AU2004201462C9 - Antibodies Recognising At Least One Epitope of Heparanase - Google Patents

Description

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AUSTRALIA

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

Level 26 239 George Street Brisbane Qld 4000 Invention Title: Antibodies Recognising At Least One Epitope of Heparanase 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: ANTIBODIES RECOGNIZING AT LEAST ONE EPITOPE OF HEPARANASE FIELD AND BACKGROUND OF THE INVENTION The present invention relates to an anti-heparanase antibody.

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 post-translation 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. e.g. the selection marker as well as the gene of interest (58).

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 non-active 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 (59).

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 ainterferon, 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 nonactive 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 Saceharomyces 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

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grams per liter culture. These advantages make Pichia pastoris suitable for an efficient production of recombinant proteins 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 Dglucosamine disaccharide units that are substituted to a varying extent with Nand 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 selfassembly 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.

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Heparanase activity has also been described in activated immune system cells and highly metastatic cancer cells but research has been handicapped by the lack of biologic tools to explore potential causative roles of heparanase in disease conditions.

Heparanase: Heparanase is a glycosylated enzyme that is involved in the catabolism of certain glycosaminoglycans. It is an endo-p3-glucuronidase that cleaves heparan sulfate at specific intrachain sites (9-10,18-19). Interaction of T- and Blymphocytes, platelets, granulocytes, macrophages and mast cells with the subendothelial extracellular matrix (ECM) is associated 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 microenvironvent (11).

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 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 (12).

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 12-myristate 13-acetate (PMA) treatment (13,14).

In contrast, various tumor cells appear to express and secrete heparanase in a constitutive manner in correlation with their metastatic potential 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 (15,27).

Purification of natural heparanase: Heparanase activity has been described in a number of cell types including cultured skin fibroblasts, human neutrophils, activated rat Tlymphocytes, 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++-cheIating 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 cDNA 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 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 cDNA 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 continuation-in-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 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 pF1 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 ofHeparanase 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 (16,17). 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 an endo-p-D-glucuronidase (heparanase) that cleaves HS at specific intrachain sites Expression of a HS degrading heparanase was found to correlate with the metastatic potential of mouse lymphoma fibrosarcoma and melanoma cells. The same is true for human breast, bladder and prostate carcinoma cells Patent Application No. 09/071,739), and primary and metastatic pancreatic duct adenocarcinoma (Koliopanos et al Cancer Res 2001;61:4655-59). Moreover, elevated levels of heparanase were detected in sera and urine Patent Application No. 09/071,739) of metastatic tumor bearing animals and melanoma patients and in tumor biopsies of cancer patients 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 (68,69). 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 0.5x10 6 labeled degradation fragments of HS side chains are eluted more toward the Vt of the column (0.5<kav<0.8, Mr 7x10 3 (18).

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 O positions. While O-desulfation abolished the heparanase inhibiting effect of heparin, O-sulfated, N-acetylated heparin retained a high inhibitory activity, provided that the N-substituted molecules had a molecular size of about 4,000 daltons or more Treatment of experimental animals with heparanase inhibitors markedly reduced 90%) the incidence of lung metastases induced by B16 melanoma, Lewis lung carcinoma and mammary adenocarcinoma cells (9,10,20). 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 Finally, heparanase externally adhered to B16-F1 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 granted 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 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 (21,22). Basic fibroblast growth factor (bFGF) has been extracted from a subendothelial ECM produced in vitro (23) 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 (24-26). 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 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 (29,30).

Moreover, the size of HS required for optimal effect was similar to that of HS fragments released by heparanase Similar results were obtained with vascular endothelial cells growth factor (VEGF) suggesting the operation of a dual receptor mechanism involving HS in cell interaction with heparinbinding 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 (7,28).

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 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 heparanase-encoding 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 (51).

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 Blymphocytes, 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 (33).

Treatment of experimental animals with heparanase inhibitors nonanticoagulant species of low molecular weight heparin) markedly reduced the incidence of experimental autoimmune encephalomyelitis (EAE), adjuvant arthritis and graft rejection (8,34) in experimental animals, indicating that the use of heparanase inhibitors or neutralizing antibodies to inhibit heparanase activity may inhibit autoimmune and inflammatory diseases Recently, heparanase activity has been correlated with leukemia. Heparanase expression has been demonstrated in human leukemia cells, restricted to acute myeloid leukemia (Bitan et al, Exp Hematol 2002;30:34-41), and inhibition of heparanase, by PI-88, has been found to significantly reduce the malignant cell load in myeloid leukemia models (Iversen, et al Leukemia 2002;16:376-81).

The involvement of heparanase in other physiological processes 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) (32,35); cell interaction with plasma lipoproteins cellular susceptibility to certain viral and some bacterial and protozoa infections (36-38); and disintegration of amyloid plaques (39).

Viral Infection: The presence of heparan sulfate on cell surfaces have been shown to be the principal requirement for the binding of Herpes Simplex (36) and Dengue (37) 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 (38).

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

Restenosis and Atherosclerosis: Proliferation of arterial smooth muscle cells (SMCs) in response to endothelial injury and accumulation of cholesterol rich lipoproteins are basic events in the pathogenesis of atherosclerosis and 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: 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 (43).

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 (44).

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

Furthermore, the intracellular distribution of the protein differed between

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invasive and non-invasive 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 (44) 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 (prouPA), 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 tumor-associated 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 (49): Large body of evidence suggests that the matrix metalloproteinases MMP-2 and MMP-9 play an important role in tumor invasion process (49,50).

Heparanase and cardiovascular disease: Much of cardiovascular disease is characterized by changes in the vasculature, particularly increased vascular permeability, associated with a loss of normally sulfated HSPG in the ECM of the affected endothelial tissues. Recent studies have revealed that lysolethecin, an atherogenic component of oxidized LDL, induces heparanase activity in endothelial cells (Sivaram P. et al, JBC 1995; 270:29760-5), leading to changes in HSPGs (Pillarisetti S. Trends Cardiovas Med 2000;10:60-65), and that the reduced HSPGs in turn modify the lipoprotein binding characteristics of the endothelium (Pillarisetti S. et al J Clin Invest ~lil -I X 18 1997;100:867-74). Thus, regulation of heparanase expression and activity in the endothelial and intimal layers can be crucial to both healthy and diseased states of the arterial vasculature. Indeed, the recent demonstration of the prevention of arterial restenosis injury in rats and rabbits by administration of the heparanase inhibitor PI-88 (Francis DJ et al Cire Res 2003;92:e70-77) suggests a role for such inhibition in treatment and prevention ofvasculopathy.

Inhibition of heparanase has been suggested as treatment for a number of vascular conditions including heart disease. International Patent Application WO 01/35967A1 to Herr et al discloses the use of heparanase inhibitor compounds, such as reduced carboxy, partially desulfated and n-acetylated derivatives of heparin, for the treatment of cardiac insufficiency, especially congestive heart failure. However, no inhibition of disease is demonstrated, and the claims are based solely on the observation of increased heparanase expression in heart tissue from a rat model of congestive heart failure.

Similarly, International Patent Application WO 03/011119A2, to Pillarisetti et al, also demonstrated heparanase expression in atherosclerotic lesions and endothelial cells in vivo and in culture, and the induction of heparanase expression with lysolecithin, advanced glycation endproducts (AGE) and TNFa. The use of biotinylated HS for assaying heparanase activity in tissues and tissue samples, and for identification of compounds inhibiting heparanase activity is disclosed, but no evidence for treatment or prevention of heart disease by inhibition of heparanase activity or expression is presented.

Heparanase structure: 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 N-terminus of the heparanase precursor (residues Gln36-Lysl08 or Glu 09) (Fairbankset al. J. Biol. Chem. 1999;274, 28587-29590). It is most likely that heparanase is expressed as a 65 kDa pre-pro form that is first processed into a 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 (SEQ ID NO: a 8 kDa small subunit (SEQ ID. NO: 11) 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 Glu' 09 -Ser 11 0 site and the Gln' 7 -Lys 15 8 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.

Despite unique substrate specificity and catalytic properties, functional and distant structural similarities were found between the 50 kDa subunit of heparanase and members of several of the glycosyl hydrolase families (8,30, 42) from glycosyl hydrolase clan A including strong local identities to regions containing the critical active-site catalytic proton donor and nucleophile residues that are conserved in this clan of enzymes. On the basis of secondary structure an TIM barrel fold, which is common to the GH-A families, has been predicted. Glu225 and Glu343 of human heparanase were identified as the likely proton donor and nucleophile residues, respectively, using sequence alignments with a number of glycosyl hydrolases from GH-A.

This was confirmed by the loss of heparan sulphate degrading activity in COS- 7 expressed mutant heparanase having substitution of residues Glu225 and Glu343 with alanine. In contrast, the alanine substitution of two other glutamic acid residues (Glu378 and Glu396), both predicted to be outside the active site, did not affect heparanase activity (Hullet et al. Biochemistry 2000, 39, 15659- 15667). These data suggest that heparanase is a member of the clan A glycosyl hydrolases and has a common catalytic mechanism that involves two conserved acidic residues, a putative proton donor at Glu225 and a nucleophile at Glu343.

A number of basic residues that are conserved in human, rat, and mouse heparanase are found in proximity to the proposed catalytic proton donor and nucleophile, KK (residues 231 and 232) near Glu225 and KK (residues 337 and 338) near Glu343. Further, three clusters of basic amino acids that conform to HS-binding protein consensus sequences (xBBBxxBx or xBBxBx) (Cardin, A. and Weintraub, H. J. R. Arteriosclerosis, 1989 9, 21-32) are present in human heparanase: QKKFKN (residues 157-162), PRRKTAKM (residues 271-278) and SKRRKLRV (residues 426-433). When these conserved residues are mapped onto the structure of endo-1,4-,-xylanase from P. simplicissimum (pdb entry 1BG4), three of these four basic clusters (residues 231 and 232, 271-278, and 157-162) can be predicted to be situated on the top of the TIM-barrel fold, in proximity to the proposed active site, potentially interacting with HS. The position of the last basic cluster (residues 426-433) could not be predicted.

Thus, specific sites within the heparanase enzyme having potential therapeutic, diagnostic and investigative interest have been suggested, however, their usefulness as antigenic determinants, and the applicability of specific antibodies to these sites has yet to be revealed.

Other potential therapeutic applications of anti-heparanase antibodies: Apart from the modulation of heparanases' involvement in tumor cell metastasis, inflammation, vasculopathy and autoimmunity, anti-heparanase antibodies may be applied to modulate: bioavailability of heparin-binding growth factors (Bashkin et al. Biochem 1989;28:1737-43); cellular responses to heparin-binding growth factors bFGF, VEGF) and cytokines (IL-8) (Rapraeger et al. Science 1991;252:1705-08; Gitay-Goren et al. J Biol Chem 1992;267:6093-98); cell interaction with plasma lipoproteins (Eisenberg, S et al. J. Clin Investig 1992;90:2013-21); cellular susceptibility to certain viral and some bacterial and protozoa infections (Shieh et al. J Cell Biol 1992;1 16:1273- 81; Chen et al. Nature Med 1997;3:866-71; Putnak et al. Nat Med 1997;3:828- 29); and disintegration of amyloid plaques (Narindrasorasak et al. J Biol Chem 1991;266:12878-83). Anti-heparanase antibodies may thus prove useful for conditions such as wound healing, angiogenesis, restenosis, atherosclerosis, inflammation, neurodegenerative diseases and viral infections. Antiheparanase antibodies may be applied for immunodetection and diagnosis of micrometastases, autoimmune and vascular lesions, thrombosis and renal failure in biopsy specimens, plasma samples, and body fluids. Common use in basic research is expected.

Use of monoclonal antibodies for clinical therapeutics: Monoclonal antibodies (Mabs) are beginning to gain a prominent role in the therapeutics arena. Approximately 80 Mabs are in clinical development which represent over 30% of all biological proteins undergoing clinical trials (76,80). Market entry of new Mab therapies is expected to be dramatically accelerated. Fueling this growth has been the emergence of technologies to create increasingly human-like (humanized) Mabs, ranging from chimerics to fully human. These new Mabs promise to overcome the human antibody to mouse antibody response (81).

Monoclonal antibodies, which can be viewed as nature's own form of "rational drug design", can offer an accelerated drug-discovery approach for appropriate targets, because producing high affinity Mabs that specifically block the activity of an antigen target is usually easier and faster than designing a small molecule with similar activity (79).

Due to their long serum half-life, low toxicity and high specificity, Mabs began to reveal their true therapeutic potential, particularly in oncology, where current therapeutic regimens have toxic side effects that, in many cases, require repetitive dosing in the respective treatment protocols (79).

II

The promise of monoclonal antibody therapy and diagnostics is reflected in the growing number of Mabs with clinical indications in late-stage clinical trials: more than 9 murine monoclonals, 2 chimeric, 9 humanized, and 8 other types of Mabs in Phase III clinical trials. FDA approval has already been granted for more than 25 Mabs, including therapeutic Mabs such as Inflixamab (anti-TNF1 for Crohn's disease) and Abcixamab (anti glycoprotein lb for prevention of clotting), Neumega (for treatment of thrombocytopenia), Rituxan (human-mouse chimeric anti CD20 for treatment of non-Hodgkin's B cell lymphoma), Herceptin, humanized Mab raised against the protooncogene HER-2/neu, for treating breast cancer patients with metastatic disease and ProstaScint (anti-PSA) and HumaSPECT (anti-CTA recombinant human antibody) for detection and monitoring of prostate and colon cancer, respectively. Many others are in Phase II and Phase I clinical trials.

In order to use anti-angiogenesis approach in preventing metastatic disease, Genentech introduced a recombinant humanized Mab to the vascular endothelial growth factor (VEGF). The anti-VEGF rhu Mab was found to be safe and well tolerated in a 25-patient pilot Phase I clinical study (79).

Specificity ofanti-heparanase antibodies: Many of the "anti-heparanase" antibodies reported in the literature have, upon careful examination, been revealed to lack anti-heparanase specificity. In most cases, this has been due to mistaken identification of the antigen as heparanase, or inadequate assessment of the purity of the heparanase antigen preparation. For example, Oosta, et al.

(Oosta, et al J. Biol. Chem. 1982, 257: 11,249-11,255) described the purification of a human platelet heparanase with an estimated molecular mass of 134 kDa expressing an endoglucuronidase activity. Hoogewerf, et al reported the purification of a 30 kDa human platelet heparanase closely related to the CXC chemokines CTAPIII, NAP-2 and P-thromboglobulin (the latter was claimed to be an endoglucosaminidase) that cleaves both heparin and heparan sulfate essentially to disaccharides (Hoogewerf, A.J. et al J. Biol.

Chem. 1995, 270: 3268-3277). Freeman and Parish (Freeman, and Parish, Biochem. 1988,330:1341-1350) have purified to homogeneity a kDa platelet heparanase exhibiting endoglucuronidase activity. Likewise heparanase enzyme purified from human placenta and from hepatoma cell line Pat. No. 5,362,641) had a molecular mass of approximately 48 kDa. A similar molecular weight was determined by gel filtration analysis of partially purified heparanase enzymes isolated form human platelets, human neutrophils and mouse B16 melanoma cells.

In contrast, heparanase purified from B16 melanoma cells by Nakajima, et al having a molecular weight of 96 kDa had been localized immunochemically to the cell surface and cytoplasm of human melanoma lesions using a polyclonal antiserum (Jin, Nakajima, M. and Nicolson, G.L.

Int. J. Cancer, 1990, 45: 1088-1095) and in tertiary granules in neutrophils using monoclonal antibodies (26a) (Jin, Nakajima, M. and Nicolson, G.L.

Int. J. Cancer, 1990, 45: 1088-1095). However, the melanoma heparanase amino terminal sequence was found to be characteristic of a 94 kDa glucoseregulated protein (GRP94/endoplasmin) lacking heparanase activity (Mollinedo, et al Biochem. 1997; 327:917-923), suggesting that the endoplasmin-like 98 kDa protein found in purified melanoma heparanase preparations is a contaminant (Mollinedo, et al Biochem. 1997; 327:917- 923, De Vouge, et al Int. J. Cancer 1994, 56: 286-294). Likewise, antiserum directed against the amino terminal sequence of CTAP III was applied to immunolocalize the heparanase enzyme in biopsy specimens of human prostate and breast carcinomas (Graham, and Underwood, P.A.

Biochem. and Mol. Biol. International, 1996; 39: 563-571, Kosir, M. et al J.

Surg. Res. 1997;67: 98-105). However, the validity of the results is questionable, since recombinant CTAPIII/NAP2 chemokines are devoid of heparanase activity while commercial preparations of CTAPIII from platelets are contaminated with heparanase and hence exhibit HS degrading activity. In addition, western blot analysis of the platelet enzyme purified by Freeman and Parish demonstrated that purported heparanase-related proteins (such as human p-thromboglobulin, platelet factor-4 CTAP-III and NAP-2) were absent from purified platelet heparanase preparations (Freeman, and Parish, C.R., Biochem. 1988,330:1341-1350).

Finally, none of the sequences published by Hoogewerf et al (platelet CTAP-III/NAP-2) (Hoogewerf, A.J. et al J. Biol. Chem. 1995, 270: 3268-3277) or Jin et al. (B16 melanoma) (Jin, Nakajima, M. and Nicolson, G.L. Int. J.

Cancer, 1990, 45: 1088-1095) nor sequences of the bacterial heparin/heparan sulfate degrading enzymes (hep I III) (Ernst, et al Critical Reviews in Biochemistry and Molecular Biology: 1995;30(5): 387-444) demonstrated homology with sequences derived from the purified human placenta and hepatoma heparanases (SEQ ID NO:4).

Several years ago rabbit polyclonal antibodies directed against a partially purified preparation of human placenta heparanase were prepared (as disclosed in U.S. Pat. No. 5,362,641), which were later found to be directed against plasminogen activator inhibitor type I (PAI-1) that was co-purified with the placental heparanase. These findings led to a modification of the original purification protocol to remove the PAI-1 contaminant.

Thus it is evident that many previous efforts to elicit anti-heparanase antibodies have resulted in antibodies which are elicited by protein contaminants, thus incapable of recognizing heparanase, and/or incapable of specifically recognizing heparanase.

SUMMARY OF THE PRESENT INVENTION The background art does not teach or suggest an anti-heparanase antibody, or a heparanase activity neutralizing monoclonal anti-heparanase antibody.

The background art also does not teach or suggest treatment of a subject in need thereof by using heparanase activity neutralizing monoclonal antiheparanase antibodies, or monitoring a heparanase related condition using such antibodies.

The present invention overcomes these disadvantages of the background art by providing polyclonal and monoclonal anti-heparanase antibodies, heparanase activity neutralizing monoclonal antibodies, and uses thereof for treatment of a subject with a pathological or heparanase related condition, as well as methods for monitoring such a heparanase related condition using the taught antibodies.

Therefore, there is clearly a widely recognized need for, and it would be highly advantageous to have, an anti-heparanase antibody and a heparanase activity neutralizing monoclonal anti-heparanase antibody, as taught in the present invention.

According to one aspect of the present invention, there is provided an isolated antibody or portion thereof, capable of specifically binding to at least one epitope of a heparanase protein.

According to an embodiment of the present invention, the heparanase protein which the isolated antibody binds is at least 60% homologous to the amino acid sequence of any of SEQ ID NOs:1-5 and 11, preferably at least homologous to the amino acid sequence of any of SEQ ID Nos: 1-5 and 11, more preferably at least 80% homologous to the amino acid sequence of any of SEQ ID Nos: 1-5 and 11 and most preferably at least 90% homologous to the amino acid sequence of any of SEQ ID Nos: 1-5 and 11.

Hereinafter, all percent homologies are given according to a determination of homology with the Smith-Waterman algorithm, using the Bioaccelerator platform developed by Compugene (gapop: 10.0, gapext: matrix: blosum62).

According to another embodiment, the heparanase protein which the isolated antibody binds comprises an amino acid sequence as set forth in any of SEQ ID NOs: 1-5 and 11.

According to another embodiment, the at least one epitope is at least homologous to the amino acid sequence of any of SEQ ID NOs: 6-10, preferably at least 80% homologous to the amino acid sequence of any of SEQ ID NOs: 6-10, more preferably at least 90% homologous to the amino acid sequence of any of SEQ ID NOs: 6-10.

According to another embodiment, the at least one epitope comprises an amino acid sequence as set forth in any of SEQ ID NOs: 6-10.

According to another embodiment, the at least one epitope comprises a sequence at least 70% homologous to the amino acid sequence of SEQ ID NO:6, preferably at least 80% homologous to the amino acid sequence of SEQ ID NO:6, more preferably at least 90% homologous to the amino acid sequence of SEQ ID NO:6.

According to another embodiment, the at least one epitope comprises a sequence at least 90% homologous to the amino acid sequence of SEQ ID NO:8.

According to another embodiment, the at least one epitope comprises a sequence at least 90% homologous to the amino acid sequence of SEQ ID NO:9.

According to another embodiment, the at least one epitope comprises a sequence at least 90% homologous to the amino acid sequence of SEQ ID According to another embodiment, the at least one epitope comprises a sequence at least 75% homologous to the amino acid sequence of SEQ ID NO:7, preferably at least 80% homologous to the amino acid sequence of SEQ ID NO:7, more preferably at least 90% homologous to the amino acid sequence of SEQ ID NO:7.

According to another embodiment, the isolated antibody comprises a polyclonal antibody.

According to another embodiment, the polyclonal antibody is selected from the group consisting of a crude polyclonal antibody and an affinity purified polyclonal antibody.

According to another embodiment, the isolated antibody comprises a monoclonal antibody.

According to another embodiment, the monoclonal antibody is selected from the group consisting of HP130, HP 239, HP 108.264, HP 115.140, HP 152.197, HP 110.662, HP 144.141, HP 108.371, HP 135.108, HP 151.316, HP 117.372, HP 37/33, HP3/17, HP 201 and HP 102.

According to another embodiment, the isolated antibody comprises a chimeric antibody.

According to another embodiment, the isolated antibody comprises a humanized antibody.

According to another embodiment, the isolated antibody comprises a Fab fragment.

According to another embodiment, the isolated antibody comprises a single chain antibody.

According to another embodiment, the isolated antibody comprises an immobilized antibody.

According to another embodiment, the isolated antibody compris'es a labeled antibody.

According to another embodiment, the at least one epitope is selected from the group consisting of a heparan-sulfate binding site flanking region, a catalytic proton donor site, a catalytic nucleophilic site, an active site and binding site linking region and a C-terminal sequence of heparanase P8 subunit.

According to an embodiment of the invention, the heparan-sulfate binding site flanking region comprises an amino acid sequence at least homologous to the amino acid sequence as set forth in SEQ ID NO:6, preferably at least 80% homologous to the amino acid sequence as set forth in SEQ ID NO:6, more preferably at least 90% homologous to the amino acid sequence as set forth in SEQ ID NO:6.

According to an embodiment, the heparan-sulfate binding site flanking region comprises an amino acid sequence as set forth in SEQ ID NO:6.

According to another embodiment, the at least one epitope comprises a heparan-sulfate binding site flanking region.

According to another embodiment, the catalytic proton donor site comprises an amino acid sequence at least 90% homologous to the amino acid sequence as set forth in SEQ ID NO:8.

According to another embodiment, the catalytic proton donor site comprises an amino acid sequence as set forth in SEQ ID NO:8.

According to another embodiment, the at least one epitope comprises a catalytic proton donor site.

According to another embodiment, the catalytic nucleophilic site comprises an amino acid sequence at least 90% homologous to the amino acid sequence as set forth in SEQ ID NO:9.

According to another embodiment, the catalytic nucleophilic site comprises an amino acid sequence as set forth in SEQ ID NO:9.

According to another embodiment, the at least one epitope comprises a catalytic nucleophilic site.

According to another embodiment, the active site and binding site linking region comprise an amino acid sequence at least 90% homologous to the amino acid sequence as set forth in SEQ ID According to another embodiment, the active site and binding site linking region comprise an amino acid sequence as set forth in SEQ ID According to another embodiment, the at least one epitope comprises an active site and binding site linking region.

According to another embodiment, the C-terminal sequence of heparanase P8 subunit comprises an amino acid sequence at least 75% homologous to the amino acid sequence as set forth in SEQ ID NO:7, preferably at least homologous to the amino acid sequence as set forth in SEQ ID NO:7, more preferably at least 90% homologous to the amino acid sequence as set forth in SEQ ID NO:7.

According to another embodiment, the C-terminal sequence of heparanase P8 subunit comprises an amino acid sequence as set forth in SEQ ID NO:7.

According to another embodiment, the at least one epitope comprises a Cterminal sequence of heparanase P8 subunit According to another embodiment, the heparanase protein is substantially free of contaminating proteins, as determined by an assay selected from the group consisting of immunodetection, gel electrophoresis and catalytic activity.

According to another embodiment, the heparanase protein is a recombinant heparanase protein.

According to another aspect of the present invention there is provided a cell line for producing a monoclonal antibody, comprising a cell line for producing the aforementioned monoclonal antibody. According to an embodiment of the present invention, the antibody or portion thereof produced by the cell line is humanized.

According to another aspect of the present invention there is provided a use of the aforementioned anti-heparanase antibodies or portion thereof for treating a subject suffering from a pathological condition, comprising administering a therapeutically effective amount of the anti-heparanase antibodies to the subject.

According to still another aspect of the present invention there is provided a use of the aforementioned anti-heparanase antibodies or portion thereof for treating or preventing a heparanase-related disorder or condition in a subject, comprising administering a therapeutically effective amount of any of the antiheparanase antibodies to the subject.

According to an embodiment of the present invention, the heparanaserelated disorder or condition is selected from the group consisting of an inflammatory disorder, a wound, a scar, a vasculopathy and an autoimmune condition. The vasculopathy is preferably selected from the group consisting of atherosclerosis, restenosis and aneurysm.

