EP0708838A1 - Utilisation de l'heparanase pour identifier et isoler un compose anti-heparanase - Google Patents

Utilisation de l'heparanase pour identifier et isoler un compose anti-heparanase

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Publication number
EP0708838A1
EP0708838A1 EP94922654A EP94922654A EP0708838A1 EP 0708838 A1 EP0708838 A1 EP 0708838A1 EP 94922654 A EP94922654 A EP 94922654A EP 94922654 A EP94922654 A EP 94922654A EP 0708838 A1 EP0708838 A1 EP 0708838A1
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Prior art keywords
seq
lys
leu
ala
heparanase
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German (de)
English (en)
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Arlene J. Hoogwerf
Steven R. Ledbetter
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Pharmacia and Upjohn Co
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Pharmacia and Upjohn Co
Upjohn Co
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01166—Heparanase (3.2.1.166)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)

Definitions

  • the present invention discloses the use of mammalian heparanase, preferably recombinant heparanase, for screening for anti-heparanase compounds. More particularly, the present invention provides a method of selecting IHA (Inhibitors of Heparanase Activity). In addition, the present invention provides a purified heparanase, particularly suitable for use to identify and isolate anti-heparanase compounds as well as for other known uses of heparanases, such as its use to accelerate wound healing or its use as an immobilized heparanase filter connected to extracorporeal devices to degrade heparin and neutralize its anticoagulant properties during surgery.
  • IHA Inhibitors of Heparanase Activity
  • Elevated heparanase activity has been documented in mobile, invasive cells. Examples include; invasive melanoma, lymphoma, mastocytoma, mammary adenocarcinoma, leukemia, and rheumatoid fibroblasts. This activity has also been documented in non-pathologic situations involving the migration of lymphocytes, neutrophils, macrophages, eosinphils and platelets. An inhibitor of heparanase would therefore broadly influence the invasive potential of these diverse cells.
  • Inhibition of heparan sulfate degradation would also inhibit the release of bound growth factors and other biologic response modifiers that would, if released, fuel the growth of adjacent tissues and provide a supportive environment for cell growth (Rapraeger, et al., Science 252: 1705-1708, 1991). Inhibitors of heparanase activity would be of value in the treatment of arthritis, vascular restenosis, tumor growth and progression, and fibro-proliferative disorders.
  • Heparanase refers to a mammalian enzyme which can degrade heparin proteoglycans (HPG) and/or heparan sulfate proteoglycans (HSPG).
  • Heparanase activity in mammalian cells is well known. It is found in various melanoma cells (Nakajima, et al, Cancer Letters 31: 277-283, 1986), mammary adenocarcinoma cells (Parish, et al, Int. J. Cancer, 40: 511-518, 1987), leukemic cells (Yahalom, et al, Leukemia Research 12: 711-717, 1988), mast cells (Ogren and Lindahl, J. Biol Chem. 250: 2690-2697, 1975), macrophages (Savion, et al, J. Cell.
  • WO 91/02977 describes a substantially, but partially, purified heparanase produced by cation exchange resin chromatography and the affinity absorbent purification of heparanase-containing cell extract.
  • WO 91/02977 also describes a method promoting wound healing utilizing compositions comprising a "purified" form of heparanase.
  • U.S. Patent 4,882,318 describes heparanase-inhibiting compositions for preventing tumor metastasis.
  • Haimovitz-Friedman et al. (Blood 78: 789-796, 1991) describe an assay for heparanase activity that involves the culturing of endothelial cells in radiolabeled 35 SO 4 to produce radiolabeled heparan sulfate proteoglycans, the removal of the cells which leaves the deposited extracellular matrix that contains the 35 S-HSPG, the addition of potential sources of heparanase activity, and the detection of possible activity by passing the supernatant from the radiolabeled extracellular matrix over a gel filtration column and monitoring for changes of the size of the radiolabeled material that would indicate that HSPG degradation had taken place.
  • This assay does not have the capability for large-scale screening of inhibitors.
  • Nakajima et al. (Anal Biochem. 196: 162-171, 1986) describe a solid-phase substrate for the assay of melanoma heparanase activity.
  • Heparan sulfate from bovine lung is chemically radiolabeled by reacting it with [ 14 C]-acetic anhydride. Free amino groups of the [ 14 C]-heparan sulfate were acetylated and the reducing termini were aminated.
  • the [ 14 C]-heparan sulfate was chemically coupled to an agarose support via the introduced amine groups on the reducing termini. This substrate is limited in that it is an extensively chemically modified form of naturally occurring heparan sulfate.
  • heparin is quantitated by its ability to interfere with the color development between a protein and the dye Coomassie brilliant blue. Heparanase activity is detected by the loss of this interference. This assay is limited in use for screening because it is so indirect that other non-heparin compounds could also interfere with the protein-dye reaction.
  • the CXC chemokine family (also called the intercrine ⁇ family) is one branch of the supergene "intercrine” cytokine family (Oppenheim, Ann. Rev. Bit :m. 9: 617-648, 1991). It's members include platelet factor 4, platelet basic protein and derivatives, ⁇ IP-10, gro( ⁇ , ⁇ , ⁇ ), NAP-l/interleukin-8, mig, and ENA-78 (for review, see Miller and Krangel, Critical Reviews in Immunology 12: 17-46, 1992).
  • the other branch, the CC chemokines or intercrine- ⁇ family includes MlPl ⁇ , MIPl ⁇ ,JE/MCP-l, RANTES, and MCAF.
  • All members of both branches of this chemokine family characteristically are basic heparin-binding polypeptides, display molecular weights between 8 and 11 kD, share 20 - 50% homology, and function broadly in pathologic sit:.* ; ons characterized by inflammation and tissue remodeling.
  • the pr ⁇ icolytically processed forms of platelet basic protein include CTAP-m, ⁇ - thromboglobulin, and NAP-2.
  • ⁇ -thromboglobulin Moore, et al, Biochim. Biophys. Acta. 379: 360-369, 1975
  • CTAP-III Castor, et al, Arthritis Rheum. 20: 859-868, 1977, were originally isolated from activated supernatants or lysates from outdated pk ⁇ ts.
