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WO1991019197A1 - Localisation immunochimique de l'heparanase dans des melanomes de la souris et de l'homme - Google Patents

Localisation immunochimique de l'heparanase dans des melanomes de la souris et de l'homme Download PDF

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Publication number
WO1991019197A1
WO1991019197A1 PCT/US1991/003832 US9103832W WO9119197A1 WO 1991019197 A1 WO1991019197 A1 WO 1991019197A1 US 9103832 W US9103832 W US 9103832W WO 9119197 A1 WO9119197 A1 WO 9119197A1
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Prior art keywords
heparanase
endoglycosidase
antibody
container means
glycosaminoglycan
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PCT/US1991/003832
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English (en)
Inventor
Garth L. Nicolson
Motowo Nakajima
Li Jin
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Board Of Regents, The University Of Texas System
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Priority to CA002083052A priority Critical patent/CA2083052A1/fr
Publication of WO1991019197A1 publication Critical patent/WO1991019197A1/fr

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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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3053—Skin, nerves, brain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/5743—Specifically defined cancers of skin, e.g. melanoma

Definitions

  • the immunochemical localization of the glycosamino endoglycosidase, heparanase, in human and murine tumor types is an object of the present invention.
  • the present invention also relates to an assay for endoglycosidase enzymic activity and a labeled substrate for use in such an assay.
  • the assay of the present invention is viewed as useful for the detection of cancerous malignancies.
  • proteoglycans form the ground substance in the extracellular matrix of connective tissues.
  • proteoglycans are polyanionic substances of high molecular weight and contain many different types of heteropolysaccharide side chains covalently linked to a polypeptide backbone. These proteoglycans may contain over 95% carbohydrates.
  • the polysaccharide groups of the proteoglycans were formerly called mucopolysaccharides but now are preferably termed glycosaminoglycans since all contain derivatives of glucosamine or galactosamine.
  • a variety of enzymes may be involved in the normal metabolic degradation of proteoglycans.
  • glycosaminoglycans initial proteo- glycan degradation often involves proteolysis to separate or digest protein components. Such proteolysis results in the production of glycosaminoglycans.
  • the glycosaminoglycans in turn are subject to glycosamino- glycan endoglycosidase enzymic action which produces smaller glycosaminoglycan fragments.
  • the glycosamino- glycans or fragments thereof are subject to glycosamino ⁇ glycan exoglycosidase enzymic action which produces monosaccharides from the non-reducing ends of glycosaminoglycans.
  • the highly metastatic line had much higher 35 S liberating activity than did the low metastatic line.
  • the background described herein involves an interest in convenient, accurate and reproducible endoglycosidase assays, particularly since endoglycosidases may play critical roles in the establishment of tumor metastases.
  • Metastasis formation occurs via a complex series of unique interactions between tumor cells and normal host tissues and cells. These processes involve several discrete and selective steps such as: invasion of surrounding tissues, penetration of lymphatics of blood vessels and transport in lymph or blood, or dissemination into a serous cavity, arrest and invasion at distant sites, and survival and growth to form secondary lesions.
  • Basement membranes are continuous sheets of extra ⁇ cellular matrix composed of collagenous and non- collagenous proteins and proteoglycans that separate parenchymal cells from underlying interstitial connective tissue. They have characteristic permeabilities and play a role in maintaining tissue architecture. Metastasizing tumor cells must penetrate epithelial and endothelial basement membranes during invasion and metastasis, and the penetration and destruction of basement membranes by invasive tumor cells has been observed using electron microscopy. Since basement membranes are rigid structures formed from unique sets of macromolecules, including type IV collagen, laminin, heparan sulfate (HS) , proteoglycan and fibronectin, the successful penetration of a basement membrane barrier probably requires the active participation of more than one tumor cell-associated enzyme.
  • HS heparan sulfate
  • HS heparan sulfate
  • metastatic B16 melanoma cells degrade matrix glycoproteins, such as fibronectin, and matrix sulfated glycosaminoglycans, such as heparan sulfate. Since heparan sulfate was released in solution as fragments approximately one-third their original size, it has been proposed that metastatic tumor cells characteristically have a heparan sulfate endoglycosidase.
  • glycosaminoglycans The abilities of B16 cells to degrade HS from various origins and other purified glycosaminoglycans (heparin, chondroitin 4- sulfate, chondroitin 6-sulfate, dermatan sulfate, keratan sulfate, and hyaluronic acid) has been studied.
  • an analytic p rocedure was developed using high-speed gel permeation chromatography (Irimura et al., (1983) Anal. Biochem. V 130, p 161; Nakajima et al., (1984) J. Biol. Chem. V 259, p 2283).
  • [ 35 S]sulfate was purified from basement membrane producing EHS sarcoma and PYS-2 carcinoma cells, and subendothelial matrices of bovine aortic endothelial (BAE) and corneal endothelial (BCE) cells (ibid) .
  • BAE bovine aortic endothelial
  • BCE corneal endothelial
  • the newly formed reducing termini of HS fragments were investigated by: labeling with NaB[ 3 H] 4 ; hydrolysis to monosaccharides; and analysis of these saccharides by paper chromatography. Since 3 H-reduced terminal mono- saccharides from HS fragments were overwhelmingly (>90%) L-gulonic acid, the HS-degrading enzyme responsible was an endoglucuronidase (heparanase) .
  • HS-degrading endoglucuronidases have been found in various tissues, such as human skin fibroblasts, rat liver cells, human placenta, and human platelets. HS- degrading endoglucuronidases in mammalian cells were reported previously by other investigators to be "heparitinases" to indicate heparitin sulfate (heparan sulfate)-specific endoglycosidase. However, heparitinase originally was used to designate an elimination enzyme (EC 4.2.2.8) in Flavobacterium heparinum.
  • HS-specific endoglycosidases in mammalian cells are endo ⁇ glucuronidases, except for one found in skin fibroblasts, it was proposed that mammalian cell endoglucuronidases capable of degrading HS should be called "heparanases", consistent with the currently used term "heparan sulfate”.
  • Glycosaminoglycan endoglycosidases have been assayed for enzyme activity by some other means.
  • Oldberg et al. (1980, Biochem. V 19, pp 5755-5762) described an assay for a platelet endoglycosidase which degraded heparin-like polysaccharide. This assay involved measuring a decreasing amount of 3 H-heparan sulfate, the decrease being a function of endoglycosidase activity.
  • Endoglycosidase assays using solid-phase substrates were described by Iverius (1971, Biochem. J. V 124, pp 677-683) and Oosta et al. (1982, J. Biol. Chem. V 257, pp 11249-11255) .
  • Iverius coupled a variety of glycosamino ⁇ glycans to cyanogen bromide-activated Sepharose 4B beads.
  • the endoglycosidase hyaluronidase was assayed for enzymic activity by incubation of the enzyme with chondroitin sulfate bound to Sepharose 4B.
  • the enzyme activity was monitored by following the production of soluble uronic acid with a colorimetric assay procedure.
  • Oosta et al. described an assay for heparitinase, an endoglycosidase from platelets which cleaves heparin and heparan sulfate.
  • the Oosta et al. system and assay comprised:
  • glycosaminoglycans were cross- linked to agarose by the reaction of free amino groups of glycosaminoglycans and amino-reactive cyanogen bromide- activated agarose. Since glycosaminoglycans, such as heparin and heparan sulfate, have several free glucosamine amino groups, this type of crosslinking results in excessive covalent linkages between substrate molecules and agarose gel, resulting in a loss of susceptibility to endoglycosidases and nonlinear rates of degradation. Thus the most desirable solid-phase substrate for glycosaminoglycan endoglycosidase is glycosaminoglycan crosslinked to a solid support at one end of the molecule such as reducing terminal.
  • Heparanase an endo-?-D-glucuronidase, has been associated with melanoma metastasis.
  • Polyclonal antibodies directed against the murine N-terminal heparanase peptide detected a M r ⁇ 97,000 protein on SDS- polyacrylamide gel electrophoresis of mouse melanoma ad human melanoma cell lysates.
  • human A375-SM and mouse B16-BL6 melanoma cells were stained with the anti-heparanase antibodies.
  • Heparanase antigen was localized in the cytoplasms of permeabilized melanoma cells as well as at the cell surface of unpermeabilized cells.
  • a solid phase substrate which yields soluble labeled products upon hydrolysis by a glycosaminoglycan endoglycosidase and methods of producing said substrate are comprised in the present invention.
  • the solid phase substrate comprises glycosaminoglycan bearing labeled N- acetyl groups and being reductively aminated at its reducing terminal end to produce an amine-terminus.
  • the substrate is further coupled to an amino-reactive solid matrix through its amine-terminus.
  • a method of producing the solid phase substrate comprises the steps of: at least partially N-desulfating or N-deacetylating a glycosaminoglycan; labeling at least partially N-deacetylated or N-desulfated glycosamino- glycan to produce labeled glycosaminoglycan; completely N-acylating the labeled glycosaminoglycan with acyl anhydride or acyl halide; reductively aminating a reducing terminal end of said labeled glycosaminoglycan to produce labeled amine-terminal glycosaminoglycan; and coupling, through its terminal amine, the labeled amine- terminal glycosaminoglycan to an amine-reactive solid phase support to produce the solid matrix substrate.