According to another embodiment, the heparanase-related disorder or condition is selected from the group consisting of angiogenesis, cell proliferation, a cancerous condition, tumor cell proliferation, invasion of circulating tumor cells and a metastatic disease. The cancerous condition is preferably selected from the group consisting of a blood, breast, bladder, rectum, stomach, cervix, ovarian, colon, renal and prostate cancer.

According to still another aspect of the present invention there is provided a method of detecting the presence of a heparanase polypeptide in a sample, comprising incubating the sample with any of the heparanase-specific antibodies detailed above, in a manner suitable for formation of a heparanase polypeptide-antibody immune complex; such that the heparanase-specific antibody is characterized by specifically binding to heparanase, and detecting the presence of the heparanase polypeptide-antibody immune complex to determine whether a heparanase polypeptide is present in the sample.

According to an embodiment of the present invention, the anti-heparanase antibody is labeled with a labeling agent that provides a detectable signal.

According to another embodiment, the labeling agent is selected from the group consisting of an enzyme, a fluorophore, a chromophore, a protein, a chemiluminescent substance and a radioisotope.

According to still another aspect of the present invention there is provided a method for detecting a heparanase-related disease or condition in a subject, comprising: obtaining a biological sample from the subject; contacting the biological sample with any of the anti-heparanase antibodies mentioned above in a manner suitable for formation of a heparanase polypeptide-antibody immune complex; and detecting the presence of the heparanase polypeptide-antibody immune complex to determine whether a heparanase polypeptide is present in the sample, such that the presence or absence of said heparanase polypeptideantibody immune complex indicates a heparanase-related disease or condition; thereby detecting a heparanase-related disease or condition in a subject.

According to an embodiment of the present invention, the subject is a vertebrate, preferably the subject is mammalian, more preferably the mammalian subject is a human subject.

According to another embodiment, the heparanase-related disorder or condition is selected from the group consisting of an inflammatory disorder, a wound, a scar, a vasculopathy and an autoimmune condition. The vasculopathy is preferably selected from the group consisting of atherosclerosis, restenosis and aneurysm.

According to another embodiment, the heparanase-related disorder or condition is selected from the group consisting of angiogenesis, cell proliferation, a cancerous condition, tumor cell proliferation, invasion of circulating tumor cells and a metastatic disease. The cancerous condition is preferably selected from the group consisting of a blood, breast, bladder, rectum, stomach, cervix, ovarian, colon, renal and prostate cancer.

According to another embodiment, the heparanase-related disorder or condition is a renal disease or disorder. The renal disease or disorder is preferably selected from the group consisting of diabetic nephropathy, glomerulosclerosis, nephrotic syndrome, minimal change nephrotic syndrome and renal cell carcinoma.

According to another embodiment, the biological sample is selected from the group consisting of serum, plasma, urine, synovial fluid, spinal fluid, tissue sample, a tissue and/or a fluid.

According to another embodiment, contacting the sample is performed in situ.

According to another embodiment, contacting the sample is performed in vitro.

According to still another aspect of the present invention there is provided a method for monitoring the state of a heparanase-related disorder or condition in a subject, the method comprising: obtaining a biological sample from the subject; contacting the biological sample with an anti-heparanase antibody in a manner suitable for formation of a heparanase polypeptide-antibody complex; detecting a presence, absence or level of the heparanase polypeptideantibody complex to determine a presence, absence or level of a heparanase polypeptide in the biological sample; repeating the three previous stages of the method at predetermined time intervals; and determining a degree of change of said presence, absence or level of the heparanase polypeptide at the predetermined time intervals, such that the change indicates a state of the heparanase-related disorder or condition in the subject; thereby monitoring the state of the heparanase-related disorder or condition in the subject.

According to an embodiment of the present invention, the subject is a vertebrate, preferably the subject is mammalian, more preferably the mammalian subject is a human subject.

According to another embodiment, the heparanase-related disorder or condition is selected from the group consisting of an inflammatory disorder, a wound, a scar, a vasculopathy and an autoimmune condition. The vasculopathy is preferably selected from the group consisting of atherosclerosis, restenosis and aneurysm.

According to another embodiment, the heparanase-related disorder or condition is selected from the group consisting of angiogenesis, cell proliferation, a cancerous condition, tumor cell proliferation, invasion of circulating tumor cells and a metastatic disease. The cancerous condition is preferably selected from the group consisting of a blood, breast, bladder, rectum, stomach, cervix, ovarian, colon, renal and prostate cancer.

According to another embodiment, the heparanase-related disorder or condition is a renal disease or disorder. The renal disease or disorder is 33 preferably selected from the group consisting of diabetic nephropathy, glomerulosclerosis, nephrotic syndrome, minimal change nephrotic syndrome and renal cell carcinoma.

According to another embodiment, the biological sample is selected from the group consisting of serum, plasma, urine, synovial fluid, spinal fluid, tissue sample, a tissue and/or a fluid.

According to another embodiment, contacting the sample is performed in situ.

According to another embodiment, contacting the sample is performed in vitro.

According to still another aspect of the present invention, there is provided a pharmaceutical composition comprising any of the previously mentioned isolated anti-heparanase antibodies or a portion thereof and a pharmaceutically acceptable carrier.

According to an embodiment of the invention, the anti-heparanase antibody is a monoclonal antibody.

According to another embodiment, the anti-heparanase antibody is a humanized antibody.

According to still another aspect of the present invention there is provided an affinity medium for binding human heparanase polypeptides, the medium comprising any of the previously mentioned anti-heparanase antibodies immobilized to a chemically inert, insoluble carrier.

According to an embodiment of the invention, the chemically inert, insoluble carrier is selected from the group consisting of acrylic and styrene based polymers, gel polymers, glass beads, silica, filters and membranes.

BRIEF DESCRPTION OF THE DRAWINGS The invention is herein described, by way of example only, with reference to the accompanying drawings, wherein: FIG 1. demonstrates epitope mapping of monoclonal antibodies HP-130 and HP-239 according to the present invention. The different polypeptides (as indicated below) were fractionated on SDS-PAGE and transferred to a nitrocellulose membrane (Schleicher and Schuell). The membrane was reacted with either antibody HP-130 or HP-239 as indicated above. Lane 1, cell extracts containing a heparanase segment of 414 amino acids of the heparanase open reading frame (amino acids 130-543, SEQ ID NO:4). Lane 2, cell extracts containing a heparanase segment of 314 amino acids of the heparanase open reading frame (amino acids 230-543, SEQ ID NO:4). Lane 3, cell extracts containing a heparanase segment of 176 amino acids of the heparanase open reading frame (amino acids 368-543, SEQ ID NO:4). Lane 4, cell extracts containing heparanase segment of 79 amino acids of the heparanase open reading frame (amino acids 465-543, SEQ ID NO:4). Lane 5, cell extracts containing heparanase segment of 229 amino acids of the heparanase open reading frame (amino acids 1-229, SEQ ID NO:4). Lane 6, cell extracts containing heparanase segment of 347 amino acids of the heparanase open reading frame (amino acids 1-347, SEQ ID NO:4). Lane 7, cell extracts containing heparanase segment of 465 amino acids of the heparanase open reading frame (amino acids 1-465, SEQ ID NO:4). Lane 8, size markers (Bio- Rad).

FIG. 2 demonstrates neutralization of recombinant heparanase expressed in insect cells with monoclonal antibodies. Heparanase activity after preincubation of the recombinant heparanase expressed in insect cells, with increasing amounts (as indicated under each bar) of antibody HP-130 (130) and antibody HP-239 (239). The percentage of activity is calculated from the control reaction, pre-incubated in the absence of the antibody.

FIG. 3 demonstrates neutralization of natural heparanase purified from human placenta with monoclonal antibodies. Heparanase activity after preincubation of heparanase isolated from human placenta with increasing amounts (as indicated under each bar) of antibody HP-130 (130) and antibody HP-239 (239). The percentage of activity is calculated from the control reaction, pre-incubated in the absence of the antibody.

FIG. 4 demonstrates the specific recognition of human heparanase by anti-heparanase monoclonal antibodies HP3/17 and HP 37/33. Purified recombinant human heparanase (lanes 1 and 6) or cell extracts from CHO cells expressing human (lanes 2 and 5) or mouse (lanes 3 and 4) heparanase were separated electrophoretically on 4-12% NuPAGE gel (Novex Ltd, USA), blotted onto PVDF membrane, and reacted with Igg/ml Mabs HP3/17 (lanes 1- 3) or HP 37/33 (lanes 4-6).

FIG. 5 demonstrates the specific immunoprecipitation of human heparanase by Mab HP3/17. Purified recombinant human heparanase (H53MC), or extracts of Si-11 cells (CHO cells expressing human heparanase) were incubated with or without 10g antibody for 2 hours at 4°C, incubated with Protein G beads and washed twice with PBS. Bound protein was released from the beads by boiling, separated electrophoretically on a 4-12% NuPAGE gel (Novex, Ltd, USA), blotted onto PVDF membrane, and reacted with affinity purified polyclonal goat anti-heparanase (anti-p45) antibodies. Lane 1recombinant heparanase (H53MC), 50ng, no immunoprecipitation. Note the presence of both a processed and higher molecular mass unprocessed form of heparanase. Lane 2- S1-l1 cell extract immunoprecipitated with antiheparanase HP3/17. Lane 3- S1-11 cell extract immunoprecipitated with antiheparanase HP37/33 (anti-pep9 monoclonal similar to HP3/17). Lane 4recombinant heparanase (H53MC) immunoprecipitated with anti-heparanase HP3/17. Lane 5- recombinant heparanase (H53MC) immunoprecipitated with anti-heparanase HP37/33. Lane 6-SI-11 cell extract without immunoprecipitation. Lane 7- recombinant heparanase (H53MC) without immunoprecipitation. Lane 8- Protein G beads alone. Note the specific immunoprecipitation of the processed (lower molecular weight) form of the purified recombinant human heparanase with both HP3/17 and HP37/33 antipep9 monoclonal antibodies (lanes 4 and 5, compared with lane 1).

FIGs. 6A-D demonstrate the detection of heparanase within human blood cells by anti-heparanase Mabs. Human blood smears were stained with 100lg/ml (Figure 6B) or 10g/ml (Figure 6C) anti-heparanase Mab HP3/17, or Mab HP 37/33 (Figure 6D). Note the strong staining of the neutrophils (brown stain), while the lymphocytes and RBCs remain unstained.

Figure 6A- unstained smear. Magnification X1000.

FIGs. 7A-B illustrate the specific detection of human heparanase in transgenic mouse liver, by anti-heparanase Mab. Sections of heparanase expressing transgenic mouse liver (Figure 7A) and normal mouse liver (Figure 7B) were stained with anti-heparanase Mab HP3/17. Note the strong response of the heparanase expressing liver (brown stain), and the absence of staining in the normal mouse liver, indicating the specificity of HP3/17 for human heparanase.

FIGs. 8A-B illustrate the detection of heparanase in normal human tissues by anti-heparanase Mab. Photomicrographs of sections of normal human placenta stained with anti-heparanase Mab HP3/17 (Figure 8B) or left unstained (Figure 8A) demonstrate detection of heparanase expression (brown stain) in the normal human placenta.

FIGs. 9A-C illustrate the detection of human heparanase in colorectal cancer. Photomicrographs of sections of normal colon tissue (Figure 9A, 100X magnification- left panel, 400X magnification -right panel) and colorectal cancer tissue (Figures 9B, 100X magnification- left panel, 400X magnification -right panel) stained with anti-heparanase Mab HP3/17 reveal a strong expression of heparanase (brown stain) in the cancerous (Figure 9B), but not normal (Figure 9A) colon tissue. The section in Figure 9C was left unstained for comparison.

FIG. 10 demonstrates the specific recognition of human heparanase by monoclonal antibodies HP201 and HP102, demonstrated by Western blot analysis. Cell extracts from CHO cells expressing human heparanase (S1-11, lanes 3 and 6) or mock transfected controls (Dhfr", lanes 2 and 5) were separated electrophoretically on 4-12% NuPAGE gel (Novex Ltd, USA), blotted onto PVDF membrane, and reacted with supernatants from hybridomas HP201 (anti-pep38) and HP102 (anti-peplO). Lanes 1 and 4 are molecular weight markers.

FIG. 11 illustrates the in vivo inhibition of tumor growth in mice by treatment with specific anti-heparanase monoclonal antibodies. Prior to injection to C57BI mice, the B16-FI melanoma tumor cells were preincubated with either monoclonal anti-heparanase antibodies HP 130 (filled squares), anti-pep9 antibody HP37/33 (filled triangles), or PBS (filled diamonds).

Beginning one day before injection of the tumor cells, intraperitoneal injections of either 200gg monoclonal anti-heparanase antibody HP 130 (group B) or anti-pep9 antibody HP37/33 (group or of 0.15ml PBS (group A) were administered every 2-3 days for 16 days. The study terminated 18 days post tumor cell injection. Tumor cell growth is expressed as mean tumor volume (X103) over time post-induction. Note the strong inhibition of tumor growth with treatment by HP 130 and HP 37/33.

FIG. 12 is a Table illustrating the in vivo inhibition of experimental arthritis in mice by specific anti-heparanase monoclonal antibody HP 3/17.

Experimental arthritis was induced in C57B1 mice by injection of a cocktail of anti-collagen type-II monoclonal antibodies (Chondrex LLC, Redmond, WA) on day 0, followed by 25gg lipopolysaccharide (LPS) administration i.p. on day 3, according to de Fougerolles et al (J Clin Invest 2000; 105:721-9). The mice were treated with 4 intravenous injections of 250j.g each of either antiheparanase monoclonal antibody (anti-pep9) HP 3/17 (group mouse antihuman IgG3 monoclonal antibody control (group or PBS control (group A), beginning at day 0, and every 2-3 days thereafter. Severity of arthritis was scored according to blinded observation of swelling in all 4 paws of each mouse, on a scale of 0-4, 4 being maximal swelling, and 0 being normal. Note the progressive anti-arthritic effect of HP 3/17, beginning as early as day 7.

FIG. 13 is a graph illustrating the protective effect of treatment with specific anti-heparanase monoclonal antibody on experimental autoimmune diabetes in non-obese diabetic (NOD) mice. Four week old female NOD mice received either 200gg specific anti-heparanase monoclonal antibody HP 3/17 (anti-pep9)(filled diamonds) or 200jRl PBS (filled squares) in intraperitoneal injections twice a week for 4 weeks, and then once a week thereafter. Diabetes was determined by blood glucose measurement. Animals having 500mg/dl blood glucose were euthanized. Note the delayed onset of disease and enhanced survival in the HP 3/17 treated mice.

FIGs 14A and 14B are graphic representations for demonstrating neutralization of recombinant heparanase activity by monoclonal antibodies HP 3/17 and HP 37/33. Neutralization is expressed as the change in heparanase activity after pre-incubation of the recombinant heparanase with increasing amounts (as indicated under each column) of antibody HP-37/37 (Figure 14A) and antibody HP 3/17 (Figure 14B), both elicited against peptide pep9 (SEQ ID NO:9, see Table compared with controls no antibody).

FIGs 15A and 15B. demonstrates epitope mapping of monoclonal antibodies HP 37/33 and HP 135.108, according to the present invention. Serial peptides of descending size, having approximately 50 amino acids intervals between them, representing amino acids 130-543 of human heparanase (SEQ ID NO were expressed in E. coli BL21 from a series of plasmids generated from a DNA fragment comprising the P45 subunit of mature heparanase polypeptide, using the Erase A Base kit (Promega). The different heparanase fragments were fractionated by gel electrophoresis and blotted onto PVDF (Schleicher and Schuell) membrane. Lane 1-Molecular weight markers. Lane 2-peptide d45 bam. Lane 3-peptide d42. Lane 4-peptide d43. Lane d63. Lane 6-peptide d84. Lane 7-peptide d123. Lane 8-peptide d142. Lane 9peptide dl86. Lane 10-peptide d207 and d22. Membranes were incubated with hybridoma medium or with IgG purified monoclonal antibodies, as indicated, in order to localize the epitope detected by a specific antibody. Interacting antibody was detected using an HRP-conjugated goat/donkey anti mouse antibody.

Figure 15A shows the mapping of heparanase epitopes recognized by monoclonal antibody HP135.108, raised against the intact active recombinant human heparanase dimer (CHO p53).

Figure 15B shows the mapping of heparanase epitopes recognized by monoclonal antibody HP37/33, raised against peptide pep9 (SEQ ID NO: 9).

Note the absence of immune interaction in lanes 7, 8 and 9 in both FIG and FIG 15B, indicating that both HP 135.108 and HP 37/33 recognize heparanase partial polypeptides of 35-50 kDa, but not >25kDa fragments. This pattern localizes the epitope to within the region of amino acids 320-410 of heparanase precursor (SEQ ID NO 4).

FIG. 16 demonstrates the specific recognition of human heparanase by anti-heparanase monoclonal antibody HP 135.108. Purified recombinant human heparanase (lane 1) or cell extracts from CHO cells expressing human (lane 2) or mouse (lane 3) heparanase were separated electrophoretically on 4- 12% NuPAGE gel (Novex Ltd, USA), blotted onto PVDF membrane, and reacted with HP 135.108 hybridoma supernatant.

FIGs. 17A-17C demonstrate the specific recognition of human and mouse recombinant heparanase by purified polyclonal anti-heparanase antibodies.

Purified recombinant human heparanase (20ng, lane or cell extracts from CHO cells expressing human (lane 2) or mouse (lane 3) heparanase were separated electrophoretically on a 4-12% Nu Page gel (Novex Ltd., USA), blotted onto PVDF membrane, and reacted with purified polyclonal goat- or rabbit- anti-heparanase antibodies. Extracts from mock-transfected dhfr" CHO cells (lane 4) served as controls.

Figure 17A shows the specificity of affinity purified polyclonal goat antiheparanase subunit (GapH45) for the large subunit of purified recombinant human (lane recombinant human from CHO extract (lane 2) heparanase.

Note the recognition of heparanase species at p45 (large subunit) and (proheparanase), and not of the small (p8) subunits.

Figure 17B shows the specificity of protein G-purified polyclonal goat anti-heparanase (GH53), raised against recombinant active (p45/p8) human heparanase, for both the large and small subunits of purified recombinant human heparanase (lane 1) and recombinant human heparanase from CHO extract (lane Note the recognition of heparanase species at p45 (large subunit) and p60 (proheparanase) as well as of the small (p8) subunits.

Figure 17C shows the specificity of protein G-purified polyclonal rabbit anti-heparanase (RH53), raised against recombinant active (p 4 5/p8) human heparanase, for both the large and small subunits of purified recombinant human heparanase (lane 1) and recombinant human heparanase from CHO extract (lane Note the recognition of heparanase species at p45 (large subunit) and p60 (proheparanase) as well as of the small (p8) subunits.

FIG. 18 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 pRSEThpaS I (two different colonies). Molecular size in kDa is shown to the left (Prestained SDS-PAGE standards, Bio-Rad, CA).

FIG. 19 is a schematic presentation of the expression vector Sheparanase. Relative positions of some restriction enzymes and genes are indicated. For the construction and utilities of pPIC3.5K-Sheparanase, see Example XI in the Examples section below.

FIG. 20 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 pPIC3.5K-Sheparanase, see Example XI in the Examples section below.

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FIG. 21 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. 22a-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.

FIGs. 23a-b show Western blot analysis of hpa transfected cells. Cell extracts (40lg of CHO cells or 8pg 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 23a CHO stable cellular clones (lanes 1-3) and transiently transfected 293 human cells (lane Figure 23b 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. 24a-b demonstrate recombinant heparanase secretion induced by calcium ionophore and PMA. Cells of a stable CHO clone (2TT1) were 42 induced with either calcium ionophore (Figure 24a) or PMA (Figure 24b).

Conditioned media were collected and 20ml 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. 24c demonstrates recombinant heparanase secretion by human 293 cells. Conditioned media of human 293 cells transfected with pSihpa (lanes 3 and pS2hpa (lanes 5 and 6) or control, untransfected cells (lanes 1 and 2), were loaded on a denaturative 4-20 polyacrylamide gel (lanes 1, 3 and or fold concentrated by 10 kDa ultrafiltration tube (Intersep (lanes 4 and Heparanase was detected by Western blot analysis with a rabbit antiheparanase 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. 25a demonstrates heparanase activity as expressed by the ability to degrade heparin. Following overnight incubation with 50ml 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 1mg/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.

FIG. 25b-c demonstrate recombinant heparanase activity following secretion induced by calcium ionophore as determined by the soluble 35

S-ECM

degradation assay. 25b 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 determnined by the soluble 3S-ECM degradation assay. 25c 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. 25c-g show the relative heparanase activity of p70 and p52 (see Figures 25b-c) by comparing the ability of diluted (x2, x4 or x8) conditioned media to degrade 35

S-ECM.

FIG. 26 demonstrates glucose consumption record of heparanase producing cells in a large scale, 0.5 liters, Spinner-Basket bioreactor.

FIG. 27 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 (n) 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 ofpF2 infected cells.

FIGs. 28a-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 (28a) or pFhpa4 (28b) viruses or with control viruses were incubated (18 h, 37°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. 29 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

I

the peak I substrate, into peak II HS degradation fragments) was found in the high (>50 kDa) but not low 50 kDa) molecular weight compartment.

FIGs. 30a-b demonstrate the effect of heparin on heparanase activity expressed by pFhpa2 and pFhpa4 infected High five cells. Culture media of pFhpa2 (30a) and pFhpa4 (30b) infected High five cells were incubated (18 h, 37°C) with sulfate labeled ECM-derived soluble HSPG (peak I, 0) in the absence or presence of 10g/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. 31a-b demonstrate degradation of sulfate labeled intact ECM by virus infected High five and Sf21 cells. High five (31a) and Sf21 (31b) cells were plated on sulfate labeled ECM and infected (48 h, 28°C) with pFhpa4 or control pF 1 viruses. Control non-infected Sf21 cells were plated on the labeled ECM as well. The pH of the cultured medium was adjusted to 6.0 6.2 followed by 24 h incubation at 37C. 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. 32a-b demonstrate degradation of sulfate labeled intact ECM by virus infected cells. High five (32a) and Sf21 (32b) cells were plated on sulfate labeled ECM and infected (48 h, 28°C) with pFhpa4 or control pF1 (c) viruses. Control non-infected Sf21 cells 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. 33a-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 pF1 viruses were incubated (48 h, 37 0 C, pH 6.0) 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 ofpFhpa4 infected cells.

FIGs. 34a-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°C, pH 6.0) with growth medium of pFhpa4 infected High five (34a) and Sf21 (34b) cells in the absence or presence of 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. 35 demonstrates the purification of recombinant heparanase by a Source-S column. Lanes 1-14, 40ml 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. 36 demonstrates Western blot analysis of fractions 1-14 of Figure 35. 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. 37 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. 38a demonstrates the production of heparanase in pSlhpa transfected BHK21 cells. Cell extracts (2 x10 5 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. 38b 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 g 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. 39a demonstrates recombinant heparanase constitutive secretion by CHO cells transfected with pSlhpa (clone S1PPT-8). Conditioned media of untreated cells (lane mock treated cells (lane 3) and calcium ionophore treated cells (0.1 g/ml for 24 hours; lane 4) were electrophoresed next to a cellular extract of 1x10 5 cells from clone 2TT1 (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 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. 39b demonstrates recombinant heparanase constitutive secretion by CHO cells transfected with pShpaCdhfr (2TT1 clones). Conditioned media (150gl, concentrated by 10 kDa ultrafiltration tube) of 2TTI-2 clone (lane 2) and of clone 2TT1-8 (lane 3) were electrophoresed next to a cellular extract of 5 cells from clone 2TT1 (lane Samples were separated on a 4-20% gradient SDS-PAGE, followed by Western blot analysis with a rabbit antiheparanase 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. 40a demonstrates purification of recombinant heparanase from a mammalian cellular extract by ion exchange chromatography. 2TT1-8 CHO cells (lx 108) were extracted in 2.5ml of 10mM 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 Pharmacia Biotech) was equilibrated with 20mM 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 1M sodium chloride in 20mM 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 214nm and fractions of Iml each were collected, starting with the first fraction which was eluted after 13 minutes and which is identified by the arrowhead mark.

FIG. 40b 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. 2 0

M

1 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.

_I FIG. 40c demonstrates the presence of catalytically active recombinant heparanase in mammalian cellular extract fractions. An aliquot (30jl) from each fraction that was collected was analyzed for heparanase activity by the DMB assay. Load extract prior to purification. 5-7 and 16-26 correspond to fraction Nos.

FIG. 40d 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. Patent No.

6,190,875.

FIG. 41a 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 2TTI-8 cells on mono-S column; B extract of 2TT1-8 cells; C unbound, flow through proteins; and D wash fraction proteins.

FIG. 41b 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 9-1 was determined using the DMB assay. Load extract prior to purification; C unbound, flow through proteins; and D wash fraction proteins.

FIGs. 42a-b demonstrates proteolytic processing of heparanase from insect cells conditioned medium by protease impurities. Figure 42a shows a

I

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 0 C in either, phosphate citrate buffer pH 7, containing 5% PEG 300 (lane phosphate citrate buffer pH 4, containing 5% PEG 300 and 1 x protease inhibitors cocktail (Boehringer Mannheim, Cat. No. 1836170, lane or phosphate citrate buffer pH 4, containing 5% PEG 300 (lane M- Molecular weight markers (NEB Cat. No. 7708S). Figure 42b shows the results of DMB heparanase activity assays for the proteins.

FIGs. 42c-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°C in 20mM 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 42c) or to heparanase DMB activity assay (Figure 42d). 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. 43a demonstrates proteolytic processing of heparanase secreted from insect cells by trypsin. 10lg 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 0 C. Following incubation, reaction tubes were placed on ice and 1.7jg/ml aprotinin (trypsin inhibitor) was added. Activity was determined using the DMB assay.