  • ⁇ -thromboglobulin was identified as an ⁇ -granule protein that could be released upon activation (Kaplan, et al, Blood 53: 604- 618, 1979).
  • Platelet basic protein itself was later isolated from fresh platelets, megakaryocytes, and HEL cells, an immortal human erythroleukemia cell line (Holt, et al, Biochemistry 25: 1988-1996, 1986; Holt, et al, Exp. Hematol. 16: 302-306, 1988).
  • Walz and Baggiolini isolated the processed form of NAP-2 from platelet-containing cultures of stimulated mononuclear cells (Walz, et al, J. Exp. Med. 170: 1745-1750, 1989).
  • Transglutaminases catalyze the posttranslational modification of proteins by transamidation of available glutamine residues. This action results primarily in the formation of epsilon-(gamma-glutamyl)lysine cross-links (Greenberg, et al, FASEB J. 5: 3071-3076, 1991). This posttranslational modification has been reported to dramatically alter the action of some small proteins. - For example, a transglutaminase produces a glutamine-lysine cross-link in the 13 kD phospholipase A 2 and increases its specific enzymatic activity (Cordella-Miele, et al, J. Biol. Chem. 265: 17180-17188, 1990).
  • a transglutaminase cross-links another small molecule, interleukin-2, and converts its activity to one that is cytotoxic to mature oligodendrocytes (Eitan and Schwartz, Science 261: 106-108,1993).
  • the glutamine-lysine cross-link in a protein would result in the loss of overall positive charge for that protein.
  • the transglutaminases are optimally active and generally used under reducing conditions such as dithiothreitol. The concept that glutamine-lysine cross-linking alters the activity of these small proteins may be applicable to other small molecules as well.
  • the present invention discloses a method of screening for compounds having anti- heparanase activity (AHA compounds), i.e. inhibitors of heparanase activity (IHA), comprising the steps of: contacting a potential AHA compound with radiolabeled heparin heparan sulfate and heparanase for a time and under such conditions sufficient to allow for inhibition of heparanase activity; detecting inhibition of heparanase activity; and selecting compounds that inhibit heparanase activity.
  • AHA compounds anti- heparanase activity
  • IHA heparanase activity
  • the present invention also discloses the amino acid sequence identity of the heparanase that has been purified to homogeneity by chromatography under reducing conditions.
  • the present invention provides a purified heparanase, and a method for producing it.
  • the present invention provides recombinant heparanase and a means for producing it.
  • the term "purified heparanase” as used in the specification and claims includes the recombinant heparanase as described in the subject application.
  • the recombinant heparanase of the subject invention can be used for the same purposes and in the same manner as the purified heparanase.
  • the purified heparanase of the present invention has an isoelectric point of less than 5.5 (preferably about 4.8 - 5.1) and preferably is activated by treatment with transglutaminase using reducing conditions.
  • the recombinant heparanase of the present invention has an isoelectric point of less than 5.5 (preferably about 4.8 - 5.1), and is isolated under reducing conditions and is activated by treatment with transglutaminase.
  • Suitable transglutaminases that may be used for this purpose include Activated Factor
  • Xma guinea pig liver transglutaminase, epidermal transglutaminase, keratinocyte transglutaminase, and tissue transglutaminase.
  • the heparanase of the present invention has the amino acid sequence (SEQ. ID. NO: 1) of:
  • the present invention provides a heparanase having the amino acid sequence (SEQ ID NO: 3) of:
  • the present invention provides a heparanase having the amino acid sequence (SEQ ID NO: 5) of: Gly Lys Glu Glu Ser Leu Asp Ser Asp Leu Tyr Ala Glu Leu Arg Cys 1 5 10 15
  • the present invention provides a heparanase having the amino acid sequence (SEQ ID NO: 7) of: Glu Leu Arg Cys Met Cys He Lys Thr Thr Ser Gly He His Pro Lys 1 5 10 15
  • the foregoing amino acid sequences correspond to the products of a single gene called platelet basic protein (Walz and Baggiolini, BBRC 159: 969-981, 1989; Castor, et al, BBRC 163: 1071-1078, 1989).
  • the complete gene sequence of platelet basic protein is well known. See, for example, Wenger et al., Blood, 73: 1498-1503, 1989 and Proc. Natl. Acad. Sci. USA, 90, 3660-3664, 1993.
  • the present invention also provides heparanase having the amino acid sequences of other members of the CXC chemokine family [including Platelet factor 4 (SEQ. ID NO. 12), ⁇ IP-10 (SEQ. ID NO. 14), gro/MGSA (SEQ. ID NO. 16), gro- /M ⁇ P-2a (SEQ. TD NO. 18), gf /'MIP-2 ⁇ (SEQ. ID NO. 20), Interleukin-8/NAP-l (SEQ. ID NO. 22) and ENA-78 (SEQ. ID NO. 24)] as well as members of the CC chemokine family [including MlP-l ⁇ (SEQ. ID NO. 26), MlP-l ⁇ (SEQ. ID NO.
  • transglutaminases that may be used for this purpose include Activated Factor Xl ⁇ a, guinea pig liver transglutaminase, epidermal transglutaminase, keratinocyte transglutaminase, and tissue transglutaminase.
