WO2003080671A1

WO2003080671A1 - Anti-rank monoclonal antibodies and pharmaceutical composition containing the same

Info

Publication number: WO2003080671A1
Application number: PCT/KR2003/000553
Authority: WO
Inventors: Zang-Hee Lee; Hong-Hee Kim
Original assignee: Komed Co., Ltd.
Priority date: 2002-03-21
Filing date: 2003-03-21
Publication date: 2003-10-02
Also published as: KR20030076448A; AU2003210054A1

Abstract

Anti-RANK monoclonal antibodies capable of inhibiting osteoclast differentiation, hybridoma producing such antibodies, and diagnostic methods of metabolic bone diseases using these antibodies are disclosed. The present invention also relates to the pharmaceutical compositions comprising anti-RANK monoclonal antibodies for the treatment of metabolic bone diseases. The monoclonal antibodies are also useful in screening for the inhibitors of osteoclast differentiation.

Description

ANTI-RANK MONOCLONAL ANTIBODIES AND PHARMACEUTICAL COMPOSITION CONTAINING THE SAME

Technical Field

The present invention relates to an anti-receptor activator of nuclear factor KB (RANK) monoclonal antibody and a pharmaceutical composition comprising the monoclonal antibody. More particularly, the present invention relates to an anti-RANK monoclonal antibody capable of selectively recognizing RANK and inhibiting osteoclastic differentiation, a hybridoma producing the monoclonal antibody, and a pharmaceutical composition for treating bone metabolism disorders by inhibiting osteoclastic differentiation, comprising the monoclonal antibody or its fragments. Also, the present invention is concerned with a method for screening inhibitors of osteoclast differentiation with the use of the monoclonal antibody.

Background Art

Bone metabolism functions reside primarily with two cells: osteoblasts_responsible for the synthesis of bone and osteoclasts involved in cell resorption. Osteoblasts and osteoclasts differ in their cellular origins and maturation process. While osteoblasts are generated from mesenchymal cells, osteoclasts originate from mesenchyte/macrophage precursor cells. Imbalance between activities of osteoblasts and osteoclasts leads to changes of various hormones, inflammation factors, and growth factors, resulting in disorders of the skeletal system, for example, bone loss (osteoporosis) or bone resorption (osteopetrosis). Particularly, menopausal osteoporosis, bone metastatic lesion caused by the migration of tumors such as breast cancer or prostatic cancer to bone, primary bone tumor (e.g., multiple myeloma), and melanoma are accompanied by bone resorption, which is due mainly to the activation of osteoclast differentiation and the enhancement of osteoclastic functions.

Osteoclasts are known to stem from hemapoietic stem cells of monocyte/macrophage lineage and to be differentiated under the influence of various hormones, growth regulation factors, cytokines and etc., but this mechanism remains unclear. It is also known that marrow cells, peripheral blood monocytes, and spleen cells, when they are mixed in vivo, can all be differentiated to osteoclasts. Representative of agents accelerating osteoclast differentiation is lα, 25-dihydroxyvitamin D₃. According to a report, factors originating from basal cells/osteoblasts are essential to the induction of the precursor cell fusion for the formation of osteoclast-like multmucleated cells. Great recent advances in molecular biology have made it possible to identify such factors, receptor activator of nuclear factor KB being representative. Receptor activator of nuclear factor KB (RANK) is a member of the tumor necrosis factor receptor (TNFR) superfamily. TNFR is involved in cell proliferation, differentiation and apoptosis in relation to inflammation and immune responses. The TNFR superfamily comprises cell-surface receptors including type 1 and 2 TNF receptors (TNFRl and TNFR2), Fas, CD27, 4-1BB and CD30. Signaling of TNFR is triggered by ligand-receptor binding (Baker and Reddy, Oncogene, 17, 3261-3270 (1998)).

RANK, which is a newly discovered member of the TNFR superfamily, identified as a dentritic cell membrane protein, stimulates T-cell proliferation and dentritic cell functions (Anderson et al., Nature, 390,175-179(1997); Green and Flavell, J. Exp. Med., 189, 1017-1020 (1999)). The cell membrane protein is also reported to be involved in bone metabolism (Nakagawa et al, Biochem. Biophys. Res. Commun., 253, 395-400 (1998)). RANK-deficient mice are characterized by profound osteopetrosis resulting from an apparent block in osteoclast differentiation, with failure of tooth eruption, and defective lymph node formation (Li et al., Proc. Natl. Acad. Sci. USA, 97, 1566-1571(2000): Dougall et al., Genes Dev. 13, 2412-2424 (1999).

Extensive research on RANK has been reported. For example, the nucleic acid sequences and polypeptide sequence of human RANK are disclosed in U. S. Pat. No. 6,271,349, and antisense sequences which inhibit RANK expression are in U. S. Pat. No. 6,171,860. An anti-RANK polyclonal antibody, acting as an agonist accelerative of osteoclast differentiation, is reported (Nakagawa N. et al., Biochem Biophys. Res. Commun.,

253, 395-400 (1988)). However, no research has been directed forward monoclonal antibodies acting as an antagonist inhibitive of osteoclast differentiation, thus far.

Disclosure of the Invention

Leading to the present invention, the intensive and thorough research on differentiation processes of osteoclasts, conducted by the present inventors, resulted in the production of monoclonal antibodies against RANK, which plays an important role in osteoclastic differentiation.

In accordance with an aspect of the present invention, there is provided an anti- RANK monoclonal antibody or its fragments, capable of selectively recognizing RANK and inhibiting osteoclastic differentiation.

In accordance with another aspect of the present invention, there is provided a method for producing an anti-RANK monoclonal antibody. In accordance with a further aspect of the present invention, there is provided a hybridoma, producing the anti-RANK monoclonal antibody.

In accordance with still a further aspect of the present invention, there is provided a kit for diagnosing bone metabolism disorders, comprising the monoclonal antibody or its fragments.

In accordance with still another aspect of the present invention, there is provided a method for diagnosing bone metabolism disorders, comprising the steps of: bringing a biological sample of an animal into contact with the antibody or its fragments; and (b) measuring the RANK expression of the biological sample and comparing with the RANK expression of a control obtained from a healthy animal.

