AU772311B2

AU772311B2 - Polynucleotide encoding a polypeptide having heparanase activity and expression of same in transduced cells

Info

Publication number: AU772311B2
Application number: AU69997/01A
Authority: AU
Inventors: Elena Feinstein; Iris Pecker; Israel Vlodavsky
Original assignee: Hadasit Medical Research Services and Development Co; Insight Biopharmaceuticals Ltd
Current assignee: Hadasit Medical Research Services and Development Co; Insight Biopharmaceuticals Ltd
Priority date: 1997-09-02
Filing date: 2001-09-11
Publication date: 2004-04-22
Anticipated expiration: 2018-08-31
Also published as: AU6999701A

Description

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AUSTRALIA

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-nsight Strategy Mnarkeing Ltd., Hadasit Medical Research Services Development Ltd.

COMPLETE SPECIFICATION DIVISIONAL PATENT Invention Title: Polynucleotide encoding a polypeptide having heparanase activity and expression of same in transduced cells The following statement is a full description of this invention including the best method of performing it known to us:- POLYNUCLEOTIDE ENCODING A POLYPEPTIDE HAVING HEPARANASE ACTIVITY AND EXPRESSION OF SAME IN TRANSDUCED CELLS FIELD AND BACKGROU ND OF THE INENTION The present invention relates to a polynucleotide, referred to hereinbelow as hpa, encoding a polypeptide having heparanase activity, vectors including same and transduced cells expressing heparanase. The invention further relates to a recombinant protein having heparanase activity.

o Heparan sulfate proteoglycans: Heparan sulfate proteoglycans (HSPG) are ubiquitous macromolecules associated with the cell surface and extra cellular matrix (ECM) of a wide range of cells of vertebrate and invertebrate tissues The basic HSPG structure includes a protein core to which several linear heparan sulfate chains are covalently attached. These polysaccharide chains are typically Scomposed of repeating hexuronic and D-glucosamine disaccharide units that are substituted to a varying extent with N- and O-linked sulfate moieties and Nlinked acetyl groups Studies on the involvement of ECM molecules in cell attachment, growth and differentiation revealed a central role of HSPG in embryonic morphogenesis, angiogenesis, neurite outgrowth and tissue repair (1- 20 HSPG are prominent components of blood vessels In large blood vessels they are concentrated mostly in the intima and inner media, whereas in capillaries they are found mainly in the subendothelial basement membrane where they support proliferating and migrating endothelial cells and stabilize the structure of the capillary wall. The ability of HSPG to interact with ECM macromolecules.

5 such as collagen, laminin and fibronectin, and with different attachment sites on plasma membranes suggests a key role for this, proteoglycan in the self-assembly and insolubility of ECM components, as well as in cell adhesion and locomotion.

Cleavage of the heparan sulfate (HS) chains may therefore result in degradation of the subendothelial ECM and hence may play a decisive role in extravasation of 30 blood-borne cells. HS catabolism is observed in inflammation, wound repair, diabetes, and cancer metastasis, suggesting that enzymes which degrade HS play important roles in pathologic processes. Heparanase activity has been described in activated immune system cells and highly metastatic cancer cells but research has been handicapped by the lack of biologic tools to explore potential causative roles of heparanase in disease conditions.

Involvement of Heparanase in Tumor Cell Invasion and Metastasis: Circulating tumor cells arrested in the capillary beds of different organs must invade the endothelial cell lining and degrade its underlying basement membrane (BM) in order to invade into the extravascular tissue(s) where they establish metastasis 10). Metastatic tumor cells often attach at or near the intercellular junctions between adjacent endothelial cells. Such attachment of the metastatic cells is followed by rupture of the junctions, retraction of the endothelial cell borders and migration through the breach in the endothelium toward the exposed s underlying BM Once located between endothelial cells and the BM, the invading cells must degrade the subendothelial glycoproteins and protcoglycans of the BM in order to migrate out of the vascular compartment. Several cellular enzymes collagenase IV, plasminogen activator, cathepsin B, elastase, etc.) are thought to be involved in degradation of BM Among these enzymes is o an endo-p-D-glucuronidase (heparanase) that cleaves HS at specific intrachain sites 8, 11). Expression of a HS degrading heparanase was found to correlate with the metastatic potential of mouse lymphoma fibrosarcoma and melanoma cells. Moreover, elevated levels of heparanase were detected in sera from metastatic tumor bearing animals and melanoma patients and in tumor biopsies of cancer patients (12).

The control of cell proliferation and tumor progression by the local microenvironment, focusing on the interaction of cells with the extracellular matrix (ECM) produced by cultured corneal and vascular endothelial cells, was investigated previously by the present inventors. This cultured ECM closely 20 resembles the subendothelium in vivo in its morphological appearance and molecular composition. It contains collagens (mostly type III and IV, with smaller amounts of types I and proteoglycans (mostly heparan sulfate- and dermatan sulfate- proteoglycans, with smaller amounts of chondroitin sulfate proteoglycans), laminin, fibronectin, entactin and elastin (13, 14). The ability of cells to degrade HS in the cultured ECM was studied by allowing cells to interact with a metabolically sulfate labeled ECM, followed by gel filtration (Sepharose 6B) analysis of degradation products released into the culture medium (11).

While intact HSPG are eluted next to the void volume of the column (Kav<0.2, Mr 0.5x106), labeled degradation fragments of HS side chains are eluted more 30 toward the Vt of the column (0.5<kav<0.8, Mr =5-7x10 3 (11).

The heparanase inhibitory, effect of various non-anticoagulant species of heparin that might be of potential use in preventing extravasation of blood-borne cells was also investigated by the present inventors. Inhibition of heparanase was best achieved by heparin species containing 16 sugar units or more and having sulfate groups at both the N and O positions. While O-desulfation abolished the heparanase inhibiting effect of heparin, O-sulfated, N-acetylated heparin retained a high inhibitory activity, provided that the N-substituted molecules had a molecular size of about 4,000 daltons or more Treatment of experimental animals with heparanase inhibitors non-anticoagulant species of heparin) markedly reduced the incidence of lung metastases induced by B16 melanoma, Lewis lung carcinoma and mammary adenocarcinoma cells 8, 16).

Heparin fractions with high and low affinity to anti-thrombin III exhibited a comparable high anti-metastatic activity, indicating that the heparanase inhibiting activity of heparin, rather than its anticoagulant activity, plays a role in the antimetastatic properties of the polysaccharide Heparanase activity in the urine of cancer patients: In an attempt to further elucidate the involvement of heparanase in tumor progression and its relevance to human cancer, urine samples for heparanase activity were screened (16a). Heparanase activity was detected in the urine of some, but not all, cancer patients. High levels of heparanase activity were determined in the urine of patients with an aggressive metastatic disease and there was no detectable activity in the urine of healthy donors.

Heparanase activity was also found in the urine of 20% of normal and microalbuminuric insulin dependent diabetes mellitus (IDDM) patients, most likely due to diabetic nephropathy, the most important single disorder leading to renal failure in adults.

Possible involvement of heparanase in tumor angiogenesis: Fibroblast 20 growth factors are a family of structurally related polypeptides characterized by high affinity to heparin They are highly mitogenic for vascular endothelial cells and are among the most potent inducers of neovascularization (17, 18).

Basic fibroblast growth factor (bFGF) has been extracted from the subendothelial ECM produced in vitro (19) and from basement membranes of the cornea suggesting that ECM may serve as a reservoir for bFGF. Immunohistochemical staining revealed the localization of bFGF in basement membranes of diverse tissues and blood vessels Despite the ubiquitous presence of bFGF in normal tissues, endothelial cell proliferation in these tissues is usually very low, suggesting that bFGF is somehow sequestered from its site of action. Studies on 30 the interaction of bFGF with ECM revealed that bFGF binds to HSPG in the ECM and can be released in an active form by HS degrading enzymes (15, 22). It was demonstrated that heparanase activity expressed by platelets, mast cells, neutrophils, and lymphoma cells is involved in release of active bFGF from ECM and basement membranes suggesting that heparanase activity may not only function in cell migration and invasion, but may also elicit an indirect neovascular response. These results suggest that the ECM HSPG provides a natural storage depot for bFGF and possibly other heparin-binding growth promoting factors (24, 25). Displacement of bFGF from its storage within 4 basement membranes and ECM may therefore provide a novel mechanism for induction of neovascularization in normal and pathological situations.

Recent studies indicate that heparin and HS are involved in binding of bFGF to high affinity cell surface receptors and in bFGF cell signaling (26, 27).

Moreover, the size of HS required for optimal effect was similar to that of HS fragments released by heparanase Similar results were obtained with vascular endothelial cells growth factor (VEGF) suggesting the operation of a dual receptor mechanism involving HS in cell interaction with heparin-binding growth factors. It is therefore proposed that restriction of endothelial cell growth 0o factors.in ECM prevents their systemic action on the vascular endothelium, thus maintaining a very low rate of endothelial cells turnover and vessel growth. On the other hand, release of bFGF from storage in ECM as a complex with HS fragment, may elicit localized endothelial cell proliferation and neovascularization in processes such as wound healing, inflammation and tumor development (24, Expression of heparanase by cells of the immune system: Heparanase activity correlates with the ability of activated cells of the immune system to leave the circulation and elicit both inflammatory and autoimmune responses.

Interaction of platelets, granulocytes, T and B lymphocytes, macrophages and 20 mast cells with the subendothelial ECM is associated with degradation of HS by a :specific heparanase activity The enzyme is released from intracellular compartments lysosomes, specific granules, etc.) in response to various activation signals thrombin, calcium ionophore, immune complexes, antigens, mitogens, etc.), suggesting its regulated involvement in inflammation and cellular immunity.

Some of the observations regarding the heparanase enzyme were reviewed in reference No. 6 and are listed hereinbelow: First, a proteolytic activity (plasminogen activator) and heparanase participate synergistically in sequential degradation of the ECM HSPG by 30 inflammatory leukocytes and malignant cells.

Second, a large proportion of the platelet heparanase exists in a latent form, probably as a complex with chondroitin sulfate. The latent enzyme is activated by tumor cell-derived factor(s) and may then facilitate cell invasion through the vascular endothelium in the process of tumor metastasis.

Third, release of the platelet heparanase from a-granules is induced by a strong stimulant thrombin), but not in response to platelet activation on

ECM.

Fourth, the neutrophil hcparanase is preferentially and readily released in response to a threshold activation and upon incubation of the cells on ECM.

Fifth, contact of neutrophils with ECM inhibited release of noxious enzymes (proteases, lysozyme) and oxygen radicals, but not of enzymes (heparanase, gelatinase) which may enable diapedesis. This protective role of the subendothelial ECM was observed when the cells were stimulated with soluble factors but not with phagocytosable stimulants.

Sixth, intracellular heparanase is secreted within minutes after exposure of T cell lines to specific antigens.

Seventh, mitogens (Con A, LPS) induce synthesis and secretion of heparanase by normal T and B lymphocytes maintained in vitro. T lymphocyte heparanase is also induced by immunization with antigen in vivo.

Eighth, heparanase activity is expressed by pre-B lymphomas and Blymphomas, but not by plasmacytomas and resting normal B lymphocytes.

Ninth, heparanase activity is expressed by activated macrophages during incubation with ECM, but there was little or no release of the enzyme into the incubation medium. Similar results were obtained with human myeloid leukemia Scells induced to differentiateto mature macrophages.

.Tenth, T-cell mediated delayed type hypersensitivity and experimental S -20 autoimmunity are suppressed by low doses of heparanase inhibiting nonanticoagulant species of heparin Eleventh, heparanase activity expressed by platelets, neutrophils and metastatic tumor cells releases active bFGF from ECM and basement membranes. Release of bFGF from storage in ECM may elicit a localized neovascular response in processes such as wound healing, inflammation and tumor development.

Twelfth, among the breakdown products of the ECM generated by heparanase is a tri-sulfated disaccharide that can inhibit T-cell mediated inflammation in vivo This inhibition was associated with an inhibitory 30 effect of the disaccharide on the production of biologically active TNFa by activated T cells in vitro (31).

Other potential therapeutic applications: Apart from its involvement in tumor cell metastasis, inflammation and autoimmunity, mammalian heparanase may be applied to modulate: bioavailability of heparin-binding growth factors cellular responses to heparin-binding growth factors bFGF, VEGF) and cytokines (IL-8) (31a, 29); cell interaction with plasma lipoproteins (32); cellular susceptibility to certain viral and some bacterial and protozoa infections (33, 33a, 33b); and disintegration of amyloid plaques Heparanase may thus prove useful for conditions such as wound healing, angiogenesis, restenosis, atherosclerosis, inflammation, neurodegenerative diseases and viral infections.

Mammalian heparanase can be used to neutralize plasma heparin, as a potential replacement of protamine. Anti-heparanase antibodies may be applied for immunodetection and diagnosis of micrometastases, autoimmune lesions and renal failure in biopsy specimens, plasma samples, and body fluids. Common use in basic research is expected.

The identification of the hpa gene encoding for heparanase enzyme will enable the production of a recombinant enzyme in heterologous expression o0 systems. Availability of the recombinant protein will pave the way for solving the protein.structure function relationship and will provide a tool for developing new inhibitors.

Viral Infection: The presence of heparan sulfate on cell surfaces have been shown to be the principal requirement for the binding of Herpes Simplex (33) and Dengue (33a) viruses to cells and for subsequent infection of the cells.

Removal of the cell surface heparan sulfate by heparanase may therefore abolish virus infection. In fact, treatment of cells with bacterial heparitinase (degrading heparan sulfate) or heparinase (degrading heparan) reduced the binding of two related animal herpes viruses to cells and rendered the cells at least partially 20 resistant to virus infection There are some indications that the cell surface heparan sulfate is also involved in HIV infection (33b).

Neurodegenerative diseases: Heparan sulfate proteoglycans were identified in the prion protein amyloid plaques of Genstmann-Straussler Syndrome, Creutzfeldt-Jakob disease and Scrape Heparanase may disintegrate these amyloid plaques which are also thought to play a role in the pathogenesis of Alzheimer's disease.

Restenosis and Atherosclerosis: Proliferation of arterial smooth muscle cells (SMCs) in response to endothelial injury and accumulation of cholesterol rich lipoproteins are basic events in the pathogenesis of atherosclerosis and 30 restenosis Apart from its involvement in SMC proliferation low affinity receptors for heparin-binding growth factors), HS is also involved in lipoprotein binding, retention and uptake It was demonstrated that HSPG and lipoprotein lipase participate in a novel catabolic pathway that may allow substantial cellular and interstitial accumulation of cholesterol rich lipoproteins The latter pathway is expected to be highly atherogenic by promoting accumulation of apoB and apoE rich lipoproteins LDL, VLDL, chylomicrons), independent of feed back inhibition by the cellular sterol content.

Removal of SMC HS by heparanase is therefore expected to inhibit both SMC proliferation and lipid accumulation and thus may halt the progression of restenosis and atherosclerosis.

There is thus a widely recognized need for, and it would be highly advantageous to have a polynucleotide encoding a polypeptide having heparanase activity, vectors including same, transduced cells expressing heparanase and a recombinant protein having heparanase activity.

SUMMARY OF THE INVENTION According to the present invention there is provided a polynucleotide, o1 referred to hereinbelow as hpa, hpa cDNA or hpa gene, encoding a polypeptide having heparanase activity, vectors including same, transduced cells expressing heparanase and a recombinant protein having heparanase activity.

Cloning of the human hpa gene which encodes heparanase, and expression of recombinant heparanase by transfected host cells is reported.

A purified preparation of heparanase isolated from human hepatoma cells was subjected to tryptic digestion and microsequencing. The YGPDVGQPR (SEQ ID NO:8) sequence revealed was used to screen EST databases for homology to the corresponding back translated DNA sequence. Two closely related EST sequences were identified and were thereafter found to be identical.

20 Both clones contained an insert of 1020 bp which included an open reading frame of 973 bp followed by a 27 bp of 3' untranslated region and a Poly A tail.

Translation start site was not identified.

Cloning of the missing 5' end of hpa was performed by PCR amplification of DNA from placenta Marathon RACE cDNA composite using primers selected according to the EST clones sequence and the linkers of the composite. A 900 bp PCR fragment, partially overlapping with the identified 3' encoding EST clones was obtained. The joined cDNA fragment (hpa), 1721 bp long (SEQ ID NO:9), contained an open reading frame which encodes a polypeptide of 543 amino acids (SEQ ID NO:10) with a calculated molecular weight of 61,192 daltons.

30 Cloning an extended 5' sequence was enabled from the human SK-hepl cell line by PCR amplification using the Marathon RACE. The 5' extended sequence of the SK-hepI hpa cDNA was assembled with the sequence of the hpa cDNA isolated from human placenta (SEQ ID NO:9). The assembled sequence contained an open reading frame, SEQ ID NOs: 13 and 15, which encodes, as shown in SEQ ID NOs:14 and 15, a polypeptide of 592 amino acids with a calculated molecular weight of 66,407 daltons.

The ability of the hpa gene product to catalyze degradation of heparan sulfate in an in vitro assay was examined by expressing the entire open reading frame of hpa in insect cells, using the Baculovirus expression system. Extracts and conditioned media of cells infected with virus containing the hpa gene, demonstrated a high level of heparan sulfate degradation activity both towards soluble ECM-derived HSPG and intact ECM. This degradation activity was s inhibited by heparin, which is another substrate of heparanase. Cells infected with a similar construct containing no hpa gene had no such activity, nor did noninfected cells. The ability of heparanase expressed from the extended 5' clone towards heparin was demonstrated in a mammalian expression system.

The expression pattern of hpa RNA in various tissues and cell lines was to investigated using RT-PCR. It was found to be expressed only in tissues and cells previously known to have heparanase activity.

A panel of monochromosomal human/CHO and human/mouse somatic cell hybrids was used to localize the human heparanase gene to human chromosome 4. The newly isolated heparanase sequence can be used to identify a chromosome region harboring a human heparanase gene in a chromosome spread.

According to further features in preferred embodiments of the invention S: described below, there is provided a polynucleotide fragment which includes a polynucleotide sequence encoding a polypeptide having heparanase catalytic S 20 activity.

According to still further features in the described preferred embodiments the polynucleotide fragment includes nucleotides 63-1691 of SEQ ID NO:9 or nucleotides 139-1869 of SEQ ID NO:13, which encode the entire human heparanase enzyme.