  • the present invention provides a heparanase having the amino acid sequence (SEQ ID NO: 12) of:
  • the present invention provides a heparanase having the amino acid sequence (SEQ ID NO: 14) of:
  • the present invention provides a heparanase having the amino acid sequence (SEQ ID NO: 16) of:
  • the present invention provides a heparanase having the amino acid sequence (SEQ ID NO: 18) of:
  • CTCTCCTCCT CGCACAGCCG CTCGAACCGC CTGCTGAGCC CCATGGCCCG 51 CGCCACGCTC TCCGCCGCCC CCAGCAATCC CCGGCTCCTG CGGGTGGCGC 101 TGCTGCTCCT GCTCCTGGTG GCCGCCAGCC GGCGCGCAGC AGGAGCGCCC
  • the present invention provides a heparanase having the amino acid sequence (SEQ ID NO: 20) of:
  • the present invention provides a heparanase having the amino acid sequence (SEQ ID NO: 22) of:
  • the present invention provides a heparanase having the amino acid sequence (SEQ ID NO: 24) of:
  • the present invention provides a heparanase having the amino acid sequence (SEQ ID NO: 26) of:
  • the present invention provides a heparanase having the amino acid sequence (SEQ ID NO: 28) of:
  • the present invention provides a heparanase having the amino acid sequence (SEQ ID NO: 30) of:
  • the present invention provides a heparanase having the amino acid sequence (SEQ ID NO: 32) of:
  • the present invention provides a heparanase having the amino acid sequence (SEQ ID NO: 34) of:
  • MCP-3 monocyte chemoattractant protein 3
  • the present invention provides a heparanase having the amino acid sequence (SEQ ID NO: 36) of:
  • the present invention provides a heparanase having the amino acid sequence (SEQ ID NO: 38) of: Met Arg He Ser Ala Thr Leu Leu Cys Leu Leu Leu He Ala Ala Ala Phe Ser He Gin Val
  • the present invention provides a heparanase having the amino acid sequence (SEQ ID NO: 40) of:
  • the purified heparanase of the present invention allows for the convenient selection of compounds having anti-heparanase activity (AHA compounds), i.e. inhibitors of heparanase activity (IHA), by measuring inhibition of heparanase activity. Inhibition of heparanase activity can be measured utilizing in vivo radiolabeled heparan sulfate/heparin. This ligand is radiolabeled to high specific activity by intraperitoneal injection of 0.5mCi of S-35 sulfate into C57 mice bearing a 1-2 cm basement membrane tumor (EHS; Engelbreth, Holm, Swarm tumor).
  • EHS Engelbreth, Holm, Swarm tumor
  • the tumor is harvested after 16 hours and the heparan sulfate proteoglycan extracted in 4 volumes of 6M urea, 20mM Tris pH 6.8, protease inhibitors, 0.15M NaCl and 0.5% triton X- 100.
  • the urea extract is chromatographed on an anion exchange column and the proteoglycan is eluted in a linear gradient of NaCl.
  • the radiolabeled proteoglycan is exchanged into a solution of 4.0M guanidine-HCl, 20mM Tris pH 7.4 and applied to a size exclusion column.
  • the proteoglycan peak is pooled and exchanged into 0.15mM NaCl and 20mM Tris pH7.4. Purified, radiolabeled proteoglycan is coupled to commercially available agarose support.
  • a quantitative assay of heparanase activity is constructed with the radiolabeled ligand in a multi- well format. Briefly, known quantities of recombinant heparanase are added to a multi-well plate containing equal amounts of radiolabeled ligand in each well. Enzyme-ligand interaction proceeds overnight and the ligand-agarose complex is recovered by centrifugation. Radioactivity in the liquid phase is determined by scintillation counting and is the measure of enzyme activity. Potential enzyme inhibitors can be evaluated by adding the compound to the solution phase or alternatively adding the assay components to multi-well plates containing preweighed amounts of test compound.
  • the purified heparanase of the subject invention can be used for therapeutic wound healing or can be immobilized onto filters and used to degrade heparin from the blood of patients post-surgery.
  • Wound treatment can be achieved by administration to an afflicted individual an effective amount of a pharmaceutical composition comprising the purified heparanase in combination with a pharmaceutically acceptable, preferably slow releasing, carrier.
  • a pharmaceutically acceptable, preferably slow releasing, carrier e.g. PCT US90/04772 incorporated herein by reference.
  • Immobilization onto filters can be achieved by the methods well known in the art including those disclosed by Langer et al. in Biomaterials: Inter-facial Phenomenon and Applications, eds. Cooper et al, pp 493-509, 1982 and those described in U.S. Patent No. 4,373,023, 4,863,611 and 5,211,850 (all incorporated herein by reference).
  • the purified heparanase of the subject invention can be prepared by the method described in procedure A or procedure B, but preferably procedure A.
  • PROCEDURE A Reverse transcription of the mRNA from activated human leukocyte-derived cells [preferably lymphocytes, neutrophils, platelets, Jurkitt lymphoma cells, Dami cells (Greenberg et al., Blood 72:1968-1977, (1988)] is used to prepare the cDNA for the desired heparanase enzyme (preferably SEQ. ID. NO: 1; optionally SEO ID. NO: 3, SEQ. ID. NO: 5, SEQ. ID. NO: 7; SEQ. ID. NO: 13, SEQ. JD. NO: 15, SEQ. ID. NO: 17, SEQ. ID. NO: 19, SEQ. JD. NO: 21, SEQ. ID. NO: 23, SEQ. ID. NO: 25, SEQ. ID. NO: 27, SEQ.
  • Serum-free medium conditioned by infected sf9 cells is collected after 72 hours. This media is the starting material for purification of recombinant heparanase. Serum-free conditioned media is adjusted to contain 20mM Sodium Acetate, pH 5.0, 0.15M NaCl, ImM reduced glutathione (GSH), ImM dithiothreitol (DTT) and lOmM beta-octylglucoside. Medium is applied to a column of cation-exchange resin (Pharmacia) and eluted from the column in a linear gradient of NaCl. Fractions containing heparanase are pooled and diluted to a final salt concentration of 0.15M NaCl.
  • ID. NO: 1 from human blood cells or cell lines (such as platelets) under reducing conditions which allow for the occurrence of post-translational modifications that increase the specific activity of heparanase and make it suitable for use in the above described screening assay.
  • the cells are treated with a suitable activator (such as, but not limited to, thrombin or histamine) which allows for the release of enzymes and cytokines from the cell.
  • a suitable activator such as, but not limited to, thrombin or histamine
  • Reducing agents are added to the supernatant from the activated cells.
  • Suitable reducing agents include dithiothreitol (DTT), dithioerythritol (DTE), reduced glutathione (GSH), and ⁇ -mercaptoethanol.
  • the reduced, activated supernatant is chromatographed on a column of immobilized heparin or heparan sulfate under reducing conditions at pH 5, using a salt gradient (such as NaCl, KC1, or other salt) to elute the bound proteins.