  • the labeling may be accomplished by substitution on amino groups of the partially N-desulfated or N- deacetylated glycosaminoglycan of a substance yielding a detectable signal.
  • This substance may be a radioisotopic label, a fluorescent label or an enzymatic label.
  • a fluorescent label is preferred for ease of assay and a radioisotopic label for similarity to the natural glycosaminoglycan.
  • Fig. 1. Synthesis of a solid-phase heparanase substrate: Chemical modification and radiolabeling of HS and its coupling to amino-reactive agarose gel bead.
  • Fig. 2 Elution profiles on high-speed gel permeation chromatography of unmodified- and chemically modified-HS before and after treatment with B16 melanoma heparanase.
  • HS heparan sulfate
  • NDS-HS N-desulfated heparan sulfate
  • NDS-HAc-HS N-desulfated N-acetylated heparan sulfate.
  • Arrows (a)-(e) indicate the elution positions of the standard glycans: (a) C6S from shark cartilage (M r 60,000); (b) HS from bovine lung (M r 34,000); (c) heparin from porcine mucosal tissue (M r 11,000); (d) monosialosyl biantennary complex-type glycopeptide from porcine thyroglobulin (M r 2190) ; (e) N-acetyl-D- glucosamine (M r 221) .
  • Fig. 3. Dose dependent degradation of partially N- desulfated N[ 14 C]acetylated heparan sulfate (PNDS-N[ 14 C]Ac- HS) immobilized on agarose by B16 melanoma cell heparanase.
  • the PNDS-N[ C]Ac-HS (4500 cpm) immobilized on agarose was incubated with various amount of B16 cell extract for 6 h (open squares), 12 h (open circles), and 24 h (open hexagons) , or with various amounts of heat inactivated (100°C, 5 min) B16 cell extract for 12 hr (open triangles) in the presence of SAL.
  • the released radioactivity in a half volume of the supernatant versus the amount of cell extract added (ug protein) was plotted.
  • Fig. 4 shows the levels of heparanase activity in the sera of controls and patients with malignant melanoma.
  • Fig. 5 shows levels of heparanase activity in sera of rats injected with a highly metastatic adenocarcinoma.
  • Fig. 6 shows the relationship between serum heparanase activity and the size of a primary metastatic tumor in rats.
  • Fig. 7 shows the relationship between rat serum heparanase levels and numbers of metastases from a malignant tumor.
  • Fig. 8 shows the position of substrate hydrolysis for melanoma heparanase.
  • FIG 9 Autoradiograph of a 7.5% SDS- polyacrylamide gel containing proteins immunoprecipitated from human A375-SM (a-d) and mouse B16-BL6 (e-h) melanoma cell lysates metabolically labeled with [ 35 S]-methionine.
  • Cell lysates were incubated with PBS (d,h) , biotinylated anti-heparanase antibodies (b,c,f,g), or biotinylated anti-heparanase antibodies preincubated with heparanase- peptide (a,e) . They were subsequently incubated with streptavidin-agarose (a-h) and samples were run in the presence of ⁇ -mercaptoethanol. The molecular weight markers at the far right are expressed in kDa.
  • FIG 10 Micrographs of the immunocytochemical localization of heparanase in human A375-SM melanoma cells.
  • Cultured cells were fixed (a-f) , or permeablized (a,c-f) . They were then incubated with anti-heparanase antibodies (a,b,e), with anti-heparanase antibodies preabsorbed with heparanase peptide (d) or with PBS (c,f). The cells were then washed and incubated with peroxidase-conjugated goat-anti-rabbit IgG (a,b,d,f) or PBS (c,e).
  • Original magnification x25; bar 30 ⁇ .
  • the micrographs are of the same magnification.
  • FIG 11 Immunohistochemical localization of heparanase in a human cutaneous melanoma (a,b,c) or murine melanoma metastasis in the lung (d,e,f) .
  • Frozen sections fixed with 2% paraformaldehyde were stained with hematoxylin and eosin (a,d) or incubated with anti- heparanase antibodies (c,f) or anti-heparanase antibodies preabsorbed by heparanase peptide (b) or PBS (e) .
  • the sections were then incubated with goat-anti-rabbit IgG conjugated with peroxidase (b,c,e,f).
  • Original Magnification x80; bar 20 urn.
  • the micrographs are of the same magnification.
  • Immunoassays such as those using antibodies raised to an glycosaminoglycan endoglycosidase such as heparan sulfate endoglycosidase, for example, are described, which measure the enzyme.
  • the present invention also describes a new assay for glycosaminoglycan endoglycosidase activity, most preferably that of the heparan sulfate endoglycosidase termed "heparanase”.
  • This new assay describes using a solid phase substrate which yields soluble labeled products upon hydrolysis by a glycosaminoglycan endoglycosidase.
  • the new assay also describes novel adaptations of this solid phase enzymatic assay to liquid-phase conditions.
  • Extracellular matrix is a tissue barrier that tumor cells must penetrate in the process of tumor invasion and metastasis.
  • Extracellular matrix includes basement membranes and connective tissue stroma and is composed of collagens, proteoglycans, laminin, fibronectin and other glycoproteins. Degradation of this matrix, an important step in tumor cell invasion and metastasis, is mediated by a variety of degradative enzymes, including proteases and glycosidases of tumor or host cell origin (Mullins et al., Biochim . Biophy ⁇ . Acta , 695, 177-214 (1983); Nakajima et al., J. Cell . Biochem .
  • Structural analogues of heparanase substrate such as heparin and its chemically modified derivatives, inhibited B16 murine melanoma pulmonary metastases in experimental metastasis assays (Irimura et al., Biochemistry, 25, 5322-5328 (1986); Villanueva et al., 1988) . Therefore, tumor cell heparanase is thought to play an important role in melanoma invasion and metastasis.
  • anti- heparanase antibodies directed against the N-terminal heparanase peptides were prepared and characterized.
  • heparanase was localized at the cell surfaces and in the cytoplasm of human and mouse melanoma cells.
  • This solid phase substrate comprises a glycosamino ⁇ glycan bearing radioisotopically labeled N-acyl groups.
  • These labeled N-acyl groups are preferably 3 H labeled or "c labeled acetyl groups although other labeled acyl groups, such as formyl or propionyl groups may be used.
  • the solid phase substrate of the present invention may comprise as the glycosaminoglycan: hyaluronic acid, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, heparin, or combinations thereof.
  • glycosamino ⁇ glycans will allow assays for the enzymic activity of endoglycosidases having a substrate specificity for the particular glycosaminoglycan being used.
  • the amino-reactive solid matrix to which the amine- terminal labeled glycosaminoglycan may be bound may have many acceptable forms, both in the basic nature of the matrix and in the a ine-reactive chemical site.
  • a preferable solid matrix is agarose-based, most preferably Sepharose or Sepharose derivatives in bead form (Pharmacia) .
  • Other solid matrices such as celluloses or polyacryla ides may be used provided that the have amine-reactive substituent functions for coupl ⁇ ing.
  • Sepharose beads may be acti ⁇ vated with cyanogen bromide and then coupled to amine- bearing molecules such as heparin, other glycosamino ⁇ glycans or glycosaminoglycan derivatives.
  • Cyanogen bromide mediated coupling is a usable coupling method for practice of the present invention.
  • Cyanogen bromide activated agarose or any other amine-reactive solid matrix may couple to more than one amine group of a glycosaminoglycan and glycosaminoglycan derivative with multiple amine functions.
  • N-hydroxy succinide ester is a preferable amine-reactive function bound to a solid matrix and is commercially available or readily synthesized.
  • Such N-hydroxysuccinide esters couple to primary amine groups at a pH between about 6 and 9. Aga ⁇ rose may be activated by periodate oxidation to contain aldehyde functions. This aldehydic agarose may be reacted with labeled amine-terminal glycosaminoglycan and the linkage stabilized by reduction with sodium cyanoborohydride. (Perikh et al., Methods in Enzym. Vol XXXIV, p 81 Acad. Press (1974)).
  • the glycosaminoglycans generally have their amine functions either sulfated or acetylated. After at least partial N-desulfation or N-deacetylation, for example, the resultant primary amino groups on the glycosaminoglycan are available for labelling.
  • Deacetylation may be accomplished by hydrazinolysis under conditions avoiding excessive alkalinity which could lead to hybrolysis of glucosaminyl linkages.
  • Desulfation may be accomplished by formation of pyridinium salts of the glycosaminoglycan followed by solvolysis in dimethylsulfoxide.
  • Amino group labeling is accomplished by reaction with a fluorescent compound such as fluorescein isothiocyanate, an enzyme such as alkaline phosphatase (and a bifunctional coupling agent) or with a radioisotopically labeled acyl anhydride or acyl halide.
  • a label is then covalently attached to at least some of the free amine groups.
  • Remaining free amine groups of the labeled glycosaminoglycan are then acylated, for example by acetic anhydride treatment.
  • the acylated labeled glycosaminoglycan is then aminated at its reducing terminal. This amination is accomplished by incubation with an amine salt to form a Schiff base with the terminal and subsequent reduction to form a terminal amine.
  • Amino group labeling may be accomplished by coupling a measurable compound or active protein to at least a few of the amino groups.
