AU2004201431B2 - Genetically Modified Cells and Methods for Expressing Recombinant Heparanase and Methods of Purifying Same - Google Patents

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AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT Names of Applicants: Address for Service: InSight Biopharmaceuticals Ltd Hadasit Medical Research Services and Development Ltd CULLEN CO.

Level 26 239 George Street Brisbane Qld 4000 Invention Title: Genetically Modified Cells and Mcthods For Exprcssing Recombinant Heparanase and Methods of Purifying Same Details of Original Applications: 69997/01 37705/99 The following statement is a full description of this invention, including the best method of performing it, known to us: 2 GENETICALLY MODIFIED CELLS AND METHODS FOR EXPRESSING RECOMBINANT HEPARANASE AND METHODS OF PURIFYING SAME FIELD AND BACKGROUND OF THE INVENTION The present invention relates to a polynucleotide, referred to herein below as hpa, encoding a polypeptide cleavable to obtain heparanase catalytic activity and to a recombinant protein cleavable to obtain heparanase catalytic activity. The present invention further relates to genetically modified cells overexpressing recombinant heparanase, to methods of overexpressing recombinant heparanase in cellular systems and to methods of purifying recombinant heparanase. Furthermore, the invention relates to nucleic acid constructs for directing the expression of modified heparanase species to which a protease recognition and cleavage sequence has been introduced, to the modified heparanase species expressed therefrom and to their proteolytic products. The invention further relates to in vivo and in vitro methods of inhibiting and/or otherwise regulating heparanase activity, for example by regulating cleavage of the above polypeptide or recombinant protein.

The extracellular matrix (ECM) acts both as a structural scaffold and as an informational medium. Its dynamic status is determined by cells that secrete both its constituent molecules and enzymes that catalyze the degradation of these molecules. A stasis between ECM degrading enzymes and their inhibitors maintains the integrity of the matrix.

While controlled ECM remodeling is fundamental to normalprocesses, uncontrolled disruption underlies diverse pathological conditions.

Among the integral constituents of basement membrane and ECM are cell adhesion molecules such as laminin and fibroncctin, structural components like collagen and ellastin, and proteoglycans including sydecan, serglican, proteoglycan I and II versican Brief overview on recombinant gene expression: For biochemical characterization of a protein and for pharmaceutical applications, it is often necessary to overproduce and purify large quantities of the protein. A major consideration when setting up a production scheme for a recombinant protein is whether the product should be expressed intracellularly or if a secretion system can be used to direct the protein to the growth medium. The inherent properties of the protein and the intended applications dictate the expression system of choice. Another consideration when attempting the production of recombinant eukaryotic proteins are the folding and post translational modification processes associated with their natural expression. Preferably, production is carried out in a cellular system that supports appropriate transcription, translation, and posttranslation modification of the protein of interest. Thus, cultured mammalian cells are widely used in applied biotechnology as well as in different disciplines of basic sciences of cellular and molecular biology for producing recombinant proteins of mammalian origin.

One of the most widely used cells for recombinant protein expression, particularly for biotechnological applications, is the Chinese hamster ovary cell line (CHO). Alternatively, baby hamster kidney cells (BHK21), Namalwa cells, Dauidi cells, Raji cells, Human 293 cells, Hela cells, Ehrlich's ascites cells, Sk-Hepl cells, MDCK1 cells, MDBK1 cells, Vero cells, Cos cells, CV-1 cells, NIH3T3 cells, L929 cells and BLG cells (mouse melanoma) have also been shown to consecutively express large quantities of recombinant proteins.

These cells are easily transfected with foreign DNA, that can integrate into the host genome to create stable cell lines, with new acquired characteristics expression of recombinant proteins). These new cell lines originate from a single cell that has undergone foreign DNA incorporation and are therefore referred to as "cellular clones." Since integration of foreign DNA in host cell genome is relatively inefficient, the isolation of cellular clones requires a selection system that discriminates between the stably transformed and the primary cells.

Dihydrofolate reductase deficiency in CHO cells (CHO dhfr- cell line) offers a particularly convenient selection system for cellular clones. Transfection of the dhfr gene along with the gene of interest, results in the survival of clones in a growth medium containing methotrexate (MTX). The higher the number of foreign dhfr gene copies in the cellular clone, the higher the MTX concentration the cells can survive. It has been demonstrated that integration events of foreign DNA into host cell genome often maintain all the components of the transfected DNA. the selection marker as well as the gene of interest (67).

In contrast to mammalian expression systems, that inherently express limited quantities of recombinant proteins, other expression systems, such as bacteria, yeast, and virus infected insect cells are widely used.

Using such cellular gene expression systems, large amounts of either active or nonactive protein can be obtained and used for biochemical analysis of protein properties, structure function relationship, kinetic studies, identification of, screening for, or production of specific inhibitors, production of poly- and monoclonal antibodies recognizing the protein, pharmaceutical applications and the like.

Bacteria are the most powerful tool for the production of recombinant proteins. A recombinant protein that is overproduced in a bacterial system might constitute up to 30% of the total protein content of the cells. The recombinant protein accumulates in inclusion bodies where it is relatively pure (comprises up to 50% of the protein content of the bodies) and protected from protease degradation.

Inclusion bodies enable the accumulation of up to 0.2 grams of protein per liter fermentation culture.

Using specific expression vectors, bacteria can also be directed to produce and secrete proteins into the periplasm and therefrom into the growth medium. Although the reported production quantities are not as high as in inclusion bodies, purification of the expressed protein may be simpler (68).

These advantages and the relative simple growth conditions required for bacteria to thrive, made bacteria a powerful and widely used cellular expression system for the production of recombinant proteins of interest human a-interferon, human P-interferon, GM-CSF, G- CSF, human LNFy, IL-2, IL-3, IL-6, TNF, human insulin, human growth hormone, etc.).

Furthermore, bacterially produced recombinant proteins that are non-active due to inappropriate folding and disulfide bonding may be reduced and/or denatured and thereafter deoxidized and/or refolded to acquire the catalytically active conformation.

However, when glycosylation of the protein is essential for its activity or uses, eukaryotic expression systems are required.

Yeasts are eukaryotic microorganisms that ,are widely used for commercial production of recombinant proteins. Examples include the production of insulin, human GM- CSF and hepatitis B antigens (for vaccination) by the yeast Saccharomyces cerevisiae. The relatively simple growth conditions and the fact that yeasts are eukaryotes make the yeast gene expression system highly suitable for the production of recombinant proteins, primarily those with pharmaceutical relevance.

In recent years methylotrophic yeasts Pichia pastoris, Hansenula polymorpha) became widely used, thus replacing in many cases the more traditionally used yeast Saccharomyces cerevisiae.

Methylotrophic yeasts can grow to a high cellular density, express and if appropriate, secrete, high levels of recombinant proteins. Quantities of the secreted, correctly folded recombinant protein can accumulate up to several grams per liter culture. These advantages make Pichiapastoris suitable for an efficient production of recombinant proteins (69).

One aspect of the present invention thus concerns the expression of recombinant heparanase in cellular systems.

Heparan sulfate proteoglycans (HSPGs): HSPGs are ubiquitous macromolecules associated with the cell surface and extracellular matrix (ECM) of a wide range of cells of vertebrate and invertebrate tissues The basic HSPG structure consists of a protein core to which several linear heparan sulfate chains are covalently attached. The polysaccharide chains are typically composed of repeating hexuronic and D-glucosamine disaccharide units that are substituted to a varying extent with N- and 0-linked sulfate moieties and N-linked acetyl groups Studies on the involvement of ECM molecules in cell attachment, growth and differentiation revealed a central role of HSPGs in embryonic morphogenesis, angiogenesis, metastasis, neurite outgrowth and tissue repair The heparan sulfate (HS) chains, which are unique in their ability to bind a multitude of proteins, ensure that a wide variety of effector molecules cling to the cell surface HSPGs are also prominent components of blood vessels In large 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 of endothelial cells and stabilize the structure of the capillary wall. The ability of HSPGs to interact with ECM macromolecules 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 HS may therefore result in disassembly of the subendothelial ECM and hence may play a decisive role in extravasation of normal and malignant 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 also been described in activated immune system cells and highly metastatic cancer cells (10-12), but research has been handicapped by the lack of biological tools to explore potential causative roles of heparanase in disease conditions.

Heparanase: Heparanase is a glycosylated enzyme that is involved in the catabolism of certain glycosaminoglycans. It is an endo-p-glucuronidase that cleaves heparan sulfate at specific intrachain sites (12-15). Interaction of T- and B-lymphocytes, platelets, granulocytes, macrophages and mast cells with the subendothelial extracellular matrix (ECM) is associated 6 with degradation of heparan sulfate by heparanase activity Connective tissue activating peptide III (CTAP), an a-chemokine, was found to have heparanase-like activity. Placenta heparanase acts as an adhesion molecule or as a degradative enzyme depending on the pH of the'microenvironment (17).

Heparanase is released from intracellular compartments lysosomes, specific granules) in response to various activation signals thrombin, calcium ionophores, immune complexes, antigens and mitogens), suggesting its regulated involvement in inflammation and cellular immunity responses (16).

It was also demonstrated that heparanase can be readily released from human neutrophils by 60 minutes incubation at 4'C in the absence of added stimuli (18).

Gelatinase, another ECM degrading enzyme that is found in tertiary granules of human neutrophils with heparanase, is secreted from the neutrophils in response to phorbol 12myristate 13-acetate (PMA) treatment (19,20).

In contrast, various tumor cells appear to express and secrete heparanase in a constitutive manner in correlation with their metastatic potential (21).

Degradation of heparan sulfate by heparanase results in the release of heparin-binding growth factors, enzymes and plasma proteins that are sequestered by heparan sulfate in basement membranes, extracellular matrices and cell surfaces (22,23).

Purification of natural heparanase: Heparanase activity has been described in a number of cell types including cultured skin fibroblasts, human neutrophils, activated rat T-lymphocytes, normal and neoplastic murine B-lymphocytes, human monocytes and human umbilical vein endothelial cells, SK hepatoma cells, human placenta and human platelets.

A procedure for purification of natural heparanase was reported for SK hepatoma cells and human placenta patent No. 5,362,641) and for human platelets derived enzymes Purification was performed by a combination of ion exchange and various affinity columns including Con-A Sepharose, Blue A-agarose, Zn+-chelating agarose and Heparin- Sepharose. Evidently, the amount of active heparanase recovered by these methods is low.

Cloning and expression of the heparanase gene: A purified fraction of heparanase isolated from human hepatoma cells was subjected to tryptic digestion. Peptides were separated by high pressure liquid chromatography (HPLC) and micro sequenced. The sequence of one of the peptides was used to screen databases for homology to the corresponding back translated DNA sequence. This procedure led to the identification of a clone containing an insert of 1020 base pairs (bp) which included an open reading frame of 963 bp followed by 27 bp of 3' untranslated region and a poly A tail. The new gene was designated hpa. Cloning of the missing 5' end of hpa was performed by PCR amplification of DNA from placenta eDNA composite. The joined hpa cDNA (also referred to as phpa) fragment contained an open reading frame which encodes a polypeptide of 543 amino acids with a calculated molecular weight of 61,192 daltons. Cloning an extended 5' sequence was enabled from the human SK-hepl cell line by PCR amplification using the Marathon RACE system. The 5' extended sequence of the SK-hepl hpa cDNA was assembled with the sequence of the hpa eDNA isolated from human placenta. The assembled sequence contained an open reading frame which encodes a polypeptide of 592 amino acids with a calculated molecular weight of 66,407 daltons. The cloning procedures are described in length in U.S.

Patent Application Nos. 08/922,170, 09/109,386, and 09/258,892, the latter is a continuationin-part of PCT/US98/17954, filed August 31, 1998, and in U.S. Patent No. 5,968,822 all of which are incorporated herein by reference.

In other experiments, it was demonstrated that the heparanase enzyme expressed by cells infected with the pFhpa virus is capable of degrading HS complexed to other macromolecular constituents fibronectin, laminin, collagen) present in a naturally produced intact ECM (see U.S. Patent Application No. 09/109,386, now US Patent No.

5,968,822, which is incorporated herein by reference), in a manner similar to that reported for highly metastatic tumor cells or activated cells of the immune system The ability of the hpa gene product to catalyze degradation of heparan sulfate (HS) in vitro was examined by expressing the entire open reading frame of hpa in High five and Sf21 insect cells, and the mammalian human 293 embryonic kidney cell line expression systems.

Extracts of infected cells were assayed for heparanase catalytic activity. For this purpose, cell lysates were incubated with sulfate labeled, ECM-derived HSPG (peak followed by gel filtration analysis (Sepharose 6B) of the reaction mixture. While the substrate alone consisted of high molecular weight material, incubation of the HSPG substrate with lysates of cells infected with hpa containing virus resulted in a complete conversion of the high molecular weight substrate into low molecular weight labeled heparan sulfate degradation fragments (see, for example, U.S. Patent Application No. 09/071,618 and U.S. Patent No. 6,426,209, which are incorporated herein by reference).

In subsequent experiments, the labeled HSPG substrate was incubated with the culture medium of infected High Five and Sf21 cells. Heparanase catalytic activity, reflected 8 by the conversion of the high molecular weight HSPG substrate into low molecular weight HS degradation fragments, was found in the culture medium of cells infected with the pFhpa virus, but not the control pF virus.

Altogether, these results indicate that the heparanase enzyme is expressed in an active form by cells infected with Baculovirus or mammalian expression vectors containing the newly identified human hpa gene.

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 (24,25). 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 underlying base membrane (BM) Once located between endothelial cells and the BM, the invading cells must degrade the subendothelial glycoproteins and proteoglycans 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 heparanase that cleaves HS at specific intrachain sites (16,11). Expression of a HS degrading heparanase was found to correlate with the metastatic potential of mouse lymphoma fibrosarcoma and melanoma (21) cells.

Moreover, elevated levels of heparanase were detected in sera from metastatic tumor bearing animals and melanoma patients (21) 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 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 (77,78). 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 While intact HSPG are eluted next to the void volume of the column (Kav<0.2, Mr 0.5x 106), labeled degradation fragments of HS side chains are eluted more toward the Vt of the column (0.5<kav<0.8, Mr =5-7 x 103) (26).

The inhibitory effect of various non-anticoagulant species of heparin on heparanase was examined in view of their potential use in preventing extravasation of blood-borne cells.

Inhibition of heparanase was best achieved by heparin species containing 16 sugar units or more and having sulfate groups at both the N and 0 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 markedly reduced 90%) the incidence of lung metastases induced by B 16 melanoma, Lewis lung carcinoma and mammary adenocarcinoma cells (12,13,28). 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 anti-metastatic properties of the polysaccharide (12).

Finally, heparanase externally adhered to B16-Fl melanoma cells increased the level of lung metastases in C57BL mice as compared to control mice (see U.S. Patent Application No. 09/260,037, entitled Introducing a Biological Material into a Patient, which is a continuation in part of U.S. Patent Application No. 09/140,888 which was later issued as U.S.

Patent No. 6,423,312, and is incorporated herein by reference).

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 (79).

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 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 (29,30). Basic fibroblast growth factor (bFGF) has been extracted from a subendothelial ECM produced in vitro (31) 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 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 (32-34). 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 (36,37).

Displacement of bFGF from its storage within 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 (38,39). 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) (41), 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 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 (36,37).

Recombinant heparanase for screening purposes: Put together, the accumulated evidence indicates that a reliable and high throughput screening system (HTS) for heparanase inhibiting compounds may be applied to identify and develop non-toxic drugs for the treatment of cancer and metastasis. Research aimed at identifying and developing inhibitors of heparanase catalytic activity has been handicapped by the lack of a consistent and constant source of a purified and highly active heparanase enzyme and of a reliable screening system. Such a HTS system is described in U.S. Patent Application No. 09/113,168 and US Patent No. 6,475,763, which are incorporated herein by reference. To this end, however, methods are required for obtaining high quantities of highly pure and active heparanase, so as to enable to study the kinetics of heparanase per se and in the presence of potential inhibitors. The recent cloning, expression and purification of the human heparanaseencoding gene offer, for the first time, a most appropriate and reliable source of active recombinant enzyme for screening of anti-heparanase antibodies and compounds which may inhibit the enzyme and hence be applied to identify and develop drugs that may inhibit tumor metastasis, autoimmune and inflammatory diseases.

Screening for specific inhibitors using a combinatorial library: A new approach aimed at rational drug discovery was recently developed for screening for specific biological activities. According to the new approach, a large library of chemically diverged molecules is screened for the desired biological activity. The new approach has become an effective and hence important tool for the discovery of new drugs.

The new approach is based on "combinatorial" synthesis of a diverse set of molecules in which several components predicted to be associated with the desired biological activity are systematically varied. The advantage of a combinatorial library over the alternative use of natural extracts for screening for desired biologically active compounds is that all the components comprising the library are known in advance In combinatorial screening, the number of hits discovered is proportional to the number of molecules tested. This is true even when knowledge concerning the target is unavailable. The large number of compounds, which may reach thousands of compounds tested per day, can only be screened, provided that a suitable assay involving a high throughput screening technique, in which laboratory automation and robotics may be applied, exist.

Expression of heparanase by cells of the immune system: Heparanase catalytic 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 mast cells with the subendothelial ECM is associated with degradation of heparan sulfate (HS) by heparanase catalytic activity The enzyme is released from intracellular compartments lysosomes, specific granules) in response to various activation signals thrombin, calcium ionophore, immune complexes, antigens, mitogens), suggesting its regulated involvement and presence in inflammatory sites and autoimmune lesions. Heparan sulfate degrading enzymes released by platelets and macrophages are likely to be present in atherosclerotic lesions (42).

Treatment of experimental animals with heparanase alternative substrates nonanticoagulant species of low molecular weight heparin (markedly reduced the incidence of experimental autoimmune encephalomyelitis (EAE), adjuvant arthritis and graft rejection (10,43) in experimental animals, indicating that heparanase inhibitors may be applied to inhibit autoimmune and inflammatory diseases (10,43).

Some of the observations regarding the heparanase enzyme were reviewed in reference No. 10 and are listed herein below: First, a proteolytic activity (plasminogen activator) and heparanase participate synergistically in sequential degradation of the ECM HSPG by 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 heparanase is preferentially and readily released in response to 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 B-lymphomas, 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 cells induced to differentiate to mature macrophages.