  • a salt gradient such as NaCl, KC1, or other salt
  • Fractions containing heparanase activity are pooled and exchanged into any buffer appropriate for the pH of 6.8 and containing 0.15 M NaCl, reducing agents, and non-ionic detergent. This is passed over any suitable anion-exchange column (bed volume of 5 ml or less).
  • the unbound material from this column is adjusted to pH 5 with acid, and is loaded onto any suitable cation-exchange column (bed volume of 5 ml or less), equilibrated in a suitable pH 5 buffer containing 0.15 M NaCl, reducing agents, and non-ionic detergents.
  • the bound protein is eluted from the column with a salt gradient, and the fractions containing heparanase activity are pooled and size fractionated to below 30,000 daltons with 30 K-cut-off membranes.
  • the protein below 30,000 daltons is concentrated by either heparin-sepharose chromatography or by centrifugation through 5 K-cut-off membranes.
  • Example 1 Use of Heparanase as a screen for AHA compounds.
  • 35 S-Heparan sulfate-Sepharose 6B is resuspended in: 0.15 M NaCl, 0.03% human serum albumin, 10 ⁇ M MgCl 2 , 10 ⁇ M CaCl 2 , antiproteolytic agents (1 ⁇ g/ml leupeptin, 2 ⁇ g/ml antipain, 10 ⁇ g/ml benzamidine, 10 units/ml aprotinin, 1 ⁇ g/ml chymostatin, and 1 ⁇ g/ml pepstatin), and 0.05 M Na acetate, pH 5.6 and 5,000 cpm, in a total volume of 200 ⁇ l, are aliquoted into each well of a 96 well plate. To each well is added 5 units of activated heparanase and the digestion allowed to proceed overnight at 37 degrees.
  • a potential inhibitor of heparanase activity would be identified by its ability to reduce the amount of radiolabeled heparan sulfate released into the supernatant by 50% at a concentration of 1 ⁇ M or less.
  • Example 2 The preparation of heparanase under reducing conditions as outlined in Procedure B. Part A:
  • Platelet-rich plasma (10 9 platelets/ml; 1800 r. l) is obtained from healthy, ---formed volunteers by plasmapheresis. The plasma is removed from the platelets by centrifugation
  • Heparin-Sepharose Chromatography Activated platelet supernatants are pooled and adjusted to contain 1 mM GSH and 1 mM DTT. This pool is loaded (2.5 ml min) onto a column of heparin-sepharose (2.6 x 7.5 cm, 40 ml) equilibrated in 1 mM GSH, 1 mM DTT, 150 mM NaCl, 10 mM NaPO 4 , pH 7.4.
  • the column After loading the sample, the column is washed with 200 ml of 0.15 M NaCl, 1 mM GSH, 1 mM DTT, 10 mM Na acetate, pH 5, foUowed by 60 ml of 0.35 M NaCl, 1 mM DTT, 1 mM GSH, 10 mM Na acetate, pH 5.
  • the column is then eluted with a 160 ml linear gradient between 0.35 M NaCl and 1.5 M NaCl in the same buffer.
  • the flow- through and wash with equilibration buffer are collected as one pool.
  • the column is then eluted with 10 ml of 0.15 M NaCl, 10 mM ⁇ -octylglucoside, 1 mM GSH, 1 mM DTT, 10 mM Na acetate, pH 5, followed by 10 ml of 1.5 M NaCl, 10 mM ⁇ -octylglucoside, 1 mM GSH, 1 mM DTT, 10 mM Na acetate, pH 5. Aliquots of each pool are used for determination of heparanase activity by the "Purification Assay".
  • Part C Properties of the purified heparanase.
  • the final yield of heparanase protein from 1850 ml platelet-rich plasma was 2.7 mg. Protein concentration was determined by the method of Lowry J. Biol. Chem. 193: 265-275, 1951), or if more precise determinations were required, by amino acid analysis on an amino acid analyzer (Beckman 6300). The overall recovery of activity was 8%, with a 4150-fold purification. The preparation was judged to be homogeneous by the presence of a single band of 9000 daltons on an 18% silver-stained SDS-polyacrylamide gel, run according to the method of Laemmli (N ⁇ t ⁇ re 227: 680-685, 1970).
  • the pH optimum of the purified heparanase was determined by conducting the "Purification assay" activity between pH 3.5 and 8.0, using a citrate buffer (pH 3.5 - 6.0), citrate-phosphate buffer (pH 6.5 - 7.0), and phosphate buffer (pH 7.5 - 8.). Heparanase was active between pH 5.0 and 8.0, with the optimum pH at 5.8. N-terminal amino acid sequencing of heparanase produced by this procedure was performed using a gas/liquid phase Protein Sequencer (Applied Biosystems Inc. Model 470).
  • Phenylthiodantoin amino acids were resolved and quantitated by an on-line HPLC system (Model 120, Applied Biosystems Inc.) with data analysis on a Nelson Analytical System.
  • N- terminal amino acid sequences of the heparanase produced in this example were 85 % SEQ. ID. NO: 9 (namely: Asn Leu Ala Lys Gly Lys Glu Glu Ser Leu Asp Ser Asp Leu Tyr Ala 1 5 10 15
  • Glu Leu Arg which is identical to CTAP-HI, and 15% SEQ. ID. NO: 10 (namely: Ser Ser Thr Lys Gly Gin Thr Lys Arg Asn Leu Ala Lys Gly Lys Glu), 1 5 10 15 which is the precursor form, platelet basic protein.
  • SEQ. ID. NO: 10 namely: Ser Ser Thr Lys Gly Gin Thr Lys Arg Asn Leu Ala Lys Gly Lys Glu
  • 1 5 10 15 which is the precursor form, platelet basic protein.
  • the N-terminal sequence of commercial ⁇ -thromboglobulin namely, Calbiochem (Cat. # 605165), Celsus Laboratories (Cat.
  • Chromatofocusing of the heparanase produced by this procedure results in two peaks of differing isoelectric points.
  • heparanase is dissolved in 0.025 M imidazole, pH 7.3.
  • the sample is loaded onto a 0.5 x 20 cm column of Polybuffer Exchanger 94 (Pharmacia), equilibrated with 0.025 M imidazole, pH 7.3.
