WO2009010957A2

WO2009010957A2 - Heparanase c-terminal domain, sequences derived therefrom, substances directed against said domain and uses thereof as modulators of heparanase biological activity

Info

Publication number: WO2009010957A2
Application number: PCT/IL2008/000952
Authority: WO
Inventors: Israel Vlodavsky; Neta Ilan; Liat Fux; Svetlana Gingis-Velitski; Itay Shafat
Original assignee: Hadasit Medical Research Services and Development Co
Current assignee: Hadasit Medical Research Services and Development Co
Priority date: 2007-07-17
Filing date: 2008-07-10
Publication date: 2009-01-22
Anticipated expiration: 2010-01-17
Also published as: WO2009010957A3; IL184671A0

Abstract

The present invention relates to an isolated and purified amino acid sequence derived from the C-terminal domain of heparanase that modulates heparanase biological activity. More specifically, the sequence of the invention comprises amino acid residues 413 to 543 of human heparanase or any fragment, peptide, mutant, derivative and variant thereof. The invention further provides modulators of heparanase biological activity that may be amino acid sequences derived from heparanase C-terminal domain or substances which bind to this sequence. The sequences of the invention as well as heparanase modulators are specifically applicable in compositions and methods for the treatment of subjects suffering of heparanase associated disorders.

Description

HEPARANASE C-TERMINAL DOMAIN, SEQUENCES DERIVED THEREFROM, SUBSTANCES DIRECTED AGAINST SAID DOMAIN AND USES THEREOF AS MODULATORS OF HEPARANASE BIOLOGICAL ACTP7ITY

Field of the invention

The invention relates to modulation of heparanase biological activity. More particularly, the invention relates to an amino acid sequence derived form the C-terminal domain of heparanase and uses thereof as heparanase modulator and as a target sequence for modulating heparanase biological activity. The invention further provides compositions, methods and uses of said sequence and substance recognizing said sequence or peptides derived therefrom for modulating heparanase activity in a subject in need thereof.

Background of the invention

All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.

Heparanase is a mammalian endo-/3-D-glucuronidase capable of cleaving HS side chains at a limited number of sites, yielding HS fragments of still appreciable size (~5-7 kDa) and biological activity [Pikas, D. et al. J. Biol. Chem. 273: 18770-18777 (1998); Vlodavsky, I. and Friedmann, Y. J Clin Invest 108:341-347 (2001)]. Heparanase activity has long been detected in a number of cell types and tissues [Vlodavsky (2001) ibid.]. Importantly, heparanase activity correlates with the metastatic potential of tumor-derived cells, attributed to enhanced cell dissemination as a consequence of HS cleavage and remodeling of the extracellular matrix (ECM) barrier [Ilan, N. et al. Int. J. Biochem Cell Biol. (2006)]. Similarly, heparanase activity is implicated in neovascularization, inflammation and autoimmunity, facilitating migration and invasion of vascular endothelial cells and activated cells of the immune system [Ilan (2006) ibid.]. A proof of concept for this notion has been provided by applying siRNA and ribozyme technologies, demonstrating a casual involvement of heparanase in tumor metastasis, angiogenesis [Edovitsky, E. et al. J. Natl. Cancer Inst. 96:1219-1230 (2004)] and inflammation [Edovitsky, E. et al. Blood (2005)]. Clinically, up regulation of heparanase mRNA and protein expression have been documented in a variety of primary human tumors, correlating with reduced postoperative survival and increased lymph node and distant metastasis, thus providing a strong clinical support for the pro- metastatic feature of the enzyme [Ilan, N. et al. Int. J. Biochem Cell Biol. 38: 2018-2039 (2006); McKenzie, E. A. Br. J. Pharmacol (2007)]. In addition, heparanase induction was noted in pathological disorders other than human neoplasm [Edovitsky (2005) ibid.; Levidiotis, V. et al. Kidney Int. 60:1287-1296 (2001); Waterman, M. et al. Mod. Pathol. (2006)], making the enzyme an attractive target for the development of anti-cancer and anti- inflammatory drugs. Attempts to inhibit heparanase enzymatic activity were initiated already at the early days of heparanase research, in parallel with the emerging clinical relevance of this activity [Bar-Ner, M. et al. Blood 70:551- 557 (1987); Nakajima, M. et al. J. Biol. Chem. 259:2283-2290 (1984)].

Involvement of heparanase was also demonstrated in different signal transduction pathways, particularly in the PI3'K/Akt pathway. More specifically, the inventors have recently demonstrated that exogenous addition of the latent 65 kDa heparanase stimulates Akt-dependent endothelial cell invasion and migration independent of heparanase enzymatic activity. Non enzymatic activities of heparanase also include enhanced adhesion of tumor- derived cells and primary T-cells, and were correlated with Akt, Pyk2, and ERK activation. Moreover, it was previously shown that heparanase over expression or exogenous addition induced the expression of VEGF in a Src- dependent manner, thus stimulating tumor angiogenesis. Protein domains responsible for signaling by heparanase have not been so far identified. More recently, with the availability of recombinant heparanase and the establishment of high-throughput screening methods, a variety of inhibitory molecules have been developed, including peptides, small molecules, modified non-anticoagulant species of heparin, as well as several other polyanionic molecules, such as laminaran sulfate, suramin and PI-88 [Ferro, V. et al. Mini Rev. Med. Chem. 4: 693-702 (2004)]. Similarly, anti-heparanase polyclonal antibodies were developed and demonstrated to neutralize heparanase enzymatic activity and to inhibit cell invasion [He, X. et al. Cancer Res. 64:3928-3933 (2004)], proteinuria [Levidiotis, V. et al. Nephrology (Carlton) 10: 167-173 (2005)] and neointima formation [Myler, H. et al. J. Biochem. (Tokyo) 139:339-345 (2006)]. Neutralizing anti-heparanase monoclonal antibodies, however, have not been so far reported.

In search for modulators of different heparanase biological activities, the inventors have utilized a structure/function approach in order to identify domains of heparanase that mediate its enzymatic activity-dependent as well as enzymatic activity-independent functions. As indicated above, domains of heparanase mediating its non-enzymatic activities have not been defined yet. Identification of such domains may provide a powerful tool for screening of heparanase modulating substances. The present invention demonstrates the role of heparanase C-domain in mediating different heparanase enzymatic and non-enzymatic activities. The invention demonstrates for the first time, the role of heparanase C-domain in secretion of the protein, in signaling pathways (PI3'K/Akt) leading to tumor progression, as well as cell survival and in stabilizing the conformation of the molecule in a manner facilitating also its enzymatic activities. Thus, specific substances directed against heparanase C- domain, are expected to act as modulators (e.g. inhibitors or enhancers) of different heparanase biological functions. More particularly, inhibitors of heparanase directed to its C-domain may be implicated in tumor progression, cell survival, cell migration, angiogenesis and metastasis. Whereas enhancers of heparanase activities may be implicated in wound healing, as well as in the treatment of cardiovascular disorders.

It is therefore one object of the invention to provide an amino acid sequence, particularly a sequence derived from heparanase C-terminal domain, as a modulator of heparanase biological activities and as a target molecule in screening of potential modulators of heparanase. Such modulating molecules specifically recognize and bind to heparanase C-terminal domain, and thereby modulate its biological activities.

Another object of the invention is to provide amino acid sequences, fragments and peptides derived from heparanase C-terminal domain, as well as compositions and uses thereof in modulating different biological activities of heparanase.

In yet another object of the invention is to provide modulators which directly and specifically recognize and bind heparanase C- terminal domain, and thereby modulate different biological activities of this protein.

The invention further provides screening methods for substances which modulate heparanase activities, utilizing its C- terminal domain and fragments thereof.

In yet another object, the invention provides compositions and methods comprising modulators of heparanase activities for the treatment of a subject in need thereof, particularly a subject suffering from heparanase associated disorders. Summary of the Invention

According to a first aspect, the invention relates to an isolated and purified amino acid sequence derived from the C-terminal domain of heparanase or any fragment, peptide, mutant, derivative and variant thereof. This domain is required for different biological activities of heparanase and therefore modulates heparanase biological activity.

According to a further aspect, the invention relates to a composition comprising as active agent an amino acid sequence derived from heparanase C-terminal domain as defined by the invention, any nucleic acid construct encoding such sequence, cells transfected or transformed by said construct, or of any combinations thereof. It should be noted that the composition of the invention optionally further comprises a pharmaceutically acceptable carrier, diluent, excipient and/or additive. According to a specific embodiment, the composition of the invention is applicable in modulation of heparanase biological activities.

According to another aspect, the invention relates to a modulator of heparanase biological activity. Such modulator according to the invention, may be an amino acid sequence derived from the C-terminal domain of heparanase, or any fragment, peptide or mutant thereof. Alternatively, the modulator of the invention may be a substance which specifically binds to an amino acid sequence within the C-terminal domain of heparanase.

The invention thus further provides a composition for the modulation of heparanase biological activity, comprising as active ingredient a modulator as described by the invention, said composition optionally further comprising a pharmaceutically acceptable carrier, diluent, excipient and/or additive. Another aspect of the invention relates to a pharmaceutical composition for the treatment of a process or a pathologic disorder associated with heparanase biological activity. The composition of the invention comprises as an active ingredient heparanase modulator molecule as described by the invention. The composition of the invention optionally further comprises a pharmaceutically acceptable carrier, diluent, excipient and/or additive.

In yet another aspect of the invention relates to a method for the modulation of heparanase biological activity. The method of the invention comprises the step of in vivo, ex vivo or in vitro contacting heparanase under suitable conditions, with a modulatory effective amount of heparanase modulator or with a composition comprising the same. The modulator according to the invention may be any one of: (i) an isolated and purified amino acid sequence derived from the C-terminal domain of heparanase; or (ii) a substance which specifically binds to an amino acid sequence derived from the C-terminal domain of heparanase and is capable of modulating heparanase biological activity.

In another aspect, the invention relates to a method of screening for heparanase modulator which specifically binds to an amino acid sequence derived from the C-terminal domain of heparanase and is therefore capable of modulating heparanase biologic activity. The screening method comprises the steps of:

(a) obtaining a candidate substances;

(b) selecting from the substances obtained in step (a) a substance which bind to the C-terminal domain of heparanase, or to any fragment thereof; and

(c) evaluating the candidate substance selected in step (b) by determining the modulatory effect of said substance on the biological activity of heparanase. These and other aspects of the invention will become apparent by the hand of the following figures and examples.

Brief Description of the Figures

The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

Figure 1A-1D

Heparanase three dimensional model and different constructs Fig. IA. Three dimensional model of heparanase. The model, including the 8 (yellow) and 50 kDa (gray) protein subunits, amino acids critical for heparanase catalysis (GIu²²⁵ and GIu³⁴³, red), and heparin binding regions (Lys¹⁵⁸-Asp^m, also denoted by SEQ ID NO. 11, and Gln²™-Lys²80; also denoted by SEQ ID NO. 12, cyan and green, respectively) is shown in the left panel. A more detailed structure of the C-domain is shown in the right panel. The model illustrates eight _/3-strands, one of which is contributed by the 8 kDa subunit (yellow), arranged in two sheets (blue and orange) which are connected by an unstructured, flexible loop (arrow). The model was constructed by a protein structure prediction server (http ://www .robetta. or g) . based on sequence and structure homology of constitutively- active (GS3) single chain heparanase to the crystal structure of α-L-arabinofuranosidase isolated from Geobacillus stearothermophilus T-6, as described in "Experimental procedures".

Fig. IB. shows schematic diagram of gene constructs utilized in this study, generated based on wild-type heparanase.

Fig. 1C. shows schematic diagram of gene constructs utilized in this study, generated based on GS3 heparanase variants.

Fig. ID. Sequence alignment of human (Homo sapience C-domain, also denoted by SEQ ID NO. 3), mouse (Mus musculus C-domain, also denoted by

SEQ ID NO. 14), and chicken (Gallus gallus C-domain, also denoted by SEQ

ID NO. 15) heparanase C-domain. β strands sequences are marked by blue and orange arrows; part of the unstructured loop and the C-terminal 17 amino acids, used for creating the deletion mutants of the invention, are marked by red boxes; point mutations with no noticeable effect are marked green; point mutations found to inhibit heparanase secretion are marked red.

Abbreviations: Tim-Bar. (TIM-barrel), C-dom. (C-domain), W.T. (Wild type), term, (terminal), muts. (mutants), po. Mut. (point mutation), Ho. Sap. (homo sapiens), Mus. Mus. (Mus musculus), Gal. gal. (Gallus gallus).

Figure 2A-2C

A critical role of the C-domain in heparanase enzymatic activity

Fig.2A. Catalytic activity of different heparanase constructs. Heparanase

(Hepa)-, TIM-barrel (TIMB)-, and heparanase deleted for the C-terminal 17 amino acids (Δ17)-, all in the backbone of GS3 gene constructs, as well as C- domain- and control (Mock)- transfected JAR cells (2xlO⁶) were subjected to three freeze/thaw cycles and cell lysates were applied onto 35 mm dishes coated with ³⁵S-labeled ECM. Release of sulfate-labeled material was evaluated by gel filtration, as described in "Experimental procedures".

Fig. 2B. Co-transfection. JAR cells were transfected with control empty vector

(Mock), heparanase (Hepa), or co-transfected with TIM-barrel (GS3) and C- domain gene constructs and heparanase activity was determined as described above.

Fig. 2C. Point mutations. JAR cells were transfected with control empty vector (Mock), heparanase, or heparanase mutated at Phe⁵³¹, VaI⁵³³, He⁵³⁴, Ala⁵³⁷, or Cys⁵⁴², all as GS3 gene constructs, and heparanase activity was determined as above. Representative activity assays are shown for the I534R and A537K mutations. Abbreviations: ReI. (released), SuI. (sulfate), lab. (labeled), mat. (material), cpm. (counts per minute), C-dom. (C-domain), Mo. (Mock), Frac. (fractions).

Figure 3A-3D

Intact C-domain is required for heparanase secretion

Fig. 3A. Deletion of the C-domain or its C-terminus abrogates heparanase secretion. HEK 293 cells were stably transfected with control, empty vector (mock), or Myc-tagged wild type (Hepa), TIM-barrel (TIMB), and C-domain heparanase gene constructs, or heparanase deleted for its 17 C-terminal amino acids (Δ17). Cells were incubated (20 h, 37⁰C) without (-) or with heparin (+, 50 μg/ml); Total cell lysates were prepared and subjected to immunoblotting applying anti-Myc antibody (left panel). Conditioned medium was collected from corresponding cultures and medium samples were similarly blotted with anti-Myc antibody (right panel).

Fig. 3B. Point mutations. HEK 293 cells were stably transfected with control (mock), heparanase (Hepa), or heparanase gene constructs mutated at lsoleucine⁵³⁴ (I534R) or alanine⁵³⁷ (A537K) and lysate (left panel) and medium (right panel) samples were similarly blotted with anti-Myc antibody. Fig. 3C. Hsp90 inhibitor attenuates heparanase and C-domain secretion. Heparanase (Hepa) and C-domain transfected 293 cells were incubated with vehicle control (DMSO; -) or Hsp90 inhibitor 17-AAG (500μM; +) for three days. Lysate and medium samples were then prepared and subjected to immuno-blotting applying anti-Myc antibody. Note, decreased heparanase secretion following Hsp90 inhibition. Cell lysates were similarly blotted with anti-phospho Akt (p-Akt, left, second panel) and anti Akt (Akt, lower panel) antibodies.

Fig. 3D. Cellular localization. Control (Mock, upper panels), heparanase (Hepa, second panels), TIM-barrel (TIMB, third panels), and C-domain (fourth panels) transfected HEK 293 cells, as well as cells transfected with heparanase deleted for its C-terminal 17 amino acids (Δ17, fifth panels) or mutated at alanine 537 (A537K, lower panels) were stained with anti- heparanase monoclonal antibody (left most panels, red) or were triple stained for Myc-tag (second left, red), the ER marker calnexin (third left, green), and merged with cell nuclei labeled with TO-PRO (fourth left, blue). Cells were similarly stained with anti-Myc-tag (third right, red), the Golgi marker wheat germ agglutinin-FITC (second right, green) and merged with cell nuclei labeled with TO-PRO (right most, blue). Note that all heparanase variants are found co-localized with the ER marker (fourth left, yellow), while only heparanase and the C-domain are co-localized with the Golgi marker (right most, yellow). Abbreviations: C-dom. (C-domain), Mo. (Mock), TIMB (TIM- barrel), Lys. (lysates), Med. (medium), Hepa (heparanase), P-Akt (phosphorylated Akt), Mer. (Merge).

Figure 4A-4D

Induction of Akt phosphorylation by the C-domain

Fig. 4A. Protein expression and secretion. HEK 293 cells were stably transfected with control (mock), heparanase (Hepa), C-domain, and TIM- barrel (TIMB) gene constructs and lysate (left) and medium (right) samples were subjected to immunoblotting applying anti-Myc antibody. Fig. 4B-4D. Akt induction. Lysate samples were similarly blotted with anti- phospho-Akt (p-Akt, upper panel) and anti-Akt (lower panel) antibodies (4B). Conditioned medium was collected from corresponding cultures and applied to parental HEK 293 cells for 30 min. Cell lysates were then prepared and subjected to immunoblotting applying anti-phospho-Akt (p-Akt, upper panel) and anti-Akt (lower panel) antibodies (4C).

Fig. 4D. Purified heparanase (Hepa) and C-domain proteins were applied to HS-deficient CHO-745 cells for 30 min and Akt phosphorylation levels were analyzed as above. Akt phosphorylation index was calculated by densitometry analysis of phosphorylated Akt levels divided by the total Akt values. Data is presented as fold increase of Akt phosphorylation compared with control, mock transfected cells, set arbitrary to a value of 1 (bottom panels). Abbreviations: C-dom. (C-domain), Mo. (Mock), TIMB (TIM- barrel), Lys. (lysates), Med. (medium), Hepa (heparanase), P-Akt (phosphorylated Akt), Tot. (total), fo. (fold), inc. (increase), indue, (induction).

Figure 5A-5E

Heparanase C-domain facilitates cell proliferation and tumor xenograft progression

Fig. 5A. Cell proliferation. Control (mock), heparanase, and C-domain transfected HEK 293 cells were incubated with BrdU for 2 h and BrdU incorporation was evaluated by immunostaining as described in "Experimental procedures". Shown are representative photomicrographs of BrdU incorporation in control (Mock, left panel), heparanase (Hepa, middle panel), and C-domain (second right) transfected cells. BrdU incorporation was quantified by counting BrdU-positive cells in high-power fields as described in "Experimental procedures" (histogram in the right panel). Fig. 5B-5C. Tumor xenograft progression. Control (Mock), heparanase (Hepa), C-domain, and TIM-barrel (TIMB) transfected U87 cells were inoculated (5xlO⁶) subcutaneously at the flank of Balb/C nude mice (n=7). Xenograft development was measured with a caliper and tumor volume was calculated as described under "Experimental procedures" (5B). At the end of the experiment on day 51 animals were sacrificed, xenografts were harvested and weighted (5C). Fig. 5D. Immunohistochemistry. Five micron sections of tumor xenograft produced by control (Mock, upper panels), heparanase (Hepa, second panels), C-domain (third panels), and TIM-barrel (TIMB, fourth panels) transfected U87 glioma cells were subjected to immunostaining applying anti-phospho-Akt (p-Akt, left) or anti-PECAM-1 (right) antibodies.

