More particularly, the invention relates to the use of the adhesion promoting non-catalytic heparanase molecules for enhancing cell-to-cell and cell-to- matrix adhesion in a tissue sealant composition and in implantable medical devices. The invention further provides nucleic acid constructs encoding the non-catalytic heparanase molecules of the invention and methods of treatment of adhesion-related pathologies.

Background of the Invention Throughout this application various publications are referenced to. It should be appreciated that the disclosure of these publications in their entireties are hereby incorporated into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

Cell adhesion to the extracellular matrix (ECM) is a tightly regulated process that plays a key role in the control of cell migration, proliferation and differentiation, associated with diverse physiological and pathological processes [Yamada, K. M. and Geiger, B. Curr. Opin. Cell Biol. 9: 76-85 (1997); Aplin, A. E. et al., Curr. Opin. Cell Biol. 11: 737-744 (1999); Eliceiri, B.

P. , and Cheresh, D. A. Curr. Opin. Cell Biol. 13: 563-568 (2001) ]. The predominant integrin receptor family mediates cell adhesion and provides a physical link between the ECM and cytoskeletal elements [Burridge, K. et al. Annu. Rev. Cell Biol. 4: 487-525 (1988); Geiger, B. et al. Nat. Rev. Mol. Cell Biol. 2: 793-805 (2001); Miranti, C. K. , and Brugge, J. S. Nat. Cell Biol. 4: E83-90 (2002)]. Integrins also initiate and regulate a variety of signaling responses, including the activation of mitogen activated protein kinase (MAPKs) and tyrosine phosphorylation of cytoplasmic molecules (e. g., paxillin) [Giancotti, F. G. , and Ruoslahti, E. Science 285: 1028-1032 (1999) ; Assoian, R. K., and Schwartz, M. A. Curr. Opin. Genet. Dev. 11: 48-53 (2001); Turner, C. E. Nat. Cell Biol. 2: E231-236 (2000) ].

Nonetheless, cell adhesion is a multi-step process that involves membrane constituents other than integrins [Zimmerman, E., Geiger, B. , and Addadi, L. hyaluronan. Biophys. J. 82: 1848-1857 (2002) ]. For example, leukocytes adhesion to the endothelium involves a rapid, low affinity, non-integrin mediated adhesion, followed by a prolonged integrin-mediated, high affinity adhesive interactions [Springer, T. A. Cell 76 : 301-314 (1994); Dwir, O., Kansas, G. S., and Alon, R. J. Cell Biol. 155: 145-156 (2001) ]. Non-integrin mechanisms may play a key role in the formation of early cell-ECM adhesions occurring prior to the formation of specific integrin mediated interactions of cells with the ECM. It was shown, for example, that hyaluronan might support the formation of such early adhesive cell-ECM interactions in adherent cells [Zimmerman (2002) ibid.]. Other cell surface, non-integrin molecules such as heparan sulfate proteoglycans (HSPGs) may also be involved in the regulation of early stages of cell-ECM interactions [Bernfield, M. et al. Annu. Rev. Biochem. 68: 729-777 (1999); Iozzo, R. V. J.

Clin. Invest. 108: 165-167 (2001) Wight, T. N., Kinsella, M. G. , and Qwarnstrom, E. E. Curr. Opin. Cell Biol. 4,793-801 (1992) ; Esko, J. D. , and Selleck, S. B. Annu. Rev. Biochem. 71: 435-471 (2002)].

Heparan sulfate proteoglycans play a major role in diverse cellular processes, and are regarded as key molecules in the self-assembly, insolubility and barrier properties of basement membranes (BM) and the ECM [Bernfield (1999) ibid ; Iozzo (2001) ibid ; Wight (1992) ibid ; Esko (2002) ibid ; David, G. and Bernfield, M. Matrix Biol. 17: 461-463 (1998) ]. The mammalian endoglycosidase (or heparanase), capable of partially depolymerizing heparan sulfate (HS) chains, has been identified in highly invasive normal and malignant cells, including cytotrophoblasts, activated cells of the immune system, as well as in lymphoma, melanoma and carcinoma cells [Dempsey, L.

A. Trends Biochem. Sci. 25: 349-351 (2000) ; Vlodavsky, I. et al. Invasion Metastasis 12: 112-127 (1992) ; Parish, C. R. et al. Biochim. Biophys. Acta 1471 : M99-108 (2001); Vlodavsky, I., and Friedmann, Y. J. Clin. Invest. 108 : 341-347 (2001); Nakajima, M. , Irimura, T. , and Nicolson, G. L. J. Cell Biochem. 36: 157-167 (1988)]. The expression of heparanase has long been correlated with the metastatic potential of tumor cells, and treatment with heparanase inhibitors markedly reduced the incidence of experimental metastasis and autoimmunity [Parish (2001) ibid ; Viodavsky (2001) ibid ; Nakajima (1988) ibid ; Vlodavsky, I. et al. Invasion Metastasis 14: 290-302 (1994); Parish, C. et al. Cancer Res. 59,3433-3441 (1999) ]. In addition, there is a significant correlation between enhanced heparanase mRNA expression and shorter postoperative survival of cancer patients [Koliopanos, A. et al.

Cancer Res. 61,4655-4659 (2001) ; Cohji, K. et al. Int. J. Cancer 95,295-301 (.-) 001)].

Apart from its involvement in the egress of cells from the vasculature, heparanase is tightly involved in normal and pathological angiogenesis, primarily by means of releasing heparin-binding angiogenic factors sequestered by heparan sulfate (HS) in BM and ECM [Vlodavsky (2001) ibid ; Elkin, M. et al. Faseb J. 15: 1661-1663 (2001); Folkman, J. et al. Am. J. Pathol. 130: 393-400 (1988); Goldshmidt, O. et al. Proc. Natl. Acad. Sci. USA 99: 10031-10036 (2002)].

The human heparanase cDNA encodes a latent enzyme of 543 amino acids, which is then cleaved at the N-terminus, yielding the mature highly active 50 kDa enzyme [Parish (2001) ibid ; Fairbanks, M. et al. J. Biol. Chem. 274: 29587-29590 (1999) ]. Only a single heparanase cDNA sequence encoding a functional enzyme has been identified so far [Vlodavsky, I. et al. Nat. Med. 5: 793-802 (1999 (a) ) ; Hulett, M. D. Nat. Med. 5: 803-809 (1999); Kussie, P. et al. Biochem. Biophys. Res. Commun. 261: 183-187 (1999); Toyoshima, M. , and Nakajima, M. J. Biol. Chem. 274 : 24153-24160 (1999) ], indicating that this enzyme is the dominant HS degrading endoglycosidase in mammalian tissues. The direct role of heparanase in all the above-mentioned processes has been further emphasized by demonstrating that a surface-associated and secreted form of the enzyme (chicken-heparanase, or chimeric heparanase composed of the chicken heparanase signal sequence fused to the human enzyme) [Goldshmidt, O. et al. J. Biol. Chem. 276 : 29178-29187 (2001)] markedly promotes tumor angiogenesis and metastasis [Goldshmidt (2002) ibid].

In a previous study by the present inventors, it was hypothesized that depending on the local pH, heparanase may function as an ECM degrading enzyme (pH < 6.8), or as a T-cell adhesion molecule (pH > 7.0) [Gilat, D. et al. J. Exp. Med. 181 : 1929-1934 (1995) ]. The inventors'recent identification of a cell surface-associated form of heparanase [Goldshmidt (2002) ibid] led to the investigation and characterization of the enzyme's involvement in cell adhesion. For this purpose, non-adhesive Eb mouse lymphoma cells were stably transfected with the human (H-hpa, localized primarily in peri-nuclear granules), the chicken (Chk-hpa) or the chimeric (chim-hpa) heparanase cDNAs, expressed predominantly on the cell surface [Goldshmidt (2002) ibid].

The transfected cells were then compared for their adhesiveness to a naturally produced subendothelial ECM and to a confluent vascular endothelial cell monolayer. The results of the present study indicate that surface-associated heparanase promotes adhesion of otherwise non-adherent lymphoma cells. Heparanase-stimulated cell adhesion was accompanied by tyrosine phosphorylation and re-organization of paxillin in ECM adhesions. Cell adhesion was augmented by cell surface heparanase regardless of whether the cells were transfected with active or point mutated inactive enzyme, indicating a novel adhesion feature of the heparanase molecule.

The adhesive effect of heparanase was further examined in endothelial cells (EC) and in glioma cells. Exogenously added heparanase rapidly interact with EC, followed by internalization and processing into the 50 kDa active form. Interestingly, heparanase uptake by EC was accompanied by Akt phosphorylation that precedes heparanase processing and exhibited time and dose dependency. Heparanase-mediated Akt phosphorylation was independent of its enzymatic activity or the presence of membranous HS proteoglycans, and was augmented by heparin. Moreover, heparanase addition stimulated PI'3 kinase-dependent endothelial cell migration and invasion. Activation of the PI3'I£/Akt signaling pathway was also observed in glioma cells (U87) stably transfected with heparanase Activation of the PI3'K/Akt pathway may contribute to adhesion exhibited by heparanase. This activation may therefore support the use of non-catalytic heparanase as an adhesion molecule for wound-healing and in promotion of cell survival and resistance to apoptosis. It should be noted that this effect is likely mediated by an as yet unidentified heparanase receptor.

Because of the critical role played by cell adhesion processes, a need exists for the ability to manipulate such processes. The present invention provides compositions and methods for manipulating cell-to-cell and cell-to-matrix adhesion mediated by non-catalytic heparanase.

It is therefore an object of the present invention to provide nucleic acid constructs encoding recombinant non-catalytic adhesion enhancing heparanase molecules.

A further object of the invention is to develop adhesive tissue sealant compositions using the adhesive heparanase molecule. This could than be applied to a skin surface injuries, for example, which would be useful in maintaining homeostasis and enhance wound healing. Yet another object of the invention is to develop an implantable medical device. Particularly a vascular graft, having improved resistance to blood flow by enhancing the attachment of endothelial cells to the luminal surface thereof, and increasing the survival of cells and their resistance to apoptosis.

These properties are achieved by using the adhesion promoting and Akt activating heparanase of the invention.

Further understanding of the nature and advantages of the invention will become apparent by reference to the remaining portion of the specification and drawings.

Summary of the Invention In a first aspect, the present invention relates to a nucleic acid construct comprising a polynucleotide sequence encoding heparanase-derived polypeptide having cell-to-cell and cell-to-matrix adhesive properties and being devoid of heparanase catalytic activity. The construct of the invention optionally further comprises operably linked regulatory elements.

In a specifically preferred embodiment, the nucleic acid construct of the invention may comprise as the heparanase-derived polypeptide devoid of heparanase catalytic activity, a human heparanase-derived polypeptide.