In accordance with yet another aspect of the present invention, there is provided a pharmaceutical composition for treating bone metabolism disorders by inhibiting osteoclastic differentiation, comprising the monoclonal antibody or its fragments in a therapeutically or prophylactically effective amount, and a pharmaceutically acceptable carrier. In accordance with yet still another aspect of the present invention, there is provided a method for screening inhibitors of osteoclast differentiation, comprising the steps of: treating an osteoclast differentiation model with a candidate inhibitor; and (b) bringing the osteoclast differentiation model into contact with the antibody or its fragments and quantitatively analyzing RANK expression to determine whether the candidate inhibitor is an inhibitor of osteoclast differentiation as defined by a decrease in RANK expression.

Brief Description of the Drawings

Fig. 1 is a schematic diagram showing the construction of the recombinant vector pGEX4T-l:RANK.

Fig. 2 shows an electrophoresis result of the recombinant GST-RANK fusion protein.

Fig. 3 shows immunofluorescence assay results demonstrating that the monoclonal antibody of the present invention specifically recognizes the RANK expressed in pig vascular endothelial cells (A), HUVEC(human umbilical vein endothelial cell) (B), differentiated osteoclasts (C), and differentiated RAW264.7 cells (D).

Fig. 4 shows Western blotting results demonstrating that the monoclonal antibody of the present invention specifically recognizes the RANK expressed in human embryonic kidney 293T (HEK293T) cells.

Fig. 5 shows FACS analysis results demonstrating that the monoclonal antibody of the present invention specifically recognizes the RANK expressed in osteoclast-like cells.

Figs. 6A-6C are histograms showing the quantitative analysis for the inhibition of osteoclastic differentiation by use of TRAP dyeing method. Fig. 7 is a schematic diagram illustrating the osteoclastic differentiation processes and a method for screening inhibitors of osteoclastic differentiation by use of the monoclonal antibody of the present invention, with resort to HTS (high throughput screening).

Best Mode for Carrying Out the Invention

In this invention, an anti-RANK monoclonal antibody and its fragments with activity to recognize RANK selectively and inhibit the differentiation of osteoclasts are disclosed.

Monoclonal antibodies are proteins which can recognize specific sites of an antigen and bind specifically to the sites. Monoclonal antibodies do not inhibit the activity of antigens without binding specifically to specific sites of antigens. Thus, not all monoclonal antibodies produced against an antigen inhibit the activity of the antigen. Although it is possible to produce monoclonal antibodies capable of recognizing RANK involved in signal transduction, the variety of the antigenic sites of RANK makes it difficult to make monoclonal antibodies which selectively inhibit a desired physiological activity. No monoclonal antibody has been found which acts as an antagonist which inhibits the differentiation of osteoclasts.

The term "antibody fragments" as used in the present invention refers to specific parts of an antibody, which are able to bind to a target antigen, e. g. RANK or its specific sites in this invention. For instance, the antibody fragments comprise fragments F(ab')2, Fab, Fab' and Fv. Generally, these fragments can be produced with resort to DNA recombinant technology or by digesting the antibody protein with papain or pepsin (CURRENT PROTOCOLS IN IMMUNOLOGY, John Wiley and Sons Coliganet et al., Eds (1991-1992)).

The monoclonal antibody fabricated according to the present invention is proved capable of selectively recognizing RANK expressed in tissues and cells as measured by immunofluorescence assay, Western blotting, and fluorescence-activated cell sorter (FACS) (Example 6 to 8). Also, research on the effect of the monoclonal antibody of the present invention on an osteoclast differentiation system has been done. Relative numbers of differentiating osteoclasts are much lower when the system is treated with the monoclonal antibody than when not treated (Example 9).

The TNFR superfamily member, RANK, is expressed in epithelial cells, osteoclast precursors, B-cells, and activated T-cells. Having 20-40 % homology in amino acid sequence with other TNFR superfamily members, RANK is a 616-amino acid protein of which the extracellular domain comprises cysteine residues (Anderson et al., Nature 390, 175-179 (1997)).

Activated by association with the ligand RANKL (also named ODF (osteoclast differentiation factor), OPGL (osteoprotegerin ligand), or TRNACE (TNF-related activation induced cytokine)), RANK induces the differentiation of hemopoietic stem cells into osteoclasts, thereby participating in bone metabolism. In addition, RANK is involved in the control of the immune system as demonstrated in the mixed lymphocyte reaction in which RANK is found to enhance allo-stimulatory activity of dentritic cells and give the co- stimulation necessary for the activation of T-helper cells in the absence of CD40.

The signal transduction pathway of RANK is so complicated that its full processes have not yet been discovered completely. According to literature, the association of RANK with its ligand initiates the signal transduction pathway. The cytoplasmic domain of the RANK-ligand composite recruits TRAF proteins, especially TRAF6, and adaptors such as c- src, and then the TRAF6 activates NF-κB and JNK(c-Jun N-terminal kinase) which are essential to the formation and activation of osteoclasts (Darnay et al., JBC 273, 20551-20555 (1998)). Additionally, TRAF6 interacts with c-src to activate Akt/PKB, a serine/threonine kinase which plays a crucial role in anti-apoptosis signal transduction and cytoskeletal system rearragement (Wong et al., Mol. Cell 4,1041-1049 (1999)). This coincides with the research results obtained with mice deficient in NF-κBl/NF-κB2, c-fos, TRAF6 and c-src in which osteoclasts are destroyed or functionally retarded (Iotsova et al., Nat Med, 3, 1285-1289 (1998); Grigoriadis et al., Science 266, 443-448 (1994); Lomaga et al., Gene Dev 13, 1015- 1024 (1999); Soriano et al, Cell 64, 693-702 (1991)).

The monoclonal antibody of the present invention can be produced by using RANK as an immunogen to immunize animals, fusing splenocytes of the immunized animals with myeloma cells to produce hybridomas, screening the hybridomas to identify those which produce anti-RANK monoclonal antibodies able to selectively recognize RANK and simultaneously inhibit osteoclast differentiation, culturing the screened hybridoma, and isolating the antibodies from the hybridoma culture.