  • the measurable compound may be one of the many known to be highly absorbent of visible light or more preferably one which is fluorescent when excited by irradiation at particular wavelengths such as fluorescein as mentioned above. Labeling by attachment of enzymes (as alkaline phosphatase mentioned above) as active proteins to the partially N-desulfated or N- deacetylated glycosaminoglycan is also a possibility. Labeling by enzymes or measurable compounds having light absorbent or fluorescent structures, however, may involve sterically bulky substituents.
  • Such sterically bulky substituents may, when substituted at too high a level, render glycosaminoglycan derivatives which are poor substrates for glycosaminoglycan endoglycosidases.
  • a partially N-desulfated heparan sulfate was coupled in a 1:1 ratio to fluoroscein isothiocyanate. This fluorescein labeled derivative was found to be a good substrate for melanoma heparanase. It is contemplated that up to a 10:1 ratio of fluorescein to HS may be produced and serve as a heparanase substrate.
  • enzyme labels generally contain free amine groups which may bind to amine-reactive solid matrices.
  • One preferred label for glycosaminoglycans is a radioisotopic label similar or identical in structure to naturally occurring N-substituents. While 35 S-sulfate N-substituents could be utilized, 14 C- or 3 H-acetyl N-substituents are preferred as readily produced.
  • a solid phase substrate for a glycosaminoglycan endoglycosidase is created. As described elsewhere herein, this solid phase substrate yields soluble radio- isotopically labeled substances as a function of glycosaminoglycan endoglycosidase enzymatic activity.
  • An alternative manner of measuring this same activity would be to observe the disappearance of radioisotopic label bound to the solid matrix as a function of enzymatic activity. This type of measurement has the disadvantage of being a negative measurement and also that incubation supernatant would have to be carefully removed from residual solid matrix substrate.
  • the solid phase substrate of the present invention is one which yields soluble products labeled with a detectable signal upon hydrolysis by a glycosaminoglycan endoglycosidase.
  • This solid phase substrate comprises a glycosaminoglycan bearing a label which does not prevent hydrolysis of the labeled glycosaminoglycan by a glycosaminoglycan endoglycosidase.
  • the labeled glycosaminoglycan is linked through a single end, preferably the reducing terminal end and by a single covalent linkage, to a solid matrix.
  • the detectable signal may be radioisotopic, light absorbent, fluorescent or enzymatically active.
  • the solid matrix is preferably hydrophilic and may include polymers such as cellulose, dextran, polyacrylates or their derivatives, alone or in combination.
  • the substrate of the present invention may be soluble if a detectable label is present along with a tagging molecule.
  • the tagging molecule may be used as a 1 handle 1 for removal of a portion of attached glycosaminoglycan.
  • the labeling of at least partially desulfated or deacylated glycosaminoglycan is most preferably accom ⁇ plished by treatment with 3 H-acetic anhydride or 14 C- acetic anhydride, although analogous acetyl halides, particularly chlorides or also alkyl bromides are contemplated as useful.
  • analogous acetyl halides particularly chlorides or also alkyl bromides are contemplated as useful.
  • other coupling methods may be used in this labelling procedure.
  • the substrate of this invention may also be a liquid phase substrate with separation of the cleaved products from the uncleaved substrate occurring after the enzymatic reaction.
  • a glycosaminoglycan such as heparan sulfate, for example, could be tagged at one end, preferably the reducing end, to another molecule.
  • the glycosaminoglycan should be labeled at additional sites by other molecules such as 125 I, fluorescein, enzymes, and the like, that may be used for detection of cleaved products in the assay.
  • a liquid substrate of the type described herein should be an assay with increased sensitivity to the action of glycosaminoglycan endoglycosidases. This increased sensitivity would at least in part relate to an enhanced availability in solution to soluble enzymes.
  • the molecular tag at one end of the glycosaminoglycan could be either a small molecule, such as fluorescein or biotin, or a larger molecule, such as a peptide or a protein.
  • the linkage of this molecule to an end of the glycosaminoglycan substrate must not significantly inhibit the hydrolysis of the tagged glycosaminoglycan by the glycosaminoglycan endoglycosidase.
  • the molecular tag should have the ability to act as a potential 'handle 1 for the labelled glycosaminoglycan chain and for the residue of the glycosaminoglycan chain remaining after cleavage by a glycosaminoglycan endoglycosidase.
  • the molecule would be able to act as a point of attachment for a protein molecule having affinity for the bound tagging molecule.
  • Such a protein-molecule relationship will enable tagged portions of the labeled glycosaminoglycan to be readily separated from labeled but untagged portions liberated by endoglycosidase- induced hydrolysis of glycosaminoglycan substrate hydrolysis.
  • the molecular tag should be either: a) a haptenic molecule capable of generating specifically binding antibodies when attached to a carrier such as a protein and immunogenically administered to an animal; b) a segment of or a whole immunogenic substance such as a protein or peptide; or c) a substance having a high binding affinity for existent proteinaceous molecules such as avidin or protein A, for example.
  • the uncleaved products may then be separated from the cleaved products by incubation with, for example, solid-phase antibodies having an affinity for the tag. Proteins other than antibodies that bind the molecular tag that has been attached to the end of the glycosaminoglycan may also be used to separate uncleaved glycosaminoglycan. If solid phase antibodies or solid phase binding proteins are used, the solid phase may be any support that can be readily coupled or absorbed to antibodies or binding proteins and that can affect a separation of cleaved product from uncleaved substrate.
  • solid phase examples include agarose; Sepharose; polymers, such as polystyrene; glass; cellulose and glass beads; and magnetizable beads.
  • the solid-phase could be in the form of large or small particles or a tube or microtiter plate or other device that is readily adaptable to the detection system.
  • the separation of cleaved from uncleaved glycosaminoglycan products can also be achieved by an immunoprecipitation reaction that does not require antibodies to be linked to a solid phase (see Morgan et al. (1962) Proc. Soc. of Exp. Biol. Med., V 110, pp 29- 35) .
  • the precipitating antibodies could be directed toward the molecule tagged at the end of the glycosaminoglycan chain.
  • the synthetic steps to produce the solid phase substrate of the present invention generally include partial N-deacylating, for example, by hydrazinolysis, or N-desulfating, for example by solvolysis in dimethyl- ⁇ ulfoxide; an N-acylating step with labeled acyl anhydride or halide for radioisotopic labeling; a reductive amination step; and coupling to a solid matrix through the newly introduced terminal amine.
  • An often preferred final step, to insure that no amine-reactive functions remain on the solid matrix is to incubate the product of the matrix -labeled amine-terminal glycos ⁇ aminoglycan coupling with sodium cyanoborohydride and a compound bearing a free amino group.
  • This latter compound may, for example, be one such as ethanolamine or glycine ethyl ester.
  • the substrates and procedures of the present invention present numerous advantages for the assay of glycosaminoglycan endoglycosidase enzymic activity.
  • the substrate of the present invention is bound to a solid matrix via a single carbohydrate-bound amino ligand and yields a linear pattern of enzymatic products.
  • proteoglycans containing glycosa ino- glycans as well as a bound protein component have been bound to a solid matrix of cyanogen bromide-activated agarose.
  • the proteoglycan was thereby likely bound to the aqarose primarily through its proteinaceous com ⁇ ponent.
  • both proteolytic as well as glycosamino- glycan endoglycosidic activity may liberate a soluble product.
  • the specificity of the assay for enzymic activity of the endoglycosidases is less than the results shown with the present invention.
  • heparanase heparan sulfate endoglycosidase obtained from a human melanoma cell line was found to only partially degrade N-desulfated, N-acetylated heparin. This same enzyme preparation was found to efficiently cleave N-desulfated heparan sulfate as well as N-desulfated N-acetylated heparan sulfate into characteristic degradation fragments.
  • glycosaminoglycan endoglycosi ⁇ dases heparan sulfate endoglycosidase or heparanase
  • the endoglycosidase utilizing heparan sulfate as a preferred substrate was chosen as a typical example to demonstrate a preferred embodiment of the present invention.
  • an N-hydroxy succinide agarose derivative was selected as a preferred solid matrix to couple labeled amine-terminal heparan sulfate to produce a solid phase substrate.
  • Heparanase activity produced soluble radioisotopically labeled products as demonstrated specifically in many of the following examples.
  • Melanoma heparanase is an endo-/3-glucuronidase which specifically cleaves HS at intrachain sites. Such melanoma heparanase specificity is illustrated in Figure 8. Thus, the separation of the reaction products from the substrates based on their size is required for the heparanase assay. Although previously established methods such as polyacrylamide gel ele ⁇ trophoresis and high-speed gel permeation chromatography are useful for the characterization of degradation fragments, they are not suitable for rapid and microscale quantitative assays of large sample numbers. To perform rapid quantitative assays, a covalently linked substrate is required.
  • the presently developed solid phase assay substrate is partially N-deacetylated or N-desulfated, N-[ 3 H or 14 C]- acetylated HS coupled with Affi-Gel 15.
  • a HS derivative is linked to agarose through only one covalent bond ( Figure 1) .
  • This product is one of the most sensitive endoglycosidase substrates to be developed.
  • This substrate has now been successfully used for mouse and human melanoma heparanase assays.