In a more specifically preferred embodiment, the heparanase-derived polypeptide comprised within the nucleic acid construct of the invention, may carry a point mutation replacing the active site proton donor Glu225 with Ala. According to this embodiment, such construct comprises a nucleic acid sequence substantially as denoted by SEQ ID No.: 1.

Alternatively, the heparanase-derived polypeptide may carry a point mutation replacing the active site nucleophile residue Glu343 with Ala. These constructs may comprise a nucleic acid sequence substantially as denoted by SEQ ID No.: 2. In yet a further embodiment, the heparanase-derived polypeptide comprised within the nucleic acid constructs of the invention, may carry both Glu22s and Glu343 to Ala mutations. Such double mutant constructs may comprise according to a specific embodiment, a nucleic acid sequence substantially as denoted by SEQ ID No.: 3. It should be appreciated that the amino acid locations Glu225 and Gluis refers to the amino acid sequence of the human heparanase as denoted by the GenBank Accession No: AF144325.

In yet another embodiment, the invention provides a nucleic acid construct encoding a secreted or membranal adhesion molecule having cell-to-cell and cell-to-matrix adhesive properties and being devoid of heparanase catalytic activity. According to this embodiment, the invention relates to a construct comprising a first polynucleotide sequence encoding a signal peptide of avian heparanase operably linked to a second polynucleotide sequence encoding a heparanase-derived polypeptide, optionally further comprising operably linked regulatory elements. The heparanase-derived polypeptides preferably carry a point mutation replacing either the active site proton donor Glu"25 with Ala, or a point mutation replacing the active site nucleophile residue Glu343 with Ala, or alternatively, it may carry both Glu2''5 and Glut's to Ala mutations. Such heparanase-derived polypeptides may be encoded by a nucleic acid sequence denoted by any one of SEQ ID No. 4,5, and 6.

The present invention further provides a host cell transformed or transfected with a nucleic acid construct of the invention.

In a second aspect, the invention relates to a recombinant protein comprising a heparanase-derived polypeptide. Such recombinant protein, according to the invention, has adhesion activity and is devoid of heparanase endoglycosidase catalytic activity. In a specific embodiment of said second aspect, the recombinant protein comprises the mutated human amino acid sequence substantially as denoted by any one of SEQ ID No. 7, 8 and 9, encoded by the nucleic acid sequence substantially as denoted by any one of SEQ ID No. 1,2 and 3.

In yet another specific embodiment, the invention provides a recombinant protein being a secreted or membranal molecule having adhesion activity and devoid of heparanase endoglycosidase catalytic activity. According to this embodiment, the recombinant protein of the invention comprises a signal peptide of avian heparanase and heparanase-derived polypeptide, comprising the amino acid sequence substantially as denoted by any one of SEQ ID No.

10,11 and 10-, encoded by the nucleic acid sequence substantially as denoted by SEQ ID No. 4,5 and 6, respectively.

In a further aspect, the present invention relates to a tissue sealant composition comprising as active ingredient an effective amount of adhesion promoting non-catalytic heparanase molecule or any functional fragment thereof, and optionally further comprising a compound capable of accelerating, enhancing, stimulating and/or mediating the healing of an injury optionally with a pharmaceutically acceptable carrier.

In a preferred embodiment, the adhesion promoting non-catalytic heparanase molecule comprised within the composition of the invention, may be any recombinant protein defined by the invention or any functional fragment thereof.

The present invention therefore provides for the use of such tissue sealant composition in the preparation of an agent for accelerating the healing of an injury, homeostasis of an injury to a skin surface or a viscus, endothelium formation of a blood vessel, adhesive activity of mammalian cells and/or adhesion and aggregation of platelets.

Still further, the invention provides a method of accelerating the healing of an injury, homeostasis of an injury to a skin surface or a viscus, endothelium formation of a blood vessel, adhesive activity of mammalian cells, and/or adhesion and aggregation of platelets. Such method comprises the step of administering to a subject in need a therapeutically effective amount of a tissue sealant composition of the invention.

Preferably, the method of the invention is particularly applicable for accelerating an external skin wound healing in a mammalian subject in need thereof. Accordingly, such method comprises the step of applying the tissue sealant composition of the invention to the wounded skin of said subject at the time of or subsequent to occurrence of said wound in a quantity sufficient to accelerate clinically detectable healing.

The invention further provides for a method for the treatment of a disorder associated with adhesion deficiency in a mammalian subject. According to the invention this method comprises the step of administering to said subject an effective amount of any one of an adhesion promoting non-catalytic heparanase recombinant molecule of the invention, a composition comprising the same, and cells expressing said adhesion molecules.

Still further, the invention provides the use of the tissue sealant composition in the preparation of an agent for promoting the endothelialization of vascular grafts.

In a fourth aspect, the invention relates to an implantable medical device comprising: (a) a synthetic tubular element having a luminal surface; (b) a cell layer directly or indirectly adhered to said luminal surface, wherein said cell layer comprises cells genetically modified to express a membranal adhesive non-catalytic heparanase molecule or any functional fragment thereof.

According to one specific embodiment, the implantable medical device of the invention may further comprise a layer of immobilized adhesion molecules interposed between said luminal surface and said cell layer. The additional adhesion molecule layer may comprise, for example, molecules selected from the group consisting of fibronectin, laminin, collagen and the non-catalytic heparanase of the invention, or any combination thereof, and is adapted for enabling indirect adhesion of said cell layer to said luminal surface.

According to a specifically preferred embodiment, the implantable medical device of the invention may be a vascular graft. The vascular graft according to the invention may comprise a tubular element composed of a biomaterial selected from the group consisting of non-soluble synthetic polymers, metals and ceramics. In a particularly preferred embodiment, such synthetic polymer may be GORE-TEX Polytetrafluoro-ethylene (PTFE).

In yet another embodiment, the genetically modified cells comprised within the vascular graft of the invention may be endothelial cells derived from a source selected from the group consisting of a segment of mammalian blood vessel, bone marrow progenitor cells, peripheral blood stem cells and circulating endothelial cells. Preferably, these endothelial cells are derived from a mammalian donor, more preferably, human and most preferably, said cells are derived from the recipient of said graft.

In a further embodiment, the cell layer comprised within the vascular graft of the invention may be a confluent monolayer of endothelial cells genetically modified to express a membranal adhesive non-catalytic heparanase molecule, or any functional fragment thereof. More particularly, these endothelial cells may be cells transformed or transfected with an expression vector comprising a nucleic acid construct encoding a membranal adhesive non-catalytic heparanase molecule according to the invention. Preferably, such genetically modified endothelial cells exhibit enhanced adherence properties and enhanced survival and resistance to apoptosis. The enhanced survival and resistance to apoptosis may be a result of activation of the PI3'K/Akt signaling pathway by said membranal adhesive non-catalytic heparanase molecule.

The present invention further provides a method for preparing an implantable medical device comprising a synthetic tubular element having a luminal surface and a cell layer directly or indirectly adhered to said luminal surface, wherein said cell layer comprises cells genetically modified to express a membranal adhesive non-catalytic heparanase molecule or any functional fragment thereof. This method comprises the steps of : (a) providing a suitable synthetic tubular element having a luminal surface; and (b) directly or indirectly adhering said cell layer onto said luminal surface.

In one specific embodiment, the method of the invention further comprises in step (b) the sub-steps of: (i) immobilizing a layer of adhesion molecules selected from the group consisting of fibronectin, laminin, collagen, the non- catalytic heparanase of the invention and any combination thereof onto said luminal surface; and (ii) adhering, or preferably, seeding a layer of cells genetically modified to express a membranal adhesive non-catalytic heparanase molecule, or any functional fragment thereof, onto said immobilized adhesion molecules layer in (i).

In a preferred embodiment, these cells overexpress an adhesive non-catalytic heparanase molecule having the amino acid sequence of any one of SEQ ID NO: 10,11 and 12, encoded by the nucleic acid sequence of any one of SEQ ID NO: 4,5, and G, respectively.

In a preferred embodiment, this method is particularly suitable for preparing an implantable medical device as defined by the invention.

In yet another aspect, the invention provides a method for screening for a substance that modulates cell-to-cell and cell-to-matrix adhesion between a cell expressing an adhesion promoting non-catalytic heparanase molecule and endothelial cells or matrix, the method comprising: (a) providing a cell expressing a recombinant adhesion promoting non-catalytic membranal heparanase molecule; (b) performing a first adhesion assay for testing the ability of said adhesive heparanase expressing cells to adhere to a naturally produced subendothelial ECM or to confluent vascular endothelial cell monolayer to obtain the first adhesion assay results; (c) performing a second adhesion assay for testing the ability of said adhesive heparanase expressing cells to adhere to a naturally produced subendothelial ECM or to confluent vascular endothelial cell monolayer, in the presence of a test substance, to obtain the second adhesion assay results; (d) comparing said first and second adhesion assay results to determine whether said test substance affects the adhesion between the adhesive heparanase expressing cells and subendothelial ECM or the confluent vascular endothelial cell monolayer ; and (e) identifying modulating substances as compounds that affect the binding between a cell expressing the membranal non-catalytic heparanase adhesive molecule and said matrix or endothelial cells.

According to a preferred embodiment of said aspect, the adhesive heparanase-expressing cells used by the screening method of the invention, are cells transformed or transfected with the nucleic acid constructs of the invention. Most preferably, these cells were anchorage-independent cells prior to transfection or transformation with the constructs of the invention.

The invention further provides the use of any one of heparanase-derived polypeptide having cell-to-cell and cell-to-matrix adhesive properties and being devoid of heparanase catalytic activity, nucleic acid construct encoding said polypeptide and host cells transfected with said construct, in the preparation of a composition for enhancing adhesion. According to a preferred embodiment, such composition may be a sealant composition, preferably, as defined by the invention.

In a specifically preferred embodiment, the invention relates to the use of the construct or the host cell of the invention in the preparation of a composition for enhancing adhesion. In yet another embodiment, the invention relates to the use of the recombinant protein of the invention in the preparation of a composition for enhancing adhesion.

The invention will be further described by the hand of the proceeding Figures.

Brief Description of the Figures Figure 1A-1F Cell surface heparanase increases cell adhesion to ECM and e1ldothelial cells Fig. 1A. Left panels: Morphology of Eb mouse lymphoma cells expressing surface associated (chimeric-hpa) vs. perinuclear (H-hpa) heparanase. Cells were grown in RPMI medium (pH 7.4) containing 10% fetal calf serum in the presence of 150 llg/ml G418 and photographed (phase contrast microscope, X200) 18 h after plating. Cells transfected with H-hpa (bottom, left) are floating, and hence appear out of focus. Right panels: Immunofluorescent staining of Eb cells transfected with chimeric-hpa (top), or H-hpa (bottom), applying rabbit anti-heparanase polyclonal antibodies (p9) and Cy2- conjugated (green) goat anti-rabbit antibodies.

Fig. 1B. Schematic presentation of the various heparanase constructs used for transfection.