Also, the monoclonal antibody of the present invention can be further produced by injecting into animals the hybridoma which selectively recognize RANK and inhibit osteoclast differentiation simultaneously, recovering abdominal dropsy from the animals after a period of time, and isolating the antibodies from the abdominal dropsy. Available as a useful immunogen is a natural RANK protein or a gene-recombinant

RANK protein. Preferably, gene-recombinant RANK is used. More preferable is a recombinant fusion protein in which RANK is fused with GST (glutathione S-transferase). The most preferable immunogen is a fusion protein in which the extracellular domain (amino acid Nos. 37-206) of RANK is fused with GST. In this invention, a 45 kD RANK-GST fusion protein is used (Examples 1 to 3).

Gene-recombinant RANK can be obtained by preparing cDNA by use of a known base sequence in accordance with a protocol well-known to the art, inserting the cDNA into an expression vector, expressing the vector in a host cell, and isolating the desired protein.

Various vectors for eucaryotic or prokaryotic cell hosts may be used as expression vectors. For use in eucaryotic cell hosts, expression control sequences may be adopted from SV40, bovine papilloma virus, adenovirus, adeno-associated virus, cytomegalovirus, or retrovirus. In the case of prokaryotic cell hosts, bacterial plasmids such as pET, pRSET, pBluescript, pGEX2T, pUC, col El, pCRl, pBR322, pMB9 and their derivatives, phage DNA exemplified by phage lamda derivatives such as λgtlO, λgtl l and NM989, and other DNA phage such as Ml 3 and filament single strand DNA phage may be used. In the present invention, a recombinant expression vector, named pGEX4T-l:RANK, is prepared.

Host cells useful for the production of the recombinant RANK may be prokaryotic or eucaryotic. Actually, hosts with high efficiency in transformation and expression are selected. Effective prokaryotic and eukaryotic hosts may be exemplified by E. coli, pseudomonas, bacilli, streptomyces, and fungi, with preference to E. coli.

Proteins expressed in the hosts can be isolated and purified from cultures by use of various conventional methods, for example, normal or reverse phase liquid chromatography such as HPLC and FPLC; affinity chromatography (inorganic ligands or monoclonal antibodies); size exclusion chromatography; immobilized metal chelate chromatography; and gel electrophoresis. Anyone who is skilled in the art can select suitable isolation and purification techniques without departing the scope of the present invention. Preferable is affinity chromatography.

In order to obtain anti-RANK monoclonal antibodies capable of not only selectively recognizing RANK but inhibiting osteoclast differentiation, animals are immunized with the immunogen illustrated above. Suitable for the immunization of the present invention are mice and rats. According to conventional immunization techniques, the antigen may be administered mfraabdominally, intravenously, intramuscularly, intraretinally, or subcutaneously. If necessary, various techniques may be adopted for boosting the immune response generated by proteins and providing greater antibody reactivity. For instance, the antigenic protein of the present invention may be used in combination with complete or incomplete Freund adjuvant to increase the immunity. The immunization regimen, though not especially limited, is performed by administering the antigen 2 to 10 times and preferably 2 to 5 times at regular intervals over days to weeks and preferably one to three weeks. Two to five days after the final round of immunization, antibody-producing cells can be obtained from the immunized animals. Examples of antibody-producing cells comprise splenocytes, lymphocytes, thymocytes, and peripheral blood cells with preference to splenocytes. When mice are used, the antigen is administered at a dose of 0.0-1,000 μg and preferably 1-300 μg per mouse.

The antibody-producing cells obtained above are fused with myeloma cells in accordance with a known technique, e.g., Koehler-Milstein method. Myeloma cells suitable to this end are mouse cell line strains, exemplified by p3/x63-Ag8, p3-Ul, NS-1,MPC-11, SP-2/0, FO, P3x63Ag8, V653 and S194. The rat cell line R-210 may be also used. The fused clones are cultured, followed by selecting ones which selectively recognize RANK.

The selection of the mono-clones selectively recognizing RANK may resort to immunochemistry, as well known in the art. Examples of the immunochemical techniques include radio immuno assay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence, Western blotting, and fluorescence-activated cell sorter (FACS), to which the present invention is not limited. Preferable is ELISA. To the antigenic RANK- GST fusion protein, the cultured clones are added as a primary antibody, followed by the addition of alkaline phosphatase-conjugated goat anti-mouse IgG as a secondary antibody. Measurement is made of absorbance to select mono clones which recognize RANK.

From among the hybridomas producing the RANK-recognizable antibodies are selected the clones which show the activity of inhibiting osteoclast differentiation. This selection can be achieved by treating a model system for osteoclast differentiation with the RANK-recognizing hybridoma culture and quantifying the expression of RANK to detect hybridomas which reduce the expression of RANK.

In the model system for osteoclast differentiation, potentially differentiable cells are induced to differentiate to osteoclasts by treatment with differentiation factors. Myelocytes, peripheral blood monocytes, and splenocytes are the cells which are potentially differentiated to osteoclasts. All of them can be differentiated to osteoclast in vitro. As described above, RANKL (also called ODF) is useful as a differentiation factor. In one specific embodiment of the present invention, mouse osteoclast or RAW264.7 cells are cultured in the presence of

ODF to induce the differentiation of osteoclasts. Since hemoto stem cells of bone marrow are differentiated to osteoclasts with the help of osteoblast/basal cells, co-culture systems, e. g., myelocyte/osteoblast, and splenocytes/osteoblast may be used as the osteoclast differentiation model system in accordance with a specific embodiment of the present invention.

The hybridoma of the present invention, which shows dual functions of selectively recognizing RANK and inhibiting osteoclast differentiation, is deposited with KCTC (Korean Collection for Type Cultures) under accession number KCTC 10192BP on Feb. 27, 2002.

The hybridoma can be subcultured according to conventional methods and cryopreserved. The hybridoma can be cultured by conventional method to recover the culture or injected into the abdominal cavity to recover ascite. Antibodies contained in the culture or the ascite can be purified by use of conventional purification techniques such as dialysis, ion exchange and gel filtration chromatography, affinity column chromatography, etc. Preferably, protein A sepharose affinity chromatography may be used for the purification of the antibodies from the culture or abdominal dropsy.

In accordance with another aspect, the present invention pertains to a kit for diagnosing bone metabolism disorders by taking advantage of the anti-RANK monoclonal antibody or its fragments. The diagnosis kit of the present invention comprises the monoclonal antibody against RANK or its fragment in addition to reagents useful for lmmuno-assay.