  • the same type of derivative has also been produced by using Reacti-Gel (NW-65F) (Pierce, Rockford, IL) .
  • Affi-Gel 15 and Reacti-Gel use quite large particles and these retain significant amounts of high molecular weight materials in the gel matrices. This may be a problem in some quantitative heparanase assays, therefore, we developed a more desirable assay substrate by using Affi-Gel 701 or 702 (Bio-Rad) which are approximately 1-3 microns in diameter with an exclusion limit of M r 10,000.
  • the specific synthetic procedure was as follows. Radiolabeled HS was reduced with sodium borohydride to form a sugar alcohol at the reducing terminal. The sugar alcohol was converted to a primary aldehyde by periodate oxidization.
  • Radiolabeled HS whose amino groups have been sulfated or acetylated should be aminated at the reducing terminal with ammonia under reducing conditions.
  • Affi-Gel 702 should be converted to an amino-reactive bead by derivatization with N-hydroxysuccinimide or N,N*-carbonyldiimidazole, and then the aminated radioactive HS should be linked to amino-reactive Affi-Gel 702.
  • the substrate may be made more radioactive by use of 125 I-labeled HS, although iodination of HS with Bolton and Hunter Reagent may be disadvantageous because of potential structural change.
  • the assay may also be improved by use of fluorescein-labeled HS for routine clinical studies, and fluorescein-labeled HS is suitable for a rapid analysis of degradation fragments on HPLC equipped with a flow fluorescence detector.
  • the assay measuring levels of a glycosaminoglycan endoglycosidase such as heparan sulfate endoglycosidase (heparanase) may also be performed in an immunoassay format using polyclonal and/or monoclonal antibodies raised to the endoglycosidase.
  • heparanase heparan sulfate endoglycosidase
  • antibodies with relatively low cross-reactivity to other endoglyco ⁇ sidases such as the platelet endoglycosidase described by Oldberg, et al. (1980) Biochem. , V 19, pp 5755-5762, can be used.
  • the antibodies may be used with a variety of immunoassay techniques to measure the endoglycosidase protein directly.
  • the endoglycosidase may be measured by either a radioimmunoassay described by Berson and Yalow (1968) Clin. Chem, Acta. r V 22, p 51 or an immunoradio- metric (IRMA) assay described by Miles, et al. (1976) Anal. Biochem. f V 61, pp 209-224 using 1251-labeled antigen or antibody.
  • IRMA immunoradio- metric
  • the endoglycosidase may also be measured by an enzyme immunoassay that uses either a competitive-binding assay or a "sandwich” assay analogous to an IRMA and using alkaline phosphatase, horse radish peroxidase, or any other enzyme coupled to an antibody or to the endoglycosidase as reviewed by Wisdom (1976) Clin. Chem.. V 22, pp 1243-1255.
  • Melanoma heparanase an endo- ⁇ -D-glucuronidase, is thought to play an important role in melanoma metastasis (Nakajima et al . , J. Cell . Biochem . , 36, 157-167 (1988). It is one of several tumor cell degradative enzymes that have been systematically studied. Heparanase activities have been found in human and mouse melanoma cell homogenates and their conditioned media as described above. The successful preparation of anti-heparanase enabled the study of the localization of murine and human heparanases in normal and tumor tissues. To ensure the specificity of the antibodies, extensive characterization was carried out.
  • Immunoprecipitates of the antibodies with melanoma cell lysates were subjected to SDS-PAGE to determine the molecular weights of the proteins the antibodies recognized.
  • the pis of the immunoprecipitated proteins were also examined using 2-dimensional gels.
  • the finding that anti-murine heparanase antibodies cross-react with human heparanase is consistent with our previous observations on human and mouse heparanase.
  • Human and mouse heparanases share a number of biochemical characteristics, such as molecular weight, substrate and inhibitor specificity, and optimal pH for enzymatic activity.
  • Heparanase antigen was localized predominantly in the cytoplasm of both B16-BL6 and A375- SM cells, as well as at the cell surface of these cells. It remains to be determined which fraction of heparanase antigen (cell surface, cytoplasmic or both) represents the active form of the enzyme. The relationship between cell surface, cytoplasmic, and secreted forms of heparanase, and the regulatory mechanisms involved are not clear.
  • the immunocytochemical staining of B16-BL6 and A375-SM melanoma cells appeared to be heterogeneous, which is consistent with the notion of tumor cell heterogeneity in the expression of gene products.
  • heparanase activity has been correlated with the metastatic potentials of murine B16 melanoma cell lines (Nakajima et al . , J. Biol . Chem . , 259, 2283-2290 (1984) .
  • heparanase antigen was examined in mouse and human melanoma tissues.
  • Metastatic lung nodules in a murine experimental metastatic model were intensely stained with the anti-heparanase antibodies, although the background staining in normal pulmonary tissues surrounding the tumor was barely noticeable.
  • the background in normal lung tissue may be due to the presence of the pulmonary alveolar macrophages in which heparanase activity was observed (Nakajima et al . , J.
  • the present invention takes advantage of the fact that the anti-mouse- heparanase antibodies cross-reacted with human heparanase and allowed study of the immunochemical localization of heparanase antigen in human melanoma tissue. Melanoma tissues from patients with metastatic diseases were examined. Melanoma cells became stained with the antibody but the surrounding normal tissues were not except that the endothelial cells of an arteriole (Fig. 3c) were slightly stained.
  • endothelial cells may express heparanase along with other degradative enzymes in the process of neo-angiogenesis and wound healing.
  • the data presented in accord with the present invention suggest that heparanase antigens in invasive melanoma cells are significantly enriched compared to surrounding normal tissues.
  • the localization of heparanase antigen at the melanoma cell surfaces supports the concept of cell surface involvement in tumor invasion and metastasis
  • the endoglycosidase may also be measured in these assays by using fluorescein or other fluorescent compounds as reviewed by Gerson (1984) J. Clin. Immunoassay, V 7, pp 73-81, by chemiluminescence as reviewed by Weeks and Woodhead (1984) , J. Clin Immunoassay, V 7, pp 82-89, or by other labels.
  • the bound endoglycosidase may be separated from the unbound endoglycosidase by a variety of techniques.
  • a primary antibody anti-endoglycosidase
  • avidin- biotin separation using a biotin-labeled antibody and solid phase avidin
  • double antibody precipitation
  • solid phase antibody against a hapten like fluorescein coupled to a primary antibody or by using a solid phase "second antibody”.
  • Double antibody is defined as a heterologous antibody that binds the anti- endoglycosidase antibody as in Midley, et al. (1969) Acta Endocrinol. , V 63, Supp. 142, p 247).
  • the solid phase systems mentioned above can include polymers, such as polystyrene; agarose; sepharose; cellulose; glass beads; and magnetizable particles of cellulose or other polymers.
  • the solid-phase can be in the form of large or small beads or particles; tubes; plates; or other forms.
  • Kits useful in the present invention include those of the general type described by Szczesniak (U.S. Pat. No. 3,899,298). Such kits comprise a carrier being compartmentalized to receive at least one, or at least two or at least three or more containers and to maintain said containers in closed confinement.
  • a first container may contain purified anti glycosaminoglycan endoglycosidase antibody (preferably monoclonal) , either in solution, in freeze-dried form or covalently bound to the inside thereof, such as for example if such container is a test tube.
  • a second container may then contain a second anti glycosaminoglycan endoglycosidase antibody (also preferably monoclonal) .
  • another container may contain detectably labeled glycosaminoglycan endoglycosidase antigen.
  • the sample is added to the first container containing the monoclonal antibody, incubated, and then antibody from the second container is added thereto to provide a "sandwich".
  • the antibody in the second container may be detectably labeled as, for example, by a radiolabel or an enzyme label.
  • Another container in the kit may contain appropriate enzyme substrate in order to carry out the "ELISA" methodology.
  • glycosaminoglycan endoglycosidase antigen can be carried out by interpolation into a standard curve, as is known in the art.
  • a multiplicity of container means, each one having a different amount of glycosaminoglycan endoglycosidase antigen can be present in the kit for such a purpose.
  • the antibody can be immobilized onto plastic strips which are then brought into contact with the samples suspected of containing glycosaminoglycan endoglycosidase antigen. Subsequently, the strip is contacted with a solution containing a second, enzyme labeled anti glycosaminoglycan endoglyco- sidase antibody; this results in a sandwich forming on the strip. Finally, introduction of the strip into a color developing solution (such as substrate for the enzyme) and detection of color, is a rapid efficient and inexpensive method for qualitatively, and even roughly quantitatively determining glycosaminoglycan endoglycosidase antigen in animal samples.
  • a color developing solution such as substrate for the enzyme
  • the immunoassays of the present invention may use antibodies which are very discriminating between the different glycosaminoglycan endoglycosidases, particularly for heparan sulfate endoglycosidase.
  • the methodology described herein should be superior in sensitivity and ease to other known methods of glycosaminoglycan endoglycosidase detection.
  • kits are easily constructed comprising labeled glycosaminoglycan affixed, preferably through its amino-terminal end to a molecular tag as described above.
  • a kit would also comprise a specific binding agent capable of removing tagged glycosaminoglycan or tagged fragments thereof from solution.
