-PAGE analysis under non-reducing conditions of about 250 kDa.
  • B SDS-PAGE purification under disulfide reducing conditions the glycoprotei yields two subunits of about 170 and 70 kDa.
  • Cell fractionation analysis o osteoclasts indicate that the 170 kDa fragment is associated with the plasm membrane enriched fraction.
  • a 45 kDa fragment is also detected i the cytosol and endoplasmic reticulum rich fractions.
  • Deglycosylation analysi of purified antigen using n-glycanase and SDS-PAGE analysis of the produc reveals a downshift of about 20 kDa in the apparent MW of the 170 and 7 kDa fractions, indicating that the marker protein is a glycoprotein.
  • 47J is found only in multinucleated giant cells formed in response to stimuli associated with bone resorption (osteoclasts). Studies in both avian an human marrow cells show that induction of the 47J osteoclast marke glycoprotein is in response to the general developmental promoters retinoi acid, 1,25 (OH) 2 D 3 , phorbol myristate acetate (PMA), 25% UMR 10 conditioned medium or dexamethasone. Induction of the 47J antigeni glycoprotein by 1 ,25 (OH) 2 D 3 is both time and dose-dependent, and correlate with increased tartrate-resistant acid phosphatase (TRAP) levels in the gian cells.
  • TRIP tartrate-resistant acid phosphatase
  • this invention presents a method for detecting increases in bone resorption indicative of any of a number of disease states referred to herein as "osteoporosis” or “osteoporotic diseases”, which are characterized by a breakdown of the homeostatic balance of bone resorption and bone generation and are therefore characterized by levels of 47 J in bodily fluids, such as blood, serum and crevicular fluids, that are increased over the general level found in homeostasis.
  • This increase in 47J indicates an imbalance wherein bone resorption outpaces bone growth, resulting in a decrease in bone mass and destruction of the supporting structures within bones.
  • monoclonal antibody 47J or any other antibody raised against an epitope of antigenic glycoprotein 47J can be used to monitor bone reso ⁇ tion processes and detect loss of homeostasis as indicated by an increase in bone resorption over the relatively constant level associated with homeostasis.
  • One at risk for osteoporotic disease in any of its varied forms can detect an increase in the bodily production of the 47J antigen by conducting periodic quantitative immunoassays of bodily fluids, such as blood, serum, or intercrevicular fluid, utilizing a labeled antibody that binds to 47J.
  • Antibodies against 47J can be utilized in any of the well known immunoassay formats in which the antibody is attached to a solid support or substrate and fluids suspected of containing the antigenic glycoprotein or a fragment thereof reactive with the particular antibody or antibody fragment used in the immunoassay is contacted with the antibody on the support.
  • osteoporositic diseases include Cushing's syndrome, certain heritabl diseases of connective tissue, such as forms of osteogenesis imperfecta periodontal gum disease, idiopathic osteoporosis, type I osteoporosis, and typ II osteoporosis.
  • the antigen was immunopurified from isolated populations of white leghor chick osteoclasts using monoclonal antibody 47J in a method well known i the art (M.J. Oursler, et al., J. Cell. Biochem., 46:219-233, 1991) as furthe described in the Examples herein.
  • Monoclonal antibody 47J is on deposi under ATCC # . This deposit was made under the provisions of th
  • hybridomas secreting monoclonal antibodies The general method used for production of hybridomas secreting monoclonal antibodies is well known (Kohler a ⁇ d Milstein, Nature, 256:495, 1975).
  • the present invention is directed to monoclonal antibodies, and hybridomas which produce them, which are reactive with 47J.
  • the isolation of hybridomas secreting monoclonal antibodies with the reactivity of the monoclonal antibodies of the invention can be accomplished using known screening techniques to determine the elementary reaction pattern of the monoclonal antibody of interest (See Oursler, J. Cell Biol., 100:1592-1600,
  • a monoclona antibody it is also possible to evaluate, without undue experimentation, a monoclona antibody to determine whether it has the same specificity as a monoclona antibody of the invention by determining whether the monoclonal antibod being tested prevents a monoclonal antibody of the invention from binding 1 to the 47J antigen, or 2) an osteoclast expressing the 47J antigen with whic the monoclonal antibody of the invention is normally reactive. If th monoclonal antibody being tested competes with the monoclonal antibody o the invention, as shown by a decrease in binding by the monoclonal antibod of the invention, then it is likely that the two monoclonal antibodies bind to th same, or a closely related, epitope.
  • Still another way to determine whether a monoclonal antibody has th specificity of a monoclonal antibody of the invention is to pre-incubate th monoclonal antibody of the invention with the 47J antigen with which it is normally reactive, and determine if the monoclonal antibody being tested is inhibited in its ability to bind the antigen. If the monoclonal antibody being tested is inhibited then, in all likelihood, it has the same, or a closely related, epitopic specificity as the monoclonal antibody of the invention.
  • the monoclonal antibodies of the invention can be used in immunoaffinity chromatography for the purification of the 47J glycoprotein, or immunoreactive fragments thereof.
  • immunoaffinity chromatography One way by which such immunoaffinity chromatography can be utilized is through the use of, for example, the binding of the monoclonal antibodies of the invention to CNBr-Sepharose-4B, Affigel (BioRad), or Tresyl-activated Sepharose (Pharmacia).
  • These solid phase- bound monoclonal antibodies can then be used to specifically bind 47J from mixtures of other proteins to enable its isolation and purification.
  • the bound 47J antigen can be eluted from the affinity chromatographic material using techniques known to those of ordinary skill in the art such as, for example, chaotropic agents, low pH, or urea.
  • Chimeric antibodies are antibodies in which th various domains of the antibodies' heavy and light chains are coded for b DNA from more than one species.
  • a chimeric antibody will compris the variable domains of the heavy (V H ) and light (V L ) chains derived from th donor species producing the antibody of desired antigenic specificity, and th constant domains of the heavy (C H ) and light (C L ) chains derived from the hos recipient species. It is believed that by reducing the exposure of the hos immune system to the antigenic determinants of the donor antibody domains especially those in the C H region, the possibility of an adverse immunoiogica response occurring in the recipient species will be reduced.
  • a chimeric antibody for in vivo clinical us in humans which comprises mouse V H and V L domains coded for by DN isolated from a hybridoma of the invention, such as the hybridoma secretin Mab 47J antibody (ATCC # ), and C H and C L domains coded fo with DNA isolated from a human leukocyte.
  • the present invention encompasses all monoclonal antibodies whic recognize the novel osteoclast antigen 47J, especially preferred ar monoclonal antibodies of human origin.