Fig 1C. Cell attachment: Eb cells expressing the various heparanase forms were prelabeled with 3H-thylllidine and seeded on ECM Fig. 1D. Cell attachment: Eb cells expressing the various heparanase forms were prelabeled with 3H-thymidine and seeded endothelial cell monolayer.

Cells suspended in RPMI medium were allowed to attach for various time periods (a 15 min ; 30 min; B 1 h; in 2 h; EIS h; B 24 h), at 37°C and the extent of cell adhesion was measured, as described in"Experimental procedures". Experiments were preformed at least 3 times and each data point is the mean S. D. of quadruplicates wells.

Fig. 1E. Cell adhesion in the presence of exogenously-added recombinant heparanase. Mock transfected Eb cells (transfected with the empty pCDNA3 plasmid) were incubated in the presence and the absence of recombinant heparanase. Cell attachment to ECM was measured as described in 1B above.

Fig. IF. Cell adhesion in the presence of exogenously-added recombinant heparanase. Human foreskin fibroblasts (HFF) were incubated in the presence or the absence of recombinant heparanase. Cell attachment to ECM was determined in the indicated time points, as described in 1B above.

Abbreviations: Chim-hoa (chimeric heparanase), Surf. Loca. (surface localization), Enz. Act. (enzymatic activity), Prot. Don. (proton donor), H-Hpa (human heparanase), Chk-hpa (chicken heparanase), Mut-chim-hpa (mutated chimeric heparanase), Si. Pep. (signal peptide), Nucleo. (nucleophil), Ce. Adh.

(cell adhesion), min. (minutes), no, ce. Fie. (number of cells/field), HFF (Human foreskin fibroblasts).

Figure 2A-2B Effect of RGD peptide ort heparariase m, ediated cell adhesior2 and spreadi7zg Fig. 2A. 3H-thymidine-labeled Eb mouse lymphoma cells over-expressing surface associated heparanase (chimeric-hpa) were preincubated (1 x 106 cells/ml, 1 h, RPMI medium, pH 7.4, 37°C) in the absence (o) or presence of 1 mg/ml RGD (in), or RAD containing peptides. The cells were then incubated (1 h, 37°C, RPMI, pH 7.4) on intact ECM or ECM that was first treated with recombinant human heparanase (2. 51lg/ml, ? 4 h pH 5.8, 37°C).

Unbound cells were washed away and the remaining firmly attached cells were solubilized in 1 M NaOH and counted in a ß-scintillation counter, as described in"Experimental procedures". Each data point represents the mean S. D. of quadruplicate wells.

Fig. 2B. Cell spreading. Eb cells were incubated on ECM for 1 h in the presence of 1 mg/ml RGD (top, Bar = 20 µm), or RAD (bottom, Bar = 50 µm) containing peptides were photographed under phase contrast microscopy.

Abbreviations: ECM (extra cellular matrix), Ce. Adh. (cell adhesion).

Figure 3A-3B Heparanase-mediated cell adhesion is associated with paxillin tyrosirze phosphorylatiori Fig. 3A. Chk-hpa transfected Eb cells were seeded on coverslips coated with ECM and subjected to double immunofluorescence staining with i) anti- paxillin monoclonal antibodies followed by Cy3-conjugated goat anti-mouse antibody (red), and ii) FITC-conjugated phalloidin (green, Bar = 20 m).

Fig. 3B. Tyrosine phosphorylation of paxillin. H-hpa-and chimeric-hpa- transfected Eb cells were seeded (1 x 10 cells/ml) on ECM in complete medium. The cells were collected after 20 and 60 min. of incubation, lysed and subjected to immunoprecipitation (IP) with anti-paxillin mAb. The immunoprecipitated material was bound to protein G-Sepharose and subjected to 10% SDS/PAGE and Western blot analysis using 4G10 anti- phosphotyrosine antibodies, as described in"Experimental procedures". Cells that were not incubated on ECM were used as zero time control. Paxillin phosphorylation was sustained for at least 1 h in the adhering cells.

Abbreviations: Chim-hpa (chimeric heparanase), pTyr (phospho tyrosine), min. (minutes).

Figure 4A-4E Heparanase-mediated cell adhesion is independent of hepara72ase actavity Fig. 4A. Effect of laminaran sulfate. The various hpa transfected cells were prelabeled with 3H-thymidine and incubated (RPMI complete medium, pH 7.4, 370C) on ECM for 30 min (o, m) or 6 h (, ) in the absence (#, #) or presence (#, #) of 10 llg/ml laminaran sulfate. The extent of cell adhesion was determined as described in the legend of Fig. 2.

Fig. 4B. Heparanase activity. Eb cells stable transfected with chimeric-hpa (C) or the mutated-hpa construct (Mut-chimeric-hpa), (o) were incubated with 35S-labeled ECM for 24 h at 37°C (pH 6. 2). 35S-labeled degradation fragments released into the incubation medium were analyzed by gel filtration on Sepharose 6B, as described in"Experimental procedures". Inset: Western blot analysis. Lysates of Chimric-hpa (lane 1)-and of Mut-chimeric-hpa (lane 2) - transfected cells were subjected to Western immunoblot analysis as described in"Experimental procedures". Recombinant heparanase preparation containing both the 50 and 65 kDa forms was used as control (lane 3). The 50 kDa heparanase protein is similarly expressed by the chimeric-hpa and mut- chimeric-hpa transfected cells.

Figs. 4C, 4D. Inactive heparanase promotes cell adhesion. 3H-thymidine labeled cells chimeric-hpa and Mut-chimeric-hpa transfected cells were incubated for various time periods (a 30 min; #2 h; # 24 h) on ECM (C), or endothelial cell monolayers (D) and evaluated for cell adhesion, as described in Fig. 2. Mock transfected cells were used as control and exhibited little or no adhesion. Inset (C): Immunofluorescent staining. Non permeabilized Mut- chimeric-hpa transfected cells were immunostained with polyclonal anti- heparanase antibodies (p9), as described in"Experimental procedures". Cell surface staining of the mutated enzyme (green), was similar to that observed with Eb cells transfected with chimeric-hpa and expressing an active cell surface associated heparanase.

Fig. 4E. Cell adhesion to poly-L-lysine. 3H-thymidine labeled Eb cells transfected with the various hpa constructs were incubated (1 x 106 cells/ml, RPMI complete medium, 3 ? OC) on poly-L-lysine coated tissue culture plastic for 15 min (#), 30 min (#), 2 h (.) and 6 h (E). Cell adhesion was determined as described in the legend to Fig. 2. Abbreviations: Ce. Adh. (cell adhesion), lab. Mat. (labeled material), Frac. (fraction), Mo. (mock), Chim-hpa (chimeric heparanase), Mut-chim-hpa (mutated chimeric heparanase), Chk-hpa (chicken heparanase), H-hpa (human heparanase).

Figure 5 Irzvasion through Matrigel of cells expressing active vs. inactive hepara7l, ase 3H-thymidine labeled Eb cells transfected with H-lzpa, chimeric-/pa, or Mut- chimeric-hpa were incubated (1 x 106 cells/ml, 6 h, 37°C, pH 7.4) in RPMI medium supplemented with 0.1% BSA on top of Matrigel-coated filters.

Following incubation, the upper surface of the filter was wiped free of cells and the extent of cell invasion was detected by counting in a ß-Scintillation counter, as described in"Experimental procedures". Each data point is the mean zt SD of triplicate filters. Abbreviations: Ce. inv. (cell invasion), Chim- hpa (chimeric heparanase), Mut-Chim-hpa (mutated chimeric heparanase) Figure 6A-6C Heparanase induces Akt phosphorylation Fig. 6A. HUVEC (left panel) and BAEC (right panel) were left untreated (0) or incubated with exogenously added heparanase (1 llg/ml). At the indicated time points, cells were washed and total cell lysates were subjected to SDS- PAGE followed by immunoblotting with anti c-Myc epitope tag (upper panel), anti heparanase #1453 (Hepa, second panel), anti phospho-Akt (p-Akt, third panel), anti Akt (fourth panel), anti phospho-MAPK (p-MAPK, fifth panel) or anti Erk 2 (bottom panel) antibodies.

Fig. 6B. Dose response. HUVEC were left untreated (0) or incubated with exogenously added heparanase at the concentrations indicated (µg/ml). Total cell lysates were prepared after 30 min of incubation and Akt phosphorylation was evaluated as above.

Fig. 6C. Densitometry analysis. 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 untreated cells set arbitrary to a value of 1. Note time and dose- responsive Akt phosphorylation upon heparanase treatment. Abbreviations: Phos. (phosphorylation), Fo. Inc. (fold increase), p-Akt (phsphorylated Akt) gg/ml (microgram/milliliter).

Figure 7A-7B Heparanase-indzcced Akt pl2osphoryl, ation is HSPG- i7ldepe7lden, t Fig. 7A. Mutated CHO-745 (left panel) and human colon tumor-derived HT- 29 (right panel) cells were left untreated (0) or incubated with exogenously added heparanase (1 ßg/ml). At the times indicated, total cell lysates were prepared and immunoblotted with anti phospho-Akt (p-Akt, upper panel), Akt (second panel), phospho-MAPK (p-MAPK, third panel) and Erk-2 (bottom panel) antibodies as described.

Fig. 7B. Densitometry analysis of Akt phosphorylation levels was determined following 30 min of heparanase incubation with HUVEC (left), BAEC (second from left), HT-29 (third from left) and CHO 745 (745, right) cells. Note comparable Akt phosphorylation levels upon heparanase addition to HSPG-positive EC (HUVEC, BAEC) and HSPG-negative (745, HT-29) cells. Abbreviations : Phos. (phosphorylation), Fo. inc. (fold increase), p-Akt (phsphorylated Akt), T. (time), con. (control).

Figure 8A-8D Hepararzase-indzcced Akt phosphorylation is augmented by hepar°i. rz Figs. SA-SB. CHO-745 cells were left untreated (Con) or incubated with heparanase (Hepa) or heparanase together with the heparanase inhibitor laminaran sulfate (LS, 10 Ag/ml). Total cell lysates were prepared after 30 min of incubation and Akt phosphorylation was analyzed by immunoblotting (A). Densitometry analysis is shown in (B). Note 4-fold increase of Akt phosphorylation levels upon heparanase treatment, and further #2-fold increase when LS is combined together with heparanase.

Figs. 8C-SD. HUVEC were left untreated (Con), incubated with heparanase (Hepa) or heparanase together with heparin (10 pg/ml). Akt phosphorylation was analyzed (C) and quantified (D) 30 min following heparanase addition as above. Abbreviations: Phos. (phosphorylation), Fo. Inc. (fold increase), p-Akt (phsphorylated Akt), con. (control).