Fig. 5D. The total number of blood vessels and blood vessels with lumen diameter above 40 microns was quantified by counting PECAM-1-positive vessels in at least eight different high power fields in each tumor xenograft. Abbreviations: C-dom. (C-domain), Mo. (Mock), TIMB (TIM- barrel), Hepa (heparanase), Po (positive), Turn, (tumor), Wei. (weight), gr. (gram), Vol. (volium), Bl. (blood), ves. (vessels), fie. (field), Lar. (large).

Figure 6A-6C

The C-domain interacts with high affinity binding site(s) / 'receptor(s) Figs. 6A, 6B. C-domain binding. HeLa (A) and HS-deficient CHO-745 (B) cells were incubated (2 h, 4⁰C) with increasing concentrations of ¹²⁵I-labeled C- domain protein without or with 100-fold excess of unlabeled heparanase and binding parameters were obtained by the Prism 4 software. Fig. 6C. Cross-linking. ¹²⁵I-labeled C-domain protein was added to the indicated cell line in the absence or presence of heparin (10 μg/ml) and 400 nM unlabeled heparanase protein. The cross linker sulfo-EGS (Pierce; 0.2 mM) was then added for 10 min, cells were washed with PBS, lysed, and subjected to SDS-PAGE followed by autoradiography. Note, the formation of two major protein complexes exhibiting molecular weights of about 130 and 170 kDa (arrows). Abbreviations: Spe. (specific), bin. (binding), Cone, (concentration), cpm. (counts per minute), C-dom. (C-domain), Mo. (Mock). Figure 7A-7C

Antibody 6F8 enhances heparanase enzymatic activity

Purified recombinant active heparanase (40 ng) was pre incubated with IEl (Fig. 7A), 44C4 (Fig. 7B) or 6F8 (Fig. 7C) anti-heparanase monoclonal antibodies, or control mouse IgG (1 μg) for 2 h in serum-free RPMI medium on ice. The mixture was then applied onto ³⁵S-labeled ECM-coated dishes and heparanase activity was determined as described under "Experimental procedures". Abbreviations: SuI. (sulfate), lab. (labeled), mat. (material), cpm. (counts per minute), Frac. (fractions).

Figure 8A-8D

Antibody 6F8 enhances the activity of cellular heparanase

Heparanase transfected HEK-293 (Fig. 8B) and MDA-231 (Fig. 7D) cells were suspended (2xlO⁶/ml) in phosphate/citrate buffer (pH 6.8) and subjected to three freeze/thaw cycles. The resultant lysates were incubated with antibody 6F8 (lμg) or control mouse IgG for two hours on ice, and were then applied onto ³⁵S-labeled ECM-coated dishes and heparanase activity was determined as above. Conditioned medium was collected from heparanase-transfected HEK-293 (Fig. 7A) and MDA-231 (Fig. 7B) cells and 1 ml was incubated with control mouse IgG or antibody 6F8 followed by determination of heparanase activity described above. Abbreviations: SuI. (sulfate), lab. (labeled), mat. (material), cpm. (counts per minute), Frac. (fractions), Med. (medium), Lys. (lysates).

Figure 9A-9D.

Antibody 6F8 facilitates cellular invasion

Heparanase transfected MDA-231 (Figs. 9A, 9B) and U87 (Figs. 9C, 9D) cells (2xlO⁵) were plated onto Matrigel-coated 8 micron Transwell filters in the presence of mouse IgG or antibody 6F8 (1 μg). Invading cells adhering to the lower side of the membrane were visualized (Figs. 9A, 9C) and counted (Figs. 9B, 9D) after 6 hours. Abbreviations: Ce. (cell), Inv. (invasion), fie. (field).

Figure 10

Antibody 6F8 enhances keratinocyte migration

HACAT cells were allowed to grow in tissue culture plates until confluence followed by a scratch made along the cell monolayer with the wide end of a 1 ml tip (time 0). Plates were washed twice with PBS to remove detached cells, incubated with complete growth medium and cell migration into the wounded area was examined for 4 days in the presence of control mouse IgG (left), anti- heparanase 6F8 (middle) or IEl (right) monoclonal antibodies (1 μg/ml).

Figure 11

Antibody 6F8 improves wound healing in a mouse punch model C57/B1 mice (n=5) were anesthetized and full-thickness 8 mm punch wounds were created on the mouse back (two wounds per mouse). Mice were injected i.p with control mouse IgG (Con) or antibody 6F8 (10 mg/kg) prior to, 3, and 5 days post wounding. The wound tissue was harvested seven days post wounding, fixed with paraformaldehyde, dehydrated, embedded in paraffin, and sectioned. 5 micron sections were rehydrated and stained with hematoxylin-eosin (Figs. 10A) or Masson/Trichome (Figs. 10C). Wound healing was calculated by measuring the distance between the epithelial edges (arrows) at the wound diameter (Figs. 10B). Abbreviations: Wou. (wound), diam. (diameter).

Figure 12

Human heparanase nucleic acid sequence and amino acid sequence as denoted by GenBank Accession No. BC051321, also denoted by SEQ ID NOs. 16 and 17, respectivelly. Detailed Description of the Invention

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

Attempts to inhibit heparanase enzymatic activity were initiated short after its discovery, in parallel with the emerging clinical relevance of this activity, and heparanase inhibitors are currently under clinical trials [Lewis, K.D. et al. Invest. New Drugs 26:89-94 (2008)]. It seems, however, that better characterization of the heparanase molecule and its three dimensional (3D) structure is required for the development of more efficacious and highly specific inhibitors in a rational manner. The inventors have now obtained a 3D model that clearly delineates a TIM-barrel fold. As shown by Example 1, this model clearly provides, for the first time, a structural appraisal of the heterodimer nature of heparanase. Notably, the 8 kDa subunit appears to enfold the 50 kDa subunit (Figure IA, middle panel), contributing β/a/β unit to the TIM-barrel fold. In addition, the model also underlines the existence of a C-terminal domain (C-domain) that appears not to participate in the TIM- barrel fold. The seemingly distinct protein domains led the inventors to hypothesize that these molecular entities mediate enzymatic (TIM-barrel) and non-enzymatic (C-domain) functions of heparanase. In fact, as shown by the following Examples 2 to 4, the C-domain was found to be critically essential for heparanase secretion, enzymatic activity, and Akt activation. Clearly, deletion of the C-domain generates enzymatically-inactive heparanase (TIM-barrel), even when constructed as a GS3 protein variant (Figure 2). As shown by the following Examples, the C-domain appears to play an important structural role although not comprising an integral part of the TIM- barrel fold, possibly stabilizing the TIM-barrel conformation. In support of this notion is the lack of heparanase secretion following deletion of the entire C- domain or its C-terminus (Δ17, Fig. 3). Furthermore, point mutations of conserved amino acids at this region eradicated heparanase secretion (Figure 3 and Table 3), suggesting that relatively modest alterations in the structure of the C-domain significantly affect the integrity/conformation of the heparanase molecule. This may be envisioned as a regulatory mechanism that prevents the secretion of mutated or misfolded protein in a manner that likely involves molecular chaperons.

Notably, the C-domain not only critically affects heparanase secretion and enzymatic activity, but also appears to mediate its signaling function. Activation of Akt was noted following exogenous addition of heparanase, or its over-expression in stably transfected cells [Zetser, A et al. Cancer Res. 63:7733-41 (2003); Gingis-Velitski, S. et al. J. Biol. Chem. 279:23536-41 (2004)], while heparanase gene silencing was associated with decreased Akt phosphorylation levels [Ben-Zaken, O. et al. Biochem. Biophys. Res. Commun. 361:829-34 (2007)], suggesting that endogenous heparanase is intimately involved in Akt regulation, likely supporting endothelial and tumor cell survival [Cohen, I. et al. Int. J. Cancer 118:1609-17 (2006)]. Protein domains responsible for Akt activation have not been so far recognized. Comparable stimulation of Akt phosphorylation following the addition of purified heparanase and C-domain proteins, or their over-expression, clearly delineates the C-domain as the molecular entity that mediates Akt induction by heparanase (Figure 4). Furthermore, xenografts produce by C-domain expressing cells appeared markedly enlarged compared with xenografts produced by control (mock) or TIM-barrel transfected cells and were similar in size to tumor xenografts produced by heparanase transfected cells (Figure 5), likely due to enhanced cell proliferation and/or decreased apoptosis, possibly through enhanced angiogenesis. These findings show, for the first time, that in some tumor systems (i.e., glioma) heparanase facilitates primary tumor progression regardless of its enzymatic activity.

The molecular mechanism utilized by heparanase to elicit signal transduction has not been resolved yet, but is thought to involve heparanase binding protein(s)/receptor(s). Employing binding studies, the inventors have recently reported the existence of low-affinity, high abundant, as well as high-affinity, low abundant binding sites for heparanase [Ben-Zaken, O. et al. Int. J. Biochem. Cell. Biol. 40:530-42 (2008)]. In contrast, only high-affinity binding sites were identified for the C-domain, exhibiting affinity comparable in magnitude to the one calculated for heparanase (Figure 6). While the low- affinity binding sites of heparanase were identified as HSPGs, high-affinity binding sites were thought to be MPR or LRP, cell surface proteins implicated in heparanase uptake [Vreys, V. et al. J. Biol. Chem. 280:33141-8 (2005)]. Akt activation by heparanase was noted, however, in MPR-, and LRP-deficient cells [Ben-Zaken, O. et al. Biochem. Biophys. Res. Commun. 361:829-34 (2007)], suggesting the existence of additional cell surface receptors that mediate the signaling function of heparanase. Indeed, cross-linking experiments consistently revealed the existence of two protein complexes of •~130 and -170 kDa, as shown by the present invention (Figure 6), representing binding sites/receptors with molecular weights of -110 and -150 kDa, respectively, following subtraction of the labeled 20 kDa C-domain. Such molecular weights are not in accordance with proteins shown to bind heparanase (i.e., MPR, LRP, CD222) [Vreys, V. et al. J. Biol. Chem. 280:33141- 8 (2005); Ben-Zaken, O. et al. Int. J. Biochem. Cell. Biol. 40:530-42 (2008); Wood, R.J. and Hulett M.D. J. Biol. Chem. 283:4165-76 (2008)], likely representing novel high affinity heparanase receptors. Taken together, the present invention have identified a protein domain critical for heparanase enzymatic activity, secretion, and signaling. Pro-tumorigenic properties of the C-domain (as denoted by SEQ ID NO. 13) clearly reveal the biological significance of this domain, and support the notion that heparanase exerts enzymatic activity-independent effects. The C-domain, and particularly its unstructured flexible loop (as denoted by SEQ ID NO. 13), appears as an attractive target for the development of a new class of heparanase inhibitors. Inhibiting heparanase enzymatic and non-enzymatic functions, applying, for example, N-acetylated glycol-split heparin [Vlodavsky, I. et al. Curr. Pharm. Des.13:2057-73 (2007)] and C-domain neutralizing antibodies, respectively, is therefore expected to profoundly affect tumor progression and metastasis.

Thus, according to a first aspect, the invention relates to an isolated and purified amino acid sequence derived from the C-terminal domain of heparanase. The invention shows for the first time that this domain is required for different biological activities of heparanase, and therefore modulates heparanase biological activity.

It should be noted that heparanase biological activity or activities as used herein include enzymatic and non-enzymatic activities of heparanase. Non limiting examples of heparanase activities shown as mediated by the C- domain are disclosed by the following Examples. Such activities include secretion of the protein, signaling, particularly through the PI3'K/Akt pathway, modulation of cell survival, apoptosis, cell migration, tumor progression, as well as signaling leading to wound healing. These were demonstrated by the present invention, as not involving heparanase enzymatic activity. It should be further noted that the present invention demonstrate that heparanase binds to a membranal receptor through its C- domain. Therefore, the term heparanase biological activities as used in the present invention, encompasses the binding of heparanase to a molecular receptor, that may be located in lipid rafts, as well as to any signaling pathway involving such interaction. It should be further appreciated that the term "heparanase biological activities" further encompasses any biologic activity known as involving heparanase, for example, neovascularization, inflammation and autoimmunity, facilitating migration of vascular endothelial cells and activated cells of the immune system and angiogenesis.

The C-terminal domain of heparanase was also demonstrated by the invention as required for heparanase catalytic activity, possibly by stabilizing conformation and mediating secretion of the molecule. Therefore, "heparanase activity" as used herein also encompasses enzymatic activity.

As used herein in the specification and in the claims section below, the phrase "heparanase catalytic activity" or its equivalent "heparanase activity" refers 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 β-elimination. Heparanase activity which is modulated according to the present invention can be of either recombinant or natural heparanase. Such activity is disclosed, for example, in US 6,177,545 and US 6,190,875.

According to one specific and preferred embodiment, the C-domain of heparanase refers to human heparanase. Therefore, according to a preferred embodiment, the amino acid sequence of the invention may comprise amino acid residues 413 to 543 of human heparanase or any functionally equivalent fragment, peptide, mutant, derivative and variants thereof. It should be indicated that the amino acid residues location mentioned herein refers to the human heparanse amino acid sequence as denoted by GenBank accession number BC051321, which is also disclosed in Figure 12 and also disclosed by SEQ ID NO. 17, as encoded by the nucleic acid sequence, denoted by SEQ ID NO. 16.

As used herein in the specification and in the claims section below, the term "C-terminus", "C-terminal domain" or "C-domain" refer to a continuous sequences involving amino acids derived from any location or locations, either continuous or dispersed, along the C-terminal amino acids of residues 413-543 of human heparanase, as also denoted by SEQ ID NO. 3.

According to a particular and preferred embodiment, the amino acid sequence of the invention comprises the amino acid sequence as denoted by SEQ ID NO: 3, or any fragment, derivative, peptide and variant thereof.

It should be further noted that the invention also relates to amino acid sequences derived from C-terminal domain of the mouse heparanase (Mus musculus, as denoted by SEQ ID NO. 14, including residues 405-535) and the chicken heparanase (Gallus gallus, as denoted by SEQ ID NO. 15, including residues 393-523).

As shown by the following Examples, heparanase C-terminal domain (probably, the C-terminal 17 amino acid residues) mediates secretion of the enzyme and thereby is required for heparanase catalytic activity. This domain has been further shown by the present invention as mediating signal transduction pathway leading to activation of Akt (particularly the "unstructured loop" sequence comprising residues 483 to 509). Therefore, the invention provides the use of an amino acid sequence comprising said C- terminal domain or any fragment, peptide or mutant therefore, as a modulator of heparanase biological activity (either enhancement or inhibition). By "functional fragments" is meant "fragments", "variants", "analogs" or "derivatives" of the molecule. A "fragment" of a molecule, such as any of the amino acid sequence of the C-domain of heparanase used by the present invention is meant to refer to any amino acid subset of the molecule, preferably of residues 413 to 543. It should be noted that these amino acid sequences are located in the heparanase amino acid sequence as presented by GenBank accession number BC051321, as also shown by Figure 12, and also denoted by SEQ ID NO. 17.

Thus, according to one specific embodiment, a fragment of heparanase C- terminal domain may be a fragment comprising thirty-eight residues 481 to 519 (the unstructured "loop") of human heparanase, as denoted by SEQ ID NO. 13. It should be noted that the unstructured loop sequence has been shown by the present invention and particularly by Example 4, as possibly involved in activation of Akt. The invention therefore provides further fragments comprising this unstructured loop sequence and fragments thereof. One fragment comprises ninety seven amino acid residues from amino acid 446 to 543, of human heparanase, as also denoted by SEQ ID NO. 18. Another fragment comprises eighty five residues from 458 to 543 of human heparanase as denoted by SEQ ID NO. 19. According to further embodiment, the invention provides another fragment that comprises the unstructured loop sequence. This fragment consists of sixty seven residues from amino acid residue 476 to residue 543 of human heparanase, as denoted by SEQ ID NO. 20. The invention further provides a fragment of the unstructured loop that is about twenty seven amino acids long, from amino acid 483 to amino acid 509, as denoted by SEQ ID. NO. 21. It should be noted that the deletion of this fragment from the heparanase molecule resulted in heparanase molecule devoid of any activity, as presented by Table 3. According to a further embodiment, another fragment of the C-domain may comprise residues 527 to 543 of human heparanase, as denoted by SEQ ID NO. 10. This 17 amino acid sequence was shown by the invention as involved in secretion of the molecule and thereby is required for heparanase catalytic activity.

According to another embodiment, the amino acid sequence of the invention may be a peptide comprising any of the C-domain fragments indicated above or any derivative or variant thereof. According to one specific embodiment, the peptide provide by the invention may comprises amino acid sequence as denoted by SEQ ID NO. 13, or any fragments or derivatives thereof. Other peptides containing the loop sequence or any part thereof may be the peptides of any one of SEQ ID NO. 18, 19, 20 and 21. In yet another embodiment, a peptide according to the invention may comprise the C-domain terminal 17 amino acid residues, as denoted by SEQ ID NO. 10.

Still further, the invention provides heparanase molecule or a C-terminal domain of heparanase carrying at least one mutation in any of heparanase residues from 413 to 543, as denoted by SEQ ID NO. 3. Such mutation may be a point mutation, deletion, insertion, nonsense mutation, missense mutation, rearrangement or any combination thereof.

According to one embodiment, the C-terminal domain carries a point mutation. More specifically, a mutant of heparanase C-domain may comprise at least one point mutation in any one of residues Phe⁵³¹, VaI⁵³³, He⁵³⁴, Ala⁵³⁷, and Cys⁵⁴². Non-limiting examples for such mutations may be I534R, A537K,

F531R, V533R and C542A.

Further mutants of heparanase provided by the invention are listed in Table

3. According to another embodiment, the invention provides heparanase molecule carrying a deletion in the C-terminal domain. These deletion mutants may carry a deletion of any of the amino acid sequences as denoted by any one of SEQ ID NO. 3, 13, 18, 19, 20, 21 and 10. Examples for such deletion mutants are the TIMB (SEQ ID NO. 2), Δ17 (also denoted by SEQ ID NO. 9) and the Δloop (also denoted by SEQ ID NO. 22), as presented by Table 3.

A "variant" of the C-terminal domain is meant to refer to a naturally occurring molecule substantially similar to either the entire molecule or a fragment thereof. An "analog" of a molecule is a homologous molecule from the same species or from different species. By "functional" is meant having same biological function, for example, required for any of heparanase biologic activities, as discussed herein before.