  • monoclonal antibodies of one isotype might b more preferable than those of another in terms of their diagnostic o therapeutic efficacy.
  • unmodified mouse monoclonal antibodies of isotyp gamma-2a and gamma-3 are generally more effective in lysing target cell than are antibodies of the gamma-1 isotype.
  • This differential efficacy i thought to be due to the ability of the gamma-2a and gamma-3 isotypes t more actively participate in the cytolytic destruction of target cells.
  • Particula isotypes of a monoclonal antibody can be prepared either directly, by selecting from the initial fusion, or prepared secondarily, from a parental hybridoma secreting a monoclonal antibody of different isotype by using the sib selection technique to isolate class-switch variants (Steplewski, et al., Proceedings of the National Academy of Science, U.S.A. , 82:8653, 1985; Spira, et al., Journal of Immunological Methods, 74:307, 1984).
  • the monoclonal antibodies of the invention would include class-switch variants having specificity for an epitope on 47 J.
  • the isolation of other hybridomas secreting monoclonal antibodies with the specificity of the monoclonal antibody 47J can also be accomplished by one of ordinary skill in the art by producing anti-idiotypict antibodies (Herlyn, et al., Science, 232:100, 1986).
  • An anti-idiotypic antibody is an antibody which recognizes unique determinants present on the monoclonal antibody produced by the hybridoma of interest. These determinants are located in the hypervariable region of the antibody. It is this region which binds to a given epitope and, thus, it is responsible for the specificity of the antibody.
  • the anti- idiotypic antibody can be prepared by immunizing an animal with the monoclonal antibody of interest.
  • the animal immunized will recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants.
  • anti-idiotypic antibodies of the second animal which are specific for the monoclonal antibodies produced by a single hybridoma which was used to immunize the second animal, it is now possible to identify other clones with the same idiotype as the antibody of the hybridoma used for immunization.
  • an anti-idiotypic monoclona antibody made to a first monoclonal antibody will have a binding domain in th hypervariable region which is the "image" of the epitope bound by the firs monoclonal antibody.
  • the anti-idiotypic monoclona antibody could be used for immunization since the anti-idiotype monoclona antibody binding domain effectively acts as an antigen.
  • any isotype can be used since amelior ation of the osteoporotic disease in these situations is not dependent upo complement-mediated cytolytic destruction of those osteoclasts bearing th 47J antigen.
  • the monoclonal antibodies of the invention can be used in any animal in whic it is desirable to administer in vitro or in vivo immunodiagnosis o immunotherapy.
  • the term "animal” as used herein is meant to include bot humans as well as non-humans.
  • antibody as used in this invention is meant to include intac molecules as well as fragments thereof, such as for example, Fab and F(ab') 2 , which are capable of binding the epitopic determinant.
  • the monoclonal antibodies of the invention are suited for use, for example, in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier.
  • the monoclonal antibodies in these immunoassays can be detectably labeled in various ways.
  • types of immunoassays which can utilize monoclonal antibodies of the invention are competitive and non-competitive immunoassays in either a direct or indirect format.
  • Examples of such immunoassays are the radioimmunoassay (RIA) and the sandwich (immunometric) assay.
  • Detection of the antigens using the monoclonal antibodies of the invention can be done utilizing immunoassays which are run in either the forward, reverse, or simultaneous modes, including immunohistochemical assays on physiological samples. Those of skill in the art will know, or can readily discern, other immunoassay formats without undue experimentation.
  • the monoclonal antibodies of the invention can be bound to many different carriers and used to detect the presence of 47 J.
  • carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite.
  • the nature of the carrier can be either soluble or insoluble for pu ⁇ oses of the invention. Those skilled in the art will know of other suitable carriers for binding monoclonal antibodies, or will be able to ascertain such, using routine experimentation.
  • labels and methods of labeling known to those of ordinary skill in the art.
  • Examples of the types of labels which can be used in the present invention include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, and bio-luminescent compounds.
  • Those of ordinary skill in the art will know of other suitable labels for binding to the monoclonal antibody, or will be able to ascertain such, using routine experimentation.
  • the binding of these labels to the monoclonal antibody of the invention can be done using standard techniques common to those of ordinary skill in the art.
  • 47 J may be detected by the monoclonal antibodies of the invention when present in biological fluids and tissues. Any sample containing a detectable amount of 47 J can be used.
  • a sample can be a liquid such as urine, saliva, cerebrospinal fluid, blood, serum or crevicular fluids, and the like, or a solid or semi-solid such as tissues, feces, and the like, or, alternatively, a solid tissue such as those commonly used in histological diagnosis.
  • Another technique which may also result in greater sensitivity consists of coupling the antibodies to low molecular weight haptens. These haptens can then be specifically detected by means of a second reaction. For example, it is common to use such haptens as biotin, which reacts with avidin, or dinitrophenyl, pyridoxal, and fluorescein, which can react with specific anti- hapten antibodies.
  • epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • the detectably labeled monoclonal antibody is given in a dose which is diagnostically effective.
  • diagnosisically effective means that the amount of detectably labeled monoclonal antibody is administered in sufficient quantity to enable detection of the site having the 47J antigen for which the monoclonal antibodies are specific.
  • the concentration of detectably labeled monoclonal antibody which is administered should be sufficient such that the binding to those cells having 47J is detectable compared to the background. Further, it is desirable that the detectably labeled monoclonal antibody be rapidly cleared from the circulatory system in order to give the best target-to-background signal ratio.
  • the dosage of detectably labeled monoclonal antibody for in vivo diagnosis will vary depending on such factors as age, sex, and extent of disease of the individual.
  • the dosage of monoclonal antibody can vary from about 0.01 mg/m 2 to about 500 mg/m 2 , preferably 0.1 mg/m 2 to about 200 mg/m 2 , most preferably about 0.1 mg/m 2 to about 10 mg/m 2 .
  • Such dosages may vary, for example, depending on whether multiple injections are given, progression of the disease, and other factors known to those of skill in the art.