FIG. 43b demonstrates a Western blot analysis of heparanase following trypsin treatment. 10g 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 42a-b, lanes J (lane and heparanase from a CHO 2TT1 cell extract (lane 5) served as controls.

FIG. 44 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 (0.5pg heparanase per reaction) in 20mM phosphate buffer, pH 6.8, was incubated with 0, 1.5, 15 or 150 units of trypsin for 10 minutes, at 37 0

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. 45a-b demonstrates proteolytic processing of p70-bac heparanase by cathepsin L. Partially purified heparanase from insect cells (10g) was subjected to proteolysis by 1.6 mU cathepsin L (Cat. No. 219412, Calbiochem) for 3 hours, at 30 0 C, in 20mM citrate-phosphate 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 45a-b, respectively.

FIG. 46a demonstrates a hydropathy plot of SEQ ID NO:4 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. 46b is a schematic depiction of modified heparanase species (prep56' and pre-p52') that contain a unique protease recognition and cleavage sequence of factor Xa Ile-Glu-Gly-ArgJ or of enterokinase Asp-Asp-Asp- Asp-LysJ (shaded regions, located between amino acids 119 and 120 or 157 and 158 of the heparanase enzyme depicted in SEQ ID NO:4, which acids are located within peaks I and II, respectively, of Figure 46a) which enable proteolytic processing by the respective proteases to obtain homogeneously processed and highly active heparanase species (p56' and p52', respectively).

FIG. 46c 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-Argi- or of enterokinase Asp-Asp-Asp-Asp-Lys FIG. 47 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. 48 presents a comparison between nucleotide sequences of the human hpa and a mouse EST cDNA fragment (SEQ ID NO:37) which is homologous to the 3' end (starting at nucleotide 1066 of SEQ ID NO:12) of the human hpa. The aligned termination codons are underlined.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is of specific anti-heparanase polyclonal and monoclonal antibodies which can be used to detect heparanase and/or to inhibit heparanase catalytic activity. In particular, the present invention is of antiheparanase antibodies which bind specifically to at least one epitope of heparanase. Optionally and preferably, the heparanase has sequence homology to human heparanase. The antibody can optionally be used to treat and diagnose conditions associated with heparanase catalytic activity, for purification of heparanase, and for drug development in heparanase associated conditions.

As shown in greater detail below, antibodies capable of binding specifically to at least one epitope may optionally be raised against one or more peptides selected from the heparanase sequence, a portion of heparanase or recombinant heparanase, in any of its forms (including but not limited to preproheparanase, proheparanase, one or both domains of active heparanase, one or both of the subunits of the heparanase heterodimer etc.). Such antibodies would be useful for immunodetection and diagnosis of micrometastases, autoimmune lesions and renal failure in biopsy specimens, plasma samples, and body fluids. Such antibodies may also serve as neutralizing agents for heparanase activity.

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 are for the purpose of description and should not be regarded as limiting.

Herein after the phrase "polynucleotide sequence" also means a nucleic acid sequence, typically a DNA sequence.

Herein after 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.

Herein after the term "polypeptide" also means a protein.

Herein after the term "protein" also refers to a polypeptide. The protein can be recombinant or natural. The protein can be a portion of the full recombinant or natural protein. The protein preparation used for vaccination can be crude, partially purified or highly purified.

Herein after 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.

Herein after the term "subunit" refers to any polypeptide which takes part in forming an active protein.

Herein after 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 3-elimination (72).

Heparanase activity which is inhibited or neutralized according to the present invention can be of either recombinant or natural heparanase. Such activity is disclosed, for example, in U.S. Patent. Application Nos. 09/071,739; 09/071,618; and 09/113,168, which are incorporated by reference as if fully set forth herein.

Herein after 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.

Herein after 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.

Herein after 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, glycosylation, alkylation (e.g.

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.

Herein after the phrase "associated with heparanase expression" refers to conditions which at least partly depend on expression of heparanase. It is being understood that the expression of heparanase under many such conditions can be normal, yet inhibition thereof in such conditions will result in improvement of the affected individual.

Herein after 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. The immunoglobulin could also be a "humanized" antibody, in which, for example animal (say murine) variable regions are fused to human constant regions, or in which murine complementarity-determining regions are grafted onto a human antibody structure (Wilder, R.B. et al., J. Clin. Oncol., 14:1383-1400, 1996). Unlike, for example, animal derived antibodies, "humanized" antibodies often do not undergo an undesirable reaction with the immune system of the subject.

The terms "sFv" and "single chain antigen binding protein" refer to a type of a fragment of an immunoglobulin, an example of which is scFv CC49 (Larson, S.M. et al., Cancer, 80:2458-68, 1997).

Monoclonal antibodies or purified fragments of monoclonal antibodies containing at least a portion of an antigen binding region, such as Fv, F(abl)2, Fab fragments single chain antibodies Patent No. 4,946,778), chimeric or humanized antibodies (55,56) and complementarily determining regions (CDR) may be prepared by conventional procedure. Purification of the serum immunoglobulin antibodies or fragments can be accomplished using a variety of methods known to those of skill in the art, 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. (57).

The term "antibody" as used in this invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab')2, and Fv that are capable of binding to macrophages. These functional antibody fragments are defined as follows: Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds; Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and Single chain antibody a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.

Herein after the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.

Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.

Herein after the term "humanized" refers to an antibody which includes any percent above zero and up to 100% of human antibody material, in an amount and composition sufficient to render such an antibody less likely to be immunogenic when administered to a human being. It is understood that the term "humanized" reads also on human derived antibodies or on antibodies derived from non human cells genetically engineered to include functional parts of the human immune system coding genes, which therefore produce antibodies that are fully human.

Herein after the term "inhibit" refers to the suppression of activity, or to the restraining of free expression.

Herein after the term "neutralize" is specifically used in context of a single heparanase molecule which can either be neutralized or active.

Herein after the term "C'-terminal portion" refers to a continuous or discontinuous epitope or epitopes involving amino acids derived from any location or locations, either continuous or dispersed, along the about 80 C'terminal amino acids of heparanase. Continuous or discontinuous epitopes typically include from about 3 to about 8 continuous or discontinuous amino acids.

Herein after the terms "treat" or "treating" include substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical symptoms of a condition or substantially preventing the appearance of clinical symptoms of a condition.

According to an aspect of the present invention there is provided an antibody comprising an immunoglobulin capable of specifically binding at least one epitope of a recombinant or natural heparanase protein. The immunoglobulin therefore recognizes and binds native non denatured) natural or recombinant heparanase.

According to a further aspect of the present invention there is provided an affinity substrate comprising a solid matrix and an immunoglobulin immobilized thereto, capable of specifically binding at least one epitope of a recombinant or natural heparanase. 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 capable of specifically binding at least one epitope of a recombinant or natural heparanase which is immobilized thereto; washing the affinity column, for example using salt solution in a low concentration, say 0- 500mM; and eluting heparanase molecules being adsorbed on the affinity column via the immunoglobulin, for example using a highly concentrated, say 0.5-1.5M, salt solution.

While reducing the present invention to practice, the present inventors have produced specific polyclonal and monoclonal antibodies capable of specifically binding at least one epitope of human and mouse heparanase, which can be used as heparanase activity neutralizing antibodies, as sitespecific anti-heparanase antibodies capable of distinguishing between mature and unprocessed forms of the enzyme, and which can be used for diagnostic or drug development applications. U.S. Patent Application No. 09/759,207, to Pecker et al, also discloses antibodies recognizing mouse heparanase protein from B 16-F10 cells, as well as human platelet heparanase and a recombinant heparanase enzyme expressed in several human tumor and CHO cell lines.

Further, U.S. Patent Application No. 09/759,207, to Pecker et al, and U.S.

Patent Application No. 09/666,390 to Goldshmidt et al, which are incorporated herein by reference as if fully set forth herein, teach the generation and characterization of polyclonal and monoclonal antibodies that are cross reactive with human, mouse and chicken heparanase. The 50 kDa human heparanase enzyme represents an N-terminal processed enzyme, which is at least 200-fold more active than the full-length 65 kDa protein (Vlodavsky I.et al Nature Med.

1999;5: 793-802). Heparanase proteins purified from different human and animal sources have also been shown to have structural similarity, aside for sharing similar substrate specificities, yielding similar oligosaccharide cleavage products and being inhibited by heparin substrate derivatives.

The similarity in structure between diverse heparanase proteins is reflected in the amino acid sequences. Table 1, brought below, shows the aligned amino acid sequences of rat, mouse, chicken and human heparanase.

mouse rat human chicken mouse rat human chicken mouse rat human chicken Table 1: HEPARANASE SEQUENCE HOMOLOGY DATA 20 30 40 50 I I I I II

-MLR-------LLLLWLWGPLGALAQGAPAGTAPTDDVVDLEFYTKRPLRSVSPSFLSIT

-MLRP------LLLLWLWGRLRALTQGTPAGTAPTKDVVDLEFYTKRLFQSVSPSFLSIT

MLLRSKPALPPPLMLLLLCPLGPLSPGALPRPAQAQDVVDLDFFTQEPLHLVSPSFLSVT

MLVLLLLVLLLAVPP--------RR-TAELQLGLREPIGAVSPAFLSLT

80 90 100 110 120 I I I I I 1

IDASLATDPRFLTFLGSPRLRALARGLSPAYLRFGGTKTDFLIFDPDKEPTSEERSYWKS

IDASLATDPRFLTFLGSPRLRALARGLSPAYLRFGGTKTDFLIFDPNKEPTSEERSYWQS

IDANLATDPRFLILLGSPKLRTLARGLSPAYLRFGGTKTDFLIFDPKKESTFEERSYWQS

LDASLARDPRFVALLRHPKLHTLASGLSPGFLRFGGTSTDFLIFNPNKUSTWEEKVLSEF

*t *t.

130 140 150 160 170 180 Ill I I QVNHDICRSEPVSAAVLRKLQVEWPFQELLLLRE YQKEFKN TYSRSSVDMLYSFAKCS QDNNDICGSERVSADVLRKLQMEWPFQELLLLRE(YQREFKN TYSRSSVDMLYSFAKCS QVNQDICKYGSIPPDVEEKLRLEWPYQEQLLLRE YQKKFKN TYSRSSVDVLYTFANCS QAK-DVCEAWPSFAVVPKLLLTQWPLQEKLLLAE SWKKHKN TITRSTLDILHTFASSS t t 190 200 210 220 230 240 I I I I GLDLIFGLNALLRTPDLRWNSSNAQLLLDYCSSKGYNISWELGNEPNS

WRKAHILIDGL

RLDLIFGLNALLRTPDLRWNSSNAQLLLNYCSSKGYNISWELGNEPNSFWKKAQISIDGL

GLDLIFGLNALLRTADLQWNSSNAQLLLDYCSSKGYNISWELGNEPNSFLKKADIFINGS

GFRLVFGLNALLRRAGLQWDSSNAKQLLGYCAQRSYNISWELGNEPNSFRKKSGICIDGF

A A. r* mous rat human chicken .1 mouse rat human chicken mouse rat human chicken 250 260 270 280 290 300 1 1 I 1I

QLGEDFVELHKLLQRS-AFQNAKLYGDIGFRXT-VKLRSFLKAGGEVIDSLWHHYY

QLGEDPVELHKLLQKS-AFONAK:YGPDIGO4PRGKTV4 LRSFLKAGGEVIDSLTWHHYY QLGEDYIQLHKLLRKS-FKNACLYGPDV~hPRRKTAK$LKSFLKAGGEVIDSVTNHHYY QLGRDFVHLRLLSHPLYRHAELYGLDvGz QH LRSFMKSGGKNLDSVTNHHYY 310 320 I I I I I I LNGRIATKEOFLSSDALDTFILSVQKI LKVTKEITFGKKVWLGETS SAYGGGAPLLSNTF LNCRVATKEDFLSSOVLDTFILSVQKI LKVTKE4T PGKE{VWLGETS SAYGGGAPLLSNTF LNGRTATREDFLNPDVLDI FIS SVQKVFQVVES DRPGKKVWLGETS SAYGGGAPLLSDTF VNGRSATREDFLSPEVLDS FATAIHDVLGIVEA'rVPGKKVWLGETOSAYGGGAPQLSNTy 370 380 390 400 410 LU mouse rat human chicken mouse 30 rat human chicken I I I I II AAGFMWLDRLGLSAQr4GTEVVMRQVFFGAGNYHLVDENFEPLPDYWLSLLFKKLVGPRVL

AAGFMWLDKLGLSAQLOIEVVMRQVFFGAGNYHLVDENFEPLPDYWLSLLFKKLVGPKVL

AAGFt4WLDKLGLSARNGI EVVtRQVFFGAGNYHLVDENF0PLPDYWLSLLFKKLVGTKVL VAGFMWLDKLGLAARRGI DVVMRQVSFGAGSYHLVDAGFKPLPDYWLSLLYKRLVGTRVL 430 440 450 460 470 480 iI I I II LSRVKG IDRSKLRV'LHCTNVYHRYQEGDLTLYVLNLHNVTKHLKVPPPLFRKPVDTYL MSRVKGI*DRSKLRVA LHCTNVYHPRYREGDLTLYVLNLHNVTKHLKLPPPMFSRPVDKYL MASVOG4 KRRKLRVA LHCTNTDNPRYEEGDLTLYAINLHNVTKYLRLPYPFSNKQVDKYL QtSVEQIThARZRV LHCTNPRHPKYREGDVTLFALNLSNVPQSLQLPKQLWSKSVD)QYL 490 500 51C 520 530 540 1 1 1 1I LKPSGPDGLLSKSVOLNGQILKMVDEOTLPALTEKPLPAGSALSLFAFSYGFFV:

RNAKI

LKPFGSDGLLSKSVQLNGQ-TZKMVDEQTLPALTEKPLPAGSSLSVPAFSYGFFVIRNAKI

LRPLGPHGLLSE{SVQLNGLTLKMVDDQTLPPLMEKPLRFGSSLGIPAFSYSFFVI

RNAKV

LLPHGKDS ILSREVQLNGRLLQMVDDETLPALEEMALAPGSTLGLPAFSYGFYVI RNAKA *AC ID* AACI (SEQ ID NO:2( AACT (SEQ ID NO:4) lACI (SEQ ID mouse rat human chicken mouse rat human chicken Muitiple alignment of heparanase from Human, Rat, Mouse and chicken generated by Clustal W. Active site residues are bolded and putative heparin binding sites are boxed.

As demonstrated in Table 1, heparanase polypeptides derived from chicken, rat, mouse and human have a range of overall amino acid sequence homology, with mouse and rat being the closest, and chicken and rat being the most distant. Overall interspecies amino acid homology for heparanase is, in ascending order: chicken (SEQ ID NO:2) and rat (SEQ ID NO:3) (66.1% similarity, 55% identity); chicken and human (SEQ ID NO:4) (68% similarity, 6 1.3% identity); chicken and mouse (SEQ ID NO:5) (67.4% similarity, 60.3%

I

identity); human and mouse (80% similarity, 75.9% identity); human and rat (80.6% similarity, 75.6% identity) and mouse and rat (94.2% similarity and 92.7% identity).

As detailed in the Background section above, several observations regarding the structure of heparanase polypeptide and related enzymes, have provided detailed structure-function correlations for a number of specific peptide sequences found within the complete heparanase amino acid sequence.

As has been demonstrated for a number of biologically active proteins, functional domains in proteins from different sources often display sequence homology, indicating similarity in the three-dimensional configuration and close structure-function homology. Thus, the functional sites identified within the heparanase polypeptide display a range of homology percentages between rat, mouse, chicken and human heparanase which is different from that of the overall homology percentage range, ranging from less than 60% (chick-human at peptide pep38, SEQ ID NO:6, coordinates 437-466 of SEQ ID NO: 4, Table 1) to greater than 85% (chicken-human at peptide pep8, SEQ ID NO:8, coordinates 219- 233 of SEQ ID NO: 4, Table 1).

The relationship between functional epitopes and specific regions having similar sequence homologies was previously investigated. Peptides representing the C-terminus of the P8 subunit, that participate in the dimerization of the 45 kDa (SEQ ID NO:l) and 8 kDa (SEQ ID NO:ll) components of the mature, processed human heparanase heterodimer (peptide p8#7, SEQ ID NO.7), a region in proximity to the heparin binding site (peptide pep38, SEQ ID NO.6), a sequence comprising the proton donor residue of the active site (peptide pep8, SEQ ID NO.8), a region comprising the nucleophilic residue of the active site (peptide pep9, SEQ ID NO.9), and a region linking the active and binding site (peptide pep 10, SEQ ID NO:10) have been identified.

While reducing the present invention to practice, peptides representing these specific sequences were used for immunization of animals to produce the specific anti-heparanase antibodies of the present invention (see Materials and Experimental Procedures hereinbelow). Without wishing to be limited to a single hypothesis, as demonstrated hereinbelow, the resultant anti-heparanase antibodies are capable of binding to the specific sequences. However, in the context of the present invention it is noted that the specific anti-heparanase antibodies disclosed herein can be used for the methods and compositions described herein regardless of the accuracy of the proposed function of the immunizing peptides.

Interspecies homology between equivalent functional domains of biologically active proteins, may be reflected in similar antigenicity of the characteristic epitopes, as anti-heparanase antibodies having cross-reactivity between human and non-human heparanases have been disclosed. US Patent Application No. 09/930,218 to Goldshmidt et al, incorporated herein by reference as if fully set forth herein, discloses cross reactivity of a monoclonal antibody generated against human heparanase (FHP130, see Materials and Experimental Procedures hereinbelow and U.S. Patent Application No.

08/922,170, incorporated herein by reference as if fully set forth herein).

HP130 effectively detected both human and mouse heparanase on Western blots Patent Application 09/759,207, incorporated herein by reference as if fully set forth herein), and also detected recombinant human, chick and chimeric chick-human heparanase expressed in C6 rat glioma and Eb lymphoma cells on Western blot analysis and cell immunostaining Patent Application No. 09/930,218 to Goldshmidt et al, now U.S. Patent No.

6,677,137 incorporated herein by reference as if fully set forth herein).

Analysis of the interspecies homology of the immunologically active heparanase peptide fragments of the present invention, discloses diverse crossspecies conservation. The site specific anti-heparanase antibodies of the present invention display interspecies cross-reactivity (see Figures 4 and 5, Example III, hereinbelow), indicating that the interspecies sequence homology is reflected in the three-dimensional configuration, conferring both immunological and functional similarity across species.

A typical monoclonal antibody may be expected to recognize a unique short stretch of amino acids (for example, about 6 amino acids, although this region may be larger or smaller) or a different structural component of similar size. Such interspecies-conserved short sequences are dispersed along the entire protein sequence, and they are specifically concentrated in functional regions.

As demonstrated in Table 1 hereinabove, the regions comprising the epitopes recognized by antibodies HP239 (coordinates 130-230 of SEQ ID NO:4, determined by epitope mapping described hereinbelow) and HP130 (coordinates 465-543 of SEQ ID NO: 4, determined by epitope mapping, described hereinbelow), demonstrate a lower level of overall homology (chickhuman less than despite the strong inter-species cross-reactivity of the monoclonal antibody HP130, which is described hereinabove. This immunological cross-reactivity, along with the conservation of functional sites, indicates that anti-heparanase antibodies of the present invention can effectively bind to and neutralize a wide range of heparanase enzymes from diverse species, having moderate levels of overall sequence homology.

Thus, according to another aspect of the present invention there is provided an isolated antibody or portion thereof capable of specifically binding to at least one epitope of a heparanase protein.

According to a preferred embodiment of the present invention, the isolated antibody or portion thereof binds specifically to a heparanase protein having a sequence at least 60% homologous, preferably at least homologous, more preferably at least 80% homologous, most preferably at least 90% homologous to the amino acid sequence of any of SEQ ID NOs: and 11. In a more preferred embodiment, the heparanase protein comprises an amino acid sequence as set forth in any of SEQ ID NOs: 1-5 and 11.

According to another preferred embodiment of the present invention the at least one epitope of a heparanase protein is at least 60% homologous, preferably at least 70% homologous, more preferably at least 80% homologous, most preferably at least 90% homologous to the amino acid sequence of any of SEQ ID NOs: 6-10. In a more preferred embodiment, the at least one epitope of a heparanase protein comprises an amino acid sequence as set forth in any of SEQ ID NOs: 6-10.

According to further preferred embodiments of the present invention, the at least one epitope of a heparanase protein comprises a sequence at least homologous, preferably at least 80% homologous, more preferably at least homologous to the amino acid sequence of SEQ ID NO: 6.

According to a still further preferred embodiment, the at least one epitope of a heparanase protein comprises a sequence at least 90% homologous to the amino acid sequence of SEQ ID NO:8.

According to a still further preferred embodiment, the at least one epitope of a heparanase protein comprises a sequence at least 90% homologous to the amino acid sequence of SEQ ID NO:9.

According to a still further preferred embodiment, the at least one epitope of a heparanase protein comprises a sequence at least 90% homologous to the amino acid sequence of SEQ ID According to still further preferred embodiments, the at least one epitope of a heparanase protein comprises a sequence at least 75% homologous, preferably at least 80% homologous, more preferably at least 90% homologous to the amino acid sequence of SEQ ID NO:7.

The isolated antibody of the present invention can be a polyclonal or a monoclonal antibody. The polyclonal and monoclonal antibodies of the present invention can be chimeric antibodies, humanized antibodies, Fab fragments, single-chain antibodies, immobilized antibodies and labeled antibodies. In one embodiment, the polyclonal antibodies of the present invention are crude antibodies, and in another preferred embodiment the polyclonal antibodies are affinity purified antibodies. In another embodiment, the monoclonal antibodies of the present invention are selected from the group consisting of HP130, HP 239, HP 108.264, HP 115.140, HP 152.197, HP 110.662, HP 144.141, HP 108.371, HP 135.108, HP 151.316, HP 117.372, HP 37/33, HP3/17, HP 201 and HP 102.

Methods for affinity purification of anti-heparanase polyclonal antibodies are described in US Patent Application No. 09/944,602, which is incorporated herein by reference as if fully set forth herein. Briefly, polyclonal antiserum raised against human recombinant heparanase was incubated with gel-purified heparanase, transferred to a nitrocellulose membrane under conditions suitable for formation of heparanase protein-antibody immune complexes. Next, the nitrocellulose membranes are washed, and the bound affinity purified antiheparanase antibodies are eluted from the membranes with 0.1N Glycine pH 2.8, pH adjusted and dialyzed with PBS.

As detailed above and in the Examples section hereinbelow, the regions and peptides comprising the epitopes recognized by the isolated antiheparanase antibodies of the present invention have been well characterized (see, for example, Table 2, hereinbelow). Thus, according to another preferred embodiment, the isolated antibody or portion thereof of the present invention binds specifically to at least one epitope selected from the group consisting of a heparan-sulfate binding site flanking region, a catalytic proton donor site, a catalytic nucleophilic site, an active site and binding site linking sequence and a C-terminus sequence of heparanase P8 subunit. Peptides were designed to elicit antibodies that would block activity either by direct interaction with functional sites (pep8 and pep9) (SEQ ID NOs: 8 and 9, respectively) or by a steric interference by binding to a structurally adjacent region (pep 38 and pep (SEQ ID NOs. 6 and 10, respectively).

According to another embodiment of the present invention, the heparan sulfate binding site flanking region comprises an amino acid sequence at least homologous to the amino acid sequence as set forth in SEQ ID NO:6 (pep38). In a preferred embodiment, the heparan sulfate binding site flanking region comprises an amino acid sequence at least 70% homologous, more preferably at least 80% homologous, most preferably at least 90% homologous to the amino acid sequence as set forth in SEQ ID NO:6. In a most preferred embodiment, the heparan sulfate binding site flanking region comprises an amino acid sequence as set forth in SEQ ID NO:6 (pep38).

According to yet another embodiment, the catalytic proton donor site comprises an amino acid sequence at least 90% homologous to the amino acid sequence as set forth in SEQ ID NO:8 (pep8). In a more preferred embodiment, the catalytic proton donor site comprises an amino acid sequence as set forth in SEQ ID NO:8 (pep8).

According to still another embodiment, the catalytic nucleophilic residue site comprises an amino acid sequence at least 90% homologous to the amino acid sequence as set forth in SEQ ID NO:9 (pep9). In a more preferred embodiment, the catalytic nucleophilic residue site comprises an amino acid sequence as set forth in SEQ ID NO:9 (pep9).

According to yet another embodiment, the active site and binding site linking sequence comprises an amino acid sequence at least 90% homologous to the amino acid sequence as set forth in SEQ ID NO:10 (pep10). In a more preferred embodiment, the active site and binding site linking sequence comprises an amino acid sequence as set forth in SEQ ID NO:10 (pep According to still another embodiment, the C-terminal sequence of heparanase P8 subunit comprises an amino acid sequence at least homologous to the amino acid sequence as set forth in SEQ ID NO:7 (pep8#7).

In a preferred embodiment, the C-terminal sequence of heparanase P8 subunit comprises an amino acid sequence at least 80% and more preferably homologous to the amino acid sequence as set forth in SEQ ID NO:7. In a most preferred embodiment, the C-terminal sequence of heparanase P8 subunit comprises an amino acid sequence as set forth in SEQ ID NO:7 (pep8#7).