  • Polybuffer 74 (Pharmacia; 1:8, pH 4) is pumped onto the column at 0.5 ml/min.
  • All of the platelet basic protein processed forms have pi's that are calculated and reported to be greater than 7.6.
  • the heparanase activity resides in the platelet basic protein and/or the processed form, CTAP-IH that is modified such that the pi is lowered to 4.8 - 5.1.
  • Heparanase obtained after rltromatofocusing exhibited a specific activity of 80 unitr g protein, using the "Purification Assay.” This represents a 1000-fold increase in the specific activity compared to the commercial protein ( ⁇ -thromboglobulin; 0.075 units/ ⁇ g protein).
  • the modification that may be responsible for the lower isoelectric point of active heparanase is ADP-ribosylation.
  • ADP-ribosylation (Adenine diphosphate-ribosylation) is a post ⁇ translational modification of proteins or D ⁇ A in which the ADP-ribose group of NAD (Nicotinamide adenine dinucleotide) is enzymatically transfe ⁇ ed to proteins or DNA.
  • the PVDF membrane was immunoblotted with the anti-Peptide C antisera (1:1500 in PBS containing 5% dry milk, 0.05% Tween-20, 0.15 M NaCl, 20 mM Tris, pH 7.4, 2 hours at room temperature, followed by incubation with peroxidase-labeled goat anti-chicken IgG (1:500 in above buffer, 1 hour room temperature), and reacted with a peroxidase substrate.
  • the immunoblot revealed that the 8000 dalton that was labeled with [ 32 P] was CTAP- ⁇ l/heparanase.
  • CTAP-m/heparanase has an auto-ADP-ribosylation activity, since the [ 32 P] -ADP-ribosylation of CTAP-HI/heparanase occurs in reactions where the only protein present is commercial CTAP-HI or purified heparanase.
  • Other chemokine family members tested which includes IP-1-, IL-8, gro- ⁇ , and MCAF, also have auto-ADP-ribosylation activity.
  • CTAP-HI/heparanase is a consequence of ADP-ribosylation of the enzyme in the presence of nitric oxide. It is further contemplated that the action of transglutaminase on the ADP-ribosylated enzyme will lead to further increase in the specific activity.
  • heparanase with transglutaminase results in a substantial (about 13-fold) increase in the specific activity of the enzyme.
  • the heparanase (2 wl at 56 nM) obtained by Example 2, Part B is treated with either transglutaminase from guinea pig liver (4 mU; Sigma) or with Factor XIH (1 ⁇ g; Celsus Laboratories, Inc.), the blood coagulation factor that is activated by treatment with 5 units of thrombin at 37 degrees for 30 minutes.
  • Heparanase is activated by incubation of either 2mU liver transglutaminase or 5 units of activated Factor XIH in the presence of 0.1 M NaAcetate buffer at pH 6.0 containing ImM reduced glutathione and ImM CaCl for 35 minutes at 37 degrees. Treatment of heparanase with either type of transglutaminase results in a substantial increase in the specific activity of the heparanase.
  • CTAP-HI ai . , lr-terleukin-8, a CXC chemokine family member assures that an essentially identical folding pattern will be shared by the two proteins. Since the 3-dimensional structure of Interleukin-8 is known (Clore, et al., Biochemistry 29: 1689-1696,1990; Baldwin, et al, J. Biol. Chem. 265: 6851-6853), one can model the same for CTAP-HI. Such a model can serve to direct r arch into rationally designed IHA and to help explain the action of transglutaminase in activating the CTAP-HI.
  • Part D Purification Assay for Heparanase Activity Heparanase activity from platelets or column fractions is detected by its ability to digest the > 70 kD S-HSPG to produce lower molecular weight products. Each digest contains 10 ⁇ l sample, 35 S-HSPG (2000 cpm), 0.15 M NaCl, 0.03% human serum albumin, 10 ⁇ M MgCl 2 , 10 ⁇ M CaCl 2 , antiproteolytic agents (1 ⁇ g/ml leupeptin, 2 ⁇ g/ml antipain, 10 ⁇ g/ml benzamidine, 10 units/ml aprotinin, 1 ⁇ g ml chymostatin, and 1 ⁇ g/ml pepstatin), and 0.05 M Na acetate, pH 5.6 in a total volume of 300 ⁇ l.
  • Digests are carried out for 3 to 21 h. The presence of lower molecular weight radiolabeled products is detected by centrifugation through 30,000 MW-cutoff filters.
  • the digests containing 2000 cpm of 35 S-HSPG (> 70 K) are centrifuged through 30,000 molecular weight cut-off filters (MiUipore Ultrafree-MC 30,000 NMWL filter units).
  • 35 S-HSPG degradation is evident by the presence of radioactivity in the filtrate that passed through the 30 K membrane; this heparanase activity is expressed as the % of total cpm ⁇ 30 K for a given digest. Analysis of heparan sulfate degradation by this method is quick and reproducible.
  • 1 unit of heparanase activity is defined as 1% cpm ⁇ 30 K per h.
  • the 0.1 M Na acetate buffer is replaced by 50 mM citrate, citrate-phosphate, or phosphate buffer at varying pH's.
  • concentration of a thiol oxidant (diamide) needed for optimum activity is determined. This concentration (100 ⁇ M diamide) is added to all assay tubes when reduced samples are assayed. Preparation of 35 S-HSPG (>70 K) for use in the 'Purification Assay.”
  • 35 S-HSPG (>70 K) is prepared from mice bearing a basement membrane tumor that overproduces HSPG (EHS tumor), using modifications of the method of Ledbetter, et. al., 1987. Briefly, the radiolabeled HSPG was prepared by injecting C57BL mice bearing the EHS tumor (Orkin, et.al., 1977) with sodium [ 35 S]sulfate (0.5 mCi/mouse) 18 h before harvesting the tumor.
  • the HSPG is extracted from the weighed tumor with 6 volumes (w/v) of Buffer A (3.4 M NaCl, 0.1 M 6-aminohexanoic acid, 0.04 M EDTA, 0.008 M N-ethylmaleimide, 0.002 M PMSF, and 0.05 M Tris-HCl, pH 6.8), by homogenization with a Polytron for 30 s, followed by stirring at 4°C for 1 h. Insoluble material is collected by centrifugation (12,000 x g for 10 min), and the supernatant is discarded. The insoluble residue is reextracted with 2 volumes (original tumor weight) of Buffer A for 30 min with stirring at 4°C.