Tenth, T-cell mediated delayed type hypersensitivity and experimental autoimmunity are suppressed by low doses of heparanase inhibiting non-anticoagulant species of heparin (43).

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 trisulfated disaccharide that can inhibit T-cell mediated inflammation in vivo This inhibition was associated with an inhibitory effect of the disaccharide on the production of biologically active TNFao by activated T-cells in vitro The involvement of heparanase in other physiological prbcesses and its 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) (44, 41); cell interaction with plasma lipoproteins cellular susceptibility to certain viral and some bacterial and protozoa infections (45-47); and disintegration of amyloid plaques (48).

Viral Infection: The presence of heparan sulfate on cell surfaces have been shown to be the principal requirement for the binding of Herpes Simplex (45) and Dengue (46) 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 resistant to virus infection There are some indications that the cell surface heparan sulfate is also involved in HIV infection (47).

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

14 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 restenosis Apart from its involvement in SMC proliferation as a low affinity receptor 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 cholesterol 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.

In summary, 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.

ECM proteases and their involvement in tumor progression and metastasis: The cooperation with pericellular proteolysis cascades is required for vascular remodeling during angiogenesis, inflammatory processes, tumor progression and metastasis. In particular, the invasive processes that occur during tumor progression local invasion, intravasation, extravasation and metastasis formation involve extracellular matrix (ECM) degradation by proteases.

Four classes of proteases, are known to correlate with malignant phenotype: (i) cysteine proteases including cathepsin B and L; (ii) aspartyl protease cathepsin D; (iii) serine proteases including plasmin, tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA); (iv) Matrix metalloproteinases (MMPs) including collagenases, gelatinases A and B (MMP2 and MMP9) and stromelysin (MMP3).

Cathepsins are a family of proteases that are found inside cells in normal physiological conditions. Secretion of cathepsins correlates with various pathological conditions, such as arthritis, Alzheimer's disease and cancer progression (52).

The lysosomal cystein proteases cathepsin B and L have been suggested to play a role in tumor cell invasion and spread, either by directly cleaving extracellular matrix proteins or indirectly by activating other proteases (53).

Cathepsin B was found to have elevated expression levels in cancer cells.

Furthermore, the intracellular distribution of the protein differed between invasive and noninvasive cancer cells. In invasive cells, cathepsin B was found in the plasma membrane, whereas in non-invasive cells it was confined to the lysosomes In human tumor cells cathepsin B was secreted from the cells (53) and was shown to degrade extracellular matrix components Cathepsin B and L have been shown to degrade type IV collagen, laminin and fibronectin in vitro at both acid and neutral pH Both enzymes are able to activate the proenzyme form of the urokinase-type plasminogen activator (pro-uPA), which is secreted by tumor cells and can bind to receptors on the tumor cell surface In this cascade mechanism, the lysosomal cysteine proteases may function as effective mediators of tumorassociated proteolysis.

MMPs are a family of zinc dependent endopeptidases. They are secreted as inactive proenzymes and are activated by limited proteolysis During human pregnancy, cytotrophoblasts adopt tumor-like properties: they attach the conceptus to the endometrium by invading the uterus and they initiate blood flow to the placenta by breaching maternal vessels.

Matrix metalloproteinase MMP-9 (a type IV collagenase/gelatinase) was shown to be upregulated during cytotrophoblast differentiation along the invasive pathway. Furthermore, it was shown that the activity of that protease specified the ability of the cells to degrade ECM components in vitro (58).

Large body of evidence suggests that the matrix metalloproteinases MMP-2 and MMP-9 play an important role in tumor invasion process (59,58).

There is clearly a widely recognized need for, and it would be highly advantageous to have, a polynucleotide encoding a polypeptide cleavable to obtain heparanase activity, a recombinant protein cleavable to obtain heparanase catalytic activity, genetically modified cells overexpressing recombinant heparanase, methods of overexpressing recombinant heparanase in cellular systems, nucleic acid constructs for directing the expression of modified heparanase species to which a protease recognition and cleavage sequence has been introduced, to the modified heparanase species expressed therefrom and to their proteolytic products, in vivo and in vitro methods of inhibiting and/or otherwise regulating heparanase activity, for example by regulating cleavage of the above polypeptide or recombinant protein and methods of purifying recombinant heparanase, so as to enable, a search for heparanase inhibitors using a high throughput assay and a combinatorial approach.

BRIEF DESCRIPTION OF THE DRAWINGS The invention herein described, by way of example only, with reference to the accompanying drawings, wherein: FIG. 1 demonstrates the expression of recombinant heparanase in E. coli BL21(DE3)pLysS cells. Insoluble fractions of induced E. coli cells containing expression constructs for heparanase were analyzed on 10% SDS-PAGE. Following electrophoresis the gel was stained with commassie blue. Lane 1 cells transformed with pRSET (negative control), lanes 2 and 3 cells transformed with pRSEThpaS1 (two different colonies).

Molecular size in kDa is shown to the left (Prestained SDS-PAGE standards, Bio-Rad, CA).

FIG. 2 is a schematic presentation of the expression vector Relative positions of some restriction enzymes and genes are indicated. For the construction and utilities of pPIC3.5K-Sheparanase, see Example 4 in the Examples section below.

FIG. 3 is a schematic presentation of the expression vector pPIC9K-PP2. Positions of some restriction enzymes and genes are indicated. For the construction and utilities of see Example 4 in the Examples section below.

FIG. 4 demonstrates the secretion of human heparanase by transformed Pichia pastoris yeast cells. Western blot analysis using a rabbit anti-heparanase polyclonal antibody (disclosed in U.S. Patent Application No. 09/071,618, and U.S. Patent No. 6,426,209 which are incorporated by reference as if fully set forth herein) was performed on culture supernatants of different transformants (with and without selection for G-418 resistance). Lane 1 Sheparanase transformant, lane 2 pPIC3.5K transformant (negative control), lanes 3-6 transformants selected on 4 mg/ml of G-418. Molecular size is shown on the right as was determined using prestained SDS-PAGE standards, Bio-Rad, CA.

FIGs. 5a-e are schematic presentations of heparanase expression vectors adapted to direct heparanase expression in animal cells. hpa containing plasmids pShpa, pShpaCdhfr, pSlhpa, pS2hpa and pChpa are of 5374 bp, 7090 bp, 6868 bp, 6892 bp and 6540 bp, respectively. SV40 prom SV40 early promoter, CMV prom Citomegalovirus promoter, dhfr mouse dihydrofolate reductase gene, PPT preprotrypsin signal peptide, hpa heparanase cDNA sequence, hpa' and hpa" truncated hpa sequences.

1 FIGs. 6a-b show Western blot analysis of hpa transfected cells. Cell extracts of CHO cells or 8pag of 293 cells) were separated on 4-20 gradient SDS-PAGE and transferred to PVDF membranes. Detection of hpa gene products was performed with a rabbit anti-heparanase polyclonal antibody (disclosed in U.S. Patent Application No. 09/071,739 and in U.S. Patent No. 6,177,545) followed by ECL detection (Amersham, UK). Figure 6a CHO stable cellular clones (lanes 1-3) and transiently transfected 293 human cells (lane Figure 6b Mock transfected CHO cells (lane CHO cells performing stable or transient expression (lanes 1 and 2, respectively). Molecular size in kDa is shown to the right, as was determined using prestained SDS-PAGE standards, Bio-Rad, CA.

FIGs. 7a-b demonstrate recombinant heparanase secretion induced by calcium ionophore and PMA. Cells of a stable CHO clone (2TT1) were induced with either calcium ionophore (Figure 7a) or PMA (Figure 7b). Condition media were collected and 20 ml loaded on SDS polyacrylamide gel followed by Western blot analysis with a rabbit anti-heparanase polyclonal antibody (disclosed in U.S. Patent Application No. 09/071,739 and in U.S. Patent No. 6,177,545) followed by ECL detection (Amersham, UK). Molecular size in kDa is shown on the right, as was determined using prestained SDS-PAGE standards, Bio-Rad, CA..

FIG. 7c demonstrates recombinant heparanase secretion by human 293 cells.

Conditioned media of human 293 cells transfected with pS 1 hpa (lanes 3 and pS2hpa (lanes and 6) or control, untransfected cells (lanes 1 and were loaded on a denaturative 4-20 polyacrylamide gel (lanes 1, 3, and or 5 fold concentrated by 10 kDa ultrafiltration tube (Intersep (lanes 4 and Heparanase was detected by Western blot analysis with a rabbit anti-heparanase polyclonal antibody (disclosed in U.S. Patent Application No.

09/071,618 and in U.S. Patent No. 6,426,209) followed by ECL detection (Amersham, UK).

Molecular size in kDa is shown on the left, as was determined using prestained SDS-PAGE standards, Bio-Rad, CA.

FIG. 8a demonstrates heparanase activity as expressed by the ability to degrade heparin. Following overnight incubation with 50 ml unconcentrated (lanes 3, 20 x concentrated (lanes 4 and 7) or 40 x concentrated (lanes 5 and 8) conditioned media, from untreated (lanes 3-5) versus treated (lanes 6-8, 2 hours of incubation with 1 mg/ml calcium ionophore) stable clones, samples were electrophoretically separated on 7.5% polyacrylamide gel. Undegraded and degraded (by purified natural human heparanase) controls are shown in lanes 1 and 2 respectively.

18 FIG. 8b-c demonstrate recombinant heparanase activity following secretion induced by calcium ionophore as determined by the soluble 35S-ECM degradation assay. 8b the heparanase activity in one ml untreated conditioned media (c60), compared to one ml conditioned media treated with 100ng/ml calcium ionophore for 24 hours (p70) from stable CHO clones was determined by the soluble 35 S-ECM degradation assay. 8c the heparanase activity in one ml untreated conditioned media (c45), compared to one ml conditioned media treated with 1 mg/ml calcium ionophore for two hours (p52) from stable CHO clones was determined by the soluble 35 S-ECM degradation assay. Degraded substrates shift to the right.

FIGs. 8c-g show the relative heparanase activity of p70 and p52 (see Figures 8b-c) by comparing the ability of diluted (x2, x4 or x8) conditioned media to degrade 3 5

S-ECM.

FIG. 9 demonstrates glucose consumption record of heparanase producing cells in a large scale, 0.5 liters, Spinner-Basket bioreactor.

FIG. 10 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 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 of pF2 infected cells.

FIGs. 1 a-b demonstrate degradation of soluble sulfate labeled HSPG substrate by the growth medium of pFhpa2 and pFhpa4 infected cells. Culture media of High five cells infected with pFhpa2 (11 a) or pFhpa4 (11b) viruses or with control viruses were incubated (18 h, 37 0 C) with sulfate labeled ECM-derived soluble HSPG (peak I, The incubation media were then subjected to gel filtration on Sepharose 6B. Low molecular weight HS degradation fragments (peak II) were produced only during incubation with the hpa gene containing viruses. There was no degradation of the HSPG substrate by the growth medium of cells infected with control viruses.

FIG. 12 presents size fractionation of heparanase activity expressed by pFhpa2 infected cells. Growth medium of pFhpa2 infected High five cells was 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) (o) molecular weight compartment.

FIGs. 13a-b demonstrate the effect of heparin on heparanase activity expressed by pFhpa2 and pFhpa4 infected High five cells. Culture media of pFhpa2 (13a) and pFhpa4 (13b) infected High five cells were incubated (18 h, 37 0 C) with sulfate labeled ECM-derived soluble HSPG (peak I, 0) in the absence or presence of 10 pg/ml heparin. Production of low molecular weight HS degradation fragments was completely abolished in the presence of heparin, a potent competitor for heparanase activity.

FIGs. 14a-b demonstrate degradation of sulfate labeled intact ECM by virus infected High five and Sf21 cells. High five (14a) and Sf21 (14b) cells were plated on sulfate labeled ECM and infected (48 h, 28 0 C) with pFhpa4 or control pFl viruses. Control noninfected Sf21 cells (R were plated on the labeled ECM as well. The pH of the cultured medium was adjusted to 6.0 6.2 followed by 24 h incubation at 37'C. 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.

FIGs. 15a-b demonstrate degradation of sulfate labeled intact ECM by virus infected cells. High five (15a) and Sf21 (15b) cells were plated on sulfate labeled ECM and infected (48 h, 28 0 C) with pFhpa4 or control pFl viruses. Control non-infected Sf21 cells (R) were plated 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 0 C. Sulfate labeled degradation fragments 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.

FIGs. 16a-b demonstrate degradation of sulfate labeled intact ECM by the growth medium of pFhpa4 infected cells. Culture media of High five (16a) and Sf21 (16b) cells that were infected with pFhpa4 or control pFl viruses were incubated (48 h, 37 0 C, pH with intact sulfate labeled ECM. The ECM was also incubated with the growth medium of control non-infected Sf21 cells (R Sulfate labeled material released into the reaction mixture was subjected to gel filtration analysis. Heparanase activity was detected only in the growth medium of pFhpa4 infected cells.

FIGs. 17a-b demonstrate the effect of heparin on heparanase activity in the growth medium of pFhpa4 infected cells. Sulfate labeled ECM was incubated (24 h, 37 0 C, pH with growth medium ofpFhpa4 infected High five (17a) and Sf21 (17b) cells in the absence or presence (v )of 10 pg/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.

FIG. 18 demonstrates the purification of recombinant heparanase by a Source-S column. Lanes 1-14, 40 ml of fractions 1-14 eluted from a Source-S column. Samples were analyzed on 8-16% gradient SDS-PAGE. Gel was stained with commassie blue.

FIG. 19 demonstrates Western blot analysis of fractions 1-14 of Figure 18. Fractions 1-14 eluted from a Source-S column were analyzed following blotting onto nitrocellulose membrane with a rabbit anti-heparanase polyclonal antibody (disclosed in U.S. Patent Application No. 09/071,739 and in U.S. Patent No. 6,177,545) followed by ECL detection (Amersham, UK).

FIG. 20 is a schematic presentation of plasmid pCdhfr that contains the mouse dhfr gene under CMV promoter regulation. This vector does not express heparanase and serves as negative control.

FIG. 21a demonstrates the production of heparanase in pSIhpa transfected BHK21 cells. Cell extracts (2 x 105 BHK21 cells) were separated on 8-16 gradient SDS-PAGE and transferred to PVDF membranes. Detection of hpa gene products was performed with a mouse anti-heparanase monoclonal antibody No. HP-117 (disclosed in U.S. Patent Application No.

09/071,739 and in U.S. Patent No. 6,177,545) followed by ECL detection (Amersham, UK).

Molecular size in kDa is shown to the right, as was determined using prestained SDS-PAGE standards, Bio-Rad, CA. Lane 1 pSlhpa transfected BHK21 cells. Lane 2 control, pCdhfr transfected, BHK21 cells.

FIG. 21b demonstrates heparanase activity in human 293 cell extract. Cells were collected and concentrated by centrifugation (2750 x g for 5 min). The pellets were passed through three cycles of 5 minutes freezing in liquid nitrogen and thawing at 37 0 C. Cell lysate was centrifuged for 15 minutes at 3000 x g, and the supernatant was collected for analysis.

Increasing amounts of supernatant, between 0 and 5 pg protein per assay were assayed using the DMB activity assay described herein (see also U.S. Patent Application No. 09/113,168 and in U.S. Patent No 6,190,875).

FIG. 22a demonstrates recombinant heparanase constitutive secretion by CHO cells transfected with pSlhpa (clone S1PPT-8). Conditioned media 2 0pl) of untreated cells (lane mock treated cells (lane 3) and calcium ionophore treated cells (0.1 pg/ml for 24 hours; lane 4) were electrophoresed next to a cellular extract of 1x10 5 cells from clone 2TTI (CHO cells transfected with pShpaCdhfr, lane Samples were separated on a 4-20% gradient SDS- PAGE, followed by Western blot analysis with a rabbit anti-heparanase polyclonal antibody (disclosed in U.S. Patent Application No. 09/071,739 and in U.S. Patent No. 6,177,545) and by 21 ECL detection (Amersham, UK). Molecular size in kDa is shown on the right, as was determined using prestained SDS-PAGE standards, Bio-Rad, CA.

FIG. 22b demonstrates recombinant heparanase constitutive secretion by CHO cells transfected with pShpaCdhfr (2TT1 clones). Conditioned media (1501pl, concentrated by kDa ultrafiltration tube) of 2TT1-2 clone (lane 2) and of clone 2TT1-8 (lane 3) were electrophoresed next to a cellular extract of Ixl0 5 cells from clone 2TT1 (lane Samples were separated on a 4-20% gradient SDS-PAGE, followed by Western blot analysis with a rabbit anti-heparanase polyclonal antibody (disclosed in U.S. Patent Application No.

09/071,739 and in U.S. Patent No. 6,177,545) and by ECL detection (Amersham, UK).

Molecular size in kDa is shown on the right, as was determined using prestained SDS-PAGE standards, Bio-Rad, CA.

FIG. 23a demonstrates purification of recombinant heparanase from a mammalian cellular extract by ion exchange chromatography. 2TT1-8 CHO cells (lx 108) were extracted in ml of 10 mM phosphate citrate buffer pH 5.4. The extract was centrifuged at 2750 x g for 5 minutes and the supernatant was collected for heparanase enzyme purification using a cation exchange chromatography column. The chromatography column (mono-S HR 5/5, Pharmacia Biotech) was equilibrated with 20 mM sodium phosphate buffer, pH 6.8, and the mixture was loaded atop thereof. Proteins were eluted from the column using a linear gradient of 0 to 1 M sodium chloride in 20 mM sodium phosphate buffer, pH 6.8. The gradient was carried out in 20 column volumes at a flow rate of one ml per minute. The elution of proteins was monitored at 214 nm and fractions of 1 ml each were collected, starting with the first fraction which was eluted after 13 minutes and which is identified by the arrowhead mark.

FIG. 23b demonstrates the presence of immunologically active recombinant heparanase in the mammalian cellular extract. An aliquot from each fraction that was collected was analyzed for the presence of the heparanase enzyme by Western blot analysis. 20pl from each fraction, numbered 1-26, were separated on a 4-20 SDS-PAGE. The proteins were transferred from the gel to a PVDF membrane and were detected with a monoclonal antibody No. HP-117 (disclosed in U.S. Patent Application No. 09/071,739 and in U.S. Patent No.