  • the type of detection instrument available is a major factor in selecting a given radioisotope.
  • the radioisotope chosen must have a type of decay which is detectable for a given type of instrument.
  • Still another important factor in selecting a radioisotope for in vivo diagnosis is that the half-life of the radioisotope be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that deleterious radiation with respect to the host is minimized.
  • a radioisotope used for in vivo imaging will lack a particle emission, but produc a large number of photons in the 140-250 keV range, which may be readil detected by conventional gamma cameras.
  • radioisotopes may be bound to immunoglobulin eithe directly or indirectly by using an intermediate functional group.
  • Intermediat functional groups which often are used to bind radioisotopes which exist a metallic ions to immunoglobulins are the bifunctional chelating agents such a diethylenetriaminepentacetic acid (DTPA) and ethylenediaminetetraacetic aci (EDTA) and similar molecules.
  • DTPA diethylenetriaminepentacetic acid
  • EDTA ethylenediaminetetraacetic aci
  • metallic ions which ca be bound to the monoclonal antibodies of the invention are 1 1 ln, 97 Ru, 67 Ga ⁇ Ga, 72 As, ⁇ 9 Zr, 90 Y, and 201 TI.
  • the monoclonal antibodies of the invention can also be labeled with paramagnetic isotope for pu ⁇ oses of in vivo diagnosis, as in magneti resonance imaging (MRI) or electron spin resonance (ESR).
  • MRI magneti resonance imaging
  • ESR electron spin resonance
  • an conventional method for visualizing diagnostic imaging can be utilized.
  • Usuall gamma and positron emitting radioisotopes are used for camera imaging an paramagnetic isotopes for MRI.
  • Elements which are particularly useful in suc techniques include 157 Gd, 55 Mn, 162 Dy, 52 Cr, and ⁇ Fe.
  • the monoclonal antibodies of the invention can be used to monitor the cours of amelioration of malignancy in an animal. Thus, by measuring the increas or decrease in the number of cells expressing 47J or changes in th concentration of 47J present in various body fluids, it would be possible t determine whether a particular therapeutic regimen aimed at ameliorating th malignancy is effective.
  • ameliorate denotes a lessening of the detrimental effect of the osteoporotic disease in the animal receiving therapy.
  • therapeutic ⁇ cally effective means that the amount of monoclonal antibody or 47J used is of sufficient quantity to ameliorate the malignancy.
  • immunologically effective amount is meant to denote that amount of 47J antigen which is necessary to induce an ameliorative immune response to the malignancy, for example, by stimulating the production of antibodies which will bind to 47J epitopes.
  • 47J can be administered parenterally by injection, rapid infusion, nasopharyngeal absorption, dermal absorption, and orally.
  • Preparations for parenteral administration include sterile or aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Carriers for occlusive dressings can be used to increase skin permeability and enhance antigen absorption.
  • Liquid dosage forms for oral administration may generally comprise a liposome solution containing the liquid dosage form.
  • Suitable forms for suspending the liposomes include emulsions, suspensions, solutions, syrups, and elixirs containing inert diluents commonly used in the art, such as purified water. Besides the inert diluents, such compositions can also include adjuvants, wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents. It is also possible for the antigenic preparations containing 47J to include a adjuvant. Adjuvants are substances that can be used to non-specifically augment a specific immune response. Normally, the adjuvant and the antigen are mixed prior to presentation to the immune system, or presented separately, but into the same site of the animal being immunized.
  • Adjuvants can be loosely divided into several groups based on their composition.
  • Thes groups include oil adjuvants (for example, Freund's Complete and Incomplete), mineral salts (for example, AIK(S0 4 ) 2 , AINa(S0 4 ) 2 , AINH 4 (S0 4 ), silica, alum, AI(OH) 3 , Ca 3 (P0 4 ) 2 , kaolin, and carbon), polynucleotides (for example, poly I and poly AU acids), and certain natural substances (for example, wax D from Mycobacterium tuberculosis, as well as substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella).
  • oil adjuvants for example, Freund's Complete and Incomplete
  • mineral salts for example, AIK(S0 4 ) 2 , AINa(S0 4 ) 2 , AINH 4 (S0 4 ), silica,
  • the physical form of the 47J antigen which is used to immunize an animal can be either aggregated or non-aggregated.
  • Aggregated 47J can be produced from non-aggregated 47J by such common techniques as, for example, treatment with glutaraldehyde or other cross-linking agents.
  • the aggregated 47J thus derived could then be used for purposes of producing a malignancy ameliorating composition effective in inducing an active immune reaction.
  • both of these forms of 47J should cause the production o antibodies to 47J.
  • these anti-47J antibodies diagnostically as, for example, in a kit to detect the presence of 47J in a specimen.
  • the 47 J antigen preparations of the invention can be used to induce th production of antibodies which will bind to epitopic determinants of 47 J.
  • particuiarly useful method in enhancing the production of antibodies to 47J is to first immunize with the 47J antigenic preparation of the invention followed by a later immunization.
  • the dosage of 47J administered to an animal will vary depending on such factors as age, condition, sex and extent of disease, if any, and other variables which can be adjusted by one of ordinary skill in the art.
  • the antigenic 47J preparations of the invention can be administered as either single or multiple dosages and can vary from about 50 mg to about 500 mg for the 47J antigen per dose, more preferably about 50 mg to about 300 mg 47J antigen per dose, most preferably about 100 mg to about 200 mg 47J antigen per dose.
  • the monoclonal antibodies of the invention can also be used, alone or in combination with effector cells or molecules (Douillard, et al., Hybridoma, 5 (Supp. 1: S139, 1986), for immunotherapy in an animal having an osteoporotic disease which triggers expression of levels of 47 J antigen increased over with epitopes reactive with the monoclonal antibodies of the invention.
  • the monoclonal antibodies of the invention may be unlabeled or labeled with a therapeutic agent. These agents can be coupled either directly or indirectly to the monoclonal antibodies of the inven ⁇ tion.
  • One example of indirect coupling is by use of a spacer moiety. These spacer moieties, in turn, can be either insoluble or soluble (Diener, et al.,
  • therapeutic agents which can be coupled to the monoclonal antibodies of the invention for immunotherapy are drugs, radioisotopes, lectins, and toxins.
  • non-proteinaceous drugs encompasses compounds which are classically referred to as drugs, for example, mitomycin C, daunorubicin, and vinblastine.
  • the proteinaceous drugs with which the monoclonal antibodies of the invention can be labeled include immunomodulators and other biological response modifiers.