According to still further features in the described preferred embodiments there is provided a polynucleotide fragment which includes a polynucleotide sequence capable of hybridizing with hpa cDNA, especially with nucleotides 1- 721 of SEQ ID NO:9.

According to still further features in the described preferred embodiments 30 the polynucleotide sequence which encodes the polypeptide having heparanase activity shares at least 60 homology, preferably at least 70 homology, more preferably at least 80 homology, most preferably at least 90 homology with SEQ ID NOs:9 or 13.

According to still further features in the described preferred embodiments the polynucleotide fragment according to the present invention includes a portion (fragment) of SEQ ID NOs:9, or 13. For example, such fragments could include nucleotides 63-721 of SEQ ID NO:9 and/or a segment of SEQ ID NO:9 which encodes a polypeptide having the heparanase catalytic activity.

According to still further features in the described preferred embodiments the polypeptide encoded by the polynucleotide fragment includes an amino acid sequence as set forth in SEQ ID NOs:10 or 14 or a functional part thereof.

According to still further features in the described preferred embodiments the polynucleotide sequence encodes a polypeptide having heparanase activity, which shares at least 60 homology, preferably at least 70 homology, more preferably at least 80 homology, most preferably at least 90 homology with SEQ ID NOs:10 or 14.

According to still further features in the described preferred embodiments o0 the polynucleotide fragment encodes a polypeptide having heparanase activity, which may therefore be allelic, species and/or induced variant of the amino acid sequence set forth in SEQ ID NOs:10 or 14. It is understood that any such variant may also be considered a homolog.

According to still further features in the described preferred embodiments there is provided a single stranded polynucleotide fragment which includes a polynucleotide sequence complementary to at least a portion of a polynucleotide strand encoding a polypeptide having heparanase catalytic activity as described above.

According to still further features in the described preferred embodiments 20 there is provided a vector including a polynucleotide sequence encoding a polypeptide having heparanase catalytic activity.

The vector may be of any suitable type including but not limited to a phage, virus, plasmid, phagemid, cosmid. bacmid or even an artificial chromosome. The polynucleotide sequence encoding a polypeptide having heparanase catalytic activity may include any of the above described polynucleotide fragments.

According to still further features in the described preferred embodiments there is provided a host cell which includes an exogenous polynucleotide fragment including a polynucleotide sequence encoding-a polypeptide having 30 heparanase catalytic activity.

The exogenous polynucleotide fragment may be any of the above described fragments. The host cell may be of any type such as prokaryotic cell, eukaryotic cell, a cell line, or a cell as a portion of a multicellular organism cells of a transgenic organism).

According to still further features in the described preferred embodiments there is provided a recombinant protein including a polypeptide having heparanase catalytic activity.

According to still further features in the described preferred embodiments there is provided a pharmaceutical composition comprising as an active ingredient a recombinant protein having heparanase catalytic activity.

According to still further features in the described preferred embodiments there is provided a medical equipment comprising a medical device containing, as an active ingredient a recombinant protein having heparanase catalytic activity.

According to still further features in the described preferred embodiments there is provided a heparanase overexpression system comprising a cell overexpressing heparanase catalytic activity.

According to still further features in the described preferred embodiments there is provided a method of identifying a chromosome region harboring a human heparanase gene in a chromosome spread comprising the steps of hyridizing the chromosome spread with a tagged polynucleotide probe encoding heparanase; washing the chromosome spread, thereby removing excess of non-hybridized probe; and searching for signals associated with said hyrbidized tagged polynucleotide probe, wherein detected signals being indicative of a chromosome region harbouring a 20 human heparanase gene.

The present invention can be used to develop new drugs to inhibit tumor cell metastasis, inflammation and autoimmunity. The identification of the hpa gene encoding the heparanase enzyme enables the production of a recombinant enzyme in heterologous expression systems.

25 The invention provides, in one aspect of the invention, a polynucleotide fragment comprising a polynucleotide sequence encoding a polypeptide having heparanase catalytic activity, wherein said polypeptide shares at least 70% homology with SEQ ID NOs: 10 or 14, as determined using default parameters of a DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the University of Wisconsin.

In another aspect, the invention provides a vector comprising a polynucleotide sequence encoding a polypeptide having heparanase catalytic activity, wherein said polypeptide shares at least 70% homology with SEQ ID NOs: 10 or 14, as determined using default parameters of a DNA sequence analysis software package developed by the Genetic Computer Group (GCG) as the University of Wisconsin.

In another aspect, the invention provides a host cell comprising an exogenous polynucleotide fragment including a polynucleotide sequence encoding a polypeptide having heparanase catalytic activity, wherein said polypeptide shares at least 70% homology with SEQ ID NOs: 10 or 14, as determined using default parameters of a DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the University of Wisconsin.

In another aspect, the invention provides a host cell comprising a polynucleotide sequence at least 70% homologous with SEQ ID NOs: 9 or 13, as determined using default parameters of a DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the University of Wisconsin, said polynucleotide sequence encoding a polypeptide having heparanase catalytic activity.

In another aspect, the invention provides a recombinant protein comprising a polypeptide having heparanase catalytic activity, said polypeptide shares at least 70% homology with SEQ ID NOs: 10 or 14, as determined using default parameters of a DNA sequence analysis software 20 package developed by the Genetic Computer Group (GCG) at the University of Wisconsin.

In another aspect, the invention provides a pharmaceutical composition comprising, as an active ingredient, a recombinant protein including a polypeptide having heparanase catalytic activity, said polypeptide shares at least 70% homology with SEQ ID NOs: 10 or 14, as determined using default parameters of a DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the University of Wisconsin.

In another aspect, the invention provides a modulator of heparinbinding growth factors, cellular responses to heparin-binding growth factors and cytokines, cell interaction with plasma lipoproteins, cellular susceptibility to viral, protozoa and bacterial infections or disintegration of neurodegenerative plaques comprising, as an active ingredient, a recombinant protein including a polypeptide having heparanase catalytic activity, said polypeptide shares at least 70% homology with SEQ ID NOs: 10 or 14, as determined using default parameters of a DNA sequence analysis software lob package developed by the Genetic Computer Group (GCG) at the University of Wisconsin.

In another aspect, the invention provides a medical equipment comprising a medical device containing, as an active ingredient, a recombinant protein including a polypeptide having heparanase catalytic activity, said polypeptide shares at least 70% homology with SEQ ID NOs: or 14, as determined using default parameters of a DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the University of Wisconsin.

In another aspect, the invention provides a host cell expressing a recombinant heparanase, wherein said recombinant heparanese shares at least 70% homology with SEQ ID NOs: 10 or 14, as determined using default parameters of a DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the University of Wisconsin.

In another aspect, the invention provides a heparanase overexpression system comprising a cell overexpressing heparanase catalytic activity, wherein said heparanase catalytic activity is effected by a heparanase sharing at least 70% homology with SEQ ID NOs: 10 or 14, as determined using default parameters of a DNA sequence analysis software package developed 20 by the Genetic Computer Group (GCG) at the University of Wisconsin.

In another aspect, the invention provides a method of identifying a chromosome region harboring a heparanase gene in a chromosome spread comprising the steps of: a) hybridizing the chromosome spread with a tagged polynucleotide probe at least 70% homologous with SEQ ID NOs: 9 or 13 as determined using default parameters of a DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the University of S• Wisconsin; b) washing the chromosome spread, thereby removing excess of nonhybridized probe; and c) searching for signals associated with said hybridized tagged polynucleotide probe, wherein detected signals being indicative of a chromosome region harboring a heparanase gene.

In another aspect the invention provides a preparation comprising a protein having heparanase (endo-P-D-glucuronidase) catalytic activity or being cleavable so as to acquire said heparanase catalytic activity, the preparation being free of non-heparanase polypeptides encoded by human nucleic sequences.

In another aspect the invention provides an isolated protein having heparanase (endo-P-D-glucuronidase) catalytic activity or being cleavable so as to acquire said heparanase catalytic activity, said isolated protein being substantially devoid of glycosilation.

In another aspect the invention provides a pharmaceutical composition comprising, as an active ingredient, the isolated protein of claim 3, and a pharmaceutically acceptable carrier.

In another aspect the invention provides a preparation comprising a protein having heparanase (endo-P-D-glucuronidase) catalytic activity or being cleavable so as to acquire said heparanase catalytic activity, the preparation being substantially free of a CXC chemokine or PAIl.

In another aspect the invention provides a pharmaceutical composition comprising, as an active ingredient, the preparation of claim 5, and a pharmaceutically acceptable carrier.

20 In another aspect the invention provides an isolated protein having heparanase catalytic (endo-P-D-glucuronidase) activity or being cleavable so as to acquire said heparanase catalytic activity, said isolated protein being characterized by non-human cell derived sugar prosthetic groups.

In another aspect the invention provides a preparation comprising a 25 protein of about 50 or about 65 kDa as determined by a denaturing polyacrylamide gel electrophoresis, said protein having- heparanase (endo-P- D-glucuronidase) catalytic activity or being cleavable so as to acquire said heparanase catalytic activity, respectively, the preparation being free of nonheparanse polypeptides encoded by human nucleic acid sequences.

In another aspect the invention provides an isolated protein of about or about 65 kDa as determined by a denaturing polyacrylamide gel electrophoresis, said protein having heparanase (endo-p-D-glucuronidase) catalytic activity or being cleavable so as to acquire said heparanase catalytic activity, respectively, said isolated protein being substantially devoid of glycosilation.

In another aspect the invention provides a preparation comprising a protein of about 50 or about 65 kDa as determined by a denaturing polyacrylamide gel electrophoresis, said protein having heparanase (endo-p- D-glucuronidase) catalytic activity or being cleavable so as to acquire said heparanase catalytic activity, respectively, the preparation being substantially free of a CXC chemokine or PAIl.

In another aspect the invention provides an isolated protein of about or about 65 kDa as determined by a denaturing polyacrylamide gel electrophoresis, said protein having heparanase (endo-p-D-glucuronidase) catalytic activity or being cleavable so as to acquire said heparanase catalytic activity, respectively, said isolated protein being characterized by insect cell derived sugar prosthetic groups.

In another aspect the invention provides an isolated protein of about or about 65 kDa as determined by a denaturing polyacrylamide gel electrophoresis, said protein having heparanase (endo-p-D-glucuronidase) catalytic activity or being cleavable so as to acquire said heparanase catalytic activity, respectively, said isolated protein being characterized by non-human cell derived sugar prosthetic groups.

In another aspect the invention provides a preparation comprising a 20 protein at least 70% homologous to SEQ ID NO:10 or 14, said protein having heparanase (endo-p-D-glucuronidase) catalytic activity or being cleavable so as to acquire said heparanase catalytic activity, the preparation being free of non-heparanase polypeptides encoded by human nucleic acid sequences.

In another aspect the invention provides an isolated protein at least 25 70% homologous to SEQ ID NO:10 or 14, the protein having heparanase *(endo-p-D-glucuronidase) catalytic activity or being cleavable so as to acquire said heparanase catalytic activity, said isolated protein being substantially devoid of glycosilation.

In another aspect the invention provides a preparation comprising a protein at least 70% homologous to SEQ ID NO:10 or 14, said protein having heparanase (endo-p-D-glucuronidase) catalytic activity or being cleavable so as to acquire said heparanase catalytic activity, the preparation being substantially free of a CXC chemokine or PAIl.

In another aspect the invention provides an isolated protein at least 70% homologous to SEQ ID NO:10 or 14, the protein having heparanase (endo-p-D-glucuronidase) catalytic activity or being cleavable so as to acquire said heparanase catalytic activity, said isolated protein being characterized by insect cell derived sugar prosthetic groups.

In another aspect the invention provides an isolated protein at least homologous to SEQ ID NO:10 or 14, the protein having heparanase catalytic (endo-P-D-glucuronidase) activity or being cleavable so as to acquire said heparanase catalytic activity, said isolated protein being characterized by non-human cell derived sugar prosthetic groups.

In another aspect the invention provides a preparation comprising a protein having a pair of glutamic acids participating in its active site and having heparanase (endo-P-D-glucuronidase) catalytic activity or being cleavable so as to acquire said heparanase catalytic activity, the preparation being free of non-heparanase polypeptides encoded by human nucleic acid sequences.

In another aspect the invention provides an isolated protein having a pair of glutamic acids participating in its active site and having heparanase (endo-P-D-glucuronidase) catalytic activity or being cleavable so as to acquire said heparanase catalytic activity, said isolated protein being substantially devoid of glycosilation.

In another aspect the invention provides a preparation comprising a protein having a pair of glutamic acids participating in its active site and having heparanase (endo-P-D-glucuronidase) catalytic activity or being cleavable so as to acquire said heparanase catalytic activity, the preparation being substantially free of a CXC chemokine or PAIl.

In another aspect the invention provides- an isolated protein having a- 25 pair of glutamic acids participating in its active site and heparanase (endo-p- D-glucuronidase) catalytic activity or being cleavable so as to acquire said heparanase catalytic activity, said isolated. protein being characterized by insect cell derived sugar prosthetic groups.

In another aspect the invention provides an isolated protein having a pair of glutamic acids participating in its active site and having heparanase catalytic (endo-P-D-glucuronidase) activity or being cleavable so as to acquire said heparanase catalytic activity, said isolated protein being characterized by non-human cell derived sugar prosthetic groups.

In another aspect the invention provides a preparation comprising a protein having heparanase (endo-P-D-glucuronidase) catalytic activity or being cleavable so as to acquire said heparanase catalytic activity, said protein being capable of eliciting an anti-heparanase antibody, the preparation being free of non-heparanase polypeptides encoded by human nucleic acid sequences.

In another aspect the invention provides an isolated protein having heparanase (endo-P-D-glucuronidase) catalytic activity or being cleavable so as to acquire said heparanase catalytic activity, said protein being capable of eliciting an anti-heparanase antibody, said isolated protein being substantially devoid of glycosilation.

In another aspect the invention provides a preparation comprising a protein having heparanase (endo-P-D-glucuronidase) catalytic activity or being cleavable so as to acquire said heparanase catalytic activity, said protein being capable of eliciting an anti-heparanase antibody, the preparation being substantially free of a CXC chemokine or PAI1.

In another aspect the invention provides an isolated protein having heparanase (endo-P-D-glucuronidase) catalytic activity or being cleavable so as to acquire said heparanase catalytic activity, said protein being capable of eliciting an anti-heparanase antibody, said isolated protein being characterized by insect cell derived sugar prosthetic groups.

*20 In another aspect the invention provides an isolated protein having heparanase catalytic (endo-P-D-glucuronidase) activity or being cleavable so as to acquire said heparanase catalytic activity, said protein being capable of eliciting an anti-heparanase antibody, said isolated protein being characterized by non-human cell derived sugar prosthetic groups.

25 In another aspect the invention provides an isolated protein having heparanase catalytic (endo-P-D-glucuronidase) activity or being cleavable so as to acquire said heparanase catalytic activity, said protein being capable of eliciting an anti-heparanase antibody.

In another aspect the invention provides a polynucleotide fragment comprising a polynucleotide sequence encoding a polypeptide having heparanase catalytic activity, wherein said polypeptide shares at least homology with SEQ ID Nos:10 or 14.

In another aspect the invention provides a polynucleotide fragment comprising a polynucleotide sequence at least 60% homologous with SEQ ID Nos:9 or 13, said polynucleotide sequence encoding a polypeptide having heparanase catalytic activity.

In another aspect the invention provides a pharmaceutical composition comprising any one of the above described preparations or isolated proteins as an active ingredient in combination with a pharmaceutically acceptable carrier.

In another aspect the invention provides a vector comprising a polynucleotide sequence encoding a polypeptide having heparanase catalytic activity, wherein said polypeptide shares at least 60% homology with SEQ ID or 14.

In another aspect the invention provides a vector comprising a polynucleotide sequence at least 60% homologous with SEQ ID Nos:9 or 13, said polynucleotide sequence encoding a polypeptide having heparanase catalytic activity.

In another aspect the invention provides a host cell comprising an exogenous polynucleotide fragment including a polynucleotide sequence encoding a polypeptide having heparanase catalytic activity, wherein said polypeptide shares at least 60% homology with SEQ ID Nos:10 or 14.

In another aspect the invention provides a host cell comprising a polynucleotide sequence at least 60% homologous with SEQ ID Nos:9 or 13, said polynucleotide sequence encoding a polypeptide having heparanase 20 catalytic activity.

In another aspect the invention provides a recombinant protein comprising a polypeptide having heparanase catalytic activity, said polypeptide shares at least 60% homology with SEQ ID Nos:10 or 14.

In another aspect the invention provides an amino acid sequence as set 25 forth in SEQ ID Nos:10 or 14.

In another aspect the invention provides the pharmaceutical composition comprising, as an active ingredient, a recombinant protein *including a polypeptide having heparanase I catalytic activity, said polypeptide shares at least 60% homology with SEQ ID Nos:10 or 14.

In another aspect the invention provides a modulator of heparinbinding growth factors, cellular responses to heparin-binding growth factors and cytokines, cell interaction with plasma lipoproteins, cellular susceptibility to viral, protozoa and bacterial infections or disintegration of neurodegenerative plaques comprising, as an active ingredient, a recombinant protein including a polypeptide having heparanase catalytic activity, said polypeptide shares at least 60% homology with SEQ ID Nos:10 or 14.

In another aspect the invention provides a medical equipment comprising a medical device containing, as an active ingredient, a recombinant protein including a polypeptide having heparanase catalytic activity, said polypeptide shares at least 60% homology with SEQ ID or 14.

In another aspect the invention provides a host cell expressing a recombinant heparanase, wherein said recombinant heparanase shares at least 60% homology with SEQ ID Nos:10 or 14.

In another aspect the invention provides a cell extract or conditioned cell media or a partially purified cell extract or conditioned cell media comprising an extract or media of the host cell as previously described.

In another aspect the invention provides a heparanase inhibitors screening system comprising the cell extract or conditioned cell media or the partially purified cell extract, conditioned cell media or recombinant proteins as previously described.

In another aspect the invention provides a heparanase overexpression system comprising a cell overexpressing heparanase catalytic activity, wherein said heparanase catalytic activity is effected by a heparanase sharing at least 60% homology with SEQ ID Nos:10 or 14.

