WO2001000798A2 - Clonage et expression de cathepsine k humaine dans des cellules mammaliennes - Google Patents

Clonage et expression de cathepsine k humaine dans des cellules mammaliennes Download PDF

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Publication number
WO2001000798A2
WO2001000798A2 PCT/US2000/016502 US0016502W WO0100798A2 WO 2001000798 A2 WO2001000798 A2 WO 2001000798A2 US 0016502 W US0016502 W US 0016502W WO 0100798 A2 WO0100798 A2 WO 0100798A2
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cat
antibody
pcat
host cell
recombinant
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PCT/US2000/016502
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WO2001000798A3 (fr
WO2001000798A9 (fr
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Mohammad S. Saedi
Timothy Hill
Cheryl Schaffer Brehme
Steven J. Roman
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Hybritech Incorporated
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Priority to AU61991/00A priority Critical patent/AU6199100A/en
Publication of WO2001000798A2 publication Critical patent/WO2001000798A2/fr
Publication of WO2001000798A3 publication Critical patent/WO2001000798A3/fr
Publication of WO2001000798A9 publication Critical patent/WO2001000798A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6472—Cysteine endopeptidases (3.4.22)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates generally to cathepsin K protein and specifically to cloning and expressing human cathepsin K in mammalian cells, and the use of such expressed cathepsin K.
  • Bone remodeling is a dynamic process involving bone resorption followed by bone formation (1).
  • the bone resorption process is carried out by the osteoclasts in bone resorption pits.
  • the process involves dissolution of mineral components of bone at low pH followed by proteolytic digestion of the proteinaceous matrix proteases.
  • Cathepsin K (Cat K) (3, 4) is a recently discovered cysteine protease of the papain superfamily.
  • the cDNA for Cat K has been cloned and isolated by different groups and referred to by different names (Cat O (2); Cat K (3); Cat O2 (4); and Cat X (5)).
  • Cat K cDNA is 1.6 kb and codes for a 329 amino acid protein.
  • This protein has a 15 amino acid N-terminal signal sequence (pre sequence) followed by a 99 amino acid pro region, followed by a 215 amino acid mature region ( Figure 1).
  • the gene for Cat K has been localized to human chromosome lq21 (6,7).
  • Northern blot (8) and immunohistochemical (9) studies indicate that Cat K is predominantly expressed in osteoclasts.
  • Cat K efficiently degrades both type I and type II collagen (10).
  • an autosomal disease, pycnodysostosis which causes dense, brittle bones and osteoporosis, has been associated with defects in Cat K (11).
  • Cat K Due to its expression pattern, protease activity, and association with bone disease, Cat K has been postulated to participate in the bone resorption process and thus has a major role in bone remodeling. Also, this protein has been identified as a potential therapeutic target for osteoporosis.
  • Cat K has been expressed in baculovirus (12, 13) and yeast (14) expression systems and the recombinant proteins have been used as a source for crystallizing Cat K (15, 16, 18, 19). However, the expression of Cat K in recombinant mammalian cells has not been reported.
  • Cat K needs to be expressed in a mammalian system. This is because the post-translational modification of proteins in baculovirus and yeast is not the same as in mammalian cells. Thus, proteins expressed in these systems do not have the same modifications as their native, mammalian counterparts. Therefore, a need exists to express Cat K in mammalian cells such that a mature and enzymatically active Cat K may be produced.
  • the present invention is based on the unexpected expression of Cat K in a recombinant mammalian system.
  • the present invention demonstrates that pro Cat K (pCat K) may be stably expressed in mammalian cells and secreted from these cells. This recombinant pCat K could then be converted to mature, enzymatically active Cat K by pepsin digestion.
  • one aspect of the present invention provides a chimeric expression vector.
  • the vector comprises a nucleic acid molecule encoding a ppCat K or pCat K polypeptide.
  • the nucleic acid molecule is operably linked to a control sequence recognized by a mammalian host cell transformed with the vector.
  • the present invention also provides a mammalian host cell transformed with a nucleic acid molecule encoding a ppCat K polypeptide or pCat K polypeptide operably linked to control sequences recognized by the mammalian host cell.
  • Another aspect of the present invention provides a method of using a nucleic acid molecule encoding a ppCat K or pCat K polypeptide.
  • the method comprises the steps of: (a) expressing the nucleic acid molecule in a cultured mammalian host cell stably transformed with a chimeric expression vector comprising the nucleic acid molecule operably linked to control sequences recognized by the host cell transformed with the vector, and