  • biological response modifiers is meant to encompass substances which are involved in modifying the immune response in such manner as to enhance the destruction of the antigen bearing osteoclast for which the monoclonal antibodies of the invention are specific.
  • immune response modifiers include such compounds as lymphokines. Lymphokines include tumor necrosis factor, interleukins 1, 2, and 3, lymphotoxin, macrophage activating factor, migration inhibition factor, colony stimulating factor, and interferon.
  • Interferons with which the monoclonal antibodies of the invention can be labeled include alpha-interferon, beta- interferon, and gamma-interferon and their subtypes.
  • certain isotypes may be more preferable than others depending on such factors as osteoclast distribution as well as isotype stability and emission.
  • the distribution of the osteoporotic disease can be evaluated by the in vivo diagnostic techniques described above. Depending on the extent and location of the condition, some emitters may be preferable to others.
  • alpha and beta particle-emitting radioisotopes are preferred in immunotherapy.
  • a high energy beta emitter capable of penetrating several millimeters of tissue such as ⁇ 0 Y, may be preferable in certain situations.
  • a short range, high energy alpha emitter such as 212 Bi
  • examples of radioisotopes which can be bound to the monoclonal antibodies of the invention for therapeutic purposes are 125 l, 131 l, ⁇ 0 Y, ⁇ 7 Cu, 212 Bi, 211 At, 212 Pb, 47 Sc, 109 Pd, and 1 ⁇ Re.
  • Lectins are proteins, usually isolated from plant material, which bind to specific sugar moieties. Many lectins are also able to agglutinate cells and stimulate lymphocytes. However, ricin is a toxic lectin which has been used immunotherapeutically. This is preferably accomplished by binding the alpha- peptide chain of ricin, which is responsible for toxicity, to the antibody molecule to enable site specific delivery of the toxic effect.
  • Toxins are poisonous substances produced by plants, animals, or microorganisms that, in sufficient dose, are often lethal.
  • Diphtheria toxin is a substance produced by Corynebacterium diphtheria which can be used therapeutically. This toxin consists of an alpha and beta subunit which under proper conditions can be separated.
  • the toxic A component can be bound to an antibody and used for site specific delivery to a 47J bearing osteoclast for which the monoclonal antibodies of the invention are specific.
  • Oth therapeutic agents which can be coupled to the monoclonal antibodies of th invention are known, or can be easily ascertained, by those of ordinary ski in the art.
  • the labeled or unlabeled monoclonal antibodies of the invention can also b used in combination with therapeutic agents such as those described abov Especially preferred are therapeutic combinations comprising the monoclon antibody of the invention and immunomoduiators and other biological respons modifiers.
  • liposomes with the monoclonal antibodies of th invention in their membrane to specifically deliver the liposome to the area the osteoclast expressing 47J.
  • These liposomes can be produced such th they contain, in addition to the monoclonal antibody, such immunotherapeuti agents as those described above which would then be released at the site osteoclast giant cells (Wolff, et al., Biochemical et Biophysical Acta, 802:25
  • the dosage ranges for the administration of the monoclonal antibodies of th invention are those large enough to produce the desired effect in which th symptoms of the osteoporotic disease are ameliorated.
  • the dosage shoul not be so large as to cause adverse side effects, such as unwanted cross reactions, anaphylactic reactions, and the like.
  • the dosage will var with the age, condition, sex and extent of the disease in the patient and ca be determined by one of skill in the art.
  • the dosage can be adjusted by th individual physician in the event of any complication. Dosage can vary fro about 0.1 mg/kg to about 2000 mg/kg, preferably about 0.1 mg/kg to abo 500 mg/kg, in one or more dose administrations daily, for one or several days.
  • lower dosages comparable to those used for in vivo immunodiagnostic imaging, can be used.
  • the monoclonal antibodies of the invention can be administered parenterally by injection or by gradual perfusion over time.
  • the monoclonal antibodies of the invention can be administered intravenously, intraperitoneally, intra ⁇ muscularly, subcutaneously, intracavity, or transdermally, alone or in combination with effector cells.
  • Preparations for parenteral administration include sterile aqueous or non- aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replen- ishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • the invention also relates to a method for preparing a medicament or pharmaceutical composition comprising the 47 J antigen, or the monoclonal antibodies of the invention, the medicament being used for therapy of osteoporotic disease.
  • the antigen is used to repeatedly detec and quantitate the amount of 47 J in the fluids of subjects over a course of tim usually at spaced intervals ranging from weeks to months or even years. B comparing the amounts of 47J at spaced intervals, it is possible to determin whether bone resorption is increasing over the patient's baseline level (th amount of bone reso ⁇ tion taking place when bone reso ⁇ tion and bon generation are in a healthy balance).
  • an increase in bone abso ⁇ tion can be detected from a single assay b detecting an increase in the amount of 47 J as compared with the patient' baseline level or with the generally accepted range consistent wit homeostasis.
  • the assay is accomplished by coating a solid substrate with th antibody, incubating the test solutions, i.e. sera of subjects suspected o having an osteoporotic disease with the antibody-coated solid substrate, an then washing away unbound test solution, for example with deionized o distilled water.
  • a solution containing a developing reagent is then incubate with the solid substrate, and the amount of binding of the developing reagen to the 47J/anti-47J complex is determined, for instance by scintillation countin if the label is a radioisotope, or by spectrophotometer or spectrofluoromete if the label is a colorimetric agent. If the solid support is a microtiter place, i is preferred that the binding of the developing agent be determined by a automatic microtiter plate reader. A detailed description of the steps used i conducting such immunometric assays is found in Current Protocols i Immunology, ed. J.E. Coligan, et al., Current Protocols, 1994.
  • solid substrate and “solid support” mean an support useful in immunometric assays made from natural or synthetic materia which is insoluble in water and can be rigid or non rigid.
  • the soli substrate should not significantly affect the desired activity of the anti-lgE antibodies.
  • Preferred solid substrates include filter paper, filtering devices such as glass membranes, porous membranes and beads, test tubes or test wells made from polyethylene, polystyrene, nylon, nitrocellulose or glass microfibers. Also useful are particulate materials such as agarose, cross- linked dextran and other polysaccharides.
  • a porous membrane can be comprised of a flexible or rigid matrix made from any of a variety of filtration or chromatographic materials, including glass fibers, micro-fibers and natural or synthetic materials.