Specific anti-heparanase antibodies of the present invention include, but are not limited to monoclonal antibodies such as HP130 (binds specifically to an epitope within the C-terminus of the heparanase polypeptide in the portion of the sequence between amino acid coordinates 465 and 543 of SEQ ID NO:4), HP 239 (binds specifically to an internal epitope of the heparanase polypeptide in the portion of the sequence between amino acid coordinates 130 and 230 of SEQ ID NO:4), HP 108.264, HP 115.140, HP 152.197, HP 110.662, HP 144.141, HP 108.371, HP 135.108, HP 151.316, and HP 117.372 which bind specifically to an epitope within the region of the heparanase precursor defined by amino acid coordinates 320 to 410 of the heparanase polypeptide (SEQ ID NO:4). Other specific anti-heparanase antibodies include monoclonal antibodies elicited against specific, defined heparanase peptides such as HP 37/33 and HP3/17 (anti-pep9, SEQ ID NO:9, amino acid coordinates 334-348 of SEQ ID NO:4), HP 201 (anti-pepl0, SEQ ID NO:10, amino acid coordinates 297-307 of SEQ ID NO:4) and HP 102 (anti-pep38, SEQ ID NO:6, amino acid coordinates 437-446 of SEQ ID NO:4), and polyclonal antibodies GH53 (goat anti-intact, active heparanase heterodimer antibody), RH53 (rabbit anti-intact, active heparanase heterodimer antibody), and GapH45 (affinity purified goat anti p45 heparanase subunit).

Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference as if fully set forth herein).

Several heparanase-specific antibodies have been previously described (see Background section hereinabove). However, analysis of a number of the early anti-heparanase antibody preparations reported, revealed the presence of contaminating, non-relevant cross reacting antibodies, such as anti-PAI-1, making their use in diagnostic and therapeutic applications impractical and unreliable. In stark contrast to such poorly defined antibodies, the antibodies and pharmaceutical compositions of the present invention comprise solely heparanase-specific antibodies, as determined by Western blot, inhibition of catalytic activity, and epitope mapping, as detailed in the Examples section hereinbelow.

Antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.

For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, enzymatic cleavage using pepsin directly produces two monovalent Fab' fragments and an Fe fragment. These methods are described, for example, by Goldenberg, U.S.

Patent. Nos. 4,036,945 and 4,331,647, and references contained therein, which are hereby incorporated by reference in their entirety. See also Porter, R. R.

[Biochem. J. 73: 119-126 (1959)]. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad.

Sci. USA 69:2659-62 (1972)]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains, connected by an oligonucleotide.

The constructed structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cell synthesizes a single polypeptide chain with a linker peptide bridging the r two V domains. Methods for producing sFvs are described, for example, by Whitlow and Filpula, Methods 2: 97-105 (1991); Bird et al., Science 242:423- 426 (1988); Pack et al., Bio/Technology 11:1271-77 (1993); and U.S. Patent.

No. 4,946,778, which is hereby incorporated by reference in its entirety.

Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction (PCR) to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].

Humanized forms of non-human murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequences derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a nonhuman species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. Optimally, the humanized antibody will also comprise at least a portion of an immunoglobulin constant region typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)]. Examples of humanized monoclonal antibodies having CDRs of murine or rat origin include Campath (Millenium Pharmaceuticals Cambridge Mass), specific for CD54, Zenapax (Protein Design Labs, Fremont, CA) specific for CD25, and D1.3 (MRC, LMB, Cambridge, UK), specific for lysozyme.

Methods for humanizing non-human antibodies are well known in the art.

Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import residues", which are typically taken from an import variable domain. Humanization can essentially be performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies Patent. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol.

Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be produced by introduction of human immunoglobulin loci into transgenic animals, for example mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed in the transgenic animals, in a process that closely resembles the process seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technology 10,: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et al. Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995).

Additional details concerning antibody humanization are found in references 81-83 which are incorporated as if fully set forth herein. Examples of human antibodies include the anti-cytokeratin anti-tumor Mab Humaspect (Organon, CA), AL-901 (Tanox Biosystems and Genentech, CA) specific for IgE; HuMax EGFR (GenMab A/S, Copenhagen, DK) specific for human EGFR and the anti-hepatitis B Ostavir (Protein Design Labs, Fremont, CA).

Thus, in accordance with one aspect of the teachings of the present invention there are provided isolated polyclonal and monoclonal antibodies elicited by at least one epitope of a heparanase protein. The polyclonal and monoclonal antibodies of the present invention can be chimeric antibodies, humanized antibodies, Fab fragments or single-chain antibodies. In one embodiment, the polyclonal antibodies of the present invention are crude antibodies, and in another, preferred embodiment the polyclonal antibodies are affinity purified antibodies. Methods for affinity purification of anti-heparanase polyclonal antibodies are described in U.S. Patent Application No. 09/071,739.

The anti-heparanase antibodies of the present invention are capable not only of specific binding to, or interacting with, heparanase, but also specifically inhibiting or neutralizing heparanase catalytic activity (see Example II hereinbelow).

According to a preferred embodiment of the present invention at least about 60%, preferably, at least about 70%, more preferably, at least about and most preferably at least about 90% of the heparanase activity is abolished by the inhibition when from about 1 to about 1-40, preferably from about 2 to about 30, more preferably from about 4 to about 20, most preferably from about 5 to about 15 ratio of heparanase to antibody is realized, either, in situ, in loco, in vivo or in vitro.

As specifically shown in the Examples section hereinunder (Example II), antibodies binding specifically the C-terminal portion (HP 130) and to the nucleophilic residue of the heparanase active site (HP 3/17 and HP 37/33) of heparanase were effective in neutralizing significant proportions of heparanase catalytic activity, indicating that the C'-terminal portion, as well as the nucleophilic residue of the active site of heparanase is involved in its catalytic activity.

According to another aspect of the present invention there is provided an in vivo or in vitro method of preparing a heparanase activity neutralizing monoclonal anti-heparanase antibody. The method is effected by implementing the following method steps, in which, in a first step, cells (either in vivo or in vitro) capable of producing antibodies are exposed to a heparanase protein or an immunogenic part thereof, and thereby generate antibody producing cells.

In a subsequent step the antibody producing cells are fused with myeloma cells to generate a plurality of cells, each producing monoclonal antibodies. The plurality of monoclonal antibodies is then screened to identify a monoclonal antibody which specifically inhibits heparanase activity. The later step is typically preceded by first screening for a monoclonal antibody which specifically binds heparanase.

According to a preferred embodiment of the present invention the method further comprises the step of humanizing the heparanase activity neutralizing monoclonal anti-heparanase antibody. Such a humanizing step can be effected following the procedures described hereinabove, which are known in the art.

Typically humanizing antibodies involves genetically modifying non-human cells to include functional genes and sequences derived from the human immune system gene complex or the system as a whole. The modification is performed prior to exposing the cells to an immunogen, as described in the above method steps.

According to yet another aspect of the present invention, there is provided a hybridoma cell line for producing a monoclonal antibody, comprising a cell line for producing the monoclonal anti-heparanase antibody of the present invention. The antibody or portion thereof produced by the hybridoma or by another cell line can be humanized (for a more detailed description of methods for hybridoma production, and humanized antibody production, see below).

In the present study, the availability of recombinant enzyme and specific antibodies enabled the demonstration of an involvement of the heparanase enzyme in tumor-associated processes such as metastasis and angiogenesis, and the therapeutic and diagnostic potential of the anti-heparanase antibodies.

It will be appreciated that, in the context of the present invention and without wishing to be limited to a single hypothesis, the anti-heparanase antibodies and methods of the present invention may also be used for therapy and/or prevention of pathological conditions and/or diseases whether or not commonly and/or previously associated with heparanase activity, alone or in combination with other therapies. Thus, according to another aspect of the present inventi6n, there is provided a method for treating a subject suffering from a pathological condition, the method comprising administering a therapeutically effective amount of an anti-heparanase antibody or portion thereof, the anti-heparanase antibody is capable of specifically binding to at least one epitope of a heparanase protein. Preferably, the heparanase protein is at least 60% homologous to the amino acid sequence of any of SEQ ID NOs: 1and 11, and/or at least 70% homologous to one or more of the epitope sequences of SEQ ID NOs: 6-10.

I

73 Inhibition of heparanase has been proposed for treatment of a variety of conditions and disorders. Reduction of heparanase activity by inhibitory heparan sulfate derivatives (see for example Ayal-Hershkovitz et al, International Patent Application Publication Nos. WO 02/060374A3 and WO 02/060375A2, and Herr et al, International Patent Application Publication No.

WO 01/35967A1, all incorporated herein by reference as if fully set forth herein), antisense and ribozyme (US Patent. Application No. 09/435,739), has been disclosed. Bohlen et al (International Patent Application Publication No.

WO 03/006645A2, incorporated herein by reference as if fully set forth herein) disclosed the use of mouse heparanase-pulsed dendritic cells (APC, antigen presenting cells), and anti-heparanase DNA vaccination to elicit an immune response against heparanase, demonstrating prolonged survival in animal metastatic tumor (Lewis lung carcinoma and melanoma) models. However, treatment or prevention of heparanase-related diseases with specific antiheparanase antibodies, and/or treatment or prevention of diseases in which heparanase activity has been implicated as a factor, was not disclosed.

The anti-heparanase antibodies of the present invention can be used to inhibit heparanase activity, and, as a result, can be used for prevention and/or treatment of heparanase-related disorders or conditions, such as inflammatory disorders, wounds, scars, vasculopathies and autoimmune conditions. While reducing the present invention to practice, immunohistochemistry of paraffinembedded sections of cancerous tissue from patients uncovered the strong reactivity of the anti-heparanase antibodies of the present invention with heparanase expressed in cancerous and malignant tissue (see Example III, Figures 8 and 9 described hereinbelow).

Therefore, according to another aspect of the present invention, there is provided a method for treating or preventing a heparanase-related disorder or condition in a subject, the method comprising administering a therapeutically effective amount of an anti-heparanase antibody or portion thereof, the antiheparanase antibody capable of specifically binding to at least one epitope of a heparanase protein. Preferably, the heparanase protein sequence is at least homologous to the amino acid sequence of any of SEQ ID NOs:1-5 and 11, and/or at least 60% homologous to one or more of the epitope sequences of SEQ ID NOs: 6-10.

Without wishing to be limited by a single hypothesis, modulation of heparanase activity may prevent activated cells of the immune system from leaving the circulation and thus inhibit elicitation of both inflammatory disorders and autoimmune responses. While reducing the present invention to practice, it was uncovered that administration of the specific anti-heparanase monoclonal antibody HP 3/17 elicited against the peptide pep9 (Table 2) (SEQ ID NO:9), effectively inhibited inflammatory arthritis (Example VI, Figure 12) in anti-collagenase- treated mice, and also delayed onset and reduced mortality in the NOD mouse model of autoimmune diabetes (IDDM) (Example VI, Figure 13).

Thus, in one embodiment of the present invention, the anti-heparanase antibodies can be used to treat or ameliorate inflammatory symptoms of any disease or condition wherein immune and/or inflammation suppression is beneficial such as, but not limited to, inflammation of the joints, musculoskeletal and connective tissue disorders, inflammatory symptoms associated with hypersensitivity, allergic reactions, asthma, otitis and other otorhinolaryngological diseases, dermatitis and other skin diseases, posterior and anterior uveitis, conjunctivitis, optic neuritis, scleritis and other immune and/or imflammatory ophthalmic diseases.

In another preferred embodiment, the anti-heparanase antibodies of the present invention can be used to prevent, treat or ameliorate an autoimmune disease such as, but not limited to Eaton-Lambert syndrome, Goodpasteur's syndrome, Graves disease, Guillain-Barre syndrome, autoimmune hemolytic anemia, hepatitis, insulin-dependent diabetes mellitus (IDDM), systemic lupus erythematosus (SLE), multiple sclerosis myaesthenia gravis, plexus disorders such as acute brachian neuritis, polyglandular deficiency syndrome, primary biliary cirrhosis, rheumatoid arthritis, scleroderma, thrombocytopenia, thyroiditis such as Hashimoto's disease, Sjogren's syndrome, allergic purpura, psoriasis, mixed connective tissue disease, polymyositis, vasculitis, dermatomyositis, polyarteritis nodosa, polymyalgia rheumatica, Wegener's granulomatosis, Reiter's syndrome, Behcet's syndrome, ankylosing spondylitis, pemphigus, bullous pemphigoid, dermatitis herpetiformis or Crohn's disease.

As detailed in the Background section hereinabove, heparanase expression and catalytic activity has been implicated in the pathogenesis of vascular disease, particularly in pathological modification of endothelial cells and arterial intima (see, for example, Sivaram P. et al, JBC 1995; 270:29760-5, Pillarisetti S. Trends Cardiovas Med 2000;10:60-65, and Pillarisetti S. et al J Clin Invest 1997;100:867-74). Recently, Pillarisetti et al (International Patent Application No: WO 03/011119A2, incorporated herein by reference as if fully set forth herein) have disclosed that heparanase mediates the effects of atherogenic factors such as oxidized LDL. Thus, in yet a further embodiment, the anti-heparanase antibodies of the present invention, capable of modulating the levels of heparanase activity in tissues, can be used for the treatment or prevention of vasculopathies such as, but not limited to atherosclerosis, aneurysm, and stenosis or restenosis following vascular trauma such as, for example, transluminal percutaneous cardiac angioplasty or stent implantation (see, for example, U.S. Patent No: 6,569,441 to Kwiz et al. for exhaustive description of stenosis and restenosis).

In yet another embodiment of the present invention, the anti-heparanase antibodies can be used for the treatment or prevention of heart disease and cardiomyopathy. Since increased heparanase activity has been demonstrated in cardiac tissue of rats genetically predisposed to cardiac insufficiency (see, for example, International Patent Application No WO 01/35967A1 to Herr et al), and heparanase inhibition has been proposed for prevention and treatment of heart failure, the anti-heparanase antibodies of the present invention can be used for treatment of congestive heart failure and related symptoms and indications such as peripheral edema, pulmonary and hepatic congestion, dyspnea, hydrothorax and abdominal dropsy.

Heparanase catalytic activity has been shown to modulate the function of HSPG associated biological effector molecules, including growth factors, chemokines, cytokines and the like Thus, without being limited to one hypothesis, modulation of heparanase activity may, for example, prevent angiogenesis caused due to the activation of growth factors such as bFGF, and inhibit cell proliferation, such as tumor cell proliferation. Further, as described in detail in the Background section hereinabove, it has been shown that metastatic potential of tumor cells (such as melanoma cells) is highly correlated with increased degradation of heparan sulfates, and increased expression of heparanase. Thus, modulation of heparanase activity may also be used to inhibit degradation of the basement membrane, as inhibition of such degradation may inhibit or block invasion of circulating tumor cells, and thus prevent metastasis.

While reducing the present invention to practice, it was determined that administration of specific anti-heparanase monoclonal antibodies HP37/33, elicited against the peptide pep9 (Table 2) (SEQ ID NO:9), or HP130, which binds to a region between amino acid coordinates 465 and 543 of human heparanase (SEQ ID NO: 4) (see Epitope Mapping in the Examples section hereinbelow), effectively inhibited the growth of primary melanoma tumors and reduced tumor-related mortality in mice (Example VI, Figure 11). Thus, the anti-heparanase antibodies of the present invention can be used to treat or prevent a condition or disorder characterized by angiogenesis, cell proliferation, a cancerous condition, tumor cell proliferation, invasion of circulating tumor cells or a metastatic disease.

In one embodiment, the anti-heparanase antibodies can be used for treatment or prevention of conditions characterized by angiogenesis and neovascularization such as, but not limited to, tumor angiogenesis, ophthalmic disorders such as diabetic retinopathy and macular degeneration, particularly age-related macular degeneration, reperfusion of gastric ulcer, and for contraception or inducing abortion at early stages of pregnancy.

In another embodiment, the anti-heparanase antibodies of the present invention can be used for treatment or prevention of a cancerous condition, tumor cell proliferation or metastatic disease such as, but not limited to nonsolid cancers, e.g. hematopoietic malignancies such as all types of leukemia: acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), myelodysplastic syndrome (MDS), mast cell leukemia, Hodgkin's disease, non-Hodgkin's lymphomas, Burkitt's lymphoma and multiple myeloma, as well as for the treatment or prevention of growth of solid tumors such as tumors in lip and oral cavity, pharynx, larynx, paranasal sinuses, major salivary glands, thyroid gland, esophagus, stomach, small intestine, colon, colorectum, anal canal, liver, gallbladder, extrahepatic bile ducts, ampulla of Vater, exocrine pancreas, lung, pleural mesothelioma, soft tissue sarcoma, carcinoma and malignant melanoma of the skin, breast, vulva, vagina, cervix uteri, corpus uteri, ovary, fallopian tube, gestational trophoblastic tumors, penis, prostate, testis, kidney, renal pelvis, ureter, urinary bladder, urethra, carcinoma of the eyelid, carcinoma of the conjunctiva, malignant melanoma of the conjunctiva, malignant melanoma of the uvea, retinoblastoma, carcinoma of the lacrimal gland, sarcoma of the orbit, brain, spinal cord, vascular system, hemangiosarcoma and Kaposi's sarcoma.

The anti-heparanase antibodies of the present invention are also useful for treating or preventing wounds, scars and cell proliferative diseases such as, but not limited to psoriasis, hypertrophic scars, acne and sclerosis/scleroderma, polyps, multiple exostosis, hereditary exostosis, retrolental fibroplasia, hemangioma, and arteriovenous malformation.

As mentioned hereinabove, heparanase catalytic activity has been shown to modulate the function of HSPG associated biological effector molecules.

These effector molecules include: growth factors, such as, but not limited to, HGH, FGF and VEGF; chemokines, such as, but not limited to, PF-4, IL-8, MGSA, IP-10, NAP-2, MCP-1, MIP-la, MIP-1p and RANTES; cytokines, such as, but not limited to, IL-3, TNFa, TNFP, GM-CSF and IFNy; and degradative enzymes, such as, but not limited to, elastase, lipoprotein lipase and cathepsin G.

The anti-heparanase antibodies and methods described herein for determining heparanase activity in vitro and in vivo can be used to determine subjects having conditions for which treatment according to the methods and antibodies of the present invention is suitable. The identification of those suitable subjects, including mammals such as rabbits, rats, mice, domesticated animals, or preferably humans suffering from such conditions for which such treatment is suitable is well within the ability and knowledge of one skilled in the art.

Thus, according to yet another aspect of the present invention, there is provided a method for detecting a heparanase-related disease or condition in a subject, the method effected by obtaining a biological sample from the subject, contacting the biological sample with an anti-heparanase antibody, the antiheparanase antibody capable of specifically binding to at least one epitope of a heparanase protein, in a manner suitable for formation of a heparanase polypeptide-antibody immune complex and detecting the presence of the heparanase polypeptide-antibody immune complex to determine whether a heparanase polypeptide is present in the sample, wherein the presence or absence of the heparanase polypeptide-antibody immune complex indicates a heparanase-related disease or condition, thereby detecting a heparanase-related disease or condition in a subject. Preferably, the heparanase protein-to which the antibody is capable of binding comprises a sequence which is at least homologous to the amino acid sequence of any of SEQ ID NOs: 1-5 and 11, and/or at least 60% homologous to any of the epitope sequences of SEQ ID NOs: 6-10.

In one embodiment, the subject is a vertebrate, preferably a mammal, most preferably a human subject. Heparanase-related disorders or condition suitable for treatment with the antibodies and methods of the present invention are detailed hereinabove.

As described in the Examples section hereinbelow, the anti-heparanase antibodies of the present invention provide sensitive and specific detection of heparanase polypeptides in diverse forms of samples: immunoprecipitation of heparanase from solution (Figure detection of heparanase antigen transferred to membranes following electrophoretic separation (Figures 4 and detection of heparanase in blood smears (Figures 6A-C), in paraffin sections of liver (Figures 7A-B), placenta (Figures 8A-B), and colon (Figures 9A-C).

Thus, according to one embodiment, the biological sample is selected from the group consisting of serum, plasma, urine, synovial fluid, spinal fluid, tissue sample, a tissue and/or a fluid. Methods for preparation of the sample for immunodetection with anti-heparanase antibodies of the present invention, and contacting the sample in a manner suitable for formation of a heparanase polypeptide-antibody immune complex are well known in the art. Suitable methods are described in detail in the Materials and Experimental Procedures section hereinbelow.

Detection of heparanase in biological samples can be effected in samples removed from the subject, as in biopsy, blood tests, pathology samples and the like, or can be performed in living tissue or bodily fluid in vivo. Thus, according to one embodiment contacting the sample is performed in situ or in vitro. The antibodies used in formation of the heparanase polypeptide-antibody immune complex can be polyclonal or monoclonal. Anti-heparanase antibodies suitable for detection using the method of the present invention are described in detail hereinbelow.

Heparanase activity has been detected in the urine of patients suffering from renal cancer, diabetes mellitus and renal disease. Screening for heparanase activity in biological samples from cancer patients revealed significant heparanase activity in the urine of 21 (renal cell carcinoma, breast carcinoma, rabdomyosarcoma, stomach cancer, myeloma) out of 157 cancer patients. High levels of heparanase activity were determined in the urine of patients with an aggressive disease (primarily breast carcinoma and multiple myeloma) and there was no detectable activity in the urine of healthy donors.

In another series of experiments, heparanase activity was measured in the urine of diabetic and healthy subjects. Urinary heparanase activity was strongly correlated with IDDM, and was even detected in the urine of normo- and microalbuminuric IDDM (insulin dependent diabetic mellitus) patients.

Heparanase activity was also detected in the urine of proteinuric patients not suffering from diabetes. These included patients with focal segmental glomerulosclerosis, minimal change nephrotic syndrome and congenital nephrotic syndrome (see U.S. Patent. Application No. 09/944,602, incorporated herein by reference as if fully set forth herein).

While not wishing to be limited to a single hypothesis, it is conceivable that heparanase may overcome the filtration barrier of the glomerular basement membrane and ECM simply by virtue of its ability to degrade the HS moieties that are responsible for their critical permeaselective properties. Urinary heparanase is therefore expected to reflect the presence of heparanase in the circulation and hence be a sensitive marker for metastatic, inflammatory and kidney disease.

Diabetic nephropathy, occurring in approximately 30% of patients with type I diabetes, is a major cause of end stage renal disease. The inability to discriminate the subpopulation that will develop renal damage prior to the appearance of microalbuminuria, 10-15 years following the diagnosis of diabetes, prevents significantly changing the devastating natural history of the disease. Urinary heparanase activity is a distinguishing feature, occurring in of normoalbuminuric females, within an otherwise homogenous group of patients. Thus, in yet a further embodiment of the present invention, the antiheparanase antibodies of the present invention can be used for detection of renal disease such as diabetic neuropathy, glomerulosclerosis, nephrotic syndrome, minimal change nephrotic syndrome and renal cell carcinoma.

According to a further aspect of the present invention, there is provided a method of detecting the presence of a heparanase polypeptide in a sample, the method effected by incubating the sample with a heparanase-specific antibody, the heparanase-specific antibody capable of specifically binding to at least one epitope of a heparanase protein, in a manner suitable for the formation of a heparanase polypeptide-antibody immune complex, wherein the heparanasespecific antibody is characterized by specifically binding to heparanase, and detecting the presence of the heparanase polypeptide-antibody immune complex to determine whether a heparanase polypeptide is present in the sample. Preferably, the heparanase protein is at least 60% homologous to the amino acid sequence of any of SEQ ID NOs: 1-5 and 11, and/or at least homologous to the epitope sequences of SEQ ID NOs: 6-10.

Detection of the heparanase polypeptide-antibody immune complex can be effected by immunoassays well known in the art. Such immunoassays include ELISA, Western blot, and immunohistological staining. Preferred methods comprise the detection of heparanase-specific antibodies with labeled second goat-anti-mouse antibodies.

It will be appreciated that, in addition to diagnosis of a disease or condition, detection of heparanase polypeptides according to the methods of the present invention can be used to monitor the progression of such a disease or condition, in a subject under observation or following treatment. Methods of monitoring a number of marker antigens by immunoassay in blood or tissue samples of patients following diagnosis or treatment are well known in the art, as described in detail, for example, for prostate cancer (PSA, see U.S. Patent.

No. 6,482,598 to Micolajczyk et al, incorporated herein by reference as if fully set forth herein) and cancerous tumors (CEA, see US Patent. No. 4,871,834 to Matsuoka et al, incorporated herein by reference as if fully set forth herein).

Thus, according to yet another aspect of the present invention, there is provided a method for monitoring the state of a heparanase-related disorder or condition in a subject, the method effected by obtaining a biological sample from the subject; contacting the biological sample with an anti-heparanase antibody of the present invention in a manner suitable for formation of a heparanase polypeptide-antibody complex; detecting a presence, absence or level of the heparanase polypeptide-antibody complex to determine a presence, absence or level of a heparanase polypeptide in the biological sample; repeating the previous stages at predetermined time intervals; and determining a degree of change of the presence, absence or level of the heparanase polypeptide at the predetermined time intervals, the change indicating a state of the heparanase-related disorder or condition in the subject; thereby monitoring the state of the heparanase-related disorder or condition in said subject. The determination of a normative standard of presence, absence or level of heparanase polypeptide-antibody complex in biological samples from subjects at risk, diagnosed or undergoing treatment, in order to monitor and assess the state of a disease, can be made by comparing data of heparanase expression from large population samples (see, for example, International Patent Application WO 03/011119A2 to Pillarisetti et al, which is incorporated herein by reference as if fully set forth herein). Monitoring the levels of heparanase periodically, for example in biological samples of a subject, following surgical removal or therapy against a metastatic cancer, may be prognostic of the prospects for short and long term survival, when compared with large scale statistical correlations. Similarly, levels of heparanase antigen in samples of different origin, such as urinary heparanase, compared to heparanase levels in biopsy samples for example, may provide further information regarding the localization and origin of disease processes. Quantitative assessment can be made when comparing to such standards. Qualitative assessment can also be made, by comparing presence, absence or levels of heparanase polypeptide over a period of time post therapy), to gauge the efficacy of or need for further treatment (as is routinely done with PSA for example- see U.S. Patent No. 6,482,598 to Micolajczyk et al).