6,177,545) followed by ECL detection (Amersham, UK). Molecular size in kDa is shown to the right, as was determined using SDS-PAGE standards St a purified recombinant heparanase enzyme from CHO cells.

FIG. 23c demonstrates the presence of catalytically active recombinant heparanase in mammalian cellular extract fractions. An aliquot (30gl) from each fraction that was collected 22 was analyzed for heparanase activity by the DMB assay. Load extract prior to purification. 7 and 16-26 correspond to fraction Nos.

FIG. 23d demonstrates a heparanase dose response. Increasing amounts from fraction No. 20, which exhibited the highest activity using the DMB assay (Figure 23c), were analyzed for heparanase activity using the tetrazolium assay, as disclosed in U.S. Patent Application No.

09/113,168 and in U.S. Pat No. 6,190,875.

FIG. 24a demonstrates the purification of heparanase from a mammalian cellular extract by an affinity column. A cellular extract from CHO 2TTI-8 cells was loaded on an affinity column containing antibodies elicited against native (non-denatured) recombinant heparanase. Western blot analysis of different fractions using a monoclonal antibody No.

HP-117 (disclosed in U.S. Patent Application No. 09/071,739 and in U.S. Patent No.

6,177,545) followed by ECL detection (Amersham, UK) is shown. Molecular size in kDa is shown to the left, as was determined using SDS-PAGE standards A recombinant heparanase enzyme purified from CHO 2TT1-8 cells on mono-S column; B extract of 2TT1-8 cells; C unbound, flow through proteins; and D wash fraction proteins.

FIG. 24b demonstrates the purification of heparanase from a mammalian cellular extract by an affinity column. A cellular extract from CHO 2TTI-8 cells was loaded on an affinity column containing antibodies elicited against native (non-denatured) recombinant heparanase. Heparanase activity in affinity column fraction Nos 1-9 .was determined using the DMB assay. Load extract prior to purification; C unbound, flow through proteins; and D wash fraction proteins FIGs. 25a-b demonstrates proteolytic processing of heparanase from insect cells conditioned medium by protease impurities. Figure 25a shows a Western blot analysis of heparanase, following processing of the enzyme expressed in insect cells. Heparanase expressed in insect cells, partially purified on a Source-S column, was incubated for one week at 4°C in either, 20 mM phosphate citrate buffer pH 7, containing 5% PEG 300 (lane mM phosphate citrate buffer pH 4, containing 5% PEG 300 and 1 x protease inhibitors cocktail (Boehringer Mannheim, Cat. No. 1836170, lane or 20 mM phosphate citrate buffer pH 4, containing 5% PEG 300 (lane M- Molecular weight markers (NEB Cat. No. 7708S).

Figure 25b shows the results of DMB heparanase activity assays for the proteins.

FIGs. 25c-d demonstrate the effect of a panel of protease inhibitors on proteolytic processing and activation of heparanase expressed in insect cells. Heparanase expressed in insect cells, partially purified on a Source-S column, was incubated for one week at 4 0 C in 23 mM phosphate citrate buffer, pH 4, containing 5 PEG 300 and one of the different protease inhibitors: A antipain; B bestatin; C chymostatin; D- E-64; E leupeptin; F pepstatin; G phosphoramidon; H EDTA; I aprotinin. The treated samples were either subjected to western blot analysis (Figure 25c) or to heparanase DMB activity assay (Figure 25d). J positive control, incubated in the absence of a protease inhibitor at pH 4; K negative control, heparanase incubated with the same buffer at pH 7. M Molecular weight marker (NEB Cat.

No. 7708S).

FIG. 26a demonstrates proteolytic processing of heparanase secreted from insect cells by trypsin. 10pg of heparanase, expressed in insect cells, and partially purified on a Source-S column, was incubated with increasing concentrations of trypsin 1.5, 5, 10, 15 units/test, Cat. No. T-8642, Sigma USA) for 10 minutes at 25°C. Following incubation, reaction tubes were placed on ice and 1.71pg/ml aprotinin (trypsin inhibitor) was added. Activity was determined using the DMB assay.

FIG. 26b demonstrates a Western blot analysis of heparanase following trypsin treatment. 10pg of heparanase, expressed in insect cells, and partially purified on a Source-S column, was incubated without (lane 1) or with 150 or 500 units of trypsin (lanes 2 and 3, respectively). A processed heparanase sample treated as described in Figure 25a-b, lanes J (lane and heparanase from a CHO 2TT1 cell extract (lane 5) served as controls.

FIG. 27 proteolytic processing of heparanase secreted from CHO cells by trypsin.

Conditioned medium of CHO cells transfected with pSlhpa (clone S1PPT-8) that secrete heparanase in a constitutive manner was subjected to proteolysis by trypsin. Unpurified CHO conditioned medium containing heparanase (0.5 pg heparanase per reaction) in 20 mM phosphate buffer, pH 6.8, was incubated with 0, 1.5, 15 or 150 units of trypsin for 10 minutes, at 37°C. Reactions were stopped by transferring the reaction tubes into ice and adding 2pg/ml aprotinin. Tryptic digest products were assayed for heparanase activity using the DMB assay.

FIG. 28a-b demonstrates proteolytic processing of p70-bac heparanase by cathepsin L. Partially purified heparanase from insect cells (10gg) was subjected to proteolysis by 1.6 mU cathepsin L (Cat. No. 219412, Calbiochem) for 3 hours, at 30 0 C, in 20 mM citratephosphate buffer, pH 5.4. Heparanase catalytic activity and immunoreactivity before and after processing with cathepsin L as were determined using the DMB heparanase activity assay and Western blot analysis with monoclonal antibody No. HP-117 (disclosed in U.S.

Patent Application No. 09/071,739 and in U.S. Patent No. 6,177,545 incorporated herein by reference) followed by ECL detection (Amersham, UK), Figures 28a-b, respectively.

24 FIG. 29a demonstrates a hydropathy plot of SEQ ID NO:2 predicted for heparanase as calculated by the Kyte-Doolittle method for calculating hydrophilicity, using the Wisconsin University GCG DNA analysis software. I and II point at peaks of most hydrophilic regions of the enzyme.

FIG. 29b is a schematic depiction of modified heparanase species (pre-p56' and pre-p52') that contain a unique protease recognition and cleavage sequence of factor Xa Ile- Glu-Gly-Arg 4 or of enterokinase Asp-Asp-Asp-Asp-Lys 4 (shaded regions, located between amino acids 119 and 120 or 157 and 158 of the heparanase enzyme depicted in SEQ ID NO:2, which acids are located within peaks I and II, respectively, of Figure 29a) which enable proteolytic processing by the respective proteases to obtain homogeneously processed and highly active heparanase species (p56' and p52', respectively).

FIG. 29c is a schematic depiction of the steps in constructing nucleic acid constructs harboring a unique protease recognition and cleavage sequence of factor Xa Ile-Glu-Gly- Arg 4- or of enterokinase Asp-Asp-Asp-Asp-Lys 4. FIG. 30 presents the 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.

FIG. 31 presents a comparison between nucleotide sequences of the human hpa and a mouse EST cDNA fragment (SEQ ID NO:27) which is 80 homologous to the 3' end (starting at nucleotide 1066 of SEQ ID NO:1) of the human hpa. The aligned termination codons are underlined.

SUMMARY OF THE PRESENT INVENTION According to one aspect of the present invention there is provided an isolated polynucleotide comprising a polynucleotide sequence encoding a polypeptide cleavable to obtain heparanase catalytic activity.

According to an embodiment of the invention, the polynucleotide sequence includes at least part of SEQ ID NOs: 1 or 28.

According to another embodiment, the polynucleotide sequence includes at least part of nucleotides 63-1691 of SEQ ID NO: t or nucleotides 139-1869 of SEQ ID NO: 28.

According to another embodiment, the polynucleotide sequence is as set forth in SEQ ID NO: 28.

According to another embodiment, the polynucleotide sequence includes a portion of SEQ ID NOs: 1 or 28, such that the portion encodes a polypeptide cleavable to obtain heparanase catalytic activity.

According to another embodiment, the polypeptide encoded by the polynucleotide sequence includes an amino acid sequence as set forth in SEQ ID NOs: 2 or 29 or includes a portion of SEQ ID NOs: 2 or 29, which comprises the heparanase catalytic activity.

According to another embodiment, the polynucleotide sequence shares at least homology with SEQ ID NOS: 1 or 28, preferably at least 70% homology with SEQ ID NOS: 1 or 28, more preferably at least 80% homology with SEQ ID NOS: 1 or 28, most preferably at least 90% homology with SEQ ID NOS: 1 or 28.

According to another embodiment, the polynucleotide sequence is selected from the group consisting of double stranded DNA, single stranded DNA and RNA.

According to another embodiment, the polynucleotide sequence comprises at least a first polynucleotide sequence encoding an N-terminal portion of a precursor heparanase polypeptide, a second, in frame, polynucleotide sequence encoding a protease recognition and cleavage sequence and a third, in frame, polynucleotide sequence encoding a C-terminal portion of said precursor heparanase polypeptide, such that the protease recognition and cleavage sequence enables cleavage of the precursor heparanase polypeptide by the protease.

According to another embodiment, this polynucleotide sequence is a nucleic acid construct.

According to another embodiment, the protease does not have a recognition and cleavage site in the N- and C- terminal portions.

According to another embodiment, the N- and C- terminal portions of the polynucleotide sequence which encodes the precursor heparanase polypeptide do not contain a protease recognition and cleavage site.

According to another embodiment, the polynucleotide sequence encodes a catalytically active heparanase when correctly folded and digested by said protease.

Additionally, according to another embodiment, the first and the third polynucleotide sequences each correspond to at least a portion of SEQ ID NO: 1.

According to a further aspect of the present invention there is provided a polypeptide cleavable to obtain a catalytically active heparanase, encoded by the aforementioned polynucleotide sequence.

26 According to an embodiment of the invention, the polypeptide shares at least homology with SEQ ID NOS: 2 or 29, preferably at least 70% homology with SEQ ID NOS: 2 or 29, more preferably at least 80% homology with SEQ ID N.OS: 2 or 29, most preferably at least 90% homology with SEQ ID NOS: 2 or 29.

According to another embodiment, the polypeptide is cleavable by a protease.

According to another embodiment, the cleavage by a protease results in at least a first polypeptide fragment and a second polypeptide fragment, such that when correctly folded each of the first and second polypeptide fragments comprises a subunit of a catalytically active heparanase.

According to another embodiment, the first polypeptide fragment mass is in the range of about 45-55 kDa, and the second polypeptide fragment mass is in the range of about 5-10 kDa.

According to another embodiment, the protease is selected from the group consisting of a cysteine protease, an aspartyl protease, a serine protease and a metalloproteinase.

Additionally, according to another embodiment, the cleavage of the polypeptide by the protease is performed at a pH- in which the protease is active, and preferably protease is most active at this pH.

According to another aspect of the present invention there is provided a polypeptide comprising a precursor protein for the aforementioned polypeptide, which fuirther comprises an upstream portion of a heparanase polypeptide, a mid portion of a protease recognition and cleavage sequence and a downstream portion of a heparanase polypeptide, such that the protease is selected having no recognition and cleavage sequences in the upstream and the downstream portions of the heparanase.

According to another aspect of the present invention there is provided a heparanase polypeptide cleaved from the aforementioned precursor protein, such that the heparanase polypeptide comprises a catalytically active polypeptide.

According to another aspect of the present invention there is provided a genetically modified cell, comprising the polynucleotide fragment mentioned above.

According to an embodiment of the invention, the polynucleotide fragment is stably integrated in the genome of the cell.

According to another embodiment, the polynucleotide fragment is external to the genome of the cell.

According to another embodiment, the genetically modified cell is a bacterial cell, and more preferably the genetically modified cell is E. coli.

According to another embodiment, the genetically modified cell is an animal cell.

According to another embodiment, the genetically modified cell is an insect cell, and more preferably the insect cell is selected from the group consisting of High five and Sf21 cells.

According to another embodiment, the genetically modified cell is a mammalian cell, and more preferably the mammalian cell is selected from the group consisting of CHO cells, BHK21 cells, Namalwa cells, Dauidi cells, Raji cells, Human 293 cells, Hela cells, Ehrlich's ascites cells, Sk-HepI cells, MDCK-1 cells, MDBK-1 cells, Vero cells, Cos cells, CV-1 cells, NIH3T cells, L929 cells and BLG cells.

According to another embodiment, the genetically modified cell is a yeast cell. More preferably, the yeast cell is a methylotrophic yeast. Most preferably, the methylotrophic yeast is selected from a group consisting of Pichia pastoris, Hansenula polymorpha and Saccharomyces cerevisiae.

According to another aspect of the present invention there is provided a method of activating the aforementioned polypeptide which is a non-catalytically active precursor heparanase polypeptide, the method comprising digesting the non-active precursor heparanase polypeptide with a protease, thereby activating the non-active precursor heparanase polypeptide.

According to an embodiment of the invention, the non-active precursor heparanase polypeptide comprises a natural precursor heparanase polypeptide.

According to another embodiment, the active precursor heparanase polypeptide comprises a natural precursor heparanase polypeptide.

According to another embodiment, the non-active precursor heparanase polypeptide comprises a recombinant precursor heparanase polypeptide.

According to another embodiment, the active precursor heparanase polypeptide comprises a recombinant precursor heparanase polypeptide.

According to another embodiment, the polypeptide comprises a purified heparanase polypeptide.

According to another embodiment, the polypeptide comprises a non-purified heparanase polypeptide.

According to another embodiment, the polypeptide comprises a partially purified heparanase polypeptide.

According to another embodiment, the digestion is performed in vivo or the digestion can be performed in vitro.

According to another aspect of the present invention there is provided a method of activating a heparanase polypeptide, the method comprising digesting the heparanase polypeptide with a protease capable of cleaving the heparanase polypeptide at a region containing its most hydrophilic sites within the first 170 N-terminal amino acids of said heparanase protein, as determined using the Kyte-Doolittle method for calculating hydrophilicity, using the Wisconsin University GCG DNA analysis software, so as to release a catalytically active portion of the heparanase.

According to another aspect of the present invention there is provided a method of activating a modified heparanase polypeptide, wherein the modified heparanase polypeptide comprises at least one introduced protease cleavage recognition sequence at a region containing the most hydrophilic sites within the first 170 N-terminal amino acids of a natural heparanase polypeptide as determined using the Kyte-Doolittle analysis software, the method comprising digesting the modified heparanase polypeptide with a matching protease being capable of cleaving the introduced protease cleavage recognition sequence.

According to another aspect of the present invention there is provided a method of inhibiting cleavage of a heparanase polypeptide, the method comprising administering a protease inhibitor to a subject in need thereof.

According to an embodiment of the invention, the administering of the protease inhibitor is performed in vivo.

According to an embodiment, the protease inhibitor is selected from the group consisting of a cysteine protease inhibitor, an aspartyl protease inhibitor, a serine protease inhibitor and a metalloproteinase inhibitor.

According to an embodiment, inhibiting cleavage of heparanase is used for medical treatment of the subject.

According to an embodiment, the medical treatment of the subject comprises treatment of a heparanase related condition or treatment of cancer. Additionally, according to another embodiment, the cancer which is treated comprises a metastatic cancer.

According to an embodiment, the need of the subject comprises being affected with a heparanase related condition or being affected with cancer.

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 being cleavable to obtain heparanase catalytic activity and a recombinant protein cleavable to obtain heparanase catalytic activity.

The present invention further relates to genetically modified cells overexpressing heparanase and of methods for overexpressing recombinant heparanase in cellular systems, which can be used to obtain purified recombinant heparanase in large quantities. Specifically, the present invention can be used to provide a scheme for biotechnological large scale recombinant heparanase production. The invention further relates to the activation of heparanase precursors by proteolysis and further to methods of in vivo and in vitro inhibition and/or other regulation of heparanase activity, for example by regulating cleavage of the above polypeptide or recombinant protein.

In fact, in the scope of the present invention are included any methods of inhibition or regulation of heparanase activity, including as a non limiting example inhibition of a natural or recombinant heparanase polypeptide or any other type of heparanase polypeptide, and inhibition of cleavage of a heparanase polypeptide in different environments, such as in vivo and in vitro. Particularly, the present invention is directed at regulation and control of heparanase catalytic activity with regard to cleavage of particular sequences, and described in further detail below.

In addition, any polynucleotide sequence which is at least 60% homologous to SEQ ID NOs:1 or 28 and which encodes a polypeptide cleavable to obtain heparanase catalytic activity, and any polynucleotide sequence encoding a polypeptide which is at least homologous to SEQ ID NOs: 2 or 29 and is cleavable to obtain heparanase catalytic activity is included in the scope of the present invention. This definition includes but is not limited to a preproheparanase sequence and a proheparanase sequence. As described in further detail below, the actual cleavage is performed in a specific part in the sequence, resulting in a plurality of smaller polypeptides which may form a catalytically active heparanase.

The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.

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.

According to the present invention there is provided a polynucleotide (either DNA or RNA, either single stranded or double stranded) which includes a polynucleotide sequence encoding a polypeptide being cleavable to obtain heparanase catalytic activity.

In another aspect, the present invention provides a genetically modified cell transduced with a polynucleotide sequence encoding a polypeptide being cleavable to obtain heparanase catalytic activity, designed to direct expression of recombinant heparanase by the cell.

In yet another aspect, the present invention provides a method of obtaining recombinant heparanase by genetically modifying a cell with an expression vector including a polynucleotide sequence encoding a polypeptide being cleavable to obtain heparanase catalytic activity, designed to direct expression of recombinant heparanase by the cell.

As used herein in the specification and in the claims section below, the phrase "genetically modified cell" refers to a cell that includes a recombinant gene. As further detailed below the cell may be a eukaryotic or prokaryotic cell.

As used herein in the specification and in the claims section below, the term "transduced" refers to the result of a process of inserting nucleic acids into cells. The insertion may, for example, be effected by transformation, viral infection, injection, transfection, gene bombardment, electroporation or any other means effective in introducing nucleic acids into cells. Following transduction the nucleic acid is either integrated in all or part, to the cell's genome (DNA), or remains external to the cell's genome, thereby providing stably transduced or transiently transduced cells.