  • the porous membrane can be used alone or as a component of more elaborate devices immunological testing devices known in the art, such as the ICON® device (Hybritech Incorporated, San Diego, CA) and like devices described in U.S. Patent Nos. 4,632,901 and 4,727,019, or a dipstick device as described in U.S. Patent No. 4,366,241, all of which are incorporated herein by reference.
  • the 47J antigen can be bound directly or indirectly to the membrane or solid support.
  • the direct binding can be accomplished by any covalent or non- covalent method known in the art such as, for example, the use of glutaraldehyde and aminosilanes as well as other methods described in Immobilized Enzymes, I Chibata, Halstead Press, New York, 1978;
  • the 47J antigen be purified or isolated, for instance by recombinant preparation, for example by expression in E. coli as described in F. Schoedel, et al., Vaccine, 6:624-628, 1993.
  • Polypeptides having the functional epitope of the 47J antigen that are bound by the 47J antibody can also be attached to a solid support for use in the practice of this invention.
  • the 47J glycoprotein includes functional fragments, as long as the biological activity of the glycoprotein remains. Therefore, fragments and polypeptides containing the biological activity of 47J are included in the invention.
  • the invention also provides polynucleotides encoding 47J and functional fragments thereof. These polynucleotides include DNA, cDNA and RNA sequences. It is understood that all polynucleotides encoding all or a portion of the 47J are also included herein, as long as they encode a polypeptide of which the cleavage product has biological activity. Such polynucleotides include naturally occurring, synthetic, and intentionally manipulated polynucleotides. For example, the polynucleotide may be subjected to site- directed mutagenesis. The polynucleotide sequence also includes antisense sequences and sequences that are degenerate as a result of the genetic code.
  • nucleotide sequences There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the invention as long as the amino acid sequence of the polypeptide encoded by the nucleotide sequence is functionally unchanged.
  • the invention also provides polynucleotides which are complementary to the nucleotide sequences of the invention.
  • a "complementary" nucleotide sequence will hybridize to a specific nucleotide sequence under conditions which allow the complementary sequence to hybridize. These conditions include temperature, pH, buffer and nucleotide composition.
  • the positive and negative strands of a double-stranded DNA molecule are complementary nucleotide sequences.
  • Polynucleotides of the invention include fragments which are at least 15 bases in length, and typically 18 bases or greater, which selectively hybridize to genomic DNA which encodes 47 J or the 47J fragment of interest. Selective hybridization denotes conditions (e.g., pH, temperature, buffer) which avoid non-specific binding of a nucleotide sequence to the target DNA which is its complement.
  • DNA sequences of the invention can be obtained by several methods.
  • the DNA can be isolated using hybridization procedures which are well known in the art. These include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect shared nucleotide sequences; 2) antibody screening of expression libraries to detect shared structural features; and 3) synthesis by the polymerase chain reaction (PCR).
  • hybridization procedures which are well known in the art. These include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect shared nucleotide sequences; 2) antibody screening of expression libraries to detect shared structural features; and 3) synthesis by the polymerase chain reaction (PCR).
  • Hybridization procedures are useful for the screening of recombinant clones by using labeled mixed synthetic oligonucleotide probes where each probe is potentially the complete complement of a specific DNA sequence in the hybridization sample which includes a heterogeneous mixture of denatured double-stranded DNA.
  • hybridization is preferably performed on either single-stranded DNA or denatured double-stranded DNA.
  • Hybridization is particularly useful in the detection of cDNA clones derived from sources where an extremely low amount of mRNA sequences relating to the polypeptide of interest are present.
  • an 47J antigen-containing cDNA library for example, can be screened by injecting the various cDNAs into oocytes, allowing sufficient time for expression of the cDNA gene products to occur, and testing for the presence of the desired cDNA expression product, for example, by using antibody specific for 47J antigen or by using functional assays for 47J activity.
  • a cDNA library can be screened indirectly for 47 J antigen polypeptides having at least one epitope using antibodies specific for 47J antigen polypeptides.
  • antibodies can be either polyclonally or monoclonally derived and used to detect expression product indicative of the presence of 47J antigen cDNA.
  • Oligonucleotide probes which correspond to a part of the sequence encoding the protein in question, can be synthesized chemically. This requires that short, oligopeptide stretches of amino acid sequence must be known.
  • the DNA sequence encoding the protein can be deduced from the genetic code, however, the degeneracy of the code must be taken into account. It is possible to perform a mixed addition reaction when the sequence is degenerate. This includes a heterogeneous mixture of denatured double-stranded DNA.
  • DNA sequences encoding a polypeptide can also be obtained by: 1) isolation of double-stranded DNA sequences from the genomic DNA; 2) chemical manufacture of a DNA sequence to provide the necessary codons for the polypeptide of interest; and 3) in vitro synthesis of a double-stranded DNA sequence by reverse transcription of mRNA isolated from a eukaryotic donor cell. In the latter case, a double-stranded DNA complement of mRNA is eventually formed which is generally referred to as cDNA.
  • the isolation of genomic DNA isolates is the least common. This is especially true when it is desirable to obtain the microbial expression of mammalian polypeptides due to the presence of introns.
  • DNA sequences are frequently the method of choice when the entire sequence of amino acid residues of the desired polypeptide product is known.
  • the direct synthesis of DNA sequences is not possible and the method of choice is the synthesis of cDNA sequences.
  • the standard procedures for isolating cDNA sequences of interest is the formation of plasmid- or phage-carrying cDNA libraries which are derived from reverse transcription of mRNA, which is abundant in donor cells that have a high level of genetic expression. When used in combination with polymerase chain reaction technology, even rare expression products can be cloned.
  • the production of labeled single or double- stranded DNA or RNA probe sequences duplicating a sequence putatively present in the target cDNA may be employed in DNA/DNA hybridization procedures which are carried out on cloned copies of the cDNA which have been denatured into a single-stranded form (Jay, et al., Nucl. Acid Res., 11:2325, 1983).