  • the specific binding agent may already be bound to a solid matrix or may be so bound by the user of the kit and assay.
  • Preferred binding agents are proteins, more preferably, antibodies and most preferably, monoclonal antibodies.
  • Melanoma heparanase was purified from cultured murine melanoma cells.
  • Murine B16-F10 melanoma cells (Fidler, Nature (London) New Biol . 242, 148-149 (1973) were cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 nutrient mixture (DME/F12; Hazleton, Lenexa, KS) supplemented with 5% heat- inactivated fetal bovine serum (FBS; Hyclone, Logan, UT) .
  • DME/F12 Dulbecco's modified Eagle's medium
  • Ham's F-12 nutrient mixture DME/F12; Hazleton, Lenexa, KS
  • FBS heat- inactivated fetal bovine serum
  • the cell extract was centrifuged at 30,000 x g for 30 min at 4°C and the supernatant containing approximately 50 mg protein was passed through a column of heparin-Sepharose (Pharmacia LKB Biotechnology, Piscataway, NJ) equilibrated with buffer 1.
  • the heparin-Sepharose column was sequentially washed with buffer 1, 20 mM sodium acetate, pH 6.0, containing 0.2% Triton X-100 (buffer 2), and 0.15 M sodium chloride, 20 mM sodium acetate, pH 6.0 (buffer 3).
  • Heparin-binding proteins were eluted with a linear sodium chloride gradient (0.15 M - 1.2 M) in 20 mM sodium acetate, pH 6.0. The eluted materials were monitored by measuring absorbance at 280 nm, and the heparanase activity was measured using heparanse solid phase substrates as previously described (Nakajima et al. JiJial . Biochem . , 157, 162-171 (1986b). Heparanase active fractions contained approximately 6 mg proteins and 90% of total activity.
  • the eluent containing approximately 0.2 mg protein and 48% of total heparanase activity was collected and extensively dialyzed against buffer 3, and then passed through a N- acetylated N-desulfated heparin-Sepharose column (Nakajima et al. Anl . Biochem . , 157, 162-171 (1986b) equilibrated with the same buffer. After washing with 0.3 M sodium chloride, 20 mM sodium acetate, pH 6.0, binding proteins were eluted with 0.6 M sodium chloride 20 mM sodium acetate, pH 6.0.
  • the proteins were eluted with 12.5 mM Tris-HCl, 0.15 M NaCl, pH 7.5 (1 ml/min, 23°C) and the elution was monitored at 280 nm with a Waters Model 490 multiple wavelength detector.
  • the second peak fractions of molecular weight range approximately 100,000 to
  • heparanase 150,000 contained heparanase (0.04 mg protein, 29% of total activity) .
  • the heparanase fraction was further subjected to chromatofocusing using a PBE94 column (Pharmacia LKB) .
  • Starting buffer and elution buffer were 25 mM imidazole-HCl, pH 7.5 and Polybuffer 74-HC1
  • the heparanase band was cut and electroeluted using an ISCO electrophoretic concentrator Model 1750 (ISCO, Lincoln, NE) and its N-terminal amino acid sequence was analyzed by a model 470A gas-phase sequencer (Applied Biosystem Inc.) with an attached model 120A-PTH analyzer.
  • the peptide was designed to have an additional cysteine residue at C-terminus to facilitate coupling to a carrier protein, keyhole limpet hemocyanin (KLH; Calbiochem, La Jolla, CA) .
  • KLH keyhole limpet hemocyanin
  • Other protein carriers are of course usable in place of KLH.
  • Coupling of the peptides with KLH was performed by the methods described by Liu et al., Biochemistry, 18, 690-697 (1979). Briefly, LKH was suspended in 10 mM sodium phosphate buffer pH 7.2 (PB) at 20 mg/ml and dialyzed using a cellulose membrane with a molecular weight cutoff of 12,000-14,000 (Spectrum, Los Angeles, CA) against 2 L of the same buffer overnight (3 changes). The dialysate was centrifuged at 10,000 x g for 10 min to remove undissolved material. The concentration of KLH was adjusted to 16 mg/ml after protein concentration was determined by the Pierce
  • Coomassie protein assay (Pierce, Rockford, IL) using bovine serum albumin (Sigma) as a standard.
  • the peptide was dissolved in PB at a concentration of 5 mg/ml, and m- maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Sigma) was dissolved in dimethylformamide (DMF; Aldrich,
  • the activated KLH was separated from remaining low molecular weight MBS by gel filtration chromatography. It appeared at the void volume of a Bio-Gel P-30 gel filtration column (2.5 x 25 cm) and was eluted with 50 mM phosphate buffer, pH 6.0.
  • the peptide solution (5 mg/ml) was then mixed with the activated KLH, and the pH was adjusted with sodium hydroxide to 7.0-7.5.
  • the peptide-KLH mixture was centrifuged at 10,000 x g to remove undissolved material.
  • the coupling efficiency was determined by the Ellman assay for free thiol (Ellman et al., Arch . Biochem . Biophys . , 82, 70-77 (1959). The coupling efficiency was greater than 78.5%.
  • the KLH- coupled peptide antigens were aliquoted and stored at - 80°C.
  • New Zealand White rabbits were immunized subcutaneously with KLH-coupled heparanase-peptide.
  • the antigen Prior to injection, the antigen was emulsified with either complete freund's adjuvant for the primary immunization or incomplete Freund's adjuvant for subse ⁇ quent boosting immunizations.
  • Dosage of antigen was 500 ug of peptide for the first injection and 250 ⁇ g for subsequent boosting injections. The interval between first and second injections was 3 weeks and boosting injections were administered when the antisera titer began to drop.
  • Antibody activity to heparanase-peptide was determined using an enzyme-linked immunosorbant assay with peptide coated 96-well plates (Costar, Cambridge, MA) .
  • the plates were prepared by coating each well with 1 ⁇ g of synthetic peptide antigen in 100 ⁇ l of 0.1 M sodium bicarbonate, pH 9.0, and allowing the buffer to evaporate at 37°C overnight.
  • the titers of antisera raised against heparanase peptides were between 1:800 and 1:1200 when preimmune sera were used as reference.
  • the antibodies directed against heparanase-peptide were further purified by antigen affinity chromatography using heparanase-peptide covalently coupled to Affi-Gel 10 beads (Bio-Rad, Richmond, CA) .
  • the sera were first precipitated with 45% (v/v) ammonium sulfate at 4°C and centrifuged at 10,000 x g for 20 min.
  • the precipitates were then dialyzed against 1000 vol of 5 mM HEPES, 150 mM NaCl, pH 7.4, overnight at 4°C with 3 changes of buffer.
  • the dialysates were loaded on Affi-Gel-heparanase-peptide affinity columns.
  • the affinity columns were washed extensively with 5 mM HEPES, 150 mM NaCl, pH 7.4, and then eluted with 50 mM sodium citrate, 150 mM NaCl, pH 5.5, and finally with 50 mM sodium citrate, 150 mM NaCl, pH 2.0.
  • the fractions were collected and protein concentrations determined by the Pierce Coomassie Protein Assay, using IgG as the protein standard.
  • Antibody activity to heparanase-peptide was determined by ELISA assay.
  • Antibody fractions eluted at pH 2.0 were collected and designated as anti-heparanase antibodies.
  • the antibodies were biotinylated according to the procedure of Updyke et al., J. Immunol . Meth . , 73, 83-95 (1984) .
  • the heparanase-peptide was alkylated with iodoacetamide to inactivate the sulfydryl group at the C- terminus of the peptide and used to compete with anti- heparanase antibodies as a control in an immunoprecipitation experiment.
  • the reaction was carried out at a concentration of 2 mM peptide, 1 mM dithiothreitol, 20 mM iodoacetamide, in 50 mM Tris, 150 mM sodium chloride, pH 7.5, at 25°C for 1 hr.
  • the sample was concentrated and the alkylated peptide was separated on a Bio-Gel P-2 column (mesh 400) .
  • the void volume was collected and lyophilized.
  • DPBS Dulbecco's phosphate buffered saline
  • ICN [ 35 S] ethionine
  • the cells were then centrifuged at 600 x g for 5 min, the pellet lysed with 0.5% Triton X-100, 50 mM Tris-HCl, 1 mM PMSF, 5 mM NEM pH 7.5, at a ratio of 1 ml per 6 x 10 6 cells, and the incubation continued at 25°C for 10 min with mixing on a Vortex machine.
  • the specific activity was then determined by measuring trichloroacetic acid- precipitable radioactivity, and the protein concentration of the cell lysate was determined. The specific activity was higher than 5 x 10 6 cpm/ ⁇ g of protein.
  • the samples were washed once with 10 mM HEPES, 150 mM NaCl, pH 7.5, containing 0.5% Triton X-100, followed by washing with 10 mM HEPES, 150 mM NaCl, pH 7.5, 4 times.
  • the precipitates were suspended in SDS sample buffer in the presence or absence of 2.5% ⁇ - mercaptoethanol and heated at 100°C for 3 min.