In one embodiment, detecting the presence, absence or level of heparanase protein in a biological sample is effected by extracting proteins from the biological sample (the protein extract may be a crude extract and can also include non-proteinacious material), size separating (for example by electrophoresis, gel filtration etc.) the proteins, interacting the proteins with an anti-heparanase antibody (either poly or monoclonal), and detecting the antibody-protein complexes. In the case of gel electrophoresis, the interaction with the antibody is typically performed following blotting of the sizeseparated proteins onto a solid support (membrane). The predetermined time intervals can be intervals of minutes, where monitoring rapidly occurring changes in the state of the disease or condition is required, such as for example when monitoring heparanase protein during surgical or emergency procedures.

Longer time intervals, such as hours, days, weeks or months, can be chosen for example for monitoring the progression of a metastatic disease following chemotherapy.

In many cases it was shown that directly or indirectly via liposomes) linking a drug anti cancerous drug, such as, for example radio isotopes) to an antibody, which recognizes a protein specifically expressed by a tissue sensitive to the drug, and administering the antibody-drug complex to a patient, resulted in targeted delivery of the drug to the expressing tissue.

Therefore, the specific anti-heparanase antibodies of the present invention can be used for targeted drug delivery to a tissue, in tissues expressing heparanase. A complex of a drug, directly or indirectly linked to an antiheparanase antibody, can be administered to a patient. External radio imaging can also be used, wherein the drug is replaced with an imageable radio isotope.

Endoscopic or laparoscopic imaging is also envisaged. In the latter cases the drug is typically replaced by a fluorescence or luminescence substance. These procedures may for example, be effective in finding and/or destroying micrometastases.

Besides the therapeutic uses of specific anti-heparanase antibodies, these antibodies may be used for research purposes, to allow better understanding of the role of heparanase in different processes.

While reducing the present invention to practice, monoclonal antiheparanase antibodies were elicited to specific regions of the heparanase polypeptide, some of the antibodies preferentially detecting the mature, processed form of the heparanase polypeptide (Figures 4, 5 and 10). Further, Western blots of human and mouse heparanase with the anti-heparanase antibodies of the present invention demonstrated interspecies immune crossreactivity. Such specific antibodies, directed against different regions of the heparanase protein, can be used for identification and purification of heparanase protein from recombinant cell cultures, for example in the reduction of contamination by inaccurate translation products and unprocessed heparanase protein taken from recombinant cell cultures. Present methods for affinity purification of heparanase protein are based on the enzyme-substrate interaction between heparin and heparanase, employing Heparin-Sepharose affinity medium (see, for example, International Patent Application No. WO 99/11789 to Pecker et al, incorporated herein by reference as if fully set forth herein), which binds all heparin-binding proteins. The anti-heparanase antibodies of the present invention, can be attached to substrates using methods well known in the art, and thus provide a simple and inexpensive method for identification and affinity purification of heparanase proteins having the specific epitopes to which the antibodies bind.

Thus, according to a further aspect of the present invention, there is provided an affinity medium for binding human heparanase polypeptides, the medium comprising an anti-heparanase antibody of the present invention immobilized to a chemically inert, insoluble carrier. The inert, insoluble carrier is optionally and preferably selected from a group consisting of acrylic and styrene based polymers, gel polymers, glass beads, silica, filters and membranes. Methods suitable for preparation of affinity media for immuneaffinity purification of recombinant protein are described in detail in, for.

example, US Patent Nos. 5,683,916 to Goffe, et al, and 5,783,087 to Vlock et al., which are incorporated herein by reference as if fully set forth herein.

Thus, the condition can be related to altered function of a HSPG associated biological effector molecule, such as, but not limited to, growth factors, chemokines, cytokines and degradative enzymes. The condition can be, or involve, angiogenesis, tumor cell proliferation, invasion of circulating tumor cells, metastases, inflammatory disorders and/or autoimmune conditions.

Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting.

Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

EXAMPLES

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

MATERIALS AND EXPERIMENTAL PROCEDURES Materials: Heparin Sepharose was purchased from Pharmacia. 1,9- Dimethylmethylene Blue was purchased from Aldrich (Cat. No. 34108).

Monoclonal antibody production: Six to eight weeks old female Balb/C mice were each intradermally immunized with 50ig (50pl) recombinant heparanase (prepared and purified essentially as described in U.S. Patent.

Application No. 09/071,618 and U.S. Patent No. 5,968,822, which are incorporated by reference as if fully set forth herein) emulsified in 50pl PBS complete Freund's adjuvant. Two to three weeks later the same amount of the emulsion was injected subcutaneously or intradermally at multiple sites, in incomplete Freund's adjuvant. After 3 weeks 25pg antigen in aqueous solution was injected intraperitoneally. Seven to ten days later the animals were bled, and the titer of the relevant antibodies was determined. Three to four weeks after the last boost, one.or two animals were injected intraperitoneally with of soluble antigen (in PBS) and 3-4 days later spleens were removed.

Fusion and cloning of monoclonal antibodies: The spleens of immunized mice were ground, splenocytes were harvested and fused with NSO myeloma cells by adding 41% PEG. Hybridoma cells were grown in HATselective DMEM growth media containing 15% HS (Beit Haemek), 2 mM glutamine, Pen-Strep-Nystatin solution (Penicillin: 10,000 units/ml, Streptomycin: 10 mg/ml, Nystatin: 1,250 units/ml), at 37 0 C in 8% CO 2 Hybridoma cells were cloned by limiting dilution. Hybridomas producing Mabs to human heparanase were identified by reactivity with solid-phase immobilized human heparanase (native and denatured (ELISA)).

Cell culturing: Hybridoma cells were cultured in T-175 flasks (Corning Costar, Cat. No. 430824) in a C0 2 -enriched incubator at 37 0 C in DMEM medium (Beit Haemek, Israel) supplemented with 10% horse serum (Beit- Haemek Cat. No. 04-124-1A). Culture volume was 80 ml.

Production of antibodies by the starvation method Cultures reaching cell density of 2x106 cells/ml or higher, were used for the production of antibodies. Cells were removed from the flasks by pipetting and were centrifuged at 1,000 rpm for 5 minutes, in order to pellet the cells. The cell pellets were suspended in basal DMEM (with no serum added), and centrifuged at 1,000 rpm for 5 minutes. This procedure was repeated once more and the cell pellets were suspended in the original volume of basal DMEM medium. Cell suspension was plated into new T-175 flasks and placed inside the incubator.

After 48 hours, cells were pelleted by centrifugation at 3,500 rpm for minutes. Culture supernatants were filtered through 0.2 micron pore-size filter (Nalgene, Cat. No. 156-4020) and were supplemented with 0.05% sodium azide. Culture supernatants were kept refrigerated until purification.

Purification of monoclonal antibodies: Purification was performed by affinity chromatography using Protein G (28,14). 2.5ml of Protein G Sepharose 4 Fast Flow (Pharmacia Cat. No. 17-0618-01) were used to pack each column (Bio Rad, Cat. No. 737-1517). The flow rate for packing the columns was 4ml/min. Each column was equilibrated with 100ml of PBS pH 7.2. Culture supernatants (filtered and supplemented with sodium azide as described above) were loaded on the column at a flow rate of 1ml/minute. After loading, each column was washed with 80 ml of PBS pH 7.2 at a flow rate of 4ml/minute.

Elution was done with 12ml of 0.1M Glycine-HCI buffer, pH 2.7, at a flow rate of 1 ml/minute. One ml fractions were collected into tubes containing 0.3ml of 1M Tris pH 9.0. Following elution each column was further washed with of the elution buffer at a flow rate of 4ml/min. Each column was then regenerated by passing 50ml of regeneration buffer (0.1M Glycine-HCI buffer pH After regeneration, the column was immediately neutralized with 100ml of PBS pH 7.2, 0.1% sodium azide was added and the column was thereafter stored in the refrigerator.

Eluted fractions were analyzed for protein content using the Bradford protein determination method. According to the obtained results, 4-6 fractions were pooled and dialyzed (Spectrum dialysis tubing, MWCO 6,000-8,000, Cat.

No. 132653) three times against 500ml of PBS buffer pH 7.2 with 0.05% sodium azide, or against PBS pH 7.2 to which 1% thimerosal (Sigma, Cat. No.

T-8784) was added. After dialysis samples were stored at 4 0

C.

Western blots: Proteins were separated on 4-20%, polyacrylamide ready gradient gels (Novex). Following electrophoresis proteins were transferred to Hybond-P nylon membrane (Amersham) (350 mA/100V for 90 minutes).

Membranes were blocked in TBS containing 0.02% Tween 20 and 5% skim milk for 1-16 hours and then incubated with antisera diluted in the same blocking solution. Blots were then washed in TBS-Tween, incubated with appropriate HRP-conjugated anti-mouse anti-rabbit IgG, and developed using ECL reagents (Amersham) according to the manufacturer's instructions.

Epitope mapping: A 1.7 Kb fragment of hpa cDNA (a hpa cDNA cloned in pfastBacHTA, see U.S. Patent Application No. 08/922,170 now U.S. Patent No. 5,968,822, which is incorporated by reference as if fully set forth herein) was digested by various restriction enzymes to create serial deletions from both the 3' and the 5' ends of the heparanase open reading frame (ORF) as follows.

3' deletions: EcoRI BstEII fragment encoding amino acids 1-465, deletion of an NdeI Xbal fragment generating an ORF of 347 amino acids (1- 347) and a deletion of AflII XbaI fragment generating an ORF of 229 amino acids (1-229).

deletions: BamHI Xhol fragment encoding 414 amino acids, (130- 543), an AflII XhoI fragment encoding 314 amino acids (230-543), an NdeI Xhol fragment encoding 176 amino acids (368-543) and a BstEII XhoI fragment encoding 79 amino acids of the heparanase open reading frame (465- 543).

The heparanase segments were expressed in Baculovirus expression system, essentially as described in U.S. Patent. Application No. 09/071,618, which is incorporated by reference as if fully set forth herein. The fragments were subcloned into the pfastBacHT vector to generate His-tagged fusion constructs. Recombinant baculovirus containing the various fragments were generated using the Bac to Bac system (GibcoBRL, Gibco Laboratories, Grand Island New York) according to the manufacturer recommendations. Extracts of Sf21 cells expressing various segments of a heparanase protein were analyzed.

The recombinant heparanase segments were detected by Western blots.

Epitope mapping of monoclonal antibodies HP 37/33 and HP 135.108 was performed by subcloning heparanase partial cDNA, containing nucleotides 511-1721 of SEQ ID NO 1, in the bacterial expression vector pRSETA. This DNA fragment encodes amino acids 130-543 of SEQ ID NO 4 comprising the P45 subunit of a mature heparanase polypeptide, a part of the P6 linker and a bacterial leader sequence generating an ORF of 453 amino acids comprising a polypeptide of approximately 50kDa. Serial deletions starting at the 3' of heparanase coding sequence were designed to generate a ladder of heparanase fragments sized 20-50kDa. Deletions were generated using the Erase A Base kit (Promega Corp, Madison WI) according to the manufacturer's recommendations. Reaction conditions were adjusted to obtain a difference of approximately 150bp between resulting DNA fragments (in descending size order) d45bam, d42, d43, d63, d84, d123, d142 d186, d207 and d22.

Heparanase fragments were expressed in E. coli BL21 and cell extracts were separated by gel electrophoresis and blotted onto PVDF membrane. The membranes were incubated with hybridoma medium or with IgG purified monoclonal antibodies, in order to localize the specific epitope detected by a specific antibody.

An interacting antibody was detected using an HRP-conjugated goat/donkey anti mouse antibody.

Heparanase activity assay: 1 0 0 pl heparin Sepharose (50% suspension in double distilled water) were incubated in 0.5ml eppendorf tubes placed on a head-over-tail shaker (37°C, 17 hours) with enzyme preparations in reaction mixtures containing 20mM Phosphate citrate buffer pH 5.4, ImM CaC12 and ImM NaC1, in a final volume of 200p1. Enzyme preparations used were either purified recombinant heparanase expressed in insect cells Patent.

Application No. 09/071,618, incorporated by reference as if fully set forth herein), or heparanase partially purified from human placenta At the end of the incubation period, the samples were centrifuged for 2 minutes at 1,000 rpm, and the products released to the supernatant due to the heparanase activity were analyzed using the calorimetric assay Dimethylmethylene Blue (DMB) as described in U.S. Patent Application No. 09/113,168 now U.S. Patent No.

6,190,875, which is incorporated by reference as if fully set forth herein.

Dimethylmethylene Blue assay (DMB): Supernatants (100pl) were transferred to plastic cuvettes. The samples were diluted to 0.5ml with PBS plus 1% BSA. 1,9-Dimethylmethylene blue (32mg dissolved in 5ml ethanol and diluted to 1 liter with formate buffer) (0.5ml) was added to each sample.

Absorbance of the samples was determined using a spectrophotometer (Cary 100, Varian) at 530nm. A control to which the enzyme was added at the end of the incubation time, was included in each sample.

Anti-heparanase antibodies recognizing specific sites: For generation of antibodies against specific sites within the human heparanase peptide, animals were immunized with peptides of defined amino acid sequence from the P8 and subunits of mature active heparanase.

Polyclonal antibodies: Polyclonal antibodies were generated against heparanase peptides by immunizing rabbits with KLH-conjugated peptides.

Conjugation of cysteine N-terminal-labeled peptide to maleimide activated KLH (Pierce Biochemicals) was done according to the manufacturer's instructions. Briefly, 2.5mg of heparanase peptide were dissolved in 250pl of Pierce conjugation buffer (Pierce Inc, Cat#77164). Lyophilized maleimideactivated BSA (Pierce Cat#77116) or maleimide-activated KLH (Pierce Cat#77606) were dissolved in 200pl of the conjugation buffer. Following mixing of the peptide and carrier solutions and overnight incubation at room temperature, conjugation efficiency was tested using DTNB. Conjugate was dialyzed against PBS, and stored frozen. Immunization of rabbits was conducted at Harlan Biotech according to their standard protocols: Two rabbits were immunized each with 150pg of peptide-KLH emulsified with equal volume of complete Freund's adjuvant. An equal amount of protein emulsified with incomplete Freund's adjuvant was injected to each rabbit two weeks after the first injection and again after another four weeks. Ten days after the third injection the rabbits were bled and serum was examined for reactivity with recombinant heparanase (Direct ELISA, see hereinbelow). Four weeks after bleeding another boost was injected and 10 days later blood was collected.

IgG fractions were purified from rabbit sera and monoclonal antibodies were purified from hybridoma medium by protein G affinity chromatography, using protein G sepharose beads (Pharmacia) according to the manufacturer's recommendations. Briefly, the antiserum was diluted with PBS, loaded onto a Protein G column and washed repeatedly with PBS. IgG antibodies were eluted with 0.1N Glycine buffer, pH 2.7, antibody containing fractions pooled, dialyzed and further analyzed. Antibody specificity for heparanase polypeptides was confirmed by Western blotting and ELISA.

Anti-heparanase antibodies raised against intact heparanase or heparanase p45 subunit: Polyclonal goat or rabbit anti-heparanase antibodies were prepared against intact active heparanase (p45/p8 dimer) (GH53 and RH53). It should be noted that by "intact" it is meant that both subunits of the heparanase heterodimer were used for the immunization process. Rabbits were immunized with 250 lg of recombinant active (p45/p8 heterodimer) heparanase mixed with 0.5ml complete Freund's adjuvant, administered initially intradermally (ID) to the clipped dorsum of the rabbits, in as many sites as possible. Goats were similarly immunized with an initial injection of 500tg recombinant human heparanase. Rabbits were boosted with 150utg antigen mixed with 0.5ml incomplete Freund's adjuvant administered subcutaneously at 3 week intervals. Goats received 250lag boosts at 3-4 week intervals. Animals were bled for antibodies one week following the last boost.

The IgG fraction was identified and purified on Protein G as described above for polyclonal antibodies. Goat polyclonal antibodies were further affinity purified on a column of heparanase p45-subunit Sepharose.

Western blot: Proteins were separated on 4-12%, polyacrylamide ready gradient gels (Nupage). Following electrophoresis proteins were transferred to PVDF membrane Membranes were blocked in TBS (Tris Buffered Saline) containing 0.02% Tween 20 detergent and 5% skim milk for 1-16 hours, and then incubated with antisera diluted in blocking solution. Blots were then washed in TBS-Tween, incubated with appropriate HRP-conjugated anti mouse/anti rabbit IgG, and developed using ECL reagents (Amersham, UK) according to the manufacturer's instructions.

Direct ELISA: Falcon polyvinyl plates were coated with 50ng/well of CHO derived human heparanase in PBS (pH 7.2) overnight at 4°C.

Hyperimmune serum or hybridoma medium samples were added to the wells, and incubated at room temperature for 2 hours. Binding of antibodies was then detected by incubation with HRP-conjugated goat anti mouse or rabbit IgG (Fab specific) (Sigma-Aldrich Corp, St Louis MO), followed by development in o-phenylenediamine substrate (Sigma-Aldrich Corp, St Louis MO) and measurement of absorbance at 450 nm. Plates were washed in PBS with 0.05% Tween between incubations.

Site-specific monoclonal antibodies: Mice were vaccinated with a KLH conjugated peptide representing a specific site in the heparanase polypeptide (see Table Eight to ten weeks old female Balb/C and NZB mice were each immunized subcutaneously with 50tl saline suspension, containing either 5 lg recombinant heparanase or 50Qg peptide-KLH emulsified in 5 0 Pl complete Freund's adjuvant. Three weeks later the same amount of antigen was injected subcutaneously in incomplete Freund's adjuvant emulsion, or intraperitoneally in saline suspension. The antigen administration was repeated three weeks later. After 7-10 days blood was collected, and the titer of the relevant antibodies was determined by direct ELISA. Four to sixteen weeks after the last boost, one or two animals were injected intravenously with 10 pg of antigen in saline suspension and 3-4 days later spleens were removed.

Fusion and cloning: The spleens of immunized mice were ground, splenocytes were harvested and fused with the NSO myeloma cells (see US Patent No. 5,565,337 to Diamond et al, incorporated herein by reference as if fully set forth herein) by adding 41% PEG. Hybridoma cells were grown in HAT-selective Dulbecco's Modified Eagle Medium (DMEM) growth media containing 15% HS (Sigma, St Louis MO), 2 mM glutamine and Gentamycin, at 37 0 C in an atmosphere containing 8% CO 2 Hybridoma cells were cloned by limiting dilution. Hybridomas were screened by direct ELISA for interaction with solid-phase immobilized recombinant purified human heparanase, and selected for further studies after two cycles of limiting dilution. Purification of monoclonal antibodies from the hybridoma medium was performed with Protein G as detailed hereinabove.

Immunohistochemistry: Paraffin sections were fixed in Acetone- Methanol 10 minutes at 20-24 0 C (room temp.). Endogenous peroxidases were blocked with 0.3% H202 in methanol, 15 minutes at 20-24°C. Slides were incubated with PBS containing 10mM glycine for 15 minutes at 20-24 0

C.

Slides were then incubated with normal horse serum block solution prepared according to the manufacturer's instructions (Vectastain, Vector Labs, Burlingame CA) for 30 minutes at 20-24 0 C, followed by an incubation with HP3/17 monoclonal antibody (diluted 1:50-200 with PBS), overnight at 4 0

C.

The slides were then further incubated for 30 minutes at 20-24 0 C with biotinylated antibody solution prepared according to manufacturer's instructions (Vectastain, Vector Labs, Burlingame CA), followed by an incubation with Vectastain (avidin) solution (prepared according to manufacturer's instructions) for 30 minutes at 20-24 0 C (RT).

Slides were then incubated with DAB solution (prepared according to the kit manufacturer's instructions) for at least 10 minutes at 20-24 0 C (until brown stain appears on slides) and counterstained with Mayer's Hematoxylin for minutes at 20-24'C. Slides were washed with H 2 0, mounted with mounting media, and covered with covering glass. Slides were washed with PBS between each step.

Immunoprecipitation: Purified recombinant heparanase, l g/501 PBS, or 50pl cell lysate (prepared from 2-5x106 cells by 3x freeze/thaw cycles in PBS) was incubated with 10lg HP3/17 monoclonal antibody for 2 hours on ice. Ten microliters of pre-blocked (lhr with 1% BSA, 0.05% Tween 20 in PBS) Protein G beads (Pharmacia Cat. #17-0618-02) were added and the mixture was incubated for 2 hours on ice. The mixture was then centrifuged for 2 minutes at 5,000 rpm, the supernatant removed and the beads washed twice with 500pl PBS (centrifuged 2 minutes at 5,000 rpm). lOl H 2 0, 25pl SB, DTT, 55pl H 2 0 were added to the washed beads, and the beads were boiled for minutes. The supernatant, containing the eluted proteins, was either frozen or loaded, 20pl/lane, onto 4-12% NuPage gel for electrophoretic separation.

Separated proteins were transferred to a PVDF membrane and subjected to Western blot analysis using l pg/ml rabbit or goat polyclonal purified antiheparanase antibodies.

Animal Models of Disease: Primary melanoma: Primary melanoma tumors were induced in C57B1 mice according to Dong Y. et al (Cancer Research 1999;59:1236-43). Briefly, 10 5 B16-F1 tumor cells, optionally preincubated up to 12 hours with monoclonal antibodies or PBS (controls), were injected via tail vein into C57B1 mice to create solid tumors. Antibody administration to the tumor-bearing mice was performed intraperitoneally. Tumor volume was expressed in mm 3 measured with a microcaliper.

Experimental inflammatory arthritis: Arthrogen-collagen-arthritis was induced in mice by anti-collagen type II antibodies and lipopolysaccharide (LPS) as previously described (de Fougerolles et al, J Clin Invest 2000;105:721-29). Briefly, mice were injected intraperitoneally with each of four anti-collagen type II monoclonal antibodies (Chondrex LLC, Redmond WA) on day 0, followed by an intraperitoneal injection of 25.tg LPS on day 3. Mice developed swelling in wrists, ankles and digits after 3-4 days.

Monoclonal antibodies (250pg) or control IgG protein (200tg) was administered intraperitoneally every 2-3 days, starting on day 0. Severity of arthritis in each limb was scored by observation as follows: 0= normal; 1= mild redness, slight ankle and wrist swelling; 2= moderate ankle and wrist swelling; 3=severe swelling including ankle, wrist and digits; 4=maximal inflammation.

Autoimmune diabetes: The non-obese diabetic mouse (NOD) (Jackson Laboratories, Maine USA) is a well-known and highly characterized model of autoimmune (IDDM) diabetes, developing islet inflammation at 4-6 weeks, progressing to overt IDDM at 4-5 months (Bendelac, A et al J Exp Med 1987;166:823-32). Female NOD mice were injected intraperitoneally with either 200 gg monoclonal antibodies or 200ial PBS (control), once or twice weekly as described, and blood glucose levels measured weekly. Diabetic mice were euthanized when they reached >500 mg/dl glucosuria.

EXPERIMENTAL RESULTS EXAMPLE I Epitope mapping with monoclonal anti-heparanase antibodies As part of the task of characterizing purified monoclonal antibodies, it is necessary to determine whether individual antibodies raised against the same antigen bind to identical or overlapping epitopes.

A linear method was used to map the epitopes within the heparanase protein that are recognized by each antibody. Serial deletions were made and assayed for the production of fragments that can be recognized by each antibody. In practice, this method can only localize the binding site to a small region.

Supernatants from two monoclonal antibodies, HP-130 and HP-239 were examined by western blot for reactivity with various segments of recombinant heparanase expressed in Baculovirus infected insect cells.

As can be seen in Figure 1, monoclonal antibody HP-130 recognized a segment of 79 amino acids at the C-terminus of the heparanase open reading frame (amino acids 465-543), binding only to peptides in lanes 1 (amino acids 130-543, SEQ ID NO:4), 2 (amino acids 230-543, SEQ ID NO:4), 3 (amino acids 368-543, SEQ ID NO:4) and 4 (amino acids 465-543, SEQ ID NO:4).

The monoclonal antibody HP-239 recognized an internal epitope localized to amino acids 130-230, binding only to peptides in lanes 1 (amino acids 130-543, SEQ ID NO:4), 5 (amino acids 1-229, SEQ ID NO:4), 6 (amino acids 1-347, SEQ ID NO:4) and 7 (amino acids 1-465, SEQ ID NO:4).

As shown in Figures 15A and 15B, monoclonal antibody HP37/33, which was raised against a specific peptide (pep9, SEQ ID NO:9) corresponding to amino acids 334-348 of SEQ ID NO 4, recognizes heparanase partial polypeptides of 35-50 kDa but not <25 kDa, confirming that the epitope is localized within the region of amino acids 320-410 of heparanase precursor (SEQ ID NO Antibody 135.108, which was raised against the intact active recombinant human heparanase dimer also recognizes an epitope within this region. Additional monoclonal antibodies HP 108.264, HP 115.140, HP 152.197, HP 110.662, HP 144.141, HP 108.371, HP-151.316, and HP 117.372, also raised against the intact active recombinant human heparanase dimer, recognized an epitope within the same region, giving an identical epitope mapping profile (results not shown).

EXAMPLE H Neutralizing anti-heparanase antibodies Neutralization of recombinant heparanase expressed in insect cells: The ability of the different monoclonal antibodies to inhibit the activity of a recombinant heparanase expressed in insect cells was examined. Reactions mixtures containing 5pg of enzyme were pre-incubated for 30 minutes at room temperature, with increasing amounts of antibodies (for example, 25 to 170p.g, forming molar ratios of 1:1.7 to 1:10 enzyme to antibody, for antibody HP-130, and 12.5 to 250pg, forming molar ratios of 1:0.85 to 1:18.5, for antibody HP- 239). For monoclonal antibodies HP 37/33, and HP 3/17, 24ng of heparanase was pre-incubated with increasing amounts of monoclonal antibody (0.072 4.6gg), forming heparanase:antibody molar ratios from 1:1 to 1:64.