The terms derivatives and functional derivatives as used herein mean any amino acid sequence, and preferably, peptides comprising the amino acid sequence of any one of any one of SEQ ID NO: 3, 10, 13, 18, 19, 20 and 21, with any insertions, deletions, substitutions and modifications to the amino acid sequence, preferably, peptide that do not interfere its ability to modulate heparanase biological activity (hereafter referred to as "derivative/s"). A derivative should maintain a minimal homology to said amino acid sequence, e.g. even less than 30%. Preferably, homology of the derivative to any of the sequences of the invention may range between 30% to 100%. More specifically, homology may be 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. It should be appreciated that the term "insertions" as used herein is meant any addition of amino acid residues to the peptides of the invention, between 1 to 50 amino acid residues, preferably, between 20 to 1 amino acid residues and most preferably, between 1 to 10 amino acid residues. More specifically, the invention encompasses insertion, addition or extension of any one of 1, 2, 3, 4, 5, 6, 7 8, 9, 10 or more, amino acid residues to the peptide or amino acid sequence of the invention. The term "deletion" as used herein is meant any reduction of amino acid residues from the peptides of the invention, between 1 to 50 amino acid residues, preferably, between 20 to 1 amino acid residues and most preferably, between 1 to 10 amino acid residues. More specifically, deletion or reduction of any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, amino acid residues from the peptide or amino acid sequences of the invention.

More particularly, wherein the amino acid sequence of the invention is a peptide, any derivative as described above, may be used.

More specifically, the term derivative further encompasses any addition, substitutions and modifications performed with the sequence of the invention.

It should be further appreciated that the peptides of the invention may be extended at the N-terminus and/or C-terminus thereof with various identical or different amino acid residues. As an example for such extension, the peptide may be extended at the N-terminus and/or C-terminus thereof with identical or different hydrophobic amino acid residue/s which may be naturally occurring or synthetic amino acid residue/s. One example for a synthetic amino acid residue is D-alanine.

An additional and preferred example for such an extension may be provided by peptides extended both at the N-terminus and/or C-terminus thereof with a cysteine residue. Naturally, such an extension may lead to a constrained conformation due to Cys-Cys cyclization resulting from the formation of a disulfide bond. Another example may be the incorporation of an N-terminal lysyl-palmitoyl tail, the lysine serving as linker and the palmitic acid as a hydrophobic anchor.

In addition, the peptides may be extended by aromatic amino acid residue/s, which may be naturally occurring or synthetic amino acid residue/s. A preferred aromatic amino acid residue may be tryptophan. Alternatively, the peptides can be extended at the N-terminus and/or C-terminus thereof with amino acids present in corresponding positions of the amino acid sequence of the naturally occurring C-terminal domain of heparanase, as denoted by SEQ ID NO. 3.

Nonetheless, according to the invention, the peptides of the invention may be extended at the N-terminus and/or C-terminus thereof with various identical or different organic moieties which are not naturally occurring or synthetic amino acids. As an example for such extension, the peptide may be extended at the N-terminus and/or C-terminus thereof with an N-acetyl group.

The lack of structure of linear peptides renders them vulnerable to proteases in human serum and acts to reduce their affinity for target sites, because only few of the possible conformations may be active. Therefore, it is desirable to optimize the peptide structure, for example by creating different derivatives of the various peptides of the invention.

In order to improve peptide structure, the amino acid sequence, particularly, peptides of the invention can be coupled through their N-terminus to a lauryl- cysteine (LC) residue and/or through their C-terminus to a cysteine (C) residue, or to other residue/s suitable for linking the peptide to adjuvant/s for immunization. The peptides of the invention, as well as derivatives thereof may all be positively charged, negatively charged or neutral and may be in the form of a dimer, a multimer or in a constrained conformation. A constrained conformation can be attained by internal bridges, short-range cyclizations, extension or other chemical modification.

For every single peptide sequence used by the invention and disclosed herein, this invention includes the corresponding retro-inverso sequence wherein the direction of the peptide chain has been inverted and wherein all the amino acids belong to the D-series.

According to another aspect, the invention further provides a nucleic acid construct comprising a nucleic acid sequence encoding the C-terminal domain of heparanase, which construct optionally further comprises operably linked regulatory elements. More specifically, the nucleic acid sequence of the invention encodes the C-terminal domain of heparanase which comprises amino acid sequence as defined by the invention.

In yet another specifically preferred embodiment, the nucleic acid construct of the invention comprises the C-terminal domain of heparanase which has the amino acid sequence as denoted by SEQ ID NO: 3. It should be noted that the C-domain is encoded by a nucleic acid sequence comprising nucleic acid residues 1376-1767 of the human heparanase encoding nucleic acid sequence as shown by Figure 12, and also by SEQ ID NO. 16.

The invention further provides nucleic acid construct comprising nucleic acid sequence encoding for any fragment of heparanase C-terminal domain. For example, nucleic acid sequence encoding any of the amino acid sequences as denoted by any one of SEQ ID NO. 9, 10, 13, 18, 19, 20 and 21. It should be appreciated that the invention further provides the expression vectors and nucleic acid constructs encoding any of the mutated heparanase derivatives of the invention and for any of the constructs of the invention, particularly any of the constructs listed in Table 2.

Still further, the invention provides a nucleic acid construct comprising a polynucleotide sequence encoding heparanase-derived polypeptide devoid of the C-domain. Specifically, such molecule is devoid of the C-domain (amino acid residues 413 to 534) of heparanase and is designated as TIM-barrel. It should be noted that this construct optionally further comprises operably linked regulatory elements. According to a particular embodiment, such C- terminal deleted polypeptide comprises the amino acid sequence (residues 36 to 417) as denoted by SEQ ID NO: 2 and is encoded by a nucleic acid sequence including nucleotides 243 to 1767, of the human heparanase sequence shown by Figure 12 and denoted by SEQ ID NO. 16. Another construct encoding heparanase deletion mutated molecule, is a construct encoding the mutant designated as Δ17. This mutated molecule is devoid of the last 17 amino acid residues of heparanase and comprises amino acid residues 36 to 526, as also denoted by SEQ ID NO. 9. In yet another embodiment, the invention provides a construct encoding heparanase molecule devoid of most of the loop sequence (the Δloop, also denoted by SQ ID NO. 22). As demonstrated by the following Examples, the heparanase-derived polypeptide of both deletion mutants is devoid of different heparanase biological activities, such as secretion, signaling, interactions and binding to membranal receptor molecule, catalytic activity, migration, cell survival and tumor progression.

As used herein, the term "nucleic acid" refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The terms should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single-stranded and double-stranded polynucleotides.

According to another embodiment, the invention provides an expression vector comprising any of the nucleic acid construct described herein.

"Construct", as used herein, encompasses vectors such as plasmids, viruses, bacteriophage, integratable DNA fragments, and other vehicles, which enable the integration of DNA fragments into the genome of the host.

Expression vectors are typically self-replicating DNA or RNA constructs containing the desired gene or its fragments, and operably linked genetic control elements that are recognized in a suitable host cell and effect expression of the desired genes. These control elements are capable of effecting expression within a suitable host. Generally, the genetic control elements can include a prokaryotic promoter system or a eukaryotic promoter expression control system. This typically includes a transcriptional promoter, an optional operator to control the onset of transcription, transcription enhancers to elevate the level of RNA expression, a sequence that encodes a suitable ribosome binding site, RNA splice junctions, sequences that terminate transcription and translation and so forth. Expression vectors usually contain an origin of replication that allows the vector to replicate independently of the host cell.

A vector may additionally include appropriate restriction sites, antibiotic resistance or other markers for selection of vector-containing cells. Plasmids are the most commonly used form of vector but other forms of vectors which serve an equivalent function and which are, or become, known in the art are suitable for use herein. See, e.g., Pouwels et al., Cloning Vectors: a Laboratory Manual (1985 and supplements), Elsevier, N.Y.; and Rodriquez, et al. (eds.) Vectors: a Survey of Molecular Cloning Vectors and their Uses, Buttersworth, Boston, Mass (1988), which are incorporated herein by reference.

Further according to the present invention there is provided a host cell comprising any of the nucleic acid constructs and expression vectors described herein (particularly, the constructs as listed in Table 2). The host cell can be of any type. It may be a prokaryotic cell, an eukaryotic cell, a cell line, or a cell as a portion of an organism. The polynucleotide encoding heparanase C-domain, polynucleotide encoding any fragment of the C-terminal domain, heparanase molecule devoid of the C-domain (the TIMB constructs), devoid of the last 17 amino acid residues (Δ17 constructs), or heparanase molecule devoid of residues 481-509 (Δloop constructs), can be permanently or transiently present in the cell. In other words, genetically modified cells obtained following stable or transient transfection, transformation or transduction, are all within the scope of the present invention. The polynucleotide can be present in the cell in low copy (say 1-5 copies) or high copy number (say, 5-50 copies or more). It may be integrated in one or more chromosomes at any location or be present as an extrachromosomal material. More particularly, a specific embodiment of the invention relates to a host cell transformed or transfected with a construct expressing heparanase C-domain, or mutants devoid of said domain (TIMB or Δ17 constructs). Suitable host cells include prokaryotes, lower eukaryotes, and higher eukaryotes. Prokaryotes include gram negative and gram positive organisms, e.g., E. coli and B. subtilis. Lower eukaryotes include yeast, S. cereυisiae and Pichia, and species of the genus Dictyostelium. Higher eukaryotes include established tissue culture cell lines from animal cells, both of non-mammalian origin, e.g., insect cells and birds, and of mammalian origin, e.g., human and other primate, and of rodent origin.

"Host cell" as used herein refers to cells which can be recombinantly transformed with vectors constructed using recombinant DNA techniques. A drug resistance or other selectable marker is intended in part to facilitate the selection of the transformants. Additionally, the presence of a selectable marker, such as drug resistance marker may be of use in keeping contaminating microorganisms from multiplying in the culture medium. Such a pure culture of the transformed host cell would be obtained by culturing the cells under conditions which require the induced phenotype for survival.

As used herein, the term "transfection" means the introduction of a nucleic acid, e.g., an expression vector, into a recipient cells by nucleic acid-mediated gene transfer. "Transformation", as used herein, refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA.

"Cells", "host cells" or "recombinant cells" are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cells but to the progeny or potential progeny of such a cell. Because certain modification may occur in succeeding generation due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. It should be noted that any host cell comprising any of the constructs or vectors described herein, is within the scope of the invention.

In a further aspect the invention provides a recombinant protein comprising an amino acid sequence derived from the C-terminal domain of heparanase, or any fragment, peptide or mutant thereof. Preferably, such recombinant protein may be encoded by any of the vectors described by the invention. The invention further provides therefore a recombinant protein comprising heparanase-derived polypeptide. Such protein is devoid of amino acid residues 413 to 543 of human heparanase, or fragments thereof, and is deficient in any of heparanase biologic activities. Still further, the invention provides a recombinant protein comprising heparanase C-domain (residues 413-543, as denoted by SEQ ID NO. 3). In yet another embodiment, the invention provides a recombinant protein comprising any of the C-domain fragments described by the invention. Particular examples are the fragments as denote by any one of SEQ ID NO. 10, 13, 18, 19, 20 and 21.

As indicated above, the invention provides isolated and purified amino acid sequences, peptides or antibodies. As used herein, the terms "isolated" and "purified" in the context of a proteinaceous agent (e.g., a peptide, polypeptide, protein or antibody) refer to a proteinaceous agent which is substantially free of cellular material and in some embodiments, substantially free of heterologous proteinaceous agents (i.e. contaminating proteins) from the cell or tissue source from which it is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of a proteinaceous agent in which the proteinaceous agent is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, a proteinaceous agent that is substantially free of cellular material includes preparations of a proteinaceous agent having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous proteinaceous agent (e.g. protein, polypeptide, peptide, or antibody; also referred to as a "contaminating protein"). When the proteinaceous agent is recombinantly produced, it is also preferably substantially free of culture medium, i.e. culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation. When the proteinaceous agent is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the proteinaceous agent. Accordingly, such preparations of a proteinaceous agent have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the proteinaceous agent of interest. Preferably, proteinaceous agents disclosed herein are isolated.

Thus, it should be further appreciated that any of the constructs, host cells and recombinant proteins of the invention may be used for compositions and methods for modulation of heparanase biologic activity and for treatment of heparanase associated disorders.

According to a further aspect, the invention relates to a composition comprising as active agent isolated and purified amino acid sequence derived from heparanase C-terminal domain as defined by the invention or any fragment, peptide, mutant, derivative and variant thereof, or any nucleic acid construct or expression vector encoding such sequence, or cells transfected or transformed by said construct, or of any combinations thereof. It should be noted that the composition of the invention optionally further comprises a pharmaceutically acceptable carrier, diluent, excipient and/or additive.

More specifically, the composition of the invention may comprises amino acid residues 413 to 543 of human heparanase or any functionally equivalent fragment, peptide, mutant, derivative and thereof. It should be indicated that the amino acid residues location mentioned herein refers to the human heparanase amino acid sequence as denoted by GenBank accession number BC051321, which is disclosed in Figure 12 and also by SEQ ID NO. 17, as encoded by the nucleic acid sequence denoted by SEQ ID NO. 16.

According to a particular and preferred embodiment, the amino acid sequence comprised within the construct of the invention may be any of the amino acid sequence as denoted by SEQ ID NO: 3, or any fragment, derivative, peptide and variant thereof. It should be further noted that the invention also relates to the use of amino acid sequences derived from C-terminal domain of the mouse heparanase (Mus musculus, as denoted by SEQ ID NO. 14) and the chicken heparanase (Gallus gallus, as denoted by SEQ ID NO. 15), for the composition of the invention.

Still further, according to one specific embodiment, the composition of the invention comprises as an active ingredient, a fragment of heparanase C- domain that may comprise residues 481 to 519 (the unstructured "loop") of human heparanase, as denoted by SEQ ID NO. 13 or any fragments of heparanase C-terminal domain comprising this "unstructured loop" sequence. For example, a fragment of ninety seven amino acid residues from amino acid 446 to 543, of human heparanase, as also denoted by SEQ ID NO. 18, a fragment of eighty five residues from 458 to 543 of human heparanase as denoted by SEQ ID NO. 19, a fragment of sixty seven residues from amino acid residue 476 to residue 543 of human heparanase, as denoted by SEQ ID NO. 20 or a fragment of twenty seven amino acids residues, from amino acid 483 to amino acid 509, as denoted by SEQ ID. NO. 21, comprising part of the loop sequence of SEQ ID NO. 13.

According to a further embodiment, the composition of the invention may comprise as an active ingredient, a fragment of the C-domain comprising residues 527 to 543 of human heparanase, as denoted by SEQ ID NO. 10.

According to another embodiment, the composition of the invention may comprise as an active ingredient, a peptide comprising any of the C-domain fragments indicated above or any derivative or variant thereof. According to one specific embodiment, the peptide may be any one of the peptides of SEQ ID NO. 3, 10, 13, 18, 19, 20 and 21. In yet another embodiment, the composition of the invention may comprises any of the mutated molecules of the invention, for example, heparanase molecule or a C-terminal domain of heparanase carrying a mutation in any of heparanase residues from 413 to 543, as denoted by SEQ ID NO. 3. More specifically, a mutant of heparanase C-domain may comprise at least one point mutation in any one of residues Phe⁵³¹, VaI⁵³³, He⁵³⁴, Ala⁵³⁷, and Cys⁵⁴². Non- limiting examples for such mutations may be I534R, A537K, I534R, A537K, F531R, V533R and C542A. Further mutants of heparanase provided by the invention are listed in Table 3.

According to one embodiment, the composition of the invention is intended for the modulation of heparanase biological activity.

In yet another embodiment, the composition of the invention is particularly applicable for modulating heparanase biological activity in a subject in need thereof.

The invention further provides the use of an isolated and purified amino acid sequence derived from the C-terminal domain of heparanase, for the modulation of heparanase biological activity, and for the preparation of compositions for modulating heparanase activity.

It should be noted that modulation may be either "enhancement" or inhibition" of different biological activities of heparanase.

The intimate involvement in angiogenesis and the ability of heparanase C- domain to induce blood vessels formation, shown here directly for the first time, may have important clinical implication. Tumor growth is angiogenic- dependent and therefore, inhibition of blood vessel formation by modulators specific for heparanase C-domain is sought as a cancer therapeutic. In the other hand, clinical situations critically suffer from severe tissue damage and induction of angiogenesis by modulating substances directed to heparanase C- domain and enhancing its biologic activities is believed to significantly improve tissue function. The most common and important example is ischemic heart damage, affecting millions of people every year.

As indicated above, modulation of heparanase activities by targeting its C- domain may be applicable for treating variety of disorders. For example, enhancing the recruitment of inflammatory cells to specific sites by the modulating composition of the invention may facilitate inflammatory response. On the other hand, inhibiting heparanase activities may be useful in preventing or reducing inflammation under several pathological conditions, including chronic and acute inflammation.

The invention therefore further provides the use of an isolated and purified amino acid sequence derived from the C-terminal domain of heparanase, as a modulator of heparanase biological activity. According to this embodiment, any of the fragments, mutants or peptides described by the invention may be used. For example, any peptide or amino acid sequence comprising the sequence of any one of SEQ ID NO. 3, 9, 10, 13, 18, 19, 20, 21and any mutant thereof.

Still further, the invention provides the use of an isolated and purified amino acid sequence derived from the C-terminal domain of heparanase or any fragment, peptide, mutant or derivative thereof, in the preparation of a composition for the modulation of heparanase biological activity.

It should be appreciated that the amino acid sequence used by the invention may be a polypeptide, and preferably, a peptide derived from human heparanase C-domain or of any fragments thereof. According to one specific embodiment, the peptide may be any one of the peptides of SEQ ID NO. 3, 9, 10, 13, 18, 19, 20 and 21.

Thus, according to one embodiment, the invention relates to the use of an amino acid sequence derived from heparanase C-domain or of any fragment, peptide, mutant or derivative thereof, for enhancing heparanase biological activities.

As used herein in the specification and in the claims section below, the term "enhance" and its derivatives refers to increase or expand free expression of activity. According to a preferred embodiment of the present invention, such enhancement may be of at least about 60-70%, preferably, at least about, 70- 80%, more preferably, at least about 80-90% and most preferably, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95% and 100%, 200%, 300%, 400%, 500% or more of the heparanase basal activity. Such enhancement is a result of the enhancing peptide or amino acid sequence of the invention.