  • Mouse metastatic melanoma tissues were prepared as follows. Subconfluent B16-BL6 cell monolayers were rinsed with DPBS and incubated with 2 mM EDTA in Ca 2+ , Mg 2+ -free PBS for 10 min, suspended as single cells in DME/F12, chilled in an ice-water bath before centrifugation, and resuspended in cold DME/F12 at a concentration of 2.5 x 10 5 cells/ml. Just prior to injection, the cell suspension was warmed to room temperature, and cell aliquots (5 x 10 4 cells in 0.20 ml) were injected into the lateral tail vein of 6- to 8-week- old C57/BL6 mice using a 27-gauge needle. Ten mice were used in each experiment. After 2-3 weeks, the mice were exsanguinated under anesthesia and the lungs were carefully taken for frozen sectioning.
  • Human melanoma tissues were obtained from seven patients diagnosed with malignant melanoma. Human nevi biopsies were contributed by normal donors. Frozen tissue sections were prepared as described below.
  • Sections were fixed with 2% paraformaldehyde in PBS at 25°C for 20 min. After 3 rinses with PBS, the slides were incubated for 1 hr with preimmune rabbit IgG and then with PBS, anti-heparanase antibodies, or anti- heparanase antibodies pre-incubated with synthetic peptide antigens for l hr at 25°C. Following 3 rinses with PBS, the slides were incubated with peroxidase- conjugated goat anti-rabbit IgG (Calbiochem, La Jolla, CA) at a 1:150 dilution in PBS or PBS for 1 hr.
  • peroxidase- conjugated goat anti-rabbit IgG Calbiochem, La Jolla, CA
  • AEC 3-amino-9-ethylcarbazole
  • Aldrich N,N-dimethylformamide
  • the slides were covered with cover slips using glycerol- gelatin mounting medium prepared by dissolving 10 g of gelatin (Fisher Scientific) in 60 ml distilled water with heating until the gelatin dissolved, and supplementing the gelatin solution with 70 ml glycerin and 1 ml phenol.
  • Bovine lung heparan sulfate was (HS) a kind gift from Dr. N. Di Ferrante (Baylor College of Medicine, Houston, TX) and its average M r was determined as 34,000 by sedimentation equilibrium (Nakajima, M. , et al., (1984) J. Biol. Chem. V 259, pp 2283-2290 and Irimura, T. , et al., (1983) Anal. Biochem. V 130, pp 461-468). Heparin (M r 11,000) from porcine mucosal tissue was kindly donated by Drs. M. B. Mathews, J. A. Cifonelli, and L.
  • C6S Chondroitin 6-sulfate
  • M r was determined as 60,000 as described previously (Irimura, T., et al., (1983) Anal. Biochem. V 130, pp 461-468).
  • Heparin from bovine lung and porcine intestinal mucosa and N-acetyl-D-glucosamine were obtained from Sigma Chemical Co. (St. Louis, MO) .
  • Monosialosyl biantennary complex-type glycopeptide UB-I-b (M r 2190) was prepared from thyroglobulin (Sigma) (Irimura, T., et al. , (1983) Anal. Biochem. V 130, pp 461-468). Heparitinase from Flavobacterium heparinum (EC4.2.2.8) was obtained from Miles Scientific.
  • High-speed gel permeation chromatography High-speed gel permeation chromatography. High ⁇ speed gel permeation chromatography was carried out using a high pressure liquid chromatograph system (LDC, Riviera Beach, FL) equipped with two sequential columns (0.7 x 75 cm) of Fractogel (Toyopearl) TSK HW-55(S) (MCB, Gibbstown, NJ) as described previously (Irimura, T. , et al., (1983) Anal. Biochem. V 130, pp 461-468). A one hundred microliter aliquot of sample solution was delivered into the injection port, and the chromatographic elution was performed with 0.2 M sodium chloride at a flow rate of 1.0 ml/min at 55°C (Irimura,
  • Titan III Zip Zone cellulose acetate plates (6.0 x 7.6 cm, Helena Laboratories, Beaumont, TX) were used, and electrophoresis was carried out at 70 V for 60 min in 0.5 M pyridine-acetate (pH 5.0), instead of 0.1 M barium acetate buffer employed by Cappelletti et al. (Cappeltti, et al., Anal. Biochem., V 99, pp 311-315). During the electrophoresis the buffer and cellulose acetate plates were kept below 4°C using petroleum ether cooled with ice.
  • N-desulfation and acetylation of HS was conducted by the methods of Nagasawa and Inoue (Nagasawa et al., (1977) Methods in Carbohyd. Chem. V 8, pp 291-294) .
  • the sodium salt of purified HS was converted to the pyridinium salt by cation exchange chromatography on a column of AG50WX8(H + form, Bio-Rad, Richmond, CA) and neutralization with pyridine.
  • Radioisotope labeling of HS was labeled with tritium at the reducing end as described previously (Nakajima, M. , et al., (1984) J. Biol. Chem. V 259, pp 2283-2290).
  • One milligram of purified HS was reduced with 2 Ci of NaB[ 3 H] 4 (340 mCi/mmol; New England Nuclear, Boston, MA) in 0.1 M sodium borate buffer, pH 8.0, for 5 h at 25°C.
  • the reductively aminated products were dissolved in 10 ml of 0.1 N sodium bicarbonate, pH 8.5, and mixed with Affi-Gel 15 (or Affi-Gel 10; Bio-Rad) prepared from the original suspension by successive washing in 2-propanol and then ice-cold distilled water. The mixture was incubated at 4°C for 24 h with gentle mixing. The pH was then adjusted to pH 8.5 with 0.1 N sodium bicarbonate and the incubation further continued.
  • Anhydrous hydrazine (0.5 mg, Pierce Chemical, Rockford, II.) was added to the dried HS, and the mixture was heated in a tightly screwed tube under nitrogen atmosphere at 100°C for 1 hr. After the reaction, the hydrazine was removed by repeated evaporation with toluene over sulfuric acid dessicant under vacuum conditions. To separate deacety ⁇ lated HS from residual reagents and partial degradation products, completely dried residue was dissolved in 0.5 ml water and subjected to gel filtration on a 0.8 x 30 cm column of BioGel p-10 (400 mesh) eluting with distilled water. The void volume fraction was collected and lyophilized. The yield was approximately 60% by weight.
  • the N-deacetylated HS was then N-acetylated with 50 uCi [ 14 C]-acetic anhydride (10 mCi/ mole: NEN, Boston, Ma.) or 5 mCi 3 H-acetic anhydride (400 mCi/mmole:NEN) in 0.5 ml of 4 M sodium acetate for 18 hrs. Complete N-Acetylation was accomplished by mixing with 0.1 ml of non-labeled acetic anhydride for 1 hr. 14 C- or 3 H-labeled HS was purified on the same BioGel P-10 column as described above.
  • 3 H-HS was aminated at the reducing terminal with 2 M ammonium acetate in the presence of 0.4 M sodium cyanoborohydride in 50% methanol at 50°C for 6 days.
  • Aminated 3 H-HS was purified by gel filtration as above, and the resulting solution was made to 0.1 M in sodium carbonate.
  • 1.0 ml Affi-Gel 15 was added after the gel beads were washed with isopropanol and chilled water according to the manufacturer's recommendations.
  • the coupling reaction was continued at 4°C for 48 hrs with continuous agitation.
  • the agarose beads were then washed with 4 M sodium chloride repeatedly to remove non- covalently attached 3 H-HS from the beads.
  • B16- F10 The high lung- colonizing metastatic murine B16 melanoma subline (B16- F10) and fourteen established cell lines of human malignant melanoma were employed in this study.
  • the human melanoma cell lines used were: SK-MEL-19, SK-MEL- 23, SK-MEL-93(DX1) , SK-MEL-93(DX3) , SK-MEL-93(DX6) ,
  • A375 Met Mix cells were prepared from spontaneous lung metastases of A375 parental cells in the athymic nude mice and both A375 cell lines were provided by Dr. I.J. Fidler (The University of Texas-M.D. Anderson Hospital and Tumor Institute, Houston, TX) .
  • DMEM/F12 Dulbecco's modified minimum essential medium and Ham's F12 medium
  • fetal bovine serum Hyclone, Sterile Systems, Inc., Logan, UT
  • All cell cultures were determined to be free of mycoplasma contamination with the use of mycoplasma detection system (BRL MycoTest; Bethesda Research Laboratories, Gaithersburg, MD) .
  • the incubation was carried out at 37°C with gentle mixing, and was terminated by chilling the mixture to 4°C and adding 20 ul of trichloroacetic acid to a final concentration of 5%.
  • the supernatant was obtained by centrifugation at 9800 x g for 5 min and it was subjected to analysis by high-speed gel permeation chromatography.
  • Heparanase assay using a solid-phase substrate A suspension of PNDS-N[ 14 C]Ac-HS coupled to Affi-Gel 15 was mixed with a melanoma cell extract and incubated in 400 ul of reaction buffer A containing 20 mM SAL. The enzyme reaction was terminated by chilling the solution to 4°C and mixing it with 40 ul of 50% trichloroacetic acid. After incubation for 10 min at 4°C, the mixture was centrifuged at 9800 x g for 5 min, and the supernatant was withdrawn.
  • HS labeled with tritium at the reducing end was N-desulfated by solvolysi ⁇ with DMSO, and N-desulfated [ 3 H]HS was then N- acetylated with acetic anhydride.