  • a cDNA expression library such as lambda gt11
  • a cDNA expression library can be screened indirectly for expression of a polypeptide having at least one epitope, using antibodies specific for the polypeptide.
  • antibodies can be either polyclonally or monoclonally derived and used to detect expression product indicative of th presence of protein encoded by the cDNA.
  • DNA sequences encoding the 47J derived polypeptide of the invention can b expressed in vitro by DNA transfer into a suitable host cell.
  • "Host cells” ar cells in which a vector can be propagated and its DNA expressed.
  • the ter also includes any progeny of the subject host cell. It is understood that al progeny may not be identical to the parental cell since there may be mutation that occur during replication. However, such progeny are included when th term "host cell” is used. Methods of stable transfer, in other words when th foreign DNA is continuously maintained in the host, are known in the art.
  • the polynucleotide sequences may be inserted int a recombinant expression vector.
  • recombinant expression vector refers to a plasmid, virus or other vehicle known in the art that has bee manipulated by insertion or incorporation of the genetic sequences for 47 antigen, for example, and a carrier peptide.
  • Such expression vectors contai a promoter sequence which facilitates the efficient transcription of the inserte genetic sequence of the host.
  • the expression vector typically contains a origin of replication, a promoter, as well as specific genes which allo phenotypic selection of the transformed cells.
  • Vectors suitable for use in th present invention include, but are not limited to the T7-based expression vector for expression in bacteria (Rosenberg, et ai, Gene, 56:125, 1987), th pMSXND expression vector for expression in mammalian cells (Lee an Nathans, J. Biol. Chem., 263:3521, 1988), and baculovirus-derived vectors fo expression in insect cells.
  • the DNA segment can be present in the vecto operably linked to regulatory elements, for example, a promoter (e.g., T7 metallothionein I, or polyhedrin promoters).
  • Poly nucleotide sequences encoding the polypeptide of the invention can be expressed in either prokaryotes or eukaryotes.
  • Hosts can include microbial, yeast, insect and mammalian organisms.
  • the preferred host of the invention is a eukaryote. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art. Such vectors are used to incorporate DNA sequences of the invention. It is preferable that the host cell of the invention naturally encodes an enzyme which recognizes the cleavage site of the fusion protein.
  • the host cell in which expression of the fusion polypeptide is desired does not inherently possess an enzyme which recognizes the cleavage site, the genetic sequence encoding such enzyme can be cotransfected to the host cell along with the polynucleotide sequence for the fusion protein.
  • Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art.
  • the host is prokaryotic, such as E. coli
  • competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCI 2 method by procedures well known in the art.
  • CaCI 2 or RbCI can be used. Transformation can also be performed after forming a protoplast of the host cell or by electroporation.
  • Eukaryotic cells can also be cotransfected with DNA sequences encoding the 47J derived polypeptide of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the he ⁇ es simplex thymidine kinase gene.
  • Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein.
  • a eukaryotic viral vector such as simian virus 40 (SV40) or bovine papilloma virus
  • SV40 simian virus 40
  • bovine papilloma virus bovine papilloma virus
  • Techniques for the isolation and purification of either microbially or eukaryoti- cally expressed polypeptides of the invention may be by any conventional means such as, for example, preparative chromatographic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibodies.
  • Non-viable osteoclasts were isolated according to the method of P. Collin- Osdoby, et al., J. Bone Min. Res., 6:1353-1365, 1991, which is inco ⁇ orated herein by reference in its entirety. Briefly, White Leghorn chick hatchlings maintained on a low calcium diet (Purina) for a minimum of 28 days were used for each osteoclast preparation. The chicks were sacrificed, and the tibia and humeri were cleaned and bone marrow was flushed out of the marrow cavities with Hank's balanced salt solution (HBSS) (GIBCO, Chagrin Falls, OH).
  • HBSS Hank's balanced salt solution
  • the bones were split and incubated at 37°C with collagenase solution 0.5 mg/ml HBSS for 30 minutes.
  • Cells were released from bone pieces by several cycles of agitation and rinsing in HBSS, sequentially passed through 350 and 110 / Nitex nylon filters (Tekmar, Sawmill Dr. NY) and pelleted by centrifugation. Cell pellets were further purified by sedimentation over 35% percol gradient. These cells were either used directly for ELISA or further purified by centrifugation over 6% percol and stored as pellet at -70°C.
  • the collagenase treated bone pieces were further subjected to trypsinization (Worthington Biochemical Corp., Freehold, NJ) to obtain viable osteoclasts. Then the osteoclasts were purified by successive sedimentation over 35% and 6% percol and cells were either used directly or stored as pellet at -70°C.
  • the marrow mononuclear cells obtained by flushing of marrow cavities as described above were purified by centrifugation through Ficoll Hypaque ® (Pharmacia, Piscataway, NJ) and then cultured in alpha minimal essential media ( ⁇ -MEM) containing 10% fetal calf serum (FCS) and 1% 100X antibiotic/antimycotic (Gibco BRL, Frederick, MD).
  • ⁇ -MEM alpha minimal essential media
  • FCS fetal calf serum
  • Gibco BRL 100X antibiotic/antimycotic
  • the human leukemic cell line HL-60 was cultured and maintained in RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented with 10% heat inactivated FCS containing 1% antibiotic/antimycotic. Cells were grown in 75 cm 2 tissue culture flasks in vertical position at 37°C in a 95% air 5% C0 2 environment and passaged every 72 hours.
  • RPMI 1640 medium Gib Island, NY
  • FCS 10% antibiotic/antimycotic
  • marrow mononuclear cells were fed on day 3 and every other day thereafter with 25% UMR 106 conditioned medium (CM) 10% fetal bovine serum (Irvine Scientific), 1% glutamine, 1% nonessential amino acids, and 1% antibiotic/antimycotic solution (GIBCO)).
  • CM fetal bovine serum
  • GEBCO antibiotic/antimycotic solution
  • mononuclear cells were treated upon plating with differentiating agent phorbol- 12-myristate-13-acetate (PMA) (10 "8 M) for three days.
  • PMA phorbol- 12-myristate-13-acetate
  • PMA phorbol- 12-myristate-13-acetate
  • HL-60 cells were treated with PMA at 10 "7 M for three days. Then the cells were harvested and washed with HBSS and stored as pellet at -70°C.
  • Marrow giant cells formed in culture following induction by PMA were labelled with 35 S methionine 5//Ci/ml of methionine free medium for 16 hours, chased for one hour with CM, and the cells were collected as pellet and stored at -70 ⁇ C until analyzed.