As used herein in the specification and in the claims section below, the phrase "polynucleotide sequence" also means a nucleic acid sequence, typically a DNA sequence.

31 As used herein in the specification and in the claims section below, the term "upstream" refers to a polynucleotide sequence which extends in the 5' direction from a different polynucleotide sequence. In addition, the term refers to a polypeptide sequence which is nearer the C terminal from a different polypeptide sequence, or encoded by a polynucleotide sequence which extends in the 5' direction from the polynucleotide sequence encoding a different polypeptide sequence.

As used herein in the specification and in the claims section below, the term "polypeptide" also means a protein.

As used herein in the specification and in the claims section below, the term "natural polypeptide" refers to any naturally occurring polypeptide, for which the encoding nucleotide sequence was not cloned or manually constructed. The term further includes polypeptides that had undergone post-translational modification, if the modification was not man-made or artificially induced.

As used herein in the specification and in the claims section below, the term "subunit" refers to any polypeptide which takes part in forming an active protein.

As used herein in the specification and in the claims section below, the phrase "heparanase catalytic activity" or its equivalent term "heparanase activity" both refer to an animal endoglycosidase hydrolyzing activity which is specific for heparin 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 (81).

As used herein in the specification and in the claims section below, the term "expression" refers to the processes executed by cells while producing and/or secreting proteins, including where applicable, but not limited to, for example, transcription, translation, folding and post translational modification and processing.

As used herein in the specification and in the claims section below, the terms "vector" and "construct" are interchangeably used herein and refer to any vehicle suitable for genetically modifying cells, including, but not limited to, viruses bacoluvirus), phages, plasmids, phagemids, bacmids, cosmids, artificial chromosomes and the like.

As used herein in the specification and in the claims section below, the phrase "a polynucleotide sequence encoding a polypeptide having heparanase catalytic activity" 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 removal of a signal peptide and of a pro- or preprotein sequence), methionine modification,

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32 glycosylation, alkylation methylation), acetylation etc. is catalytically active or has potential to be catalytically active in degradation of, for example, ECM and cell surface associated HS. Thus, this phrase refers to any catalytically active or inactive conformant of a polypeptide which may acquire at least one active conformation having heparanase catalytic activity.

In a preferred embodiment of the present invention, the polynucleotide sequence encodes, in addition to a polypeptide cleavable to obtain heparanase catalytic activity, a signal peptide for protein secretion. The signal peptide may be the natural signal peptide of heparanase or any other suitable signal peptide, one non-limiting example is given under the Examples section hereinunder.

As described in further detail in the Examples section below, expression of the full length heparanase polypeptide in mammalian cells 293 kidney cells, CHO) yielded a major protein of about 50 kDa and a minor of about 65 kDa in cell lysates. Comparison of the enzymatic activity of the two forms, revealed that the 50 kDa enzyme is at least 100-200 fold more active than the 65 kDa form. A similar difference was observed when the specific activity of the recombinant 65 kDa baculovirus enzyme was compared to that of the 50 kDa heparanase preparations purified from human platelets, SK-hep-1 cells, or placenta. It was suggested that the 50 kDa protein is a mature processed form of a latent heparanase precursor. Amino terminal sequencing of the platelet heparanase indicated that cleavage of the sequence had occured in a site which is located within a hydrophillic peak, which is likely to be exposed and hence accessible to proteases.

Although the 3D structure of heparanase has not yet been completely resolved, significant structure-function relationships have been revealed for portions of the enzyme. The active enzyme has been claimed to exist as a heterodimer, comprising the previously described 45 kDa polypeptide which is noncovalently linked to an 8 kDa peptide derived from the Nterminus of the heparanase precursor (residues Gln36-Lysl08 or Glul09) (Fairbanks et al. J.

Biol. Chem. 1999;274, 28587-29590). It is most likely that heparanase is expressed as a kDa pre-pro form that is first processed into a 60 kDa pro form (also referred to herein as latent heparanase or mature heparanase) upon cleavage of the signal peptide. The 60 kDa latent/mature heparanase is activated into an active heparanase as follows: The 60 kDa latent/mature heparanase is proteolytically cleaved twice into a 45 kDa major subunit, a 8 kDa small subunit and a 6 kDa linker that links the 45 kDa major subunit and the 8 kDa small subunit in the latent enzyme. The 45 kDa major subunit and the 8 kDa small subunit heterocomplex to form the 53 kDa active form of heparanase. The heparanase activation cleavages occur at the Glul 0 9 -Ser 11 0 site and the Gln' 5 7 -Lys l58 site.

The heterodimeric structure of the enzyme was found to be essential for its catalytic activity (McKenzie et al., Biochemical Journal 2003;373:423-35). In-vitro processing studies with cathepsin B and D have indicated that heparin is required for the cleavage steps of the processing to occur. In addition to proteolytic processing described herein, the 45 kDa subunit is further glycosylated, forming the large component of the mature heparanase heterodimer referred to as the 50 kDa subunit.

It is clear from the above results that cleavage of a heparanase precursor is necessary for reaching maximal activity of the heparanase protein, and therefore inhibition of such cleavage would result in inhibition of heparanase activity. As described in further detail below, inhibition of heparanase activity may be used to treat individuals with heparanase related conditions, or where a certain condition is intensified or made more severe by heparanase activity.

In a preferred embodiment of the present invention the polynucleotide fragment includes at least a segment of SEQ ID NOs: 1 or 28. In a more preferred embodiment of the present invention the polynucleotide fragment includes nucleotides 63-1691 of SEQ ID NO: 1, or nucleotides 139-1869 of SEQ ID NO: 28, which encode the entire human heparanase enzyme.

The present invention is 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, E.F., Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York.) with hpa cDNA, especially with nucleotides 1-721 of SEQ ID NO:1.

In fact, any polynucleotide sequence which encodes a polypeptide being cleavable to obtain 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: 1 or 28 is within the scope of the present invention. The polynucleotide fragment according to the present invention may include any part of SEQ ID NOs: 1 or 28. For example, it may include nucleotides 63-721 of SEQ ID NO: 1, which is a novel sequence.

However, it may include any segment of SEQ ID NOs: 1 or 28 which encodes a polypeptide cleavable to obtain heparanase catalytic activity.

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According to another preferred embodiment of the present invention, the polynucleotide sequence is as set forth in SEQ ID NO:1 or a functional part thereof. The functional part encodes a polypeptide cleavable to obtain heparanase catalytic activity.

However, the scope of the present invention is not limited to SEQ ID NO:1 or a functional part thereof, as natural and man made innocuous variations thereof mutations, such as point mutations) may also encode a polypeptide cleavable to obtain heparanase catalytic activity.

Furthermore, it is shown hereinunder that a 52 kDa (formerly referred to as 45-50 kDa) protein, naturally processed from a 70 kDa (formerly referred to as 60 or 60-70 kDa) protein encoded by SEQ ID NO: 1, has heparanase catalytic activity.

The polynucleotide sequence may be a cDNA, a genomic DNA and a composite DNA (including at least one intron derived from heparanase or any other gene) as further detailed in U.S. Patent Application No. 09/258,892 and in U.S. Patent No. 6,664,105, which are incorporated herein by reference. Similarly it can be derived from any animal including mammals and avians because, as shown in U.S. Patent Application No. 09/258,892 and in U.S.

Patent No. 6,664,105, heparanase sequences derived from species other than human beings are readily hybridizeable with the human sequence, allowing for isolation of such sequences by methods known in the art.

The functional part may be either man induced by genetic engineering or post translation artificial processing by a protease) or naturally processed, depending on the cellular system employed.

In another preferred embodiment of the invention the polypeptide encoded by the polynucleotide fragment includes an amino acid sequence as set forth in SEQ ID NOs: 2 or 29 or a functional part thereof, i.e. a portion harboring heparanase catalytic activity.

However, any polynucleotide fragment which encodes a polypeptide being cleavable to obtain heparanase catalytic activity is within the scope of the present invention. Therefore, the polypeptide may be allelic, species and/or induced variant, or a natural or man-made innocuous variation mutation, such as single amino acid substitution) of the amino acid sequence set forth in SEQ ID NOs: 2 or 29 or functional part thereof. Polypeptides corresponding to species other than human and having heparanase catalytic activity are also within the scope of the present invention.

In fact, any polynucleotide sequence which encodes a polypeptide being cleavable to obtain heparanase catalytic 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: 2 or 29 is within the scope of the present invention.

As used herein in the specification and in the claims section below, the term "functional part thereof" refers to a part of a nucleic acid sequence which encodes a polypeptide being cleavable to obtain heparanase catalytic activity or a part of a polypeptide sequence having heparanase catalytic activity or being cleavable to obtain heparanase catalytic activity.

In this context, it is important to remember that in many cases truncated or naturally processed polypeptides exhibit a catalytic activity similar to that of the natural polypeptide of the preprocessed polypeptide, respectively. Apparently, in many cases, not all of the amino acids of a protein are essential for its catalytic function, some may be responsible for other features, such as secretion, stability, interaction with other macromolecules, etc., whereas other may be replaced without affecting activity to a great extent. In many cases the processed protein exerts higher catalytic activity as compared with its unprocessed counterpart.

According to yet another preferred embodiment of the present invention, the polynucleotide sequence is selected from the group consisting of double stranded DNA, single stranded DNA and RNA.

As mentioned above, the present invention is further directed at providing genetically modified cells overexpressing heparanase and methods for overexpressing heparanase in cellular systems, which can be used to obtain purified recombinant heparanase in large quantities. Specifically, the present invention can be used to provide a scheme for biotechnological large scale recombinant heparanase production. In addition, the aforementioned cleavage of a polypeptide to obtain heparanase catalytic activity may occur in such recombinant cells, or by cleaving recombinant heparanase, since as mentioned above any polypeptide cleavable to obtain heparanase catalytic activity is in the scope of the present invention.

According to a preferred embodiment of the present invention, the genetically modified cell is a bacterial cell, preferably E. coli.

According to another preferred embodiment of the present invention, the genetically modified cell is an animal cell.

The animal cell may be a mammalian cell, such as, but not limited to, Chinese hamster ovary cell line (CHO), baby hamster kidney cells (BHK21), Namalwa cells, Dauidi cells, Raji cells, Human 293 cells, Hela cells, Ehrlich's ascites cells, Sk-Hepi cells, MDCK1

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cells, MDBK1 cells, Vero cells, Cos cells, CV-1 cells, NIH3T3 cells, L929 cells or BLG cells (mouse melanoma).

Alternatively, the animal cell may be an insect cell, such as, but not limited to, High five or Sf21.

According to another preferred embodiment of the present invention, the genetically modified cell is a yeast cell, preferably a methylotrophic yeast, such as, but not limited to, Pichia pastoris and Hansenula polymorpha. Another preferred yeast is Saccharomyces cerevisiae.

The specified bacterial, yeast and animal cells are of specific advantage and interest since they are widely used in large scale biotechnological production of proteins and therefore knowledge has accumulated with respect to their large scale propagation, maintenance and with respect to recombinant protein purification therefrom. Use of these cells for large scale production of DNA or proteins and methods for such production are well known in the art, see for example US Patent No. 6,140,086.

The invention is further directed at providing a recombinant protein including a polypeptide being cleavable to obtain heparanase catalytic activity.

According to a more preferred embodiment of the present invention, the recombinant heparanase is human recombinant heparanase. The recombinant heparanase may be cloned in any type of cell, including but not limited to the aforementioned genetically modified cells.

The recombinant protein may be purified by any conventional protein purification procedure close to homogeneity and/or be mixed with additives. The 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 anti-heparanase antibodies, either poly or monoclonal antibodies, and as a screening active ingredient in an anti-heparanase inhibitors or drugs screening assay or system.

According to yet another preferred embodiment of the present invention, the method is further affected by purifying the recombinant heparanase. As further detailed hereinunder efficient purification 90% purified) of recombinant heparanase may be effected by a single step ion exchange Source-S) column.

The purification may be from the cells themselves. To this end the cells are collected, by centrifugation, homogenated and the recombinant heparanase is purified from the homogenate. If the recombinant heparanase is secreted by the cells to the growth medium, then purification is preferably from the growth medium itself.

According to yet another preferred embodiment of the present invention, the method further includes a step of subjecting the cell to a substance which induces secretion into the growth medium of secretable proteins, thereby inducing secretion of the recombinant heparanase into the growth medium. Preferably, the substance is selected from the group consisting of thrombin, calcium ionophores, immune complexes, antigens and mitogens, all are known to induce secretion of native heparanase from expressing cells. As shown in the Examples section below, the calcium ionophore calcimycin (A23187) and phorbol 12myristate 13-acetate, are effective in inducing secretion of recombinant heparanase from transduced cells into their media.

According to yet another preferred embodiment of the present invention, the cell is grown to a large biotechnological scale of at least half a liter, preferably at least 5, 7 or 35 liters of growth medium, in a bioreactor, such as but not limited to, Spinner-Basket bioreactor.

Further according to the present invention there is provided a method of purifying a recombinant heparanase from overexpressing cells or growth medium in which they grow by adsorbing the recombinant heparanase on a Source-S column under low salt conditions about 50mM NaCI), washing said column with low salt solution thereby eluting other proteins, and eluting the recombinant heparanase from the column by a salt gradient 50mM to IM NaCI) or a higher concentration of salt about 0.4M).

According to a further aspect of the present invention there is provided an antibody comprising an immunoglobulin elicited against recombinant native heparanase. The immunoglobulin therefore recognizes and binds native non denatured) natural or recombinant heparanase.

As used herein in the specification and in the claims section below, the term "antibody" includes serum immunoglobulins, polyclonal antibodies or fragments thereof or monoclonal antibodies or fragments thereof. The antibodies are preferably elicited against a surface determinant of the particulate. Monoclonal antibodies or purified fragments of the monoclonal antibodies having at least a portion of an antigen binding region, including such as Fv, F(abl)2, Fab fragments single chain antibodies Patent 4,946,778), chimeric or humanized antibodies (64-65) and complementarily determining regions (CDR) may be prepared by conventional procedure. Purification of the serum immunoglobulins antibodies or fragments can be accomplished by a variety of methods known to those of skill including, but not limited to, precipitation by ammonium sulfate or sodium sulfate followed by dialysis against saline, ion exchange chromatography, affinity or immunoaffinity chromatography as well as gel filtration, zone electrophoresis, etc. (see 66).

According to a further aspect of the present invention there is provided an affinity substrate comprising a solid matrix and an immunoglobulin elicited against recombinant native heparanase being immobilized thereto. Methods of immobilizing immunoglobulins to solid matrices, such as cellulose, polymeric beads including magnetic beads, are well known in the art. One such method is described in the Examples section that follows. The solid support according to the present invention can be packed into an affinity column.

According to a further aspect of the present invention there is provided a method of affinity purifying heparanase. The method is effected by loading a heparanase preparation on an affinity column including a solid matrix and an immunoglobulin elicited against recombinant native heparanase being immobilized thereto; washing the affinity column, using low, say 0-500 mM, salt solution; and eluting heparanase molecules being adsorbed on the affinity column via the immunoglobulin, e.g. using a highly concentrated, say 0.5-1.5M, salt solution.

According to a further aspect of the present invention there is provided a method of activating a polypeptide to obtain heparanase catalytic activity comprising the step of digesting a heparanase precursor polypeptide by a protease. The heparanase precursor polypeptide according to this aspect of the present invention can be natural or recombinant, purified, partially purified or non-purified. The protease can be of any type, including, but not limited to, a cysteine protease, an aspartyl protease, a serine protease and a metalloproteinase. Examples of specific proteases associated with the above listed protease families are provided in the Background section.

The use of other proteases for which heparanase includes a recognition and cleavage sequence is envisaged. According to a preferred embodiment digesting the heparanase enzyme by the protease is effected at a pH in which the protease is active, preferably most active. It is known that some proteases are most active in acidic pH values whereas other proteases are most active in basic pH values. The pH value at which a specific protease is most active can be readily determined by one ordinarily skilled in the art.

According to a further aspect of the present invention there is provided a method of in vivo or in vitro inhibition of proteolytic processing of heparanase. The method according to this aspect of the present invention is affected by administering a protease inhibitor. The protease inhibitor can be, for example, a cysteine protease inhibitor, an aspartyl protease inhibitor, a serine protease inhibitor or a metalloproteinase inhibitor. Examples of suitable inhibitors are provided in the Examples section that follows. The inhibition of proteolytic processing of a heparanase precursor polypeptide may be for any type of heparanase precursor polypeptide including but not limited to natural heparanase precursor polypeptides and recombinant heparanase precursor polypeptides as defined above. In fact, inhibition of proteolytic processing of any polypeptide sequence which is cleavable to obtain heparanase catalytic activity is envisaged, and specifically for polypeptide sequences that share at least homology with SEQ ID NOs: 2 or 29. Such inhibition of proteolytic processing may be performed in vivo or in vitro, and may be performed in any type of cell, including but not limited to mammalian cell lines, and the genetically modified cells discussed herein above.

Some protease inhibitors are used pharmaceutically for treatment or prevention of various conditions. Inhibition of proteolytic processing of heparanase precursor polypeptide by a protease inhibitor can be used for treatment of cancer, metastatic cancers in particular, in which heparanase catalytic activity is involved, because, as further exemplified in the Examples section that follows, the preheparanase (non-processed, p 7 0 heparanase) is characterized by lower activity as compared to its processed counterpart (p5 2 heparanase).

According to a further aspect of the present invention there is provided a nucleic acid construct comprising a first nucleic acid sequence encoding for an upstream (N terminal) portion of a heparanase polypeptide, a second, in frame, nucleic acid sequence encoding a protease recognition and cleavage sequence and a third, in frame, nucleic acid sequence encoding for a downstream portion (C terminal) of a heparanase polypeptide, wherein the second nucleic acid sequence is in between the first nucleic acid sequence and the third nucleic acid sequence. Examples of such constructs are provided in the Examples section that follows.

Preferably, the protease is selected having no recognition and cleavage sequences in the upstream and the downstream portions of heparanase, such that when expressed the modified heparanase is digested only at the introduced recognition and cleavage sequence of the protease. Preferably, the third nucleic acid sequence encodes for a catalytically active heparanase when correctly folded. However, embodiments wherein the second nucleic acid sequence is so positioned such that when expressed the modified heparanase protein is digestible into portions lacking catalytic activity are also envisaged. Such embodiments can be used to provide a heparanase species having a shorter half life, in, for example, physiological conditions, as compared with the non-modified enzyme. One ordinarily skilled in the art would know how to select locations for introduction of the recognition and cleavage sequence such that the sequence will not hamper the catalytic activity of the enzyme prior to cleavage thereof by the protease.