Figure 9A-9E Heparanase-indu, ced Akt phosphorylatiorz is ivdependerzt of heparanase enzymatic activity Fig. 9A. Heparanase activity assay. Gel filtration profile of intact soluble sulfate-labeled ECM (peak I,) incubated (1 h, 37oC) with heparanase before (A) and after (+) immobilization. Note heparanase activity in the heparanase preparation and complete loss of activity upon exposure to high pH conditions during immobilization.

Fig. 9B. Immobilized heparanase facilitate cell adhesion. HUVEC (2x105) were plated onto BSA-, or heparanase-coated dishes and following incubation (1 h, 37°C) adhering cells were fixed with 4% PFA and photographed.

Figs. 9C-9E. Immobilized, inactive, heparanase induces Akt phosphorylation. HUVEC were plated onto heparanase-coated dishes or were left in suspension as control (Con). After incubation for the indicated time points (min), total cell lysates were subjected to immunoblotting applying anti phospho-Akt (p-Akt, upper panel), Akt (second panel), phospho-MAPK (p-MAPK, third panel) or ERK-2 antibodies (C). Akt (D) and MAPK (E) phosphorylation was quantified by densitometry as described. Note a marked Akt as well as MAPK phosphorylation upon HUVEC adhesion to the immobilized inactive heparanase protein. Abbreviations: Phos.

(phosphorylation), Fo. Inc. (fold increase), p-Akt (phsphorylated Akt), con.

(control), lab. mat. (labeled material), Frac. (fraction).

Figure 10A-lOG Heparanase stimulates EC migration.

Fig. 10A. Tube-like structure formation on Matrigel-coated dishes. HUVEC (4x105) were plated onto Matrigel-coated dishes in the absence (Con) or presence of heparanase (Hepa, 1 llg/ml) and EC organization was evaluated after 24 h.

Figs. 10B-10E. Heparanase stimulates EC migration. Confluent HUVEC cultures were scraped with the wide end of 1 ml pipette tip and cell migration into the wounded areas was evaluated after 7 days in the absence (Con) or presence of heparanase (Hepa, 1 Fg/ml) (B). HUVEC (2x105) were plated onto fibronectin-coated inserts and were left untreated (Con) or incubated with heparanase (Hepa, 1 ßg/ml) alone or combined with the PI 3-kinase inhibitor LY 294002 (10 Rg/ml ; Hepa +LY) or the heparanase inhibitor laminaran sulfate (Hepa +LS). Cells migrating into the lower compartment were visualized by crystal violet staining (C) and quantified by counting of at least 6 random fields (D, E). Note-2. 5-fold increase in cell migration upon heparanase treatment and inhibition of the heparanase effect by LY (D), but not LS (E) treatment.

Figs. 1OF-IOG. Heparanase stimulates EC invasion. HUVEC (2x105) were plated onto Matrigel-coated inserts and were left untreated (Con) or incubated with heparanase (Hepa) or with heparanase and LY (Hepa +LY).

Cell invasion was visualized (F) and quantified (G) after 5 h. Abbreviations: Con. 9control0, ce. Mig. (cell migration), ce. Inv. (cell invasion), ce. P. fie. (cell per field).

Figure 11A-11C Increased cell spreading and migration of heparanase. transfected U87 cells FIG. 11A. Vo, Low and Hi cells were plated on glass coverslips for 1 h and visualized under a microscope (left panel), or immunostained with antibodies directed against phosphotyrosine (P-Y, second panel), phospho FAK (p-FAK, third panel) and paxillin (fourth panel). Actin was visualized by phalloidin staining (right most panel). Note cortical actin and cytoplasmic paxillin in the control Vo cells compared with formation of actin stress fibers and localization of paxillin, phospho FAK and other tyrosine-phosphorylated proteins, in areas of focal contacts in Low and Hi cells. Original magnification: X10 (left panel), X60 (all other images).

Fig. 11B. Vo, Low and Hi cells were plated on culture dishes for 1 h and total cell lysates were analyzed for phospho FAK (p-FAK, upper panel), phospho AKT (p-Akt, second panel), phospho p38 (p-p38, third panel), phospho JNK (p-JNK, fourth panel) and phospho ERK (p-ERK, fifth panel) by immunoblotting. Blots were striped and re-probed with a relevant control antibody recognizing the total amount of protein in each panel. Note the marked increase of FAK and Akt phosphorylation levels in the heparanase- transfected Low and Hi cells. The amount of GTP-bound Rac1 was analyzed by incubating total cell lysates (200 ptg) with 30 ig of the p21-binding domain of PAK fused to glutathione S-transferase (GST). Following 30 minutes incubation, the beads were washed and, after electrophoresis, membranes were probed with anti Rac antibodies (bottom panel). Total Rac expression is shown below. Note a significant increase in Rac-GTP in the heparanase- transfected cells.

Fig. 11C. Vo, Low and Hi cells were allowed to grow for 2 days on tissue culture plates and were then scraped 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 photographed after 24 h and 48 h. Note the appearance of cell clamps and reduced migration in the Vo cell culture, compared with the heparanase-transfected Low and Hi cells. Abbreviations: Ph. Contra. (phase contrast), p-Tyr (phospho tyrosine), Lo. (low), Hi (high), h (hour).

Detailed Description of the Invention Heparan sulfate and its cleavage by heparanase participate in diverse normal and pathological processes such as development, morphogenesis, tissue repair, inflammation, metastasis and angiogenesis. Heparanase activity and localization must therefore be tightly regulated [Parish (2001) ibid ; Fairbanks (1999) ibid ; Goldshmidt (2001) ibid; Nadav, L. et al. J. Cell Sci.

115: 2179-2187 (2002)]. For example, the enzyme is highly sensitive to changes in the local pH, exerting a high enzymatic activity under acidic conditions that exists in the vicinity of tumors and in inflammatory sites vs. little or no activity at a physiological pH [Toyoshima (1999) ibid; Gilat (1995) ibid.]. Since the enzyme degrades primarily an extracellular component, regulation by the cell microenvironment is highly probable [Gilat, (1995) ibid.].

The occurrence of heparanase on cell surfaces [Goldshmidt (2001) ibid] and its secretion into the extracellular space within tissues [Nadav (2002) ibid; Dempsey, L. et al. Glycobiology 10: 467-475 (2000) ] suggest that at a physiological pH (pH > 7.2) heparanase may exert functions other than enzymatic degradation of heparan sulfate [Gilat, (1995) ibid.]. To investigate this possibility, anchorage-independent mouse Eb-lymphoma cells were transfected with various constructs containing heparanase, and their adhesion to ECM or to endothelial cell monolayers examined. The results of the present invention demonstrate that surface associated heparanase promotes a firm adhesion of otherwise non-adherent lymphoma cells, as compared to cells over-expressing the intracellular form of the enzyme, which remain floating. Cell adhesion is not affected by laminaran sulfate, a potent inhibitor of heparanase activity and experimental metastasis [Miao, H. et al.

Int. J. Cancer 83: 424-431 (1999) ]. These results indicate that heparanase- mediated cell adhesion does not necessarily require its enzymatic activity.

To further investigate this feature, i. e., the heparanase-mediated cell adhesion independent of its enzymatic activity, a mutated heparanase was generated, using the chimeric-hpa cDNA as a template for a point mutation and replacing the active site proton donor Glu225 with Ala. Examination of the mutated enzyme cellular localization and enzymatic activity confirmed the generation of a surface-associated, and enzymatically inactive form of heparanase. Evaluation of cell adhesion revealed, however, that cells over- expressing the mutated heparanase adhere to ECM to the same extent as cells expressing the surface associated active enzyme. These results clearly indicate that heparanase-mediated cell adhesion is independent of its HS- degrading activity, provided that the enzyme is expressed on the cell surface.

Heparanase molecules having a point mutation in the nucleophile residue Glu343 to Ala, and double-mutated molecules having both Glu225 and Glu343 mutations, showed similar results (not shown). It should be noted that the Glu225 and Glu343 location refers to the amino acid sequence of the human heparanase as denoted by the GenBank Accession No. AF144325.

The results of the present invention indicate that apart from its well- established role as a HS-degrading enzyme, heparanase may function as a pro-adhesive molecule, independent of its endoglycosidase activity. The combined feature of heparanase as an enzyme and cell adhesion molecule, further emphasizes its potential significance in processes involving cell adhesion, migration and invasion, such as tumor metastasis, neovascularization, inflammation and autoimmunity. In fact, the inventors have recently demonstrated that lymphoma cells over-expressing cell surface heparanase elicit a markedly increased angiogenic response and metastatic dissemination i77, vivo [Goldshmidt (9002) ibi. d.]. The significance of cell surface localization and secretion of ECM-degrading enzymes in cancer metastasis was previously demonstrated by showing a correlation between the metastatic potential of breast and bladder carcinoma cells and translocation of cathepsin D and B from within lysosomes to the plasma membrane [Rochefort, H. et al. Enzyme Protein 49: 106-116 (1996) ]. Clearly, enzymes expressed on the cell surface and/or secreted are more effective then intracellular enzymes, in mediating cell adhesion and invasion. Once localized on the cell surface, heparanase may function as an endoglycosidase, or as a cell adhesion molecule, depending on the local pH. At relatively acidified pH conditions, heparanase performs as a HS-degrading enzyme, while at the physiological pH of a quiescent tissue, it may function primarily as an adhesion molecule.

Thus, the local state of a tissue can regulate the activities of heparanase and can determine whether it will function as an enzyme and/or as a pro-adhesive molecule. Without being bound by any theory, the adhesion properties of heparanase may be a result of interaction with a yet undefined receptor molecule and subsequent initiation of different signal transduction pathways.

As shown by Examples 6,7, 8 and 9, exogenously added heparanase, as well as overexpressed heparanase in transfected cells (Fig. 11), stimulated Akt phosphorylation in human, bovine and mouse (data not shown), EC (Fig. 6; Fig. 7B; Fig. 8E, F; Fig. 9D) while other signaling pathways such as the MAPK were not induced (Fig. 6A, fifth panel; Fig. 7A, third panel). However, the MAPK pathway was clearly induced where wound healing process was examined using the migration assay (Fig. 9).

The results of the present invention ascribe a new function to heparanase, paving the way for studies focusing on non-enzymatic activities of the heparanase molecule, similar to those exerted by other ECM-degrading enzymes (i. e., thrombin, plasminogen activator, MMPs) [Preissner, IS :. et al.

Curr. Opin. Cell Biol. 12: 621-628 (2000); Sternlicht, M. D., and Werb, Z.

Annu. Rev. Cell Dev. Biol. 17: 463-516 (2001)].

Thus, in a first aspect, the present invention relates to a nucleic acid construct comprising a polynucleotide sequence encoding a heparanase- derived polypeptide having cell-to-cell and cell-to-matrix adhesive properties and being devoid of heparanase catalytic activity. The construct of the invention optionally further comprises operably linked regulatory elements.

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 (such as sense or antisense) and double-stranded polynucleotides.