20 In another aspect the invention provides a method of identifying a chromosome region harboring a heparanase gene in a chromosome spread comprising the steps of: hybridizing the chromosome spread with a tagged polynucleotide probe at least 60% homologous with SEQ ID NOs: 9 or 13 or a portion 25 thereof; washing the chromosome spread, thereby removing excess of nonhybridized probe; and searching for signals associated with said hybridized tagged polynucleotide probe, wherein detected signals being indicative of a chromosome region harboring a heparanase gene.

In another aspect the present invention provides a single stranded polynucleotide fragment comprising a polynucleotide sequence complementary to at least a portion of a polynucleotide strand defined by nucleotides 226 to 721 of SEQ ID NO: 9 or its complementary strand.

In another aspect, the present invention provides a method of amplifying a heparanase polynucleotide comprising: contacting at least a pair of amplification primers with a polynucleotide sample, each of said amplification primers comprises a sequence complementary to at least a portion of a polynucleotide strand defined by nucleotides 226 to 721 of SEQ ID NO: 9 or its complementary strand; and amplifying the heparanase polynucleotide defined between said amplification primers.

BRIEF DESCRIPTION OF THE DRAWINGS The invention herein described, by way of example only, with reference to the accompanying drawings, wherein: FIG. 1 presents nucleotide sequence and deduced amino acid sequence of hpa cDNA. A single nucleotide difference at position 799 (A to T) between the EST (Expressed Sequence Tag) and the PCR amplified cDNA (reverse transcribed RNA) and the resulting amino acid substitution (Tyr to Phe) are indicated above and below the substituted unit, respectively. Cysteine residues and the poly adenylation consensus sequence are underlined. The asterisk denotes the stop codon TGA.

20 FIG. 2 demonstrates degradation of soluble sulfate labeled HSPG substrate by lysates of High Five cells infected with pFhpa2 virus. Lysates of High Five cells that were infected with pFhpa2 virus or control pF2 virus were incubated (18 h, 37 0 C) with sulfate labeled ECM-derived soluble HSPG (peak The incubation medium was then subjected to gel filtration 25 on Sepharose 6B.

Low molecular weight HS degradation fragments (peak II) were produced only during incubation with the pFhpa2 infected cells, but there was no degradation of the HSPG substrate by lysates ofpF2 infected cells.

FIGs. 3a-b demonstrate degradation of soluble sulfate labeled HSPG substrate by the culture medium of pFhpa2 and pFhpa4 infected cells. Culture media of High Five cells infected with pFhpa2 (3a) or pFhpa4 (3b) viruses or with control viruses were incubated (18 h, 37 OC) with sulfate labeled ECMderived soluble HSPG (peak I, The incubation media were then subjected to gel filtration on Sepharose 6B. Low molecular weight HS degradation fragments to (peak II) were produced only during incubation with the hpa gene containing viruses. There was no degradation of the HSPG substrate by the culture medium of cells infected with control viruses.

FIG. 4 presents size fractionation of heparanase activity expressed by pFhpa2 infected cells. Culture medium of pFhpa2 infected High Five cells was is applied onto a 50 kDa cut-off membrane. Heparanase activity (conversion of the peak I substrate, into peak II HS degradation fragments) was found in the high 50 kDa) but not low 50 kDa) molecular weight compartment.

FIGs. 5a-b demonstrate the effect of heparin on heparanase activirt expressed by pFhpa2 and pFhpa4 infected High Five cells. Culture media of 20 pFhpa2 (5a) and pFhpa4 (5b) infected High Five cells were incubated (18 h, 37 C) with sulfate labeled ECM-derived soluble HSPG (peak I, o) in the absence or presence of 10 pig/ml heparin. Production of low molecular weight HS degradation fragments was completely abolished in the presence of heparin, a potent inhibitor of heparanase activity 7).

FIGs. 6a-b demonstrate degradation of sulfate labeled intact ECM by virus infected High Five and Sf21 cells. High Five (6a) and Sf21 (6b) cells were plated on sulfate labeled ECM and infected (48 h, 28 OC) with pFhpa4 or control pFl viruses. Control non-infected Sf21 cells were plated on the labeled ECM as well. The pH of the cultured medium was adjusted to 6.0 6.2 followed 30 by 24 h incubation at 37 oC. Sulfate labeled material released into the incubation medium was analyzed by gel filtration on Sepharose 6B. HS degradation fragments were produced only by cells infected with the hpa containing virus.

FIG. 7a-b demonstrate degradation of sulfate labeled intact ECM by virus infected cells. High Five (7a) and Sf21 (7b) cells were plated on sulfate labeled ECM and infected (48 h, 28 oC) with pFhpa4 or control pFl viruses.

Control non-infected Sf21 cells were plate on labeled ECM as well. The pH of the cultured medium was adjusted to 6.0 6.2, followed by 48 h incubation at 28 oC. Sulfate labeled degradation fragments released into the incubation 12 medium was analyzed by gel filtration on Sepharose 6B. HS degradation fragments were produced only by cells infected with the hpa containing virus.

FIGs. 8a-b demonstrate degradation of sulfate labeled intact ECM by the culture medium of pFhpa4 infected cells. Culture media of High Five (8a) and Sf21 (8b) cells that were infected with pFhpa4 or control pF viruses were incubated (48 h, 37 OC, pH 6.0) with intact sulfate labeled ECM. The ECM was also incubated with the culture medium of control non-infected Sf21 cells Sulfate labeled material released into the reaction mixture was subjected to gel filtration analysis. Heparanase activity was detected only in the culture medium o of pFhpa4 infected cells.

FIGs. 9a-b demonstrate the effect of heparin on heparanase activity in the culture medium of pFhpa4 infected cells. Sulfate labeled ECM was incubated (24 h, 37 oC, pH 6.0) with culture medium of pFhpa4 infected High Five (9a) and Sf21 (9b) cells in the absence or presence of 10 1g/ml heparin. Sulfate labeled material released into the incubation medium was subjected to gel filtration on Sepharose 6B. Heparanase activity (production of peak II HS degradation fragments) was completely inhibited in the presence of heparin.

FIGs. 10a-b demonstrate purification of recombinant heparanase on heparin-Sepharose. Culture medium of Sf21 cells infected with pFhpa4 virus 20 was subjected to heparin-Sepharose chromatography. Elution of fractions was performed with 0.35 2 M NaCI gradient Heparanase activity in the eluted fractions is demonstrated in Figure 10a Fractions 15-28 were subjected to 15% SDS-polyacrylamide gel electrophoresis followed by silver nitrate staining.

A correlation is demonstrated between a major protein band (MW 63,000) in fractions 19 24 and heparanase activity.

FIGs. la-b demonstrate purification of recombinant heparanase on a Superdex 75 gel filtration column. Active fractions eluted from heparin- Sepharose (Figure 10a) were pooled, concentrated and applied onto Superdex 'FPLC column. Fractions were collected and aliquots of each fraction were tested for heparanase activity Figure 1 la) and analyzed by SDS-polyacrylamide gel electrophoresis followed by silver nitrate staining (Figure 1 A correlation is seen between the appearance of a major protein band (MW 63,000) in fractions 4 7 and heparanase activity.

FIGs. 12a-e demonstrate expression of the hpa gene by RT-PCR with total RNA from human embryonal tissues (12a), human extra-embryonal tissues (12b) and cell lines from different origins (12c-e). RT-PCR products using hpa specific primers primers for GAPDH housekeeping gene and control reactions without reverse transcriptase demonstrating absence of genomic DNA or other 13 contamination in RNA samples M- DNA molecular weight marker VI (Bochringer Mannheim). For 12a: lane 1 neutrophil cells (adult), lane 2 muscle, lane 3 thymus, lane 4 heart, lane 5 adrenal. For 12b: lane 1 kidney, lane 2 placenta (8 weeks), lane 3 placenta (11 weeks), lanes 4-7 mole (complete hydatidiform mole), lane 8 cytotrophoblast cells (freshly isolated), lane 9 cytotrophoblast cells (1.5 h in vitro), lane 10 cytotrophoblast cells (6 h in vitro), lane 11 cytotrophoblast cells (18 h in vitro), lane 12 cytotrophoblast cells (48 h in vitro). For 12c: lane 1 JAR bladder cell line, lane 2 NCITT testicular tumor cell line, lane 3 SW-480 human hepatoma cell line, lane 4 no HTR (cytotrophoblasts transformed by SV40), lane 5 HPTLP-I hepatocellular carcinoma cell line, lane 6 EJ-28 bladder carcinoma cell line. For 12d: lane 1 SK-hep-1 human hepatoma cell line, lane 2 DAMI human megakaryocytic cell line, lane 3 DAMI cell line PMA, lane 4 CHRF cell line PMA. lane 5 CHRF cell line. For 12e: lane 1 ABAE bovine aortic endothelial cells, lane 2 1063 human ovarian cell line, lane 3 human breast carcinoma MDA435 cell line, lane 4 human breast carcinoma MDA231 cell line.

FIG. 13 presents a comparison between nucleotide sequences of the human hpa and a mouse EST cDNA fragment (SEQ ID NO:12) which is 80 homologous to the 3' end (starting at nucleotide 1066 of SEQ ID NO:9) of the 20 human hpa. The aligned termination codons are underlined.

FIG. 14 demonstrates the chromosomal localization of the hpa gene. PCR products of DNA derived from somatic cell hybrids and of genomic DNA of hamster, mouse and human of were separated on 0.7 agarose gel following.

amplification with hpa specific primers. Lane 1 Lambda DNA digested with BstEII, lane 2 no DNA control, lanes 3 29, PCR amplification products.

Lanes 3-5 human, mouse and hamster genomic DNA, respectively. Lanes 6- 29, human monochromosomal somatic cell hybrids representing chromosomes 1- 22 and X and Y, respectively. Lane 30 Lambda DNA digested with BstEII. An amplification product of approximately 2.8 Kb is observed only in lanes 5 and 9, representing human genomic DNA and DNA derived from cell hybrid carrying human chromosome 4, respectively. These results demonstrate that the hpa gene Sis localized in human chromosome 4.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is of a polynucleotide, referred to hereinbelow interchangeably as hpa, hpa cDNA or hpa gene, encoding a polypeptide having heparanase activity, vectors including same, transduced cells expressing heparanase and a recombinant protein having heparanase activity.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

The present invention can be used to develop treatments for various diseases, to develop diagnostic assays for these diseases and to provide new tools for basic research especially in the fields of medicine and biology.

Specifically, the present invention can be used to develop new drugs to inhibit tumor cell metastasis, inflammation and autoimmunity. The identification of the hpa gene encoding for the heparanase enzyme enables the production of a recombinant enzyme in heterologous expression systems.

Furthermore, the present invention can be used to modulate bioavailability of heparin-binding growth factors, cellular responses to heparin-binding growth factors bFGF, VEGF) and cytokines cell interaction with plasma 20 lipoproteins, cellular susceptibility to viral, protozoa and some bacterial infections, and disintegration of neurodegenerative plaques. Recombinant heparanase is thus a potential treatment for wound healing, angiogenesis, restenosis, atherosclerosis, inflammation, neurodegenerative diseases (such .as, for example, Genstmann-Straussler Syndrome, Creutzfeldt-Jakob disease, Scrape and Alzheimer's disease) and certain viral and some bacterial and protozoa infections. Recombinant heparanase can be used to neutralize plasma heparin, as a potential replacement of protamine.

As used herein, the term "modulate" includes substantially inhibiting, slowing or reversing the progression of a disease, substantially ameliorating clinical symptoms of a disease or condition, or substantially preventing the appearance of clinical symptoms of a disease or condition. A "modulator" therefore includes an agent which may modulate a disease or condition.

Modulation of viral, protozoa and bacterial infections includes any effect which substantially interrupts, prevents or reduces any viral, bacterial or protozoa activity and/or stage of the virus, bacterium or protozoon life cycle, or which reduces or prevents infection by the virus, bacterium or protozoon in a subject, such as a human or lower animal.

As used herein, the term "wound" includes any injury to any portion of the body of a subject including, but not limited to, acute conditions such as thermal bums, chemical bums, radiation burns, burns caused by excess exposure to ultraviolet radiation such as sunburn, damage to bodily tissues such as the perineum as a result of labor and childbirth, including injuries sustained during medical procedures such as episiotomies, trauma-induced injuries including cuts, those injuries sustained in automobile and other mechanical accidents, and those caused by bullets, knives and other weapons, and post-surgical injuries, as well as chronic conditions such as pressure sores, bedsores, conditions related to diabetes and poor circulation, and all types of acne, etc.

Anti-heparanase antibodies, raised against the recombinant enzyme, would be useful for immunodetection and diagnosis of micrometastases, autoimmune lesions and renal failure in biopsy specimens, plasma samples, and body fluids.

Such antibodies may also serve as neutralizing agents for heparanase activity.

Cloning of the human hpa gene encoding heparanase and expressing recombinant heparanase by transfected cells is herein reported. This is the first mammalian heparanase gene to be cloned.

A purified preparation of heparanase isolated from human hepatoma cells was subjected to tryptic digestion and microsequencing.

20 The YGPDVGQPR (SEQ ID NO:8) sequence revealed was used to screen EST databases for homology to the corresponding back translated DNA sequences. Two closely related EST sequences were identified and were thereafter found to be identical.

Both clones contained an insert of 1020 bp which includes an open reading frame of 973 bp followed by a 3' untranslated region of 27 bp and a Poly A tail, whereas a translation start site was not identified.

Cloning of the missing 5' end was performed by PCR amplification of DNA from placenta Marathon RACE cDNA composite using primers selected according to the EST clones sequence and the linkers of the composite.

30 A 900 bp PCR fragment, partially overlapping with the identified 3' encoding EST clones was obtained. The joined cDNA fragment (hpa), 1721 bp long (SEQ ID NO:9), contained an open reading frame which encodes, as shown in Figure 1 and SEQ ID NO:11, a polypeptide of 543 amino acids (SEQ ID NO: 10) with a calculated molecular weight of 61,192 daltons.

A single nucleotide difference at position 799 (A to T) between the EST clones and the PCR amplified cDNA was observed. This difference results in a single amino acid substitution (Tyr to Phe) (Figure Furthermore, the published EST sequences contained an unidentified nucleotide, which following DNA sequencing of both the EST clones was resolved into two nucleotides (G and C at positions 1630 and 1631 in SEQ ID NO:9, respectively).

The ability of the hpa gene product to catalyze degradation of heparan sulfate in an in vitro assay was examined by expressing the entire open reading frame in insect cells, using the Baculovirus expression system.

Extracts and conditioned media of cells infected with virus containing the hpa gene, demonstrated a high level of heparan sulfate degradation activity both towards soluble ECM-derived HSPG and intact ECM, which was inhibited by heparin, while cells infected with a similar construct containing no hpa gene had 0 no such activity, nor did non-infected cells.

The expression pattern of hpa RNA in various tissues and cell lines was investigated using RT-PCR. It was found to be expressed only in tissues and cells previously known to have heparanase activity.

Cloning an extended 5' sequence was enabled from the human SK-hepl cell line by PCR amplification using the Marathon RACE. The 5' extended sequence of the SK-hepl hpa cDNA was assembled with the sequence of the hpa cDNA isolated from human placenta (SEQ ID NO:9). The assembled sequence contained an open reading frame, SEQ ID NOs: 13 and 15, which encodes, as shown in SEQ ID NOs:14 and 15. a polypeptide of 592 amino acids, with a o calculated molecular weight of 66,407 daltons. This open reading frame was shown to direct the expression of catalitically active heparanase in a mammalian cell expression system. The expressed heparanase was detectable by anti :heparanase antibodies in Western blot analysis.

A panel of monochromosomal human/CHO and human/mouse somatic cell hybrids was used to localize the human heparanase gene to human chromosome 4. The newly isolated heparanase sequence can therefore be used to identify a chromosome region harboring a human heparanase, gene in a chromosome spread.

Thus, according to the present invention there is provided a polynucleotide 30 fragment (either DNA or RNA, either single stranded or double stranded) which includes a polynucleotide sequence encoding a polypeptide having heparanase *catalytic activity.

The term "heparanase catalytic activity" or its equivalent term "heparanase activity" both refer to a mammalian endoglycosidase hydrolyzing activity which is specific for heparan or heparan sulfate proteoglycan substrates, as opposed to the activity of bacterial enzymes (heparinase I, II and III) which degrade heparin or heparan sulfate by means of p-elimination (37).

17 In a preferred embodiment of the invention the polynucleotide fragment includes nucleotides 63-1691 of SEQ ID NO:9, or nucleotides 139-1869 of SEQ ID NO:13, which encode the entire human heparanase enzyme.

However, the scope of the present invention is not limited to human s heparanase since this is the first disclosure of an open reading frame (ORF) encoding any mammalian heparanase. Using the hpa cDNA, parts thereof or synthetic oligonucleotides designed according to its sequence will enable one ordinarily skilled in the art to identify genomic and/or cDNA clones including homologous sequences from other mammalian species.

The present invention is therefore further directed at a polynucleotide fragment which includes a polynucleotide sequence capable of hybridizing (base pairing under either stringent or permissive hybridization conditions, as for example described in Sambrook, Fritsch, Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Ne\w York.) with hpa cDNA, especially with nucleotides 1-721 of SEQ ID NO:9.

In fact, any polynucleotide sequence which encodes a polypeptide having heparanase activity and which shares at least 60 homology, preferably at least homology, more preferably at least 80 homology, most preferably at least 90 homology with SEQ ID NOs:9 or 13 is within the scope of the present 20 invention.

The polynucleotide fragment according to the present- invention may include any part of SEQ ID NOs: 9 or 13. For example, it may include nucleotides 63-721 of SEQ ID NO:9, which is a novel sequence. However, it may include any segment of SEQ ID NOs:9 or 13 which encodes a polypeptide having the heparanase catalytic activity.

When the phrase "encodes a polypeptide having heparanase catalytic activity" is used herein and in the claims section below it refers to the ability of directing the synthesis of a polypeptide which, if so required for its activity, following post translational modifications, such as but not limited to, proteolysis 30 removal of a signal peptide and of a pro- or preprotein sequence), methionine modification, glycosylation, alkylation methylation), acetylation, etc., is catalytically active in degradation of, for example, ECM and Scell surface associated HS.

In a preferred embodiment of the invention the polypeptide encoded by the polynucleotide fragment includes an amino acid sequence as set forth in SEQ ID or 14 or a functional part thereof, a portion harboring heparanase catalytic activity.