As to the immuno-assay reagents, they are exemplified by a suitable carrier, a marker generating a detectable signal, a solubihzer, and a detergent. In the case that the marker is an enzyme, a substrate with which the activity of the enzyme can be measured, and a reaction quencher may be further used.

Illustrative and non-limitative examples of the carrier include soluble carriers such as physiologically acceptable buffers well known to the art (e. g. PBS), and insoluble carriers such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluorine resin, crosslinked dextran, polysaccharide, magnetic particles (metal-coated latex), paper, glass, metals, agarose and combinations thereof.

Enzymes, fluorescent materials, luminescent materials, and radioactive materials can be used as the markers which can generate detectable signals. Peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose oxidase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, and invertase fall into the range of the markers useful in the present invention. Fluoric acid isothiocyanate and picoviliprotein are useful as the fluorescent materials. Isolucinol or lucigenin is an example of the luminescent materials. Being radioactive, I₁₃₁, C₁₄ and H₃ also can be used as markers. However, any material, if it be applied to immuno-assay, can be used, instead of the above-illustrated materials.

The term "bone metabolism disorders" as used in the present invention means disorders caused by an increase in osteoclast differentiation and an enhancement of osteoclast function on the basis of an increase in RANK expression. For instance, bone metastatic lesion caused by the migration of tumors such as breast cancer cells or prostatic carcinoma cells to bone, primary bone tumor (e.g., multiple myeloma), and melanoma fall into the category of the bone metabolism disorders. h a further embodiment, the present invention pertains to a method for diagnosing bone metabolism disorders, comprising the steps of (a) bringing a biological specimen of an animal into contact with the antibody of the present invention or its fragments and (b) measuring the RANK expression of the specimen and comparing with the RANK expression of a control obtained from a healthy animal.

Tissues, cells, whole blood, phlegm, serum, plasma, saliva, cerebrospinal fluid, and urine may be the biological specimen.

The bone metabolism disorders mentioned in the diagnosis method are as defined above. Quantification of the RANK expression by use of the antibody of the present invention or its fragments may resort to immunochemical methods well known in this art.

Illustrative and non-limiting examples include RIA, ELISA, immunofluorescence, Western blotting, and FACS.

In the present invention, an immunofluorescence method is conducted (Example 6), in which vascular endothelial tissues, vascular endothelial cells and osteoclasts are brought into contact with a monoclonal antibody conjugated with a fluorescent marker and then observed under a fluorescent microscope to detect RANK expression.

Also, Western blotting is conducted to specifically detect RANK proteins expressed in cells (Example 7), in which cells transformed with a vector are cultured to overexpress RANK and treated with monoclonal antibodies of the present invention. With the aid of FACS, the detection of the RANK protein expressed in cells can be achieved (Example 8), in which osteoclasts are treated with monoclonal antibody fragments labeled with a fluorescent marker and fluorescent peaks are observed.

In still another aspect, the present invention pertains to a pharmaceutical composition for treating bone metabolism disorders, which is characterized by the activity of inhibiting osteoclast differentiation, comprising the monoclonal antibody of the present invention or its fragments in a therapeutically or prophylactically effective amount, and a pharmaceutically acceptable carrier. The bone metabolism disorders mentioned are as defined above. Carriers used in the pharmaceutical composition of the present invention comprise pharmaceutically acceptable carriers, adjuvant, and vehicles, which are all generically called "pharmaceutically acceptable carriers". Concrete examples of the pharmaceutically acceptable carriers suitable for the pharmaceutical composition, to which the scope of the present invention is not limited, include alumina, aluminum stearate, recitin, serum proteins (e. g., human serum albumin), buffers (various phosphates, glycin, sorbic acid, potassium sorbate, partial glyceride mixture of saturated vegetable fatty acid), water, salts or electrolytes (e. g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salts), viscous silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substrates, polyethylene glycol, sodium carboxymethyl cellulose, polyarylate, wax, polyethylene-polyoxypropylene-block polymer, polyethylene glycol, and lanolin.

Administration for the pharmaceutical composition of the present invention may be conducted locally or systematically via oral and parenteral routes, of which the illustrative but non-limiting examples include intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transcutaneous, hypodermic, intraabdominal, intranasal, intraintestinal, sublingual, and intrarectal administration.

The term "therapeutically effective dose" means an amount suitable to treat the disorders, ranging from 50 to 500 mg per kg of patient per day.

The term "prophylactically effective dose" means an amount suitable for the prevention of the disorders, ranging from about 1 to 50 mg per kg of the patient per day. In a still further aspect, the present invention pertains to a method for screening inhibitors of osteoclast differentiation, comprising the steps of (a) treating an osteoclast differentiation model with inhibitor candidates of osteoclast differentiation and (b) bringing the osteoclast differentiation model into contact with an antibody of the present invention or its fragment and quantifying RANK expression to determine whether the candidates act as inhibitors.

In a preferable version, there is provided a method for screening inhibitors of osteoclast differentiation, comprising the steps of (a) treating an osteoclast differentiation model with a candidate with resort to HTS (high throughput screening) and (b) bringing the osteoclast differentiation model into contact with an antibody of the present invention or its fragment and quantitatively analyzing RANK expression to determine the candidate as an inhibitor of osteoclast differentiation in the case of a decrease in RANK expression.

As described above, the osteoclast differentiation model used for screening inhibitors of osteoclast differentiation is a differentiation induction system. Cells differentiable to osteoclasts comprise myelocytes, mononucleates of peripheral blood, and splenocytes. They can all be differentiated to osteoclasts in vitro. RANKL may be used as a differentiating factor, as explained above, hi a specific embodiment, mouse osteoclast or RAW264.7 cells are cultured in the presence of ODF to induce the differentiation to osteoclasts. Since hematopoietic stem cells of bone marrow are differentiated to osteoclasts with the help of osteoblast/basal cells, a co-culture system of myelocytes and osteoblasts may be used as the osteoclast differentiation model system in accordance with a specific embodiment of the present invention. Quantitative analysis of the RANK expression using the monoclonal antibody of the present invention or its fragment may resort to immunochemical techniques, as described above.