Accordingly, the amino acid sequence of the invention, or any fragment, peptide, mutant, derivative and variant derived therefrom or any compositions comprising the same, exhibiting enhancing effect on heparanase activities, may be used for treating pathologic disorder associated with heparanase catalytic activity. More specifically, the C-domain of heparanase was shown by the invention as essential in mediating heparanase activation of Akt. Therefore, it should be appreciated that enhancing Akt activation directly or indirectly (for example by interaction with a membranal receptor, agonist or antagonist), by the amino acid sequence or peptide of the invention may facilitate and enhance cell survival. This may be applicable for example in the treatment of cardiovascular disorders. Myocardial Akt signaling enhances coronary angiogenesis through the induction of angiogenic growth factors, and impaired angiogenesis in the presence of growth promoting stimuli plays a causal role in contractile dysfunction. Thus, enhanced coronary angiogenesis induced by Akt signaling may contribute to Akt-mediated improvement of cardiac function. However, it is likely that Akt signaling exerts beneficial actions on the heart through additional mechanisms. Several lines of evidences support the notion that cardiomyocyte apoptosis plays a causal role in the development of heart failure, and that inhibition of cardiomyocyte apoptosis attenuates contractile dysfunction in heart failure. Transgenic mice over expressing IGF-I, an upstream effector of Akt signaling, displays less myocyte apoptosis following myocardial infarction. IGF-I administration also reduces myocardial apoptosis in response to ischemia/reperfusion injury in rats, and IGF-I functions as a survival factor for cultured cardiac myocytes exposed to the cardiotoxin doxorubicin. The cytoprotective effect of IGF-I on cultured cardiomyocytes can be abrogated by the PI3K inhibitor wortmannin or by the transduction of dominant-negative Aktl, whereas constitutively- active Aktl protects cardiomyocytes from apoptosis in the absence of IGF-I. Thus, Akt signaling is both essential and sufficient for IGF-I survival signals in cardiomyocytes in vitro. Furthermore, adenovirus-mediated Aktl gene transfer in the heart diminishes cardiomyocyte apoptosis and limits infarct size following ischemia/reperfusion injury, and ameliorate doxorubicin-induced contractile dysfunction. Thus, inhibition of cardiomyocyte apoptosis may be one of the mechanisms by which Akt signaling attenuates contractile dysfunction in the failing myocardium.

In the other hand, inhibition of heparanase biological activities using the amino acid sequence of the invention, or any fragment, peptide, mutant, derivative and variant derived therefrom, may be applicable in the treatment or the inhibition of a process or a pathologic disorder associated with heparanase biological activity. For example, processes requiring inhibition of cell survival, such as angiogenesis, tumor formation, tumor progression and tumor metastasis. As used herein in the specification and in the claims section below, the term "inhibit" and its derivatives refers to suppress or restrain from free expression of activity. According to a preferred embodiment of the present invention at least about 60-70%, preferably, at least about, 70-80%, more preferably, at least about 80-90% and most preferably, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95% and 100%, of the heparanase activity is abolished by the inhibitory peptide or amino acid sequence of the invention or the modulating molecule of the invention as well as the antibody of the invention described hereinafter.

Accordingly, the amino acid sequence of the invention, or any fragment, peptide, mutant, derivative and variant derived therefrom, exhibiting inhibitory effect on heparanase activities, may be used for treating pathologic disorder associated with heparanase catalytic activity, for example, a malignant proliferative disorder.

More specifically, the amino acid sequence of the invention or any fragment, peptide or mutant thereof, may be used for treating malignant proliferative disorders such as, solid and non-solid tumor that may be any one of carcinoma, sarcoma, melanoma, leukemia, lymphoma and glioma.

In yet another embodiment, the amino acid sequence of the invention or a peptide derived therefrom, which particularly possess inhibitory effect on some heparanase biological activities, may be used for treating pathologic disorders such as inflammatory disorder, kidney disorders and autoimmune disorder, which were shown as associated with heparanase catalytic and non-catalytic activities.

According to another aspect, the invention relates to a modulator of heparanase biological activity. The modulator or modulating molecule according to the invention may be an amino acid sequence derived from the C- terminal domain of heparanase or any fragment, peptide, mutant, derivative and variant thereof. Alternatively, such modulator may be a substance which specifically binds to an amino acid sequence within the C-terminal domain of heparanase or of any fragment, peptide, mutant, derivative and variant thereof. More specifically, the C-terminal domain of heparanase may be as defined by the invention, preferably, a sequence comprising all or part of the amino acid sequence as denoted by SEQ ID NO. 3. Fragments and peptides of such sequences are as described herein before. Of particular interest, is a fragment comprising the "unstructured loop" sequence which contains residues 481 to 519, as denoted by SEQ ID NO. 13, or any C-domain fragments comprising this "loop" sequence or fragments thereof, for example, any one the amino acid sequences of SEQ ID NO. 18, 19, 20 or 21.

According to one specific and preferred embodiment, the modulator of the invention possesses inhibitory action on heparanase biologic activity. More specifically, such modulator may serve as heparanase inhibitor.

It should be appreciated that inhibition of heparanase biologic activities for example, through Akt signaling, may be applicable in treating pathologic disorders particularly different malignancies. In malignant disorders, activation of cell survival by Akt/PKB may not be desired, and therefore, an inhibitory molecule directed to or derived from heparanase C-domain, may specifically inhibit signaling leading to Akt activation and thereby inhibition of cell survival and tumorogenicity.

In yet another alternative and specifically preferred embodiment, the modulator of the invention possesses enhancing effect on heparanase and therefore may be used for enhancing any of the heparanase biologic activities described herein above. Molecules enhancing heparanase biologic activities may be applicable in wound healing, and may also have beneficial application in cell adhesion. In addition, enhancement of Akt signaling, which was demonstrated by the invention as one of heparanase biological activities mediated by its C-domain, may be applicable in treating cardiovascular disorders.

According to a specific embodiment, a fragment of heparanase C-domain may be any of the fragments described by the invention.

According to a specifically preferred embodiment, the modulator may be an isolated peptide comprising an amino acid sequence derived from a C-terminal domain of heparanase.

According to another specifically preferred embodiment, such peptide is capable of competing with the corresponding sequence within the heparanase molecule and thereby inhibiting heparanase biological activity. Of particular interest, is a peptide comprising the amino acid sequence of residues 481-519 of heparanase, as denoted by SEQ ID NO. 13 (also indicated as the "unstructured loop" sequence). Sequences comprising such "loop" or parts thereof may be any on of SEQ ID NO. 18, 19, 20 or 21. It should be indicated that this "loop" sequence has been shown as involved in Akt signaling.

The invention therefore further provides a composition comprising as active agent a modulator that may be a peptide comprising an amino acid sequence derived from the C-terminal domain of heparanase, said composition optionally further comprising a pharmaceutically acceptable carrier, diluent, excipient and/or additive. According to a specifically preferred embodiment, such composition may be used for the modulation of heparanase biological activity.

As indicated above, compositions for modulating heparanase activity include inhibitory compositions which may be applicable in treating malignant disorders, and enhancing compositions that may be applicable in cardiovascular disorders, wound healing and also other cosmetic applications.

In yet another alternative embodiment of this aspect, the modulator of the invention, may be a substance which specifically binds to an amino acid sequence within the C-terminal domain of heparanase. More specifically, the amino acid sequence of heparanase C-domain, as defined by the invention.

According to one specific and preferred embodiment, such substance may be for example an antibody which specifically recognizes an amino acid sequence derived from the C-terminal domain of heparanase or any fragment, peptide, mutant, derivative and variant thereof.

According to another embodiment, such antibody may be a polyclonal or a monoclonal antibody.

According to another specific embodiment, the antibody used as a modulator by the invention specifically binds heparanase C-domain as defined by the invention. The invention thus provides monoclonal antibodies designated #1E1, 44C4 and 6F8, which were identified by the invention and were shown (Table 1), as specifically recognizing heparanase C-domain.

According to one specifically preferred embodiment, the invention provides as a modulatory molecule, an antibody, preferably, the monoclonal antibody designated #6F8, which specifically binds to heparanase C-domain and thereby enhances heparanase catalytic activity, as demonstrated by Examples 8 to 10.

In yet another aspect, the invention relates to an antibody which specifically recognizes an amino acid sequence derived from the heparanase C-domain. By the term "specifically recognizes" is meant that the amino acid sequence of the invention or any fragment or derivative thereof, serves as an epitope for such antibody.

The term "epitope" as used herein is meant to refer to that portion of any molecule capable of being bound by an antibody that can also be recognized by that antibody. Epitopes or "antigenic determinants" usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics.

According to a preferred embodiment, any of the antibodies of the invention specifically recognizes an amino acid sequence derived from heparanase C- domain or any fragment, peptide, mutant, derivative and variant thereof. It should be appreciated that the antibody of the invention may be a polyclonal or a monoclonal antibody.

The generation of polyclonal antibodies against proteins is described in Chapter 2 of Current Protocols in Immunology, Wiley and Sons Inc.

Monoclonal antibodies may be prepared from B cells taken from the spleen or lymph nodes of immunized animals, in particular rats or mice, by fusion with immortalized B cells under conditions which favor the growth of hybrid cells. The technique of generating monoclonal antibodies is described in many articles and textbooks, such as the above-noted Chapter 2 of Current Protocols in Immunology. Spleen or lymph node cells of these animals may be used in the same way as spleen or lymph node cells of protein-immunized animals, for the generation of monoclonal antibodies as described in Chapter 2 therein. The techniques used in generating monoclonal antibodies are further described in by Kohler and Milstein, Nature 256; 495-497, (1975), and in USP 4,376,110.

The term "antibody" is meant to include intact molecules as well as fragments thereof, such as, for example, Fab and F(ab')2, which are capable of binding antigen.

It will be appreciated that Fab and F(ab')2 and other fragments of the antibodies are within the scope of the present invention and may be used for the compositions and the methods disclosed herein for intact antibody molecules. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')₂ fragments).

For future clinical applications, where the anti-heparanase C-domain antibody is a monoclonal antibody, it may be improved, through a humanization process, to overcome the human antibody to mouse antibody response. Rapid new strategies have been developed recently for antibody humanization which may be applied for such antibody. These technologies maintain the affinity, and retain the antigen and epitope specificity of the original antibody [Rader, C, et ai, Proc. Natl. Acad. Sci. 95, 8910-8915 (1998); Mateo, C, et al, Immunothechnology 3, 71-81 (1997)]. 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. Unlike, for example, animal derived antibodies, "humanized" antibodies often do not undergo an undesirable reaction with the immune system of the subject.

Thus, as used herein, the term "humanized" and its derivatives 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 being 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 which are fully human.

According to another preferred embodiment, the invention provides a monoclonal antibody which specifically recognizes and binds to heparanase C- domain, as defined by the invention. Such antibody may be any of the antibodies prepared by the invention, for example, the antibodies designated #1E1, 44C4 and 6F8.

According to one embodiment, the antibody provided by the invention which binds heparanase C-domain, is capable of modulating heparanase biological activity. More specifically, an antibody possessing modulatory effect on heparanase activities was shown by the invention as a monoclonal antibody designated #6F8.

More particularly, as demonstrated by Examples 8 to 10, this antibody enhances heparanase catalytic activity, as well as other heparanase mediated biological activities (wound healing).

The invention thus further provides a composition for the modulation of heparanase biological activity, comprising as active ingredient a modulator of heparanase biological activity, wherein said modulator being any one of (i) an amino acid sequence derived from the C-terminal domain of heparanase, and (ii) a substance which specifically binds to an amino acid sequence within the C-terminal domain of heparanase. The composition of the invention may optionally further comprises a pharmaceutically acceptable carrier, diluent, excipient and/or additive. More specifically, the C-terminal domain of heparanase may be as defined by the invention.

According to a specifically preferred embodiment, the invention provides a pharmaceutical composition for the treatment of a process or a pathologic disorder associated with heparanase biological activity. The composition of the invention therefore comprises as an active ingredient, any of the heparanase modulator substances defined by the invention, in an amount sufficient for the modulation of heparanase biologic activity. It should be mentioned that the composition of the invention optionally further comprises a pharmaceutically acceptable carrier, diluent, excipient and/or additive.

It should be noted that wherein indicated a condition, process or pathology "associated with" heparanase catalytic or non-catalytic activity, this term may also include any condition that is "caused by", "related to", "linked to", "usually occurring together with", "believed to have an impact on" etc.

As indicated above, the terms "modulator", "modulation" and "modulating" as used herein encompass either inhibition or enhancement of heparanase activities. Therefore, compositions and methods inhibiting heparanase biologic activities, provided by the invention may be applicable in treating malignant disorders as well as immune-related disorders. Compositions and methods enhancing heparanase biological activities through its C-terminal domain, may be applicable in wound healing, as well as in the treatment of cardiovascular disorders, possible through activation of the Akt pathway. According to one preferred embodiment, the therapeutic composition of the invention is intended for treating a process associated with heparanase biologic activity, for example, angiogenesis, cell survival, cell signaling, tumor formation, tumor progression or tumor metastasis. More specifically, such composition may comprises a modulator that inhibits heparanase biological activities.

According to one embodiment, the composition of the invention may be specifically applicable for treating a malignant proliferative disorder.

According to one embodiment, a malignant proliferative disorder may be any one of solid and non-solid tumor such as, carcinoma, sarcoma, melanoma, leukemia and lymphoma.

According to another embodiment, the composition of the invention may be applicable for treating an inflammatory disorder, a kidney disorder or an autoimmune disorder.

According to one specific embodiment, heparanase inhibitor may be a peptide derived from heparanase C-terminal domain, as described by the invention. A particular example may- be a peptide comprising the "loop" sequence as denoted by SEQ ID NO 13 or any parts thereof, for example, the sequences as denoted by SEQ ID NO. 13, 18, 19, 20 and 21. It should be noted that the invention encompasses any other heparanase inhibitors, that may be small molecules or any other substance directed to heparanase C-terminal domain. Optionally such molecule may be identified by the screening method of the invention described herein after. According to an alternative and preferred embodiment, the invention further provides a pharmaceutical composition for the treatment of a process or a pathologic disorder associated with heparanase biological activity. Such composition comprising as active ingredient heparanase enhancing modulator directed to or derived from heparanase C-terminal domain, in an amount sufficient for the enhancement of heparanase biologic activity. The composition of the invention may optionally further comprises a pharmaceutically acceptable carrier, diluent, excipient and/or additive.

According to one preferred embodiment, the composition of the invention may be applicable in the treatment of a cardiovascular disorder or a process of wound healing, which may benefit heparanase associated activities.

Another aspect of the invention relates to a method for the modulation of heparanase biological activity. The method of the invention comprises the step of in vivo, ex vivo or in vitro contacting heparanase under suitable conditions, with a modulatory effective amount of heparanase modulator or with a composition comprising the same. This modulator may be any one of (i) an amino acid sequence derived from the C-terminal domain of heparanase, and (ii) a substance which specifically binds to an amino acid sequence within the C-terminal domain of heparanase.

According to one embodiment, the modulator used by the method of the invention may be an amino acid sequence or a peptide derived from heparanase C-terminal domain. Such amino acid sequence may be as denoted by SEQ ID NO. 3 or any fragments, peptides mutants, derivatives or variants thereof. According to a specific embodiment, a fragment may comprise the amino acid sequence of any one of SEQ ID NO. 10, 13, 18, 19, 20 and 21. According to another embodiment, such modulator may be any of the mutated heparanase molecules disclosed by the invention. According to another particular embodiment, the modulatory substance used by the method of the invention may be a substance which bind to the C- terminal domain of heparanase and thereby modulates its biological activity. According to a specific embodiment, such substance may be an antibody which specifically recognizes an amino acid sequence derived from the C-terminal domain of heparanase and is capable of modulating heparanase biological activity.

It should be noted that any modulator which binds heparanase C-domain and thereby modulates any of heparanase biologic activities is encompasses by the present invention. This include in addition to specific antibodies directed against the C-terminal domain of heparanase, also any small molecule, or any modulator, optionally a modulator isolated and identified by the screening method of the invention, as will be described herein after.

The invention further provides a method for the modulation of heparanase biological activity in a subject in need thereof. This method comprises the step of administering to said subject a modulatory effective amount of heparanase modulator being any one of: (i) an isolated and purified amino acid sequence derived from the C-terminal domain of heparanase; or (ii) a substance which specifically binds to an amino acid sequence derived from the C-terminal domain of heparanase and is capable of modulating heparanase biological activity, or a composition comprising the same.

According to one embodiment, the modulator used by the method of the invention may be an amino acid sequence or a peptide derived from heparanase C-terminal domain. Such amino acid sequence may be as denoted by SEQ ID NO. 3 or any fragments, peptides mutants, derivatives or variants thereof. According to a specific embodiment, a fragment may comprise the amino acid sequence of anyone of SEQ ID NO. 10, 13, 18, 19, 20 and 21. According to another embodiment, such modulator may be any of the mutated heparanase molecules disclosed by the invention.

According to another particular embodiment, the modulatory substance used by the method of the invention may be a substance which bind to the C- terminal domain of heparanase and thereby modulates its biological activity. According to a specific embodiment, such substance may be an antibody which specifically recognizes an amino acid sequence derived from the C-terminal domain of heparanase and is capable of modulating heparanase biological activity.

According to another aspect, the invention relates to a method for the inhibition or the treatment of a process or a pathologic disorder associated with heparanase biological activity. The method of the invention comprises the step of administering to a subject in need thereof a therapeutically effective amount of a modulator of heparanase or a composition comprising the same. Such inhibitory modulator may be for example, an isolated and purified peptide comprising an amino acid sequence derived from the C-terminal domain of heparanase. Such peptides may inhibit heparanase activity mediated by the C-terminal domain, possibly, by competing with the active corresponding sequences. Alternatively, an inhibitory modulator of heparanase used by the method of the invention may be a substance which specifically binds to an amino acid sequence derived from C-terminal domain of heparanase and is thereby capable of inhibiting heparanase biological activity, or a composition comprising the same.

According to one particular embodiment, such modulator may be an antibody which specifically recognizes an amino acid sequence derived from the C- terminal domain of heparanase and is capable of inhibiting heparanase biological activity.

As used herein in the specification and in the claims section below, the term "treat" or treating and their derivatives includes 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.

As used herein in the specification and in the claims section below, the phrase "associated with heparanase biologic activity" refers to conditions which at least partly depend on the biologic activity of heparanase. It is understood that the catalytic, as well as non-catalytic activity of heparanase under many such conditions may be normal, yet inhibition thereof in such conditions will result in improvement of the affected individual.

It should be further noted that disorders or said conditions may 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, autoimmune conditions, kidney disorder, cardiovascular disorder and/or a condition involving wound.

It is to be therefore understood that the inhibiting compositions and methods of the invention are useful for treating or inhibiting tumors at all stages, namely tumor formation, primary tumors, tumor progression or tumor metastasis. Thus, in one embodiment of the present invention, the compositions and methods of the invention can be used for inhibition of angiogenesis, and are thus useful for the treatment of diseases and disorders associated with angiogenesis or neovascularization such as, but not limited to, tumor angiogenesis, opthalmologic disorders such as diabetic retinopathy and macular degeneration, particularly age-related macular degeneration, and reperfusion of gastric ulcer.

As used herein to describe the present invention, "malignant proliferative disorder", "cancer", "tumor" and "malignancy" all relate equivalently to a hyperplasia of a tissue or organ. If the tissue is a part of the lymphatic or immune systems, malignant cells may include non- solid tumors of circulating cells. Malignancies of other tissues or organs may produce solid tumors. In general, the composition as well as the methods of the present invention may be used in the treatment of non-solid and solid tumors, for example, carcinoma, melanoma, leukemia, and lymphoma.