  • the pCat K is recovered from the host cell culture medium, and the pCat K may be converted to matured, enzymatically active Cat K.
  • the present invention also provides a method of producing a recombinant mature enzymatically active human Cat K.
  • the method comprises the steps of:
  • a chimeric expression vector comprising a nucleic acid molecule encoding a ppCat K or pCat K polypeptide operably linked to a control sequence recognized by a mammalian host cell transformed with the vector; (b) transforming the mammalian host cell with the chimeric expression vector;
  • a recombinant pCat K that is capable of converting to a recombinant mature, enzymatically active Cat K produced, and a recombinant, mature, enzymatically active Cat K produced by methods of the present invention are also provided.
  • a further aspect of the present invention provides antibodies against Cat K, and an immunoassay for detecting and determining Cat K in a sample.
  • the method includes the steps of:
  • the present invention also provides an immunoassay kit for detecting Cat K in a sample.
  • the kit includes a known amount of an agent which specifically binds to Cat K to be detected, wherein the agent is detectably labeled or binds to a detectable label.
  • Yet another aspect of the present invention provides a method for ameliorating a condition associated with a defect in Cat K.
  • the method includes administering to a subject in need thereof a sufficient amount of the recombinant Cat K of the present invention.
  • the conditions include, but are not limited to, an autosomal disease, pycnodysostosis, bone resorption dysfunction, and osteopetrosis.
  • the present invention also provides a pharmaceutical composition comprising a recombinant Cat K by the method of claim 8, and a pharmaceutically acceptable carrier.
  • FIGURE 1 is the DNA sequence of the expression plasmid with proCat K insert.
  • FIGURE 2 shows Western blot analysis of spent media from AV12-Cat K single cell clones using CP1A-003 mAb.
  • FIGURE 3 shows Cat K purified from AV12-Cat K spent media stained with coomassie blue.
  • FIGURE 4 shows the results of Cat K enzyme assay.
  • FIGURE 5 shows the results of zymogram.
  • FIGURE 6 shows the results of Western blot.
  • the vector comprises a nucleic acid molecule encoding a ppCat K or pCat K polypeptide operably linked to a control sequence recognized by a mammalian host cell transformed with the vector.
  • Cat K and Cat K polypeptide'' as used herein are used interchangeably and include recombinant prepro, pro and mature Cat K polypeptides.
  • preproCat K "proCat K,” “ppCat K,” “pCat K,” “ppCat K polypeptide” and “pCat K polypeptide” are used interchangeably and preferably encompass all inactive precursor forms of Cat K.
  • chimeric means that a vector comprises DNA from at least two different species, or comprises DNA from the same species, which is linked or associated in a manner which does not occur in the "native" or wild type of the species.
  • Control sequences is defined to mean DNA sequences necessary for the expression of a operably linked coding sequence in a particular host organism.
  • the control sequences that are suitable for prokaryotic cells include a promoter (and, optionally, an operator sequence) and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals and enhancers.
  • operably linked means that the nucleic acids are placed in a functional relationship with another nucleic acid sequence.
  • DNA encoding a presequence or secretory leader is operably linked to DNA encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked” means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic ohgonucleotide adaptors or linkers are used in accordance with conventional practice.
  • the recombinant DNA can be readily introduced into the target cells by transfection or transformation with an expression vector comprising cDNA encoding Cat K, for example, by the modified calcium phosphate precipitation procedure of C. Chen et al., Mol. Cell. Biol., 1, 2745 (1987). Transfection can also be accomplished by lipofection, using commercially available kits, e.g., provided by BRL Life Technologies, Inc.
  • Suitable host cells for the expression of CAT K are derived from multicellular organisms. Such host cells are capable of complex processing and glycosylation activities.
  • mammalian cells are the prefened host for expression of mammalian protein, since these cells modify and process the recombinant protein in a manner closely related to the natural host of the protein.
  • any higher eukaryotic cell culture can be employed in the practice of the invention, whether from vertebrate or invertebrate culture.
  • invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and conesponding permissive insect host cells have been identified.
  • PCR Polymerase chain reaction
  • RNA and/or DNA are amplified as described in U.S. Patent No. 4,683,195.
  • sequence information from the ends of the region of interest or beyond is employed to design ohgonucleotide primers. These primers will be identical or similar in sequence to opposite strands of the template to be amplified.
  • PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage or plasmid sequences, and the like. See, generally, Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51, 26') (1987); Erlich, ed., PCR Technology (Stockton Press, NY, 1989).
  • Cat K polypeptide When Cat K is expressed in a recombinant cell of other than human origin, the Cat K polypeptide is completely free of proteins or polypeptides of human origin. However, it is necessary to purify Cat K polypeptide from recombinant cell proteins or polypeptides to obtain preparations that are substantially homogeneous as to Cat K polypeptide. For example, the culture medium or lysate can be centrifuged to remove particulate cell debris. The membrane and soluble protein fractions are then separated. The Cat K polypeptide may then be purified from the soluble protein fraction and, if necessary, from the membrane fraction of the culture lysate.
  • Cat K polypeptide can then be purified from contaminant soluble proteins and polypeptides by fractionation on immunoaffinity or ion exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica or on an anion exchange resin such as DEAE; chromatofocusing; SDS-PAGE- ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; or ligand affinity chromatography.
  • Cat K is recovered from the host cell culture medium, particularly as proCat K.
  • the proCat K may be converted to matured, enzymatically active Cat K by pepsin digestion.
  • both the proCat K and or mature Cat K may be used to produce anti- Cat K antibodies.
  • the present invention provides an antibody which consists essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations.
  • Monoclonal antibodies are made from an antigen containing the recombinant Cat K of the present invention or fragments thereof by methods well known in the art (E. Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).
  • this method involves preparing an antibody- producing fused cell line, e.g., from primary spleen cells fused with a compatible continuous line of myeloma cells, growing the fused cells either in mass culture or in an animal species from which the myeloma cell line used was derived or is compatible.
  • Such antibodies offer many advantages in comparison to those produced by inoculation of animals, as they are highly specific and sensitive and relatively "pure” immunochemically.
  • Immunologically active fragments of antibodies are also within the scope of the present invention, e.g., the f(ab) fragment, as are partially humanized monoclonal antibodies.
  • polyclonal antibodies can be further purified, for example, by binding to and elution from a matrix to which a polypeptide or a peptide to which the antibodies were raised is bound.
  • a matrix to which a polypeptide or a peptide to which the antibodies were raised is bound.
  • Those skilled in the art will know of various techniques common in the immunology arts for purification and/or concentration of polyclonal antibodies, as well as monoclonal antibodies. (See, for example, Coligan et al., Unit 9, Current Protocols in
  • antibody as used in this invention includes intact molecules as well as fragments thereof, such as Fab, F(ab') 2 and Fv, which are capable of binding the epitopic determinant. These antibody fragments retain some ability to selectively bind with its antigen or receptor and are defined as follows:
  • Fab the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of the whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;
  • Fab' the fragment of an antibody molecule that can be obtained by treating the whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain — two Fab' fragments are obtained per antibody molecule;
  • F(ab') the fragment of the antibody that can be obtained by treating the whole antibody with the enzyme pepsin without subsequent reduction —
  • F(ab') 2 is a dimmer of two Fab' fragments held together by two disulfide bonds;
  • Fv defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains
  • SCA Single chain antibody
  • epitopic determinants means any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • Epitopic determinants usually consist of chemically active groupings of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural charactenstics, as well as specific charge charactenstics
  • Antibodies of the present invention may be used to detect Cat K m patient fluid samples by in vitro immunoassay procedures or m patient tissues by immunohistochemical assay procedures Detection of the presence or amount of Cat K m a particular patient sample may be of significant diagnostic utility and may be an indicator of, or conelate with, the development of a condition associated with Cat K deficiency Examples of such a condition include, but are not limited to, an autosomal disease, pycnodysostosis, bone resorption dysfunction and osteoporosis Immunohistochemical methods for the detection of antigens m patient tissue specimens are well known in the art and need not be descnbed m detail herein For example, methods for the immunohistochemical detection of antigens are generally descnbed in Taylor, Arch Pathol_Lab Med 702 113 (1978) Bnefly, m the context of the present invention, a tissue specimen obtained from a patient suspected of having a Cat K deficiency-related problem is contacted with an antibody