In a specifically preferred embodiment, the nucleic acid constructs of the invention may comprise a human heparanase-derived polypeptide devoid of heparanase catalytic activity, as heparanase-derived polypeptide.

The construct of the invention may encode the precursor 65Kd heparanase molecule, which devoid of the heparanase catalytic activity, but however still exhibit adhesive properties, as demonstrated by Fig. IE.

Alternatively and preferably, a heparanase-derived polypeptide may be encoded by a nucleic acid sequence comprising at least one mutation, point mutation, nonsense mutation, missense mutation, deletion, insertion or rearrangement.

In a specifically preferred embodiment, the heparanase-derived polypeptide comprised within the nucleic acid constructs of the invention, may carry a point mutation replacing the active site proton donor GlU22s with Ala.

According to this embodiment, such construct comprises a nucleic acid sequence substantially as denoted by SEQ ID No.: 1. Alternatively, the heparanase-derived polypeptide may carry a point mutation replacing the active site nucleophile residue Glu3 with Ala. These constructs may comprise a nucleic acid sequence substantially as denoted by SEQ ID No.: 2. In a yet further embodiment, the heparanase-derived polypeptide comprised within the nucleic acid construct of the invention, may carry both Glu225 and Glu343 to Ala mutations. Such double mutant constructs may comprise, according to a specific embodiment, a nucleic acid sequence substantially as denoted by SEQ ID No.: 3.

In an alternative embodiment, the invention provides a nucleic acid construct encoding a secreted or membranal adhesion molecule having cell-to-cell and cell-to-matrix adhesive properties and being devoid of heparanase catalytic activity. According to this embodiment, the invention relates to a construct comprising a first polynucleotide sequence encoding a signal peptide of avian (preferably, chicken) heparanase operably linked to a second polynucleotide sequence encoding heparanase-derived polypeptide and optionally further comprising operably linked regulatory elements. These heparanase-derived polypeptides preferably may carry a point mutation replacing the active site proton donor Glu925 with Ala, a point mutation replacing the active site nucleophile residue Glu3'l3 with Ala, or may carry both GIU'. 25 and Glu343 to Ala mutations. Such heparanase-derived polypeptides may be encoded by a nucleic acid sequence denoted by any one of SEQ ID No. 4,5, and 6.

It should be noted that the locations Glu225 and Glu343 refer to the position of said amino acids within the amino acid sequence of human heparanase as denoted by the sequence of GenBank Accession No. AF144325.

It is to be appreciated that fragments of the nucleic acid encoding the subject heparanase are also within the scope of the invention. As used herein, a fragment encoding the active portion of adhesion promoting heparanase refers to a nucleic acid having fewer nucleotides than the nucleotide sequence encoding the entire amino acid sequence of heparanase but which nevertheless encodes a peptide which has cell-to-cell and cell-to-matrix adhesion properties, i. e. the fragment retains the ability to enhance adhesion. The nucleic acid constructs of the present invention are preferably comprised within expression vectors.

"Expression Vectors", as used herein, encompass plasmids, viruses, bacteriophages, integratable DNA fragments, and any 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 gene. These control elements are capable of effecting expression within a suitable host. Generally, the genetic control elements can include a prokaryotic promoter system or an eukaryotic promoter expression control system. Such systems typically include 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 serves 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 fully incorporated herein by reference. In general, such vectors contain in addition specific genes, which are capable of providing phenotypic selection in transformed cells. The use of prokaryotic and eukaryotic viral expression vectors to express the genes coding for the polypeptides of the present invention is also contemplated.

The vector is introduced into a host cell by methods known to those skilled in the art. Introduction of the vector into the host cell can be accomplished by any method that introduces the construct into the cell, including for example, calcium phosphate precipitation, microinjection, electroporation or transformation. See, e. g. , Current Protocols in Molecular Biology, Ausubel, F.

M. , ed. , John Wiley & Sons, N. Y. (1989).

Thus, the present invention further provides for a host cell transformed or transfected with the nucleic acid constructs and expression vectors of the invention.

"Cells", "host cells"or"recombinant host 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 modifications 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 is still included within the scope of the term as used herein.

"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.

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. cerevisiae and Pichia, and species of the genus Dictyostelium,. Higher eukaryotes are preferably mammalian cells.

In a particular specific embodiment, the host cells of the invention may be endothelial cells. The nucleic acid constructs of the present invention can be introduced into the endothelial cells via any standard mammalian transformation method. Such methods include, but are not limited to, direct DNA uptake techniques, and virus or liposome mediated transformation (for further detail see, for example,"Methods in Enzymology"Vol. 1- 317, Academic Press).

In a second aspect, the invention relates to a recombinant protein comprising a heparanase-derived polypeptide. Such recombinant protein, according to the invention, has adhesion activity and is devoid of heparanase endoglycosidase catalytic activity.

In a specific embodiment of said aspect, the recombinant protein comprises the amino acid sequence substantially as denoted by any one of SEQ ID No.

7,8 and 9, encoded by the nucleic acid sequence substantially as denoted by SEQ ID No. 1, 2 and 3, respectively.

In yet another specific embodiment, the invention provides a recombinant protein being any one of a secreted and membranal molecule having adhesion activity and devoid of heparanase endoglycosidase catalytic activity. According to this embodiment, the recombinant protein of the invention comprises a signal peptide of avian (preferably chicken) heparanase and heparanase-derived polypeptide, and comprises the amino acid sequence substantially as denoted by any one of SEQ ID No. 10,11 and 12, encoded by the nucleic acid sequence substantially as denoted by any one of SEQ ID No.

4,5 and 6.

It should be appreciated that also the precursor 65Kd heparanase molecule, which is devoid of catalytic activity, can activate Akt, as demonstrated by Fig. 6, and therefore may be applicable as an adhesive molecule.

The unexpected significant adhesive properties of heparanase, revealed by the inventors, and particularly the creation of the non-catalytic adhesive heparanase molecule, enabled the inventors to use the recombinant molecules of the invention in promotion and enhancement of adhesion. For example, the use of such novel molecule as a tissue sealant molecule.

Therefore, in a further aspect, the present invention relates to a tissue sealant composition comprising as active ingredient an effective amount of adhesion promoting non-catalytic heparanase molecule or any functional fragments thereof, and optionally further comprising a compound capable of accelerating, enhancing, stimulating and/or mediating the healing of an injury, optionally combined with a pharmaceutically acceptable carrier.

In a preferred embodiment, the adhesion promoting non-catalytic heparanase molecule comprised within the composition of the invention, may be any of the membranal, secreted or cytoplasmic non-catalytic recombinant heparanase molecules defined by the invention or any functional fragments thereof.

It should be noted however that in addition to the mutated heparanase molecule, also the precursor 65Kd molecule, which is devoid of the heparanase catalytic activity, may be used.

The present invention therefore provides the use of such tissue sealant composition in the preparation of an agent for: accelerating the healing of an injury, homeostasis of an injury to a skin surface or a viscus, endothelium formation of a blood vessel, adhesive activity of mammalian cells and/or adhesion and aggregation of platelets.

By the term viscus is meant a main organ that is situated inside the body, an internal organ.

It should be noted that the invention further provides the use of any one of heparanase-derived polypeptide having cell-to-cell and cell-to-matrix adhesive properties and being devoid of heparanase catalytic activity, nucleic acid construct encoding said polypeptide and host cells transfected with said construct, in the preparation of a composition for enhancing adhesion.

According to a preferred embodiment, such composition may be a sealant composition, preferably, as defined by the invention.

In a specifically preferred embodiment, the invention relates to the use of the construct or the host cell of the invention in the preparation of a composition for enhancing adhesion. In yet another embodiment, the invention relates to the use of the recombinant protein of the invention in the preparation of a composition for enhancing adhesion. It should be appreciated that for preparation of adhesive promoting composition, heparanase precursor molecule which devoid of the heparanase catalytic activity, or mature heparanase in the presence of an inhibitor such as LS for example, or in basic pH conditions, may also be used.

Shortly after an injury occurs, a healing process, i. e. , a recovery of lesions, starts with an adhesion and agglutination of platelets or thrombocytes to the injury. It is necessary to closely control the consecutively conjugated functions of various cells, and the degradation and regeneration processes.

These include formation of fibrin clots, absorption of blood clots, and epithelialization. The injury-healing process comprises the formation of many blood capillaries, active fibroblasts, and collagen fibrils, but it is not followed by the formation of a particular skin structure. A process of healing of an injury starts with adhesion and aggregation of platelets to an injured tissue, and with thromboplastin liberated from the injured cells at the same time.

Thromboplastin activates coagulation factors, and finally converts prothrombin to thrombin.

It is therefore desired to develop adhesive compositions which can be applied to an injury on the skin surface or an intracorporeal tissue, in order to enhance adhesion as well as platelet aggregation, and thus be. useful in maintaining hemostasis and enhancing the curing or healing of the injury.

The invention, therefore, provides a tissue sealant composition which can seal injuries, reduce blood loss, maintain hemostasis, and promote healing of an injured site on a skin surface, organs or the like.

Thus, an object of the present invention is to provide a tissue sealant composition which can promote the healing of an injury, endothelium formation, or cellular adhesion of animal cells. By applying the sealant. said composition is capable of agglutinating platelets or thrombocytes to the injured tissue, and to maintain the contact of growth factors induced by platelets or thrombocytes with the injured tissue, the blood vessels or animal cells for a long time.

The tissue sealant of the present invention may contain, in addition to heparanase, for example, one or more compounds capable of accelerating the healing of an injury, e. g. one or more compounds capable of strengthening, stimulating or mediating biological activities of growth factors derived from platelets or thrombocytes in the tissue sealant.

Thus, the tissue sealant composition may be used in the preparateion of an agent for accelerating the healing of an injury, homeostasis of an injury to a skin surface or a viscus, endothelium formation of a blood vessel, adhesive activity of mammalian cells and/or adhesion and aggregation of platelets.

Specifically, the tissue sealant of the present invention may contain, for example, antibiotics, activated protein C, heparin, prostacyclin (PGI3), antithrombin III, ADPase, anticoagulants such as a plasminogen activator, an anti-inflammatory steroids such as dexamethasone, cardiovascular agents such as a calcium channel-blocking agent as well as local anaesthetics, such as bupivacaine.

The tissue sealant of the present invention may further contain polyclonal, monoclonal or chimera antibodies, or functional derivatives or fragments thereof. These may be antibodies that inhibit the growth of smooth muscles or the growth of undesired cells within or around the site where the tissue sealant is applied.

The tissue sealant composition of the present invention can be applied to an injured lesion in a form suitable for the application purposes. For example, the tissue sealant may be liquid, gel or powder. When a liquid sealant is prepared, water or buffer may be used as solvent.

The tissue sealant of the present invention may provide a matrix filling gaps, which is necessary for transfering cell components and to cause bone induction in a living human body.