Therefore, according to a specific embodiment, the C-domain specific modulator of the invention, specifically modulators inhibiting heparanase activity or a composition comprising the same, can be used for the treatment or inhibition of non-solid cancers, e.g. hematopoietic malignancies such as all types of leukemia, e.g. acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), myelodysplastic syndrome (MDS), mast cell leukemia, hairy cell leukemia, Hodgkin's disease, non-Hodgkin's lymphomas, Burkitt's lymphoma and multiple myeloma, as well as for the treatment or inhibition 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, extraliepatic bile ducts, ampulla of vater, exocrine pancreas, lung, pleural mesothelioma, bone, 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.

As indicated above, heparanase activity further correlates with the ability of activated cells of the immune system to leave the circulation and elicit both inflammatory and autoimmune responses. Interaction of platelets, granulocytes, T and B lymphocytes, macrophages and mast cells with the subendothelial ECM is associated with degradation of heparan sulfate (HS) by heparanase catalytic activity [Vlodavsky, I. et al., Invasion & Metastasis 12, 112-127 (1992)]. The enzyme is released from intracellular compartments (e.g., lysosomes, specific granules) in response to various activation signals (e.g., 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 [Campbell, K. H. et al. Exp. Cell Res. 200, 156-167 (1992)]. Treatment of experimental animals with heparanase alternative substrates (e.g., non- anticoagulant species of low molecular weight heparin) markedly reduced the incidence of experimental autoimmune encephalomyelitis (EAE), adjuvant arthritis and graft rejection [Vlodavsky (1992) ibid. Lider, O. et al, J. Clin. Invest. 83:752- 756 (1989)] in experimental animals, indicating that heparanase inhibitors may be applied to inhibit autoimmune and inflammatory diseases. Therefore, in a further embodiment, the C-domain specific inhibitory modulators of the invention, or any compositions and methods thereof, may be useful for treatment of or amelioration of inflammatory symptoms in any disease, condition or disorder where immune and/or inflammation suppression is beneficial such as, but not limited to, treatment of or amelioration of inflammatory symptoms in the joints, musculoskeletal and connective tissue disorders, or of inflammatory symptoms associated with hypersensitivity, allergic reactions, asthma, atherosclerosis, otitis and other otorhinolaryngological diseases, dermatitis and other skin diseases, posterior and anterior uveitis, conjunctivitis, optic neuritis, scleritis and other immune and/or inflammatory ophthalmic diseases.

In another preferred embodiment, the C-domain specific inhibitory modulators of the invention or any compositions thereof, are useful for treatment of or amelioration of an autoimmune disease such as, but not limited to, inflammatory bowel disease, ulcerative colitis and Crohn's disease, Eaton- Lambert syndrome, Goodpasture's syndrome, Greave's disease, Guillain-Barr syndrome, autoimmune hemolytic anemia (AIHA), hepatitis, insulin- dependent diabetes mellitus (IDDM), systemic lupus erythematosus (SLE), multiple sclerosis (MS), myasthenia gravis, plexus disorders e.g. acute brachial neuritis, polyglandular deficiency syndrome, primary biliary cirrhosis, rheumatoid arthritis, scleroderma, thrombocytopenia, thyroiditis e.g. Hashimoto's disease, Sjbgren's syndrome, allergic purpura, psoriasis, mixed connective tissue disease, polymyositis, dermatomyositis, vasculitis, polyarteritis nodosa, polymyalgia rheumatica, Wegener's granulomatosis, Reiter's syndrome, Behget's syndrome, ankylosing spondylitis, pemphigus, bullous pemphigoid, dennatitis herpetiformis and insulin dependent diabetes.

Another alternative embodiment relates to the treatment of heparanase associated disorders, where enhancement of heparanase activity is desired. Such conditions may be related for example to cardiovascular disorders or a process of wound healing.

Accordingly, the invention provides a method for the enhancement of a process for the treatment of a pathologic disorder associated with heparanase biological activity. The method of the invention comprises the step of administering to a subject in need thereof a therapeutically effective amount of one of an enhancing modulator of heparanase or of any composition comprising the same. According to a specific embodiment, such enhancing modulator may be an isolated and purified amino acid sequence derived from the C-terminal domain of heparanase, any fragments, peptides, mutants and derivatives hereof, as described by the invention. Alternatively, such enhancing modulator may be a substance which specifically binds to an amino acid sequence derived from C-terminal domain of heparanase and is capable of enhancing heparanase biological activity. In a particular embodiment, such substance may be an antibody, specifically the monoclonal 6F8 antibody, which was shown by the invention as enhancing different biological activities of heparanase.

According to one specific embodiment, the method of the invention which involves enhancement of heparanase activity may be applicable in treating wounds and for cosmetic applications. It should be noted that where the substance used by the method of the invention, enhances heparanase activation of Akt, this may be applicable for treating cardiovascular disorders.

While reducing the present invention to practice, the ability of heparanase C- domain, as specifically demonstrated by using the C-domain specific antibody, 6F8, to induce angiogenesis and wound healing were put to test. As is further demonstrated below, the results were striking (particularly, Figures 10 and 11), rendering heparanase C-domain specific antibody highly likely to become a medication for induction and/or acceleration of wound healing and/or angiogenesis. Cosmetic applications are also envisaged by the present invention, for example, enhancement of hair growth.

According to a specifically preferred embodiment, the invention provides a method of inducing or accelerating a healing process of a wound. According to a specific embodiment, the method of the invention involves administering to the wound, or to the subject in need thereof, a therapeutically effective amount of at least one of enhancing modulator which enhances heparanase activities. Such enhancing modulator may be an isolated and purified amino acid sequence derived from the C-terminal domain of heparanase, or any fragments, peptides, mutants, derivatives and variants thereof. Alternatively, such enhancing modulator may be a substance which specifically binds to an amino acid sequence derived from the C-terminal domain of heparanase and is capable of enhancing heparanase biological activity, so as to induce or accelerate the healing process of the wound.

It should be noted that any modulators which specifically binds (the 6F8 antibody), or derived from heparanase C-domain, as well as compositions comprising the recombinant heparanase which can be used for inducing and/or accelerating wound healing and/or angiogenesis, as well as for cosmetic treatment of hair and skin, are also within the scope of the invention.

According to a specific embodiment, the method of the invention may use any of the enhancing modulator defined by the invention, any modulator identified by the screening method of the invention, or any antibody described by the invention. It should be mentioned that the use of the monoclonal 6F8 antibody may be preferred. Thus, the invention provides the use of any of the antibodies of the invention for the modulation of heparanase biologic catalytic activity. ^v

Still further, the invention provides the use of any of the antibodies of the invention in the preparation of a composition for the enhancement of heparanase glycosidase catalytic activity.

According to another specifically preferred embodiment, the invention relates to the use of any of the antibodies of the invention, and particularly, the 6F8 antibody, in the preparation of a pharmaceutical composition for enhancing a wound healing process.

The primary goal in the treatment of wounds is to achieve wound closure. Open cutaneous wounds represent one major category of wounds and include burn wounds, neuropathic ulcers, pressure sores, venous stasis ulcers, and diabetic ulcers. Open cutaneous wounds routinely heal by a process which comprises six major components: (i) inflammation; (ii) fibroblast proliferation; (iii) blood vessel proliferation; (iv) connective tissue synthesis; (v) epithelialization; and (vi) wound contraction. Wound healing is impaired when these components, either individually or as a whole, do not function properly. Numerous factors can affect wound healing, including malnutrition, infection, pharmacological agents (e.g., actinomycin and steroids), advanced age immunodeficiency and diabetes.

With respect to diabetes, diabetes mellitus is characterized by impaired insulin signaling, elevated plasma, glucose and a predisposition to develop chronic complications involving several distinctive tissues. Among all the chronic complications of diabetes mellitus, impaired wound healing leading to foot ulceration is among the least well studied. Yet skin ulceration in diabetic patients takes a staggering personal and financial cost. Moreover, foot ulcers and the subsequent amputation of a lower extremity are the most common causes of hospitalization among diabetic patients. In diabetes, the wound healing process is impaired and healed wounds are characterized by diminished wound strength.

The term "wound" refers broadly to injuries to the skin and subcutaneous tissue initiated in any one of a variety of ways (e.g., pressure sores from extended bed rest, wounds induced by trauma, cuts, ulcers, burns and the like) and with varying characteristics. Wounds are typically classified into one of four grades depending on the depth of the wound: (i) Grade I: wounds limited to the epithelium; (ii) Grade II wounds extending into the dermis; (iii) Grade III: wounds extending into the subcutaneous tissue; and (iv) Grade IV (or full- thickness wounds): wounds wherein bones are exposed (e.g., a bony pressure point such as the greater trochanter or the sacrum). The term "partial thickness wound" refers to wounds that encompass Grades I-III; examples of partial thickness wounds include burn wounds, pressure sores; venous stasis ulcers, and diabetic ulcers. The term "deep wound" is meant to include both Grade III and Grade IV wounds.

The term "healing" in respect to a wound refers to a process to repair a wound as by scar formation.

The phrase "inducing or accelerating a healing process of a wound" refers to either the induction of the formation of granulation tissue of wound contraction and/or the induction of epithelialization (i.e., the generation: of new cells in the epithelium). Wound healing is conveniently measured by decreasing wound area. Hereinafter, the term "treating a wound" includes inducing or accelerating a healing process of a wound, as well as ameliorating a condition of the wound, and/or a complication (complicating condition) associated with the wound.

The present invention contemplates treating all wound types, including deep wounds and chronic -wounds.

The term "chronic wound" refers to a wound that has not healed within thirty days.

The anti-heparanase C-domain antibody 6F8, or any modulating substance or agent directed to heparanase C-domain as disclosed by the invention, may be used as a therapeutic for a wide variety of wounds under pathological conditions. These include diabetic and pressure ulcers, burns and incisional wounds, and may expand further to tissue damage caused by ischemia, mainly in the context of heart and kidney diseases. Moreover, accelerated healing may contribute to the aesthetically appearance of the wounds, implicating a potential cosmetic benefit. Other cosmetic application of the antibody of the invention may include induction of hair growth.

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

According to exemplary but preferred embodiments of the present invention, anti-heparanase C-domain antibody 6F8 preferably has a concentration in a range of from about 0.005 microgramper per Kg to about 500 microgram per Kg of body weight. More preferably, anti-heparanase C- domain antibody 6F8 has a concentration in a range of from about 0.5-microgram per Kg, to about 50 microgram per Kg of the treated mammal. Optionally and preferably, anti- heparanase C-domain antibody 6F8, or any other C-domain directed modulator, may be present in a concentration in a range of from about 1 microgram to about 150 micrograms per dose. It should be noted that the presence of one or more protein stabilizing agents, which are well known in the art and which could easily be selected by one of ordinary skill in the art, may increase the potential overall activity of anti-heparanase C-domain antibody 6F8, during treatment by up to two orders of magnitude. Also, dosing may vary according to whether a single dose is administered or a plurality of doses is administered. The anti-heparanase C-domain antibody 6F8 may be preferably provided in a suitable therapeutic/pharmaceutical composition, preferably with a suitable carrier and more preferably with one or more stabilizing agents.

Formulations for topical administration are applicable for wound healing as well as for cosmetic use. Such formulations may include, but are not limited to, lotions, ointments, gels, creams, suppositories, drops, liquids, sprays and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, stents, active pads, and other medical devices may also be useful. Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, sachets, capsules or tablets. Thickeners, diluents, flavorings, dispersing aids, emulsifiers or binders may be desirable. Formulations for parenteral administration may include, but are not limited to, sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. The magnitude of therapeutic dose of any of the compositions of the invention will of course vary with the group of patients (age, sex, etc.), the nature of the condition to be treated and with the route administration, all of which shall be determined by the attending physician.

Although the method of the invention is particularly intended for the treatment of disorders associated with heparanase biologic activity in humans, other mammals are included. By way of non-limiting examples, mammalian subjects include monkeys, equines, cattle, canines, felines, rodents such as mice and rats, and pigs.

The pharmaceutical compositions of the invention may be administered by the methods of the invention, systemically, for example by parenteral, e.g. intravenous, intraperitoneal or intramuscular injection. In another example, the pharmaceutical composition can be introduced to a site by any suitable route including intravenous, subcutaneous, transcutaneous, topical, intramuscular, intraarticular, subconjunctival, or mucosal, e.g. oral, intranasal, or intraocular administration.

Local administration to the area in need of treatment may be achieved by, for example, local infusion during surgery, topical application, directs injection into the inflamed joint, directly onto the eye, etc.

For oral administration, the pharmaceutical preparation may be in liquid form, for example, solutions, syrups or suspensions, or in solid form as tablets, capsules and the like. For administration by inhalation, the compositions are conveniently delivered in the form of drops or aerosol sprays. For administration by injection, the formulations may be presented in unit dosage form, e.g. in ampoules or in multidose containers with an added preservative. The compositions of the invention can also be delivered in a vesicle, for example, in liposomes. In another embodiment, the compositions can be delivered in a controlled release system.

The amount of the therapeutic or pharmaceutical composition of the invention which is effective in the treatment of a particular disease, condition or disorder will depend on the nature of the disease, condition or disorder and can be determined by standard clinical techniques. In addition, in vitro assays as well in vivo experiments may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease, condition or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

As used herein, "effective amount" means an amount necessary to achieve a selected result. For example, an effective amount of the composition of the invention useful for modulating (inhibition or enhancement) of heparanase activity and thereby for the treatment of said pathology.

In an attempt to apply a more rational approach, the inventors sought for functional domains of heparanase that would serve as targets for drug development.

Interestingly, functional domains other than the basic heterodimer structure [Levy-Adam, F. et al. (2003) ibid.] and amino acids (GIu²²⁵ and GIu³⁴³) critical for the enzyme catalytic activity [Hulett, M. D., et al., Biochemistry 39, 15659- 15667 (2000)], have not yet been elucidated in the heparanase protein. In the present study, heparanase C-domain was demonstrated as mediating different biological activities of heparanase, such as, cell survival (possibly through signaling involving the Akt pathway), association with a membranal receptor, tumor progression and cell migration. The identification of this domain by the invention provides a target for screening of modulators of heparanase.

Hence, in another aspect, the invention relates to a method of screening for a test substance which specifically binds to an amino acid sequence derived from heparanase C-domain and is capable of modulating different heparanase biological activities. Such substance may be particularly useful for the treatment of heparanase-associated pathologic disorders.

Key to the application of high-throughput screening for high-affinity binding of substances, preferably antibodies, small molecules or peptides, is the development of a sensitive and convenient screening assay.

Development of a robust screening assay for substances, through their affinity for heparanase C-domain, will be the first step in said screening method.

Thus, according to one embodiment, the screening method of the invention comprises the steps of:

(a) obtaining candidate substances;

(b) selecting from the substances obtained in step (a) a substance which bind to the C-terminal domain of heparanase, or to any fragment, peptide, mutant, derivative and variant thereof; and

(c) evaluating the candidate substance selected in step (b) by determining the modulatory effect of said substance on the biological activity of heparanase. According to one embodiment, the candidate substance may be selected by the steps of:

(a) providing a mixture comprising the C-terminal domain of heparanase or any functional fragment, peptide, fragment, peptide, mutant, derivative and variant thereof;

(b) contacting said mixture with said candidate substance under suitable conditions for said binding; and

(c) determining the effect of the test substance on an end-point indication, whereby modulation of said end point is indicative of binding of the C-terminal domain of heparanase to said test substance.

According to a specifically preferred embodiment, the C-terminal domain of heparanase may be an amino acid sequence comprising all or part of residues 413 to 543 of heparanase, preferably of human heparanase as denoted by SEQ ID NO. 3, or any fragment, peptide derivative or variant thereof. According to one embodiment particular fragments are any of the fragments denoted by amino acid SEQ ID NO. 13, 10, 18, 19, 20 21 and any other derivatives disclosed by the invention.

According to one embodiment, the mixture used by the screening method of the invention may comprise the C-terminal domain of heparanase or any fragment or peptide thereof, and optionally, solutions, buffers and/or compounds which provide suitable conditions for interaction of said C-terminal domain of heparanase with the candidate substance. This mixture may further comprise solutions suitable for the detection of an end-point indicating the interaction of the tested candidate substance with the C-terminal domain of heparanase. According to one embodiment, the mixture may be a cell-free mixture. Thus, the C-terminal domain of heparanase or any fragment, peptide, mutant, derivative, homologue and variant thereof may be provided as any one of a purified recombinant protein, and a cell lysates or a preparation of a transformed host cell expressing said C-terminal domain of heparanase.

In yet another embodiment, the mixture may be a cell mixture, such as transfected cell culture.

It should be appreciated that the mutated TIMB heparanase construct, which encodes heparanase molecule devoid of the C-domain or alternatively, the Δ17 construct encoding heparanase molecule devoid of the last 17 amino acid residues of the C-domain, or the Δloop construct encoding heparanase molecule devoid of most of the loop sequence, may be also used in the screening method of the invention. Accordingly, comparative binding of the test substance to a mutated (TIMB, Δ17, or Δloop constructs) and wild type molecule will distinguish between test substances which bind the particular sequence of the invention, and those which bind other regions of heparanase.

The candidate substance examined by the screening method of the invention may be further evaluated by determining its ability to modulate at least one of heparanase catalytic or non-catalytic activities, induing for example, secretion of heparanase, interaction with a membranal receptor shown by the invention as located to lipid rafts, activation of Akt, and thereby modulation of cell survival and potentially, cell apoptosis, as well as involvement in processes leading to wound healing, cell migration, cell adhesion etc.

Therefore, in a preferred embodiment, the third step of the screening method involves evaluation of the selected test substance ability of to modulate heparanase activity, which evaluating method comprises the steps of: (a) providing a test system comprising an active heparanase molecule or any functional fragments thereof, and a heparanase substrate; (b) contacting said system with a candidate substance obtained and selected by the method of the invention, under conditions suitable for heparanase biologic activity; and (c) determining the effect of the candidate substance on an end-point indication as compared to a control. Such effect is indicative of the capability of the candidate substance to inhibit the examined heparanase biologic activity.

According to one embodiment, the test system may be any one of cell free mixture and in vitro /ex vivo cell culture.

According to a specifically preferred embodiment, the test system may be a cell-free mixture. Accordingly, is such system heparanase may be provided as any one of a purified recombinant protein, and a cell lysates or membrane preparation of a transformed host cell.

In an alternative embodiment, the test system may be an in-υitro I 'ex-υiυo cell culture comprising an endogenously expressed heparanase or exogenously expressed heparanase.

Using these systems, any other biologic activities of heparanase may be analyzed. These may include examination of heparanase secretion, signaling, particularly through the PI3'K/Akt pathway, modulation of cell survival, apoptosis, cell migration, tumor progression, cell adhesion, neovascularization, angiogenesis as well as signaling leading to wound healing.

The candidate substance obtained and selected by the screening method of the invention, may be any one of protein based, carbohydrates based, lipid based, nucleic acid based (particularly, haptamers), natural organic based, synthetically derived organic based, inorganic based, and peptidomimetics based substances. Such substance may be for example a product of positional scanning of combinatorial libraries of peptides, libraries of cyclic peptidomimetics, and random or dedicated phage display libraries.

Thus, the invention further provides any modulating substance identified by the screening method of the invention.