  • Buffer A 3.4 M NaCl, 0.1 M 6-aminohexanoic acid, 0.04 M EDTA, 0.008 M N-ethylmaleimide, 0.002 M PMSF, and 0.05 M Tris-HCl, pH 6.8
  • Buffer A 3.4 M NaCl, 0.1
  • Insoluble material is again collected by centrifugation, and the supernatant fraction is discarded.
  • the insoluble material is then suspended in 6 volumes of Buffer B (6 M urea, 0.1 M 6-aminohexanoic acid, 0.04 M ethylenediaminetetraacetic acid (EDTA), 0.002 M PMSF, and 0.05 M Tris-HCl, pH 6.8), homogenized with an electric homogenizer (Polytron) for 30 s, and stirred for 2 h at 4°C.
  • the mixture is centrifuged to remove insoluble material, and the supernatant is retained.
  • the insoluble material is reextracted with 2 volumes of Buffer B.
  • the mixture is centrifuged, and the supernatant is combined with the previous supernatant.
  • 35 S-HSPG is isolated from the Buffer B supernatant by sequential chromatography on anion exchange and gel filtration columns.
  • the Buffer B supernatant is dialyzed overnight against 10 volumes of 6 M urea, 0.15 M ⁇ aCl, 0.05 M Tris-HCl, pH 6.8, and is adjusted to contain 0.5% non-ionic detergent (Triton X-100).
  • This supernatant (from 11 g tumor) is chromatographed on a 30 ml column of anion exchange resin (DEAE-Sephacel) equilibrated with 6 M urea, 0.15 M ⁇ aCl, 0.05% Triton X-100, 0.05 M Tris-HCl, pH 6.8.
  • Example 3 Preparation of cD ⁇ A encoding Heparanase. Media is removed from cultured HEL (HEL 92.1.7; Human erythroleukemia; ATCC No.
  • TIB 180 cells stimulated with lOnM phorbol 12-myristate 13-acetate (Sigma Chemical Co., St. Louis, MO) and the cells scraped from the dish and pelleted by centrifugation. The pellet is extracted with 200wl of TRI reagent (Molecular Research Center Inc. Cincinnati, OH) and the total cellular RNA is prepared according to the manufacturer's instructions.
  • TRI reagent Molecular Research Center Inc. Cincinnati, OH
  • the reverse transcriptase reaction was performed with lOwl of total cellular RNA in the presence of 4 «1 of 5x transcripiase buffer (Bethesda Research Laboratories, Gaithersburg, MD), l ⁇ l 0.2mM DTT, 4 ⁇ l random hexanucleotides (Amersham Corp.
  • Media is removed from cultured leukocyte-derived cells [e.g., lymphocytes, neutrophils, platelets, Jurkatt lymphoma cells, Dami ceUs (Greenberg et al., Blood 72:1968-1977, (1988)], stimulated 7'ith Concanavalin A or phorbol 12-myristate 13 acetate (Sigma Chemical Co., St. Louis, MO) and the cells scraped from the dish and pelleted by centrifugation. The pellet is extracted with 200 «1 of TRI reagent (Molecular Research Center Inc. Cincinnati, OH) and the total cellular RNA is prepared according to the manufacturer's instructions.
  • leukocyte-derived cells e.g., lymphocytes, neutrophils, platelets, Jurkatt lymphoma cells, Dami ceUs (Greenberg et al., Blood 72:1968-1977, (1988)
  • 7'ith Concanavalin A or phorbol 12-myristate 13 acetate Sigma Chemical Co., St. Louis
  • the reverse transcriptase reaction was performed with 10 ⁇ l of total cellular RNA in the presence of 4 ⁇ l of 5x transcriptase buffer (Bethesda Research Laboratories, Gaithersburg, MD), lwl 0.2mM DTT, 4wl random hexanucleotides (Amersham Corp. Arlington Heights, ELL), and lwl lOmM dNTP (BRL).
  • 5x transcriptase buffer Bethesda Research Laboratories, Gaithersburg, MD
  • lwl 0.2mM DTT 4wl random hexanucleotides
  • BNL lwl lOmM dNTP
  • the polymerase chain reaction is carried out as follows. To 3wl of the first strand (above) is added lwl of each Primer (see below), 77wl of water lOwl lOx PCR buffer (Perkin Elmer Cetus, Norwalk CT) and 2wl each dNTP. This solution is heat denatured at 95 degrees C and lwl Amplitaq DNA polymerase (Perkin Elmer Cetus) is added. Hybridization temperature begins at 72 degrees and is lowered by one degree per cycle until reaching 55 degrees. Each hybridization step is followed with a constant elongation temperature of 72 degrees. Upon completion the solution is left at 0 degrees until storage at -20 degrees.
  • the cDNA encoding heparanase is preferably cloned into a vector designed for expression in eukaryotic cells, rather than into a vector designed for expression in prokaryotic cells (e.g. E. coli).
  • Eukaryotic cells are preferred for expression of genes obtained from higher eukaryotes because the signals for synthesis, processing, and secretion of these proteins are usually recognized, whereas this is often not true for prokaryotic hosts (Ausubel, et al, ed., in Short Protocols in Molecular Biology, 2nd edition, John Wiley & Sons, publishers, pg.16-49, 1992.).
  • Eukaryotic hosts may include, but are not limited to, the foUowing: insect cells, African green monkey kidney cells (COS cells), Chinese hamster ovary cells (CHO cells), and Murine 3T3 fibroblasts.
  • the N-terminal peptide has the following sequence (SEQ ID NO: 41: Asn Leu Ala Lys Gly Lys Glu Glu Ser Leu Asp Ser Asp Leu Cys, in which the final Cys residue was added to regions of known sequence (SEQ ID NOS: 1,3) for the purpose of conjugation to a carrier protein.