The tissue sealant of the present invention may be applied to any injury, that is, any injured tissue in living organisms. The injured tissue may be an intra- corporeal tissue, such as an inside wall of a stomach, a fracture, or the like, a skin surface or the like, and also a soft tissue, such as a spleen, or a hard tissue, such as bone. The injury may be a lesion, trauma or wound, or one formed by an infection or from a surgical operation.

By the terms"injury"or"wound"is meant any surgery, trauma, burns, diabetes or pressure ulcers, various forms of dermatitis and vascular ulcers.

For instance, diabetic patients and those on systemic steroids often have impaired healing mechanisms affecting their ability to resist tissue breakdown. Still further, patients suffering from pressure or diabetes related ulcers or inflicted with surface or shallow wounds often experience delayed healing. Acute or chronic wounds are also contemplated, for example in cases of patients suffering from chronic post-surgical wounds, impaired-healing burns and ulcers.

The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated, and the particular mode of administration. It should be understood, however, that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated. The amount of active ingredient may also depend upon the therapeutic or prophylactic agent, if any, with which the ingredient is co- administered.

As mentioned above, the tissue sealant composition of the invention may further comprise a pharmaceutically acceptable carrier. The term"carrier"as used herein includes acceptable adjuvants and vehicles. Pharmaceutically acceptable carriers that may be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium. chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

The invention therefore provides a tissue sealant which can seal injuries, reduce loss of blood, maintain hemostasis, and promote healing of an injured site on a skin surface, organs or the like.

Still further, the invention provides a method of accelerating the: healing and homeostasis of an injury to a skin surface or a viscus, endothelium formation of a blood vessel, adhesive activity of mammalian cells, and/or adhesion and aggregation of platelets. Such method comprises the step of administering to a subject in need a therapeutically effective amount of a tissue sealant composition of the invention.

More specifically, the method of the invention may be particularly applicable for accelerating an external skin wound healing in a mammalian subject in need thereof. Such method comprises the step of applying a tissue sealant composition of the invention to the wounded skin of said subject, at the time of or subsequent to occurrence of said wound, in a quantity sufficient to accelerate clinically detectable healing.

The adhesive heparanase molecule of the invention clearly enhances cell-to- cell adhesion. Such adhesive molecule may be particularly applicable for the treatment of different adhesion deficiencies in human subjects. Therefore, the invention further provides a method for the treatment of a disorder associated with adhesion deficiency in a mammalian subject. According to the invention, this method comprises the step of administering to said subject an effective amount of any one of the adhesion promoting non-catalytic heparanase recombinant molecules of the invention, a composition comprising the same and cells expressing said adhesion molecules.

Such adhesion deficiency disorder may be caused for example by impaired/defective leukocytes or platelets adhesion.

The physiologic importance of leukocyte adhesion. proteins is underscored by the existence of a human genetic disease, leukocyte adhesion deficiency (LAD) [Anderson et al. , J. Infect. Dis. 152: 668 (1985); Arnaout et al. , Fed.

Proc. 44: 2664 (1985) ]. Various studies have indicated that the molecular defect associated with LAD results in either lack of synthesis of the common B chain or normal rate of synthesis followed by rapid degradation [Liowska- Grospierre et al., Eur. J. Immunol. 16 : 205 (1986) ; Dimanche et al. , Eur. J.

Immunol. 17: 417 (1987). In the severe form of LAD, neither LFA-1, Mac-1, nor pl50/95 are expressed on the leukocyte membrane. Low levels of leukocyte membrane expression have been observed in patients suffering from the moderate form of the disease. This leads to a defective mobilization of polymorphonuclear leukocytes and monocytes from the vasculature to the tissues during the inflammatory response, with consequent recurrent bacterial infections [Anderson (1985) ibid. ; Arnaout (1985) ibid.].

Platelet receptors which mediate platelet adhesion and aggregation are located on the two major platelet surface glycoprotein complexes. These complexes are the glycoprotein Ib-IX complex, which facilitates platelet adhesion by binding the von Willebrand factor (vWF), and the glycoprotein IIb-IIIa complex which links platelets into aggregates by binding to fibrinogen. Patients with the Bernard-Soulier syndrome, a congenital bleeding disorder, show deficient platelet adhesion due to a deficiency in the glycoprotein Ib-IX complex which binds vWF, as well asmild thrombocytopenia, and large lymphocoid platelets.

Thus, a specific embodiment of the method of the invention is particularly intended for the treatment of adhesion deficiency associated disorders, such as, for example, LAD, Glanzmann's thrombasthenia (defective platelets function) and Bernard-Soulier syndrome (deficient platelets adhesion).

It should be appreciated that any non-catalytic heparanase molecule may be applicable as an adhesive enhancing molecule. For example, and in addition to the mutated heparanase molecules of the invention, the precursor 65Kd heparanase molecule, which is devoid of heparanase catalytic activity, may be used. Moreover, the use of heparanase inhibitors, for example LS, or the use of basic pH conditions may also be used for providing non-catalytic heparanase.

As shown by the Examples, over-expression of the adhesion promoting non- catalytic heparanase molecule significantly enhances cell-to-cell and cell-to- matrix adhesion. Such properties may be therefore employed in improving adhesion of cell to matrix. A particular example of such application may be improved implantable devices.

In particular, the invention relates to methods for improving the performance of medical devices when implanted in a biological environment. Particularly, the invention relates to devices such as implantable vascular grafts.

Currently, ilaplanted devices are considered successful when any undesirable surface responses that may occur neither unduly affect the host, nor significantly interfere with the primary function of the device. For example, the formation of a thrombus layer on the luminal surface does not typically affect the function of a large diameter vascular graft, whereas it may occlude a small diameter graft.

Initial, research into the development of materials having improved biocompatibility focused largely on the generation of those showing minimal reaction with tissue. Although this approach has improved the function of several devices, further improvements in the compatibility and performance of artificial implants are desired.

To improve function and durability of synthetic grafts, autologous endothelial cells (EC) have been used for seeding grafts before implantation.

Endothelial cells provide the optimal biocompatible surface, and may provide long term protection from thrombosis due to their thrombolytic capacity. In addition, EC coverage can also prevent neo-intimal proliferation and inflammatory reaction in the graft. However, to date, randomized large scale studies have not proven that EC seeding can improve graft durability.

As indicated herein, autologous endothelial cell seeding can reduce the thrombogenicity of synthetic graft surfaces. However, vascular graft surfaces tend to be poor substrates for cell adhesion, and the fraction of seeded cells which adhere in the time frame dictated by graft implantation procedures is relatively low. Some investigators pre-coat vascular prostheses with various extracellular matrix proteins in an attempt to improve cell adhesion.

Nonetheless, the poor binding of many of such proteins to the graft material also results in poor cell adhesion.

Plasma discharge, the exposure of biomaterials to a plasma or ionized gas, which results in the generation of functional groups or surface coatings on a material surface, has also been used in the preparation of vascular grafts. In U. S. Pat. No. 4,718, 907, for example, vascular prosthesis material is provided, having fluorine-containing polymer coating on its luminal surfaces and fluorine to carbon ratio greater than 1.5 to improve the potency of the implanted graft. Plasma discharge treatment of certain vascular prosthesis material in the presence of non-polymerizing gases, such as ammonia plasma treatment of polystyrene and polytetrafluoroethylene graft material, has been shown to increase protein binding and endothelial cell adhesion.

One of the limitations of the artificial vascular grafts, as well as some natural vascular grafts currently available, is the lack of durability over extended periods of time. Although the use of endothelial cells greatly enhances the quality of coated artificial grafts, by improving both performances in general and thrombolytic activity in particular, such grafts typically suffer from loss of cell coating under blood flow forces since the coated cells cannot withstand the shear forces exerted thereupon by flowing blood.

There is, therefore a great need for new methods of improving endothelial cells adhesion to implantable medical devices. Moreover, in Examples 6 to 9 presented herein, the activation of the PI3'K/Akt pathway suggests that heparanase may have an effect on cell survival and reduced apoptosis, and therefore may increase the resistance of endothelial cells. Thus, the present invention provides a novel technology for improved endothelial cell adhesion to implantable medical devices. Thus, the invention further provides for the use of. the tissue sealant compositiom of the invention in the preparation of an agent for promoting the endothelialization of vascular grafts.

In a forth aspect, the invention relates to an implantable medical device comprising : (a) a synthetic tubular element having a luminal surface; (b) a cell layer directly or indirectly adhered to said luminal surface, wherein said cell layer comprises cells genetically modified to express a membranal adhesive non-catalytic heparanase molecule or any functional fragment thereof As used herein the term"genetically modified"refers to the introduction of exogenous polynucleotide sequences into a cell. Such sequences can integrate into the genome of the cell, or alternatively, such exogenous sequences can be in the cell nucleus or cytoplasm in a transient manner.

According to one specific embodiment, the implantable medical device of the invention may further comprise a layer of immobilized adhesion molecules interposed between said luminal surface and said cell layer. This implantable medical device is formed of a biomaterial that provides a surface bearing immobilized adhesion molecules in an amount and type suitable to promote endothelialization through the surface and into the device. The additional layer of adhesion molecuis may comprise preferably an extracellular matrix protein which promotes endothelial cell attachment such as laminin or fibronectin or mixtures of such proteins. However, collagen type I, III or IV, other proteins such as fibrin, vitronectin, tenascin, basic fibroblast growth factor, proteins containing the arginine-glycine-aspartic acid (RGD) sequence, as well as the non-catalytic heparanase molecule of the. invention, may also be useful. The adhesion molecule may be bound to the luminal surface through contacting a concentrated solution of the adhesion molecule with the luminal surface, for a period of time effective to provide a desired amount of the protein on the surface. if vivo, adhesion molecules are typically able to bind to specific cell surface receptors, mechanically attaching cells to the substrate or to adjacent cells. In addition to promoting cell attachment, suitable adhesion molecules can promote other cell responses like cell migration (as shown by Example 8) and differentiation (which in turn can include the formation of capillary tubes by endothelial cells).

It is to be appreciated that the density of adhesion molecules carried by the device supporting luminal surface should be sufficient to enhance endothelial cell adhesion and migration. This density can be provided in the form of a plurality of different molecule types and/or a plurality of molecules of a particular type.

As used herein, the following terms and words shall have the following ascribed meanings :"implantable medical device", which for brevity will be referred to as a"device"or a"medical device", will refer to an object fabricated, at least in part, from a biomaterial and intended for use in contact with body tissues, including body fluids ;"biomaterial"shall refer to the chemical composition of the material used to prepare a device, and which provides one or more of its tissue contacting surfaces;"surface"shall refer to the interface between the biomaterial and its environment. The surface is capable of serving as an immobilization site for cell adhesion molecules, as well as for the attachment and migration of endothelial cells;"adhesion molecules"shall refer to peptides, proteins and glycoproteins capable of binding to substrates and/or cells in order to attach cells to the substrate or to adjacent cells.