SThe above construct, when introduced into a cell expression system can be used to provide a precursor heparanase protein comprising an upstream portion of heparanase, a mid portion of a recognition and cleavage sequence of a protease and a downstream portion of heparanase, wherein the protease is selected having no recognition and cleavage sequences in the upstream and the downstream portions of heparanase. The recognition and cleavage sequence of the protease is composed either entirely from amino acids which are not present in natural heparanase, or from amino acids which are not present in natural heparanase in part, and further from adjacent amino acids which are present in natural heparanase. Further according to the present invention there is provided a heparanase protein resulting by digesting the precursor heparanase protein described herein.

According to a further aspect of the present invention there is provided a method of obtaining a homogeneously processed, active heparanase. The method according to this aspect of the present invention is effected by expressing the precursor heparanase protein in a cell which secretes the precursor heparanase protein into the growth medium to obtain a conditioned growth medium, the precursor heparanase protein including an upstream portion of heparanase, a mid portion of a recognition and cleavage sequence of a protease and a downstream portion of heparanase, wherein the protease is selected having no recognition and cleavage sequences in the upstream and the downstream portions of heparanase; treating the precursor heparanase protein with the protease; and purifying a proteolytic heparanase product having heparanase catalytic activity.

It will be appreciated that the various heparanase species described herein, either activated and/or precursors can be used to produce pharmaceutical compositions, including, in addition to heparanase, a pharmaceutically acceptable carrier. Affinity purified and protease treated, modified, recombinant heparanase is of particular interest for pharmaceutical applications due to its homogeneity and purity.

The present invention has advantages because it provides means for expressing, purifying and activating recombinant/natural heparanase. Such heparanase can be used in pharmaceutical compositions (see, for example, U.S. Patent Application No. 09/046,465 or U.S. Patent No. 5,984,997, in which heparanase is used in the treatment of CF), or as a source of enzyme for high throughput heparanase activity assay, which can be used for efficient screening of specific heparanase inhibitors (see, for example, U.S. Patent Application No.

09/113,168 and U.S. Patent No 6,190,875). By identifying the heparanase proteolytic activation process, novel indirect methods of in vivo heparanase inhibition by administration of protease inhibitors were conceived and tested in vitro. By identifying the heparanase proteolytic activation process, novel constructs encoding novel heparanase species has been constructed and can be used to direct the expression of a heparanase which is homogeneously processed and activated or alternatively neutralized by a dedicated protease.

EXAMPLES

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

Generally, the nomenclature used herein and the laboratory procedures in recombinant DNA technology described below are those well known and commonly employed in the art. Standard techniques are used for cloning, DNA and RNA isolation, amplification and purification. Generally enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like are performed according to the manufacturers' specifications. Similarly, standard techniques are used for the proteolysis of heparanase by various proteases. These techniques and various other techniques used while reducing the present invention to practice are generally performed according to Sambrook et al., Molecular Cloning--A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

(1989), which is incorporated herein by reference. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

EXAMPLE 1 Cloning of the hpa gene Purified fraction of heparanase isolated from human hepatoma cells (SK-hep-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:26). These two sequences i

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were derived from clones 257548 and 260138 (I.M.A.G.E Consortium) prepared from 8 to 9 weeks 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:1), contained an open reading frame which encodes, as shown in Figure 30 and SEQ ID NO:3, a polypeptide of 543 amino acids (SEQ ID NO:2) 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 G721 of SEQ ID NO:1 and Figure As further shown in Figure 30, there was a single sequence discrepancy between the EST clones and the PCR amplified sequence, which led to an amino acid substitution from Tyr246 in the EST to Phe246 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.

As stated above, the 3' end of the partial cDNA inserts contained in EST clones 257548 and 260138 started at nucleotide 721 of hpa (SEQ ID NO:1). 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:1). 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.

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.

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EXAMPLE 2 hpa homologous genes EST databases were screened for sequences homologous to the hpa gene. Three mouse ESTs were identified (accession No. Aal 77901, from mouse spleen, Aa067997 from mouse skin, Aa47943 from mouse embryo), assembled into a 824 bp cDNA fragment which contains a partial open reading frame (lacking a 5' end) of 629 bp and a 3' untranslated region of 195 bp (SEQ ID NO:27). As shown in Figure 3 1, 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, Tyro sine -protein kinase-RYK, Fibulin- 1, Insulin-like growth factor binding protein and several others. The amino terminus is 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.

EXAMPLES

Expression of recombinant human heparanase in bacteria Experimental Methods Construction of expression vector: A 1.6 kb fragment of hpa cDNA (SEQ ID NO: 1) was amplified from pfasthpa (hpa cDNA cloned in pFastflac, see U.S. Patent Application No.

08/922,170 and U.S. Patent No. 5,968,822) by PCR using specific sense primer: Hpu-55ONde 5'-CGCATATGCAGGACGTCGTGGACCTG-3' (SEQ ID NO:4) and a vector specific antisense primer: 3'pFast 5'-TATGATCCTCTAGT ACTTCTCGAC-3' (SEQ ID NO:5). PCR conditions were: denaturation 94 0 C, 40 seconds, first cycle 3 minutes; annealing 58 0 C, seconds; and elongation 72 0 C, 2.5 minutes, total of 5 cycles, and then denaturation 94 0 C, seconds; annealing 68 0 C, 60 seconds; and elongation 72 0 C, 2.5 minutes, for additional cycles. The Hpu-55OA~de primer introduced an NdeI site and an in frame ATG codon preceding nucleotide 168 of hpa. The PCR product was digested by NdeI and BamNI and its sequence was confirmed with vector specific and gene specific primers, using an automated DNA sequencer (Applied Biosystems, model 373A).

A 1.3 kb BamHI-KpnI fragment was cut out of pFasthpa. The two fragments were ligated with the pRSET bacterial expression vector (Invitrogen, CA.).

The resulting plasmid, designated pRSEThpaSl, encoded an open reading frame of 508 amino acids (36-543, SEQ ID NO:2) of the heparanase protein, lacking the N-terminal amino acids which are predicted to be a signal peptide.

Transformation: Transformation of E. coli BL21(DE3)pLysS cells (Stratagene) was performed following Stratagene's protocol. Briefly, using p-mercaptoethanol in the transformation buffer cells were transformed by five seconds of heat shock at 42 0

C.

Expression of recombinant heparanase: E. coli BL21(DE3)pLysS cells transformed with the recombinant plasmid were grown at 37 0 C overnight in Luria broth (LB) medium containing 100tg/ml ampicillin and 34 pg/ml chloramphenicol. Cells were diluted 1/10 in the same medium, and the cultures were grown to an OD600 of approximately 0.5. Isopropylthiogalactoside (IPTG) (Promega) was added to a final concentration of ImM and the culture was incubated at 37 0 C for 3 hours. Cells from IPTG induced cultures were cooled on ice and sedimented by centrifugation at 4,000 x g for 20 minutes at 4'C, and resuspended in 0.5ml of cold phosphate-buffered saline (PBS). Cells were lysed by sonication, and cell debris were sedimented by centrifugation at 10,000 x g for 20 minutes. The resulting pellet was analyzed for proteins by 10% SDS-PAGE, essentially as described in Harlow, E. and Lane, D. Eds. in Antibodies, a laboratory manual. CSH Laboratory press. New-York.

Experimental Results The expression of recombinant heparanase in E. coli BL21(DE3)pLysS cells containing the pRSEThpaSl was analyzed by SDS-PAGE followed by commassie blue staining for proteins. Bacterial cells were fractionated and a protein of approximately 70 kDa, which is the expected size of the recombinant heparanase, was observed in the insoluble fraction (Figure 1, lanes That band did not appear when negative control cells transformed with pRSET were employed (Figure 1, lane 1).

The identification of the recombinant heparanase expressed in E. coli was confirmed by a Western blot (data not shown) using a rabbit anti-heparanase polyclonal antibody (disclosed in U.S. Patent Application No. 09/071,739 and in U.S. Patent No. 6,177,545), followed by ECL detection (Amersham, UK).

As compared to known quantities of co-size separated and stained BSA, the estimated yield of the heparanase recombinant protein under the conditions described was about 0.2mg/ml of culture (not shown). The protein was found in the insoluble fraction (inclusion bodies) and had no enzymatic activity, as was determined by the soluble 35 S-ECM degradation assay (not shown), however, the recombinant heparanase protein expressed in E. coli could provide a source for large quantities of heparanase.

It will be appreciated that solubilization and refolding of recombinant proteins expressed in E. coli are well known in the art (see, for example, for insulin, 70; others are reviewed in 71) and these procedures should be applied in order to obtain a functional protein having heparanase activity.

The expression of the recombinant heparanase in bacterial cells is thus demonstrated in this Example. It will be further appreciated that changes in protein length and/or amino acid composition might affect the efficiency of expression, correct folding and the potential yield of functional enzyme.

EXAMPLE 4 Expression of recombinant human heparanase in yeast Experimental Methods Construction of expression vectors for expression in yeast: Two expression vectors were constructed for the expression of hpa in Pichia pastoris. The first vector, designated (Figure 2) contains nucleotides 63-1694 of the hpa sequence (SEQ ID NO:1) cloned into the expression vector pPIC3.5K (Invitrogen, CA) using a multistep procedure as follows.

A pair of primers: HPU-6641 AGGAATTCACCATGCTGCT GCGCTCGAAGCCTGCG-3' (SEQ ID NO:6) and HPL-209 ATTGCTCCTGGTAG-3' (SEQ ID NO:7) were used in PCR amplification to introduce an EcoRI site just upstream to the predicted methionine. PCR conditions were: denaturation 94 0 C, 40 seconds; annealing 50 0 C, 80 seconds; and elongation 72 0 C, 180 seconds, total of cycles.

The resulting PCR product was digested with EcoRI and BamHI and cloned into the EcoRI-BamHI sites of the vector phpa2 (described in U.S. Patent Application No. 08/922,170 and in U.S. Patent No. 5,968,822). The hpa coding region was then removed as an EcoRI-NotI fragment and cloned into the EcoRI-NotI sites of the expression vector pPIC3.5K to generate the vector pPIC3.5K-Sheparanase (Figure 2).

The second vector, designated pPIC9K-PP2 (Figure includes the hpa coding region except for the predicted signal sequence (N-terminal 36 amino acids, see SEQ ID NO:2). The hpa was cloned in-frame to the a-factor prepro secretion signal in the Pichia pastoris expression vector pPIC9K (Invitrogen, CA). A pair of primers: HPU-559S, GTCTCGAGAAAAGACAGGACGT CGTGGACCTGGAC-3' (SEQ ID NO:8) and HPL-209 (SEQ ID NO:7, described above) were used in PCR amplification under the conditions described above.

The resulting PCR product was digested with Xhol and BamHI and inserted into the Xhol-BamHI sites of the vector phpa2 Patent Application No. 08/922,170 and U.S.

Patent No. 5,968,822).

Thereafter, the Xhol-NotI fragment containing the hpa sequence was removed and cloned into an intermediate vector harboring the Sacl-NotI sites of pPIC9K.

The hpa was removed from the later vector as a Sacl-NotI fragment and cloned into the Sacl-NotI sites of pPIC9K, thus creating the vector pPIC9K-PP2 (Figure 3).

Transformation and screening: Pichia pastoris strain SMD1168 (his3, pep4) (Invitrogen, CA) was used as a host for transformation. Transformation and selection were carried out as described in the Pichia expression Kit protocol (Invitrogen, CA). In all transformations the expression vectors were linearized with Sall prior to their introduction into the yeast cells.

Multiple copies integration clones were selected using G-418 (Boehringer Mannheim, Germany). Following transformation the top agar layer containing the yeast cells was removed and re-suspended in 10ml of sterile water. Aliquots were removed and plated on YPD plates yeast extract, 2% peptone, 2% glucose) containing increasing concentrations of G-418 (up to 4mg/ml). Single isolates were picked and streaked on YPD plates. G-418 resistance was then further confirmed by streaking isolates on YPD-G-418 plates.

Expression experiments: Single colonies were inoculated into 6ml BMGY Buffered Glycerol-complex Medium yeast extract, 2% peptone, 100mM potassium phosphate pH 1.34% yeast nitrogen base with ammonium sulfate without amino acids, 4x1 0 5 biotin and 1% glycerol) and incubated at 30 0 C at 250 RPM for 24 hours. Cells were harvested using clinical centrifuge and re-suspended in 2.5ml of BMMY Buffered Methanol-complex Medium (The same as BMGY except that 0.5% methanol replaces the 1% glycerol). Cells were then incubated at 30°C at 250 RPM agitation for 48 hour. Culture supernatants were separated on SDS-PAGE and electrophoretically transferred to a nitrocellulose membrane using the Hoeffer-Pharmacia apparatus, according to manufacturer protocol. A rabbit anti-heparanase polyclonal antibody (disclosed in U.S. Patent Application No. 09/071,739 and in U.S. Patent 47 No. 6,177,545) was used as a primary antibody in detection of heparanase. Horseradish peroxidase-labeled anti-rabbit antibodies and ECL Western blotting detection reagents (Amersham, UK) were used in subsequent detection steps.

Experimental Results Both pPIC3.5K-Sheparanase and pPIC9K-PP2 Pichia pastoris transformants secreted a protein with a similar molecular weight of about 70 kDa, as expected for heparanase. These results indicate that the heparanase contains a signal sequence which efficiently functions as a secretion signal in Pichiapastoris.

G-418 resistance was used to select isolates characterized by multiple gene integration events. A faint heparanase band was observed in the supernatant of Sheparanase transformant isolated without selection on G-418 (Figure 4, lane whereas no band was observed in the corresponding position in pPIC3.5K transformant, which served as negative control (Figure 4, lane A profound increase in the secretion of heparanase was observed in isolates resistance to 4mg/ml of G-418 (Figure 4, lanes 3-6).

EXAMPLE Expression and secretion of recombinant human heparanase in mammalian cells Experimental Methods Construction of hpa DNA expression vectors: A hpa gene fragment was cloned under the control of either SV40 early promoter (pShpa, Figure 20a) or the CMV promoter (pChpa, Figure 20e). One construct (pShpaCdhfr, Figure 20b) also includes a selection marker, the mouse dhfr gene.

Specifically, a 1740 bp hpa gene fragment encoding for a 543 amino acid protein was introduced into pSI (Promega, USA) or pSI-Cdhfr vectors to yield vectors pShpa and pShpaCdhfr, respectively (Figures 5a and 5b, 20a and 20b). In both cases the gene was inserted under the SV40 early promoter regulation. pShpaCdhfr also carries an expression unit of mouse dhfr gene under the regulation of CMV promoter. Another plasmid, pCdhfr (Figure included expression unit of mouse dhfr gene under the regulation of CMV promoter and served as control.

A vector designated pSlhpa (Figure 5c, 20c) was constructed by ligating a truncated hpa gene fragment (nucleotides 169-1721 of SEQ ID NO:1) to a heterologous signal peptide as follows. Preprotrypsin (PPT) signal peptide (72) was generated by chemically synthesizing two complementary oligonucleotides corresponding to the signal peptide encoding DNA sequence, the first having a sequence AATTCACCATGTCTGCACTTCTGATCCTAGCTCTTGTTGG

AGCTGC

AGTTGCTCAGGAC-3' (SEQ ID NO:9), whereas the second having a complementary sequence GAGCTAGGATCAGAAGTGCAGACATGGTG-3' (SEQ ID NO:10). Annealing of the complementary oligonucleotides produced the double strand sequence encoding to the PPT signal peptide flanked by a sticky end of an EcoRI restriction site on the 5' end thereof and a sticky end of an AatlI restriction site on the 3' end thereof. Following restriction by EcoRI and AatII of the pfasthpa vector, a 145 bp fragment was removed, and replaced by the 52 bp PPT DNA sequence to yield plasmid pSihpa. The insert thereof was cut out with EcoRI and DotI and ligated into the vector pSI.

A vector designated pS2hpa (Figure 5d and 20d) was constructed by ligating a truncated hpa gene fragment (nucleotides 144-1721) to the PPT signal peptide as follows.

Preprotrypsin (PPT) signal peptide (72) was generated by chemically synthesizing two complementary oligonucleotides corresponding to the signal peptide encoding DNA sequence, the first having a sequence AATTCACCATGTCTGCACTTCTGATCCTAGCTCTTGTTGG AGCTGAGTTGC-3' (SEQ ID NO:11), whereas the second having a complementary sequence CGGCAACTGCAGCTCCAACAAGAGCTAG GATCAGAAGTGCAGACATGGTG-3' (SEQ ID NO:12). Annealing of the complementary oligonucleotides produced the double strand sequence encoding to the PPT signal peptide flanked by a stick end of an EcoRI restriction site on the 5' end thereof and a sticky end of a Narl restriction site on the 3' end thereof.

Following restriction by EcoRI and NarI of pSlhpa plasmid, a 112 bp fragment was removed therefrom and replaced by the PPT DNA sequence to give plasmid pS2hpa (Figure Transfection of vectors into cells: DNA constructs were introduced into animal cells using the calcium-phosphate co-precipitation technique essentially as described in (73).

Selection for dhfr expressing stable cellular clones: Following transfection, cells were incubated for 48 hours in a non-selective growth medium (F12 medium supplemented with 10% fetal calf serum). Then, the medium was changed to a selection medium (DMEM supplemented with 10% dialyzed calf serum) and cells were propagated to confluence at 37 0

C,

under 8% CO 2 aeration. Methotrexate (MTX, 5000nM) was added to the growth selection 49 medium and resistant cellular clones were isolated. Alternatively, cells were transferred after transfection directly to a selection medium containing MTX (100 100OnM).

SDS polyacrylamide gel electrophoresis and Western blot analysis: Denatured and reduced samples were loaded on ready made gradient gels (Novex, USA) and separated under standard gel running conditions (as described in Protein Electrophoresis Application Guide, Hoeffer, Transfer of proteins onto a PVDF membrane was performed electrophoretically by a protein transfer apparatus (Hoeffer- Pharmacia). Detection of specific protein was accomplished by a rabbit anti-heparanase polyclonal antibody (disclosed in U.S. Patent Application No. 09/071,739 and in U.S. Patent No. 6,177,545) (x2000 dilution), followed by ECL detection (Amersham, UK).