  • osteoclast cells were extracted with CHAPS detergent solution (0.15M NaCl, 0.01 M Tris-Hcl, 0.001 M EDTA, 0.5% CHAPS (3- [(cholamidopropyl)-dimethylammonio]-1-propanesulfonate), pH 7.4, containing a protease inhibitor cocktail (0.03% soybean trypsin inhibitor, 0.1 M epsilon- amino-N-caproic acid, 0.002 M PMSF, 0.4 trypsin inhibitor units/ml aprotinin, and 10 //g/ml each of leupeptin chymostatin, antipain, and pepstatin) (Sigma Chemical Co., St.
  • ELISA reader Spent medium from induced marrow giant cells was diluted with equal volume of phosphate buffered saline (PBS), fixed with 1 :100 volume 25% aqueous glutaraldehyde and used for ELISA as above. Absorbance values were normalized for total protein, which was obtained from additional wells carried throughout the ELISA with PBS alone, using the microtiter assay based on original Lowry's method (Lowry, ef al., J. Biol. Chem., 193:215-229, 1951). For purposes of comparison, monoclonal antibody 121 F, which is specific for a 150kDA osteoclast plasma membrane-associated glycoprotein with a neutral pi of 6.95 (M.J. Oursler, ef al., J. Cell. Biochem., 46:219-233, 1991), was included in select ELISAs.
  • PBS phosphate buffered saline
  • the reaction was terminated by acidifying the mixture with 0.1 N HCl and the digestion product was applied to SDS-polyacrylamide gel described above.
  • This firmly indicated that 170, 130, 110, and 48 kDa molecules produce common fragments upon proteolysis with lysozyme C, and therefore are either proteolytic products formed during the purification procedure or are unprocessed forms of antigen present in the cells.
  • increase in concentration, or alteration in composition of protease inhibitors used throughout the purification procedure had very little effect on the presence of these protein bands.
  • the purified antigen had an apparent MW of ⁇ 250 kDa as shown in Figure 4.
  • Disulfide reduction of this component generated a major protein band of 170 kDa and relatively faint bands of 180-190 and 95 kDa.
  • Western blot analysis of subcellular fractions from osteoclasts indicated that the 170 kDa band was associated with the plasma membrane enriched fraction, while 48, and 130 kDa Mab reactive bands were detected in the cytosol and endoplasmic reticulum-rich fraction as shown in Figure 5.
  • CHAPS detergent extract of osteoclasts was subjected to centricon fractionation using membranes with 100kDa molecular weight as a cut off. ELISA of the retained and flowthrough fractions, for the possible presence of
  • 47J Mab reactive antigen was carried out as described above. Centricon fractionation analysis also indicated that 65% of the 47J Mab reactive antigen was in the retained portion ()100kDa), and 35% was in the flowthrough ((100kDa) fraction.
  • x 10 6 non-viable osteoclasts were homogenized in ice cold 10mM Tris-HCI buffer, pH 7.4, containing the protease inhibitor cocktail described in Example 4 with 20 strokes of hand driven teflon homogenizer, followed by sonication at 40% for 30 seconds in a Polytron sonicator (Brinkman.
  • the homogenate was centrifuged at 10,000 x g to remove the cell debris and the supernatant was subjected to further centrifugation in a sucrose gradient of 32- 42% in the presence of the protease inhibitor cocktail at 100,000 x g using a
  • Beckman tabletop TL100 ultracentrifuge (Beckman Instruments, Columbia, MD). The 32-38% interface enriched in plasma membrane, and 38-42% interface containing endoplasmic reticulum, and the pellet (mitochondrial and nuclear fraction) were each tested for appropriate enzyme markers and their antigen level was estimated by ELISA.
  • a membrane fraction was prepared by centrifugation of cell homogenate at 10,000 x g, for 30 minutes in 0.01 M Tris-HCI, pH 7.4, containing 0.25M sucrose and the protease inhibitor cocktail. The pellet was resuspended in PBS and either used directly for ELISA or sorted with magnetic beads coupled to 47J Mab. The membrane coupled to beads was recovered by vortexing and used for antigen purification.
  • Antigen reactive with monoclonal antibody 47J was purified from isolated non- viable osteoclasts, marrow giant cells, from PMA induced HL-60 cells, and/or from spent marrow giant cell media. Fractions eluted from the 47J Mab immunoaffinity column were analyzed for the osteoclast antigen by ELISA as described by Oursler, et al, J. Cell Biochem, 47:219-233, 1991, which is hereby incorporated herein by reference. 47J Mab was coupled to cyanogen bromide activated CL4B Sepharose (5mg 47J Mab/ml matrix).
  • the membrane fraction recovered by magnetic sorting as described above of osteoclast cells (5-7 osteoclast preps * 20 x 10 6 cells) or marrow giant cells ( x10 6 cells) was extracted with CHAPS detergent solution and mixed with the matrix overnight.
  • PMA induced HL-60 cells (24 x 10 6 cells) were extracted with CHAPS detergent solution and the extract after desalting was mixed with immunoaffinity matrix overnight.
  • Spent media, after removal of cell debris was mixed with sepharose coupled matrix.
  • Bound antigen in each case was eluted from the washed matrix using a gradient of 0-8 M urea in PBS with protease inhibitors. Fractions were dialyzed and concentrated using Amicon concentrator, and tested by ELISA for the presence of antigen.
  • Affinity purified and 125 l labelled antigen was subjected to SDS-PAGE and the band corresponding to antigen was excised from the gel and treated with or without N-glycanase (Boehringer, Mannheim, Indianapolis, IN) at 22°C for 4 hours.
  • SDS-PAGE analysis of the product was performed as described above. The gels were dried and exposed to Kodak X-Omat film at -80°C with intensifying screen.
  • ELISA of CHAPS detergent extract of osteoclasts, marrow derived giant cells, macrophages, fibroblasts and osteoblasts indicated that 47J Mab reactive antigen was expressed on osteoclasts with little or no expression on the other cell types. Moreover, the 47J antigen did not appear to be a major component of osteoclast membrane, and was far less abundant than other osteoclast specific antigens, such as the 121 F Mab specific antigen as shown in Table 1 above.
  • OC precursors avian or human marrow mononuclear cell preparation (OC precursors) were cultured with or without modulators. Expression of antigens characteristic o mature OCs was monitored using osteoclast-specific monoclonal antibodies 121F and 75B.
  • Induction of OC correlated with increased tartrate-resistant acid phosphatase (TRAP) levels in the giant cells.
  • TRIP tartrate-resistant acid phosphatase
  • a panel of cytokines was tested for induction potential and yielded dose-dependent (10 '12 - 10 "9 M) expression of OC antigens by IL-1 ⁇ , IL-16, IL-2, IL-3, IL-4, or IL-6, but not with TNF ⁇ or TNF6.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Cell Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Antigène associé aux ostéoclastes (47J) et anticorps monoclonaux, et procédé de détection et d'amélioration d'une maladie ostéoporotique. Les anticorps monoclonaux réagissent de manière spécifique avec des épitopes présents sur l'antigène 47J, lequel est spécifique aux ostéoclastes.
PCT/US1994/010700 1994-09-21 1994-09-21 Glycoproteine specifique aux osteoclastes pour le diagnostic et le traitement de l'osteoporose WO1996009067A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/US1994/010700 WO1996009067A1 (fr) 1994-09-21 1994-09-21 Glycoproteine specifique aux osteoclastes pour le diagnostic et le traitement de l'osteoporose