  • the C-terminal peptide has the following sequence (SEQ ID NO: 42): Cys Asn Gin Val Glu Val He Ala Thr Leu Lys Asp Gly Arg Lys He Cys Leu Asp Pro Asp Ala Pro Arg He Lys Lys He Val Gin Lys Lys encoded by the cDNA sequence (SEQ ID NO: 43) of TGCAACCAAG TCGA GTGAT AGCCACACTG AAGGATGGGA GGAAAATCTG CCTGGACCCA GAT TCCCA GAATCAAGAA AATTGTACAG
  • AAAAAA AAAAAA.
  • These peptides (SEQ ID NOS: 41 and 42) were produced by stepwise solid phase peptide synthesis on an Applied Biosystems 430A Peptide Synthesizer.
  • the 9- fluoroenylmethyloxycarbonyl (Fmoc) group was used as the N ⁇ amino protecting group, and temporary side-chain protectin groups were as follows: Arg (Pmc), Asn (Trt), Asp (OtBu), Gin (Trt), Glu (OtBu), His (Trt), Lys (Boc), Ser (tBu), Thr (tBu).
  • Each residue was single coupled using a HBTU/NMP protocol and capped with acetic anhydride before the next synthesis cycle.
  • the crude peptides were purified by preparative reverse phase chromatography on a Phenomenex C-18 column (22.5 x 250 mm) using a water/acetonitrile gradient, each phase containing 0.1% trifluoracetic acid (TFA). Clean fractions, as determined by analytical HPLC, were pooled, the acetonitrile was evaporated under reduced pressure, and the aqueous solution was lyophillized.
  • the purified peptides were characterized by time of flight or FAB mass spectroscopy.
  • SEQ ID NO: 42 can be produced by recombinant DNA methodology as stated in Procedure A (page 21).
  • the synthetic peptides of CTAP-HI/NAP-2 were conjugated to keyhole limpet hemocyanin utilizing a maleimide-activated carrier protein (Pierce Chemical Co. #77107). 300 ⁇ g of conjugated peptides were injected into chickens using Freunds complete adjuvant. The antisera were collected 5 weeks after initial immunization. Specific recognition by the antisera of commercial CTAP-IH (2.5 ⁇ g, (Celsus Laboratories Inc., Cincinnati, Ohio; Cat #.
  • isolated heparanase 1.5 ⁇ g
  • 10 ⁇ l of the platelet supernatant used for purification was achieved by separating the proteins on a reducing 18% polyacrylamide gel (Novex), transferring to nitrocellulose, and incubating with the pre-immune or antisera (1:1500), followed by incubation with a peroxidase labeled goat anti-chicken IgG (1:500; Kierkegaard and Perry) in the presence of PBS containing 5% dry milk and 0.05% Tween-20.
  • the pre-immune sera did not recognize 7 - 10 kD proteins in the commercial CTAP-HI, isolated heparanase, or platelet supernatants.
  • Computer assisted modeling can be accomplished using programs for automated docking of molecules within 3D databases, as described in DesJarlais, R.L., Sheridan, R.P., Seibel, G.L., Dixon, J.S., Kuntz, I.D., Venkataraghavan, R., "Using shape complementarity as an initial screen in designing ligands for a receptor binding site of known three-dimensional structure"; J. Med. Chem. 31:722-729, 1988.
  • Val Glu Val lie Ala Thr Leu Lys Asp Gly Arg Lys lie Cys Leu Asp 50 55 60 Pro Asp Ala Pro Arg lie Lys Lys lie Val Gin Lys Lys Leu Ala Gly 65 70 75 80
  • Lys Thr Thr Ser Gly lie His Pro Lys Asn lie Gin Ser Leu Glu Val 35 40 45 lie Gly Lys Gly Thr His Cys Asn Gin Val Glu Val lie Ala Thr Leu 50 55 60
  • Lys Asp Gly Arg Lys lie Cys Leu Asp Pro Asp Ala Pro Arg lie Lys 65 70 75 80 Lys lie Val Gin Lys Lys Leu Ala Gly Asp Glu Ser Ala Asp
  • AAAAACATCC AAAGTTTGGA AGTGATCGGG AAAGGAACCC ATTGCAACCA AGTCGAAGTG 180
  • Pro Asp Ala Pro Arg lie Lys Lys lie Val Gin Lys Lys Leu Ala Gly 50 55 60
  • Lys lie Cys Leu Asp Leu Gin Ala Pro Leu Tyr Lys Lys lie lie Lys 85 90 95 Lys Leu Leu Glu Ser
  • Glu Lys Leu Glu lie lie Pro Ala Ser Gin Phe Cys Pro Arg Val Glu 50 55 60 lie lie Ala Thr Met Lys Lys Lys Gly Glu Lys Arg Cys Leu Asn Pro 65 70 75 80 Glu Ser Lys Ala lie Lys Asn Leu Leu Lys Ala Val Ser Lys Glu Met
  • CCTCTCCCAT CACTTCCCTA CATGGAGTAT ATGTCAAGCC ATAATTGTTC TTAGTTTGCA 480
  • AAGCTTGCCT CAATCCTGCA TCCCCCATAG TTAAGAAAAT CATCGAAAAG ATGCTGAACA 360 GTGACAAATC CAACTGACCA GAAGGGAGGA GGAAGCTCAC TGGTGGCTGT TCCTGAAGGA 420
  • CTCTCCTCCT CGCACAGCCG CTCGAACCGC CTGCTGAGCC CCATGGCCCG CGCCACGCTC 60 TCCGCCGCCC CCAGCAATCC CCGGCTCCTG CGGGTGGCGC TGCTGCTCCT GCTCCTGGTG 120
  • GTGTTTGAGC ATCACTTAGG AGAAGTCTTC TATTTATTTA TTTATTTATT TATTTGTTTG 540
  • ATTATCTCAC ATTATGTGTT CAACATTTTT ATGCTGAAGT TTCCCTTAGA CATTTTATGT 900
  • Gin Thr Thr Gin Gly Val His Pro Lys Met lie Ser Asn Leu Gin Val 20 25 30
  • Lys Asn Gly Lys Glu lie Cys Leu Asp Pro Glu Ala Pro Phe Leu Lys 50 55 60
  • GACTCTGCCT CAGCTGGGCC TCCACTGCCC ACCCATCTAT AGATGCCTAA ATCCCGGGCA 180 GTTATCCAGA CACAACTAAA GTTCCATCCC TTCCATGAAG CCTTCCCCAA CCCTCTGGTG 240