  • NDS-HS N- desulfated HS
  • NDS-NAc-HS N-desulfated N-acetylated HS
  • N- desulfation of HS resulted in a significant loss of negative charge; however, the total negative charge was partially recovered by acetylation of free amino groups.
  • the average molecular size of NDS-HS and NDS-NAc-HS were determined by high-speed gel permeation chromatography, and were found to be 30,000 and 31,500, respectively (Fig. 2).
  • Each of HS, NDS-HS and NDS and NDS-NAc-HS was incubated with B16 melanoma cell extracts in the presence of SAL (a potent exo-3-glucuronidase inhibitor) , and the incubation products were analyzed by high-speed gel permeation chromatography.
  • N-Desulfated and N-acetylated heparin The known structures of HS and heparin suggested that N-desulfation and subsequent N-acetylation of heparin may generate local structures similar to those present in HS.
  • Heparin is a potent inhibitor of B16 melanoma heparanase (Nakajima, M. , et al., (1984) J. Biol. Chem. V 259, pp 2283-2290) ; however, its heparanase inhibitory activity is lost by the removal of N-sulfate (Irimura, et al. (1985) J. Cell. Biochem. V 9A, p 148) .
  • N-sulfate in HS is unnecessary for its cleavage by melanoma heparanase
  • N-desulfated N- acetylated heparin was used as a heparanase substrate.
  • NDS-NAc-HS N-desulfated N-acetylated heparin
  • NDS-NAc- heparin had an apparent M r of about 10,500 as determined by high-speed gel permeation chromatography; and its relative electrophoretic mobility on cellulose acetate in 0.2 M pyridine-acetate buffer, pH 5.0, was 0.87 when the electrophoretic mobility of 3 H-labeled heparin was taken as 1.00.
  • NDS-NAc-heparin was not further cleaved and the major peak of NDS-NAc-heparin on high- speed gel permeation chromatography did not shift to a lower M r even after a prolonged incubation. This suggested that N-desulfation and subsequent N-acetylation of heparin can result in the generation and/or exposure of heparanase-susceptible glucuronosyl linkages in a part of the heparin molecule. Thus, NDS-NAc-heparin cannot be utilized as a melanoma heparanase assay substrate.
  • the reducing terminal saccharides of PNDS-N[ 14 C]Ac-HS were reductively aminated with 2M ammonium formate and 0.4 M sodium cyanoborohydride in 50% methanol.
  • the products having free amino groups only on the reducing termini were then coupled to aminoreactive agarose beads such as Affi-Gel 10 or Affi-Gel 15.
  • PNDS-N[ 1 C]Ac-HS was also conjugated to Affi-Gel 10 under the same conditions used for the coupling of PNDS- N[ 14 C]Ac-HS to Affi-Gel 15.
  • the coupling efficiency was low, less than 1%, between pH 7.5 and 8.5. Therefore, a positive charge spacer at the aminoreactive site of Affi-Gel 15 may be important in the effective coupling of PNDS-N[ 14 C]Ac-HS to Affi-Gels.
  • Affi-Gel 15 one of the best heparanase assay substrates was produced: PNDS-N[ 14 C]Ac-HS immobilized on agarose through only one covalent linkage at the reducing terminal end.
  • N[ 14 C]Ac-HS immobilized on agarose gel to HS degrading enzymes was examined by incubating the substrates (4500 cpm, 15 ug) with bacterial heparitinase (EC.4.2.2.8) at a concentration of 5 units/ml in 0.1 M sodium acetate buffer, pH 7.0, containing 1 mM calcium acetate (Linker, et al. (1972) Methods Enzymol. V 28, pp 902-911). Most of 1A C activity (82%) appeared in the supernatant of the incubation mixture after a 24 h incubation, indicating that PNDS-N[ 14 C]Ac-HS immobilized on agarose is very susceptible to HS degrading enzymes.
  • the remainder of the PNDS-N[ 14 C]Ac-HS was not released from the gel, even after prolonged incubation. This could be explained by the limitation of using a Flavobacterium heparitinase.
  • the same amount of substrate (4500 cpm) was incubated with B16 cell extract for various periods in the presence of 20 mM SAL to prevent the sequential degradation by exogylcosidas ⁇ s.
  • the relationships between the amounts of cell extract (ug protein) added and the release 14 C activity are shown in Fig. 3. In this case, the maximum amount of released 14 C activity were 56% of the total 14 C activity present in the solid-phase substrates.
  • heparin The effect of heparin on the degradation of the solid-phase substrates was investigated by addition of substrate-equal amount (15 ug) of heparin from porcine intestinal mucosa or heparin from bovine lung to the incubation mixture containing B16-F10 cell extract (80 ug protein) and PNDS-N[ 14 C]Ac-HS immobilized on agarose.
  • substrate-equal amount 15 ug
  • B16-F10 cell extract 80 ug protein
  • PNDS-N[ 14 C]Ac-HS immobilized on agarose.
  • the addition of either heparin caused up to 80% inhibition of the degradation of the solid-phase substrates, consistent with our previous results (Nakajima, M. , et al., (1984) J. Biol. Chem. V 259, pp 2283-2290).
  • All the human melanoma cells showed the ability to degrade HS in the presence of SAL as shown in Table 1.
  • Six of these human malignant melanoma cell lines such as SK-MEL-93(DX1) , SK-MEL- 93(DX6), Hs939, M40, A375 Met Mix, and A375 M6 demonstrated significantly greater ability to degrade HS than did mouse B16 melanoma subline F10.
  • A375 Met Mix and A375 M6 cells were selected from A375 parental cells by their ability to colonize the lung in athymic nude mice. They were reported to have a high metastatic potential, while A375 parental cells had a very low metastatic potential (Kozlowski, et al. (1984) J. Natl. Cancer Inst. V 72, pp 913-917) . Therefore, the heparanase activity of A375 cells may correlate with their spontaneous lung metastatic potential.
  • B16 melanoma cells or B16 cell extracts from sublines of high lung colonization potential degrade purified HS at higher rates than B16 cells of poor lung colonization potential
  • B16 melanoma HS degrading endoglycosidase is an endo-3-glucuronidase (heparanase) (Nakajima, M. , et al., (1984) J. Biol. Chem. V 259, pp 2283-2290) .
  • Heparanase assay was carried out by the incubation of a Triton X-100 cell extract (2.4 x 10 5 cells) with PNDS- N[ 14 C]Ac-HS immobilized on agarose beads (4500 cpm) at 37°C for 12 h. The details of experiment are described in the materials and methods section. The radioactivity released in the presence of heat inactivated enzymes was subtracted from the raw data.
  • A375 Met Mix and A375 M6 cells derived from lung metastases of A375 parental cells in athymic nude mice possess highly spontaneous lung metastatic potential, while A375 parental cells have very low spontaneous metastatic potential.
  • Heparanase activity in the sera from malignant melanoma patients The sera from 20 melanoma patients at the various stages of the disease and from 15 normal adults were assayed for heparanase, and the results are shown in Fig. 4. The mean value and standard deviation of heparanase activity in the sera from melanoma patients and normal adults were 0.0177 + 0.0075 and
  • melanoma cells (murine B16 melanoma subline B16-BL6 or human melanoma Hs 939 cells) were grown in a 1:1 mixture of DME/F12 medium supplemented with 5% heat-inactivated fetal bovine serum. Subconfluent cells were harvested by a treatment for 10 min with 2 mM EDTA in PBS and then washed twice in 0.14 M NaCl, 10 mM Tris-HCl buffer, pH 7.2. The following steps were performed at 4°C.
  • Cells (2 x 10 8 ) were extracted in 30 ml of 50 mM Tris-HCl buffer, pH 7.2, containing 0.2% Triton X-100, 10 uM PMSF (buffer A) for 1 hr.
  • the supernatant (approximately 1.5 mg protein/ml) was collected after centrifugation at 30,000 x g for 30 min, and was loaded on a column of concanavalin A-Sepharose 4B (2 x 10 cm) equilibrated with buffer A. After washing with 10 ml of buffer A, the absorbed material was eluted with 1 M alpha-methyl-D-mannoside in buffer A.
  • the eluents were filtered through a heparin-sepharose CL-6B column (2 x 10 cm) equilibrated with 50 mM Tris HCl buffer, pH 7.2, containing 0.15 M sodium chloride 0.2% Triton X-100.
  • the column was washed with 100 ml of the same buffer and 100 ml of 0.15 M sodium chloride 50 mM Tris-HCl, pH 7.2, and then heparin-bound proteins were eluted with a linear salt gradient (0.15 M-1.2 M sodium chloride).
  • the heparanase active fractions were collected and dialyzed against 0.15 M sodium chloride and 0.01 M potassium phosphate, pH 6.0.
  • Melanoma heparanase is active between pH 5.5 and 7.5 and degrades heparan sulfate but not other glycosaminoglycans. Heparin and dextransulfate are potent inhibitors of melanoma heparanase.
  • 1 3 H-labeled glycosaminoglycan was incubated with a cell extract (80 ug of protein) in 0.1 M sodium acetate buffer (pH 6.0) containing 0.15 M NaCl, 0.2% Triton X- 100 and 0.05% NaN 3 for 6 hr at 37°C in the presence of 20 mM D-saccharic acid 1,4-lactone (SAL) and was then subjected to high-speed gel-permeation chromatography. Percent of degradation was determined by measuring the decrease in area of the high M r half of the glycosaminoglycan peak (see Figure 2) . +, more than 80%; +, 5% to 15%; -, less than 5% (S.D. ⁇ 5.0%) .