  • an immunoassay for detecting Cat K of the present invention in a biological sample comprises the steps of: (a) contacting an amount of an agent which specifically binds to the Cat K to be detected with the sample under a condition that allows the formation of a binary complex comprising the agent and the Cat K; and (b) detecting or determining the presence or amount of the complex as a measure of the amount of the Cat K contained in the sample.
  • the biological sample can be any human physiological fluid sample that contains the Cat K of the present invention. Examples of the human physiological fluid sample include, but are not limited to, serum, urine, plasma, and synovial fluid.
  • both monoclonal antibodies and polyclonal antibodies may be used as long as such antibodies possess the requisite specificity for the antigen provided by the present invention. Preferably, monoclonal antibodies are used.
  • Monoclonal antibodies can be utilized in liquid phase or bound to a solid phase carrier. Monoclonal antibodies can be bound to many different carriers and used to determine the novel form of Cat K of the present invention.
  • Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetites.
  • the nature of the carrier can be either soluble or insoluble for purposes of the invention. Examples of insoluble carriers include, but are not limited to, a bead and a microtiter plate. Those skilled in the art will know of other suitable carriers for binding monoclonal antibodies, or will be able to ascertain such under routine experimentation.
  • monoclonal antibodies in these immunoassays can be detectably labeled in various ways.
  • monoclonal antibodies of the present invention can be coupled to low molecular weight haptens. These haptens can then be specifically detected by means of a second reaction.
  • haptens as biotin, which reacts with avidin, or dinitrophenyl, pyridoxal and fluorescein, which can react with specific antihapten antibodies.
  • monoclonal antibodies of the present invention can also be coupled with a detectable label such as an enzyme, radioactive isotope, fluorescent compound or metal, chemiluminescent compound or bioluminescent compound.
  • the binding of these labels to the desired molecule can be done using standard techniques common to those of ordinary skill in the art.
  • One of the ways in which the antibody can be detectably labeled is by linking it to an enzyme. This enzyme, in turn, when later exposed to its substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected by, for example, spectrophotometric or fluorometric means (ELISA system).
  • ELISA system spectrophotometric or fluorometric means
  • enzymes that can be used as detectable labels are horseradish peroxidase, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholine esterase.
  • the procedures described can be modified using biotinylated antibodies reacting with avidin-peroxidase conjugates.
  • the amount of antigen can also be determined by labeling the antibody with a radioactive isotope.
  • the presence of the radioactive isotope would then be determined by such means as the use of a gamma counter or a scintillation counter.
  • Isotopes which are particularly useful are 3 H, 125 I, 123 1, 32 P, 35 S, 14 C, 51 Cr, 36 C1, 57 Co, 58 Co, 59 Fe, 75 Se, , n N, "mTc, 67 Ga and 90 Y.
  • Determination of the antigen is also possible by labeling the antibody with a fluorescent compound.
  • the fluorescently labeled molecule is exposed to light of the proper wave length, its presence can then be detected due to fluorescence of the dye.
  • fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • Fluorescence-emitting metal atoms such as Eu (europium) and other lanthanides, can also be used. These can be attached to the desired molecule by means of metal- chelating groups, such as DTPA or EDTA.
  • Another way in which the antibody can be detectably labeled is by coupling it to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged immunoglobulin is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • particularly useful chemiluminescent labeling compounds are luminol, isoluminol, aromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a bioluminescent compound may also be used as a label.
  • Bioluminescence is a special type of chemiluminescence which is found in biological systems and in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent molecule would be determined by detecting the presence of luminescence.
  • Important bioluminescent compounds for purposes of labeling are luciferin, lucif erase and aequorin.
  • Qualitative and/or quantitative determinations of the novel form of CAT K of the present invention in a sample may be accomplished by competitive or non-competitive immunoassay procedures in either a direct or indirect format.
  • immunoassays examples include the radioimmunoassay (RIA) and the sandwich (immunometric) assay.
  • RIA radioimmunoassay
  • sandwich immunometric assay.
  • Detection of the antigens using the monoclonal antibodies of the present invention can be done utilizing immunoassays which are run in either the forward, reverse or simultaneous modes, including immunohistochemical assays on physiological samples.