  • ATCTAAGCAC TATTCATTCT TTAAGGCATG TATTTCCAAG CCTTTTAATT TTTTCATGCC 900 TAGAGTTGGC ATGGCATATA TATATCTTTA TACAATTCTT CAAATTTTAT AGAATTTGTA 960
  • TCTCAAACTC CTGACCTCAG GTGATCTGCA GCCTCGGCCT CCAAAGTGTT GGGATTACAG 1800 GTGTGAGCGA CCATGCCTGG CTGCATAGAC CAGTTCTTAT GAGAAGGGAT CAACTAAGAA 1860
  • TTCCCCCTGT CCCCTCCACC TTCCCTCACA GTGTGTCTGG TGACAACCGA GTGGCTGTCA 3900 TCGGCCTGTG TAGGCAGTCA TGGCACCAAA GCCACCAGAC TGACAAATGT GTATCAGATG 3960
  • CTGTGATCTT CAAGACCATT GTGGCCAAGG AGATCTGTGC TGACC( AAG CAGAAGTGGG 300
  • TTTTATTTTA TTATAATGAA TTTTGTTTGT TGATGTGAAA
  • CATTATGCCT TAAGTAATGT 480 TAATTCTTAT TTAAGTTATT GATGTTTTAA GTTTATCTTT CATGGTACTA GTGTTTTTTA 540
  • TATTTTAATT TAATCTTCCA TGGATTTTGG TGGGTTTTGA ACATAAAGCC TTGGATGTAT 600

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Abstract

Une héparanase purifiée présente une activité supérieure à 20 et de préférence, à 50 unités/νg de protéine. On décrit l'utilisation de l'héparanase pour cribler des composés anti-héparanase. De plus, on décrit l'utilisation d'héparanase à forte dose destinée à accélérer la guérison des blessures ou comme filtre à héparanase immobilisée relié à des dispositifs extra-corporels afin de dégrader l'héparine et d'en neutraliser les propriétés anticoagulantes pendant une intervention chirurgicale.
EP94922654A 1993-07-29 1994-07-26 Utilisation de l'heparanase pour identifier et isoler un compose anti-heparanase Withdrawn EP0708838A1 (fr)

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TW565614B (en) * 1996-07-18 2003-12-11 Univ Australian Detection of mammalian heparanase activity and purification of mammalian heparanase
CA2265923A1 (fr) * 1996-09-13 1998-03-19 Sagami Chemical Research Center Proteines humaines comportant des sequences de signaux secretoires et adn codant ces proteines
US6737252B2 (en) 1997-07-25 2004-05-18 Schering Corporation 7 transmembrane receptor family member BLRX
US5968822A (en) * 1997-09-02 1999-10-19 Pecker; Iris Polynucleotide encoding a polypeptide having heparanase activity and expression of same in transduced cells
AU772311B2 (en) * 1997-09-02 2004-04-22 Hadasit Medical Research Services & Development Company Ltd Polynucleotide encoding a polypeptide having heparanase activity and expression of same in transduced cells
US6348344B1 (en) * 1997-09-02 2002-02-19 Insight Strategy & Marketing Ltd. Genetically modified cells and methods for expressing recombinant heparanase and methods of purifying same
JP2002510462A (ja) * 1997-10-28 2002-04-09 ジ・オーストラリアン・ナショナル・ユニバーシティー 哺乳類のエンドグルクロニダーゼをコードする単離された核酸分子およびその使用
US6121023A (en) * 1998-01-22 2000-09-19 Akzo Nobel N.V. Isothermal transcription based assay for the detection and quantification of the chemokine rantes
GB9802725D0 (en) 1998-02-09 1998-04-08 Ciba Geigy Ag Organic compounds
US6387643B1 (en) 1998-02-24 2002-05-14 Pharmacia And Upjohn Company Human platelet heparanase polypeptides, polynucleotide molecules that encode them, and methods for the identification of compounds that alter heparanase activity
AU2878600A (en) 1999-03-01 2000-09-21 Hadasit Medical Research Services & Development Company Ltd Polynucleotide encoding a polypeptide having heparanase activity and expression of same in genetically modified cells
EP1176200A3 (fr) 2000-06-20 2005-01-12 Switch Biotech Aktiengesellschaft Utilisation des polypeptides ou leurs acides nucléiques pour le diagnose ou traitement des maladies de la peau ou de la cicatrisation de blessures et leurs utilisations pour l'identification des substances pharmacologiquement actives
US6656699B2 (en) * 2000-09-15 2003-12-02 Reddy Us Therapeutics, Inc. Methods and compositions for glycosidase assays
CH696701A5 (de) 2001-12-18 2007-10-15 Hoffmann La Roche Verfahren zum Testen eines Mittels auf dessen Fähigkeit, die Heparanaseaktivität zu hemmen.
CN101243319B (zh) * 2005-06-22 2016-01-06 约翰·霍普金斯大学 卵巢癌的生物标记：ctap3-相关蛋白质
US7713521B2 (en) 2005-08-12 2010-05-11 Schering Corporation MCP1 fusions
US8524217B2 (en) 2010-05-11 2013-09-03 Merck Sharp & Dohme Corp. MCP1-Ig fusion variants
US11448657B2 (en) 2011-01-18 2022-09-20 Rambam Med-Tech Methods and kits for assessing Heparanase procoagulant activity, compositions comprising Heparanase, and methods for the treatment of coagulation-related disorders
EP2665488B1 (fr) 2011-01-18 2018-05-09 Health Corporation - Rambam Méthodes et kits pour l'évaluation de l'activité procoagulante de l'héparanase, compositions comprenant de l'héparanase, et méthodes pour le traitement de troubles associés à la coagulation
WO2021128255A1 (fr) * 2019-12-27 2021-07-01 深圳市海普瑞药业集团股份有限公司 Charge d'affinité, son procédé de préparation et son utilisation

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