However, any polynucleotide fragment which encodes a polypeptide having heparanase activity is within the scope of the present invention.

Therefore, the polypeptide may be allelic, species and/or induced variant of the amino acid sequence set forth in SEQ ID NOs:10 or 14 or functional part thereof.

In fact, any polynucleotide sequence which encodes a polypeptide having heparanase activity, which shares at least 60 homology, preferably at least homology, more preferably at least 80 homology, most preferably at least homology with SEQ ID NOs:10 or 14 is within the scope of the present invention.

o The invention is also directed at providing a single stranded polynucleotide fragment which includes a polynucleotide sequence complementary to at least a portion of a polynucleotide strand encoding a polypeptide having heparanase catalytic activity as described above. The term "complementary" as used herein refers to the ability of base pairing.

The single stranded polynucleotide fragment may be DNA or RNA or even include nucleotide analogs thioated nucleotides), it may be a synthetic oligonucleotide or manufactured by transduced host cells, it may be of any desired length which still provides specific base pairing 8 or 10, preferably more, nucleotides long) and it may include mismatches that do not hamper base pairing.

The invention is further directed at providing a vector which includes a polynucleotide sequence encoding a polypeptide having heparanase catalytic activity.

The vector may be of any type. It may be a phage which infects bacteria or a virus which infects eukaryotic cells. It may also be a plasmid, phagemid, cosmid, bacmid or an artificial chromosome. The polynucleotide sequence encoding a polypeptide having heparanase catalytic activity may include any of the above described polynucleotide fragments.

The invention is further directed at providing a host cell which includes an exogenous polynucleotide fragment encoding a polypeptide having heparanase catalytic activity.

The exogenous polynucleotide fragment may be any of the above described fragments. The host cell may be of any type. It may be a prokaryotic cell, an eukaryotic cell, a cell line, or a cell as a portion of an organism. The exogenous polynucleotide fragment may be permanently or transiently present in the cell. In other words, transduced cells obtained following stable or transient transfection, transformation or transduction are all within the scope of the present invention. The term "exogenous" as used herein refers to the fact that the 19 polynucleotide fragment is externally introduced into the cell. Therein it may be present in a single of any number of copies, it may be integrated into one or more chromosomes at any location or be present as an extrachromosomal material.

The invention is further directed at providing a heparanase overexpression system which includes a cell overexpressing heparanase catalytic activity. The cell may be a host cell transiently or stably transfected or transformed with any suitable vector which includes a polynucleotide sequence encoding a polypeptide having heparanase activity and a suitable promoter and enhancer sequences to direct overexpression of heparanase. However, the overexpressing cell may also be a product of an insertion via homologous recombination) of a promoter and/or enhancer sequence downstream to the endogenous heparanase gene of the expressing cell, which will direct overexpression from the endogenous gene. The term "overexpression" as used herein in the specification and claims below refers to a level of expression which is higher than a basal level of expression typically characterizing a given cell under otherwise identical conditions.

The invention is further directed at providing a recombinant protein including a polypeptide having heparanase catalytic activity.

The recombinant protein may be purified by any conventional protein purification procedure close to homogeneity and/or be mixed with additives. The 20 recombinant protein may be manufactured using any of the cells described above.

The recombinant protein may be in any form. It may be in a crystallized form, a dehydrated powder form or in solution. The recombinant protein may be useful in obtaining pure heparanase, which in turn may be useful in eliciting antiheparanase antibodies, either poly or monoclonal antibodies, and as a screening active ingredient in an anti-heparanase inhibitors or drugs screening assay or system.

The invention is further directed at providing a pharmaceutical composition which include as an active ingredient a recombinant protein having heparanase catalytic activity.

Formulations for topical administration may include, but are not limited to, lotions, ointments, gels, creams, suppositories, drops, liquids, sprays and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, stents, active pads, and other medical devices may also be useful. In fact the scope of the present invention includes any medical equipment such as a medical device containing, as an active ingredient, a recombinant protein having heparanase catalytic activity.

Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, sachets, capsules or tablets. Thickeners, diluents, flavorings, dispersing aids, emulsifiers or binders may be desirable.

Formulations for parenteral administration may include, but are not limited to, sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.

Dosing is dependent on severity and responsiveness of the condition to be treated, but will normally be one or more doses per day, with course of treatment lo lasting from several days to several months or until a cure is effected or a diminution of disease state is achieved. Persons ordinarily skilled in the art can easily determine optimum dosages, dosing methodologies and repetition rates.

Further according to the present invention there is provided a method of identifying a chromosome region harboring a human heparanase gene in a chromosome spread. the method is executed implementing the following method steps, in which in a first step the chromosome spread (either interphase or metaphase spread) is hybridized with a tagged polynucleotyde probe encoding heparanase. The tag is preferably a fluorescent tag. In a second step according to the method the chromosome spread is washed, thereby excess of non-hybridized 20 probe is removed. Finally, signals associated with the hybridized tagged polynucleotyde probe are searched for, wherein detected signals being indicative of a chromosome region harboring the human heparanase gene. One ordinarily skilled in the art would know how to use the sequences disclosed herein in suitable labeling reactions and how to use the tagged probes to detect, using in situ hybridization, a chromosome region harboring a human heparanase gene.

o Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non-limiting fashion.

30

EXAMPLES

The following protocols and experimental details are referenced in the Examples that follow: Purification and characterization of heparanase from a human hepatoma cell line and human placenta: A human hepatoma cell line (Sk-hep- 1) was chosen as a source for purification of a human tumor-derived heparanase.

Purification was essentially as described in U.S. Pat. No. 5,362,641 to Fuks, 21 which is incorporated by reference as if fully set forth herein. Briefly, 500 liter, 5x10" cells were grown in suspension and the heparanase enzyme was purified about 240,000 fold by applying the following steps: cation exchange (CM- Sephadex) chromatography performed at pH 6.0, 0.3-1.4 M NaCI gradient; (ii) s cation exchange (CM-Sephadex) chromatography performed at pH 7.4 in the presence of 0.1% CHAPS, 0.3-1.1 M NaCl gradient; (iii) heparin-Sepharose chromatography performed at pH 7.4 in the presence of 0.1% CHAPS, 0.35-1.1 M NaCI gradient; (iv) ConA-Sepharose chromatography performed at pH 6.0 in buffer containing 0.1 CHAPS and 1 M NaCI, clution with 0.25 M a-methyl mannoside; and HPLC cation exchange (Mono-S) chromatography performed at pH 7.4 in the presence of 0.1 CHAPS, 0.25-1 M NaCI gradient.

Active fractions were pooled, precipitated with TCA and the precipitate subjected to SDS polyacrylamide gel electrophoresis and/or tryptic digestion and reverse phase HPLC. Tryptic peptides of the purified protein were separated by reverse phase HPLC (C8 column) and homogeneous peaks were subjected to amino acid sequence analysis.

The purified enzyme was applied to reverse phase HPLC and subjected to N-terminal amino acid sequencing using the amino acid sequencer (Applied Biosystems).

20 Cells: Cultures of bovine corneal endothelial cells (BCECs) were established from steer eyes as previously described (19, 38). Stock cultures were maintained in DMEM (1 g glucose/liter) supplemented with 10 newborn calf serum and 5 FCS. bFGF (1 ng/ml) was added every other day during the phase of active cell growth (13, 14).

Preparation of dishes coated with ECM: BCECs (second to fifth passage) were plated into 4-well plates at an initial density of 2 x 10 5 cells/ml, and cultured in sulfate-free Fisher medium plus 5 dextran T-40 for 12 days.

Na 2 35

SO

4 (25 uLCi/ml) was added on day 1 and 5 after seeding and the cultures were incubated with the label without medium change. The subendothelial ECM was exposed by dissolving (5 min., room temperature) the cell layer with PBS containing 0.5 Triton X-100 and 20 mM NH 4 OH, followed by four washes with PBS. The ECM remained intact, free of cellular debris and firmly attached to the entire area of the tissue culture dish (19, 22).

To prepare soluble sulfate labeled proteoglycans (peak I material), the ECM was digested with trypsin (25 p.g/ml, 6 h, 37 OC the digest was concentrated by reverse dialysis and the concentrated material was applied onto a Sepharose 6B gel filtration column. The resulting high molecular weight 22 material (Kav< 0.2, peak I) was collected. More than 80 of the labeled material was shown to be composed of heparan sulfate proteoglycans (11, 39).

Heparanase activity: Cells (1 x 10 6 /35-mm dish), cell lysates or conditioned media were incubated on top of 35 S-labeled ECM (18 h, 37 oC) in the 3 presence of 20 mM phosphate buffer (pH Cell lysates and conditioned media were also incubated with sulfate labeled peak I material (10-20 11). The incubation medium was collected, centrifuged (18,000 x g, 4 3 min.), and sulfate labeled material analyzed by gel filtration on a Sepharose CL-6B column (0.9 x 30 cm). Fractions (0.2 ml) were eluted with PBS at a flow rate of 5 ml/h o and counted for radioactivity using Bio-fluor scintillation fluid. The excluded volume (Vo) was marked by blue dextran and the total included volume (Vt) by phenol red. The latter was shown to comigrate with free sulfate 11, 23).

Degradation fragments of HS side chains were eluted from Sepharose 6B at 0.5 Kay 0.8 (peak II) 11, 23). A nearly intact HSPG released from ECM by Strypsin and, to a lower extent, during incubation with PBS alone was eluted next to Vo (Kay 0.2, peak Recoveries of labeled material applied on the columns ranged from 85 to 95 in different experiments Each experiment was performed at least three times and the variation of elution positions (Kay values) did not exceed 15 o Cloning of hpa cDNA: cDNA clones 257548 and 260138 were obtained from the I.M.A.G.E Consortium (2130 Memorial Parkway SW, Hunstville, AL 35801). The cDNAs were originally cloned in EcoRI and NotI cloning sites in the plasmid vector pT3T7D-Pac. Although these clones are reported to be somewhat different, DNA sequencing demonstrated that these clones are identical to one another. Marathon RACE (rapid amplification of cDNA ends) i human placenta (poly-A) cDNA composite was a gift of Prof. Yossi Shiloh of Tel Aviv University. This composite is vector free, as it includes reverse transcribed cDNA fragments to which double, partially single stranded adapters.are attached on both sides. The construction of the specific composite employed is described in reference 39a.

Amplification of hp3 PCR fragment was performed according to the protocol provided by Clontech laboratories. The template used for amplification o*o was a sample taken from the above composite. The primers used for amplification were: First step: 5'-primer: API: SEQ ID NO:1; 3'-primer: HPL229: ATC-3', SEQ ID NO:2.

23 Second step: nested 5'-primcr: AP2: ACTCACTATAGGGCTCGAGCG GC-3', SEQ ID NO:3; nested primer: HPL171: 5'-GCATCTTAGCCGTCT TTCTTCG-3', SEQ ID NO:4. The HPL229 and HPL171 were selected according to the sequence of the EST clones. They include nucleotides 933-956 and 876-897 of SEQ ID NO:9, respectively.

PCR program was 94 °C 4 min., followed by 30 cycles of 94 °C sec., 62 °C 1 min., 72 OC 2.5 min. Amplification was performed with Expand High Fidelity (Boehringer Mannheim). The resulting ca. 900 bp hp3 PCR product was digested with Bfr- and Pvull. Clone 257548 (phpal) was digested o with EcoRI, followed by end filling and was then further digested with Bfrl.

Thereafter the Pvull BfrI fragment of the hp3 PCR product was cloned into the blunt end Bfi- end of clone phpal which resulted in having the entire cDNA cloned in pT3T7-pac vector, designated phpa2.

DNA Sequencing: Sequence determinations were performed with vector specific and gene specific primers, using an automated DNA sequencer (Applied Biosystems, model 373A). Each nucleotide was read from at least two independent primers.

Computer analysis of sequences: Database searches for sequence similarities were performed using the Blast network service. Sequence analysis '20 and alignment of DNA and protein sequences were done using the DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the University of Wisconsin.

RT-PCR: RNA was prepared using TRI-Reagent (Molecular research center Inc.) according to the manufacturer instructions. 1.25 pg were taken for reverse transcription reaction using MuMLV Reverse transcriptase (Gibco.BRL) and Oligo (dT) 1 5 primer, SEQ ID NO:5, (Promega). Amplification of the S resultant first strand cDNA was performed with Taq polymerase (Promega). The following primers were used: HPU-355: 5'-TTCGATCCCAAGAAGGAATCAAC-3', SEQ ID NO:6, 30 nucleotides 372-394 in SEQ ID NO:9 or 11.

HPL-229: 5'-GTAGTGATGCCATGTAACTGAATC-3', SEQ ID NO:7, nucleotides 933-956 in SEQ ID NO:9 or 11.

PCR program: 94 OC 4 min., followed by 30 cycles of 94 OC 40 sec., 62 OC 1 min., 72 oC 1 min.

Expression of recombinant heparanase in insect cells: 'Cells, High Five and Sf21 insect cell lines were maintained as monolayer cultures in SF900II-SFM medium (GibcoBRL).

Recombinant Baculovirus: Recombinant virus containing the hpa gene was constructed using the Bac to Bac system (GibcoBRL). The transfer vector pFastBac was digested with Sall and Notl and ligated with a 1.7 kb fragment of phpa2 digested with XhoI and Notl. The resulting plasmid was designated pFasthpa2. An identical plasmid designated pFasthpa4 was prepared as a duplicate and both independently served for further experimentations.

Recombinant bacmid was generated according to the instructions of the manufacturer with pFasthpa2, pFasthpa4 and with pFastBac. The latter served as a negative control. Recombinant bacmid DNAs were transfected into Sf21 insect 0o cells. Five days after transfection recombinant viruses were harvested and used to infect High Five insect cells, 3 x 10 6 cells in T-25 flasks. Cells were harvested 2 3 days after infection. 4 x 10 6 cells were centrifuged and resuspended in a reaction buffer containing 20 mM phosphate citrate buffer, 50 mM NaCI. Cells underwent three cycles of freeze and thaw and lysates were stored at -80 C.

Conditioned medium was stored at 4 °C.

Partial purification of recombinant heparanase: Partial purification of recombinant heparanase was performed by heparin-Sepharose column chromatography followed by Superdex 75 column gel filtration. Culture medium (150 ml) of Sf21 cells infected with pFhpa4 virus was subjected to heparin- 20 Sepharose chromatography. Elution of 1 ml fractions was performed with 0.35 2 M NaCI gradient in presence of 0.1 CHAPS and 1 mM DTT in 10 mM sodium acetate buffer, pH 5.0. A 25 ul sample of each fraction was tested for

S

heparanase activity. Heparanase activity was eluted at the range of 0.65 1.1 M NaCl (fractions 18-26, Figure 10a). 5 il of each fraction was subjected to 15 SDS-polyacrylamide gel electrophoresis followed by silver nitrate staining.

Active fractions eluted from heparin-Sepharose (Figure 10a) were pooled and concentrated (x 6) on YM3 cut-off membrane. 0.5 ml of the concentrated material was applied onto 30 ml Superdex 75 FPLC column equilibrated with mM sodium acetate buffer, pH 5.0, containing 0.8 M NaCI, 1 mM DTT and 0.1 30 CHAPS. Fractions (0.56 ml) were collected at a flow rate of 0.75 ml/min.

Aliquots of each fraction were tested for heparanase activity and were subjected to SDS-polyacrylamide gel electrophoresis followed by silver nitrate staining (Figure lb).

EXAMPLE 1 Cloning of the hpa gene Purified fraction of heparanase isolated from human hepatoma cells (SKhep-1) was subjected to tryptic digestion and microsequencing. EST (Expressed Sequence Tag) databases were screened for homology to the back translated DNA sequences corresponding to the obtained peptides. Two EST sequences (accession Nos. N41349 and N45367) contained a DNA sequence encoding the peptide YGPDVGQPR (SEQ ID NO:8). These two sequences were derived from clones 257548 and 260138 (I.M.A.G.E Consortium) prepared from 8 to 9 weeks to placenta cDNA library (Soares). Both clones which were found to be identical contained an insert of 1020 bp which included an open reading frame (ORF) of 973 bp followed by a 3' untranslated region of 27 bp and a Poly A tail. No translation start site (AUG) was identified at the 5' end of these clones.

Cloning of the missing 5' end was performed by PCR amplification of DNA from a placenta Marathon RACE cDNA composite. A 900 bp fragment (designated hp3), partially overlapping with the identified 3' encoding EST clones was obtained.

The joined cDNA fragment, 1721 bp long (SEQ ID NO:9), contained an open reading frame which encodes, as shown in Figure 1 and SEQ ID NO:11, a 20 polypeptide of 543 amino acids (SEQ ID NO:10) with a calculated molecular weight of 61,192 daltons. The 3' end of the partial cDNA inserts contained in clones 257548 and 260138 started at nucleotide G 72 1 of SEQ ID NO:9 and Figure As further shown in Figure 1, there was a single sequence discrepancy between the EST clones and the PCR amplified sequence, which led to an amino acid substitution from Tyr 246 in the EST to Phe 246 in the amplified cDNA. The nucleotide sequence of the PCR amplified cDNA fragment was verified from two independent amplification products. The new gene was designated hpa.

S As stated above, the 3' end of the partial cDNA inserts contained in EST 30 clones 257548 and 260138 started at nucleotide 721 of hpa (SEQ ID NO:9). The ability of the hpa cDNA to form stable secondary structures, such as stem and loop structures involving nucleotide stretches in the vicinity of position 721 was investigated using computer modeling. It was found that stable stem and loop structures are likely to be formed involving nucleotides 698-724 (SEQ ID NO:9).

In addition, a high GC content, up to 70 characterizes the 5' end region of the hpa gene, as compared to about only 40 in the 3' region. These findings may explain the immature termination and therefore lack of 5' ends in the EST clones.

26 To examine the ability of the hpa gene product to catalyze degradation of heparan sulfate in an in vitro assay the entire open reading frame was expressed in insect cells, using the Baculovirus expression system. Extracts of cells, infected with virus containing the hpa gene, demonstrated a high level of heparan sulfate degradation activity, while cells infected with a similar construct containing no hpa gene had no such activity, nor did non-infected cells. These results are further demonstrated in the following Examples.