  • Human and Murine Heparanase Are Antigenically Related Anti-heparanase antibodies directed against the amino terminus of murine melanoma heparanase were prepared as described above and some of the antibodies were subsequently biotinylated.
  • human A375-SM Fig. 9a- d
  • mouse B16-BL6 Fig. 9e-h
  • melanoma cell lysates metabolically labeled with [ 34 S]methionine were first incubated with biotinylated anti-heparanase antibodies (Fig. 9b,c,f,g) and then with streptavidin-agarose.
  • the antibody specifically immunoprecipitated a major M r ⁇ 97,000 protein or a doublet of M r ⁇ 97,000 proteins from mouse B16-BL6 (Fig. 9f,g) and human A375-SM (Fig. 9b,c) melanoma cell lysates, respectively.
  • the protein profiles on SDS-PAGE appeared to be the same under nonreducing and reducing conditions.
  • Heparanase Molecules are Immunochemicallv Localized at the Melanoma Cell Surface and in the Cytoplasm
  • Acetone-permeabilized human A375-SM (Fig. 10a) and mouse B16-BL6 melanoma cells were intensely stained with the anti-heparanase antibodies in an indirect immunochemical assay.
  • the immunocytochemical staining of A375-SM and B16-BL6 cells with anti-heparanase antibodies appeared to be heterogeneous from cell to cell (Fig. 10a, b) .
  • Nonpermeabilized A375SM (Fig. 10b) and B16-BL6 cells, in which the antibodies can only interact with antigens on the cell surface were also stained although not as intensely as their permeablized counterparts.
  • the B16-BL6 cells were stained essentially the same as the A375SM cells; however, the presence of melanin deposits sometimes interfered with the immunoprecipitates. In contrast to the intense staining of the melanoma cells by anti-heparanase antibodies, there was no detectable immunoprecipitates. In contrast to the intense staining of the melanoma cells by anti-heparanase antibodies, there was no detectable immunocytochemical staining in any of the controls (Fig. lOc-f) , including one where anti- heparanase antibodies were preincubated with the N- terminal heparanase peptide (Fig. lOd) .
  • heparanase was localized in experimental mouse metastatic melanoma tumors. Frozen sections of mouse lung tissue containing micrometastatic melanoma nodules were specifically stained with anti-heparanase antibodies. Examination showed heparanase antigen localized in the tumor, not in normal tissue (Fig. llf) . The sections presented in
  • Fig. lld-f were taken from different levels of the same micrometastatic tumor nodule.
  • Serial sections show the morphology of the micrometastatic lesion in the upper right corner of the micrograph (Fig. lid) .
  • the micrometastatic melanoma nodule appeared to contain large quantities of melanin, and the melanoma cells appeared to be heterogeneous in size and have larger nuclei than the adjacent pulmonary alveolar epithelial cells.
  • the micrometastatic melanoma nodules were intensely stained with anti-heparanase antibodies, and the intensity of staining in the metastatic melanoma tissue was higher than that in the adjacent lung tissue (Fig. llf) .
  • the same set of controls as those shown in Fig. 2 was carried out, but only a representative control is presented (Fig. lie) . No staining was found in any of the controls, and the presence of melanin in melanoma nodules was profound.
  • Metastatic and Invasive Melanoma Cells are Stained with Anti-heparanase Antibodies in Human Metastatic Melanoma Tissues
  • Human melanoma tissues from seven patients with metastatic melanomas were examined for the presence of heparanase using immunohistochemical techniques.
  • the melanoma cells were stained with anti-heparanase antibodies, and in each case the surrounding tissues, including connective tissue, were essentially unstained.
  • a cutaneous melanoma is presented (Fig. lla-c) .
  • the malignant melanoma cells, which had invaded dermis connective tissue, are shown adjacent to an arteriole and remnants of collagen fibers (Fig. 11a) .
  • the tumor cells were amelanotic and heterogeneous in size.
  • Anti-heparanase antibodies that stained melanoma cells did not stain surrounding normal parenchymal cells except slightly the endothelial cells in a blood vessel (Fig. lie) .
  • the same controls as those shown in Fig. 10 were performed, but only a representative one is presented (Fig. lib) .
  • human nevi were examined for the presence of heparanase antigen using the same procedure as the melanoma tissues. The melanocytes in the nevi and adjacent normal skin were not stained by the anti- heparanase antibodies.

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Abstract

Production d'anticorps dirigés contre le peptide d'héparanase à terminaison N. Ces anticorps sont utilisés pour la détection d'endoglycosidase de sulfate d'héparane dans des tumeurs de la souris et de l'homme. La localisation et les quantités de ces antigènes d'héparanase pourraient finalement servir à la définition de traitements thérapeutiques appropriés.
PCT/US1991/003832 1990-05-31 1991-05-30 Localisation immunochimique de l'heparanase dans des melanomes de la souris et de l'homme WO1991019197A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998003638A1 (fr) * 1996-07-18 1998-01-29 The Australian National University Detection de l'activite de l'heparanase mammifere et purification de cette heparanase
EP1060252A2 (fr) * 1998-02-24 2000-12-20 PHARMACIA & UPJOHN COMPANY Polypeptides d'heparanase des plaquettes humaines, molecules polynucleotidiques les codant et procedes d'identification de composes modifiant l'activite de l'heparanase
EP1073682A4 (fr) * 1998-05-01 2001-02-07 Insight Strategy & Marketing Sondes moleculaires specifiques pour heparanase et leur mise en application dans la medecine et dans la recherche
US6242238B1 (en) 1997-10-28 2001-06-05 The Australian National University Isolated nucleic acid molecule encoding mammalian endoglucuronidase and uses therefor
US6656699B2 (en) * 2000-09-15 2003-12-02 Reddy Us Therapeutics, Inc. Methods and compositions for glycosidase assays
US6699672B1 (en) * 1997-09-02 2004-03-02 Insight Biopharmaceuticals Ltd. Heparanase specific molecular probes and their use research and medical applications
US7666651B2 (en) 1998-08-31 2010-02-23 Insight Biopharmaceuticals Ltd. Polypeptide having heparanase activity

Citations (2)

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Publication number Priority date Publication date Assignee Title
EP0244932A2 (fr) * 1986-03-10 1987-11-11 Board Of Regents, The University Of Texas System Un substrat en phase solide qui donne sur hydrolyse par une glycosaminoglycan-endoglycosidase des produits marqués
WO1991002977A1 (fr) * 1989-08-23 1991-03-07 Hadassah Medical Organization Preparations de cicatrisations de blessures contenant de l'heparanase

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
EP0244932A2 (fr) * 1986-03-10 1987-11-11 Board Of Regents, The University Of Texas System Un substrat en phase solide qui donne sur hydrolyse par une glycosaminoglycan-endoglycosidase des produits marqués
WO1991002977A1 (fr) * 1989-08-23 1991-03-07 Hadassah Medical Organization Preparations de cicatrisations de blessures contenant de l'heparanase

Non-Patent Citations (2)

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Title
International Journal of Cancer, vol. 45, 15 June 1990, Wiley-Liss, Inc. (US), Li Jin et al.: "Immunochemical localization of heparanase in mouse and human melanomas", pages 1088-1095, see the whole article *
Journal of Cellular Biochemistry, vol. 36, 1988, Tumor Progression and Metastasis, Alan R. Liss, Inc. M. Nakajima et al.: "Heparanases and tumor metastasis", pages 49-59, see the whole article (cited in the application) *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998003638A1 (fr) * 1996-07-18 1998-01-29 The Australian National University Detection de l'activite de l'heparanase mammifere et purification de cette heparanase
US6207402B1 (en) 1996-07-18 2001-03-27 The Australian National University Act Detection of mammalian heparanase activity and purification of mammalian heparanase
US6352852B1 (en) 1996-07-18 2002-03-05 The Australian National University Method for the purification of human platelet heparanase
US6699672B1 (en) * 1997-09-02 2004-03-02 Insight Biopharmaceuticals Ltd. Heparanase specific molecular probes and their use research and medical applications
US6242238B1 (en) 1997-10-28 2001-06-05 The Australian National University Isolated nucleic acid molecule encoding mammalian endoglucuronidase and uses therefor
EP1060252A2 (fr) * 1998-02-24 2000-12-20 PHARMACIA & UPJOHN COMPANY Polypeptides d'heparanase des plaquettes humaines, molecules polynucleotidiques les codant et procedes d'identification de composes modifiant l'activite de l'heparanase
EP1073682A4 (fr) * 1998-05-01 2001-02-07 Insight Strategy & Marketing Sondes moleculaires specifiques pour heparanase et leur mise en application dans la medecine et dans la recherche
US7666651B2 (en) 1998-08-31 2010-02-23 Insight Biopharmaceuticals Ltd. Polypeptide having heparanase activity
US6656699B2 (en) * 2000-09-15 2003-12-02 Reddy Us Therapeutics, Inc. Methods and compositions for glycosidase assays

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