Following pre-incubation, heparanase activity was determined using DMB assay as described in experimental procedures. The percentage of activity measured in the presence of each antibody amount, as compared to the activity of a control reaction, lacking the antibodies, is presented in Figure 2.

As can be seen in Figure 2, monoclonal antibody HP-130 which is directed against a sequence in the C-terminus of the heparanase enzyme, was capable of almost completely inhibiting recombinant heparanase activity at a molar ratio of 1:10. Pre-incubation of the heparanase with increasing concentrations of antibody resulted in dose-dependent inhibition of the activity (Figure 2).

The other antibody examined, HP-239, which is directed against an internal epitope of heparanase, caused no inhibition of heparanase activity even at a higher molar ratio of antibody to enzyme as compared to the ratio that gave almost complete inhibition with antibody HP-130. These two antibodies were prepared and purified in the same manner, indicating that inhibition of heparanase activity by antibody HP-130 is specific. The molar ratios of enzyme to antibody in which antibody HP-130 inhibited heparanase activity are similar to molar ratios reported in the literature that are used for neutralization of other enzymes (77,78). The fact that an antibody formed against the C-terminus of the enzyme was capable of almost completely inhibiting its activity, while an antibody directed against an internal epitope had absolutely no effect could suggest the possible role of the C-terminus in the heparanase activity, and may indicate the possibility that other antibodies directed against this region may also have a neutralizing effect on heparanase activity.

Neutralization of natural heparanase activity purified from human placenta: To examine whether anti-heparanase antibodies raised against defined epitopes of the heparanase protein, such as the monoclonal antibody HP-130, could inhibit a natural heparanase in the same manner that they inhibit the recombinant enzyme, a similar experiment to the one described above was designed using heparanase purified from human placenta.

As the specific activity of the natural enzyme is much higher than its recombinant counterpart, 5ng of enzyme, were used for this experiment. The activity of this amount of enzyme is in the linear range of the DMB heparanase activity assay. The enzyme was pre-incubated with increasing amounts of antibody while maintaining similar molar ratios as used for the recombinant enzyme (20 to 450ng of antibody HP-130 forming molar ratios of 1:4 to 1:95 enzyme to antibody, and 225ng of antibody HP-239 forming a molar ratio of 1:20). The percentage of activity remained in the presence of each antibody amount, as compared to the activity of a control reaction lacking the antibodies is presented in Figure 3.

As shown in Figure 3, 225ng of antibody HP-130 were capable of inhibiting 90% of the activity in heparanase purified from human placenta. This amount of antibody forms a molar ratio of 1:20 enzyme to antibody, similar to the ratio that almost completely inhibited the recombinant heparanase expressed in insect cells. Antibody HP-239, on the other hand, used at the same molar ratio, did not have any effect on heparanase activity.

Neutralization of recombinant heparanase activity with site-specific anti-heparanase antibodies HP3/17 and HP37/33: Monoclonal antibodies elicited against specific sites in the heparanase protein were tested for their ability to neutralize heparanase activity. Preincubation of heparanase enzyme protein with the site specific monoclonal anti-heparanase antibodies HP 37/33 and 3/17 also neutralized the activity of the enzyme. As shown in Figure 14B, monoclonal antibody HP 3/17, elicited against peptide pep9 (SEQ ID NO:9, see Table 2 hereinbelow), which binds to the catalytic nucleophilic residue of the active site of heparanase, was capable of neutralizing more than 65% of heparanase activity at a heparanase:antibody molar ratio of 1:64. Monoclonal antibody HP 37/33 (Figure 14A), also elicited against peptide pep9 (SEQ ID NO:9), neutralized heparanase catalytic activity even more efficiently, achieving more than 40% reduction in activity at a heparanase:antibody molar ratio of 1:32, and more than 80% inhibition at a molar ratio of 1:64. The ability of monoclonal antibodies HP-130, HP 33/37 and HP 3/17 to inhibit natural and recombinant human heparanase enzyme exemplifies the possible use of I r recombinant heparanase to screen for neutralizing agents against naturally occurring enzymes.

EXAMPLE III Site-specific anti-recombinant human heparanase antibodies Peptide-specific anti-heparanase antibodies: In order to generate antibodies recognizing specific sites in the human heparanase polypeptide, animals were immunized with peptides representing regions of catalytic importance. Table 2 hereinbelow details a few of the peptides used as antigens, their precise location along the human heparanase amino acid sequence (SEQ ID NO:10), and the proposed function of each portion of the sequence in catalytic activity. Below the Table is the amino acid sequence of preproheparanase, with the two subunits of the mature active heparanase (P8 and P50) highlighted in bold. Note the two Glutamic acid residues comprising the active site are marked by arrowheads and the putative heparin binding domains are indicated in boxes.

Table 2- FUNCTIONAL PEPTIDE EPITOPES OF HEPARANASE

HEPARANASE

Location in Peptide Amino acid sequence SEQ ID NO Property p8 #7 SE ID 879-107 C-terminus of P8 SEQ ID PAYLRFGGTKTDFLIFDPK 89-107 Dimerization NO:7 pep38 located 5 amino SEQ ID CTNTDNPRYK 437-446 acids downstream of NO:6 a heparin binding site pep8 contains the proton ep8 Idonor residue of the SEQ ID SWELGNEPNSFLKKA 219-233 donor residue of the NO:8 heparanase active site pep9 contains the SEQ ID RPGKKVWLGETSSAY 334-348 nucleophilic residue NO:9 of the active site Pep 10 TWHHYYLNGRTATR 294-307 Designed according

I

SEQ ID to a 3D model as a surface exposed sequence, which bridges substrate binding and active site.

*NOTE: Specific peptide sequences are underlined in the sequence below *NOTE: Mature heparanase dimer sequences in bold face:

MLLRSKPALPPPLMLLLLGPLGPLSPGALPRPAQAQDVVDLDFFTQEPLH

LVSPSFLSVTIDANLATDPRFLILLGSPKLRTLARGLSPAYLRFGGTKTD

FLIFDPKKESTFEERSYWQSQVNQDICKYGS[PPDVEEKLRLEWPYQEQL

LLREH QKKFKN TYSRSSVDVLYTFANCSGLDLIFGLNALLRTADLQWN

SSNAQLLLDYCSSKGYNISWELGNAPNSFLKKADIFINGSQLGEDFIQLH

KLLRKSTFKNAKLYGPDVGQPRRKTALKSFLKAG GEV DSVTWHYYL

NGRTATREDFLNPDVLDIFISSVQKVFQVVESTRPGKKVWLGETSSAYGG

GAPLLSDTFAAGFMWLDKLGLSARMGIEVVMRQVFFGAGNYHLVDENFDP

LPDYWLSLLFKKLVGTKVLMASVQSKRRKLR

LHCTNTDNPRYKEGDL

TLYAINLHNVTKYLRLPYPFSNKQVDKYLLRPLGPHGLLSKSVQLNGLTL

KMVDDQTLPPLMEKPLRPGSSLGLPAFSYSFFVIRNAKVAACI (SEQ ID NO:4) Polyclonal site specific anti-heparanase antibodies: Peptides representing specific amino acid sequences indicated hereinabove were used to prepare polyclonal antibodies, as detailed in the Materials and Experimental Procedures section hereinabove. Antigenicity of the peptides was enhanced by conjugation to Keyhole Limpet Hemocyanin (KLH). Peptides pepS, pep9 and P8#7 demonstrated significant antigenicity, producing rabbit anti-heparanase antibodies recognizing purified human heparanase on both Western blot analysis, and according to ELISA. When reacted with denatured heparanase, the specificity of anti-pep8 and anti-pep9 for the P50 subunit of mature heparanase, and that of anti-P8#7 for the P8 subunit of mature heparanase, was clear. Thus, functional domains of SEQ ID NO:4 constitute antigenic determinants useful in producing specific anti-heparanase antibodies for therapeutic, diagnostic and research applications.

Polyclonal subunit-specific, and anti-active heterodimer antiheparanase antibody: Goats or rabbits immunized with intact, active (p45/p8 heterodimer) recombinant human heparanase protein (Fig. 17B and 17C, respectively) and purified on either protein G or on purified large (p 4 5 subunit of recombinant human heparanase (Fig. 17A) produced polyclonal antiheparanase antibodies specifically recognizing the corresponding protein on Western blots [Figs. 17A (GapH45), 17B(GH53-), and 17C (RH53)]. Both the IgG fractions of goat GH53 (Fig. 17B) and rabbit RH53 (Fig 17C) anti-intact active (p45/p8) heparanase heterodimer and the affinity purified goat anti-large subunit GapH45 (Fig. 17A) recognized the unprocessed (p 6 0) and mature subunit of purified recombinant human heparanase (lane and recombinant human heparanase from transfected CHO cell extract (lane The unprocessed precursor is considerably less abundant in the CHO cell extract (lane 2).

The specificity of the affinity purified goat anti-large subunit (p45) antiheparanase for the p45 and p60 species, compared to the anti-intact, active (p45/p8 heterodimer) anti-heparanase is clearly seen upon comparison of lanes 1 and 2 of Figs. 17A, 17B and 17C. Note the absence of reaction of goat antianti-heparanase with the small p8 subunit in Fig. 17A, and the weak interaction with recombinant mouse heparanase (lane 3).

Monoclonal site-specific anti-heparanase antibody: Mice vaccinated with the KLH-conjugated peptide RPGKKVWLGETSSAY (peptide pep9, SEQ ID NO:9, Table which contains the nucleophilic residue of the catalytically active site on human heparanase, TWHHYYLNGRTATR (peptide pepl0, SEQ ID NO:10, Table a surface exposed sequence, which bridges substrate binding and active site, and CTNTDNPRYK (peptide pep38, SEQ ID NO:6, Table located at a heparin binding site flanking region, were used to produce hybridomas which, when screened by ELISA, were positive for interaction with purified recombinant human heparanase. Following two cycles of limiting dilution, a hybridoma secreting mouse anti-pep9 IgG termed HP3/17, a hybridoma secreting anti-pep38 termed HP102, and a hybridoma r secreting anti-pepl0 termed HP201 were selected. HP3/17 and HP 37/33 antibody protein was purified from the hybridoma medium with Protein G affinity chromatography as detailed hereinabove.

The specificity of the anti-heparanase HP3/17, HP 33/17, HP210 and HP102 monoclonal antibodies was demonstrated by Western blotting with human and mouse heparanase (Figures 4, 5 and 10). HP3/17 recognizes and reacts strongly with the unprocessed P60 heparanase protein (Figure 4, lane 1) and the P45 50kDa human (Figure 4, lane 2) and the 49kDa mouse heparanase (Figure 4, lane 3) expressed by transfected CHO cells. Monoclonal antibody HP 37/33, elicited against the same peptide (pep9, SEQ ID NO:9) exhibited a similar pattern of recognition of human and mouse heparanase proteins on a Western blot (Figure 4, lanes Further analysis of immunoprecipitation of recombinant human heparanase from CHO cells or SI-11 cells with monoclonal anti-pep9 HP3/17 or HP37/33 revealed the antibody's specificity for the processed form of the recombinant enzyme (see Figure 5, lanes 4 and compared to lane Western blots of cell extracts from heparanase expressing (Si-11) and non-transformed control (Dhfr") cells show specific immunodetection of the mature 50 kDa recombinant heparanase, and of the kDa detected by both antibodies secreted by the hybridomas HP201 and HP102 (Figure 10, lanes 2, 3, 5 and 6).

Similarly, Western blots of cell extracts of CHO cells expressing recombinant human (Figure 16, lane 2) or mouse (Figure 16, lane 3) heparanase, or purified recombinant human mature heparanase (Figure 16, lane 1) with monoclonal antibodies HP 135.108 show specific immunodetection of the mature 50 kDa recombinant heparanase, and of the 65 kDa detected by both antibodies secreted by the hybridomas. Immunodetection using additional monoclonal antibodies HP 108.264, HP 115.140, HP 152.197, HP 110.662, HP 144.141, HP 108.371, HP 151.316, and HP 117.372 demonstrated an identical pattern of detection to that for HP 135.108.

The utility of such specific anti-human heparanase monoclonal antibodies is demonstrated by the accurate detection of heparanase in tissues and conditions known associated with heparanase expression. Sections of transgenic mouse liver expressing human heparanase are stained with the HP3/17 monoclonal anti-heparanase, while sections of normal mouse liver show no staining (Figure In human tissues, HP3/17 and HP 33/37 strongly detected heparanase expression in neutrophils and platelets, with none evident in normal lymphocytes (Figures 6 and strong expression is detected in human placenta (Figure Further immunohistochemistry with HP3/17 demonstrated detection of strong expression of heparanase in the cells lining the ducts of normal salivary gland, gall bladder, prostate and tubuli of the kidney medulla, while surrounding tissue showed no staining (results not shown).

As described hereinabove, heparanase catalytic activity is associated with a number of cancerous conditions, particularly metastatic disease.

Immunohistochemical analysis of normal and cancerous human tissue with the pep9-specific HP3/17 anti-heparanase monoclonal antibody demonstrate detection of strong heparanase expression in squamous cell carcinoma of the esophagus, cervix, and lung (stage II) (not shown), adenocarcinoma of the colon (Figure 9B), rectum, stomach and cervix, infiltrating duct carcinoma of the breast, transitional cell bladder carcinoma, and papillary serous ovarian cystadenocarcinoma. No false positive staining was detected in corresponding normal tissue sections (see, for example, Figure 9A).

Thus, the peptide-specific anti-heparanase monoclonal antibody HP3/17 raised against a specific region containing the active site of human heparanase, recognizes an epitope specific to the processed form of recombinant heparanase, and can be used to reliably distinguish between tissues and cell types expressing heparanase, and non-heparanase expressing tissue. Such specificity and accuracy of detection are particularly important for diagnostic, therapeutic and industrial applications of site-specific anti-heparanase 104 monoclonal antibodies such as HP130, HP 239, HP 108.264, HP 115.140, HP 152.197, HP 110.662, HP 144.141, HP 108.371, HP 135.108, HP 151.316, HP 117.372, HP 37/33, HP3/17, HP 201 and HP 102 described herein.

EXAMPLE IV Detection of disease using anti-heparanase antibodies As previously reported and also demonstrated herein, heparanase expression in biological samples is strongly indicative of metastatic disease, diabetes and diabetic neuropathy, atherosclerosis and other vasculopathies, heart disease, tumor angiogenesis, autoimmune and inflammatory diseases, renal disease and cancer. The anti-heparanase antibodies of the present invention are capable of detecting heparanase polypeptides in tissue and other biological samples. Thus, it will be appreciated that the anti-heparanase antibodies of the present invention can optionally and preferably be used to diagnose and monitor diverse diseases and conditions. The method is suitable for detecting the presence of metastatic disease, for determining the metastatic potential of cancerous growths and cells, for early distinction between types of cancer, such as blood cell cancer, for location of micrometastases in situ and in biopsy samples, for drug targeting to metastatic tissue, and for prevention and/or treatment of metastatic disease in subjects.

Tissue and other biological samples from subjects can be obtained and prepared as described hereinabove, according to methods well known in the art.

For example, tissue samples may be embedded in paraffin and sectioned, whereas blood samples may be prepared as a smear (see, for example, "Manual of Histological Staining Method of the Armed Forces Institute of Pathology," 3rd edition (1960) Lee G. Luna, HT (ASCP) Editor, The Blackstone Division McGraw-Hill Book Company, New York; The Armed Forces Institute of Pathology Advanced Laboratory Methods in Histology and Pathology (1994) Ulreka V. Mikel, Editor, Armed Forces Institute of Pathology, American Registry of Pathology, Washington, Detection of the formation of immune complex between an anti-heparanase antibody of the present invention and heparanase protein in the samples can be performed using any of a number of methods well known in the art, such as measurement of catalytic activity, radioactive, fluorescent, magnetic or spin labeling of primary antibody, or use of a specific, detectable second antibody, or protein G, as described hereinabove. Briefly, paraffin sections are fixed and blocked with normal serum block solution followed by overnight incubation with a heparanase specific antibody including, but not limited to HP130, HP 239, HP 108.264, HP 115.140, HP 152.197, HP 110.662, HP 144.141, HP 108.371, HP 135.108, HP 151.316, HP 117.372, HP 37/33, HP3/17, HP 201 and HP 102. The slides are then incubated with second, labelled antibody (such as biotinylated antiantibody, Vectastain, Vector Labs, Burlingame CA), washed and developed for visualization.

In one embodiment, detection of heparanase protein is performed in biopsy samples of subjects at risk for a metastatic disease, for example, colon cancer, by staining with one or more HRP linked anti-heparanase antibody specific for defined epitopes of human heparanase, such as, but not limited to, monoclonal antibodies HP130, HP 239, HP 108.264, HP 115.140, HP 152.197, HP 110.662, HP 144.141, HP 108.371, HP 135.108, HP 151.316, HP 117.372, HP 37/33, HP3/17, HP 201 and HP 102 disclosed hereinabove.

Immunohistopathological evidence of abnormal levels of heparanase in such samples, as demonstrated in the colon cancer cell lines described hereinabove, can be used to distinguish between malignant and benign growths, and to aid in timely determination of treatment, for example, chemotherapy or surgery. Periodic monitoring of changes in heparanase levels, as described hereinabove, can aid in determining duration, intensity, character or frequency of treatment, or prognosis in post-treatment subjects. For example, reduction in immunohistopathological detection of specific heparanase epitopes in colon biopsy samples following resection, can be indicative of the successful removal of foci of metastatic spread. Further, immunohistopathological detection of heparanase protein in the resected tissue could also aid in more accurate determination of the amount of tissue to be removed surgically.

It will be appreciated that the methods of detection and monitoring of heparanase-related and other diseases or conditions described hereinabove can be used for detection of heparanase in fluid samples as well as in tissue samples. Using the anti-heparanase antibodies of the present invention, heparanase can been detected (quantitatively and qualitatively) in urine, blood, plasma, serum, stool samples and the like. For example, elevated levels of heparanase in urine has been correlated with the presence of diabetic neuropathy, glomerulosclerosis, nephrotic syndrome and renal cell carcinoma.

As described hereinabove (see Examples I, II and III), heparanase-heparanase antibody immune complexes can be detected by an immobilized assay, such as the ELISA described in detail hereinabove, or immunoprecipitated from solution, and optionally further analyzed using gel electrophoresis.

Detection of marker antigens in fluids such as urine is well known in the art (see, for example US Patent No. 6,566,076 to Dobbs, et al, incorporated herein by reference as if fully set forth herein). Briefly, urine is filtered, and samples incubated with 5, 10 or 50pg specific anti-heparanase antibody such as HP130, HP 239, HP 108.264, HP 115.140, HP 152.197, HP 110.662, HP 144.141, HP 108.371, HP 135.108, HP 151.316, HP 117.372, HP 37/33, HP3/17, HP 201 and HP 102 monoclonal antibodies. Immune complexes are then bound by immunoglobulin specific ligands, such as Protein G beads (Pharmacia Cat. #17-0618-02), the mixture is precipitated by centrifugation and washed with PBS. The bound antibody-heparanase complexes are released by boiling, and the supernatant, containing the eluted proteins is analyzed by electrophoretic separation, transferred to a membrane and analyzed by Western blot analysis with anti-heparanase antibodies. Quantitative analysis of heparanase in urine samples can be performed by ELISA, as detailed hereinabove. Since urine of healthy subjects is normally substantially or completely free of heparanase activity and protein, detectable levels above a predetermined background level of heparanase-heparanase-antibody complexes in urine can be a strong indication for the presence of a renal disease, and the need for further investigation or initiation of treatment.

Example V Treatment of disease using specific anti-heparanase antibodies As described hereinabove, inhibition of heparanase activity has been correlated with alteration of pathological processes in a number of diseases, and even prevention of disease onset in others. For example, carcinoma cells are regarded as the main source of heparanase in the tumor microenvironment (Vlodavsky, I. et al. Nat Med 1999;5, 793-802), and the alteration of ECM of the basement membrane is a prerequisite for extravasation of tumor cells.

Treatment of experimental animals with heparanase inhibitors markedly reduces the incidence of metastases (9,10,20), indicating that inhibition of heparanase activity may inhibit tumor cell invasion and metastasis. Further, it has been shown that treatment with heparanase inhibitor PI-88 can prevent arterial restenosis injury (Francis et al, Circ Res 2003;92:e70-77).

These results show that the specific anti-heparanase antibodies according to the present invention can be used for treatment of a subject suffering from a pathological condition, in which the pathological condition is characterized by heparanase activity, which may optionally and preferably be over expression of heparanase. The method preferably includes administering the anti-heparanase antibody of the present invention to the subject.

Non-limiting examples of the pathological condition may optionally include types of cancers which are characterized by impaired (over) expression of heparanase, and are dependent on the expression of heparanase for proliferating or forming metastases. Therefore, the present invention also encompasses the treatment of cancer, particularly a heparanase-dependent cancer, in which the latter may optionally include any type of cancer for which proliferation and/or metastatic formation is affected by heparanase.

According to another embodiment of the present invention, the specific anti-heparanase antibody is used to treat other pathological conditions, including but not limited to, autoimmune reactions, inflammation, heart disease, renal disease, and the like. For example, administration of heparanase activity neutralizing antibodies, to subjects having diagnosed early-stage cancer, contained to a specific tissue, can decrease the likelihood of tumor cell proliferation and metastatic transformation. Administration of specific antibodies for passive immunotherapy is well known in the art (see, for example, U.S. Patent No: 6,254,867 to Reisner and Dagan, and U.S. Patent No: 6,254,869 to Petersen et al, both incorporated herein by reference as if fully set forth herein). In another embodiment of the present invention, specific antiheparanase antibodies of the present invention can be administered along with other therapy, including, but not limited to, chemotherapy.

It should be noted that the term "treatment" also includes amelioration or alleviation of a pathological condition and/or one or more symptoms thereof, curing such a condition, or preventing the genesis of such a condition.

The specific anti-heparanase antibodies of the present invention can be used to produce a pharmaceutical composition. Thus, according to another aspect of the present invention there is provided a pharmaceutical composition which includes, as an active ingredient thereof, a specific anti-heparanase antibody elicited by a heparanase protein or an immunogenic portion thereof and a pharmaceutical acceptable carrier. The antibody can specifically inhibit heparanase activity. As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein, either protein or physiologically acceptable salts or prodrugs thereof, with other chemical components such as traditional drugs, physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound or cell to an organism. Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g. by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. Hereinafter, the phrases "physiologically suitable carrier" and "pharmaceutically acceptable carrier" are interchangeably used and refer to a an approved carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered conjugate.

The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions 110 will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should be suitable for the mode of administration.

Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate processes and administration of the active ingredients. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Further techniques for formulation and administration of active ingredients may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference as if fully set forth herein.

While various routes for the administration of active ingredients are possible, and were previously described, for the purpose of the present invention, the topical route is preferred, and is assisted by a topical carrier. The topical carrier is one, which is generally suited for topical active ingredients administration and includes any such materials known in the art. The topical carrier is selected so as to provide the composition in the desired form, e.g. as a liquid or non-liquid carrier, lotion, cream, paste, gel, powder, ointment, solvent, liquid diluent, drops and the like, and may be comprised of a material of either naturally occurring or synthetic origin. Clearly, it is essential that the selected carrier does not adversely affect the active agent or other components of the topical formulation, and which is stable with respect to all components of the topical formulation. Examples of suitable topical carriers for use herein include water, alcohols and other nontoxic organic solvents, glycerin, mineral oil, silicone, petroleum jelly, lanolin, fatty acids, vegetable oils, parabens, waxes, and the like. Preferred formulations herein are colorless, odorless ointments, liquids, lotions, creams and gels.

Ointments are semisolid preparations, which are typically based on petrolatum or other petroleum derivatives. The specific ointment base to be used, as will be appreciated by those skilled in the art, is one that will provide for optimal active ingredient delivery and, preferably, will provide for other desired characteristics as well, e.g. emolliency or the like. As with other carriers or vehicles, an ointment base should be inert, stable, nonirritating and nonsensitizing. As explained in Remington: The Science and Practice of Pharmacy, 19th Ed. (Easton, Pa.: Mack Publishing Co., 1995), at pages 1399- 1404, ointment bases may be grouped in four classes: oleaginous bases; emulsifiable bases; emulsion bases; and water-soluble bases. Oleaginous ointment bases include, for example, vegetable oils, fats obtained from animals, and semisolid hydrocarbons obtained from petroleum. Emulsifiable ointment bases, also known as absorbent ointment bases, contain little or no water and include, for example, hydroxystearin sulfate, anhydrous lanolin and hydrophilic petrolatum. Emulsion ointment bases are either water-in-oil emulsions or oil-in-water emulsions, and include, for example, cetyl alcohol, glyceryl monostearate, lanolin and stearic acid. Preferred water-soluble ointment bases are prepared from polyethylene glycols of varying molecular weight; again, reference may be made to Remington: The Science and Practice of Pharmacy for further information.

Lotions are preparations to be applied to the skin surface without friction, and are typically liquid or semiliquid preparations, in which solid particles, including the active agent, are present in a water or alcohol base. Lotions are usually suspensions of solids, and may comprise a liquid oily emulsion of the oil-in-water type. Lotions are preferred formulations herein for treating large body areas, because of the ease of applying a more fluid composition. It is generally necessary that the insoluble matter in a lotion be finely divided.

Lotions will typically contain suspending agents to produce better dispersions as well as active ingredients useful for localizing and holding the active agent in contact with the skin, e.g. methylcellulose, sodium carboxymethylcellulose, or the like.

Creams containing the selected active ingredients are, as known in the art, viscous liquid or semisolid emulsions, either oil-in-water or water-in-oil.