Determination of heparanase activity: ECM-derived soluble HSPG assay was performed by incubating cell extracts with solubillized 35S-labeled ECM (18 hours, 37 0 C) in the presence of 20mM phosphate buffer (pH and size fractionation of the hydrolyzed fraction of the ECM by gel filtration on a Sepharose CL-6B column. Radio labeling of degradation fragments eluted at 0.5 Kav 0.8 (peak II) was determined (61).

Alternatively, degradation of soluble high molecular weight heparan sulfate or heparin molecules to smaller fragments was detected by polyacrylamide gel electrophoresis analysis. Polyacrylamide gels were loaded with 2.5mg heparin that was either untreated or incubated with heparanase containing cell extracts or media. Staining by methylene blue (74) enabled detection of the heparin molecules and its degradation products. The mobility of the molecules on the gel reflects their size. Therefore, activity of heparanase is reflected in a larger quantity of rapidly migrating heparin degradation products.

Induction of secretion: CHO stable clones and untransfected CHO cells were induced for secretion of proteins by either calcium ionophore calcimycin (A23187) (Sigma) or phorbol 12-myristate 13-acetate (PMA, Sigma), at different concentrations (0.01, 0.1 and for various incubation times 8, 24, 48 hours). Induction was performed in the absence of serum. Conditioned medium was collected with 10% buffer citrate pH 5.6 and 200KIU/ml aprotinin (Protosol, Rad Chemicals, Israel), centrifuged to remove floating cells, and kept at -200 0 C. The amount of secreted protein(s) was detected by Western blot analysis, and its activity was determined by 35 S-ECM degradation assay and soluble heparan sulfate substrate hydrolysis assay. When necessary conditioned medium was concentrated by ultrafiltration through a 10 kDa filter (Millipore).

Large scale propagation of animal cells in a Spinner-Basket bioreactor: The structure and mode of operation of the bioreactor is described in detail in reference 75. A Spinner Basket bioreactor (500ml, New Brunswick Scientific) embedded with 10 grams of Fibracel discs (Sterillin, was inoculated with seeding inoculum of 1.5x 108 cells of a stable clone of CHO cells designated GGG11 that constitutively produces recombinant heparanase. Propagation of cells was performed in a medium containing 10% serum and cell proliferation was monitored by measurement of glucose consumption.

Then growth medium was replaced with medium without serum, suitable for the production of the recombinant protein. This medium served as a source for recombinant heparanase for later purification.

Experimental Results Expression of hpa DNA in animal cells: Expression of recombinant hpa gene products was detected in a human kidney fibroblasts cell line (293), baby hamster kidney cells (BHK21) and Chinese hamster ovary (CHO dhfr-) cells, following transfection with the hpa gene (Figures 6a-b).

Analysis of recombinant heparanase by Western blotting revealed two distinct specific protein products: a large protein of about 70 kDa and a predominant protein of about 52 kDa (Figures 6a-b).

Transient expression of heparanase proteins was monitored 24-72 hours post transfection in various cell types.

Human fibroblasts (293 cell line) transfected with pShpa (Figure 5a) or pChpa constructs (Figure 5e) exhibited heparanase activity (Figure 6a, lane 4, Table 1 below).

Transfection of CHO cells with the expression vector pShpaCdhfr (Figure 5b) and subsequent selection for MTX resistant clones resulted in the isolation of numerous clones.

These cellular clones express hpa gene products in a constitutive and stable manner (Figure 6a, lanes 1-3).

Several CHO cellular clones have been particularly productive in expressing hpa proteins, as determined by protein blot analysis and by activity assays (Figures 6a, Figure 6b, lane 1, and Table Although the hpa DNA encodes for a large 543 amino acids protein (expected molecular weight about 70 kDa) the results clearly demonstrate the existence of two proteins, one of about 70 kDa and another of about 52 kDa. These observations are similar to the results of the transient hpa gene expression in human 293 cells (Figure 6a, lane 4).

51 Transient expression of pShpaCdhfr in CHO cells revealed predominantly a 52 kDa heparanase protein (Figure 6b, lane 2).

It has been previously shown that a 52 kDa protein with heparanase activity was isolated from placenta (61) and from platelets, It is thus likely that the 70 kDa protein is naturally processed in the host cell to yield the 52 kDa protein.

Heparanase secretion into the growth medium: For large scale production and purification purposes, secretion of the recombinant protein into the growth medium is highly desirable. Therefore, expression vectors were constructed (pSlhpa and pS2hpa, Figures that would drive translation of heparanase attached to the PPT signal peptide.

Both pSIhpa and pS2hpa plasmids directed the expression of protein product with heparanase activity in human 293 or CHO cells (Table The. heparanase was not secreted to the medium in CHO cells. However, transient expression of heparanase encoded by pSihpa and pS2hpa in human 293 cells resulted in the appearance of a single size (about 65 kDa) heparanase protein (Figure 7c, lanes 3-6).

Table 1: Determination of Heparanase activity in transfected animal cells Heparanase Cell type Transfected DNA Activity Human 293 cells PChpa Human 293 cells PShpa Human 293 cells PSIhpa Human 293 cells PS2hpa CHO pShpaCdhfr Cell extracts were assayed for heparanase activity using ECM-derived soluble HSPG assay or direct hydrolysis of soluble substrate Activity detected either in transiently expressing cells (293, CHO) or stable cellular clones (CHO).

In order to induce secretion of the recombinant protein(s) into the medium, stable clones and untransfected CHO cells were induced with either calcium ionophore or PMA. The results show that induction with 1mg/ml calcium ionophore for 2 hours stimulates the secretion of protein of about 52 kDa from stable clones but. not from untransfected cells (data not shown) or untreated stable clones, while longer (24-48 hours) incubation time with 100ng/ml of calcium ionophore induces predominantly the secretion of protein of about 70 kDa from stable clones (Figures 7a-b). The conditioned medium obtained from the treated stable clone, which exhibited the 52 kDa protein, had strong heparanase activity in ECM-derived soluble HSPG assay (Figures 8b-c), and in concentrated conditioned medium, in the gel shift assay (Figure 8a). The heparanase activity in the conditioned medium from the treated stable clone, which exhibited the 70 kDa, is lower than that of the 52 kDa fraction (Figures 8d-g), since it was active when diluted eight fold while the 70 kDa protein failed to show activity in this dilution. It is thus possible that the 52 kDa protein is the active form of a less active pre heparanase of 70 kDa, which is naturally processed to yield the mature-active 52 kDa heparanase.

Large scale production of heparanase: Large scale propagation of heparanase expressing cells was set up in a 500ml volume Spinner-Basket bioreactor to demonstrate the ability to obtain a dense adherent cell culture for large scale production of heparanase.

Heparanase constitutively producing cell line was propagated in the Spinner-Basket bioreactor and at the end of the proliferation phase the medium was replaced with production medium which has the same composition as the growth medium but without serum. Cell proliferation and viability were constantly monitored by daily measurements of glucose concentration in the medium. Level of glucose was also the parameter used to determine the frequency of medium refreshment in the bioreactor, as described in reference 76. Results of a typical "batch run" that includes proliferation and maintenance of heparanase producing cells in a 500 ml Spinner- Basket are shown in Figure 9.

A "batch run" in a Spinner-Basket bioreactor can last about four weeks, when serum is omitted from the culture medium. The apparatus can be linearly enlarged to bioreactors of 7 or 35 liters. Accordingly, larger amounts of Fibraccl can be packed in those vessels and accommodate, proportionally, larger numbers of cells. The bioreactors can support cell growth for weeks, or even months, depending on the nature of the cell line and the composition of medium.

EXAMPLE 6 Expression of recombinant heparanase in virus infected insect cells: Experimental Methods 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. (see U.S.

Patent Application No. 08/922,180) was digested with Sail and Nod and ligated with a 1.7 kb fragment of phpa2 digested with XhoI and NotI. 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 baemid DNAs were transfected into Sf21 insect cells. Five days after transfection recombinant viruses were harvested and used to infect High five insect cells, 3 x 106 cells in T-25 flasks. Cells were harvested 2-3 days after infection. 4 x 106 Cells were centrifuged and resuspended in a reaction buffer containing phosphate citrate buffer, 50mM NaCl. Cells underwent three cycles of freeze and thaw and lysates were stored at -80'C. Conditioned medium was stored at 4 0

C.

Experimental Results Degradation of soluble ECM-derived HSPG:- Monolayer cultures of High ive cells were infected (72 h, 28CC) with recombinant bacoluvir-us 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 0 C) with sulfate labeled, ECM-derived I-SPG (peak followed by gel filtration analysis (Sepharose 6B3) of the reaction mixture.

As shown in Figure 10, the substrate alone included almost entirely high molecular weight (Mr) material eluted next to Vo (peak 1, fractions 5-20, Kay 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 It, fractions 22-35, 0.5 Kay <c 0.75).

Fragments eluted in peak I1 were shown to be degradation products of heparan sulfate, as they were 5- to 6-fold smaller than intact heparan sulfate side chains (Kay approx. 0.33) released from ECM by treatment with either alkaline borohydride or papain; and (11) resistant to further digestion with papain or chondroitinase ABC, and susceptible to deamination by nitrous acid. Similar results (not shown) were obtained with Sf21 cells. Again, heparanase activity was detected in cells infected with the hpa containing virus (p~hpa), 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 1 a-b, heparanase activity, reflected by the conversion of the high Mr peak I substrate into the low Mr peak II which represents HS degradation fragments, was found in the growth 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 growth medium of control non-infected High five or Sf21 cells.

The medium of cells infected with the pFhpa4 virus was passed through a 50 kDa cut off membrane to obtain a crude estimation of the molecular weight of the recombinant heparanase enzyme. As demonstrated in Figure 12, all the 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 competition effect of heparin, additional substrate of heparanase was examined.

As demonstrated in Figures 13a-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 in an active form by insect cells infected with Baculovirus containing the newly identified human hpa gene.

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°C) 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 48h at 28°C or 24h at 37 0 C. Sulfate labeled material released into the incubation medium was analyzed by gel filtration on Sepharose 6B.

As shown in Figures 14a-b and 15a-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 Vo. 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 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 (Figure 17). On the other hand, incubation of the labeled ECM with cells infected with the pFhpa4 virus resulted in release of 60-70% of the ECM-associated radioactivity in the form of low Mr sulfate-labeled fragments (peak II, 0.5 <Kav< 0.75), regardless of whether the infected cells were incubated with the ECM at 28 0 C or 37 0 C. Control intact non-infected Sf21 or High five cells failed to degrade the ECM HS side chains.

In subsequent experiments, as demonstrated in Figures 16a-b, High five and Sf21 cells were infected (96h, 28 0 C) with pFhpa4 or control pF1 viruses and the growth 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 17, production of these fragments was abolished in the presence of heparin, due to its competitors nature. No heparanase activity was detected in the growth medium of control, noninfected 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 tumor cells or activated cells of the immune system.

Thus, insect cells of several origins (such as Sf21 from Spodoptera frugiperda and High five from Trichoplusia ni) may be infected productively with baculovirus. Insect cells are infected with recombinant baculovirus in which viral DNA sequences have been replaced with DNA sequences coding for a protein of interest. The protein of interest is expressed during the very late phase of infection. A major advantage of the baculovirus expression system is that it can be used for expressing large amounts of recombinant protein compared to other popular expression systems in eukaryotes expression in CHO cells). Another advantage of the system is that insect cells have most of the protein processing capabilities of higher eukaryotic cells. Thus, proteins produced in the recombinant baculovirus-infected cells can undergo coand post- translational processing yielding proteins which are similar to the natural protein.

Scaling up the process of culturing and infecting insect cells with baculovirus is required for the production of recombinant protein of choice, in milligram and up to gram quantities. These quantities may be required both for research and for commercial use. Scaling up the process involves a variety of fields, such as medium development, metabolic studies, protein purification and quantification.

56 Several problems are inherent to this system and effect the process of scaling up.

Upon infection, insect cells become increasingly fragile and sensitive to the physiochemical environment of the culture. One of the primary goals of the bioengineer is to oxygenate large scale, high-density culture sufficiently, at low shearing rates. Although oxygen uptake rates of insect cells are similar to mammalian cell lines, it was found that after infection oxygen uptake rates doubles. An optimization process, aimed for setting-up of bioreactor parameters is required, for supplying oxygen to the cells without damaging them.

The spinner Bellco, Cat. 1965-56001 was used for scaling up as described. This is a double-wall type spinner. Temperature was controlled by water circulated from a 12 liter water bath (Fried Electric, Model TEPi) equipped with a heater and a thermostat. The spinner was aerated with both air, using an aquarium pump (Rena 301) and oxygen. An oxygen cylinder (medical grade) was connected to the spinner through a two stage regulator set to a pressure of 2 psi. Both air and oxygen were connected to the spinner through a T-connector equipped with valves that enabled a control over the flow rates of air and of oxygen. A tubing for delivering air mixed with oxygen was connected to the sparger of the spinner through a 0.2j size filter.

The sparger used was of an open type, releasing air-oxygen mixture through an orifice of 3mm inner diameter. The stirring function was provided by a low-RPM magnetic stirrer (LH, type LH fermentation placed beneath the spinner.

High five and Sf21 cells were used alternatively for large scale production of heparanase. Cell culture was gradually built up to 1.2x1010 cells. Eight shake flasks of 500mlsize were used for culturing cells to 3x106 cells/ml. Cells were cultured with protein-free medium (Insect-Xpress, Bio Whittaker). 1.5 liters of the above culture was used for seeding a 6 liters-size spinner. At the time of seeding, culture was diluted to 3 liters with fresh medium. Air was sparged into the culture at 0.5 liters/min. Stirring rate was 50 RPM and temperature was set to 28 0 C. Two days after seeding, culture volume was doubled again, from 3 liters to 6 liters.

Cell density was adjusted at that time to lx 106 cells/ml. At that time pure oxygen was sparged at 1.5 liters/min in addition to the sparging of air (at 0.5 liters/minute).

Infection of the culture took place one day after doubling the culture volume from 3 liters to 6 liters, as described. Cells were counted and infected with the heparanase-coding recombinant virus pFhpa2 at a multiplicity of infection (MOI) of 0.1 or 1.0. The infected culture was maintained for approximately 72 hours under conditions set for 6 liters-size culture: Oxygen 1.5 liters/min, air 0.5 liters/min, temperature 28 0 C, agitation at 50 RPM.

Viability of cells in culture was tested every 4 hours, starting from 62 hours after virus infection and on. Viability of cells was determined by staining cells with Trypan Blue dye. The culture was harvested when viability reached 70-80%. Cells and cell debris were removed by centrifugation (IEC B-22M, Rotor Cat. 878, 20 min. at 4 0 C at 7,000 RPM).

Supernatants were filtered through 0.2 size cartridge (Millipore, Cat. KV0304HB3). Virus and small-size debris were removed with a 300 kDa-size cross-flow cartridge (Millipore, Cat.

CDUF006LM). Heparanase was concentrated from filtrate obtained from the 300 kDa-size cartridge with 10 kDa size cross-flow cartridge (Millipore, Cat. SK1P003W4). The final concentrated solution had a volume of between 0.5 liters and 1 liters. Heparanase was purified from the concentrated solution on HPLC. Table 2 below summarizes the results of two large scale heparanase production by insect cells experiments.

TABLE 2 Volume Harvest time Heparanase in Batch Cell MOI Cell viability of culture post infection harvest No. used used at harvest (hours) (mg/ml) 78 76 0.44 0 Sf21 0.1 75 76 0.16 1 Hi-5 0.1 EXAMPLE 7 Purification of recombinant heparanase Experimental Methods and Results Methods and Results: Baculovirus infected insect cells (1 or 5 liter of High five cell suspension) were harvested by centrifugation. The supernatant was passed through 0.2 micron filter (Millipore), then filtered through 300K cartridge (Millipore). The <300 kDa retentate (about 300 ml)-was washed by further filtration with 2 volumes of phosphate buffered saline (PBS). The <300 kDa filtrate was then concentrated by 10K cellulose cartridge (Millipore).

The >10 kDa retentate was diluted three fold with 10 mM phosphate buffer pH 6.8 to prepare for applying the crude enzyme preparation onto a Source-S column (Pharmacia).

The diluted >10 kDa retentate was subjected to a Source-S column (2.5 x 10 cm) pre equilibrated with 10 mM phosphate buffer pH 6.8, 50 mM NaCi. Most of the contaminating proteins did not bind to the column while heparanase bound tightly. Heparanase activity was eluted by a linear gradient of 0.05 M NaCl 1 M NaCl in phosphate buffer pH 6.8 and fractions of 5 ml were collected.

The fractions having the highest activity in degrading sulfate labeled ECM were combined. The 0.4 M NaCI fractions were about 90% pure and exhibited the highest activity (Figure 18, lane A rabbit anti-heparanase polyclonal antibody detected the purified enzyme in Western blot ECL analysis (Figure 19, lane 9).

These results demonstrate a powerful single step purification of recombinant heparanase from culture supernatants. Obviously, other purification methods, such as affinity purification using, for example, solid support bound heparanase substrates, heparanase inhibitors or anti-heparanase antibodies, size exclusion, hydrophobic interactions, etc. can be additionally employed.

EXAMPLE 8 Purification of heparanase and production of highly active heparanase species by proteolytic processing Experimental Methods Construction of hpa DNA expression vectors, transfection thereof into cells, selection for dhfr expressing stable cellular clones, induction of secretion and SDS polyacrylamide gel electrophoresis and Western blot analyses were all performed as described hereinabove under Example Heparanase activity using DMB assay: For each sample, 100 pl heparin sepharose suspension in 1 x buffer A containing 20 mM Phosphate citrate buffer pH 5.4, 1 mM CaCl 2 and 1 mM NaCl) were incubated in 0.5 ml eppendorf tube for 17 hours with a tested enzyme preparation. At the end of the incubation period, samples were centrifuged for 2 minutes at 1000 rpm and the supernatants were analyzed for sulfated polyanions (heparin) using the colorimetric dimethylmethylene blue assay as follows.