Devices of the present invention include medical devices intended for prolonged contact with body fluids or tissues, and, in particular, to those devices that can benefit from endothelialization when used in either i7T vivo or in Vit7'0 applications.

Preferred devices are implantable in the body, and include vascular grafts and artificial organs, such as the pancreas, liver, and kidney. Other suitable implant devices include, but are not limited to, devices used to implant genetically modified cells that deliver recombinant proteins for therapeutic use, and artificial tissue or organ implants, such as replacement skin, joints, or ears.

According to a specifically preferred embodiment, the implantable medical device of the invention may be a vascular graft. For this purpose, devices of the present invention can be prepared from a variety of rigid biomaterials capable of providing a surface for the adhesion and migration of endothelial cells. A wide variety of suitable materials can be employed as the support, primary considerations being that they provide an optimal combination of properties such as strength, surface area, ease of preparation and use, and cost.

Preferred support materials are synthetic polymers, including oligomers, homopolymers, and copolymers resulting from either addition or condensation polymerizations. Examples of suitable addition polymers include, but are not limited to, acrylic such as those polymerized from methyl acrylate, methyl methacrylate, acrylic acid, methacrylic acid, acrylamide, hydroxyethyl acrylate, hydroxyethyl methacrylate, glyceryl acrylate, glyceryl methacrylate, methacrylamide and ethacrylamide; vinyls such as styrene, vinyl chloride, vinyl pyrrolidone, polyvinyl-alcohol, and vinyl acetate; polymers formed of ethylene, propylene, and tetrafluoroethylene.

Examples of condensation polymers include, but are not limited to, nylons such as polycaprolactam, polylauryl lactam, polyhexamethylene adipamide, and polyhexamethylene dodecanediamide, and also polyurethanes, polycarbonates, polyamides, polysulfones, poly (ethylene terephthalate), polylactic acid, polyglycolic acid, polydimethylsiloxanes, and polyetherketone.

Other suitable support materials include metals and ceramics. Metals include, but are not limited to, titanium, stainless steel and cobalt chromium.

Ceramics include, but are not limited to, silicon nitride, silicon carbide, zirconia, and alumina, as well as glass and silica. Numerous synthetic biomaterials have been developed as vascular substitutes. Studies have demonstrated that selected materials, e. g. Dacron and ePTFE (expanded polytetrafluoroethylene) grafts, can be successfully incorporated in both large and small caliber host arteries of animal models. ePTFE is the preferred biomaterial'for use in fabricating implantable devices of the present invention, particularly for fabricating vascular grafts. Suitable ePTFE is available in the form of vascular grafts from such sources as IMPRA, Inc. , Tempe, Ariz. The commercially available grafts are constructed of ePTFE and supplied in sterile form in a variety of configurations, including straight, tapered and stepped configurations.

In a particularly preferred embodiment, the implantable device is in the form of a vascular graft and the biomaterial is selected from the group consisting of tetrafLuoroethylene polymers (such as ePTFE), aromatic/aliphatic polyester resins (such as polyethylene terephthalate ("PET") and poly (butylene terephthalate) ('PET"), polyurethanes, and silicone rubbers (such as heat cured rubbers and those formed from"room temperature vulcanizing" (RTV) silicone elastomers).

In yet another embodiment, the genetically modified cells comprised within the vascular graft of the invention may be endothelial cells said EC shall be derived from a source selected from the group consisting of a segment of mammalian blood vessel, bone marrow progenitor cells, peripheral blood stem cells and circulating endothelial cells. Endothelial cells used for seeding grafts before implantation are typically harvested from short venous segments or from adipose tissue. Other sources may be cells from transgenic animals having human characteristics or autologous EC from the individual patient. Another possible source for EC are progenitor cells isolated from the peripheral blood or bone marrow. Preferably, these endothelial cells may be derived from a mammalian donor, more preferably, human and most preferably, said cells may be derived from the recipient of said graft.

In a further embodiment, the cell layer comprised within the vascular graft of the invention may be a confluent monolayer of endothelial cells, wherein said cells are genetically. modified to express a membranal adhesive non-catalytic heparanase molecule or any functional fragment thereof. More particularly, these endothelial cells may be cells transformed or transfected with an expression vector comprising a nucleic acid construct encoding a membranal adhesive non-catalytic heparanase molecule according to the invention or any fragment thereof. Such endothelial cells exhibit enhanced adherence properties and enhanced survival and resistance to apoptosis. The enhanced survival and resistance to apoptosis may be a result of activation of the PI3'K/Akt signaling pathway by said membranal adhesive non-catalytic heparanase molecule.

Akt is a serine threonin protein kinase which plays a major role in regulating cell growth and survival. Three isoforms of Akt have been identified to date: Aktl, Akt2 and Akt3 [Datta et al., Genes and Development 13: 2905-2927 (1999) ]. Growth factors and survival factors initially activate PIS'kinase, which then phosphorylates PIP2 [PtdIns (4) P or PtdIns (4,5) P2] into PIPS [PtdIns (3,4) P2 or PtdIns (3,4, 5) Pst which recruits PDK1, ILK. 1 and Akt to the plasma membrane. This cascade facilitates the activation of Akt by PDK1 and ILK1. PTEN, on the other hand, is a PIP3 phosphatase that antagonizes the function of PI3'K and prevents Akt activation.

Activated Akt suppresses apoptosis by phosphorylating and inhibiting different target proteins required for this process. In particular, Akt phosphorylates Bad protein and prevents it from binding the mitochondrial membrane [Datta et al. , Cell 91: 231-41 (1997); del Peso et al. , Science 278: 687-689 (1997) ]. Akt also phosphorylates and inactivates capsase 9 [Cardone et al. , Science 282: 1318-1321 (1998) ], as well as a forehead transcription factor, which facilitates the expression of cell death factors such as Fas ligand [Brunet et al. , Cell 9G : 857-868 (1999)]. In addition, Akt phosphorylates IKKa, a IKB kinase subunit. In turn, the activated IKK phosphorylates IKB and targets it for degradation, resulting in increased NFKB activity and the expression of proteins required for cell survival [Romashkova et al. , Nature 401: 86-90 (1999); Ozes et al. , Nature 401: 82-82 (1999)].

The endothelial cells may be transformed with the expression vector using any convenient method. Techniques known in the art include calcium phosphate precipitated DNA transformation, electroporation, protoplast and liposome mediated fusion, DNA-coated particles, transfection, and infection, where the chimeric construct is introduced into an appropriate virus.

The vector may be episomal, e. g. plasmid or virus, or integrated into the host cell genome. Examples of expression vectors for mammalian cells known in the art and commercially available include plasmids, retrovirus-based vectors, herpes simplex virus-based vectors [see Mesri et al. Circ. Res. 76 : 161- 167 (1995) ] and adenovirus-based vectors [see Muhlhauser et al. Circ. Res.

77: 1077-86 (1995) ]. Plasmid vectors may include sequences from SV-40 or Epstein Barr virus, such as those that increase the stability of the vector after transfection.

The vector shall include regulatory sequences for the expression of the non- catalytic heparanase in endothelial cells, including a promoter, optional enhance, termination sequences, and the like. The adhesive heparanase coding sequence shall be inserted 3'to the promoter, and to the sequences for initiation of translation operable in mammalian cells. A wide variety of promoters have been described in the literature. There are constitutive or inducible promoters, where induction may be associated with a specific cell type or a specific level of maturation. Of particular interest are constitutive high level promoters, including the B-actin promoter, SV40 early and late promoters, immunoglobulin promoter, human cytomegalovirus (CMV) promoter, etc. , as well as regulatable promoters such as the tetracycline- regulated promoter system or the metallothionine promoter. The promoters may or may not be associated with enhancers, where the enhancers may be naturally associated with the particular promoter or associated with a different promoter.

A termination region is provided 3'to the adhesive heparanase coding region, where the termination region may, be naturally associated with the cytoplasmic domain or may be derived from a different source. A wide variety of termination regions may be'employed without adversely affecting expression.

The recipient of the implant may be any mammalian species, including canines, felines, equines, bovines, ovines, etc. , and primates, particularly humans. Animal models, particularly small mammals, e. g. murine, lagomorpha, etc. are of interest for experimental investigations.

The present invention further provides a method for preparing an improved implantable medical device comprising a synthetic tubular element having a, luminal surface and a cell layer directly or indirectly adhered to said luminal surface, wherein said cell layer comprises cells genetically modified to express a membranal adhesive non-catalytic heparanase molecule or any functional fragment thereof. The grafts are to be used as a replacement for natural vessels, e. g. arteries and veins, during vascular bypass or other replacement procedures. The thrombogenicity of synthetic materials conventionally used in such prostheses shall be reduced by the deposition of host or donor endothelial cells in the interstices of the grafts interior wall.

The growth and survival of such endothelial cells after deposition will be increased by genetically modifying the cells to express the adhesion promoting non-catalytic heparanase, which in addition to adherence, also induces activation of the PI3'K/Akt pathway, leading to attenuation of apoptosis.

The method of the invention comprises the steps of : (a) providing a suitable synthetic tubular element having a luminal surface ; and (b) directly or indirectly adhering said cell layer onto said luminal surface.

In one specific embodiment, the method of the invention further comprises in step (b) the sub-steps of : (i) immobilizing a layer of adhesion molecules selected from the group consisting of fibronectin, laminin, collagen, the non- catalytic heparanase of the invention, and any combination thereof onto said luminal surface; and (ii) adhering or preferably seeding a layer of cells onto said immobilized adhesion molecules layer of (i). Said cells being genetically modified to express a membranal adhesive non-catalytic heparanase molecule or any functional fragment thereof.

Preferably, the adhesion molecules are covalently bound to the device by either one of two approaches. In one embodiment, the adhesion molecules are covalently bonded to the biomaterial surface. In an alternative embodiment, the adhesion molecules are covalently bound to adjacent adhesion molecules in a manner that produces a crosslinked network of adhesion molecules, with the network being physically entrapped within the interconnecting porosity of the biomaterial. Preferred devices provide the attached adhesion molecules in a form that provides effective activity after implantation or in the cell culture conditions used for seeding. Desirably, the covalent bonding with either approach is achieved with latent reactive groups.

In the embodiment in which adhesion molecules are covalently bound to the biomaterial surface, the molecules are desirably covalently linked to the surface through a linking group, the linking group including the residue of a latent reactive group employed to covalently bond to the surface.

In another preferred embodiment, the coating of adhesion molecule is generated by covalent linkage of adjacent adhesion molecules, resulting in a network of crosslinked adhesion molecules being physically entrapped within the biomaterial.

Preferably, an adequate density of adhesion molecule is uniformly and homogeneously distributed on the material surfaces to provide a continuous surface of adhesion molecule upon which endothelial cells can attach and migrate.