Any of the anti-heparanase C-domain antibodies of the invention, preferably, monoclonal antibodies directed to the amino acid sequence of the invention, may also provide the basis for a sensitive screening assay able to detect heparanase in body fluids. This will enable a comprehensive study aimed to establish heparanase as a diagnostic marker for human pathologies.

Therefore, in a further aspect, the invention relates to a method for the diagnosis of a process or a pathologic disorder associated with heparanase biologic activity in a mammalian subject. The diagnostic method of the invention comprises the steps of: (a) providing a sample of said subject; (b) contacting said sample with an antibody which specifically recognizes and binds the C-terminal domain of heparanase;

(c) removing any unbound antibody; and

(d) detecting the extent of reaction between the antibody and the heparanase present in the tested sample, by suitable means.

Wherein increased reaction as compared to a suitable control is indicative of that said subject suffers from said disorder.

According to one embodiment, any of the antibodies defined by the invention may be applicable in the diagnostic method. More particularly, any one of the monoclonal antibodies designated IEl, 44C4 and 6F8, may be used for the diagnostic method of the invention. According to a specific embodiment, the sample used by the diagnostic method of the invention may be as a non-limiting example, body fluids, tissue specimens, tissue extracts, cells, cell extracts and cell lysates. More specifically, the sample used by the diagnostic method of the invention may be a body fluid sample such as blood, lymph, milk, virine, faeces, semen, brain extracts, spinal cord fluid (SCF), appendix, spleen and tonsillar tissue extracts

According to another preferred embodiment, a suitable means used by the diagnostic method of the invention for the detection of the active form of heparanase may be a protein based detection assay selected from the group consisting of immunohistochemical staining, Western blot analysis, immunoprecipitation, flow cytometry, ELISA and competition assay.

More particularly, as indicated above, the antibodies, including fragments of antibodies, useful in the present invention, may be used to quantitatively and/or qualitatively detect heparanase in a sample. This can be accomplished by immunofluorescence techniques employing a fluorescently or color-labeled antibody coupled with light microscopic, flow cytometric, or fluorometric detection.

Another specifically preferred embodiment relates to the antibodies of the invention conjugated to a detectable moiety. One of the ways in which an antibody in accordance with the present invention can be detectably labeled is by linking the same to an enzyme and used in an enzyme immunoassay (EIA). This enzyme, in turn, when later exposed to an appropriate substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometry, fluorometric or by visual means. Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha- glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta- galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection can be accomplished by colorimetric methods, which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.

Detection may be accomplished by using any of a variety of other immunoassays. For example, by radioactive labeling the antibodies of the invention or antibody fragments, it is possible to detect the active form of heparanase through the use of a radioimmunoassay (RIA). A good description of RIA may be found in Laboratory Techniques and Biochemistry in Molecular Biology, by Work, T.S. et al., North Holland Publishing Company, NY (1978) with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" by Chard, T., incorporated by reference herein. The radioactive isotope can be detected by such means as the use of a γ counter or a scintillation counter or by autoradiography.

It is also possible to label an antibody in accordance with the present invention with a fluorescent compound, fluorescence emitting metals, a chemi- luminescent compound or a bioluminescent compound.

A number of methods of the art of molecular biology are not detailed herein, as they are well known to the person of skill in the art. Such methods include site-directed mutagenesis, PCR cloning, expression of cDNAs, analysis of recombinant proteins or peptides, transformation of bacterial and yeast cells, transfection of mammalian cells, and the like. Textbooks describing such methods are e.g., Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory; ISBN: 0879693096, 1989, Current Protocols in Molecular Biology, by F. M. Ausubel, ISBN: 047150338X, John Wiley & Sons, Inc. 1988, and Short Protocols in Molecular Biology, by F. M. Ausubel et al, (eds.) 3rd ed. John Wiley & Sons; ISBN: 0471137812, 1995. These publications are incorporated herein in their entirety by reference. Furthermore, a number of immunological techniques are not in each instance described herein in detail, as they are well known to the person of skill in the art. See e.g., Current Protocols in Immunology, Coligan et al., (eds), John Wiley & Sons. Inc., New York, NY.

Disclosed and described, it is to be understood that this invention is not limited to the particular examples, methods steps, and compositions disclosed herein as such methods steps and compositions may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise.

Throughout this specification and the Examples and claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The following examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention.

Examples

Experimental procedures

Antibodies

Monoclonal anti-hep aranase antibodies were generated by immunizing Balb/C mice with the entire 65 kDa heparanase protein. Hybridomas were obtained by routine procedure and were selected by ELISA using the 65 kDa heparanase for coating [Shafat, I. et al. Biochem. Biophys. Res. Commun. 341:958-963 (2006)]. Several hybridomas that reacted positively with heparanase were selected for further characterization. Hybridoma subclass was determined by isotyping kit according to the manufacturer's (Serotec, Oxford, UK) instructions. Mouse IgG (Sigma, St. Louis MI) was used as control for all experiments. Anti-heparanase #1453 rabbit polyclonal antibody has previously been characterized [Zetser, A. et al. J. Cell. Sci.117:2249-58 (2004)]. Anti-heparanase monoclonal antibody was kindly provided by ImClone Systems. Anti-Myc-tag (sc-40), anti-Akt (sc-5298), and anti-calnexin (sc-11397) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-Akt (Ser473) antibody was purchased from Cell Signaling Technologies (Beverly, MA). Anti mouse platelet endothelial cell adhesion molecule (PECAM)-I (CD31) polyclonal antibody was kindly provided by Dr. Joseph A. Madri (Yale University, New Haven, CT). IEl, 44C4 and 6F8 monoclonal antibodies were examined and shown as directed against the C-domain of heparanase in ELISA experiments as demonstrated by Table 1.

Table 1 ELISA results of antibodies recognizing the C-domain

Dilutio 1:100 1:200 1:500 1:1000 n

6F8 1.332 0.958 1.059 0.807 0.798 0.835 0.453 0.318 0.688 0.232 0.22 0.228

IEl 0.679 0.87 0.832 0.465 0.338 0.69 0.212 0.219 0.286 0.101 0.116 0.109

44C4 1.988 2.212 2 0.677 0.797 0.778 0.453 0.478 0.461 0.211 0.25 0.235

CONTROL 0.022 0.003 0.004 0.002 0.004 0.005 0.006 0.003 0.004 0.004 0.006 0.004

CONTROL 0.004 0.005 0.004 0.008 0.003 0.006 0.006 0.004 0.005 0.008 0.004 0.003

CONTROL

0.003 0.006 0.008 0.005 0 002 0.006 0.005 0.003 0.003 0.003 0.004 0.004 Reagents

*Bromodeoxyuridine (BrdU) was purchased from GE Healthcare

(Buckinghamshire, England) and anti-BrdU monoclonal antibody-HRP conjugated was purchased from Roche (Mannheim, Germany).

*Hsp90 inhibitor lV-Allylamino-lT-demethoxygeldanamycin (17-AAG) was purchased from Alomone Labs (Jerusalem, Israel) and was dissolved in DMSO as stock solution. DMSO was added to the cell culture as a control.

*Fluorescein wheat germ agglutinin was purchased from Vector Laboratories

Inc (Burlingame, CA).

*Lovastatin, methyl-B-cyclodextrin (M®CD), Nycodenz, and heparin were purchased from Sigma (St. Louis, MO). Cholera toxin-® subunit-HRP conjugate was purchased from Calbiochem (San Diego, CA).

Heparanase activity assay

Preparation of ECM-coated 35 mm dishes and determination of heparanase activity were performed as described in detail elsewhere [Levy-Adam, F. et al. Biochem. Biophys. Res. Commun. 308:885-891 (2003)]. Briefly, cells (IxIO⁶) were lysed by three freeze/thaw cycles and cell extracts were incubated (18 h, 37⁰C, pH 5.8) with ³⁵S-labeled ECM. The reaction mixture (1 ml) containing sulfate-labeled degradation fragments, was subjected to gel filtration on a Sepharose CL-6B column. Fractions (0.2 ml) were eluted with PBS and their radioactivity counted in a /3-scintillation counter. Degradation fragments of HS side chains are eluted at 0.5<K_av<0.8 (fractions 15-30) and represent heparanase generated degradation products.

To evaluate the effect of hybridomas on heparanase activity, purified active heparanase (40 ng) or cell lysates prepared from 2xlO⁶ cells were pre- incubated (2 h, 4⁰C) with protein A-purified monoclonal antibody (1 μg) or control IgG in 1 ml serum-free RPMI medium. Subsequently, the incubation medium was applied onto ³⁵S-labeled ECM (2 h, 37⁰C) and the reaction mixture (1 ml) containing sulfate labeled degradation fragments was subjected to gel filtration on a Sepharose CL-6B column. Fractions (0.2 ml) were eluted with PBS and their radioactivity counted in a β-scintillation counter. Degradation fragments of HS side chains are eluted at 0.5< Kav<0.8 (fractions 15-30) and represent heparanase generated degradation products. RPMI medium conditioned by 2xlO⁶ cells was similarly evaluated.

Construction of plas raids:

Plasmids and viral gene constructs that were used in this study are listed in

Table 2.

Plasmid pSecTag2B-Hepa, which codes for heparanase with a leader sequence of Igk, was kindly provided by Dr Hua-Quan Miao (ImClone Systems, New York, NY). It was constructed by replacing the EcoRI-Apal fragment in pSecTag2B (Invitrogen) with heparanase segment lacking its signal peptide (including residues 36-543 of heparanase as denoted by SEQ ID NO. 1).

Plasmid pSecTag2B-Hepa (GS3) encodes for a constitutively active, single chain heparanase in which the linker sequence was replaced by a three glycine-serine repeats (GS3) [Nardella, C. et al. Biochemistry 43:1862-73 (2004)]. It was constructed by replacing the SacH-Aflϊl fragment of pSecTag2B-Hepa with a respective fragment from pcDNA3-Hepa (GS3), kindly provided by Dr. Christian Steinkuhler (IRBM/Merck research laboratories, Pomezia, Italy) [Nardella (2004) ibid.].

Plasmids pSecTag2A-TIMB, pSecTag2A-C-domain and pSecTag2A-HepaΔ17 which encode for the heparanase TIM-barrel domain (residues 36-417 of heparanase as denoted by SEQ ID NO. 2), C-domain (residues 413-543 of heparanase as denoted by SEQ ID NO.3) and heparanase deleted for its last 17 amino acids (residues 36-526 of heparanase as denoted by SEQ ID NO. 9 and designated as Δ17), respectively, were constructed by replacing the EcoRΪ- Xhoϊ fragment of the pSecTag2A plasmid with the respective heparanase segment.

Plasmids pSecTag2A-TIMB (GS3) and pSecTag2A-HepaΔ17 (GS3) encode for the heparanase TIM-barrel domain (including residues 36-417 of heparanase and comprising the amino acid sequence as denoted by SEQ ID NO. 4), and heparanase deleted for its last 17 amino acids (including residues 36-526 of heparanase and comprising the amino acid sequence as denoted by SEQ ID NO. 9; Δ17), respectively, in the GS3 backbone. These plasmids were constructed by replacing the EcoRI-XhoI fragment in pSecTag2A with the respective segment. Heparanase point mutations, listed in Table 3, were constructed using the Quickchange Site-directed Mutagenesis Kit (Stratagene, San Diego, CA). The lentiviral expression plasmids pTK-C domain, which encode for heparanase C-terminal domain (residues 413-543 of heparanase as denoted by SEQ ID NO.3), was constructed by replacing the BamΗI-Xhόl fragment of the lentiviral vector pTK208 (kindly provided by Dr. TaI Kafri, Gene Therapy Center, University of North Carolina, Chapel Hill, NC, USA) with a bgllϊ-XhoI Myc-tagged C-domain segments. DNA cloning was carried out by standard procedures. All heparanase derivatives were confirmed by sequencing.

The following primer pairs were used: pSecTag2A-TIMB and pSecTag2A- TIMB(GS3): forward primer 5'-GGA-ATT-CAG-GAC-GTC-GTG-GAC-CTG-S' (as denoted by SEQ ID NO. 5) and reverse primer δ'-GCC-GCT-CGA-GCC- TTG-GTG-CCC-ACC-AAT-TTC-3'; of heparanase as denoted by SEQ ID NO. 6), pSecTag2A-C-domain: forward primer δ'-GGA-ATT-CTG-GTG-GGC-ACC- AAG-GTG-TTA-ATG-3' (as denoted by SEQ ID NO. 7), and reverse primer 5'- GCC-GCT-CGA-GAG-ATG-CAA-GCA-GCA-ACT-TTG-G-S' (as denoted by SEQ ID NO. 8). Table 2. Plasmids encoding heparanase derivatives

Cells and cell culture

HEK 293, U87-MG human glioma, Chinese hamster ovary (CHO) Kl cells, human cervical adenocarcinoma HeLa cells, human choriocarcinoma JAR, lung carcinoma A549 and MDA-MB-231 human breast carcinoma cells were purchased from the American Type Culture Collection (ATCC). Cells were grown in Dulbecco's modified Eagle's medium (Biological Industries, Beit Haemek, Israel) supplemented with 10% fetal calf serum and antibiotics. Subconfluent U87, MDA-231, and HEK 293 cells were stably transfected with human heparanase gene constructs using FuGENE 6 reagent according to the manufacturer's (Roche Applied Science, Indianapolis, IL) instructions. Transfection proceeded for 48 hours, followed by selection with Zeocin (Invitrogen, Carlsbad, CA) for 2 weeks. Stable transfectant pools were further expanded and analyzed. Mutant CHO cells (pgs A-745) deficient of xylosyltransferase and unable to initiate glycosaminoglycan synthesis, were kindly provided by Dr. J. Esko (University of California, San Diego) and grown in RPMI 1640 medium (Biological Industries, Beit Haemek, Israel) supplemented with 10% FCS and antibiotics.

Cell lysates and protein blotting

Cell cultures were incubated for 24 h under serum-free conditions without or with 50 μg/ml heparin, pretreated with 1 mM orthovanadate for 10 min at 37⁰C, washed twice with ice cold PBS containing 1 mM orthovanadate and scraped into lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton- XlOO, 1 mM orththovanadate) containing a cocktail of protease inhibitors (Roche, Mannheim, Germany). Total cellular protein concentration was determined by the BCA assay, according to the manufacturer's (Pierce, Rockford, IL) instructions. Medium and lysate samples (thirty micrograms) were concentrated and pre-absorbed on Fractogel resin (Merck) and subjected to SDS polyacrylamid gel electrophoresis (SDS-PAGE) and immunoblotting, as described [Gingis-Velitski, (2004) ibid].

Binding and cross-linking

Binding experiments were carried out essentially as described [Ben-Zaken, O. et al. Int. J. Biochem. Cell. Biol. 40:530-42 (2008)]. Briefly, recombinant C- domain protein was iodinated to a high specific activity by the chloramine T method. Cells were grown in 24-well multidishes and incubated (2 h on ice) with binding buffer (RPMI 1640, 10 mM HEPES, 0.2% BSA) containing increasing concentrations of ¹²⁵I-C-domain in the absence or presence of 400- 2000 nM unlabeled heparanase or C-domain proteins. Cells were then washed with ice-cold PBS, solubilized with 200 μl of 1 M NaOH, and counted in a γ- counter. Binding parameters (Kd, .Bmax) were obtained by the Prism 4 software (GraphPad Software, San Diego, CA) [Ben-Zaken (2008) ibid.]. For Cross- linking experiments, cells were grown in 60-mm dishes and incubated for 2 h on ice with binding buffer containing 4 nM ¹²⁵I-C-domain in the absence or presence of heparin (10 μg/ml) and 400 nM unlabeled heparanase or C-domain proteins. The cross linker sulfo-EGS (Pierce; 0.2 niM) was then added for 10 min, followed by quenching with 50 mM Tris-HCl, pH 7.5. Cells were then washed with PBS₁ scraped and collected in an eppendorf tube, lysed, and subjected to SDS-PAGE followed by autoradiography.

Transfection and recombinant proteins

Transient and stable transfections were performed using FuGENE 6 reagent, according to the manufacturer's (Roche) instructions. Recombinant wild type heparanase and heparanase C-domain proteins were purified from the conditioned medium of HEK 293 cells transfected with a plasmid expressing heparanase (pSecTag2B-Hepa) or infected with lentivirus expressing the heparanase C-domain, essentially as described [Zetser, A et al. Cancer Res. 63:7733-41 (2003)].

Lentivirus production and infection of cells

Virus was produced by calcium-mediated co-transfection of the lentiviral expression plasmid (20 μg), packaging vector pCMV-dR8.91 (15 μg), and plasmid encoding the vesicular stomatitis virus coat envelope pMD2-VSVG (10 μg) into HEK 293T cells. Conditioned medium containing infective particles was collected 48 and 72 h post transfection and viral particles were added to sub-confluent cells for 24 h.

Immunocytochemistry

For immunofluorescent staining, cells were fixed with cold methanol for 10 min, washed with PBS and subsequently incubated in PBS containing 10% normal goat serum for 1 h at room temperature, followed by 2 h incubation with the indicated primary antibody. Cells were then extensively washed with PBS and incubated with the relevant Cy2/Cy3-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA) for 1 h together with TO-PRO (Invitrogen) to visualize the cell nucleus, washed and mounted (Vectashield, Vector). Staining was observed under a fluorescent confocal microscope, as described [Goldshmidt, O. et al. Exp. Cell. Res. 281:50-62 (2002)].

Cell proliferation

Cell proliferation was analyzed by BrdU incorporation using cell proliferation labeling reagent (GE Healthcare). Briefly, sub-confluent cells grown on glass cover slips were incubated for 2 h with BrdU (1:1000) in serum free medium and were then fixed with cold methanol for 15 min. Following washes with PBS, cells were incubated for 1 h with 2 N HCl, washed (3x10 min) with 0.1 M borate buffer, pH 8.5, and twice with PBS. The cells were then incubated with HRP-conjugated anti-BrdU monoclonal antibody (1:20; Roche) for 2 h, washed, and visualized using AEC staining kit (Sigma). Following hematoxylin counter staining and mounting, the mitotic index was calculated by counting BrdU- positive nuclei as percentage of total cells in at least eight different microscopic fields, as described [Zetser, A. et al. Cancer Res. 63:7733-41 (2003)]. At least 1000 cells were counted for each cell type.

Cell migration assay

The human keratinocytes cell line HACAT was kindly provided by Dr. Norbert E. Fusenig (DKFZ, Heidelberg, Germany) and was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum. Cell migration was evaluated applying the in vitro scratch assay essentially as described [Liang, C. et al. Nature protocols 2:329-333 (2007]. Briefly, cells were allowed to grow in tissue culture plates until confluence followed by creating a 'scratch' along the cell monolayer diameter with the wide end of a 1 ml tip (time 0). Plates were washed twice with PBS to remove detached cells, incubated with complete growth medium and cell migration into the wounded area was examined for 4 days in the presence of antibody 6F8 (1 μg/ml) or control mouse IgG.