Cream bases are water-washable, and contain an oil phase, an emulsifier and an aqueous phase. The oil phase, also sometimes called the "internal" phase, is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol; the aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant. The emulsifier in a cream formulation, as explained in Remington, supra, is generally a nonionic, anionic, cationic or amphoteric surfactant.

Gel formulations are preferred for application to the scalp. As will be appreciated by those working in the field of topical active ingredients formulation, gels are semisolid, suspension-type systems. Single-phase gels contain organic macromolecules distributed substantially uniformly throughout the carrier liquid, which is typically aqueous, but also, preferably, contain an alcohol and, optionally, an oil.

Various additives, known to those skilled in the art, may be included in the topical formulations of the invention. For example, solvents may be used to solubilize certain active ingredients substances. Other optional additives include skin permeation enhancers, opacifiers, anti-oxidants, gelling agents, thickening agents, stabilizers, and the like.

As has already been mentioned hereinabove, topical preparations for the treatment of heparanase-related diseases, conditions, and/or wounds according to the present invention may contain other pharmaceutically active agents or ingredients, those traditionally used for the treatment of such conditions. These include immunosuppressants, such as cyclosporine, antimetabolites, such as methotrexate, corticosteroids, vitamin D and vitamin D analogs, vitamin A or its analogs, such etretinate, tar, coal tar, anti pruritic and keratoplastic agents, such as cade oil, keratolytic agents, such as salicylic acid, emollients, lubricants, antiseptic and disinfectants, such as the germicide dithranol (also known as anthralin) photosensitizers, such as psoralen and methoxsalen and UV irradiation. Other agents may also be added, such as antimicrobial agents, antifungal agents, antibiotics and anti-inflammatory agents. Treatment by oxygenation (high oxygen pressure) may also be co-employed.

The topical compositions of the present invention may also be delivered to the skin using conventional dermal-type patches or articles, wherein the active ingredients composition is contained within a laminated structure, that serves as a drug delivery device to be affixed to the skin. In such a structure, the active ingredients composition is contained in a layer, or "reservoir", underlying an upper backing layer. The laminated structure may contain a single reservoir, or it may contain multiple reservoirs. In one embodiment, the reservoir comprises a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to affix the system to the skin during active ingredients delivery. Examples of suitable skin contact adhesive materials include, but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates, polyurethanes, and the like. The particular polymeric adhesive selected will depend on the particular active ingredients, vehicle, etc., i.e. the adhesive must be compatible with all components of the active ingredients-containing composition. Alternatively, the active ingredients-containing reservoir and skin contact adhesive are present as separate and distinct layers, with the adhesive underlying the reservoir which, in this case, may be either a polymeric matrix as described above, or it may be a liquid or hydrogel reservoir, or may take some other form.

The backing layer in these laminates, which serves as the upper surface of the device, functions as the primary structural element of the laminated structure and provides the device with much of its flexibility. The material selected for the backing material should be selected so that it is substantially impermeable to the active ingredients and to any other components of the active ingredients-containing composition, thus preventing loss of any components through the upper surface of the device. The backing layer may be either occlusive or non-occlusive, depending on whether it is desired that the skin become hydrated during active ingredients delivery. The backing is preferably made of a sheet or film of a preferably flexible elastomeric material.

Examples of polymers that are suitable for the backing layer include polyethylene, polypropylene, and polyesters.

During storage and prior to use, the laminated structure includes a release liner. Immediately prior to use, this layer is removed from the device to expose the basal surface thereof, either the active ingredients reservoir or a separate contact adhesive layer, so that the system may be affixed to the skin. The release liner should be made from an active ingredient/vehicle impermeable material.

Such devices may be fabricated using conventional techniques, known in the art, for example by casting a fluid admixture of adhesive, active ingredients and vehicle onto the backing layer, followed by lamination of the release liner.

Similarly, the adhesive mixture may be cast onto the release liner, followed by lamination of the backing layer. Alternatively, the active ingredient reservoir may be prepared in the absence of active ingredients or excipient, and then loaded by "soaking" in an active ingredient/vehicle mixture.

As with the topical formulations of the invention, the active ingredients composition contained within the active ingredients reservoirs of these laminated system may contain a number of components. In some cases, the active ingredients may be delivered "neat", i.e. in the absence of additional liquid. In most cases, however, the active ingredients will be dissolved, dispersed or suspended in a suitable pharmaceutically acceptable vehicle, typically a solvent or gel. Other components, which may be present, include preservatives, stabilizers, surfactants, and the like.

The pharmaceutical compositions described herein may also comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium 115 phosphate, various sugars, starches, cellulose derivatives, gelatin and polymers such as polyethylene glycols.

Other suitable routes of administration include for example, oral, rectal, transmucosal, transdermal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, inrtaperitoneal, intranasal, or intraocular injections.

Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more pharmaceutically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the active ingredients of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the active ingredients can be formulated readily by combining the active ingredients with pharmaceutically acceptable carriers well known in the art. Such carriers enable the active ingredients of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.

Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.

Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active ingredient doses.

Pharmaceutical compositions, which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with a filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g. gelatin for use in an inhaler or 117 insufflator may be formulated containing a powder mix of the active ingredient and a suitable powder base such as lactose or starch.

The active ingredients described herein may be formulated for parenteral administration, e.g. by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g. in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, pharmaceutical compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the pharmaceutical composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The pharmaceutical compositions of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine etc.

The active ingredients of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g.

conventional suppository bases such as cocoa butter or other glycerides.

The pharmaceutical compositions herein described may also comprise suitable solid of gel phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin and polymers such as polyethylene glycols.

Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredient effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For any active ingredient used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from activity assays in animals. For example, a dose can be formulated in animal models to 119 achieve a circulating concentration range that includes the ICs 0 as determined by activity assays. Such information can be used to more accurately determine useful doses in humans. In general, dosage is from about 0.01[ig to about 100g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years.

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in experimental animals, e.g. by determining the IC 5 0 and the LDs 0 (lethal dose causing death in 50% of the tested animals) for a subject active ingredient. The data obtained from these activity assays and animal studies can be used in formulating a range of dosage for use in human. For example, therapeutically effective doses suitable for treatment of autoimmune, inflammatory and cancerous conditions can be determined from the experiments with animal models of these diseases described hereinbelow (see Example VI).

The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.1).

Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the modulating effects, termed the minimal effective concentration (MEC). The MEC will vary for each preparation, but can be estimated from in vitro data; e.g. the concentration necessary to achieve 50-90% inhibition of a heparanase may be ascertained using the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. HPLC assays or bioassays can be used to determine plasma concentrations.

Dosage intervals can also be determined using the MEC value.

Preparations should be administered using a regimen, which maintains plasma 120 levels above the MEC for 10-90% of the time, preferable between 30-90% and most preferably 50-90%.

Depending on the severity and responsiveness of the condition to be treated, dosing can also be a single administration of a slow release composition described hereinabove, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.

The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, suitable dosage ranges for intravenous administration are generally about from about 20 to about 500p.g active compound per kg of body weight. Suitable dosage ranges for intranasal administration are generally from about 0.01lpg/kg body weight to about lmg/kg body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

Suppositories generally contain active ingredient in the range of from about 0.5% to about 10% by weight; oral formulations preferably contain from about 10% to about 95% active ingredient.

For antibodies, the preferred dosage is from about 0.1mg/kg to about 100mg/kg of body weight (generally from about 10mg/kg to about 20mg/kg). If the antibody is to act in the brain, a dosage of from about 50mg/kg to about 100mg/kg is usually appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration are often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration into the brain). A method for lipidation of antibodies is described by Cruikshank et al., 1997, J.

Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193).

I

The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

Compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising an active ingredient of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

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

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

Example VI Specific examples of treatment of disease using neutralizing anti- heparanase antibodies Anti-heparanase antibodies may be produced which have neutralizing activity, and/or other types of activity. For the purpose of this Example only and without wishing to be limited in any way, the antibody may be a neutralizing antibody.

The neutralizing antibody is preferably administered in a pharmaceutical composition. Such compositions preferably comprise a prophylactically or therapeutically effective amount of one or more anti-heparanase antibodies, and a pharmaceutically acceptable carrier.

In order to determine the efficacy of the. antibody of the present invention, preferably it is first tested in an animal model which may be selected according to good laboratory practice (GLP). The animal model is one which is able to develop the pathological condition against which the antibody is to be tested, for example by grafting of cancerous tissue (particularly for a neutralizing antiheparanase antibody) or induction of inflammatory disease. The number of animals to be selected and the dosing range to be tested could all be easily determined by one of ordinary skill in the art. A wide dosing range is preferably tested in order to determine whether there are any toxic effects.

In order to investigate the ability of specific anti-heparanase antibodies of the present invention to treat or prevent cancerous, inflammatory, autoimmune and other conditions, specific anti-heparanase antibodies were administered in mouse models of autoimmune diabetes (IDDM) (NOD mice), experimental arthritis (Arthrogen-anti-collagen type II mAb induced arthritis), and tumorigenesis (primary melanoma).

Specific antiheparanase antibodies inhibit tumor growth and tumorrelated mortality in vivo: Production of tumors by injection of melanoma cells (B 16-Fl) in mice is a well known in vivo model for testing the effectiveness of anti-cancer drugs and monoclonal antibodies in preventing or inhibiting tumorigenicity and metastatic proliferation (see, for example, Dong et al, Cancer Research 1999;59:1236-43, and Furge et al PNAS USA 2001, 98:10722-27). As shown in Figure 11, treatment with 200ptg of either the monoclonal anti-heparanase antibody HP130 (filled squares; elicited against a 79 amino acid long (coordinate 465-543) of portion of SEQ ID NO:4), or the monoclonal anti-heparanase antibody HP 37/33 (filled triangles; elicited against pep9, SEQ ID NO:9) effectively inhibited tumor growth (expressed as mean tumor volume in mm 3 in mice injected with 105 B 16-F I melanoma cells.

Antibody treated mice developed tumors consistently at least 50% smaller than those of their PBS-treated controls (filled diamonds), from day 8 until day 18.

Surprisingly, the anti-heparanase monoclonal antibodies were further found to protect against tumor-related mortality. At day 18, more than 50% of the PBScontrol animals had died, whereas no mortality was observed in the HP 130 or HP 37/33 mice.

Thus, specific anti-heparanase monoclonal antibodies of the present invention, administered in vivo, can effectively reduce the metastatic potential of tumor cells, inhibit tumor growth, and inhibit tumor-related mortality in treated animals.

Specific anti-heparanase antibodies inhibit induced inflammatory arthritis in viva: Injection of mice with anti-collagen type II monoclonal antibodies, followed by LPS, results in development of inflammatory disease having many characteristics of the clinical presentation of inflammatory arthritis: joint effusion, multi-joint involvement, pain, etc. (for details see de Fougerolles, et al J Clin Invest 2000;105:721-729). As shown in the Table of Figure 12, mice treated with both sham injection (PBS, group and 200lg of control monoclonal antibodies (anti-human IgG3, group B) developed significant arthritic symptoms 7 days after induction of arthritis. In contrast, intravenous administration of 250gg of specific anti-heparanase monoclonal antibody HP 3/17 (anti-pep9, SEQ ID NO: 9) (group reduced the symptoms by more than 30% at 11 days post induction, the effect persisting at even 14 days post induction.

Thus, specific anti-heparanase monoclonal antibodies of the present invention, administered in vivo, can effectively inhibit inflammatory arthritis in treated animals.

Specific antiheparanase antibodies inhibit autoimmune diabetes (IDDM) in vivo: The non-obese diabetic mouse (NOD) (Jackson Laboratories, Maine USA) is a well-known and highly characterized model of autoimmune (IDDM) diabetes, developing islet inflammation at 4-6 weeks, progressing to overt IDDM at 4-5 months (Bendelac, A et al J Exp Med 1987;166:823-32).

As shown in Figure 13, in mice receiving administration of 2 0 0 ktg specific antiheparanase monoclonal antibody HP 3/17 (anti-pep9, SEQ ID NO:9) (filled diamonds), the onset of diabetic symptoms (glucosuria) was delayed, and symptoms less severe than in the PBS-treated control animals (filled squares).

Further, animals in the group receiving the anti-heparanase antibody treatment showed greatly improved survival, many weeks after onset of symptoms, than the PB S-treated controls.

Thus, specific anti-heparanase monoclonal antibodies of the present invention, administered in vivo, can be used to effectively suppress the onset of diabetic symptoms in autoimmune diabetes. Further, the results described hereinabove demonstrate that in vivo administration of specific anti-heparanase monoclonal antibodies can enhance survival in autoimmune conditions such as

IDDM.

Testing in animal models, as described hereinabove, can provide the basis for determining the range of therapeutically effective doses, effective and contraindicated routes of administration, dosing schedules, formulations, compositions, combinations with additional drugs, and other parameters of administration and therapeutic guidelines for human testing. Next, the antibody is preferably tested in humans suffering from the pathological condition, according to good clinical practice (GCP). The dosage range is then preferably adjusted according to the most effective range, which may differ depending upon such factors as age, overall physical condition of the patient, weight and disease state.

EXAMPLE VII General heparanase characteristics The antibodies taught in the present invention are capable of specifically binding to at least one epitope of a heparanase protein. As all proteins heparanase is encoded by a polynucleotide fragment, and the fragment preferably includes at least a segment of SEQ ID NOs: 12 or 38. More preferably the polynucleotide fragment includes nucleotides 63-1691 of SEQ ID NO: 12, or nucleotides 139-1869 of SEQ ID NO: 38, which encode the entire human heparanase enzyme.

In addition, the polynucleotide fragment preferably includes a polynucleotide sequence capable of hybridizing (base pairing under either stringent or permissive hybridization conditions, as for example described in Sambrook, Fritsch, Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York.) with heparanase encoding cDNA, especially with nucleotides 1-721 of SEQ ID NO:12.

The polynucleotide fragment comprises a polynucleotide sequence which encodes a polypeptide being cleavable to obtain heparanase activity. The polynucleotide sequence may be as set forth in SEQ ID NO: 12 or a functional part thereof. The functional part encodes a polypeptide cleavable to obtain heparanase catalytic activity. This includes also natural and man-made innocuous variations of the aforementioned sequence mutations, such as point mutations) as such variations may also encode a polypeptide cleavable to obtain heparanase catalytic activity.

Furthermore, as shown in the background section above, 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:12, has heparanase catalytic activity, and therefore may also be elicited by the antibodies of the present invention.

The polynucleotide sequence that encodes the heparanase protein 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, and may be in the form of double stranded DNA, single stranded DNA or RNA. Similarly it can be derived from any animal including mammalians 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 hybridizable 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.

The polypeptide encoded by the aforementioned polynucleotide fragment preferably includes an amino acid sequence as set forth in SEQ ID NOs: 4 or 39 or a functional part thereof, i.e. a portion harboring heparanase catalytic activity.

However, 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: 4 or 39 or functional part thereof.

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 others 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.

The described polypeptide may be a recombinant polypeptide, and preferably the recombinant heparanase polypeptide may be human recombinant heparanase. The recombinant heparanase may be cloned in any type of cell.

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 a plurality of different cell types. 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 antiheparanase inhibitors or drugs screening assay or system.

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, for example 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.

In additions, the genetically modified cell may be subjected to a substance that induces secretion of secretable proteins into the growth medium, 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 heparanase from expressing cells. The calcium ionophore calcimycin (A23187) and phorbol 12-myristate 13-acetate, are effective in 128 inducing secretion of recombinant heparanase from transduced cells into their media as shown in the Examples below.

The genetically modified cell is preferably 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.

Purification of a recombinant heparanase from overexpressing cells or growth medium in which they grow may be achieved by adsorbing the recombinant heparanase on a Source-S column under low salt conditions (e.g.

about 50mM NaCI), washing the column with low salt solution thereby eluting other proteins, and eluting the recombinant heparanase from the column by a salt gradient 50mM to 1M NaCI) or a higher concentration of salt (e.g.

about 0.4M).

As described in the background section above, the heparanase polypeptide may be activated to obtain heparanase catalytic activity by digesting a heparanase precursor polypeptide by a protease. The heparanase precursor polypeptide can be natural or recombinant, purified, partially purified or nonpurified. 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 also possible. Preferably, 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.

One ordinarily skilled in the art can readily determine the pH value at which a specific protease is most active.

Due to the aforementioned characteristic of the heparanase polypeptide, administering of a protease inhibitor in vivo or in vitro results in the inhibition 129 of the proteolytic processing of heparanase. 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 that follow. 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. 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 that follow, the preheparanase (non-processed, p70 heparanase) is characterized by lower activity as compared to its processed counterpart (p52 heparanase).

A nucleic acid construct can be used to provide a precursor heparanase protein, when introduced into a cell expression system. The nucleic acid construct may optionally comprise a first nucleic acid segment encoding 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 a downstream portion (C terminal) of a heparanase polypeptide, such that 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 that follow. 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.

The precursor heparanase protein expressed from the above construct comprises 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. A heparanase protein is obtained by digesting the precursor heparanase protein described herein.

Additionally, a method of obtaining a homogeneously processed, active heparanase may be performed using the aforementioned precursor heparanase protein. The method 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; treating the precursor heparanase protein with a protease; and purifying a proteolytic heparanase product having heparanase catalytic activity.

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.

EXAMPLE VIII Cloning of the hpa gene Purified fraction of heparanase isolated from human hepatoma cells (SKhep-1) was subjected to tryptic digestion and microsequencing. EST (Expressed Sequence Tag) databases were screened for homology to the back translated DNA sequences corresponding to the obtained peptides. Two EST sequences (accession Nos. N41349 and N45367) contained a DNA sequence encoding the peptide YGPDVGQPR (SEQ ID NO:36). These two sequences 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:12), contained an open reading frame which encodes, as shown in Figure 47 and SEQ ID NO:13, a polypeptide of 543 amino acids (SEQ ID NO:4) with a calculated molecular weight of 61,192 daltons. The 3' end of the partial cDNA inserts contained in clones 257548 and 260138 started at nucleotide G' m of SEQ ID NO:12 and Figure 47.

As further shown in Figure 47, there is a single sequence discrepancy between the EST clones and the PCR amplified sequence, which led to an amino acid substitution from Tyr 24 6 in the EST to Phe 2 4 6 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:12).

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:12). 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.

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

Three mouse ESTs were identified (accession No. Aal77901, from mouse spleen, 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:37). As shown in Figure 48, the coding region is 80% similar to the 3' end of the hpa cDNA sequence. These ESTs are probably cDNA fragments of the mouse hpa homolog that encodes for the mouse heparanase.

Searching for consensus protein domains revealed an amino terminal homology between the heparanase and several precursor proteins such as Procollagen Alpha 1 precursor, Tyrosine-protein kinase-RYK, Fibulin-1, 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.

EXAMPLE X Expression of recombinant human heparanase in bacteria Experimental Methods Construction of expression vector: A 1.6 kb fragment of hpa cDNA (SEQ ID NO:12) was amplified from pfasthpa (hpa cDNA cloned in pFastBac, see U.S. Patent Application No. 08/922,170 and U.S. Patent No. 5,968,822) by PCR using specific sense primer: IIpu-550Nde CGCATATGCAGGACGTCGTGGACCTG-3' (SEQ ID NO:14) and a vector specific antisense primer: 3'pFast 5'-TATGATCCTCTAGT

ACTTCTCGAC-

3' (SEQ ID NO:15). PCR conditions were: denaturation 94 0 C, 40 seconds, first cycle 3 minutes; annealing 58 0 C, 60 seconds; and elongation 72°C, minutes, total of 5 cycles, and then denaturation 94°C, 40 seconds; annealing 68 0 C, 60 seconds; and elongation 72 0 C, 2.5 minutes, for additional 25 cycles.

The Hpu-550Nde primer introduced an NdeI site and an in frame ATG codon preceding nucleotide 168 of hpa. The PCR product was digested by Ndel and BamHI 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 pRSEThpaS 1, encoded an open reading frame of 508 amino acids (36-543, SEQ ID NO:4) of the heparanase protein, lacking the N-terminal 35 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 pmercaptoethanol 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 100[ig/ml ampicillin and 34 gg/ml chloramphenicol. Cells were diluted 1/10 in the same medium, and the cultures were grown to an OD600 of approximately 0.5. Isopropyl-thiogalactoside (IPTG) (Promega) was added to a final concentration of ImM and the culture was incubated at 37C 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 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 pRSEThpaS1 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 18, lanes That band did not appear when negative control cells transformed with pRSET were employed (Figure 18, 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 solubillization and refolding of recombinant proteins expressed in E. coli are well known in the art (see, for example, for insulin, 61; others are reviewed in 62) 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 XI 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 pPIC3.5K-Sheparanase (Figure 19) contains nucleotides 63-1694 of the hpa sequence (SEQ ID NO: 12) cloned into the expression vector pPIC3.5K (Invitrogen, CA) using a multistep procedure as follows.

A pair of primers: HPU-664I AGGAATTCACCATGCTGCT GCGCTCGAAGCCTGCG-3' (SEQ ID NO:16) and HPL-209 ATTGCTCCTGGTAG-3' (SEQ ID NO:17) were used in PCR amplification to introduce an EcoRI site just upstream to the predicted methionine. PCR conditions were: denaturation 94°C, 40 seconds; annealing 50°C, seconds; and elongation 72°C, 180 seconds, total of 30 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 (Figure 19).

The second vector, designated pPIC9K-PP2 (Figure 20), includes the hpa coding region except for the predicted signal sequence (N-terminal 36 amino acids, see SEQ ID NO:4). 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, CGTGGACCTGGAC-3' (SEQ ID NO:18) and HPL-209 (SEQ ID NO:17, described above) were used in PCR amplification under the conditions described above.

The resulting PCR product was digested with XhoI and BamHI and inserted into the XhoI-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 SacI-NotI sites ofpPIC9K.

The hpa was removed from the later vector as a SacI-NotI fragment and cloned into the Sacl-NotI sites of pPIC9K, thus creating the vector pPIC9K- PP2 (Figure 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 6.0, 1.34% yeast nitrogen base with ammonium sulfate without amino acids, 4 x 10- 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 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 kDa, as expected for heparanase. These results indicate that the heparanase contains a signal sequence which efficiently functions as a secretion signal in Pichia pastoris.

I

G-418 resistance was used to select isolates characterized by multiple gene integration events. A faint heparanase band was observed in the supernatant of pPIC3.5K-Sheparanase transformant isolated without selection on G-418 (Figure 21, lane whereas no band was observed in the corresponding position in pPIC3.5K transformant, which served as negative control (Figure 21, lane A profound increase in the secretion of heparanase was observed in isolates resistance to 4mg/ml of G-418 (Figure 21, lanes 3-6).

EXAMPLE XII 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 37a) or the CMV promoter (pChpa, Figure 37e). One construct (pShpaCdhfr, Figure 37b) 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 22a-b, 37a-b). 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 37f), included expression unit of mouse dhfr gene under the regulation of CMV promoter and served as control.

A vector designed pSlhpa (Figure 22c, 37c) was constructed by ligating a truncated hpa gene fragment (nucleotides 169-1721 of SEQ ID NO:12) to a heterologous signal peptide as follows. Preprotrypsin (PPT) signal peptide (63) was generated by chemically synthesizing two complementary oligonucleotides corresponding to the signal peptide encoding DNA sequence, the first having a sequence AGCTGC AGTTGCTCAGGAC-3' (SEQ ID NO:19), whereas the second having a complementary sequence GAGCTAGGATCAGAAGTGCAGACATGGTG-3' (SEQ ID Annealing of the complementary oligonucleotides produced the double strand sequence encoding to the PPT signal peptide flanked by a sticky end of an EcoRl restriction site on the 5' end thereof and a sticky end of an AatII restriction site on the 3' end thereof. Following restriction by EcoRl and AatII of the pfasthpa vector, a 145 bp fragment was removed, and replaced by the 52 bp PPT DNA sequence to yield plasmid pSlhpa. The insert thereof was cut out with EcoRI and DotI and ligated into the vector pSI.

A vector designed pS2hpa (Figure 22d and 37d) was constructed by ligating a truncated hpa gene fragment (nucleotides 144-1721) to the PPT signal peptide as follows. Preprotrypsin (PPT) signal peptide (63) was generated by chemically synthesizing two complementary oligonucleotides corresponding to the signal peptide encoding DNA sequence, the first having a sequence AGCTGAGTTGC-3' (SEQ ID NO:21), whereas the second having a complementary sequence GATCAGAAGTGCAGACATGGTG-3' (SEQ ID NO:22). 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 22d, 37d).

Transfection of vectors into cells: DNA constructs were introduced into animal cells using the calcium-phosphate co-precipitation technique essentially as described in (64).

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 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 medium and resistant cellular clones were isolated. Alternatively, cells were transferred after transfection directly to a selection medium containing MTX (100 1000nM).

SDS polyacrylamide gel electrophoresis and Western blot analysis: Denatured and reduced samples were loaded on ready made gradient (4-20%) 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 ofheparanase activity: ECM-derived soluble HSPG assay was performed by incubating cell extracts with solubilized 35 S-labeled ECM (18 hours, 37°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 Kav 0.8 (peak II) was determined (52).

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 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 1.Omg/ml), 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 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 142 hamster kidney cells (BHK21) and Chinese hamster ovary (CHO dhfr-) cells, following transfection with the hpa gene (Figures 23a-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 23 a-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 22a) or pChpa constructs (Figure 22e) exhibited heparanase activity (Figure 23a, lane 4, Table 3 below).

Transfection of CHO cells with the expression vector pShpaCdhfr (Figure 22b) 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 23a, 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 23a, Figure 23b, lane 1, and Table Although the hpa DNA encodes for a large 543 amino acids protein (expected molecular weight about 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 23a, lane Transient expression of pShpaCdhfr in CHO cells revealed predominantly a 52 kDa heparanase protein (Figure 23b, lane 2).