HTS is a potential method by which a number of candidate materials can be tested concurrently and screened for their biological activities simultaneously or almost simultaneously. According to one embodiment of the screening method using HTS, the differentiation of osteoclasts is induced in 48-, 96- or 192-well microtiter plates to which a number of candidate inhibitors are then added, followed by bringing the cells into contact with the antibody of the present invention or its fragment to measure the expression of RANK (Fig. 7). For example, as many as 96 independent experiments can be simultaneously conducted on a single plastic plate of 8x12 cm size comprising 96 wells. Typically, each well requires an assay volume ranging from 50 to 500 μl. In addition to the plate, various equipments such as meters, tools, pipettes, robots, plate washers, plate readers, which are all commercially available, are needed to suit 96-well formats to a wide homogeneous and nonhomogeneous assay.

Having generally described this invention, a further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified. EXAMPLE: Preparation of Antigen 1-1 Construction of Recombinant Vector

A RANK-GST fusion protein in which a recombinant RANK was fused with GST (glutathione-S-transferase) was prepared as an antigen for producing anti-RANK monoclonal antibodies.

First, RANK'S extracellular domain (amino acid residue No. 37 to No. 206) was amplified by PCR using a set of the following primers in order to prepare cDNA of RANK. In this regard, 1.5 mM MgCl₂, 20 pmol of each primer, 0.2 mM of each of dATP, dGTP and dTTP, 5 μM of dCTP, 1 μCi of [α-³²P]dCTP (3000 Ci/mmol; DuPontNew England Nuclear, Boston, MA), 50 ng of sample DNA, lx PCR buffer and 1.25 units of Taq DNA polymerase (Life Technologies, Inc., Grand Island, NY) were mixed together. The mixture was first heated at 95 °C for 15 min and subjected to PCR in which a PCR cycle consisting of a denaturation at 94 °C for 1 in, an annealing step at 55 °C for 1 min and an extension step at

72 °C for 10 min was conducted 30 times, followed by extension at 72 °C for an additional 1 min in the final round. As a result, about 500 bp cDNA was produced.

Primer 1: TR8-GF:5'-CGGATCCGAGAAGCATTATGAGCAT-3'(sense) (Seq. 1) Primer 2: TR8-GR: 5 '-CGAATTCTTCATTTGGTGGTTTTCT-3 "(antisense) (Seq.2)

After being digested with BamEλ and EcøRl, the amplified cDNA of RANK was subcloned into pGΕX4T-l (Pharmacia) at a site digested with the same enzymes to construct the recombinant vector pGEX4T-l:RANK (Fig. 1).

1-2 Expression and Isolation of RANK-GST Fusion Protein

E. coli DH5α was transformed with the recombinant vector pGEX4T-l:RANK and cultured in an LB medium (tryptone lOg/L, yeast extract 5g/L, NaCl 5g/L) for 16 hours at 37 °C. Cells in dense suspension were diluted with a fresh LB medium in the ratio 20:1 and cultured for 2 hours at 37 °C. In order to induce the expression of the RANK-GST fusion protein, isopropyl-1-β-D-thiogalactopyranoside (IPTG) was added in a concentration of 0.1 mM to the culture, after which incubation was performed for 2 hours at 37 °C to express the fusion protein of interest. Cells, after being withdrawn from the culture, were suspended in a dissolving buffer (50 mM Tris-Cl, 100 mM NaCl, 0.1 % Triton X-100, 0.01 % 2- mercaptomethanol, 0.5 mM EDTA, 0.2 mM PMSF) and disrupted by sonication. The cell homogenate thus obtained was centrifuged at 12,000 rpm at 4 °C for 30 min, and the supernatant was decanted and mixed with Sepharose 4B beads (Pharmacia) for 2 hours at 4 °C to adsorb the fusion protein to the beads. Re-centrifugation at 12,000 rpm for 30 min at 4 °C was performed to isolate the beads. These beads were filled in a column through which an eluent (50 mM Tris-Cl, 50 mM glutathione, pH 7.0) was then passed to elute the RANK-GST fusion protein only.

1-3 Identification and Quantification of RANK-GST Fusion Protein

The eluate obtained in Example 1-2 was reacted in a protein quantification system (Bio-Rad), and the amount of the fusion protein was determined by measuring absorbance at

750 nm by use of an automatic enzyme-linked immunoabsorbent assay reader (BIO-TEK).

Quantitative analysis of the size of the RANK-GST fusion protein was achieved by electrophoresis, with resort to a vertical electrophoresis apparatus (Bio-Rad). After being loaded on an SDS-polyacrylamide gel (12 %), the eluate was electrophoresed at 80 V. For comparison, a low molecular weight calibration kit (Pharmacia), which served as a marker, was also run on the same gel. After electrophoresis, the gel was dyed with Coomassie brilliant blue R-250 to visualize the proteins run on the gel.

The RANK-GST fusion protein was determined to be about 45 kD in size as shown in Fig. 2.

EXAMPLE 2: Immunization of Mouse Using RANK-GST Fusion Protein

10 μg of the RANK-GST fusion protein prepared in Example 1 was well mixed with the same volume of complete Freund's adjuvant (GIBCO BRL). The resulting suspension was injected in a dose of 0.2 ml into the abdominal cavity of Balb/C mice (6 weeks aged, male). After the primary injection, booster vaccination was conducted two times at intervals of 2 weeks. For the booster vaccinations, a suspension prepared by mixing the fusion protein with the same volume of incomplete Freund's adjuvant (GIBCO BRL) was mfraabdominally injected at a dose of 0.2 ml. Following the third injection, blood samples were taken from mice by tail vein puncture. Sera obtained form the blood samples were measured for antibody titer, with resort to ELISA using the RANK-GST fusion protein as an antigen. First, the RANK-GST fusion protein was added in an amount of 100 μl to each well of 96-well plates and incubated for 1 hour at room temperature with agitation. After washing the wells with phosphate buffer three times, 200 μl of bovine serum albumin was added to give a final concentration of 1 mg/ml. Washing with phosphate buffer three times made the plates suitable for ELISA. The sera taken from immunized mice were added to each well of the plates, incubated for 1 hour at room temperature with agitation, and washed with phosphate buffer three times. 5000-fold dilutions of alkaline phosphatase- conjugated goat anti-mouse IgG (Sigma), functioning as a secondary enzyme, were added in an amount of 100 μl. The plates were incubated for 1 hour at room temperature with agitation, followed by washing with phosphate buffer three times. PNPP, a substrate of alkaline phosphatase, was added in an amount of 100 μl each well to induce color development. After color developed, absorbance (OD) was read at 405 nm. Immunization was regarded to be sufficient when the sera whose absorbance (OD) were two or more times greater than were those of normal mouse serum showed an inverse dilution of 30,000 or higher. After a sufficient antibody titer was obtained, the fusion protein was injected into the tail vein of mice once more. EXAMPLE 3: Preparation of Hybridoma