Matrigel invasion assay

Invasion assay was performed using modified Boyden chambers with Matrigel- coated polycarbonate Nucleopore membrane (Corning, Corning, NY), essentially as described [Albini, A. et al. Nature protocols 2:504-511 (2007)]. Briefly, cells were serum-starved for 20 h and were then detached with trypsin-EDTA solution. Cells (2xl0⁵/0.2 ml) were added to the upper chamber in the presence of 6F8, or control mouse IgG antibodies (1 μg/ml), and invading cells adhering to the lower side of the membrane were visualized after 6 h by crystal violate staining and counted.

Flotation analysis

Detergent-insoluble complexes were analyzed on flotation gradients, as previously described. To detect the rafts-resident ganglioside GMl on dot blots, 5 μl of each flotation fraction were blotted onto nitrocellulose paper. The paper was air dried, blocked with 5% BSA in PBS, and incubated with cholera toxin jS-subunit (CT^B)-HRP (12.5 ng/ml, 2 h). Dot blots were developed with

ECL.

Animal housing and wound healing protocol

All procedures were conducted using facilities and protocols approved by the Animal Care and Use Committee of the Technion School of Medicine. Male C57BL mice (25-30 gram, Harlan, Jerusalem, Israel) were anesthetized by intraperitoneal (i.p) injection of ketamine (50 mg/kg) and xylazine (5 mg/kg), shaved and two 8 mm-diameter full thickness excisions were created with a sterile biopsy punch on the mouse back (n=5), yielding 10 wounds in each group. Wounds were left undressed and monoclonal 6F8 antibody, or control mouse IgG were injected i.p prior to, three, and five days post wounding. Since wound healing is less efficient in older animals, relatively old mice (8-10 month of age) were employed for the wound healing experiments.

Histology

Wounds were harvested seven days post wounding, fixed with 4% formaldehyde in PBS, embedded in paraffin and sectioned. Following deparaffinization and rehydration, 5 micron sections were washed (3x) with PBS and stained with hematoxyline/eosine or Mason-Trichrom, as described [Zcharia (2005) ibid]. Tissue sections were then mounted and visualized with a Zeiss axioscope microscope. Wound healing was calculated by measuring the distance between the epithelial edges at the wound diameter [Zcharia (2005) ibid.].

A model of heparanase structure

A three dimensional structure of constitutively active single chain GS3 heparanase [Nardella, C. et al. Biochemistry 43:1862-73 (2004)] was generated by a protein structure prediction server (http://www.robetta.org) [Kim, D. E. et al. Nucleic Acids Research 32 (Web Server issue): W526-31 (2004)], based on sequence and structure homology to the crystal structure of α-L- arabinofuranosidase isolated from Geobacillus stearothermophilus T-6 [Hovel, K. et al. Embo. J. 22:4922-32 (2003)].

Statistics

Data are presented as mean + SE. Statistical significance was analyzed by the two-tailed Student's t-test. The value of P<0.05 is considered significant. All experiments were repeated at least twice, with similar results. Experiments evaluating heparanase activity were repeated at least five times. Example 1

Defining the C-terminal domain of Heparanase and its role in secretion of the protein and in conferring stable conformation required for its enzymatic activity

In order to study heparanase structure, and define functional domains responsible for the variety biological functions exhibited by heparanase, the inventors utilized a structure prediction server (http://www.robetta.org) [Kim, D. E. et al. Nucleic Acids Research 32 (Web Server issue):W526-31 (2004)] that provides a prediction of the three dimensional structure of proteins. The sequence of single-chain heparanase, in which the linker segment was replaced with three Gly-Ser repeats (GS3) combining the 8 and 50 kDa protein subunits and resulting in a constitutively active enzyme, was therefore introduced [Nardella, C. et al. Biochemistry 43:1862-73 (2004)] (Fig. 1C). The model obtained, based on sequence and structure resemblance to α-L- arabinofuranosidase enzyme from Geobacillus stear other mophilus T-6 [Hovel, K. et al. Embo. J. 22:4922-32 (2003)], is shown in Figure 1. As expected and clearly illustrated by Figure IA, the predicted structure delineates a heterodimer composed of 8 and 50 kDa subunits (yellow and gray, respectively) [Levy-Adam, F. et al. Biochem. Biophys. Res. Commun. 308:885- 91 (2003); McKenzie, E. et al. Biochem. J. 373:423-35 (2003)]. Furthermore, the structure clearly illustrates a TIM-barrel fold that has previously been predicted for the enzyme [Hulett, M.D. et al. Biochemistry 39:15659-67 (2000)] . In addition, the conserved glutamic acid residues critical for heparanase catalysis (GIu²²⁵ and GIu³⁴³) (Fig. IA, red), as well as the heparin/HS binding regions (Lys^-Asp¹⁷¹, as also denoted by SEQ ID NO. 11 and Gln²™-Lys²⁸⁰, as also denoted by SEQ ID NO. 12; Fig. IA, cyan and green, respectively) [Levy- Adam, F. et al. J. Biol. Chem. 280:20457-66 (2005)] were situated in close proximity to the micro-pocked active site, corroborating the relevance of the model (Fig. IA, left panel). Notably, the catalytic site of the active single-chain heparanase appears completely exposed. This is in agreement with previous studies signifying that the linker segment, which masks the micro-pocket fold, must be completely removed in order to convert the latent heparanase into an active enzyme. Interestingly, the structure also delineates a C-terminal fold positioned next to the TIM-barrel fold. The C-terminal domain (C-domain) appears to comprise of eight β strands arranged in two sheets (Fig. IA, right panel), as well as a flexible, unstructured loop (Fig. IA, right, arrow) that lies in-between. The two sheets are packed against each other and are stabilized by hydrophobic interactions between the upper and lower β sheets. Notably, one of the C-domain β strands is contributed, apparently, by the 8 kDa protein subunit (Fig. IA, right panel, yellow), suggesting that the heparanase N- terminus plays a structural role in the establishment of the C-domain (containing residues 413-543 of heparanase, also denoted by SEQ ID NO. 3) and TIM-barrel (containing residues 36-417 of heparanase, also denoted by SEQ ID NO. 2) folds [Hulett, M.D. et al. Biochemistry 39:15659-67 (2000)].

Example 2

The C-terminal domain is essential for heparanase enzymatic activity

Apart of the well documented catalytic activity of the enzyme, heparanase was also noted to exert enzymatic activity-independent functions, facilitating the phosphorylation of selected protein kinases and inducing gene transcription Plan, N. et al. Int. J. Biochem. Cell. Biol. 38:2018-39 (2006)]. Without being bound by any theory, the inventors hypothesized that the seemingly distinct protein domains observed in the three dimensional model, namely the TIM- barrel and C-domain regions, mediate enzymatic and non-enzymatic functions of heparanase, respectively. In order to examine this possibility, the inventors designed gene constructs carrying Myc-tagged wild type (containing residues 36-543 of heparanase as also denoted by SEQ ID NO. 1), TIM-barrel (containing residues 36-417 of heparanase, also denoted by SEQ ID NO. 2), and C-domain (containing residues 413-543 of heparanase, also denoted by SEQ ID NO. 3) heparanase variants, as illustrated by FigurelB. Heparanase activity of these constructs was evaluated in stably transfected human choriocarcinoma JAR cells. This cell line was selected since it is devoid of endogenous heparanase activity [Shteper, P.J. et al. Oncogene 22:7737-49 (2003)]. All protein variants were expressed at high levels. Release from the ECM of sulfate-labeled HS degradation fragments was readily detected in JAR cells following transfection with wild type heparanase. In striking contrast, cells transfected with the TIM-barrel construct failed to display heparanase enzymatic activity (data not shown). The inventors suspected that the lack of enzymatic activity is due to impaired protein secretion shown previously to be required for the delivery of latent heparanase to late endosomes/lysosomes and its subsequent processing and activation by lysosomal cathepsins [Gingis- Velitski, S. et al. J. Biol. Chem. 279:44084-92 (2004); Abboud-Jarrous, G. et al. J. Biol. Chem. 280:13568-75 (2005); Zetser, A. et al. J. Cell. Sci.117:2249-58 (2004)]. In order to overcome impaired trafficking, the inventors applied the GS3 gene construct to generate the TIM-barrel variant, thus bypassing the requirement for protein secretion. As shown by Figure 2A, JAR cells transfected with the GS3-TIM-barrel construct failed to yield heparanase activity, while the full length GS3-heparanase was highly active. Furthermore, co-transfection of the GS3-TIM-barrel and C-domain gene constructs yielded no enzymatic activity (Fig. 2B), suggesting that the two domains ought to be expressed as a single polypeptide chain in order to fold properly and function as an active enzyme. These results thus imply that the C-domain is required for the establishment of an active heparanase enzyme, possibly by stabilizing the TIM-barrel fold.

Deletion and site directed mutagenesis approaches were next employed to identify regions and amino acids critical for this function of the C-domain. Notably, deletion of the last C-ter minus 17 amino acids (Phe⁵²⁷-Ile⁵⁴³; also denoted by SEQ ID NO. 10 Δ17) completely abolished heparanase enzymatic activity. As shown by Figure 2A, similar result was observed once the deletion was constructed in the GS3 backbone [Δ17 (GS3)]. Likewise, as also demonstrated by Table 3, deletion of part of the unstructured loop (Leu⁴⁸³- Pro⁵⁰⁹, as also denoted by SEQ ID NO. 21 and shown in Fig. ID, red box) resulted in an inactive heparanase enzyme, comprising the sequence as denoted by SEQ ID NO. 22. Moreover, point mutations of evolutionary conserved amino acid residues Phe⁵³¹, VaI⁵³³, He⁵³⁴, Ala⁵³⁷, and Cys⁵⁴² (Fig. ID, red labeled amino acid residues) resulted in inactive heparanase as shown by Figure 2C, and summarized by Table 3, thus supporting a critical role of the C-domain in the establishment of active heparanase enzyme.

Table 3: Secretion, processing and enzymatic activity of heparanase mutants

a AU heparanase mutants were expressed applying the pSecTag2B plasmid b^c HEK 293 cells were stably transfected with plasmids expressing WT heparanase or its C- termmal mutants Medium and lysates samples were subjected to immunoblottmg with anti-

Myc^b or anti-heparanase^c (1453) antibodies d The GS3 derivatives, in which the linker segment was replaced by a spacer of three Gly-Ser pairs (GS3), were utilized to study the enzymatic activity of heparanase domains and mutants JAR cells were transiently transfected with plasmids expressing WT heparanase or its C-termmal mutants and cell lysates were subjected to heparanase activity assay ^e ND - not determined.

Example 3

The C-domain is critical for heparanase secretion

The involvement of the C-terminal domain of heparanase in mediating its enzymatic activity encouraged the inventors to further investigate the potential involvement of this domain in mediating the secretion of this enzyme. The inventors next examined the expression, secretion, and cellular localization of the heparanase variants. As shown by Figure 3A, Immunoblot analysis applying anti-Myc-tag antibody revealed that the heparanase variants were all readily detected, exhibiting the expected molecular weight (Fig. 3A, left). The relatively low signal observed in the lysates of cells transfected with wild type heparanase (Fig. 3A, left, Hepa) is due to efficient secretion of the latent 65 kDa protein (Fig. 3A, right) and accumulation of the processed, 50 kDa protein subunit in the cell lysates (not shown). It should be noted that the Myc-tag is cleaved off the 65 kDa latent heparanase short after its internalization and is thus absent from the processed 50 kDa subunit [Gingis-Velitski, S. et al. J. Biol. Chem. 279:44084-92 (2004)]. In striking contrast, the TIM-barrel protein variant appeared unprocessed (not shown) and was undetected in the cell conditioned medium even in the presence of heparin which enhances the accumulation of heparanase extracellularly (Fig. 3A, right, TIMB +) [Gingis-Velitski (2004) ibid.]. Likewise, deletion of 17 amino acid residues (as denoted by SEQ ID NO. 10) from the heparanase C- terminus markedly attenuated protein secretion (Fig. 3A, right, Δ17, the deletion mutant also denoted by SEQ ID NO. 9)/ Moreover, also point mutations (Phe⁵³¹, VaI⁵³³, He⁵³⁴, Ala⁵³⁷, Cys⁵⁴²) at this region that yielded proteins devoid of enzymatic activity (Fig. 2C) also failed to get secreted (Fig. 3B right, and data not shown). In contrast, the C-domain protein variant was noted to be readily secreted (Fig. 3A, right). These findings imply that intact C-domain is critical for the establishment of active heparanase enzyme (Fig. 2) and for heparanase secretion, possibly due to interaction of the C-domain with as yet unknown protein(s) such as a molecular chaperon.

To test this hypothesis, heparanase- and C-domain-transfected HEK293 cells were incubated with the Hsp90 inhibitor 17-AAG [Sharp, S. and Workman P. Advances in Cancer Research 95:323-48 (2006)], lysates (Fig. 3C, left) and medium (Fig. 3C, right) samples were subjected to immunoblotting applying anti-Myc-tag antibody. Treatment with 17-AAG did not affect the protein levels of wild type heparanase or the C-domain variant found in cell lysates (Fig. 3C, left, +). Interestingly, however, 17-AAG treatment significantly attenuated the secretion of heparanase and even more so of the C-domain (Fig. 3C, right, +), suggesting that active Hsp90 is required for efficient secretion of heparanase. Notably, decreased heparanase and C-domain proteins secretion was associated with a comparable decrease in phospho-Akt levels (Fig. 3C, left, second panel), a protein kinase shown to be regulated by Hsp90 [Solit, D.B. et al. Cancer Res. 63:2139-44 (2003)].

Next, the inventors examined the cellular localization of the heparanase variants by confocal microscopy (Fig. 3D). Control (mock; upper panels), heparanase (Hepa; second panels), TIM-barrel (TIMB; third panels), C-domain (fourth panel), Δ17 (fifth panels), and heparanase mutated at alanine⁵³⁷ (A537K; lower panels) transfected HEK 293 cells were first stained with anti heparanase monoclonal antibody (left most panels, red). Heparanase transfected cells exhibited the typical vesicular, peri-nuclear staining, in agreement with previous reports documenting processing and accumulation of active heparanase in lysosomes [Zetser A. et al. J. Cell. Sci.ll7:2249-58 (2004); Goldshmidt O. et al. Exp. Cell. Res. 281:50-62 (2002)]. In contrast, all other heparanase variants exhibited a more diffused staining that resembled ER localization. TIM-barrel transfected cells did not stained with this antibody, indicating that its epitope is confined within the C-domain. In order to further determine the sub-cellular localization of the heparanase species, transfected cells were triple stained applying anti-Myc-tag (second left, red), anti-calnexin (an ER marker, third left, green), and TO-PRO, which labels the cell nucleus (merge, blue, fourth left). As expected, all heparanase protein variants were noted to be sorted to the ER, co-localizing with calnexin (fourth left, yellow). In striking contrast, only the heparanase and C-domain proteins, shown to be secreted (Fig. 3A, right), were noted to co-localize with the Golgi marker, wheat germ agglutinin (right most second and fourth panels; yellow). These findings thus indicate that the C-domain and more specifically its C-terminus are critically important for the shuttling of heparanase from the ER to the Golgi apparatus and subsequent secretion.

Example 4

Heparanase C-domain induces Akt phosphorylation

The inventors have previously reported that heparanase induces Akt phosphorylation independently of its enzymatic activity [Gingis-Velitski, S. et al. J. Biol. Chem. 279:23536-41 (2004); Ben-Zaken, O. et al. Biochem. Biophys. Res. Commun. 361:829-34 (2007)]. Protein domains that mediate Akt activation by heparanase have not been so far characterized. The inventors speculated that the C-domain, seemingly comprising a protein entity which is not an integral part of the TIM-barrel fold, and its apparent unstructured flexible loop (Fig. IA, arrow), mediates the activation of Akt. To examine this possibility, HEK 293 cells were stably transfected with control empty vector (mock), wild type heparanase (Hepa), TIM-barrel (TIMB), or C-domain gene constructs. Lysates (Fig. 4A, left panel) and medium (Fig. 4A, right panel) samples were subjected to immunoblotting applying anti-Myc (Fig. 4A), anti- phospho-Akt (p-Akt; Fig. 4B, upper panel) and anti-Akt (Fig. 4B, lower panel) antibodies. As demonstrated in Figure 4B, Akt phosphorylation was stimulated by cells over expressing wild type heparanase (Hepa) 2±0.3 folds as quantified by densitometry (Fig. 4B, lower panel), in agreement with our previous findings. Notably, as demonstrated in the lower panel of Figure 4B, Akt phosphorylation was markedly (over 2.7 folds) stimulated by cells over- expressing the C-domain, as determined by densitometry analysis, while the TIM-barrel protein variant yielded no Akt activation compared with control, mock transfected cells (Fig. 4B), suggesting that signaling functions of heparanase are mediated by its C-domain. To further substantiate these findings, conditioned medium was collected from stably transfected cells (Fig. 4A, right panel) and was applied onto control HEK 293 cells for 30 min. Lysate samples were then subjected to immunoblotting with anti-phospho-Akt (p-Akt; Fig. 4C, upper panel) and anti-Akt (Fig. 4C, lower panel) antibodies. Indeed, as shown by Figure 4C, medium conditioned by C-domain expressing cells yielded a marked, 4±1.5 fold induction of Akt phosphorylation while no phosphorylation was shown using medium conditioned by TIM-barrel expressing cells.

To examine the possible involvement of HS (Heparan sulphate) in Akt activation by the C-domain, HS deficient CHO-745 cells were left untreated or incubated (30 min) with recombinant purified heparanase or C-domain proteins (1 μg/ml). Lysate samples were then subjected to immunoblotting with anti-phospho-Akt (p-Akt; Fig. 4D, upper panel) and anti-Akt (Fig. 4D, lower panel) antibodies. As was noted following the application of conditioned medium, exogenous addition of purified heparanase and C-domain proteins stimulated Akt activation to comparable levels (Fig. 4D). Furthermore, Akt activation in HS-deficient CHO-745 cells following heparanase and C-domain addition appeared comparable in magnitude to Akt activation in HEK 293 (Fig. 4C) and HeLa cells (data not shown), suggesting that activation of Akt is HS-independent, as previously noted for heparanase [Gingis-Velitski, S. et al. J. Biol. Chem. 279:23536-41 (2004)]. These findings clearly indicate that non- enzymatic, signaling function of heparanase leading to activation of Akt is mediated by the C-domain.