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1994/010700 WO1996009067A1 (fr) 1994-09-21 1994-09-21 Glycoproteine specifique aux osteoclastes pour le diagnostic et le traitement de l'osteoporose

Publications (1)

Publication Number Publication Date
WO1996009067A1 true WO1996009067A1 (fr) 1996-03-28

Family

ID=22243011

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1994/010700 WO1996009067A1 (fr) 1994-09-21 1994-09-21 Glycoproteine specifique aux osteoclastes pour le diagnostic et le traitement de l'osteoporose

Country Status (1)

Country Link
WO (1) WO1996009067A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999066910A3 (fr) * 1998-06-24 2000-06-15 Peter Dietsch Agents pour l'inhibition specifique de la resorption osteoclastique

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0630787A (ja) * 1992-07-17 1994-02-08 Maruho Kk 対破骨細胞成分親和性単クローン抗体、破骨細胞検出 剤及び骨吸収抑制剤

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0630787A (ja) * 1992-07-17 1994-02-08 Maruho Kk 対破骨細胞成分親和性単クローン抗体、破骨細胞検出 剤及び骨吸収抑制剤

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF BONE AND MINERAL RESEARCH, Volume 6, Number 10, issued 1991, TEUVO et al., "Inhibition of Bone Resorption by a Monoclonal Antibody that Reacts with a 150 kD Membrane Protein in Chicken Osteoclasts", pages 1091-1097. *
JOURNAL OF CELLULAR BIOCHEMISTRY, Volume 46, issued 1991, OURSLER et al., "Evidence for an Immunological and Functional Relationship Between Superoxide Dismutase and a High Molecular Weight Osteoclast Plasma Membrane Glycoprotein", pages 331-344. *
JOURNAL OF CELLULAR BIOCHEMISTRY, Volume 46, issued 1991, OURSLER et al., "Purification and Characterization of an Osteoclast Membrane Glycoprotein with Homology to Manganese Superoxide Dismutase", pages 219-233. *
THE JOURNAL OF CELL BIOLOGY, Volume 100, issued May 1985, OURSLER et al., "Identification of Osteoclast-Specific Monoclonal Antibodies", pages 1592-1600. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999066910A3 (fr) * 1998-06-24 2000-06-15 Peter Dietsch Agents pour l'inhibition specifique de la resorption osteoclastique

Similar Documents

Publication Publication Date Title
US5977316A (en) Monoclonal antibody 1A7 and related polypeptides
US5935821A (en) Polynucleotides related to monoclonal antibody 1A7 and use for the treatment of melanoma and small cell carcinoma
Ohara et al. Production of a monoclonal antibody to and molecular characterization of B-cell stimulatory factor-1
US6207156B1 (en) Specific antibodies and antibody fragments
AU724856B2 (en) Anti-Fas ligand antibody and assay method using the anti-Fas ligand antibody
US5977315A (en) Murine anti-idiotype antibody 3H1
Mason et al. LGL-1: a non-polymorphic antigen expressed on a major population of mouse natural killer cells.
US7517967B2 (en) Monoclonal antibody 1A7 and use for the treatment of melanoma and small cell carcinoma
EP0770089B1 (fr) Facteur 12 de croissance et de differenciation
US5637493A (en) Carcinoma associated antigen (SK1) monoclonal antibodies against SK1, methods of producing these antibodies and use therefor
JP2008194052A (ja) 単離された哺乳動物単球細胞遺伝子；関連する試薬
US6114507A (en) Anti-Fas ligand antibody and assay method using the anti-Fas ligand antibody
WO1996022373A9 (fr) Anticorps monoclonal 1a7 et son utilisation pour le traitement des melanomes et des carcinomes des petites cellules
US5480974A (en) Antibodies to human C5a receptor
EP0872488B1 (fr) ANTICORPS MONOCLONAL REAGISSANT SPECIFIQUEMENT AVEC UN LIGAND Fas ET SON PROCEDE DE PRODUCTION
CN87100927A (zh) 人类非小细胞肺癌及其它几种癌症的单克隆抗和抗原
Seon et al. Monoclonal antibody that defines a unique human T-cell leukemia antigen
WO1995012409A1 (fr) Anticorps specifiques des cellules dendritiques et leurs procedes de preparation
CA2295375A1 (fr) Fragments de liaison antigeniques designes par 4b5, qui detectent specifiquement les cellules cancereuses, nucleotides codant lesdits fragments et leur utilisation pour la prophylaxie et la detection des cancers
US5770387A (en) Antibodies to mammalian NK antigens and uses
US5024946A (en) Human monoclonal antibody to antigen of gastric cancer and B-cell line for producing this antibody, method for preparing this B-cell line and antibody, antigen and method of preparation of this antigen
JPH085918B2 (ja) モノクロ−ナル抗体及び分析
WO1996009067A1 (fr) Glycoproteine specifique aux osteoclastes pour le diagnostic et le traitement de l'osteoporose
JP3757220B2 (ja) エプスタイン−バーウィルスからの免疫反応性ペプチド
JPH09502358A (ja) 哺乳動物のチモカイン遺伝子

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA US

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载