Four days after the vein injection in Example 2, splenocytes were removed from the mice and fused with the myeloma cell line SP2/0(ATCC CRL-581). For example, the spleen was removed aseptically from immunized mice and only cellular components were isolated in a Dulbecco's Modified Eagle Medium (DMEM, Gibco BRL). The splenocytes thus obtained were mixed in the ratio of 1:1 with SP2/0 cells and addition of polyethylene glycol (PEG) facilitated fusion between them. To the fused cells was added an HAT medium (Gibco BRL). 100 μl of the cell suspension was added to each well of 96-well plates over which mouse abdominal macrophages had been spread, followed by the incubation for 7 days at 37 °C. After completion of the incubation, there were found wells in which cells survived the HAT medium to form colonies. ELISA was conducted as in Example 2 to select clones in which antibodies capable of selectively recognizing RANK were produced.

EXAMPLE 4: Selection of Positive Clones

The clones obtained in Example 3 were cultured and analyzed by ELISA to select positive single clones. In this regard, the clones obtained in Example 3 were inoculated on 96-well culture plates in such a manner that 10, 5 or 0.5 cells were contained in each well, followed by incubation at 37 °C. HAT medium was used and refreshed every three days.

Culture supernatants were taken from the wells on which single colonies were found under a microscope, and analyzed by ELISA in the same manner as in Example 2. Clones which were found to be positive were diluted with HAT medium and so inoculated at as few as 0.5 cells per well. After incubation, three monoclonal hybridomas were obtained, named BBL1-1,

BBL1-2 and BBL1-3, respectively. Analysis with an isotype kit (Pierce) revealed that the immunoglobulin isotype of the monoclonal hybridomas was IgG2. The hybridoma BBL 1-1 of the present invention was deposited with KCTC (Korean Collection for Type Cultures) with accession No. KCTC 10192BP on Feb. 27, 2002.

EXAMPLE 5: Production of Asctite

The hybridoma of Example 4 was intraabdominally injected into mice to give ascite containing a high concentration of monoclonal antibodies. To this end, first, 0.5 ml of incomplete Freund's adjuvant (Gibco BRL) was intraabdominally injected into Balb/c mice, followed by the mfraabdominal injection with 2x10⁷ hybridoma cells, three days later. After two weeks, ascite filled in the abdominal cavity was sampled by use of a syringe lest the samples be contaminated with blood. Centrifugation of the sample at 12,000 rpm for 5 min at 4 °C gave a supernatant which was then subjected to protein A sepharose affinity chromatography to isolate monoclonal antibodies. A column was filled with 2 ml of protein sepharose and washed many times with an IgG binding buffer (ImmunoPure). 5 ml of the supernatant was mixed in the ratio of 1:1 with the binding buffer and passed at a speed of 2 ml/min through the equilibrated column. Afterwards, 20 ml of the IgG binding buffer was loaded onto the column to wash out proteins which remained unbound. In order to separate

IgG bound to beads, 5 ml of an IgG elution buffer (ImmunoPure) was passed through the column. The eluate was taken in an amount of 1 ml per 1.5 ml microtube and immediately mixed with 100 μl of 1.0 M Tris (pH 7.5). EXAMPLE 6: Immunofluorescence Assay Using the Anti-RANK Monoclonal Antibody

To determine whether the anti-RANK monoclonal antibody of the present invention recognizes RANK expressed in tissues and cells, vascular endothelial tissues, vascular endothelial cells and osteoclasts were reacted with fluorolabeled monoclonal antibodies and analyzed with resort to immunofluorescence assay.

Used were pig vascular endothelial tissue and HUVEC (human umbilical vascular endothelial cell). As for the osteoclasts used, they were osteoclasts that were differentiated in mice and from RAW264.7 cells (ATCC number: TIB-71, Strain: BALB/c, Tissue: Abelson murine leukemia virus-induced tumor; Macrophage; monocyte).

Osteoclasts differentiated in mice were obtained as follows.

A mouse BALB/c (5 to 6 weeks old) was sacrificed and sufficiently soaked in 70% ethanol. Tibia and femur were separated from the body by use of scissors and a pincette.

From the tibia and femur, tissues and muscles were removed by use of a pincette in HBSS containing 3x antibiotic. The tibia and femur were immersed in a fresh 3x HBSS. The bones were cut at their both ends, and α-MEM was injected into the bones with a 1ml syringe to completely extract bone marrow cells which were then suspended well. The cell suspension was centrifuged for 3 min at 1600 rpm. After removing the supernatant, the cell pellet was re-suspended and added with 20 ml of a buffer for removing red blood cells. The suspension was let to stand for 1 to 2 min. Immediately after the addition of 30 ml of PBS to the suspension, centrifugation was performed for 3 min at 1600 rpm and the supernatant was decanted. The cell pellet was suspended in 10% α-MEM from which only osteoclasts were recovered through a cell filter. The osteoclasts were inoculated at a density of lxl 0⁶ cells in 500 μl of a medium per well of 48-well plates. To the wells, ODF was added at a final concentration of 50 ng/ml. Also, M-CSF was added at a final concentration of 30ng/ml. After three days of incubation, the medium was changed with a fresh one and incubation was carried out for an additional three days to obtain differentiated osteoclasts. RAW264.7 cells differentiated to osteoclasts were obtained as follows. RAW264.7 cells stored at -80 °C were thawed and suspended in an α-MEM (c-

FBS10%, penicillin 100 U/ml, streptomycin 100 ug/ml). The RAW264.7 cell suspension was transferred to 48-well plates in the density of lxlO⁴ cells/500 μl of medium per well. In the presence of ODF at a concentration of 50 ng/ml, the cells were cultured for 3 days. The medium was changed with a fresh one, followed by culturing for an additional 3 days to obtain differentiated RAW264.7 cells.