Example 5

Heparanase C-domain stimulates cell proliferation and facilitates tumor xenograft development

Having demonstrated a signaling function of the C-domain, the inventors next examined the cellular consequences of such induction. As shown by Figure 5A, HEK 293 cells that were stably transfected with the different constructs of the invention (disclosed by Table 2), were incubated with BrdU and its incorporation was evaluated by immunocytochemistry as an indication of cellular proliferation (Fig. 5A). Heparanase (Hepa) and C-domain expressing cells were noted to incorporate twice as much BrdU compared with control (mock transfected) cells (P<0.005; Fig. 5A), suggesting that induced cell proliferation by heparanase is mediated by its C-domain. In order to further substantiate this finding, the inventors examined the progression of tumor xenografts produced by mock-, heparanase-, TIM-barrel-, and C-domain- transfected U87 glioma cells. As shown by Figure 5B, tumor xenografts produced by heparanase-expressing cells (hepa), assumed a higher growth rate and generated increasingly bigger tumors compared with tumor xenografts produced by control, mock-transfected cells (Fig. 5B), in agreement with previous studies utilizing this model system [Zetser A et al. Cancer Res. 63:7733-41 (2003)]. At the end of the experiment on day 51, xenograft produced by heparanase-transfected cells were 6.5-fold bigger in volume (p<0.017; Fig. 5B) and 5.5-fold higher in weight (p<0.02; Fig. 5C), compared with control cells (mock). Notably, tumor xenografts produced by C-domain- transfected cells appeared comparable or even slightly bigger than those produced by heparanase-transfected cells, yielding tumors that were 8-fold bigger in volume (p<0.0007; Fig. 5B) and 6.5-fold higher in weight (pO.OOl; Fig. 5C) compared with controls. As expected, the progression of tumors produced by TIM-barrel-transfected cells appeared comparable with controls, in agreement with the lack of enzymatic activity, secretion, and signaling capabilities of this protein variant shown herein before by Figures 2 to 4.

In order to correlate the in vivo finding with the stimulation of Akt phosphorylation noted in vitro, five micron histological sections were subjected to immuno-staining applying anti-phospho-Akt antibodies (Fig. 5D, left panels). Phosphorylated Akt was highly abundant in xenografts produced by heparanase- and C-domain- transfected cells (Fig. 5D, left, second and third panels), compared with xenograft produced by mock- and TIM-barrel- transfected cells (Fig. 5D, left, upper and fourth panels).

In addition, tumor angiogenesis was markedly stimulated in xenografts produced by heparanase and C-domain-expressing U87 cells compared with mock and TIM-barrel-transfected cells, as demonstrated by the CD31 staining shown in Figure 5D (right panels) and the histograms of Figure 5E.

These results imply that the pro-tumorigenic and pro- angiogenic capacity of heparanase is mediated, at least in part, by the C-domain, clearly supporting a clinical relevance of heparanase enzymatic-independent functions.

Example 6

The heparanase C-domain interacts with cell surface proteins

The secreted nature of the C-domain and its capacity to elicit signaling cascades and to accelerate tumor growth suggest its possible interaction with cell membrane protein(s)/receptor(s). In order to explore this possibility, purified recombinant C-domain was iodinated to a high specific activity and binding experiments were performed. As shown by Figure 6A, binding of ¹²⁵I- C-domain to HeLa cells exhibited linearity at low concentrations, saturating at high levels of ¹²⁵I-C-domain. Analyses of C-domain binding by the Prism 4 software suggested the existence of high affinity (UTd= 7.2 nM), low abundant

binding sites. Such affinity is similar to that observed for wild type heparanase (Kd = 2-4 nM) [Ben-Zaken, O. et al. Int. J. Biochem. Cell. Biol. 40:530-42 (2008)]. Unlike heparanase, however, the C-domain does not seem to associate with low affinity, high abundant binding sites such as HSPG. Indeed, as shown by Figure 6B, binding of ¹²⁵I-C-domain to HS-deficient CHO- 745 cells exhibited a comparable pattern in term of affinity (Kd =12.8 nM) and binding sites abundance (B_max- 166.3), indicating HS-independent association with cell membrane protein(s).

The inventors next assessed the existence of C-domain binding protein(s)/receptor(s) by cross-linking experiments. Cells were incubated with ¹²⁵I-C-domain in the absence or presence of a large excess of unlabeled recombinant heparanase or C-domain proteins. Cross-linking of the C-domain followed by SDS/PAGE revealed the existence of two major cell surface protein(s)/receptor(s) complexes, exhibiting molecular weights of about 130 and about 170 kDa that interact with the heparanase C-domain, as shown by Figure 6C (indicated by arrows). The molecular weight of cell surface proteins shown previously to bind heparanase, i.e., LRPl (85 kDa) [Vreys, V. et al. J. Biol. Chem. 280:33141-8 (2005); Ben-Zaken, O. et al. Int. J. Biochem. Cell. Biol. 40:530-42 (2008)]; cation-dependent MPR (46 kDa) and cation- independent MPR (300 kDa) [Wood, R.J. and Hulett M.D. J. Biol. Chem. 283:4165-76 (2008)] is not in accordance with the molecular weights of the protein complexes found by cross-linking. These findings suggest the existence of novel heparanase/C-domain binding proteins/receptors that bind the heparanase/C-domain with high affinity and likely mediate Akt activation.

Example 7

Heparanase modulators directed against the C-domain

As demonstrated above, the C-domain of heparanase is sufficient to elicit signaling cascades (i.e., Akt), leading to tumor progression. This domain was also implicated as mediating the secretion of heparanase and stabilizing conformation which is crucial for its enzymatic activity. Since these functions are important for tumor progression, the inventors next used the C-domain as a target for developing heparanase inhibitors. For an efficient development of a screening program of small molecules, a crystal structure of the C-domain target is highly beneficial. Therefore, the C-domain is expressed to high levels in bacteria as GST-fusion protein, followed by the removal of the GST tag by thrombin. Screening method utilizing this C-domain as a target is performed for identifying substances which bind the C-domain and thereby modulate heparanase biological activity.

It should be noted that such potential C-domain specific modulators may be for example, antibodies. Polyclonal antibodies are raised in rabbit against the C-domain purified from the conditioned medium of 293 cells infected cells. Monoclonal antibodies are raised as specifically described in Experimental procedures.

Different substances identified using the C-domain as a target, are subsequently evaluated for their potential modulating effect on different biological activities of heparanase (for example, in case of antibodies are identified, IgG fraction or affinity-purified anti- C- domain antibodies are evaluated for their modulatory effect), using different approaches.

For example, ability of the candidate modulating substance to inhibit tumor progression induced by heparanase is evaluated. In order to evaluate the efficiency of all heparanase inhibitors directed against the C-domain as tumor inhibitors, viral (lentivirus, adenovirus) and non viral-based (pSecTag, pcDNA3) gene construct are utilized to infect or transfect tumor-derived cell lines with heparanase variants. Breast carcinoma cells (MDA-MB-231, MDA- MB-435) are inoculated into the mammary fat pad as an orthotopic model, whereas ARF 77 multiple myloma cells are inoculated sub-cutanously. Cell over expressing the wild type, inactive (double mutant) and C-domain heparanase variants are inoculated and tumor xenograft development is followed. Such xenograft model in which the C-domain enhances tumor progression, is further utilized for inhibition studies by the candidate modulating substances, for example, polyclonal and monoclonal anti- C-domain antibodies as single drugs, or in combination with inhibitors of heparanase enzymatic activity (i.e., chemically modified species of heparin).

Another model used for evaluating the modulating effect of the C-domain targeted molecules utilizes the U87 human glioma cells. These cells were previously shown by the inventors as leading to development of tumors twice as big compared with tumor xenografts produce by control cells. Thus, U87 bearing mice, are treated for example with anti-C-domain antibodies or any other candidate modulator (to inhibit heparanase signaling), glycol-split heparin (to inhibit heparanase enzymatic activity), or both.

Different candidate modulating substances, such as anti-C-domain antibodies are further examined in inflammation models established recently by the inventors and demonstrated to be inhibited by anti-heparanase siRNA oligonucleotides. These include delayed-type hypersensitivity (DTH) and inflammatory bowel disease (IBD) mouse models.

The different potential modulators are also evaluated for their modulatory effect on angiogenesis. Endothelial cell organization on Matrigel into tube-like structures is utilized as a model for angiogenesis. Similarly, infected tumor, and endothelial cells are embedded in Matrigel and plug angiogenesis are evaluated by histology and immunohistochemistry.

The different modulators are further evaluated for their ability to inhibit Akt activation by the C-domain in vitro.

Example 8

Monoclonal antibody 6F8 directed against the C-domain, enhances heparanase enzymatic activity

As indicated above, the involvement of C-domain in mediating its secretion, signaling and stability, as reflected by different biological activities of the protein, may be applicable in using this C-domain as a target for screening of heparanase modulators. Such potential different modulators, specifically bind to this domain and thereby increase or inhibit heparanase biological activity. Therefore, possible modulating molecules which specifically bind the C-domain may be as indicated herein above, antibodies specific for the C-domain. As described in Experimental procedures, monoclonal antibodies were prepared, isolated, purified, and tested for their properties as heparanase modulators. First, the inventors examined the ability of several monoclonal antibodies to inhibit the enzymatic activity of recombinant heparanase. These studies were carried out at natural pH (~7.2), optimal for the interaction between antibodies and their antigens. Although performs best under acidic pH conditions [Dempsey, L. et al. Trends Biochem. Sci. 25:349-351 (2000)], heparanase activity was readily detected under these experimental settings (Figure 7). Nevertheless, none of the antibodies tested (i.e., IEl and 44C4) inhibited heparanase activity (Figure 7A, 7B). On the contrary, one monoclonal antibody, 6F8 (IgGi), exhibited the opposite trend and significantly enhanced heparanase activity, while control mouse IgG had no stimulatory or inhibitory effect (Figure 7C). The ability of antibody 6F8 to enhance the activity of cellular heparanase was next confirmed and validated. To this end, conditioned medium was collected from heparanase transfected HEK 293 (Figure 8A) and MDA-231 (Figure 8C) cells and was applied onto ³⁵S-labeled ECM-coated dishes in the presence of antibody 6F8 or control mouse IgG (lμg/ml) and heparanase activity was evaluated (Figure 8). Similarly, heparanase activity was examined in the corresponding cell lysates (Figure 8B, 8D). A consistent 2-3 fold increase was observed in heparanase activity by antibody 6F8 (Figure 8), suggesting that this antibody can be utilized to enhance heparanase activity in cellular models. Similar results were obtained with U87 glioma and HT-29 colon carcinoma cells (data not shown).

Example 9

Antibody 6F8 facilitates cellular invasion

Heparanase activity is well correlated with the metastatic potential of tumor- derived cells, a feature best recapitulated in vitro by cellular invasion through a reconstituted-basement membrane matrix (Matrigel). The inventors therefore, examined invasive capacity of heparanase transfected MDA-231 (Figure 9, left) and U87 cells (Figure 9, right) in the presence of antibody 6F8 or control mouse IgG, utilizing Matrigel-coated transwell inserts. Matrigel invasion by 231-Hepa and U87 cells was increased two-fold in the presence of antibody 6F8 (Figure 9), consistent with the observed enhancement of heparanase activity (Figure 9). Unlike the migration of individual tumor cells through eight micron pore transwell filters, epithelial cells exhibit characteristic sheet migration on a solid support. The inventors have reported previously that exogenous addition of heparanase stimulates sheet migration of human HACAT keratinocytes in a wound scratch assay [Zcharia (2005) ibid.]. Similarly, as shown by Figure 10, sheet migration of HACAT cells and wound closure in vitro was significantly enhanced by antibody 6F8 (Figure 10, middle) compared with control mouse IgG (Figure 10, left panel) and anti- heparanase IEl monoclonal antibody (Figure 10, right panel) that does not affect heparanase activity (Figure 7). These results imply that antibody 6F8 is capable of enhancing the activity of endogenous cellular heparanase and may thus be utilized in υiυo as modulator of heparanase activity.

Example 10

Antibody 6F8 improves wound healing in a mouse punch model

Relatively high levels of heparanase were detected in the skin tissue [Bernard, D. et al. J. Invest. Dermatol 117:1266-1273 (2001)] and further elevation in heparanase expression was observed in the leading edge of migrating keratinocytes and in the wound granulation tissue [Zcharia (2005) ibid.]. Moreover, elevated levels of bFGF were found in the wound fluid of heparanase transgenic mice compared with control mice, likely due to enhanced heparanase activity and the associated release of HS-bound polypeptides [Zcharia (2005) ibid.]. The inventors therefore, examined the ability of antibody 6F8 to enhance wound healing in a mouse punch model. Histological examination of 8 mm punch wounds revealed a significant improvement of wound healing in mice that were injected with antibody 6F8 (10 mg/kg) compared with mice injected with control mouse IgG (Figure HA). Thus, in two separate experiments, 37.5 and 60% of wounds treated with antibody 6F8 appeared completely closed, while only 0 and 37.5% of control wounds were healed, respectively (Figure HA, 6F8; Table 4). Moreover, significant improvement of wound closure was measured in wounds that were not completely healed. Wound diameter (i.e., granulation tissue lacking epidermal keratinocytes) of control wounds was 1104+189 and 1355+139 μM compared with 493±169 and 381+139 μM of wounds treated with antibody 6F8 (Fig. HB; Table 4), differences that are statistically highly significant (p=0.02). In addition, granulation tissue of wounds treated with antibody 6F8 appeared thicker and denser compared with control wounds (Figure HA), in agreement with the improved wound healing. In fact, wounds treated with antibody 6F8 appeared to undergo remodeling and maturation, replacing the wound granulation tissue with dense collagen matrix. This is best demonstrated by Masson/Trichome staining, clearly revealing enhanced collagen deposition in 6F8 (Figure HC, 6F8) vs. IgG (Figure HC, Con.) treated wounds.

Table 4. Wound diameter and closure upon antibody 6F8 or mouse IgG treatment

These Examples illustrates the feasibility of using heparanase C-terminal domain as a target for substances such as antibodies that modulate heparanase catalytic and non-catalytic activities. These C-domain targeted substances may therefore used as modulators and thereby as therapeutic agents for the treatment of heparanase associated conditions.

The following Table 5 summarizes all the sequences use by the present invention as presented by the following sequence listing.

Table 5 Sequences and SEQ ID NOs.

Claims

Claims:

1. An isolated and purified amino acid sequence derived from the C- terminal domain of heparanase, wherein said sequence comprises amino acid residues 413 to 543 of human heparanase or any fragment, peptide, mutant, derivative and variant thereof, and wherein said sequence modulates heparanase biological activity.

2. The amino acid sequence according to claim 1, wherein said sequence comprises the amino acid sequence of SEQ ID NO: 3, or any fragment, peptide, mutant, derivative and variant thereof.

3. The amino acid sequence according to claim 2, wherein a fragment of said sequence comprises residues 481 to 519 of human heparanase, as denoted by SEQ ID NO. 13, and is designated as the loop sequence.

4. The amino acid sequence according to claim 2, wherein a fragment of said sequence comprises residues 527 to 543 of human heparanase, as denoted by SEQ ID NO. 10.

5. The amino acid sequence according to claim 2, wherein a mutant of said sequence may comprise at least one point mutation in any one of residues Phe⁵³¹, Vaisas,

AIa⁵S⁷, _and Cys**².

6. An expression vector comprising a nucleic acid sequence encoding the amino acid sequence as defined by claim 1, which vector optionally further comprises operably linked regulatory elements.

7. A composition comprising as active ingredient an isolated and purified amino acid sequence derived from the C-terminal domain of heparanase, any nucleic acid expression vector encoding said sequence, or cells transfected or transformed by said construct, wherein said sequence comprises amino acid residues 413 to 543 of human heparanase or any fragment, peptide, mutant, derivative and variant thereof, said composition optionally further comprising a pharmaceutically acceptable carrier, diluent, excipient and/or additive.

8. The composition according to claim 7, for the modulation of heparanase biological activity in a subject in need thereof.

9. A modulator of heparanase biological activity, wherein said modulator is any one of: (i) an amino acid sequence derived from the C-terminal domain of heparanase; and (ii) a substance which specifically binds to an amino acid sequence within the C-terminal domain of heparanase.

10. The modulator according to claim 9, wherein said C-terminal domain of heparanase or any fragment, peptide, mutant, derivative and variant thereof is as defined in any one of claims 1 to 5.

11. The modulator according to claim 10, wherein said modulator is an isolated amino acid sequence derived from a C-terminal domain of heparanase.

12. The modulator according to claim 10, wherein said modulator is a substance which specifically binds to an amino acid sequence within the C- terminal domain of heparanase.

13. The modulator according to claim 12, wherein said substance is an antibody which specifically recognizes an amino acid sequence derived from the C-terminal domain of heparanase.

14. The modulator according to claim 13, wherein said antibody is a monoclonal antibody designated #6F8, wherein said antibody enhances heparanase catalytic activity.

15. A composition for the modulation of heparanase biological activity, comprising as active ingredient a modulator of heparanase biological activity, wherein said modulator being any one of (i) an amino acid sequence derived from the C-terminal domain of heparanase, and (ii) a substance which specifically binds to an amino acid sequence within the C-terminal domain of heparanase, said composition optionally further comprising a pharmaceutically acceptable carrier, diluent, excipient and/or additive.

16. A pharmaceutical composition for the treatment of a pathologic disorder associated with heparanase biological activity comprising as active ingredient a modulator of heparanase biological activity, in an amount sufficient for the modulation of heparanase biologic activity, wherein said modulator being any one of (i) an amino acid sequence derived from the C-terminal domain of heparanase, and (ii) a substance which specifically binds to an amino acid sequence within the C-terminal domain of heparanase as defined by claim 9, said composition optionally further comprising a pharmaceutically acceptable carrier, diluent, excipient and/or additive.

17. A method for the modulation of heparanase biological activity comprising the step of in vivo, ex vivo or in vitro contacting heparanase under suitable conditions, with a modulatory effective amount of a modulator or with a composition comprising the same, wherein said modulator being any one of: (i) an isolated and purified amino acid sequence derived from the C-terminal domain of heparanase; or (ii) a substance which specifically binds to an amino acid sequence derived from the C-terminal domain of heparanase and is capable of modulating heparanase biological activity.

18. A method for the modulation of heparanase biological activity in a subject in need thereof comprising the step of administering to said subject a modulatory effective amount of a modulator or of a composition comprising the same, wherein said modulator being any one of (i) an isolated and purified amino acid sequence derived from the C-terminal domain of heparanase, or a composition comprising the same or (ii) a substance which specifically binds to an amino acid sequence derived from the C-terminal domain of heparanase and is capable of modulating heparanase biological activity.

19. A method for the treatment of a pathologic disorder associated with heparanase biological activity comprising the step of administering to a subject in need thereof a therapeutically effective amount of a modulator or of a composition comprising the same, wherein said modulator being any one of (i) an isolated and purified amino acid sequence derived from the C-terminal domain of heparanase or (ii) a substance which specifically binds to an amino acid sequence derived from C-terminal domain of heparanase and is capable of modulating heparanase biological activity.

20. The method according to claim 19, wherein said modulator is the modulator according to claim 9 and wherein said disorder is any one of cancer and inflammatory disorder.

21. Use of a modulator according to claim 9, for the modulation of heparanase biologic catalytic activity.

22. A method of screening for heparanase modulator which specifically binds to an amino acid sequence derived from the C- terminal domain of heparanase and is capable of modulating heparanase biologic activity, which screening method comprises the steps of: a. obtaining a candidate substance; b. selecting from the substances obtained in step (a) a substance which bind to the C-terminal domain of heparanase, or any fragment thereof; and c. evaluating the candidate substance selected in step (b) by determining the modulatory effect of said substance on the biological activity of heparanase.