EXAMPLE 2 o Degradation of soluble ECM-derived

HSPG

Monolayer cultures of High Five cells were infected (72 h, 28 OC) with recombinant Bacoluvirus containing the pFasthpa plasmid or with control virus containing an insert free plasmid. The cells were harvested and lysed in heparanase reaction buffer by three cycles of freezing and thawing. The cell lysates were then incubated (18 h, 37 oC) with sulfate labeled, ECM-derived HSPG (peak followed by gel filtration analysis (Sepharose 6B) of the reaction mixture.

As shown in Figure 2, the substrate alone included almost entirely high molecular weight (Mr) material eluted next to V o (peak I, fractions 5-20, Kav 20 0.35). A similar elution pattern was obtained when the HSPG substrate was incubated with lysates of cells that were infected with control virus. In contrast, incubation of the HSPG substrate with lysates of cells infected with the hpa containing virus resulted in a complete conversion of the high Mr substrate into low Mr labeled degradation fragments (peak II, fractions 22-35, 0.5 Kav 0.75).

:I Fragmen'ts eluted in peak II were shown to be degradation products of heparan sulfate, as they were 5- to 6-fold smaller than intact heparan sulfate side chains (Kav approx. 0.33) released from ECM by treatment with either alkaline borohydride or papain; and (ii) resistant to further digestion with papain or chondroitinase ABC, and susceptible to deamination by nitrous acid 11).

Similar results (not shown) were obtained with Sf21 cells. Again, heparanase activity was detected in cells infected with the hpa containing virus (pFhpa), but not with control virus This result was obtained with two independently generated recombinant viruses. Lysates of control not infected High Five cells failed to degrade the HSPG substrate.

In subsequent experiments, the labeled HSPG substrate was incubated with medium conditioned by infected High Five or Sf21 cells.

As shown in Figures 3a-b, heparanase activity, reflected by the conversion of the high Mr peak 1 substrate into the low Mr peak 11 which represents lHS degradation fragments, was found in the culture medium of cells infected with the pFhpa2 or pFhpa4 viruses, but not with the control pFl or pF2 viruses. No heparanase activity was detected in the culture medium of control non-infected High Five or Sf21 cells.

The medium of cells infected with the pFhpa4 virus was passed through a kDa cut off membrane to obtain a crude estimation of the molecular weight of the recombinant heparanase enzyme. As demonstrated in Figure 4, all the o enzymatic activity was retained in the upper compartment and there was no activity in the flow through (<50 kDa) material. This result is consistent with the expected molecular weight of the hpa gene product.

In order to further characterize the hpa product the inhibitory effect of heparin, a potent inhibitor of heparanase mediated HS degradation (40) was examined.

As demonstrated in Figures 5a-b, conversion of the peak I substrate into peak II HS degradation fragments was completely abolished in the presence of heparin.

Altogether, these results indicate that the heparanase enzyme is expressed :o in an active form by insect cells infected with Baculovirus containing the newly identified human hpa gene.

EXAMPLE 3 Degradation of HSPG in intact ECM Next, the ability of intact infected insect cells to degrade HS in intact, naturally produced ECM was investigated. For this purpose, High Five or Sf21 cells were seeded on metabolically sulfate labeled ECM followed by infection (48 h, 28 oC) with either the pFhpa4 or control pF2 viruses. The pH of the medium was then adjusted to pH 6.2-6.4 and the cells further incubated with the labeled ECM for another 48 h at 28 OC or 24 h at 37 OC. Sulfate labeled material released into the incubation medium was analyzed by gel filtration on Sepharose 6B.

As shown in Figures 6a-b and 7a-b, incubation of the ECM with cells infected with the control pF2 virus resulted in a constant release of labeled material that consisted almost entirely of high Mr fragments (peak I) eluted with or next to V o It was previously shown that a proteolytic activity residing in the ECM itself and/or expressed by cells is responsible for release of the high Mr material This nearly intact HSPG provides a soluble substrate 28 for subsequent degradation by heparanase, as also indicated by the relatively large amount of peak I material accumulating when the heparanase enzyme is inhibited by heparin 7, 12, Figure On the other hand, incubation of the labeled ECM with cells infected with the pFhpa4 virus resulted in release of 70% of the ECM-associated radioactivity in the form of low Mr sulfate-labeled fragments (peak 11, 0.5 <Kav< 0.75), regardless of whether the infected cells were incubated with the ECM at 28 °C or 37 Control intact non-infected Sf21 or High Five cells failed to degrade the ECM HS side chains.

In subsequent experiments, as demonstrated in Figures 8a-b, High Five and Sf21 cells were infected (96 h, 28 with pFhpa4 or control pF1 viruses and the culture medium incubated with sulfate-labeled ECM. Low Mr HS degradation fragments were released from the ECM only upon incubation with medium conditioned by pFhpa4 infected cells. As shown in Figure 9, production of these fragments was abolished in the presence of heparin. No heparanase activity was detected in the culture medium of control, non-infected cells. These results indicate that the heparanase enzyme expressed by cells infected with the pFhpa4 virus is capable of degrading HS when complexed to other macromolecular constituents fibronectin, laminin, collagen) of a naturally produced intact ECM, in a manner similar to that reported for highly metastatic 20 tumor cells or activated cells of the immune system 7).

EXAMPLE 4 Purification of recombinant heparanase The recombinant heparanase was partially purified from medium of pFhpa4 infected Sf21 cells by Heparin-Sepharose chromatography (Figure i followed by gel filtration of the pooled active fractions over an FPLC Superdex column (Figure 1 la). A 63 kDa protein was observed, whose quantity, as was detected by silver stained SDS-polyacrylamide gel electrophoresis, correlated with heparanase activity in the relevant column fractions (Figures and 1 Ib, respectively). This protein was not detected in the culture medium of cells infected with the control pFl virus and was subjected to a similar fractionation on heparin-Sepharose (not shown).

EXAMPLE Expression of the hpa gene in various cell types, organs and tissues Referring now to Figures 12a-e, RT-PCR was applied to evaluate the expression of the hpa gene by various cell types and tissues. For this purpose, total RNA was reverse transcribed and amplified. The expected 585 bp long cDNA was clearly demonstrated in human kidney, placenta (8 and 11 weeks) and mole tissues, as well as in freshly isolated and short termed (1.5-48 h) cultured human placental cytotrophoblastic cells (Figure 12a), all known to express a high heparanase activity The hpa transcript was also expressed by normal human neutrophils (Figure 12b). In contrast, there was no detectable expression of the hpa mRNA in embryonic human muscle tissue, thymus, heart and adrenal (Figure 12b). The hpa gene was expressed by several, but not all, human bladder carcinoma cell lines (Figure 12c), SK hepatoma (SK-hep-1), ovarian carcinoma (OV 1063), breast carcinoma (435, 231), melanoma and megakaryocytic (DAMI, o1 CHRF) human cell lines (Figures 12d-e).

The above described expression pattern of the hpa transcript was determined to be in a very good correlation with heparanase activity levels determined in various tissues and cell types (not shown).

EXAMPLE 6 hpa homologous genes EST databases were screened for sequences homologous to the hpa gene.

Three mouse ESTs were identified (accession No. Aal77901, from mouse spleen, 20 Aa067997 from mouse skin, Aa47943 from mouse embryo), assembled into a 824 bp cDNA fragment which contains a partial open reading frame (lacking a end) of 629 bp and a 3' untranslated region of 195 bp (SEQ ID NO:12). As shown in Figure 13, the coding region is 80% similar to the 3' end of the hpa cDNA sequence. These ESTs are probably cDNA fragments of the mouse hpa homolog that encodes for the mouse heparanase.

Searching for consensus protein domains revealed an amino terminal homology between the heparanase and several precursor proteins such as Procollagen Alpha 1 precursor, Tyrosine-protein kinase-RYK, Fibulin-1, Insulinlike growth factor binding protein and several others. The amino terminus is S 30 highly hydrophobic and contains a potential trans-membrane domain. The homology to known signal peptide sequences suggests that it could function as a signal peptide for protein localization.

EXAMPLE 7 Isolation of an extended 5' end of hpa cDNA from human SK-hepl cell line The 5' end of hpa cDNA was isolated from human SK-hepl cell line by PCR amplification using the Marathon RACE (rapid amplification of cDNA ends) kit (Clontech). Total RNA was prepared from SK-hepl cells using the TRI-Reagent (Molecular research center Inc.) according to the manufacturer instructions. Poly A+ RNA was isolated using the mRNA separator kit (Clonetech).

The Marahton RACE SK-hepl cDNA composite was constructed according to the manufacturer recommendations. First round of amplification was performed using an adaptor specific primer AP1: ACTCACTATAGGGC-3', SEQ ID NO:1, and a hpa specific antisense primer hpl-629: 5'-CCCCAGGAGCAGCAGCATCAG-3', SEQ ID NO:17, io corresponding to nucleotides 119-99 of SEQ ID NO:9. The resulting PCR product was subjected to a second round of amplification using an adaptor specific nested primer AP2: 5'-ACTCACTATAGGGCTCGAGCGGC-3',

SEQ

ID NO:3, and a hpa specific antisense nested primer hpl-666 AGGCTTCGAGCGCAGCAGCAT-3'. SEQ ID NO:18, corresponding to nucleotides 83-63 of SEQ ID NO:9. The PCR program was as follows: a hot start of 94 °C for 1 minute, followed by 30 cycles of 90 °C 30 seconds, 68 "C 4 minutes. The resulting 300 bp DNA fragment was extracted from an agarose gel and cloned into the vector pGEM-T Easy (Promega). The resulting recombinant plasmid was designated pHPSKl.

20 The nucleotide sequence of the pHPSK1 insert was determined and it was found to contain 62 nucleotides of the 5' end of the placenta hpa cDNA (SEQ ID NO:9) and additional 178 nucleotides upstream, the first 178 nucleotides of SEQ ID NOs:13 and A single nucleotide discrepancy was identified between the SK-hepl cDNA and the placenta cDNA. The derivative at position 9 of the placenta cDNA (SEQ ID NO:9), is replaced by a derivative at the corresponding position 187 of the SK-hepl cDNA (SEQ ID NO:13).

The discrepancy is likely to be due to a mutation at the 5' end of the placenta cDNA clone as confirmed by sequence analysis of sevsral additional 30 cDNA clones isolated from placenta, which like the SK-hep cDNA contained C at position 9 of SEQ ID NO:9.

The 5' extended sequence of the SK-hep 1 hpa cDNA was assembled with the sequence of the hpa cDNA isolated from human placenta (SEQ ID NO:9).

The assembled sequence contained an open reading frame which encodes, as shown in SEQ ID NOs:14 and 15, a polypeptide of 592 amino acids with a calculated molecular weight of 66,407 daltons. The open reading frame is flanked by 93 bp 5' untranslated region (UTR).

31 EXAMPLE 8 Isolation of the upstream genomic region of the hpa gene The upstream region of the hpa gene was isolated using the Genome Walker kit (Clontech) according to the manufacturer recommendations. The kit includes five human genomic DNA samples each digested with a different restriction endonuclease creating blunt ends: EcoRV, Scal, Dral, Pvull and Sspl.

The blunt ended DNA fragments are ligated to partially single stranded adaptors. The Genomic DNA samples were subjected to PCR amplification using the adaptor specific primer and a gene specific primer. Amplification was o1 performed with Expand High Fidelity (Boehringer Mannheim).

A first round of amplification was performed using the apl primer: TAATACGACTCACTATAGGGC-3', SEQ ID NO:19, and the hpa specific antisense primer hpl-666: 5'-AGGCTTCGAGCGCAGCAGCAT-3', SEQ ID NO:18. corresponding to nucleotides 83 63 of SEQ ID NO:9. The PCR program was as follows: a hot start of 94 °C 3 minutes, followed by 36 cycles of 94 °C 40 seconds, 67 °C 4 minutes.

The PCR products of the first amplification were diluted 1:50. One pil of the diluted sample was used as a template for a second amplification using a nested adaptor specific primer ap2: 5'-ACTATAGGGCACGCGTGGT-3',

SEQ

20 ID NO:20, and a hpa specific antisense primer hpl-690, TGGCTGCTC-3', SEQ ID NO:21, corresponding to nucleotides 62-42 of SEQ ID NO:9. The resulting amplification products were analyzed using agarose gel electrophoresis. Five different PCR products were obtained from the five amplification reactions. A DNA fragment of approximately 750 bp which was obtained from the SspI digested DNA sample was gel extracted. The purified fragment was ligated into the plasmid vector pGEM-T Easy (Promega). The resulting recombinant plasmid was designated pGHP6905 and the nucleotide sequence of the hpa insert was determined.

A partial sequence of 594 nucleotides is shown in SEQ ID NO:16. The last nucleotide in SEQ ID NO:13 corresponds to nucleotide 93 in SEQ ID:13.

The DNA sequence in SEQ ID NO:16 contains the 5' region of the hpa cDNA and 501 nucleotides of the genomic upstream region which are predicted to contain the promoter region of the hpa gene.

32 EXAMPLE 9 Expression of the 592 amino acids HPA polypeptide in a human 293 cell line The 592 amino acids open reading frame (SEQ ID NOs:13 and 15) was constructed by ligation of the I 10 bp corresponding to the 5' end of the SK-hep 1 hpa cDNA -with the placenta cDNA. More specifically the Marathon RACE PCR amplification product of the placenta hpa DNA was digested with Sacl and an approximately 1 kb fragment was ligated into a Sacl-digested pGHP6905 plasmid. The resulting plasmid was digested with Earl and Aatll. The Earl sticky ends were blunted and an approximately 280 bp Earl/blunt-AatlI fragment i0 was isolated. This fragment was ligated with pFasthpa digested with EcoRl which was blunt ended using Klenow fragment and further digested with Aatll.

The resulting plasmid contained a 1827 bp insert which includes an open reading frame of 1776 bp, 31 bp of 3' UTR and 21 bp of 5' UTR. This plasmid was designated pFastLhpa.

A mammalian expression vector was constructed to drive the expression of the 592 amino acids heparanase polypeptide in human cells. The hpa cDNA was excised prom pFastLhpa with BssHII and NotI. The resulting 1850 bp BssHII-NotI fragment was ligated to a mammalian expression vector pSI (Promega) digested with MluI and NotI. The resulting recombinant plasmid, 20 pSIhpaMet2 was transfected into a human 293 embryonic kidney cell line.

Transient expression of the 592 amino-acids heparanase was examined by western blot analysis and the enzymatic activity was tested using the gel shift assay. Both these procedures are described in length in U.S. Pat. application No.

:..'09/071,739, filed May 1, 1998, which is incorporated by reference as if fully set forth herein. Cells were harvested 3 days following transfection. Harvested cells :i were re-suspended in lysis buffer containing 150 mM NaCI, 50 mM Tris pH 1% Triton X-100, 1 mM PMSF and protease inhibitor cocktail (Boehringer Mannheim). 40 pg protein extract samples were used for separation on a SDS- PAGE. Proteins were transferred onto a PVDF Hybond-P membrane (Amersham). The membrane was incubated with an affinity purified polyclonal anti heparanase antibody, as described in U.S. Pat. application No. 09/071,739.

A major band of approximately 50 kDa was observed in the transfected cells as well as a minor band of approximately 65 kDa. A similar pattern was observed in extracts of cells transfected with the pShpa as demonstrated in U.S. Pat.

application No. 09/071,739. These two bands probably represent two forms of the recombinant heparanase protein produced by the transfected cells. The kDa protein probably represents a heparanase precursor, while the 50 kDa protein is suggested herein to be the processed or mature form.

The catalytic activity of the recombinant protein expressed in the pS/hpaMet2 transfected cells was tested by gel shift assay. Cell extracts of transfected and of mock transfected cells were incubated overnight with heparin (6 pg in each reaction) at 37 in the presence of 20 mM phosphate citrate buffer pH 5.4, 1 mM CaCI 2 1 mM DTT and 50 mM NaCI. Reaction mixtures were then separated on a 10 polyacrylamide gel. The catalytic activity of the recombinant heparanase was clearly demonstrated by a faster migration of the heparin molecules incubated with the transfected cell extract as compared to the control. Faster migration indicates the disappearance of high molecular weight to heparin molecules and the generation of low molecular weight degradation products.

EXAMPLE Chromosomal localization of the hpa gene Chromosomal mapping of the hpa gene was performed utilizing a panel of monochromosomal human/CHO and human/mouse somatic cell hybrids, obtained from the UK HGMP Resource Center (Cambridge, England).

ng of each of the somatic cell hybrid DNA samples were subjected to PCR amplification using the hpa primers: hpu565 20 TATACAC-3', SEQ ID NO:22, corresponding to nucleotides 564-586 of SEQ ID NO:9 and an antisense primer hpll71 5'-GCATCTTAGCCGTCTTTCTTCG-3', SEQ ID NO:23, corresponding to nucleotides 897-876 of SEQ ID NO:9.

The PCR program was as follows: a hot start of 94 °C 3 minutes, followed by 7 cycles of 94 °C 45 seconds, 66 °C 1 minute, 68 °C 5 minutes, followed by 30 cycles of 94 °C 45 seconds, 62 °C 1 minute, 68 °C minutes, and a 10 minutes final extension at 72 °C.

The reactions were performed with Expand long PCR (Boehringer Mannheim). The resulting amplification products were analyzed using agarose gel electrophoresis. As demonstrated in Figure 14, a single band of 30 approximately 2.8 Kb was obtained from chromosome 4, as well as from the control human genomic DNA. A 2.8 kb amplification product is expected based on amplification of the genomic hpa clone (data not shown). No amplification products were obtained neither in the control DNA samples of hamster and mouse nor in somatic hybrids of other human chromosome.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and scope of the appended claims.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.

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17. Burgess, and Maciag, T. (1989). The heparin-binding (fibroblast) growth factor family of proteins. Annu. Rev. Biochem., 58, 575-606.

18. Folkman, and Klagsbrun, M. (1987). Angiogenic factors.

Science, 235, 442-447.

19. Vlodavsky, Folkman, Sullivan, Fridman, Ishai- Michaelli, Sasse, and Klagsbrun, M. (1987). Endothelial cell-derived basic fibroblast growth factor: Synthesis and deposition into subendothelial extracellular matrix. .Proc. Natl. Acad. Sci. USA, 84, 2292-2296.

Folkmnian, Klagsbrun, Sasse, Wadzinski, Ingber, D., and Vlodavsky, 1. (1980). A heparin-binding angiogenic protein basic fibroblast growth factor is stored within basement membrane. Am. J. Pathol., 130, 393-400.