To label the monoclonal antibody of the present invention with a fluorescent marker, 2 mg/ml of the IgG obtained in Example 5 was reacted with 1 mg/ml of an FITC (fluorescein isothiocyanate, Sigma) solution in dimethyl sulfoxide for 8 hours at 4 °C in a dark room. Then, an additional reaction was carried out for 2 hours at 4 °C in the presence of 50 mM of NH CI. Unbound FITC was removed through gel filtration. The IgG of the present invention was treated with pepsin to yield the antigen-binding site entity F(ab')2, which was then conjugated with FITC in the same manner as above described.

Fluorescence was detected in all of the samples of pig vascular endothelial tissues, HUVEC, differentiated osteoclasts and RAW264.7 cells differentiated to multinuclear mature osteoclasts. Therefore, the monoclonal antibody against RANK of the present invention was found to specifically recognize RANK in tissues and cells (Fig. 3 A: pig vascular endothelial tissue, B: HUVEC, C: differentiated osteoclast, D: RAW264.7 cells differentiated to osteoclasts). EXAMPLE 7: Western Blotting Using the Anti-RANK Monoclonal Antibody

Western blotting was carried out to determine whether the monoclonal antibody of the present invention specifically recognizes RANK expressed in cells. The expression vector pSRa-RANK-T7 (Kim et al, FEBS Letters 443, 297-302 (1999)) was transfected into human embryonic kidney 293T (HEK293T) cells with the aid of Superfect (Qiagen) added at 2 μl per ml of the vector. The transfected cells were cultured to overexpress RANK and lysed. The cell lysate was loaded onto an SDS-polyacrylaniide gel (10%) on a vertical electrophoresis apparatus (Bio-Rad) and run at 30 mA. After completion of the electrophoresis, the developed proteins were transferred onto a PVDF film which was then treated with the anti-RANK monoclonal antibody of the present invention as a primary antibody. Thereafter, an HRP (Horse-radish peroxidase, Sigma)-conjugated anti-mouse IgG antibody was used as a secondary antibody. A RANK protein 90 kDa in size was detected by a chemiluminescent method (Fig. 4), which demonstrates that the monoclonal antibody of the present invention specifically recognizes RANK.

EXAMPLE 8: FACS Analysis Using the Anti-RANK Monoclonal Antibody

In order to determine whether the monoclonal antibody of the present invention recognizes the RANK existing in osteoclasts, osteoclasts which were differentiated from

RAW264.7 cells in the same way as in Example 6 were analyzed by FACS. FITC- conjugated BBLl-3(Fab')2 was used as a primary antibody for the FACS analysis. lxlO⁶ RAW264.7 cells were put in a microtube and centrifuged. The cells were washed twice with 1.5 ml of an FACS analysis solution (PBS+0.1% BSA+0.1% NaN₃ solution passed through 0.45 μm filter) and the suspension was centrifuged for 5 min at 1,200 rpm. After removal of the supernatant, the cell pellet was reacted with 50 μg/ml of the primary antibody conjugated with FITC for 1 hour. The cells were washed twice with 1.5 ml of the FACS analysis solution, followed by centrifuging the suspension at 1 ,200 rpm for 5 min. 500 μl of 1% paraformaldehyde/PBS (pH 7.3) or the FACS analysis solution was added to suspend the cells and the expression of RANK on cell surface was quantitatively analyzed by use of FACS.

The results are given in Fig. 5. As seen in Fig. 5, fluorescent peaks of the cells dyed with BBL1-3 F(ab')2-FITC are shifted rightwards compared with those of a control. Therefore, the expression of RANK in RAW264.7 cells differentiated to osteoclasts was revealed by BBL1-3 F(ab')2-FITC.

EXAMPLE 9: Inhibition Effect of the Anti-RANK Monoclonal Antibody on Differentiation of Osteoclast

To examine the effect of the monoclonal antibody of the present invention on osteoclastic differentiation, the monoclonal antibodies BBL1-1, BBL1-2 and BBL1-3 were applied to osteoclast differentiation model systems. In this regard, there were used three osteoclast differentiation model systems:. 1) RAW264.7 cells alone, 2) co-culture of myelocyte and osteoblast, and 3) co-culture of splenocyte and osteoblast.

9-1 Dyeing RAW264.7 Cells with TRAP

RAW264.7 cells described in Example 6 were dispensed into 48-well plates for tissue culture and induced to differentiation in the presence of RANKL. To each well, BBL1-1, BBL1-2 or BBL1-3 was added at a final concentration of 50 μg/ml. For comparison, RAW264.7 cells were not treated with any of the antibodies (Non-treated control) or treated with IgGl (Sigma; Mouse IgGl, κ(MOPC31C)) (Isotype control). After three days of incubation, the medium was changed with a fresh one. At this time, RANKL addition and monoclonal antibody treatment were also conducted as described above. After six days of incubation, the medium was removed from the culture plate in which osteoclast had completed differentiation. The cells were immobilized by treatment with a 10% formalin solution for 5 min. After removal of the formalin solution, the cells were treated with 0.1%> Triton X-100 for 10 sec. Then, the cells were dyed with TRAP (tarrrate-resistant acid phosphatase) for 5min in the absence of Triton X-100. Dyeing with TRAP resorted to a leukocyte acid phosphatase kit (Sigma, cat. No. 387- A). After removal of the TRAP dyeing solution, the cells were washed twice with distilled water, dried, and observed with an optical microscope (xlOO) to count TRAP-positive osteoclasts (Fig. 6A).