Supernatants (100 pl) were transferred to plastic cuvettes and diluted to 0.5 ml with PBS supplemented with 1% BSA. 1,9-Dimethylmethylene blue (32 mg dissolved in 5 ml ethanol and diluted to 1 liter with formate buffer) (0.5 ml) was added to each cuvette.

Absorbency at 530 nm was determined using a spectrophotometer (Cary 100, Varian). For each sample a control, to which the enzyme was added at the end of the incubation period, was

I

59 included. For further details, see U.S. Patent Application No. 09/113,168 and U.S. Patent No 6,190,875, which are incorporated by reference as if fully set forth herein.

Heparanase activity using the tetrazolium assay: Heparanase activity was determined in reactions containing buffer A and 50 jig heparan sulfate in a final volume of 100 gl. Reactions were performed in a 96 well microtiterplate at 37°C for 17 hours. Reactions were thereafter stopped by the addition of 100 gl tetrazolium blue reagent (0.1 tetrazolium blue in 0.1 M NaOH) to each well. Color was developed following incubation at 60 0 C for 40 minutes.

Color intensity was quantitatively determined at 580 rim using a microtiterplate reader (Dynatech). For each sample a control, to which the enzyme was added at the end of the incubation period, was included. A glucose standard curve of 1-15 tg glucose was included in each assay. Heparanase activity was calculated as AO.D. of the sample containing the substrate minus the O.D of the control sample. The result was converted to gg glucose equivalent. One unit is defined as tg glucose equivalent produced per minute. For further details, see U.S.

Patent Application No. 09/113,168 and U.S. Patent No. 6,190,875, which are incorporated by reference as if fully set forth herein.

Production of rabbit anti heparanase polyclonal antibodies: Rabbits were immunized in three two weeks intervals with 200 mg of purified human recombinant heparanase protein produced in baculovirus infected Sf21 insect cells (see Examples 6-7 above) emulsified with an equal volume of complete Freund's adjuvant. Ten days after the third immunization rabbits were bled and serum was examined for reactivity with recombinant heparanase. Four weeks after bleeding another boost was injected and 10 days later blood was collected.

Purification of heparanase from mammalian cell extract using ion exchange chromatography: 2TTI CHO cells (2 x 108 cells stably transfected with pShpaCdhfr, Figure 20b) were extracted in 2.5 ml of 10 mM phosphate citrate buffer, pH 5.4. The extract was centrifuged at 2,750 x g for 5 minutes and the supernatant was collected for heparanase enzyme purification using cation exchange chromatography as follows. An HPLC column (mono-S HR Pharmacia Biotech) was equilibrated with 20 mM sodium phosphate buffer, pH 6.8, and the supernatant was loaded thereon. Proteins were eluted from the column using a linear gradient of 0 to 1 M sodium chloride in 20 mM sodium phosphate buffer, pH 6.8. The gradient was carried out in 20 column volumes at a flow rate of one ml per minute. Elution of proteins was monitored at 214 nm (Figure 23a) and fractions of 1 ml each were collected. An aliquot from each fraction was analyzed for heparanase activity using the DMB assay and for immunoreactivity using a mouse anti-heparanase monoclonal antibody (see U.S. Patent Application No. 09/071,739 and U.S. Patent No. 6,177,545, which are incorporated herein by reference). Most of the heparanase was eluted in fractions 19-20.

Preparation of an affinity column with anti heparanase antibodies: An affinity column was prepared using the Immunopure Protein G IgG Orientation Kit (Pierce). To this end, 17 mg of the above described rabbit anti heparanase polyclonal antibody, purified on protein G sepharose, were bound to a column containing 2 ml Immunopure immobilized protein G. The antibody was cross linked to the protein G with DMP. Unreacted immediate groups were blocked and the column was equilibrated with 20 mM phosphate buffer, pH 6.8.

Purification of heparanase using the affinity column: 0.5 x 108 2TT1 CHO cells were suspended in 2.5 ml of 20 mM phosphate citrate buffer, pH 5.4. Cells were frozen in liquid nitrogen and subsequently thawed at 37 0 C. Freezing and thawing were repeated two more times. The extract was then centrifuged for 15 minutes at 4000 g and the resulting supernatant was loaded onto the affinity column and was incubated, to allow binding of the enzyme to the column, at 4 0 C for 17 hours under head-over-tail shaking. Thereafter, unbound proteins were washed until absorbency at 280 nm reached zero. Proteins were eluted from the column with 0.1 M glycine HCI buffer, pH 3.5. 900 jl fractions were collected into eppendorf tubes each containing 100 ul of 1 M phosphate buffer, pH 8. The presence of heparanase in the eluted fractions was determined by Western blotting following gradient 4-20% SDS-PAGE of 20 pl samples using anti-heparanase monoclonal antibody (see U.S. Patent Application No.

09/071,739 and U.S. Patent No. 6,177,545). Heparanase activity was determined in 30 gl samples using the above described DMB assay.

Construction of heparanase expression vectors with a unique protease cleavage sequence: Expression vectors for the production of a heparanase protein species carrying a unique proteolytic cleavage site were designed and constructed. Two independent sites, just upstream of amino acids 120 or 158 (SEQ ID NO:2), both are peaking on the hydropathy plot, as calculated by the Kyte-Doolittle method for calculating hydrophilicity, using the Wisconsin University GCG DNA analysis software (Figure 29a), were selected for insertion of either one of two protease recognition and cleavage sequences within the hpa cDNA sequence to yield two heparanase species designated herein as pre-p56' and pre-p52', which, following digestion with their respective protease, yield truncated proteins designated herein p52' and p56', respectively. A first sequence included 4 amino acids (Ile-Glu-Gly-Arg., SEQ ID NO:13) which constitute a factor Xa recognition and cleavage sequence. An alternative, second,

I

sequence included 5 amino acids (Asp-Asp-Asp-Asp-LystI, SEQ ID NO: 14) which constitute a enterokinase recognition and cleavage sequence. These sequences do not appear in the natural enzyme (SEQ ID NO:2).

To this end, the following PCR primers were constructed: 52-Xa CCATCGATAGAAGGACGAAAAAAGTTCAAGAACAGCA CCTAC-3' (SEQ ID 52x-Cla 5'-GGATCGATTGGTAGTGTTCTCGGAGTAG-3' (SEQ ID NO: 16); 56-Xa GGATCGATAGAAGGACGATCTCAAGTC AACCAGGATATT-3' (SEQ ID NO:17); 56x- Cla 5'-CCATCGATGCCCAG TAACTTCTCTCTTCAAAG-3' (SEQ ID NO:l18); hpl 967 AAGCAGCAACTTTGGC-3' (SEQ ID NO:19); hpu 685 AGGTGAGCCCAAGAT-3' (SEQ ID NO:20); 52-EK CGACAAGAAAAAGTTCAAGAACAGC ACCTAC-3' (SEQ ID NO:2 52e-Cla GGATCGATCTGGTAGTGTTCTCGGAGTAG-3' (SEQ ID NO:22); 56-EK GGATCGATGACGACGACAAGTCTCAAG TCAACCAGGATATTTG-3' (SEQ ID NO:23); and 56e-Cla 5'-CCATCGATTTGG GAGTAACTTCTCTCTTCAAAG-3'(SEQ ID NO:24).

The following constructs were prepared (Figure 29b): Construction of pre-p52'-Xa hpa in pFast: A first PCR reaction was performed with a pFasthpa2 template and with primers 52-Xa and hpl 967. The resulted 11 80 bp fragment was digested with ClaI and AflIl and a 220 bp fragment was isolated. A second PCR reaction was performed with a pFasthpa2 template and with primers 52x-Cla and hpu 685. The resulting 500 bp fragment was digested with Cial and AatII and a 370 bp fragment was isolated. The ClaI-AfiII 220 bp and the ClaI-AatII 370 bp fragments were ligated to a 5,900 AarII-AfiI fragment of the pFasthpa2 plasmid.

(ii) Construction of pre-p56-'Xa hpa in pFast: A first PCR reaction was performed with a pFasthpa2 template and with primers 56-Xa and hpl 967. The resulted 1290 bp fragment was digested with ClaI and AflII and a 340 bp fragment was isolated. A second PCR reaction was performed with a pFasthpa2 template and with primers 56x-Cla and hpu 685. The resulting 380 bp fragment was digested with ClaI and AatII and a 250 bp fragment was isolated. The ClaI-AflII 340 bp and the ClaI-AatII 250 bp fragments were ligated to a 5,900 AatII-AflhI fragment of the pfasthpa2 plasmid.

(iii) Construction of pre-p52'-Enterokinase hpa in pFast: A first PCR reaction was performed with a pFasthpa2 template and with primers 52-EK and hpl 967. The resulted 1180 bp fragment was digested with ClaI and Afil and a 220 bp fragment was isolated. A second PCR reaction was performed with a pFasthpa2 template and with primers 52e-Cla and hpu 62 685. The resulting 500 bp fragment was digested with Clal and AatlI and a 370 bp fragment was isolated. The Clal-AflII 220 bp and the Clal-AatlI 370 bp fragments were ligated to a 5,900 AatlI-AfllI fragment of the pFasthpa2 plasmid.

(iv) Construction of pre-p56'-Enterokinase hpa in pFast: A first PCR reaction was performed with a pFasthpa2 template and with primers 56-EK and hpl 967. The resulted 1290 bp fragment was digested with ClaI and AflIl and a 340 bp fragment was isolated. A second PCR reaction was performed with a pFasthpa2 template and with primers 56e-Cla and hpu 685. The resulting 380 bp fragment was digested with Clal and AatlI and a 250 bp fragment was isolated. The Clal-AflII 340 bp and the Clal-AatlI 250 bp fragments were ligated to a 5,900 AatlI-AflIl fragment of the pFasthpa2 plasmid.

Construction of plasmids for expression of heparanase with protease digestion sequence: Each one of the four constructs (i to iv) described hereinabove includes an AatlI- AflII fragment which includes a factor Xa or enterokinase recognition and cleavage sequence positioned at one of the described alternative sites, i.e. upstream amino acids 120 or 158 (SEQ ID NO:2). The hpa constructs described in Figures 5a-e and 20 a-e, as well as the pFasthpa constructs, each includes a single Aatll site and a single AflII site within the hpa cDNA sequence, thus enabling the insertion by replacement of the 220 or 340 AatlI-AflII fragments as desired.

Experimental Results Expression of hpa DNA in animal cells: As already shown and discussed under Example 5 above, in order to drive transient or stable expression of the hpa gene in animal cells, the hpa gene was cloned into expression vectors, where transcription is regulated by promoters of viral origin (SV40, CMV) to ensure efficient transcription (Figure 5a-e). All vectors were suitable for transient expression of hpa in animal cells, but only vectors that include an expression cassette for the mouse dhfr gene (Figures 5b and 20f, the latter serves as a negative control) could be subjected to selection by mrthotrexate (MTX). Selection enables the establishment of cell lines that constitutively produce high levels of recombinant heparanase.

Cell lines of different origins have been transfected and expressed human heparanase gene: Transient expression of recombinant heparanase was detected in a human kidney fibroblasts cell line 293 (Figure 6a), baby hamster kidney cells (BHK21; Figure 21 a) and Chinese hamster ovary cells (CHO; Figure 6b). Stable expression of heparanase in CHO cells is shown in Figures 6a-b.

Transfection of CHO cells with the expression vector pShpaCdhfr (Figure5 b) or cotransfection with pSlhpa and pCdhfr (Figure 5c and 20f), followed by selection for MTX resistant clones resulted in the isolation of numerous clones. These cellular clones express hpa gene products in a constitutive and stable manner (Figure 6a, lanes 1-3).

Analysis of expression of recombinant heparanase in mammalian cells revealed two distinct specific protein products: a large protein of about 70 kDa (which is referred to herein as p70) and a predominant protein of about 50 kDa, which is referred to herein as p5 2 (Figures 6a, 21a).

Although the hpa DNA encodes a large 543 amino acids protein (expected molecular weight about 61 kDa), the results clearly demonstrate the existence of two proteins. These observations are similar to the results of the transient hpa gene expression in human 293 cells (Figure 6a, lane 4).

BHK21 cells, transiently transfected with pSlhpa (Figure 5c) express predominantly the p52 form of recombinant heparanase (Figure 21a, lane 1 marked by an arrow). Stable CHO clones express predominantly the p52 protein (Figure 6b, lane 2).

The presence of both p70 and p52 heparanase was detected in all cells that expressed the hpa gene, although the relative concentrations of the proteins varied between different cell types.

Cells transfected with pSlhpa (Figure 5c) expressed p5 2 (Figure 21a) indicating that the replacement of the putative heparanase signal peptide by the PPT signal sequence did not affect the expression and processing of the protein.

All cell extracts exhibited high heparanase activity following the introduction of the hpa gene. Human 293 cells transfected with pShpa (Figure 5c) exhibited high heparanase activity (Figure 21 b).

It has been previously shown that a 52 kDa protein with heparanase activity was isolated from placenta (61) and platelets (62).

It is thus concluded that the p70 protein is a preheparanase that is naturally processed in the host cell to yield the p52 protein.

Heparanase secretion into the growth medium: For large scale production and purification purposes, secretion of the recombinant protein into the growth medium is highly desirable. Therefore, expression vectors were constructed (pSlhpa and pS2hpa, Figures to direct translation of heparanase attached to the PPT signal peptide, a secretion signal peptide.

64 Both p5 1hpa and pS2hpa plasmids directed the expression of protein product with heparanase activity in human 293 or CHO cells (Figures 7c, 22a-b). Transient expression of heparanase from pSl1hpo and pS2hpa resulted in the appearance of a single size (about 70 kDa) heparanase protein in the' medium (Figure 7c, lanes similar to the larger form of recombinant heparanase detected in the cells.

CHO cells, stably transfected with either pShpaCdhfr (2TT1 clones) or pSlhpa (S I PPT clones) were further subcloned to yield stable clones which maintain their genetic and cellular characteristics stability in the absence of MTX selection. To this end, the limiting dilution procedure was employed, in which cells were cloned under non-seletive conditions and clones exhibiting the above stability were selected for further analysis.

2TT1 and S1PPT clones under (clones 2TT1 and S1PPT-p) or after (clones 2TTl-2, 2TT1-8, S1PPT-4 and S1PPT-8) selection with high MTX yielded stable clones exhibiting moderate (clones 2TTl (Figure 22b), 2TTL-2, 2TT1-8) or high (clones S1PPT-p, S1PPT-4, S1PPT-8 (Figure 22a)) constitutive secretion of heparanase into the growth medium. The secreted protein was of about 70 kDa, similar to p70, the larger heparanase form found within the cells (Figures 22a-b). Only when a large amount of p70 protein are found in the medium, a residual amount of the smaller heparanase form, p52, could be detected (Figure 22a, lane 4).

In the conditioned medium containing heparanase, some heparanase activity could be detected, although not as high as the activity measured in the respective cell extracts which, as determined immunologically, have comparable heparanase concentrations. Some improvement in secretion could be detected by calcium ionophore treatment, but the effect was transient (Figure 22a, lane 4).

The parijfication of recombinant heparan use from 2TTJ CHO cells by ion exchange chromatography: Clone 2TT1-8 was used for large scale production of heparanase.

In this cell line, the p52 form of heparanase is predominantly expressed within the cells. The cells are grown adherent to the tissue culture flask surface and were harvested when the cell culture reaches confluency.

Purification of a non-abundant protein from cells is a challenging task, where only a carefully designed and accurately discriminating protocol enables purification. See U.S. patent No. 5,362,641 and references 61 and 62 describing the purification of heparanase from placenta and platelets.

Here, a cation exchange chromatography procedure was selected for purification based on successful use thereof in the purification of insect cells produced recombinant heparanase, as described in Example 7 hereinabove.

Separation of the total protein content of 2TT1-8 cell extract on a mono-S cation exchange column is shown in Figure 23a. The vast majority of cellular proteins were eluted from the column prior to the elution of heparanase (Figure 23b). It is important to note that the p52 and the p70 were co-eluted under these conditions. Furthermore, a tight correlation was found between the presence of heparanase, as detected immunologically (Figure 23b), and its activity, as measured by the DMB (Figure 23c) and the tetrazolium (Figure 23d) activity assays.

Thus, using the above described purification protocol, one obtains ample amounts of highly active and purified heparanase which is highly suitable for use in a high throughput screening assay for heparanase activity, e.g. in the presence of candidate heparanase inhibitors, for example, combinatorial inhibitor libraries. Further details relating to a heparanase high throughput assay are provided in U.S. Patent Application No. 09/113,168 and U.S. Patent No 6,190,875, which are incorporated herein by reference.

The purification of heparanase by an anti-heparanse affinity column: Partially purified, active recombinant heparanse produced in SF21 insect cells infected with a baculovirus containing the hpa cDNA, was used to immunize rabbits for the production of polyclonal antibodies against the native recombinant heparanase protein. This antibody was thereafter purified and was used to construct a heparanase affinity column.

Cellular extract of CHO 2TT1-8 cells was loaded on the column for affinity separation. Figure 24 a-b clearly show that heparanase was specifically and efficiently bound to the affinity column. Moreover, high salt elution of the bound heparanase from the column was efficient and the activity of the eluted heparanase (Figure 24b) was tightly correlated with the presence of the recombinant enzyme (Figure 24a). Thus, using an affinity column as herein described, one can obtain a highly purified and highly active recombinant or natural heparanase in single step purification, which can be used in pharmaceutical applications. Furthermore, combining the Mono-S and affinity columns into a two step purification procedure will ensure even better results in terms of both purification and yield.

In addition, the tetrazolium assay is based on the detection of free reducing sugar ends. As such. it requires heparanase preparations devoid of such reducing ends. Heparanase purified using the above described affinity column is devoid of such reducing ends, and is therefore highly applicable for the tetrazolium activity assay.

Proteolytic processing of heparanase by protease from insect cells: Production of human recombinant heparanase in insect cells (Sf21), via baculovirus infection, and the subsequent purification of that protein are described in U.S. Patent Application Nos.

08/922,170; 09/071,618; 09/109,386; in U.S. Patent Nos. 5,968,822; 6,426,209; 6,664,105 and in PCT/US98/17954, all of which are incorporated herein by reference.