21. Cardon-Cardo, Vlodavsky, Haimovitz-Friedman, Hicklin, and Fuks, Z. (1990). Expression of basic fibroblast growth factor in normal human tissues. Lab. Invest., 63, 832-840.

22. Ishai-Michaeli, Svahn, Chajek-Shaul, Korner, G., Ekre, and Vlodavsky, 1. (1992). Importance of size and sulfation of heparin in release of basic fibroblast factor from the vascular endothelium and extracellular matrix. Biochemistry, 31, 2080-2088.

23. Ishai-Michaeli, Eldor, and Vlodavsky, I. (1990).

Heparanase activity expressed by platelets, neutrophils and lymphoma cells releases active fibroblast growth factor from extracellular matrix. Cell Reg., 1, 833-842.

24. Vlodavsky, Bar-Shavit, Ishai-Michaeli, Bashkin, and Fuks, Z. (1991). Extracellular sequestration and release of fibroblast growth factor: a regulatory mechanism? Trends Biochenm. Sci., 16, 268-271.

Vlodavsky, Bar-Shavit, Komer, and Fuks, Z. (1993).

Extracellular matrix-bound growth factors, enzymes and plasma proteins. In Basement membranes: Cellular and molecular aspects (eds. D.H. Rohrbach and R. Timpl), pp327-3 4 3 Academic press Inc., Orlando, Fl.

26. Yayon, Klagsbrun, Esko, Leder, and Ornitz, D.M.

(1991). Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell, 64, 841-848.

27. Spivak-Kroizman, Lemmon, Dikic, Ladbury, J.E., Pinchasi, Huang, Jaye, Crumley, Schlessinger, and Lax, I.

(1994). Heparin-induced oligomerization of FGF molecules is responsible for FGF receptor dimerization, activation, and cell proliferation. Cell, 79, 1015-1024.

28. Omitz, Herr, Nilsson, West, Svahn, and Waksman, G. (1995). FGF binding and FGF receptor activation by synthetic heparan-derived di- and trisaccharides. Science, 268, 432-436.

29. Gitay-Goren. Soker, Vlodavsky, and Neufeld, G. (1992).

Cell surface associated heparin-like molecules are required for the binding of vascular endothelial growth factor (VEGF) to its cell surface receptors. J. Biol.

Chem., 267. 6093-6098.

Lider, Baharav, Mekori, Miller, Naparstek, Y., Vlodavsky, and Cohen, I.R. (1989). Suppression of experimental autoimmune diseases and prolongation of allograft survival by treatment of animals with heparinoid inhibitors of T lymphocyte heparanase. J. Clin. Invest., 83, 752-756.

31. Lider, Cahalon, Gilat, Hershkovitz, Siegel, D., Margalit, Shoseyov, and Cohn, I.R. (1995). A disaccharide that inhibits tumor necrosis factor a is formed from the extracellular matrix by the enzyme heparanase. Proc. Natl. Acad. Sci. USA., 92, 5037-5041.

31a. Rapraeger, Krufka, and Olwin, B.R. (1991). Requirement of heparan sulfate for bFGF-mediated fibroblast growth and myoblast "differentiation. Science, 252, 1705-1708.

32. Eisenberg, Sehayek, Olivecrona, and Vlodavsky, 1.

S:"i (1992). Lipoprotein lipase enhances binding of lipoproteins to heparan sulfate on cell surfaces and extracellular matrix. J. Clin. Invest., 90, 2013-2021.

Vt I 7 Ia I 1 0 39 33. Shieh, Wundunn, Montgomery, Esko, and Spear, P.G. J. (1992). Cell surface receptors for herpes simplex virus are heparan sulfate proteoglycans. J Cell Biol., 116, 1273-1281.

33a. Chen, Maguire, Hileman, Fromm, Esko, J.D., Linhardt, and Marks, R.M. (1997). Dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate. Nature Medicine 3, 866- 871.

33b. Putnak, Kanesa-Thasan, and Innis, B.L. (1997). A putative cellular receptor for dengue viruses. Nature Medicine 3, 828-829.

34. Narindrasorasak, Lowery, Gonzalez-DeWhitt, Poorman, Greenberg, Kisilevsky, R. (1991). High affinity interactions between the Alzheimer's beta-amyloid precursor protein and the basement membrane form of theparan sulfate proteoglycan. J. Biol. Chem.. 266, 12878-83.

Ross, R. (1993). The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 362:801-809.

36. Zhong-Sheng, Walter, Brecht, Miranda, Mahmood Hussain, Innerarity, T.L. and Mahley, W.R. (1993). Role of heparan sulfate proteoglycans in the binding and uptake of apolipoprotein E-enriched remnant lipoproteins by cultured cells. J. Biol. Chem., 268, 10160-10167.

37. Ernst, Langer, Cooney, Ch.L., and Sasisekharan, R. (1995).

Enzymatic degradation of glycosaminoglycans. Critical Reviews in Biochemistry and Molecular Biology, 30(5), 387-444.

38. Gospodarowicz, Mescher, AL., Birdwell, CR. (1977).

Stimulation of corneal endothelial cell proliferation in vitro by fibroblast and epidermal growth factors. Exp Eye Res 25, 75-89.

39. Haimovitz-Friedman, Falcone, Eldor, Schirrmacher,

V.,

Vlodavsky, and Fuks, Z. (1991) Activation of platelet heparitinase by tumor cell-derived factors. Blood, 78, 789-796.

39a. Savitsky, Platzer, Uziel, Gilad, Sartiel, Rosental, Elroy-Stein, Siloh, Y. and Rotman, G. (1997). Ataxia-telangiectasia: structural diversity of untranslated sequences suggests complex post-translational regulation of ATM gene expression. Nucleic Acids Res. 25(9), 1678-1684.

Bar-Ner, Eldor, Wasserman, Matzner, and Vlodavsky, 1. (1987). Inhibition of heparanase mediated degradation of extracellular matrix heparan sulfate by modified and non-anticoagulant heparin species. Blood. 70, 551-557.

41. Goshen, Hochberg, Korner, Levi, Ishai- Michaeli, R., Elkin, de Grot, and Vlodavsky, I. (1996). Purification and characterization of placental heparanase and its expression by cultured cvtotrophoblasts. Mol. Human Reprod. 2, 679-684.

e e EDITORIAL NOTE APPLICATION NUMBER 69997/01 The following Sequence Listing pages 1 to 11 are part of the description. The claims pages follow on pages "41" to "44".

(1) GENERAL INFORMATION:

APPLICANT:

(ii) TITLE OF INVENTION: (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE ADDRESS:

ADDRESSEE:

STREET:

CITY:

STATE:

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NAME:

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TELEX:

SEQUENCE LISTING Iris Pecker, Israel Vlodavsky and Elena Feinstein POLYNUCLEOTIDE ENCODING A POLYPEPTIDE HAVING HEPARANASE ACTIVITY AND EXPRESSION OF SAME IN TRANSDUCED CELLS 23 Mark M. Friedman c/o Robert Sheinbein 2940 Birchtree lane Silver Spring Maryland United States of America 20906 1.44 megabyte, 3.5" microdisk Twinhead* Slimnote-890TX EM: MS DOS version 6.2, Windows version 3.11 Word for Windows version 2.0 converted to an ASCI file rA:

MBER:

MBER: 08/922,170 2 SEP 1997

TION:

Friedmam, Mark M.

UMBER:

ET NUMBER: RMAT ION: 33,883 910/1 972-3-5625553 972-3-5625554 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 27 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: CCATCCTAAT ACGACTCACT ATAGGGC 27 INFORMATION FOR SEO ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 24 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GTAGTGATGC CATGTAACTG AATC 24 INFORMATION FOR SEQ 10 NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 23 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ACTCACTATA GGGCTCGAGC GGC 23 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 22 TYPE: nucleic acid SIRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ.ID NO:4: GCATCTTAGC CGTCTTTCTT CG 22 11 SEQUENCE CHARACTERISTICS: LENGTH: TYPE: nucticC acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID TTTTTTTTTT TTTI INFORMATION FOR SEQ ID NO:6: SEQUENCE

CHARACTERISTICS-

LENGTH: 23 TYPE: nucleic acid STRANDEONESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: TTCGATCCCA AGAAGGAATC AAC 23 INFORMATION FOR SEQ ID NO:7: i) SEQUENCE

CHARACTERISTICS:

LENGTH: 24 TYPE: nucleic acid STRANOEONESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GTAGTGATGC CATGTAACTG AATC 24 INFORMATION FOR SEQ ID NO:B: SEQUENCE CHARACTERISTICS:9 LENGTH: amn9 ai TYPE: aioai STRANDEDNESS: siflgte TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ I0 NO:B: 9 INFORMATION FOR SEC ID NO:9: SEQUENCE

CHARACTERISTICS:

LENGTH: 1721 TYPE: nucteic acid STRANOEDNESS: double TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CTAGAGCTTT CGACTCTCCG CTGCGCGGCA GCTGGCGGGG GGAGCAGCCA GGTGAGCCCA AGATGCTGCT GCGCTCGAAG CCTGCGCTGC CGCCGCCGCT GATGCTGCIG CTCCTGGGGC 120 CGCTGGGTCC CCTCTCCCCT GGCGCCCTGC CCCGACCTGC GCAAGCACAG GACGTCGTGG 180 ACCTGGACTT cTTCACCCAG GAGCCGCTGC ACCTGGTGAG, CCCCTCGTTC CTGTCCGTCA 240 CCATTGACGC CAACCTGGCC ACGGACCCGC GGTTCCTCAT CCTCCTGGGT TCTCCAMAGC 300 TTCGTACCTT GGCCAGAGGC TTGTCTCCTG CGTACCTGAG GTTTGGTGGC ACCAAGACAG 360 ACTTCCTAAT TTTCGATCCC AAGAAGGAAT CAACCTTTGA AGAGAGAAGT TACTGGCAAT 420 CTCAAGTCAA CCAGGATATT TGCAAATATG GATCCATCCC TCCTGATGTG GAGGAGMAGT 480 TACGGTTGGA ATGGCCCTAC CAGGAGCAAT TGCTACTCCG AGMACACTAC CAGAAAAAGT 540 TCAAGAACAG CACCTACTCA AGAAGCTCTG TAGATGTGC-T ATACACTTTT GCAAACTGCT 600 CAGGACTGGA CTTGA7CTTT GGCCTAMATG CGTTATTAAG AACAGCAGAT TTGCAGTGGA 660 ACAGTTCTAA TGCTCAGTTG CTCCTGGACT ACTGCTCTTC CAAGGGGTAT AACATTTCTT 720 GGGMACTAGG CAATGAACCT AACAGTTTCC TTAAGAAGGC TGATATTTTC ATCAATGGGT 780 CGCAGTTAGG AGAAGATTAT ATTCAATTGC ATAAACTTC1 AAGAAAGTCC ACCTTCMAAA 840 ATGCAAAACT CTATGGTCCT GATGTTGGTC AGCCTCGA.AG AAAGACGGCT AAGATGCTGA 900 AGAGCTTCCT GAAGGCTGGT GGAGAAGTGA TTGATTCAGT TACATGGCAT CACTACTAIT 960 TGAATGGACG GACTGCTACC AGGGAAGATT TTCTAAACCC TGATGTATTG GACATTTTTA 1020 TTTCATCTGT GCAAAAAGTT TTCCAGGTGG TTGAGAGCAC CAGGCCTGGC AAGAAGGTCT 1080 GGTTAGGAGA AACAAGCTCT GCATATGGAG GCGGAGCGCC CTTGCTATCC GACACCTTTG 1140 CAGCTGGCTT TATGTGGCTG GATAAATTGG GCCTGTCAGC CCGAATGGGA ATAGMAGTGG 1200 IGATGAGGCA AGTATTCTTT GGAGCAGGAA ACTACCATTI AGTGGATGAA AACTTCGATC 1260 CFIIACCTGA TTATTGGCTA TCTCTTCTG7 TCAAGAAATT GGTGGGCACC AAGGTGTTAA 1320 TGGCAAGCGT GCAAGGTTCA AAGAGAAGGA AGCTTCGAGT ATACCTTCAT IGCACAAACA 1380 CTGACAATCC AAGGTATAAA GAAGGAGATT TAACTCTGTA TGCCATAAAC CTCCATAACG 1440 ICACCAAGTA CTTGCGGTTA CCCTATCCTT TTTCTAACAA GCAAGTGGAI AAATACCTTC 1500 JAAGACCTTT GGGACCTCAT GGATTACTTI CCAAATCTG7 CCAACTCAAT GGTCTAACTC 1560 IAAAGATGGT GC.ATGATCAA ACCTTGCCAC CITTTMTGGA AAA.ACCTCTC CGGCCAGGAA 1620 GTrCACIGGG CITGCCAGCT TTCICAIATA GTTTTTTT1GT GAIAAGAAAT GCCAAAGTTG 1680 CIGCTIGCA7 CIGAAAAIAA A.AYATACTAG rCCTGACACT G 1721 ,ILtOID4A1Ifl 4 fOR SLO ID NO:

LENGTH:

TYPE:

STRANDEDNESS:

TOPOLOGY:

(xi) SEQUENCE DESCRIPTION: 543 amino acid single ti near SEQ I0 Met Leu Lcu Arg Ser Lys Pro ALa LCu Pro Pro Pro Leu Met Leu Leu 10 Leu Leu Gly Pro Lcu Gly Pro Lcu Ala Gin Ala Gin Asp Vat Vat Asp 40 Leu His Leu Vat Sen Pro Ser Phe 55 Leu ALa Thr Asp Pro Arg Phe Leu 70 Ang Thr Leu Ala Arg Gly Leu SerI 85 Thr Lys Thr Asp Phe Leu Ile Phe 100 Giu Giu Arg Sen Tyr Trp Gin Scr 115 120 Tyr Gly Ser lie Pro Pro Asp Vat 130 135 Pro Tyr Gin GLu Gin LeU Leu Leu 145 150 Lys Asn Ser Thr Tyr Son Anrg Sen' 165 Ala Aso Cys Sen Gty Leu ASP Leu 180 Arg Thr Ala Asp Lou Gin Trp Asn 195 200 Asp Tyr Cys Son Sen Lys Gly Tyn 210 215 Giu Pro Asn Ser Phe Leu Lys Lys 225 230 Gin Leu Gly Giu Asp Tyr lie Gin 24.5 Thr Phe Lys Asn Ala Lys Leu Tyr 260 Ang Lys Thr Ala Lys Met Lou Lys 275 280 Scn Pro Gly 25 Leu Asp Phe Leu Sen Vat ie Leu Leu 75 Pro ALa Tyn 90 Asp Pro Lys 105 Gin Vai Asn Glu Glu Lys Ang Glu His 155 Sen Vat Asp 170 lie Phe Gly 185 Sen Sen Asn Asn Ile Sen Ala Asp Ile 235 Leu His Lys 250 Gly Pro Asp 265 Sen Plie Let.