9-2 Dyeing Co-Culture of Osteoblast and Myelocyte with TRAP

Osteoblasts were isolated from one-day-old mice. ICR mice were immersed in 70%) ethanol and the skull was separated by use of scissors and pincettes and cut into several pieces which were then collected in a 6 cm culture plate containing 3x HBSS. They were incubated five times for 15 min at 37 °C in the presence of 0.1% collagcnasc (Gibco BRL) and 0.2% dispase (Boehringer Mannheim). From the second round of the incubation, cell suspensions were gathered, and centrifuged for 5 min at 1,600 rpm to obtain osteoclasts. These were aliquoted into l-2xl0⁶ cells and cultured for three days in a 10 cm culture plate containing 15 ml of α-MEM supplemented with 10% FBS. Thereafter, the cultured cells were aliquoted in cryovials and stored in a nitrogen tank until use in co-culture experiments. Myelocytes were isolated from ICR female mice 6-7 weeks old. The mice were sacrificed by cervical angulation and the hind legs were sterilized with 70% ethanol after which the tibia was aseptically separated. In 3x HBSS (Gibco BRL), soft tissues were neatly removed from the tibia. The bone was cut at its opposite ends, and α-MEM was injected into the bone with a 1ml syringe to completely extract bone marrow cells which were then suspended sufficiently by pipetting several times. Cells (myelocytes and red blood cells) were obtained as a pellet by centrifugation (1,600 rpm, 5 min). The pelletized cells were treated for 2 min with about 15-20 ml of ACK buffer (155 mM NH4Cl,llmM KHCO3, 0.01 mM EDTA) and added with phosphate buffer to lyse the red blood cells with caution to minimize damage to the myelocytes. After centrifugation (1600 rpm, 5 min), the cells were suspended in 10% α-MEM.

Osteoblasts and myelocytes were dispensed in 48-well cell culture plates and induced to differentiate in the presence of 1,25-dihydroxy vitamin D3 and prostaglandin E2. To each well, BBL1-1, BBL1-2 or BBL1-3 was added to a final concentration of 50 μg/ml. For comparison, the cell mixture was not treated with any of the antibodies (Non-treated control) or treated with IgGl (Sigma; Mouse IgGl, κ(MOPC31C)) (Isotype control). After three days of incubation, the medium was changed with a fresh one. At this time, addition of 1,25-dmydroxyvitamin D3 and prostaglandin E2 and treatment with the monoclonal antibody were also conducted as described above. After six days of incubation, the medium was removed from the culture plates in which osteoclasts had completely differentiated. The cells were immobilized by treatment with a 10% formalin solution for 5 min. After removal of the formalin solution, the cells were treated with 0.1 % Triton X-100 for 10 sec. Then, the cells were dyed with TRAP (tartrate-resistant acid phosphatase) for 5min in the absence of Triton X-100. Dyeing with TRAP resorted to a leukocyte acid phosphatase kit (Sigma, cat. No. 387- A). After removal of the TRAP dyeing solution, the cells were washed twice with distilled water, dried, and observed with an optical microscope (xlOO) to count TRAP-positive osteoclasts (Fig. 6B).

9-3 Dyeing Co-Culture of Splenocyte and Myelocyte with TRAP

Splenocytes (Example 3) and myelocytes (Example 9-2) were dispensed in 48-well cell culture plates and induced to differentiate in the presence of RANKL. To wells, BBL1- 1, BBL1-2 and BBL1-3 were added at final concentrations of 50, 10 and 1 μg/ml, respectively. For comparison, the cell mixture was not treated with any of the antibodies (Non-treated control) or treated with IgGl (Sigma; Mouse IgGl, κ(MOPC31C)) (Isotype control). After three days of incubation, the medium was changed with a fresh one. At this time, RANKL addition and monoclonal antibody treatment were also conducted as described above.

After six days of incubation, the medium was removed from the culture plates in which osteoclast had completed differentiation. The cells were immobilized by treatment with a 10% formalin solution for 5 min. After removal of the formalin solution, the cells were treated with 0.1% Triton X-100 for 10 sec. Then, the cells were dyed with TRAP (tartrate-resistant acid phosphatase) for 5 min in the absence of Triton X-100. Dyeing with TRAP resorted to a leukocyte acid phosphatase kit (Sigma, cat. No. 387- A). After removal of the TRAP dyeing solution, the cells were washed twice with distilled water, dried, and observed with an optical microscope (xlOO) to count TRAP-positive osteoclasts (Fig. 6C).

Taken together, the experiment results of Figs. 6A, 6B and 6C demonstrate that the monoclonal antibodies against RANK according to the present invention can inhibit osteoclastic differentiation.

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Claims

What is claimed is:

1. An anti-RANK monoclonal antibody of IgG2 type, or its fragments, capable of selectively recognizing RANK and inhibiting osteoclastic differentiation.

2. The anti-RANK monoclonal antibody according to Claim 1, wherein the fragments comprise F(ab')2.

3. A hybridoma, producing an anti-RANK monoclonal antibody of IgG2 type capable of selectively recognizing RANK and inhibiting osteoclastic differentiation.

4. The hybridoma according to Clam 3, wherein the hybridoma is deposited under accession No. KCTC 10192BP.

5. A method for producing an anti-RANK monoclonal antibody, comprising the steps of: immunizing animals using RANK protein ; fusing splenocytes from the immunized animals with myeloma cells to give hybridoma; screening the hybridomas which produce anti-RANK monoclonal antibodies able to selectively recognize RANK and simultaneously inhibit osteoclastic differentiation; and culturing the screened hybridoma to isolate the antibody from the hybridoma culture or injecting the screened hybridoma into the abdominal cavity of animals to isolate the antibody from the ascite of the animals.

6. A kit for diagnosing bone metabolism disorders, comprising the monoclonal antibody or its fragments of claim 1 or 2.

7. A method for diagnosing bone metabolism disorders, comprising the steps of:

(a) bringing a biological sample of an animal into contact with the antibody or its fragments of claim 1 or 2; and

(b) measuring the RANK expression of the biological sample and comparing with the RANK expression of a control obtained from a healthy animal.

8. The method according to claim 7, wherein the biological sample is selected from the group consisting of tissues, cells, whole blood, phlegm, serum, plasma, saliva, cerebrospinal fluid, and urine.

9. A pharmaceutical composition for treating bone metabolism disorders by inhibiting osteoclastic differentiation, comprising the monoclonal antibody or its fragments of claim 1 or 2 in a therapeutically or prophylactically effective amount, and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 9, wherein the bone metabolism disorders comprise osteoporosis, bone metastatic lesion caused by the migration of tumors, primary bone tumor, and melanoma.

11. A method for screening inhibitors of osteoclast differentiation, comprising the steps of:

(a) treating an osteoclast differentiation model with a candidate inhibitor; and

(b) bringing the osteoclast differentiation model into contact with the antibody or its fragments of claim 1 or 2 and quantitatively analyzing RANK expression to determine whether the candidate inhibitor is an inhibitor of osteoclast differentiation as defined by a decrease in RANK expression.