  • immunoassays which are run in either the forward, reverse or simultaneous modes, including immunohistochemical assays on physiological samples.
  • One aspect of the present invention provides an immunoassay kit for detecting or determining in a sample a Cat K polypeptide of the present invention.
  • the kit comprises a known amount of an agent which specifically binds to the Cat K, wherein the agent is detectably labeled or binds to a detectable label.
  • the sample may be a sample of human physiological fluid such as, but not limited to, urine, serum, plasma or synovial fluid.
  • the sample may also be a tissue specimen coming from a patient with a condition associated with Cat K deficiency.
  • the agent may be an antibody that specifically binds to the Cat K of the present invention.
  • the agent is a monoclonal antibody, although a polyclonal antibody may also be used.
  • the present invention also provides a method of ameliorating a condition associated with a defect in Cat K. The method comprises administering to a subject in need thereof a sufficient amount of the recombinant Cat K of the present invention.
  • ameliorate denotes a lessening of the detrimental effect of defect in Cat K-associated disease in the subject receiving therapy.
  • sufficient amount means that the amount of a composition of the present invention administered is of sufficient quantity to prevent or ameliorate to some beneficial degree a disorder or condition associated with a defect in Cat K. For example, a clinically relevant lack of Cat K activity may give rise to pyenoldysostosis or osteopetrosis.
  • the amount of Cat K administered to ameliorate the condition must be large enough to produce the desired effect, but not so large as to cause any adverse side effects.
  • compositions that contain recombinant Cat K of the present invention may be administered to a subject parentally by injection or by gradual perfusion over time.
  • the Cat K polypeptide may be administered intravenously, intraperitoneally, intra-muscularly, subcutaneously, intracavity, transdermally, intranasally or enterally.
  • compositions are administered in a manner compatible with the dosage formulation and in a therapeutically effective amount.
  • quantity to be administered and timing of administration depends on the subject to be treated, capacity of the subject's system to utilize the active ingredient, and degree of therapeutic effect desired. Precise amounts of the active ingredient required to be administered depend on the judgement of the practitioner and are peculiar to each individual.
  • Suitable dosage ranges for systematic application are variable and depend on the route of administration. Suitable regimens for administration are also variable but are typified by an initial administration followed by repeated doses at intervals by subsequent injection or other administration.
  • compositions of the present invention may also be administered with a pharmaceutically acceptable carrier.
  • Preparations for parental administration of a composition of the invention 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.
  • Parental vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, dextrose and water lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, 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, antioxidants, chelating agents, and inert gases and the like.
  • a subject may be any subject that is in need of the treatment.
  • the subject is a mammal. More preferably, the subject is human.
  • FIG. 1 shows the DNA sequence of the resulted plasmid.
  • pGtl2 is a modified version of the plasmid pGTd (17) and genes clones under the control of the GBMT promoter in this plasmid are inducible by adenovirus El a.
  • pCPlA was transfected into the hamster cell line AV12-664 (ATCC CRL9595) and stable clones (AV12-Cat K) were selected in 400 nM methotrexate.
  • Avl2 cells are derived from an adenovirus-induced tumor in Syrian hamsters and constitutively express El a tumor antigen.
  • the GBMT promoter is activated by the El a antigen, leading to the overexpression of the protein cloned under this promoter.
  • FIG. 2 shows the Western blot analysis of spent media from AV12-Cat K single cell clones using CP1A-003 mAb.
  • Lane 1 is ppCat K expressed in bacteria (60 ng) as a positive control; lane 2 is MW markers (not visible by Western blot); lane 3 is pGtl2 9 (empty vector as negative control); lane 4 is AV12-Cat K pool (spent media from a pool of transfected cells); lane 5 is AV12-Cat K-02; lane 6 is AV12 Cat-K-06; lane 7 is AV12- Cat-K-07; lane 8 is AV12-Cat K-08; lane 9 is AV12-CatK-16. As shown in Figure 2, all five clones secrete a ⁇ 42 kDa protein immunoreactive with CPIA-003. This protein has about the same MW as bacterial Cat K. Clone AV12-Cat K-08 was selected for further study.
  • Digestion reactions were characterized by cathepsin fluorometric enzyme assay ( Figure 4) performed as follows: (a) 5 ⁇ L each digestion reaction added to 45 ⁇ L assay buffer (100 mM
  • Running gel 12% acrylamide in 100 mM sodium phosphate/TRIS, 0.1% SDS, pH 8.