His Tyr Tyl Ala Leu Pro Arg Pro Phe Thn Gin Glu Pro Thn lI e Asp Ala Asn Gty Son Pro Lys Leu Leu Arg Phe Gly Gly Lys Giu Sen Thn Phe 110 Gin Asp Ilc Cys Lys 125 Leu Ang Leu Glu Trp 140 Tyn Gin Lys Lys Phe -160 Va L Leu Tyr Thn Pho 175 Leu Asn Ala Lou Leu 190 Ala Gin Leu Lou Leu 205 Trp Giu Leu Gly Asn 220 *Pho lie Asn GLy Ser 240 *Leu Leu Ang Lys Ser 255 Vat Gty Gin Pro Arg 270 iLys Ala Gly Gly Gtu 285 rLeu Asn Gty Ang Thr 300 His Vai I e Asp Ser Vat Thr 290 Ala lhr Ang Giu Asp Phe Leu Asn Pro Asp Vai LoU ASP Ile Phe 305 310 315 Scr Vat LYS Vat ICU teu Lys Vat Phe 325 Leu Gly Giu Asp Thr Phe Al. MA1No Vat Gtu 330 Ser Ala Gty Phe (Aut V.I1 335 Gty Gly Gly 350 ?rp Leu Asp 365 Met Arg Gin 370 Phe Phc Gly 385 375 Ala Gly Asn Tyr 390 380 His Lcu Vat Asp Glu Asn Phe Asp Pro 395 400 Leu Pro Asp Tyr Trp Lcu Ser Leu Lcu Phe Lys Lys LeU Vat Gly Thr 405 410 415 Lys Vat Leu Met Ala 5cr Vat Gin Cly Ser Lys Arg Arg Lys Leu Arg 420 425 430 Vat Tyr Leu His Cys Thr Asn Thr Asp Asn Pro Arg Tyr Lys Gtu Gly 435 440 445 Asp Leu Thr Leu Tyr Ala lie Asn Leu His Asn Vat Thr Lys Tyr Leu 450 455 460 Arg Leu Pro Tyr Pro Phe Ser Asn Lys Gin Vat Asp Lys Tyr LCu Leu 465 470 475 480 Arg Pro Leu Gly Pro His Cly Leu Leu 5cr Lys Ser Vat Gin Leu Asn 485 490 495 CLY Lcu Ihr Leu Lys Het Vat Asp Asp Gin Thr Leu Pro Pro Leu Met 500 505 510 Gtu Lys Pro Leu Arg Pro GLy Ser Ser Leu Cly Leu Pro ALa Pho Ser 515 520 525 Tyr Ser Phe Phc Vat, ILe Arg Asn Ala Lys Vat Ala Ala Cys lie 530 535 540 543 INFORMATI[ON FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 1718 .TYPE: nucleic. acid STRANDEONESS: doubl e TOPOLOGY: I.i near (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: CT AGA GC TTC GAC 14 IdT CCG CTG CGC CGC AGC AIC CTG CTG CGC TCG MCG Met Leu Leu Arg Ser Lys 5 CIC CTG GCG CCG CTC CGT Leu Leu Cly Pro Leu.GLy GCG CAA GCA CAG GAd GCC Ala Gin Ala Gin Asp Vat 35 CIG CAC CIC GTG ACC CCC Leu His Lou Vat Ser Pro CTG GCC ACC GAC CCC CCC Leu Ala Thr Asp Pro Arg 70 CGT ACC TIC CCC AGA GGC Arg Ihr Leu Ala Arg Cly 85 ACC MAG AdA GCd TIC CIA Yhr Ly-, Thr Asp Phe Leu 100 TCC CCC CCC GAG CAC CCA CCI GAG CCC MAG CC CTC CCC CCC CCC Ala Leu Pro Pro Pro 10 CTC TCC CCT GGC GCC Lou Ser Pro Gly Ala 25 GAC CTC GAC TTC TIC Asp Leu Asp Phe Phe 40 TIC CIC TCC CIC ACC Phe Leu Ser Val Thr CTC AIC CIC CTG CCI LCU Ile Leu Lou Cly 75 TCI CCI GC TAC CIG Ser Pro Ala Tyr Leu 90 TIC CAT CCC AAG MCG Phe Asp. Pro Lys Lys 105 CIC AIC CIC CTG 110 Leu Met Leu Lou CIC CCC CCA CCI 158 Leu Pro Arg Pro ACC CAC GAG CCC 206 Thr Gin Clu Pro AlT GCd CCC M~C 254 Ilie Asp Ala Asn TC! CCA MCG CII 302 Sor Pro Lys Lou AGC ITT CCI GC 350 Arg Phe Ciy Clv 95 GAA ICA ACC TTT 398 Gtu Ser Thr Phe 110 CAA GAG AGA AGT rAC IGC CAA IC! CMA GTC AAC CAG GAT All TGC MAA 446 Gt. Glu Arg 5cr Tyr Trp Gin Ser 170l 1 ?S TAT GGA Tyr Giy 00 CCC TAC Pro Tyr 145 MAG AAC Lys Asn GCA AAC Ala Asn AGA ACA Arg Thr GAC TAC Asp Tyr 210 GA-A CCT Giu Pro 225 CAG TTA Gin Lou ACC TTC Thr Phe ACGA AAG Ara Lys GTG AT Val lie 290 OCT ACC Ala Thr 305 TCA TC1 Ser Ser MAG M( Lys Ly~ ccc tII Pro Le' TO GG Leu Gi1 37 TCC ATC CCT CCT GAT GIG G Ser lie Pro Pro Asp Val G 135 CAG GAG CMA TTG CIA CTC C Gin Gtu Gin Leu Leu Leu A .150 AGC ACC TAC TCA AGA AGC 1 Scr Thr Tyr Ser Arg Ser S 165 TGC TCA GGA CTO GAC TO i Cys Ser Gty Leu Asp Lou1 180 GCA OAT TTG CAG TOG MAC ALa Asp Leu Gin Trp Asn 195 200 TG TCT TCC MAG 000 TAT Cys ser Ser Lys Oly Tyr 215 MAC AGT TIC CTT MAG MAG Asn Ser Phe Leu Lys Lys 230 OGA GMA OAT TAT ATT CMA OLy Giu Asp Tyr lit Gin 245 AAA M.T OCA AAA CIC TAT Lys Asn Ata Lys Leu Tyr, 260 ACG OCT MAG AIG CTG MAG Thr Ala Lys Met Leu LYS 275 280 OAT ICA OTT ACA TOO CAT Asp Ser vat Thr Irp His 295 AGO GMA OAT ITT CIA MAC *Arg Glu Asp Phe Leu Asn 310 OTO CMA AAA OTT TIC CAG r Val Gin Lys Val Phe Gin 325 G GIC TOO TIA OGA GMA ACA sVat Irp Leu Oly Gtu Thr 340 G CIA TCC GAC ACC ITT GCA ui Leu Ser Asp Thr Phe Ala 355 360 C CIG TCA 0CC CGA ATO OGA y Leu Ser Ala Arg Met Gly 0 375 AG GAG Lu Oiu GA GMA ~rg GLu CT GTA jet Vat

V

MAG TTA COG ITO GMA LYS Leu Arg Leu Gtu 140 CAC TAC CAG AMA MG His Tyr Gin Lys Lys 155 OAT GIG CIA TAC ACT Asp vat Leu Tyr Thr 175 IIC TTT GOC CIA MAT OCO ITA TTA 638 185 AOT TCO Ser Ser AAC ATI Asn lie GCT OAT A',a Asp TO CAT Leu His 250 GOT CCI Oty Pro 265 AGC TTC Ser Phe CAC TAC His Tyr CCI OAT Pro Asp GIG GTl Val Val 330: AGC ICT 190 MAT OCT CAG TO CTC CTO 686 Asn Ala Gin Leu Leu Leu 205 TCT TOO GMA CIA GOC MAT 734 Ser Trp Glu Lcu Gty Asn 220 ATT TIC AIC MIT 000 ICO 782 lie Phe lie Asn Oly Ser 235 240 AAA CIT CIA AGA MAG ICC 830 Lys Leu Lcu Arg Lys Ser 255 OAT OTT GOT CAG CCT COA 878 Asp Vat Oly Gin Pro Arg 270 CIG MOG OCT GOT GGA GMA 926 Lou Lys Ala Oty Gty GIL., 285 TAT TO MAT OGA COG ACT 974 Tyr Leu Asn Gly Arg Thr 300 GIA TIC GAC AlT ITT ATT 1019 Val Leu Asp lie Phe Ile 315 320 *GAO AGC ACC AGO CCI GOC 1067 GlOu Ser Ihr Arg Pro Gty 335 OCA TAT OGA GOC OGA OCG 1115 r Ala Tyr Gly Oty Oly Ala 350 345 Se OCT GOC III AIG Ala Gty Phe Met 100 CIG OAT MAA 1163 Trp Leu Asp Lys 365 ATA GMA GIG GIG AIG AGO CMA GTA1?11 lie Giu Vat Val Met Arg Gin Vat 380 TIC TIT OGA OCA OGA AAC Phe Phe Gty Ala Gly Asn 385 390 TTA CCI GAT TAT TOG CIA Leu Pro Asp Tyr Irp Leu 40', MAG GIG TTA AIG OCA ACC TAC CAT TTA GIG OAT GMA AAC TIC ,GAT -CCI 1259 Tyr His Leu Val Asp Glu Asn Phe Asp Pro 395 -4.00 IC? CIT CdO TIC A.AG MAA 110 010 GGC ACC 1307 Ser Lcu Leu Phe Lys Lys IOu Val Gly Ihr 410 -415 GIG CMA GOT ICA AAG AGA AGO A.AO CIT COA 13S 420 CIA TAC CYT CAT Val Tyr Leu His 435 425 TGC ACA AAC ACT GAC A.AT CCA AGG Cys Thr Asn Ihi- Asp ASn Pro Arg GAT ThA Asp Leu 450 ACT CTG TAT 0CC Ihr Leu Tyr Ala ATA AAC CIC CAT MAC GTC Ile Asn Leu His Asn Vat 455 460 ICI AAC MAG CMA GTG CAT Sot Asn Lys Gin Val Asp 475 TAT AAA GMA OGA 1403 Tyr Lys Gtu Gly 445 ACC MAG TAC TTG 1451 Thr Lys Tyr Leu AAA TAC CIT CTA 1499 Lys Tyr Leu Leu 480 COG TTA CCC Arg Lou Pro 465 TAT CCI TTT Tyr Pro Phe 470 AGA CCI 110 GGA Arg Pro Leu Gty OCT CIA ACT CIA Cly Leu Thr Leu 500 CCI CAT OCA TIA CIT ICC Pro His Cly Lou Lou Ser 485 490 AAA ICI GTC CAA TC MAT 1547 Lys Sot Vat Gin Lou Asn 495 ACC TIC CCA CCI ITA AIC 1595 Thr Leu Pro Pro Lou Met 510 MAG AIC GIG GAT Lys Met Val Asp OAT CMA Asp Gin 505 CAA AAA CCI Ctu Lys Pro 515 CIC COO CCA OGA Lou Arg Pro CLy TCA CIG GCC TTG CCA OCT TIC ICA 1643 Sot Leu Oty Leu Pro Ala Phe Sot 525 TAT ACT Tyr Ser 530 TTT ITT GTG ATA Phe Pho Vat Ile MIT 0CC AA.A GTT Asn Ala Lys Val OCT CI ICC AT TCA 1691 Ala Ala Cys Ile 540 543 AAA TAA MT ATA CIA OTt CIG ACA CIG 1718 INFORMATION FOR SEC ID 140:12: 0I) SEQUENCE CHARACTERISTICS: LENGTH: 824 TYPE: nuc Iei c acid STRANDEONESS: doub Le TOPOLOGY: inear, (xi) SEQUENCE DESCRIPTION: SEQ 10, No:12 CIGGCCMCM GGICTGTO IGICCAACAC CTITGCAGCI IGGOCATAGA AGICOIGATG ATGAAAACTT IGAGCCTTTA CICCCAGOGT GITACIGICA ICCACIOCAC TAACGTCTAI TGMACCTCCA TMITGICACC TGGATACOTA CCTTCTOGA TGAMCGOTCA MATTCTGAAG

GGAGACACGA

GGCTTTGT

AGOCAGGTGT

CCI GA I IAC

AGAGTGAAG

CACCCACGAT

MOGCACTTGA

CCI ICOGOGC

AGTGGATG

CICTCCCCGC

GAMITGCCMA

MOGCCGAGGG

GAGTTCCAGA

CTCTAMGMA

AGGAAOTGCA CTMOGCCTGC MATCOCTGCT IGIATATOMA GGGGTATI CAIAAAACAA GCTTCOOGAG GGOCOTAC MITACTGCAG GIGOTOACAG OCICAGCTA COGTGGCOGT CCACCCTTGC GGCIGGATAA ATTOGGCCTC ICAGCCCAGA 120 TCIICOGACC AGGCMACTAC CACI TAGTOG 180 GGOCCTCTCT TCTGTICMAG MACIGOTAG 240 GCCCAGACAC GAGCMAACIC COACTOTAIC 300 AICAGOMOCG AOAICIAACT CIOTATOTCC 360 AGGIACCOCC TCCGIIOT IC AGGAAACCAG, 420 COGATGGATT ACTTTCCAAA TCTGICCMAC 480 AGCAGACCCT GCCAGCTTTG ACAGAAAAAC 540 CTGCCTTTTC CTATOCTTTI TTOICATAM 600 MATAAMAGGC A7ACGCIACC CCTGAOACMA 660 MCCCTAOIT TAGOACOCCA CCTCCTTGCC 720 ACTTCAGTAT TACATICAGT GIGGITTCT 780 TTMATAGCAC 1010 824 0:13: ERISI ICS: 1899 nucleic acid )NESS: double Y inear )T!ON: SEQ ID NO0-13 INFORMATION FOR SEQ ID N 0i) SEQUENCE CHARACT

LENGTH:

TYPE:

STRANDEI

TOPOLOG'

(xi) SEQUENCE DESCRIF

CGGAAAGCGA

CAGIGGGAGG

ACCCGTMACG

A CAGCCCGC AT OCTOCT OC CTG0001C CCC

CIGOACTICT

AT IGACGCCA CGTACCT TOO T ICC TAT I CMGI CAACC rr.r. I I A T GCMAGGMAGI AGGAGAOAOC COGGCAGOCC GCOGGIGT GOATIGGOAG GATGCAGAAG AGOAGIGOGA GOOATGOAGC GCOCACGGC AGGGTOAGO GOOCOGAGGA MOGGAGAAAA GOOCOCTOGG OCTCOCOG

AGOMOGTOCT

ACICICCOCI OCGCGOCAOC IGCGGGO AGCAGCCAGG

TGAGCCCMAG

GCTCGMAGCC TOCOCIOCCG CCGCCGCIOA TGCTGCTGCI CCTGGGOCCO TCTCCCCTOO COCCCTOCCC COACCIOCOC AAGCACAGGA

CGICGTGGAC

ICACCCAGGA OCCOCTGCAC CTGGTGAGCC CCICOTTCCT

OTCCGTCACC

ACCTGGCCAC GGACCCGCOO TTCCICATCC TCCTGGITC TCCAAAGCIT CCAOAGGCTI GTCTCCIGCG TACCTGAGOT TTGGIGOCAC

CMAGACAGAC

TCOATCCCAA GAAGGCMTCA ACCTTIGAAG AGAGMOGTTA

CIGGCMATCT

AGGATATTO CAAArATGGA TCCATCCCIC CTGATOTOCA

GGOAAOTA

r.r.rttITACCA CCACCAAVIG CTACTCCGAC. AACACTACCA

CAAAAAGTTC

AAGAACAGCA

G GA C tGGAC I

AGTTCTAATG

GAACTAGGCA

CAGTTAGGAG

GCAAAACTCT

AGCTTCCTGA

AATGGACGGA

TCATCTGTGC

ITTAG GAGAAA

GCTGGCTTTA

ATGAGGCAAG

TTACCTGATI

C CAACC G TGC

GACAATCCAA

A CC AAGCT AC A GAC C IT I C AA CATGGC I C

TCACTGGGCT

GCTTGCATCT

CCTACT CAAG ICAT CT TTGGC CTCACT TGCT

ATGAACCTAA

AAGATTATAY

AT GGT CC TGA

AGGCTGCJGG

CTGCTACCAG

AAAAACT TIT

CAAGCTCTGC

TGTGGCTGGA

TAT TCTTTGG

ATTGGCTATC

AAGGT TCAAA

GGTATAAAGA.

TGCGGTTACC

GACCTCATGG

ATCAT CAAA C

TGCCAGCTIT

GAAAATAAAA

vii A.AGCTCTGTA GATGTGCTAT ACACTTTTGC AAACTGCTCA CCTAAATC TTATTAAGAA CAGCAGATTT CCAGTGGAAC CCTGGACTAC TGCTCTTCCA AGGCGTATAA CATTTCTTCG CAGTTTCCTT AAGAACGCTG ATATTTTCAT CAATGGGTCG TCAATTGCAT AAACTTCTAA GAAAGTCCAC CTTCAAA&T TGTTGGTCAG CCTCGAAGAA AGACGGCTAA GATGCTGAAG AGAAGTGATT GATTCAGTTA CATGGCATCA CTACTATTTG GGAMGATTTT CTAAACCCTG ATGTATTGGA CATTTITATT CCAGGTGGTT GAGAGCACCA GGCCTGGCAA GAAGGTCTCG ATATGGAGGC GGAGCGCCCT TGCTATCCGA CACCTTTGCA TAAATTGGGC CTGTCAGCCC GAATGGGAAT AGAAGTGGTG AGCAGCAAAC TACCATTTAG TGGATGAAAA CTTCGATCCT TCTTCICTTC AAGAAATTCG TGGGCACCAA GGTGTTAATC GAGAAGGAAG CTTCGAGTAT ACCTTCATTG CACAAACACT AGGAGATTTA ACTCTGTATG CCATAAACCT CCATAACGTC CTATCCTTTT TCTAACAAGC AAGTGGATAA ATACCTTCTA ATTACTTTCC AAATCIGTCC AACTCAATCG TCTAACTCTA CTTGCCACCT TTAATCGAAA AACCTCTCCG GCCAGGAAGT CICATATAGI TTTTTTGTCA TA.AGAAATGC CAAAGTTGCT TATACTAGTC CTGACACTC 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1899 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 592 TYPE: amino acid STRANDEONESS: sing[ TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N40:14 Met Gtu Cly Ala Giu Arg Arg Lys Ala Lcu Aso Ser Cly Glu Pro LysO Vat Gly Gly Vat Ang Arg Arg Asn Cly Ala Glu 10 Cly Arg Trp, Gly Sen Pro Lou Ang Giy Ser Met Leu Leu Arg Ser, .50 25 T!rp Arg Cly Glu Gin Pro 40 Lys Pro Ala Leu Pro Pro 55 Leu Cly Pro Leu Sen Pro 70 a Pro Leu Met Leu Leu Leu Leu Gty Pro Gly Ata Leu Pro Asp Phe Phe Ihr Leu Sen Val Thn Leu lie Leu Leu Leu Ser Pro Ala Leu Ile Phe Asp Tyr Irp Gin Sen Ile Pro Pro Asp GIn G~u Gin Leu Asn Sen Thn Tyr Ala Asn Cys Sen Leu Arg Thr Ala Leu Leu Asp Tyr Leu Gly Asn Clu lite Asn Gty Sen Leu Lcu Ang Lys Asp Vat Gty Gin Phe leu Lys Ala Ang Pno ALa Gin Ala G~n Asp Vat Vat Asp Leu 85 Gin GLu Pro Leu His Leu Vat Sen Pno Sen Phe 100 105 Ite Aso Ala Asn Lou Ala Thr Asp Pro Ang Phe 110 115 120 GIl' Sen Pro Lys Leu Ang Thr Leu Ala Ang Gly 125 130 135 Tyr Leu Ang Phe GLy Gly Thr Lys Thr Asp Phe 140 145 150 Pro Lys Lys Glu Sen Thn Phe Gtu Glu Ang Sen 155 160 165 Gin Val Asn GIn Asp Ilie Cys Lys Tyr Gly Sen 170 175 180 Vat Glu Ctu Lys Leu Ang Leu Ciu Trp Pro Tyr 185 190 195 Leu Leu Arg Gtu His Tyn Gin Lys Lys Pho Lys 200 205 210 Sen Ang Sen Son Vat Asp Vat Leu Tyr 7hr Phe 215 220 225 Gly Lou Asp Lou lito Phe Gly Leu Asn Ala Leu 230 235 240 Asp Leu Gin Tnp, Asn Sen Son Asn Ala GIn Lou 245 250 255 Cys Sen Scr Lys Gly Tyr Asn Ile Sen Trp Glu 260 265 270 Pro Asn Sen Phe Lou Lys Lys Ala Asp Ile Phe 275 280 285 Gin Leu Cly Clu Asp Tyr lie Gin Leu His Lys 290 295 300 Sen Thn Pho LysAsn AlIa Lys Leu Tyr Cly Pro 305 310 315 Pro Arg Ang Lys Thr Ala tlys Met Lcu Lys Sen 320 325 330 pGly Gly Gtu Vai lie Asp Sen Vdi Thn irp, His 33c, 3410 345 His Tyr Tyr Leu Asn Gly Arg Thr 350 viii AMa Thr Arg Glu Asp Phe Leu 355 360 Asn Pro Asp Vat Phe Gin Vat Vat G~y GLu Thr Ser Asp Thr Phe Mla Ser Ala Arg Met Gty Ala Gly Asn Pro Asp Tyr Trp Lys Vat Leu Met Arg Vat Tyr LCU Gtu Gty Asp 1Cu Lys Tyr Leu Arg 370 Thr Arg Pro Gty 385 Tyr Gly Gty Gly 400 Phe Met Trp Leu 415 Giu Vai Vat Met 430 Leu Vat Asp Gtu 445 Leu Leu Phe Lys 460 Vai Gin Gly Ser 475 Thr Asn Thr Asp 490 Tyr Ala Ile Asn 505 Tyr Pro Phe Ser 520 Ser Va Gin y375a s Lys Vat Trp Leu 390 a Pro Leu Lcu Ser 405 p Lys Leu Gly Leu 420 g Gin Vat Phe Phe 435 n Phe Asp Pro Leu 450 s Leu Vat Gly Thr 465 s Arg Arg Lys Leu 480 n Pro Arg Tyr Lys 495 u His Asn Vat Thr 510 n Lys Gin Vat Asp 525 y Leu Leu Ser Lys 540 tVat Asp Asp Gin 555 g Pro GLy Ser Scr 570 c Vat lie Arg Asn 585 Thr Leu 500 Leu Pro 515 Lys Tyr Lcu Lcu Arg Pro Leu Gly Pro His G1 530 535 Ser Vat Gin Leu Asn Gty Leu Thr Leu Lys Me 545 550 Thr Leu Pro Pro Leu Met GLU Lys Pro Leu Ar 560 565 Leu Gly Leu Pro Ala Phe Ser Tyr Ser Phe Ph 575 580 Ala Lys Vat Ala Ala Cys lie 590 592 INFORMAT!ON FOR SEQ I0 SEQUENCE CHARACTERISTICS:

LENGTH:

TYPE:

STRANOEDNESS:

TOPOLOGY:

(xi) SEQUENCE DESCRIPTION: 1899 nucleic acid double linear SEO ID NO: AMA GCG TTG GAT ATG GAG Met Gtu GGtG AGC MAG GMA GTA GGA GAG AGC CGG GCA GGC GGG GCG GGG TGG GAG CAG TGG GAG GGA TGC AGA AGA GGA GTG GGA GGG GGC GCA GIG GGA GGG GIG AGG AGG CGT AAC GGG GCG GAG Gty Ala Vai Gly GiY Vat Arg Arg Arg Asn Giy Ala Gtu 10 GMA AGG AGA AAA GGG CGC TGG GGC TCG GCG GGA GGA AGT GCT AGA Gtu Arg Arg Lys Gly Arg Trp GLy ser Ala Gty Gly Ser Ala Arg 25 GCT CTC GAC TCT CCG CIG CGC GGC AGC TGG CGG GGG GAG CAG CCA Ala Leu Asp Ser Pro Leu Arg Gly Ser Trp Arg Gty Giu Gin Pro 35 40 GGT GAG CCC MAG ATG CIG CTG CGC TCG MAG CCT GCG CTG CCG CCG Gly Gtu Pro Lys Met Leu Leu Arg Ser Lys Pro Ala Leu Pro Pro 55 CCG CTG ATG CTG CIG CTC CTG GGG CCG CTG GGT CCC CTc TCC CCT Pro Leu Met Leu Leu Leu Leu Gly Pro ieu Gty Pro Leu Ser Pro 70 GCC GCC CIG CCC CGA CCI GC CMA GCA CAG GAC GTc GIG GAC CIG Gty Ata Leu Pro Arg Pro Ala Gin Ala Gin Asp Vat Vat Asp Leu 85 GAC 1ic TIC ACC CAG-GAG CCC C CAC CTG GIG AGC CCC TCC TIC Asp Phe Phe Thr Gin Clu Pro Leu His Leu Vat Scr Pro Scr Phe 95100 105 Cif. ICC GIC ACC All GC 6CC AAC CIG G,:C ACC CAr CCC, CGG TIC Leu Ser Val rhr lie Asp Ala Asn 110 CTC ATC CTC CIG CCI IdT CCA MAG Leu lie Leu Leu Gly Ser Pro Lys 125 TTG TCT CCT GCG TAC CTG AGG TIT Lcu Ser Pro Ala Tyr Leu Arg Phe 140 CIA ATT TTC GAT CCC MAG MAG GMA Leu tie Phe Asp Pro Lys Lys Giu 155 TAC IGG CMA TCT CMA GTC MAC CAG Tyr Trp Gin Ser Gin Val Asn Gin 170 ATC CCT CCT GAT GTG GAG GAG AAG lie pro Pro Asp Vai Giu GLu Lys 185 CAC GAG CMA ITC CIA CTC CCA GMA Gin Giu Gin Lcu Leu Leu Arg Gtu 200 MAC AGC ACC TAC TCA AGA AGC TCT Asn Ser Thr Tyr Scr Arg Scr Scr 215 GCA MAC TGC ICA GGA CTG GAC TTG Ala Asn Cys Scr Cly Leu Asp Lcu 230 HTA AGA ACA GCA GAT TIC CAG TGG Leu Arg Thr Ala Asp Leu GI i Irp 21.5 CTC CIG GCd TAC TGC TCT TCC MAG Lcu Leu Asp Tyr Cys Ser Ser Lys 260 CIA GGC MAT GMA CCI MAC AGI TIC Lcu Gly Asn Clu Pro Asn Scr Phe 275 ATC MAT GGG TCC CAG TTA GGA GMA Ike Asn Gly Ser Gin Leu Gly Giu 290 CTT CTA AGA MAG TCC ACC TIC AAAi Leu Leu Arg Lys Ser Thr Phe Lys 305 GAT Gil GGT CAG CCI CGA AGA AA( Asp Val Gly Gin Pro Arg Arg Ly5 320 TIC CTG MAG GCI GGT GGA GMA GT( Phe Leu Lys Ala Gly GLy Giu Val 335 ix Leu Ala Thr Asp Pro Arg Phe 115 120 CTT CCI ACC HTG GCC AGA GCC Leu Arg Thr Lcu Ala Arg Gly 130 135

,AC

GGT GGC ACC MAG ACA Cly Giy Thr Lys ThrJ 145 TCA ACC Ill GMA GAG Scr Thr Phe GLu GLu 160 GAT ATT TCC AAA TAT Asp lie Cys Lys Tyr 175 TIA CGG TIC GAA IGG Leu Arg Lcu Giu Trp 190 CAC TAG CAG AAM MG His Tyr Gin Lys Lys 205 GIA GAT GIG CIA TAC Vat Asp Vat Leu Tyr 220 ATC ITT GGC CIA MAT lie Phe Gly Leu Asn 235 MAC AGI TCI A-AT GCT Asn Ser Scr Asn Ala 250 GGG TAT MAC ATT ICI Gly Tyr Asn lie Ser 265 CIT MAG MAG GC GAT Leu Lys Lys Ala Asp 280 CAT TAT All CMA TIC i Asp Tyr lie Gin Leu 295 MIT GCA AAA CTC TAT Asn Ala LYS Leu Tyr 310 iACG GCT MAG ATG CTG Thr Ala LYS Met Leu 325 1All CAT ICA Gil ACA I e Asp 5cr Val Thr 340 Iksp kGA i rg

GGA

Gly

CCC

Pro

TTC

Phe

ACTI

Thr

C

Ala Phe 150

ACT

Ser 165

TCC

Ser 180

TAC

Tyr 195

MAG

Lys 210

ITT

Phe 225

TIA

Leu 240 CAG TIC Gin Leu 255 TOGG GMA Trp Ciu 270 ATT IT lie Phe 285 CAT A His Lys 300 CCI CCI Cly Pro 315 MAG AC LYS Ser 330 TGG CAT Trp His 345 Ill CIA Phe Leu 360 MAA CII I Lys Val 375 C Vi TA t Irp Leu 390 993 1038 1083 1128 1173 1218 1263 TAC TAT Tyr Tyr CCI GAT Pro Asp CAG GIG Gin Val MlT CGA Asn Gly 350 IG GCd Lcu Asp 365 GAG AGC Giu Sc' 380 CCC ACT CI ACC AGG Arg Thr Ala Thr Arg 355 All TIT All ICA TO lie Phc lie Ser Scr 370 ACC AGC CCI CCC MCG Thr Arg Pro Cly LYS 385 CMk CAT Glu Asp GIG CAA vat Gir MCG GI( L YS Va 1 GGA CMA ACA ACC ICT GCA TAT GGA Gty Clu Ihr Ser Ser Ala Tyr Gty 395 GGC GGA GC CCC Gly Gly Ala Pro 400 CTA ICC Leu Set 405 CAC ACC TIT GCA GCT GGC TIT ATG TGC CTG GAT AAA TTG GGC CVC Asp Thr Phe Ala Ala Gty Phe Met Trp Leu Asp Lys Leu Gly Leu 410 415 420 TCA CCC CCA AIG GGA ATA.

Ser Ala Arg Met GLy I e 425 gAA GIG GTG ATG Gtu Vat Vat Met 430 AGG CAA GTA TIC Arg Gin Vat Phe CCA CCA GGA MAC Giy Ala Cly Asn CAT ITA GIG GAT CMA MC TIC GAT CCT His Lcu Vat Asp GLu Asn Pho Asp Pro 445 CCI CAT TAT TCC CTA Pro Asp Tyr Irp Leu 455 MCG GIG ITA AIG GCA Lys Vat Leu Met Ala 470 CGA CIA TAC CII CAT Ara Vat Tyr LeU His 485 GAM CCA CAT TIA ACT Ciu Cly Asp Leu Ihr 500 TCI CII CTC TIC Ser Leu Lou Phe MCG AAA Lys Lys 460 TTG GIG CCC ACC LeU Vat Cty Ihr 465 AGC CIG CMA CCI ICA Ser Vat Gin Cly Set 475 MAG AGA AGG MCG Lys Arg Arg Lys A.AT CCA ACC TAT Asn Pro Arg Tyr TGC ACA MAC ACT Cys Thr Asn Ihr CIC TAT CCC AlA Lcu Tyr Ala Ile CCC TAT CCI ITT Pro Tyr Pro Phe A.AG TAC TIC CCC L~s Tyr Leu Arg AAA TAC CTT CIA Lyc Tyr Leu Leu CTC CAT MAC CIC ACC Leu His Asn Vat Thr 510 AAC AAG CMA GIG CAT Asn Lys Gin V31. Asp 525 GGA TTA CII TCC AAA Gly Leu Leu Ser Lys 540 ATG GIG GAT CAT CMA Met Vat Asp Asp Gin 555

III

AGA CCI Arg Pro 530 TIC GGA CCI CAT Leu Cly Pro His 535 1668 1713 1758 1803 1848 TCI CIC CMA CIC MAT CCI CIA ACT CIA MAG Ser Vat Gin Leu Asn GLy Leu Thr Leu Lys 545 550 ACC TIC CCA CCI ITA AIC Tmr Lou Pro Pro Leu Met 560 CIG CCC TIC CCA GC TTC Leu Gly Leu Pro Ala Phe 575 GMA AAA CCI CIC CCC CCA GLU Lys Pro Leu Arg Pro 565 GGA ACT ICA Gty Set Set 570 TCA TAT ACT ITT Set Tyr Set Phe 580 ITT GIG ATA AGA Phe Vat Ile Arg CCC AAA CIT GCC GCT ICC ATC ICA AAA TMA MT ATA CIA GIC CIC 1893 Ala Lys Vat Ala Ala Cys Ile 590 592 19 ACA CIC 89 INFORMATION FOR SEQ ID 14:16: 0i) SEQUENCE CHARACTERISTICS: LENGTH: 594 TYPE: nucleic acid SIRANODONESS: double TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID 140:16 ATTACTATAG CCCACGCCIG CTCGACCCCC CGGCCGGTA IIGTCIIAAT GAGAACIIGA TAMCMATTT TGGCTCCIIG ATCTCIITCC AGCICACTI TACCGIATGC ICACCCCAGA 120 TTITTTCAGC CAAAACIAA.A ATACCTCAGA MACTCCIG CCAGAGCACA AICACATTI 180 CGCICCCICA ACIGACMAGC MAGTGTTTAI MCGCTAGATC GCAGAGGAG, CCATCMATAC 240 TCCATICCAG GCTTTACICC ACCGTCACAC GCATACCCGG CCCCATCACA AICCCATCTG 300 CGACICGCMA ACCCTGCGTT CCCACCACAC CGCGCAGAAC ACGTGCCICA GCAAGCCIGG 360 TCCCCCAICC CCACCGCTGC TCCCCGCCCG CTCCICCCCG CCCGCICCTC CCCAGGCCTC 420 CCGCCCGCIT GGATCCCGGC CATCICCGCA CCCTTCAAGT CCGTGTCGI GATTTCCIMA 480 GTGMACCTCA CCGCCACCCG GCCGAMAGCC AGCCMGGAAC TAGGACACAG CCCCCCACGC 540 CCGCCCCGT TCCAIICCCA CCACTGCCAG GGAICCAGAA GACGACTCCC AGCC 9' INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 21 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17 CCCCAGGAGC AGCAGCATCA G 21 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 21 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18 AGGCTTCGAG CGCAGCAGCA T 21 INFORMATION FOR SEQ ID N0O:19: SEQUENCE CHARACTERISTICS: LENGTH: 22 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19 GTAATACGAC TCACTATAGG GC 22 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 19 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID ACTATAGGGC ACGCGTGGT 19 INFORMATION FOR SEQ ID N0:21: SEQUENCE CHARACTERISTICS: LENGTH: 21 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: i near (xi) SEQUENCE DESCRIPTION: SEQ ID N0:21 CTTGGGCTCA CCTGGCTGCT C 21 INFORMATION FOR SEO ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 23 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22 AGCTCTGTAG ATGTGCTATA CAC 23 INFORMATION FOR SEQ ID N0O:23: SEQUENCE CHARACTERISTICS: LENGTH: 22 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEO ID N0:23 GCATCTTAGC CGTCTTTCTT CG 22 oo* oo 00.

004C .00 o fotoo 04O 0 009* *000 r

Claims

1. An isolated, purified and recombinant protein having heparanase (endo-P- D-glucuronidase) catalytic activity or being cleavable so as to acquire said heparanase catalytic activity.

2. The protein of claim 1, wherein said protein is mammalian.

3. The protein of claim 2, wherein said protein is isolated from an insect cell or a mammalian cell.

4. The protein of claim 1, wherein said protein is purified by Heparin- Sepharose, gel filtration and pooling of active column fractions

5. The protein of claim 4, wherein a quantity of said protein after said purification correlates with heparanase activity in said pooled active column fractions.

6. The protein of claim 1, wherein said protein is about 50 kDa or about kDa as determined by denaturing polyacrylamide gel electrophoresis.

7. The protein of any one of claims 1 to 6, wherein said protein is capable of eliciting anti-heparanase antibodies.

8. A polynucleotide encoding the protein of any one of the preceding claims.

9. The polynucleotide of claim 8, wherein said polynucleotide is mammalian. 30 A vector comprising the polynucleotide of claim 8 or claim 9.

11. The vector of claim 10, wherein said polynucleotide is selected from the group consisting of double stranded DNA, single stranded DNA and RNA. 42

12. The vector of claim 10, wherein said vector is a phage vector.

13. The vector of claim 10, wherein said vector is a viral vector.

14. The vector of claim 13, wherein said vector is a baculovirus vector. The vector of claim 10, wherein said vector is selected from the group consisting of a plasmid, a phagemid, a cosmid, a bacmid, and an artificial chromosome.

16. A host cell comprising the vector of claim

17. The host cell of claim 16, wherein said polynucleotide is selected from the group consisting of double stranded DNA, single stranded DNA and RNA.

18. The host cell of claim 16, wherein said cell is a prokaryotic cell.

19. The host cell of claim 16, wherein said cell is a eukaryotic cell. The host cell of claim 19, wherein said cell is an animal cell.

21. The host cell of claim 20, wherein said cell is an insect cell.

22. The host cell of claim 21, wherein said insect cell is selected from the group consisting of High five and Sf21 cells.

23. The host cell of claim 20, wherein said cell is a mammalian cell.

24. The host cell of claim 23, wherein said mammalian cell is a Human 293 cell. The host cell of claim 16, wherein said cell was lacking said heparanase activity before acquiring said vector. 1

26. The host cell of claim 16, wherein said polynucleotide is stably integrated in the genome of the cell.

27. The host cell of claim 16, wherein said polynucleotide is external to the genome of the cell, rendering the cell a transiently transduced cell.

28. A pharmaceutical composition comprising, as an active ingredient, the protein of claim 1 or claim 2.

29. The pharmaceutical composition of claim 28, wherein said pharmaceutical composition is administered topically, orally or intravenously. A modulator of heparin-binding growth factors, cellular responses to heparin-binding growth factors and cytokines, cell interaction with plasma lipoproteins, cellular susceptibility to viral, protozoa and bacterial infections or disintegration of neurodegenerative plaques comprising, as an active ingredient, a theraputically effective amount of the protein of claim 1 or claim 2.

31. Medical equipment comprising a medical device containing, as an active ingredient, the protein of claim 1 or claim 2.

32. A cell extract or conditioned cell medium produced from the host cell of I 2 claim 16.

33. A heparanase overexpression system comprising the host cell of claim 16, wherein said host cell overexpresses heparanase catalytic activity.

34. The overexpression system of claim 33, wherein overexpression is effected by insertion of at least one of a promoter or enhancer sequence into said host cell. 44 The overexpression system of claim 33, wherein overexpression is achieved by said.host cell containing said vector including suitable promoter and enhancer sequences.

36. An isolated, purified and recombinant protein as defined in claim 1 and substantially as hereinbefore described with reference to the examples.

37. A polynucleotide as defined in claim 8 and substantially as hereinbefore described with reference to the examples.

38. A pharmaceutical composition comprising, as an active ingredient, the protein of claim 36.

39. A modulator of heparin-binding growth factors, cellular responses to heparin-binding growth factors and cytokines, cell interaction with plasma lipoproteins, cellular susceptibility to viral, protozoa and bacterial infections or disintegration of neurodegenerative plaques comprising, as an active ingredient, a theraputically effective amount of the protein of claim 36.

40. Medical equipment comprising a medical device containing, as an active ingredient, the protein of claim 36. /T R4 SEFC 104 DATED this 27 th day of November 2003 o 25 nrght Orara'cec l-d S Hadasit Medical Research Services Development Ltd. By their Patent Attorneys CULLEN CO. 0 *8