Briefly, conditioned medium of Sf21 cells that were infected with recombinant baculovirus, secrete heparanase to the medium. This heparanase is a glycosylated protein with an apparent molecular weight of 70 kDa. The size of that protein is similar to the produced by mammalian cells, and it possesses limited heparanase activity. This heparanase protein is referred to herein as p70-bac heparanase.

Purification of p70-bac heparanase from insect cells conditioned medium involved sequential filtration steps and a cation exchange column (Source-S). Fractions that contain predominantly p70-bac heparanase protein are collected. This purification protocol and results are described hereinabove.

The effect of different pH values on the activity and intactness of p70-bac heparanase was examined in an attempt to establish a pH optimum for heparanase activity. It was found that exposure of p70-bac heparanase to pH 4.0 for one week at 4 0 C resulted in significant (seven fold) increase in activity (Figure 25b). This activation was protease dependent as is evident form the inhibition of activation caused by a protease inhibitors cocktail (Figure The fate of the p70-bac heparanase following exposure to acidic pH was uncovered by Western-blot analysis (Figure 25a). Following exposure to pH 4, p70-bac heparanase was converted into a lower molecular weight form of about 56 kDa, which is referred to herein as p56 (Figure 25a, lane Proteolysis was inhibited in the presence of protease inhibitors (Figure 25a, lane B).

This is the first record demonstrating in vitro proteolytic processing of recombinant heparanase, (ii) associated with a significant increase in heparanase activity.

To further characterize the protease(s) involved in processing and activation of bac heparanase, a collection of individual protease inhibitors was employed (Figures The inhibitors antipain, E-64, leupeptin and chemostatin were most effective in preventing the activation of p70-bac heparanase at low pH. The effect was due to inhibition of the proteolytic processing of the p70-bac heparanase as is evident from the Western blot analysis of Figure Antipain and leupeptin are known to inhibit serine and cysteine proteases, while E-64 inhibits only cysteine proteases. These results therefore indicate that a cysteine protease(s) present in the conditioned medium of insect cells are responsible for the activation of heparanase, by processing the enzyme into a lower and more active p56 molecular weight form.

N-terminal sequencing of gel separated and PVDF transferred p56 heparanase revealed the sequence Ser-Gln-Val-Asn-Gln (SEQ ID NO:25), which corresponds to a new heparanase species that starts at Ser 120 of the full length enzyme (SEQ ID NO:2).

Proteolytic processing of heparanase by trypsin and cathepsin L: The activation of p70-bac heparanase by protease(s) from insect cells conditioned medium could be reproduced by mild digestion with trypsin (Figures 26a-b). Trypsin, 1.5 to 500 units per 10ig heparanase, gradually activated the protein, reaching maximal activation of five-fold already at units' trypsin (Figure 26a). Activation of p70-bac heparanase correlated with the expected cleavage of a portion of the p70-bac heparanase into smaller heparanase species, of about 56 kDa (Figure 26b). Smaller fragments of heparanase were also obtained by trypsinization (Figure 26b, lanes 2-3).

Similarly, recombinant heparanase processing and activation occurred when mild trypsin digestion was employed on a crude conditioned medium of CHO cells that secrete mammalian p70 heparanase (Figure 27). Activation was dose dependent.

Processing and activation of recombinant CHO produced and secreted heparanase was also obtained by mild treatment with Cathepsin L, which is a known cysteine protease (Figures 28a-b). Processing by this protease resulted in several digestion products, of about 56, 34 and 21 kDa (Figure 28b, lane 2).

It is shown herein that proteolytic digestion of recombinant heparanase from a variety of sources and by a variety of proteases results in processing of the enzyme into a lower molecular weight species; and (ii) increased catalytic activity. Processing and activation of heparanase in a similar fashion is anticipated to take place in vivo as well and therefore in vivo inhibition of proteases can be used to indirectly inhibit heparanase processing and activation.

Design of expression vectors to express heparanase precursor species adapted for in vitro activation by proteases: The p52 heparanase protein (as characterized in CHO, 293 and BHK21 cells, placental and platelets heparanase) and the p56 heparanase protein (as characterized after processing of the p70-bac heparanase) are presently the forms of heparanase that exhibit the highest enzymatic activity. It is shown herein that these heparanase 68 species are the result of proteolytic cleavages of heparanase. As was determined by solid phase microsequencing the cleavage site of p70-bac heparanase is effected between amino acids 119 and 120 (SEQ ID NO:2, see above) within the first peak of hydrophilicity (Figure29 a, peak I).

The second peak of hydrophilicity (Figure 29a, peak II) is expected to contain the cleavage site yielding the p52 heparanase species. This is not surprising, considering the fact that these regions, are positioned at the surface of the heparanase molecule and are thus susceptible to proteolysis.

Figure 29c demonstrates the steps undertaken in constructing four basic nucleic acid constructs harboring a unique protease recognition and cleavage sequence of factor Xa Ile- Glu-Gly-Argj or of enterokinase Asp-Asp-Asp-Asp-Lysi downstream amino acids 119 or 157. AatlI-AflllI restriction fragments derived from these four basic constructs can be used to replace a corresponding region in any of the hpa constructs described herein (Figures 5a-e) and for that effect, any other construct harboring a hpa derived sequence. Figure 29b shows the modified heparanase species (pre-p56' and pre-p52') that contain these unique protease recognition and cleavage sequences (shaded regions) which enable proteolytic processing by the respective proteases to obtain homogeneously processed and highly active heparanase species (p56' and p52', respectively).

The above described constructs are highly suitable for expression of heparanase in any expression system which is characterized by secretion of the recombinant heparanase to the growth medium. Such a precursor enzyme can be readily and precisely processed into a mature active form of heparanase p56' or p52'.

EXAMPLE 9 Latent and active forms of the heparanase protein The apparent molecular size of the recombinant enzyme produced in the baculovirus expression system was about 65 kDa. This heparanase polypeptide contains 6 potential N-glycosylation sites. Following deglycosylation by treatment with peptide Nglycosidase, the protein appeared as a 57 kDa band. This molecular weight corresponds to the deduced molecular mass (61,192 daltons) of the 543 amino acid polypeptide encoded by the full length hpa cDNA after cleavage of the predicted 3 kDa signal peptide. No further reduction in the apparent size of the N-deglycosylated protein was observed following concurrent 0-glycosidase and neuraminidase treatment. Deglycosylation had no detectable effect on enzymatic activity.

Unlike the baculovirus enzyme, expression of the full length heparanase polypeptide in mammalian cells 293 kidney cells, CHO) yielded a major protein of about 0 50 kDa and a minor of about 65 kDa in cell lysates. Comparison of the enzymatic activity of the two forms, using a semi-quantitative gel filtration assay, revealed that the 50 kDa enzyme is at least 100-200 fold more active than the 65 kDa form. A similar difference was observed when the specific activity of the recombinant 65 kDa baculovirus enzyme was compared to that of the 50 kDa heparanase preparations purified from human platelets, SK-hep-1 cells, or placenta. These results suggest that the 50 kDa protein is a mature processed form of a latent heparanase precursor. Amino termninal sequencing of the platelet heparanase indicated that cleavage occurs between amino acids Gln157 and Lys 158. As indicated by the hydropathic plot of heparanase, this site is located within a hydrophillic peak, which is likely to be exposed and hence accessible to proteases.

According to Fairbank et al. (57) the precursor is cleaved at three sites to form a heterodimer of a 50 kDa polypeptide (the mature form) that is associated with a 8 kDa peptide.

Although mammalian heparanase can be expressed in vitro in a variety of cell lines of human and non-human origin, there are significant drawbacks to the use of mammalian tissue culture systems for the production of human heparanase in clinically useful quantities such as the expense of growth media, potential contamination with host cell proteins and the limited production capacity of mammalian tissue culture systems.

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 broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification. are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is part of the common general knowledge or available as prior art to the present invention.

The term "comprise" and variants of the term such as "comprises" or "comprising" are used herein to denote the inclusion of a stated integer or stated integers but not to exclude any other integer or any other integers, unless in the context or usage an exclusive interpretation of the term is required.

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Claims (56)

1. An isolated polynucleotide comprising a polynucleotide sequence encoding a polypeptide cleavable to obtain heparanase catalytic activity.

2. The polynucleotide of claim 1, wherein said polynucleotide sequence includes at least a portion of SEQ ID NOs: 1 or 28.

3. The polynucleotide of claim 1, wherein said polynucleotide sequence includes nucleotides 63-1691 of SEQ ID NO: 1 or nucleotides 139-1869 of SEQ ID NO: 28.

4. The polynucleotide of claim 3, wherein said polynucleotide sequence includes nucleotides 63-721 of SEQ ID NO: 1.

5. The polynucleotide of claim 1, wherein said polynucleotide sequence is as set forth in SEQ ID NO: 28.

6. The polynucleotide of claim 1, wherein said polynucleotide sequence includes a portion of SEQ ID NOs: 1 or 28, said portion encoding said polypeptide cleavable to obtain heparanase catalytic activity.

7. The polynucleotide of claim 1, wherein said polypeptide includes an amino acid sequence as set forth in SEQ ID NOs: 2 or 29.

8. The polynucleotide of claim,l, wherein said polypeptide includes a portion of SEQ ID NOs: 2 or 29.

9. The polynucleotide of any one of claims 1-8, wherein said polynucleotide sequence shares at least 60% homology with SEQ ID NOs: 1 or 28. The polynucleotide of any one of claims 1-9, wherein said polynucleotide sequence shares at least 70% homology with SEQ ID NOs: 1 or 28. 1

11. The polynucleotide of any one of claims 1-10, wherein said polynucleotide sequence shares at least 80% homology with SEQ ID NOs: 1 or 28. 0

12. The polynucleotide of any one of claims 1-11, wherein said polynucleotide sequence shares at least 90% homology with SEQ ID NOs: 1 or 28.

13. The polynucleotide of any one of claims 1-12, wherein said polynucleotide sequence is selected from the group consisting of double stranded DNA, single stranded DNA and RNA. 0 14. The polynucleotide of any one of claims 1-13, wherein said polynucleotide sequence comprises at least a first polynucleotide sequence encoding an N-terminal portion of a precursor heparanase polypeptide, a second, in frame, polynucleotide sequence encoding a protease recognition and cleavage sequence and a third, in frame, polynucleotide sequence encoding a C-terminal portion of said precursor heparanase polypeptide, wherein said protease recognition and cleavage sequence enables cleavage of said precursor heparanase polypeptide by said protease. The polynucleotide of any one of claims 2-13, wherein said polynucleotide sequence comprises at least a first polynucleotide sequence encoding an N-terminal portion of a '0 precursor heparanase polypeptide, a second, in frame, polynucleotide sequence encoding a protease recognition and cleavage sequence and a third, in frame, polynucleotide sequence encoding a C-terminal portion of said precursor heparanase polypeptide, wherein said protease recognition and cleavage sequence enables cleavage of said precursor heparanase polypeptide by said protease.

16. The polynucleotide of claim 15, wherein said polynucleotide sequence comprises a nucleic acid construct.

17. The polynucleotide of claim 15 or claim 16, wherein said protease does not have a recognition and cleavage site in said N- and C- terminal portions. IND -r

18. The polynucleotide of claim 14, wherein said N- and C- terminal portions of said polynucleotide sequence encoding said precursor heparanase polypeptide do not contain a 0 protease recognition and cleavage site.

19. The polynucleotide of any one of claims 14-1 8, wherein said polynucleotide sequence encodes a catalytically active heparanase after cleavage by said protease. The polynucleotide of any one of claims 14-19, wherein said first and said third polynucleotide sequences each correspond to at least a portion of SEQ ID NO: 1. 810

21. A polypeptide cleavable to obtain a catalytically active heparanase, encoded by the polynucleotide sequence of claim 1.

22. The polypeptide of claim 21, wherein said polypeptide shares at least 60% homology with SEQ ID NOs: 2 or 29.

23. The polypeptide of claim 21 or claim 22, wherein said polypeptide shares at least homology with SEQ ID NOs: 2 or 29. !0 24. The polypeptide of any one of claims 21-23, wherein said polypeptide shares at least homology with SEQ ID NOs: 2 or 29. The polypeptide of any one of claims 21-24, wherein said polypeptide shares at least homology with SEQ ID NOs: 2 or 29.

26. The polypeptide of any one of claims 2 1-25, wherein said polypeptide is cleavable by a protease.

27. The polypeptide of claim 26, wherein said cleavage by said protease results in at least a first polypeptide fragment and a second polypeptide fragment, such that each of said first and second polypeptide fragments comprise a subunit of a catalytically active heparanase. IND 7-)

28. The polypeptide of claim 27, wherein said first polypeptide fragment mass is in the range of about 45 55 kDa.

29. The polypeptide of claim 27, wherein said second polypeptide fragment mass is in the range of about 5-10 kDa. The polypeptide of claim 26, wherein said protease is selected from the group consisting of a cysteine protease, an aspartyl protease, a serine protease and a metal loproteinase. 810

31. The polypeptide of claim 30, wherein cleavage of said polypeptide by said protease is performed at a pH wherein said protease is active.

32. The polypeptide claim 3 1, wherein said protease is most active at said pH.

33. A polypeptide comprising a precursor protein for the polypeptide of claim 2 1, further comprising an upstream portion of a heparanase polypeptide, a mid portion of a recognition and cleavage sequence of a protease and a downstream portion of a heparanase polypeptide, wherein said protease is selected having no recognition and cleavage sequences in said Z0 upstream and said downstream portions of said heparanase.

34. A heparanase polypeptide cleaved from the precursor protein of claim 33, wherein said heparanase polypeptide comprises a catalytically active polypeptide.

35. A genetically modified cell, comprising the polynucleotide of claim

36. The genetically modified cell of claim 35, wherein said polynucleotide is stably integrated in the genome of the cell.

37. The genetically modified cell of claim 35, wherein said polynucleotide is external to the genome of the cell. O

38. The genetically modified cell of any one of claims 35-37, wherein said cell is a bacterial cell.

39. The genetically modified cell of claim 38, wherein said cell is E. coli. The genetically modified cell of any one of claims 35-37, wherein said cell is an animal cell.

41. The genetically modified cell of claim 40, wherein said cell is an insect cell.

42. The genetically modified cell of claim 41, wherein said insect cell is selected from the group consisting of High five and Sf21 cells.

43. The genetically modified cell of claim 40, wherein said cell is a mammalian cell.

44. The genetically modified cell of claim 43, wherein said mammalian cell is selected from the group consisting of CHO cells, BHK21 cells, Namalwa cells, Dauidi cells, Raji cells, Human 293 cells, Hela cells, Ehrlich's ascites cells, Sk-HepI cells, MDCK-1 cells, MDBK-1 cells, Vero cells, Cos cells, CV-1 cells, NIH3T cells, L929 cells and BLG cells. cell. The genetically modified cell of any one of claims 35-37, wherein said cell is a yeast

46. yeast. The genetically modified cell of claim 45, wherein said yeast cell is a methylotrophic

47. The genetically modified cell of claim 46, wherein said methylotrophic yeast is selected from the group consisting of Pichia pastoris, Hansenula polymorpha and Saccharomyces cerevisiae.

48. A method of activating the polypeptide of claim 21 wherein said polypeptide is a non- catalytically active precursor heparanase polypeptide, the method comprising digesting said non-active precursor heparanase polypeptide with a protease, thereby activating said non-active precursor heparanase polypeptide.

49. The method of claim 48, wherein said protease is selected from a group consisting of a cysteine protease, an aspartyl protease, a serine protease and a metalloproteinase. The method of claim 48 or 49, wherein said digesting is effected at a pH at which said protease is active.

51. pH. The method of any one of claims 48-50, wherein said protease is most active at said

52. The method of claim 48, wherein said non-active precursor heparanase polypeptide comprises a natural precursor heparanase polypeptide.

53. The method of claim 48, wherein said active heparanase polypeptide comprises a natural heparanase polypeptide.

54. The method of claim 48, wherein said non-active precursor heparanase polypeptide comprises a recombinant precursor heparanase polypeptide. The method of claim 48, wherein said active heparanase polypeptide comprises a recombinant heparanase polypeptide.

56. The method of claim 48, wherein said polypeptide comprises a purified heparanase polypeptide.

57. The method of claim 48, wherein said polypeptide comprises a non-purified heparanase polypeptide.

58. The method of claim 48, wherein said polypeptide comprises a partially purified heparanase polypeptide.

59. The method of claim 48, wherein said digestion is performed in vivo. 0 60. The method of claim 48, wherein said digestion is performed in vitro.

61. A method of activating a heparanase polypeptide, the method comprising digesting the heparanase polypeptide with a protease capable of cleaving said heparanase polypeptide at a region containing its most hydrophilic sites within the first 170 N-terminal amino acids of said heparanase protein, as determined using the Kyte-Doolittle method for calculating hydrophilicity, using the Wisconsin University GCG DNA analysis software, so as to release a catalytically active portion of said heparanase.

62. A method of activating a recombinant heparanase polypeptide, wherein the modified heparanase polypeptide comprises at least one introduced protease cleavage recognition sequence at a region containing the most hydrophilic sites within the first 170 N-terminal amino acids of a natural heparanase polypeptide as determined using the Kyte-Doolittle analysis software, the method comprising digesting the modified heparanase polypeptide with a matching protease being capable of cleaving said introduced protease cleavage recognition sequence. !0 63. A method of inhibiting cleavage of a heparanase polypeptide, the method comprising administering a protease inhibitor to a subject in need thereof.

64. The method of claim 63, wherein said administering said protease inhibitor is performed in vivo. The method of claim 63 or claim 64, wherein said protease inhibitor is selected from the group consisting of a cysteine protease inhibitor, an aspartyl protease inhibitor, a serine protease inhibitor and a metalloproteinase inhibitor.

66. The method of claim 63, wherein said inhibiting cleavage of heparanase is used for medical treatment of said subject. IO 99

67. The method of claim 66, wherein said medical treatment of said subject comprises treatment of a heparanase related condition.

68. The method of claim 66, wherein said medical treatment comprises treatment of cancer.

69. The method of claim 68, wherein said cancer comprises a metastatic cancer. O The method of claim 63, wherein said need of said subject is related to a heparanase 0 10 related condition.

71. The method of claim 63, wherein said need of said subject comprises said subject being affected with cancer. DATED: 17 November 2006