It has been previously shown that a 52 kDa protein with heparanase activity was isolated from placenta (52) 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 (pS lhpa and pS2hpa, Figures 22c-d) that would drive translation of heparanase attached to the PPT signal peptide.

Both pSlhpa 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 pSlhpa and pS2hpa in human 293 cells resulted in the appearance of a single size (about 65 kDa) heparanase protein (Figure 24c, lanes 3-6).

Table 3: Determination of heparanase activity in transfected animal cells Cell type Transfected DNA Heparanase Activity Human 293 cells PChpa Human 293 cells PShpa Human 293 cells PSlhpa 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 Img/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 24a-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 and in concentrated conditioned medium, in the gel shift assay (Figure 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 25d-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 67. Results of a typical "batch run" that includes proliferation and maintenance of heparanase producing cells in a 500ml Spinner-Basket are shown in Figure 26.

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 5, 7 or 35 liters. Accordingly, larger amounts of Fibracel 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 XIII 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 SalI and NotI 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 bacmid 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 10 6 cells in T-25 flasks. Cells were harvested 2-3 days after infection. 4 x 10 6 cells were centrifuged and resuspended in a reaction buffer containing 20mM phosphate citrate buffer, 50mM NaCI. Cells underwent three cycles of freeze and thaw and lysates were stored at -80 0 C. Conditioned medium was stored at 4°C.

Experimental Results Degradation of soluble ECM-derived HSPG: Monolayer cultures of High five cells were infected (72 h, 28 0 C) with recombinant bacoluvirus containing the pFasthpa plasmid or with control virus containing an insert free plasmid. The cells were harvested and lysed in heparanase reaction buffer by three cycles of freezing and thawing. The cell lysates were then incubated (18 h, 37°C) with sulfate labeled, ECM-derived HSPG (peak followed by gel filtration analysis (Sepharose 6B) of the reaction mixture.

As shown in Figure 27, the substrate alone included almost entirely high molecular weight (Mr) material eluted next to Vo (peak I, fractions 5-20, Kav 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, 146 incubation of the HSPG substrate with lysates of cells infected with the hpa containing virus resulted in a complete conversion of the high Mr substrate into low Mr labeled degradation fragments (peak II, fractions 22-35, 0.5 Kay 0.75).

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

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

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

As shown in Figures 28a-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 pF 1 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 kDa cut off membrane to obtain a crude estimation of the molecular weight of the recombinant heparanase enzyme. As demonstrated in Figure 29, 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 30a-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 0 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 31 la-b and 32a-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 34). 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 37C. Control intact non-infected Sf21 or High five cells failed to degrade the ECM HS side chains.

In subsequent experiments, as demonstrated in Figures 33a-b, High five and Sf21 cells were infected (96h, 28°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 34, production of these fragments was abolished in the presence of heparin, due to its competitor's nature. No heparanase activity was detected in the growth medium of control, non-infected cells. These results indicate that the heparanase enzyme expressed by cells infected with the pFhpa4 virus is capable of degrading HS when complexed to other macromolecular constituents fibronectin, laminin, collagen) of a naturally produced intact ECM, in a manner similar to that reported for highly metastatic 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 co- and 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.

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 TEPl) 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 0.2g 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 20, 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 1.2x101 0 cells. Eight shake flasks of 500ml-size 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 150 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 Ixl06 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 0.2t 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 liters and 1 liters. Heparanase was purified from the concentrated solution on HPLC. Table 4 below summarizes the results of two large scale heparanase production by insect cells experiments.

TABLE 4 Batch Cell MOI Volume of Harvest time Cell viability Heparanase in No. used used culture post infection at harvest harvest (mg/ml) (hours) Sf21 0.1 4.5 78 76 0.44 31 Hi-5 0.1 6.0 75 76 0.16 r EXAMPLE XIV 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 cm) pre equilibrated with 10 mM phosphate buffer pH 6.8, 50 mM NaCl.

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 NaCl fractions were about 90% pure and exhibited the highest activity (Figure 35, lane A rabbit anti-heparanase polyclonal antibody detected the purified enzyme in Western blot ECL analysis (Figure 36, 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.

152 EXAMPLE XV 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 XII.

Heparanase activity using DMB assay: For each sample, l00pl heparin sepharose (50% suspension in I x buffer A containing 20mM Phosphate citrate buffer pH 5.4, ImM CaC12 and ImM NaCI) were incubated in eppendorf tube for 17 hours with a tested enzyme preparation. At the end of the incubation period, samples were centrifuged for 2 minutes at 1,000 rpm and the supematants were analyzed for sulfated polyanions (heparin) using the colorimetric dimethylmethylene blue assay as follows.

Supematants (100[l) were transferred to plastic cuvettes and diluted to with PBS supplemented with 1% BSA. 1,9-Dimethylmethylene blue (32 mg dissolved in 5ml ethanol and diluted to 1 liter with formate buffer) was added to each cuvette. Absorbency at 530nm 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 included. For further details, see U.S. Patent 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|tg heparan sulfate in a final volume of 100l. Reactions were performed in a 96 well microtiterplate at 37°C for 17 hours. Reactions were thereafter stopped by the addition of l00pl tetrazolium blue reagent tetrazolium blue in 0.1M NaOH) to each well.

Color was developed following incubation at 60°C for 40 minutes. Color intensity was quantitatively determined at 580nm 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 1-15gg 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 200mg of purified human recombinant heparanase protein produced in baculovirus infected Sf21 insect cells (see Examples XIII-XIV 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: 2TT1 CHO cells (2 x 108 cells stably transfected with pShpaCdhfr, Figure 37b) were extracted in 2.5ml of 10mM 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 20mM 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 IM sodium chloride in 20mM 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 40a) and fractions of lml 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 154 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, 17mg of the above described rabbit anti heparanase polyclonal antibody, purified on protein G sepharose, were bound to a column containing 2ml 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 20mM 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 20mM phosphate citrate buffer, pH 5.4.

Cells were frozen in liquid nitrogen and subsequently thawed at 37°C.

Freezing and thawing were repeated two more times. The extract was then centrifuged for 15 minutes at 4,000 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 280nm reached zero.

Proteins were eluted from the column with 0.1 M glycine HC1 buffer, pH 900gl fractions were collected into eppendorf tubes each containing 100gl of IM phosphate buffer, pH 8. The presence of heparanase in the eluted fractions was determined by Western blotting following gradient 4-20% SDS-PAGE of 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 30gl 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:4), 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 46a), 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-ArgJ, SEQ ID NO:23) which constitute a factor Xa recognition and cleavage sequence.

An alternative, second, sequence included 5 amino acids (Asp-Asp-Asp-Asp- Lysj, SEQ ID NO:24) which constitute a enterokinase recognition and cleavage sequence. These sequences do not appear in the natural enzyme (SEQ ID NO:4).

To this end, the following PCR primers were constructed: 52-Xa CCATCGATAGAAGGACGAAAAAAGTTCAAGAACAGCA CCTAC-3' (SEQ ID NO:25); 52x-Cla 3' (SEQ ID NO:26); 56-Xa AACCAGGATATT-3' (SEQ ID NO:27); 56x-Cla TAACTTCTCTCTTCAAAG-3' (SEQ ID NO:28); hpl 967 AAGCAGCAACTTTGGC-3' (SEQ ID NO:29); hpu 685 AGGTGAGCCCAAGAT-3' (SEQ ID NO:30); 52-EK CGACAAGAAAAAGTTCAAGAACAGC ACCTAC-3' (SEQ ID NO:31); 52e-Cla 5'-GGATCGATCTGGTAGTGTTCTCGGAGTAG-3' (SEQ ID NO:32); 56-EK TCAACCAGGATATTTG-3' (SEQ ID NO:33); and 56e-Cla CCATCGATTTGG GAGTAACTTCTCTCTTCAAAG-3' (SEQ ID NO:34).

The following constructs were prepared (Figure 46b): 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 resulting 1180 bp fragment was digested with Clal and AflII 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 Clal and AatII and a 370 bp fragment was isolated. The ClaI-AflII 220 bp and the Clal-AatII 370 bp fragments were ligated to a 5,900 AatlI-AflII 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 Clal 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 Clal and AatII and a 250 bp fragment was isolated. The Clal-AflII 340 bp and the ClaI-AatlI 250 bp fragments were ligated to a 5,900 AatlI-AflII 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 resulting 1180 bp fragment was digested with Clal and AflII and a 220 bp fragment was isolated. A second PCR reaction was performed with a pFasthpa2 template and with primers 52e-Cla and hpu 685. The resulting 500 bp fragment was digested with Clal and AatII and a 370 bp fragment was isolated. The ClaI-AflII 220 bp and the ClaI-AatlI 370 bp fragments were ligated to a 5,900 AatII-AflII fragment of the pFasthpa2 plasmid.

(iv) Construction ofpre-p56'-Enterokinase hpa in pFast: A first PCR reaction was performed with a pFasthpa2 template and with primers 56-EK and hpl 967. The resulting 1290 bp fragment was digested with Clal and AflII 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 AatII and a 250 bp fragment was isolated. The ClaI-AflII 340 bp and the Clal-AatlI 250 bp fragments were ligated to a 5,900 AatlI-AflII 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

I

hereinabove includes an AatII-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:4). The hpa constructs described in Figures 22a-e and 37a-e, as well as the pFasthpa constructs, each includes a single AatI site and a single AflII site within the hpa cDNA sequence, thus enabling the insertion by replacement of the 220 or 340 AatII-AflII fragments as desired.

Experimental Results Expression of hpa DNA in animal cells: As already shown and discussed under Example XII 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 22a-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 22b and 37f, the latter serves as a negative control) could be subjected to selection by methotrexate (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 23a), baby hamster kidney cells (BHK21; Figure 38a) and Chinese hamster ovary cells (CHO; Figure 23b). Stable expression of heparanase in CHO cells is shown in Figures 23a-b.

Transfection of CHO cells with the expression vector pShpaCdhfr (Figure 22b) or co-transfection with pS hpa and pCdhfr (Figure 22c and 37f), 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 23a, 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 p52 (Figures 23a, 38a).

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 23a, lane 4).

BHK21 cells, transiently transfected with pSlhpa (Figure 22c) express predominantly the p52 form of recombinant heparanase (Figure 38a, lane 1 marked by an arrow). Stable CHO clones express predominantly the p52 protein (Figure 23b, 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 22c) expressed p52 (Figure 38a) 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 22e) exhibited high heparanase activity (Figure 38b).

It has been previously shown that a 52 kDa protein with heparanase activity was isolated from placenta (52) and platelets (53).

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 22c-d) to direct translation of heparanase attached to the PPT signal peptide, a secretion signal peptide.

Both pSlhpa and pS2hpa plasmids directed the expression of protein product with heparanase activity in human 293 or CHO cells (Figures 24c, 39a- Transient expression of heparanase from pSlhpa and pS2hpa resulted in the appearance of a single size (about 70 kDa) heparanase protein in the medium (Figure 24c, lanes similar to the larger form of recombinant heparanase detected in the cells.

CHO cells, stably transfected with either pShpaCdhfr (2TTI clones) or pSlhpa (S1PPT 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-selective conditions and clones exhibiting the above stability were selected for further analysis.

2TT1 and S1PPT clones under (clones 2TT1 and SIPPT-p) or after (clones 2TTI-2, 2TTI-8, S1PPT-4 and SIPPT-8) selection with high MTX yielded stable clones exhibiting moderate (clones 2TT1 (Figure 39b), 2TT1-2, 2TT1-8) or high (clones S1PPT-p, S1PPT-4, S1PPT-8 (Figure 39a)) 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 39a-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 39a, 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 39a, lane 4).

The purification of recombinant heparanase from 2TT1 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 XIV hereinabove.

Separation of the total protein content of 2TT1-8 cell extract on a mono-S cation exchange column is shown in Figure 40a. The vast majority of cellular proteins were eluted from the column prior to the elution of heparanase (Figure 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 40b), and its activity, as measured by the DMB (Figure 40c) and the tetrazolium (Figure 40d) 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-heparanase affinity column: Partially purified, active recombinant heparanase 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 2TTI-8 cells was loaded on the column for affinity separation. Figure 41a-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 41b) was tightly correlated with the presence of the recombinant enzyme (Figure 41a). 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 p70 produced by mammalian cells, and it

I

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 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 42b). This activation was protease dependent as is evident form the inhibition of activation caused by a protease inhibitors cocktail (Figure 42b).

The fate of the p70-bac heparanase following exposure to acidic pH was uncovered by Western-blot analysis (Figure 42a). 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 42a, lane Proteolysis was inhibited in the presence of protease inhibitors (Figure 42a, 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 p70-bac heparanase, a collection of individual protease inhibitors was employed (Figures 42c-d). The inhibitors antipain, E-64, leupeptin and chemostatin were most effective in preventing the activation of 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 42c. 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 p70-bac 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 which corresponds to a new heparanase species that starts at Ser 120 of the full length enzyme (SEQ ID NO:4).

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 43a-b).

Trypsin, 1.5 to 500 units per 10 g p70-bac heparanase, gradually activated the protein, reaching maximal activation of five-fold already at 15 units trypsin (Figure 43a). 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 43b). Smaller fragments of heparanase were also obtained by trypsinization (Figure 43b, 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 44). Activation was dose dependent.

Processing and activation of recombinant CHO produced and secreted heparanase (p70) was also obtained by mild treatment with Cathepsin L, which is a known cysteine protease (Figures 45a-b). Processing by this protease resulted in several digestion products, of about 56, 34 and 21 kDa (Figure 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 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:4, see above) within the first peak of hydrophilicity (Figure 46a, peak The second peak of hydrophilicity (Figure 46a, 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 46c 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-Arg4 or of enterokinase Asp-Asp-Asp- Asp-Lysj downstream amino acids 119 or 157. AatII-AflI 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 22a-e) and for that effect, any other construct harboring a hpa derived sequence. Figure 46b 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.'

EXAMPLEXVI

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 N-glycosidase, the protein appeared as a 57 kDa band.

This molecular weight corresponds to the deduced molecular mass (52,192 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 O-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 50 kDa and a minor of about 65 kDa in cell lysates.

Comparison of the enzymatic activity of the two forms, using a semiquantitative 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 terminal sequencing of the platelet heparanase indicated that cleavage occurs between amino acids Gin 15 7 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.

ND 166

O

NAccording to Fairbank et al. (48) the precursor is cleaved at three sites to O 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 0 heparanase in clinically useful quantities such as the expense of growth media, Spotential contamination with host cell proteins and the limited production Scapacity of mammalian tissue culture systems.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

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 available as prior art to the present invention, or that such reference forms part of the common general knowledge.

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 r IND 166a Sstated 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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Editorial Note 2004201462 The sequence listing pages 178-203 are located after the drawings pages 1/42-42/42.

The amended claims are from pages 204-212.

Claims (11)

1. 3. The isolated antibody or portion thereof of claim 1, wherein said heparanase protein is at least 70% homologous to the amino acid sequence of SEQ ID NO:4.
2. 4. The isolated antibody or portion thereof of claim 1, wherein said heparanase protein is at least 80% homologous to the amino acid sequence of SEQ ID NO:4. The isolated antibody or portion thereof of claim 1, wherein said heparanase protein is at least 90% homologous to the amino acid sequence of SEQ ID NO:4.
3. 6. The isolated antibody or portion thereof of claim 1, wherein said heparanase protein comprises an amino acid sequence as set forth in SEQ ID NO:4.
4. 7. The isolated antibody or portion thereof of any one of claims 1-6, wherein said at least one epitope is at least 70% homologous to the amino acid sequence of SEQ ID NO:4.
5. 8. The isolated antibody or portion thereof of any one of claims 1-6, wherein said at least one epitope is at least 80% homologous to the amino acid sequence of SEQ ID NO:4. 205 O
6. 9. The isolated antibody or portion thereof of any one of claims 1-6, wherein Ssaid at least one epitope is at least 90% homologous to the amino acid sequence of 00 SEQ ID NO:4. The isolated antibody or portion thereof of any one of claims 1-6, wherein IDO said at least one epitope comprises an amino acid sequence as set forth in SEQ ID NO:4. I The isolated antibody or portion thereof of claim 1, wherein said C heparanase protein comprises an amino acid sequence as set forth in SEQ ID NO:4, provided that there is a phenylalanine residue instead of a tyrosine residue at position 246.
7. 12. The isolated antibody or portion thereof of any one of claims 1-6, wherein said at least one epitope comprises a sequence as set forth in SEQ ID NO:4, provided that there is a phenylalanine residue instead of a tyrosine residue at position 246.
8. 13. The isolated antibody or portion thereof of any one of claims 1-12 comprising a polyclonal or monoclonal antibody.
9. 14. The isolated antibody or portion thereof of claim 13, wherein said polyclonal antibody is selected from the group consisting of a crude polyclonal antibody and an affinity purified polyclonal antibody. The isolated antibody or portion thereof of claim 13, wherein said monoclonal antibody is selected from the group consisting of HP 108.264, HP
10. 115.140, HP 152.197, HP 110.662, HP 144.141, HP 108.371, HP 135.108, HP
11. 151.316, HP 117.372, HP 37/33, HP3/17, HP 201 and HP 102. 16. The isolated antibody or portion thereof of any one of claims 1 to comprising a chimeric antibody. 00 oo 17. The isolated antibody or portion comprising a humanized antibody. 18. The isolated antibody or portion comprising a Fab fragment. 19. The isolated antibody or portion comprising a single chain antibody. The isolated antibody or portion comprising an immobilized antibody. 21. The isolated antibody or portion comprising a labeled antibody. thereof of any one of claims 1 to thereof of any one of claims 1 to thereof of any one of claims 1 to thereof of any one of claims 1 to thereof of any one of claims 1 to 22. The isolated antibody or portion thereof of any one of claims 1 to wherein said at least one epitope is selected from the group consisting of a heparan-sulfate binding site flanking region, a catalytic proton donor site, a catalytic nucleophilic site, an active site and binding site linking region and a C- terminal sequence of heparanase P8 subunit. 23. The isolated antibody or portion thereof of any one of claims 1 to wherein said at least one epitope comprises a heparan-sulfate binding site flanking region. 24. The isolated antibody or portion thereof of any one of claims 1 to wherein said at least one epitope comprises a catalytic proton donor site. The isolated antibody or portion thereof of any one of claims 1 to wherein said at least one epitope comprises a catalytic nucleophilic site. 207 26. The isolated antibody or portion thereof of any one of claims 1 to wherein said at least one epitope comprises an active site and binding site linking 00 region. 27. The isolated antibody or portion thereof of any one of claims 1 to INO wherein said at least one epitope comprises a C-terminal sequence of heparanase P8 subunit. 28. The isolated antibody of any one of claims 1 to 12, wherein said heparanase protein is a recombinant heparanase protein. 29. A cell line when used for producing the monoclonal antibody or portion thereof, as claimed in claim 13. The cell line of claim 29, wherein said antibody or portion thereof is humanized. 31. A method of treating a subject suffering from a pathological condition, said method comprising the step of administering a therapeutically effective amount of the antibody or portion thereof of any one of claims 1 to 12 to said subject. 32. A method of treating or preventing a heparanase-related disorder or condition in a subject, said method comprising the step of administering a therapeutically effective amount of the antibody or portion thereof of any one of claims 1 to 12 to said subject. 33. The method of claim 32, wherein said heparanase-related disorder or condition is selected from the group consisting of an inflammatory disorder, a wound, a scar, a vasculopathy and an autoimmune condition. 34. The method of claim 33, wherein said vasculopathy is selected from the group consisting of atherosclerosis, restenosis and aneurysm. ZU6 C 35. The method of claim 32, wherein said heparanase-related disorder or 0 condition is selected from the group consisting of angiogenesis, cell proliferation, oO a cancerous condition, tumor cell proliferation, invasion of circulating tumor cells and a metastatic disease. (N IND 36. The method of claim 35, wherein said cancerous condition is selected from the group consisting of a blood, breast, bladder, rectum, stomach, cervix, ovarian, O 1 colon, renal and prostate cancer. 1 37. A method of detecting the presence of a heparanase polypeptide in a sample, the method comprising incubating said sample with a heparanase-specific antibody according to any one of claims 1 to 12 in a manner suitable for formation of a heparanase polypeptide-antibody immune complex; wherein said heparanase- specific antibody is characterized by specifically binding to heparanase, and detecting the presence of said heparanase polypeptide-antibody immune complex to determine whether a heparanase polypeptide is present in the sample. 38. The method of claim 37, wherein said anti-heparanase antibody is labeled with a labeling agent that provides a detectable signal. 39. The method of claim 38, wherein said labeling agent is selected from the group consisting of an enzyme, a fluorophore, a chromophore, a protein, a chemiluminescent substance and a radioisotope. A method for detecting a heparanase-related disease or condition in a subject, the method comprising: obtaining a biological sample from the subject; contacting said biological sample with an anti-heparanase antibody according to any one of claims 1 to 12 in a manner suitable for formation of a heparanase polypeptide-antibody immune complex; and 1 209 detecting the presence of said heparanase polypeptide-antibody immune complex to determine whether a heparanase polypeptide is present in the 00 sample, wherein the presence or absence of said heparanase polypeptide-antibody immune complex indicates a heparanase-related disease or condition; (N I thereby detecting a heparanase-related disease or condition in a subject. 41. The method of claim 40, wherein said subject is a vertebrate. 42. The method of claim 41, wherein said subject is a mammal. 42. The method of claim 41, wherein said mammasubject is a hummal. (N 43. The method of claim 42, wherein said mammal is a human. 44. The method of claim 40, wherein said heparanase-related disorder or condition is selected from the group consisting of an inflammatory disorder, a wound, a scar, a vasculopathy and an autoimmune condition. The method of claim 44, wherein said vasculopathy is selected from the group consisting of atherosclerosis, restenosis and aneurysm. 46. The method of claim 40, wherein said heparanase-related disorder or condition is selected from the group consisting of angiogenesis, cell proliferation, a cancerous condition, tumor cell proliferation, invasion of circulating tumor cells and a metastatic disease. 47. The method of claim 46, wherein said cancerous condition is selected from the group consisting of a blood, breast, bladder, rectum, stomach, cervix, ovarian, colon, renal and prostate cancer. 48. The method of claim 40, wherein said heparanase-related disorder or condition is a renal disease or disorder. 210 O 49. The method of claim 48, wherein said renal disease or disorder is selected Sfrom the group consisting of diabetic nephropathy, glomerulosclerosis, nephrotic 00 syndrome, minimal change nephrotic syndrome and renal cell carcinoma. The method of claim 40, wherein said biological sample is selected from (N D the group consisting of serum, plasma, urine, synovial fluid, spinal fluid, tissue sample, a tissue and/or a fluid. 51. The method of claim 40, wherein said contacting said sample is performed ¢Ci in situ. 52. The method of claim 40, wherein said contacting said sample is performed in vitro. 53. A method for monitoring the state of a heparanase-related disorder or condition in a subject, the method comprising: obtaining a biological sample from the subject; contacting said biological sample with an anti-heparanase antibody according to any one of claims 1 to 12 in a manner suitable for formation of a heparanase polypeptide-antibody complex; detecting a presence, absence or level of said heparanase polypeptide-antibody complex to determine a presence, absence or level of a heparanase polypeptide in said biological sample; repeating steps through at predetermined time intervals; and determining a degree of change of said presence, absence or level of said heparanase polypeptide at said predetermined time intervals, said change indicating a state of the heparanase-related disorder or condition in said subject; S211 thereby monitoring the state of the heparanase-related disorder or condition 0 in said subject. 00 54. The method of claim 53, wherein said subject is a vertebrate. N 55. The method of claim 54, wherein said subject is a mammal. 56. The method of claim 55, wherein said mammal is a human. 057. The method of claim 53, wherein said heparanase-related disorder or CN condition is selected from the group consisting of an inflammatory disorder, a wound, a scar, a vasculopathy and an autoimmune condition. 58. The method of claim 57, wherein said vasculopathy is selected from the group consisting of atherosclerosis, restenosis and aneurysm. 59. The method of claim 53, wherein said heparanase-related disorder or condition is selected from the group consisting of angiogenesis, cell proliferation, a cancerous condition, tumor cell proliferation, invasion of circulating tumor cells and a metastatic disease. The method of claim 59, wherein said cancerous condition is selected from the group consisting of a blood, breast, bladder, rectum, stomach, cervix, ovarian, colon, and prostate cancer. 61. The method of claim 53, wherein said heparanase-related disorder or condition is a renal disease or disorder. 62. The method of claim 61, wherein said renal disease or disorder is selected from the group consisting of diabetic nephropathy, glomerulosclerosis, nephrotic syndrome, minimal change nephrotic syndrome and renal cell carcinoma. 212 63. The method of claim 53, wherein said biological sample is selected from Sthe group consisting of serum, plasma, urine, synovial fluid, spinal fluid, tissue 00oO sample, a tissue and/or a fluid. 64. The method of claim 53, wherein said contacting said sample is performed (-i situ. The method of claim 53, wherein said contacting said sample is performed in vitro. 66. A pharmaceutical composition comprising the isolated anti-heparanase antibody or portion thereof of any one of claims 1 to 28 and a pharmaceutically acceptable carrier. 67. The pharmaceutical composition of claim 66, wherein said anti-heparanase antibody is a monoclonal antibody. 68. The pharmaceutical composition of claim 66, wherein said anti-heparanase antibody is a humanized antibody. 69. An affinity medium for binding human heparanase polypeptides, the medium comprising an anti-heparanase antibody according to any one of claims 1 to 12 immobilized to a chemically inert, insoluble carrier. The affinity medium of claim 69, wherein said chemically inert, insoluble carrier is selected from the group consisting of acrylic and styrene based polymers, gel polymers, glass beads, silica, filters and membranes. Date: 17 September 2007