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Abstract

Cette invention se rapporte à un procédé servant à produire une protéine Cat K recombinée, mature et active comme enzyme, exprimée dans des cellules hôtes mammaliennes, ainsi qu'à un vecteur d'expression permettant d'exprimer de façon stable la protéine Cat K, sous la forme de pCat K, dans un système mammalien. Cette invention se rapporte en outre à des procédés de détection de la protéine Cat K dans un échantillon, ainsi qu'à un kit d'immunodosage et à des procédés d'utilisation de la protéine Cat K recombinée dans le domaine pharmaceutique.
PCT/US2000/016502 1999-06-25 2000-06-15 Clonage et expression de cathepsine k humaine dans des cellules mammaliennes WO2001000798A2 (fr)

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AU61991/00A AU6199100A (en) 1999-06-25 2000-06-15 Cloning and expressing of human cathepsin k in mammalian cells

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005093421A1 (fr) * 2004-03-10 2005-10-06 Biovendor Laboratory Medicine, Inc. Dosage immuno-enzymatique pour mettre en evidence la cathepsine k

Citations (3)

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Publication number Priority date Publication date Assignee Title
EP0445939A1 (fr) * 1990-02-23 1991-09-11 Eli Lilly And Company Unité de contrôle de la transcription modifiée et son usage
WO1996013523A1 (fr) * 1994-10-27 1996-05-09 Khepri Pharmaceuticals, Inc. Protease cathepsine o2
WO1997047642A1 (fr) * 1996-06-14 1997-12-18 Smithkline Beecham Corporation Gene de la cathepsine k

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Publication number Priority date Publication date Assignee Title
EP0445939A1 (fr) * 1990-02-23 1991-09-11 Eli Lilly And Company Unité de contrôle de la transcription modifiée et son usage
WO1996013523A1 (fr) * 1994-10-27 1996-05-09 Khepri Pharmaceuticals, Inc. Protease cathepsine o2
WO1997047642A1 (fr) * 1996-06-14 1997-12-18 Smithkline Beecham Corporation Gene de la cathepsine k

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Title
BROEMME DIETER ET AL: "Human cathepsin O2, a matrix protein-degrading cysteine protease expressed in osteoclasts: Functional expression of human cathepsin O2 in Spodoptera frugiperda and characterization of the enzyme." JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 271, no. 4, 1996, pages 2126-2132, XP002162040 ISSN: 0021-9258 cited in the application *
SAEDI ET AL.: "Recombinant human cathepsin K: expression in mammalian cells, purification, and activation of the enzyme." JOURNAL OF BONE AND MINERAL RESEARCH, vol. 14, no. Suppl., September 1999 (1999-09), pages S356-Abstr.SA192, XP000982610 *
SHI G -P ET AL: "MOLECULAR CLONING OF HUMAN CATHEPSIN O, A NOVEL ENDOPROTEINASE AND HOMOLOGUE OF RABBIT OC2" FEBS LETTERS,NL,ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, vol. 357, 1995, pages 129-134, XP002068032 ISSN: 0014-5793 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005093421A1 (fr) * 2004-03-10 2005-10-06 Biovendor Laboratory Medicine, Inc. Dosage immuno-enzymatique pour mettre en evidence la cathepsine k

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