The genetically modified endothelial cells are placed onto the luminal surface of a synthetic vessel, where the vessel is a tubular member having a substantially uniform diameter. As described above, suitable materials for the vessel include expanded polytetrafluoroethylene (e-PTFE) and Dacron.

The vessel may generally be of small caliber, usually at least about 0. 5 mm in internal diameter, more usually at least about 1 mm in diameter, and not more than about 5 mm in diameter, more usually not more than about 3 mm in diameter. The vessel may be cut into lengths prior to deposition of the endothelial cells, generally ranging from 1 to 25 cm in length. The vessel may be pre-incubated with the media used for cell deposition for a period of from about 0. 5 to about 3 hours.

The cells are deposited on the vessel by sodding or seeding methods, as known in the art. Sodding procedures place the cells directly onto the polymeric internal surface as well as into the interstices of the vessel, generally under mild pressure. For example, one terminus of the vessel may be clamped, and the cells injected with a syringe through the open end. The vessel is permeable to water, and so the medium is forced through the interstices of the wall, while the cells are retained within the lumen.

Seeding procedures mix the cells with blood or plasma, and then add them to the vessel during the pre-clotting period. There are several versions of the technique known as seeding. The synthetic graft is then incubated in vitro with rotation to allow the binding of the endothelial cells to the luminal surface. After several hours or days in culture, the graft can be implanted.

Alternatively, autologous blood can be forced under pressure through the interstices of the synthetic graft to allow retention of blood cells and protein onto and into the graft prior to addition of the endothelial cells (either passively, or actively under pressure). A third alternative is to mix the endothelial cells with the blood prior to the application onto and into the graft.

After deposition, the vascular graft may be implanted immediately into the recipient, or maintained in culture for a period of time. The culture conditions are conventional for endothelial cells. For example, the prosthesis may be deposited in a plate or well containing medium and fetal calf serum, then incubated at 37° C. The culturing medium is changed at least every 2-3 days, usually daily. The vascular graft will generally be maintained in culture for not more than about three weeks, usually not more than about two weeks, and, preferably, it will be used within about one week.

In a particularly preferred embodiment, the cells used by the vascular graft of the invention preferably overexpress adhesive non-catalytic heparanase molecule having the amino acid sequence of any one of SEQ ID NO: 10,11 and 12, encoded by the nucleic acid sequence of any one of SEQ ID NO: 4,5, and 6, respectively.

In a preferred embodiment, this method is particularly suitable for preparing an implantable medical device as defined'by the invention.

The implantable device of the invention and particularly the vascular graft may be useful for any vascular bypass or replacement surgery, as well as in any situation in which'the flow of blood through a vessel has been compromised and needs repair. Such compromise may result from atherosclerosis with lesion formation and intimal hyperplasia or from vascular injury, including coronary artery disease, peripheral vascular disease or other forms of occlusive arterial disease. The method of surgical replacement is conventional for synthetic material vascular grafts.

The adhesion properties of heparanase revealed by the present invention may further serve as a tool for the identification of novel substances that modulate adhesion. Such screening may particularly be applied for identification of adhesion inhibitors that may be used in cases where adhesion is not desired.

Therefore, in yet another aspect, the invention provides a method for screening for a substance that modulates cell-to-cell and cell-to-matrix adhesion between a cell expressing an adhesion promoting non-catalytic heparanase molecule and endothelial cells or matrix, the method comprising: (a) providing a cell expressing a recombinant adhesion promoting non- catalytic membranal heparanase molecule; (b) performing a first adhesion assay for testing the ability of said adhesive heparanase expressing cells to adhere to a naturally produced subendothelial ECM or to confluent vascular endothelial cell monolayer to obtain the first adhesion assay results; (c) performing a second adhesion assay for testing the ability of said adhesive heparanase expressing cells to adhere to a naturally produced subendothelial ECM or to confluent vascular endothelial cell monolayer, in the presence of a test substance, to obtain the second adhesion assay results; (d) comparing said first and second adhesion assay results to determine whether said test substance affects the adhesion between the adhesive heparanase expressing cells and subendothelial ECM or the confluent vascular endothelial cell monolayer ; and (e) identifying modulating substances as compounds that affect the binding between a cell expressing the membranal non-catalytic heparanase adhesive molecule and said matrix or endothelial cells.

According to a preferred embodiment of the screening method, said adhesive heparanase expressing cells, are cells transformed or transfected with the nucleic acid constructs of the invention. Most preferably, these cells were anchorage-independent cells prior to transfection or transformation with the constructs of the invention.

It should be appreciated that the findings of the invention may also be applicable for screening for a substance inhibiting cell-to-cell and'cell-to- matrix adhesion mediated by heparanase, and'hence potentially inhibiting a cell adhesion-mediated pathology. Such screening may comprise the steps of : (a) providing a cell expressing a recombinant adhesion promoting non- catalytic membranai heparanase molecule ; (b) performing a first adhesion assay for testing the ability of the adhesive heparanase expressing cells to adhere to a naturally produced subendothelial ECM or to confluent vascular endothelial cell monolayer to obtain first adhesion assay results; (c) performing a second adhesion assay testing the ability of the said adhesive heparanase expressing cells to adhere to a naturally produced subendothelial ECM or to confluent vascular endothelial cell monolayer, in the presence of a test substance, to obtain second adhesion assay results; (d) comparing the first and second adhesion assay results to determine whether said test substance inhibits adhesion between the adhesive heparanase expressing . cells and subendothelial ECM or the confluent vascular endothelial cell monolayer; and (e) identifying an inhibiting substance as a compound that reduces binding between a cell expressing the membranal non-catalytic adhesive heparanase molecule and said matrix or endothelial cell monolayer, wherein reduction in attachment of said cell to said matrix or endothelial cells is indicative of inhibition of cell-to-cell or cell-to-matrix adhesion mediated by membranal non-catalytic heparanase.

Adhesive interactions are known to be extremely important in the immune system, in which the localization of immune mediator cells is likely to be due, at least in part, to adhesive interactions between cells. Re-circulation of lymphoid cells is non-random; lymphocytes demonstrate a preference for the type of secondary lymphoid organ that they will enter. In trafficking through a secondary lymphoid organ, lymphocytes must first bind to the vascular endothelium in the appropriate post-capillary venules, then open up the tight junctions between endothelial cells, and finally migrate into the underlying tissue. Migration of recirculating lymphocytes from blood into specific lymphoid tissues, called homing, has been associated with complementary adhesion molecules on the surface of the lymphocytes and on the endothelial cells of the high endothelial venules.

Likewise, the adherence of polymorphonuclear leukocytes to vascular endothelium is believed to be a key event in the development of an acute inflammatory response, and appears to be required for an effective chemotactic response as well as certain types of neutrophil-mediated vascular injury. When stimulated by specific agonist substances, the polymorphonuclear leukocytes [Tonnensen et al. J. Clin. Invest. 74: 1581-1592 (1984) ], endothelial cells [Zimmerman et al. J. Clin. Invest. 76 : 2235-2246 (1985); Bevilacque et al. J. Clin. Invest. 76: 2003-2011], or both [Gamble et al.

Proc. Natl. Acad. Sci. U. S. A.-8ll : 8667-8671 (1985)] become adhesive; as a result, polymorphonuclear leukocytes accumulate on the endothelial cell surface.

Therefore, the invention may further relate to a, method for inhibiting the adherence of lymphocytes to cytokine-activated endothelial cells, comprising exposing the lymphocytes to an effective amount of a substance which inhibits cell-to-cell or cell-to-matrix adhesion mediated by heparanase.

The method of the invention may therefore be useful in preventing the egress of lymphocytes through the vascular endothelium and into the tissue.

Accordingly, the present invention may provide for a method of suppressing the immune response in human patients in need of such treatment. In particular embodiments, the present invention provides methods of treatment of diseases associated with chronic or relapsing activation of the immune system, including collagen vascular diseases and other autoimmune diseases (such as systemic lupus erythematosis and rheumatoid arthritis), multiple sclerosis, asthma, and allergy, to name but a few. The present invention also provides methods of treatment of relatively acute activations of the immune system in patients in need of such treatment, including, for example, and not by way of limitation, graft versus host disease, allograft rejection, or transfusion reaction.

Depending on the nature of the patient's disorder, it may be desirable to inhibit lymphocyte migration into tissues systemically or, alternatively, locally. For example, in diseases involving multiple organ systems, such as systemic Lupus Erythematosus, it may be desirable to inhibit lymphocyte adhesion systemically during a clinical exacerbation. However, for a localized contact dermatitis, it may be preferable to restrict migration of lymphocytes only into those tissues affected.

Still further, the findings of the invention may be applicable for a method for preventing lymphocytes migration into a tissue comprising contacting said lymphocytes with an effective amount of a substance which inhibits cell-to- cell adhesion or cell-to-matrix adhesion mediated by heparanase, and thereby preventing the adherence of lymphocytes to cytokine activated endothelial cells mediated by membranal non-catalytic heparanase.

According to a preferred embodiment, the substance used in both methods of the invention may be identified by the screening method described herein above.

The findings of the present invention may enable specific intervention in the migration of lymphocytes through the vascular endothelium and into tissues, by preventing the cell-to-cell and cell-to-matrix interactions mediated by non- catalytic heparanase. The present invention, therefore, has particular clinical utility in suppression of the immune response. In various specific embodiments of the invention, the adherence of lymphocytes to endothelium may be inhibited systemically, or may, alternatively, be localized to particular tissues or circumscribed areas. Accordingly, the present invention may be applicable for determination of a substance useful in the treatment of diseases involving autoimmune responses as well as other chronic or relapsing activations of the immune system, including allergy, asthma, and chronic inflammatory skin conditions.

Cell adhesion is a process by which cells associate with each other, migrate towards a specific target or localize within the extra-cellular matrix (ECM).

As such, cell adhesion constitutes one of the fundamental mechanisms underlying, numerous biological phenomena. For example, cell adhesion is responsible for the adhesion of hematopoietic cells to endothelial cells and the subsequent migration of those hemopoietic cells out of blood vessels and to the site of injury. As such, cell adhesion plays a role in pathologies such as inflammation and immune reactions in mammals.

Thus, the findings of the present invention may further be applicable for a method of treating a mammalian subject suffering from a cell adhesion mediated pathology. Such method may include administering to said subject a therapeutically effective amount of a substance which inhibits cell-to-cell adhesion or cell-to-matrix adhesion mediated by a non-catalytic membranal heparanase.

More specifically, such cell adhesion mediated pathology may be any one of tumor metastasis, autoimmunity and inflammatory diseases.

Finally, the findings of the invention may be applicable in a method for the inhibition of platelet aggregation thereby preventing thrombotic disease, said method comprises the step of administering to said subject a therapeutically effective amount of a substance which inhibits cell-to-cell adhesion or cell-to- matrix adhesion mediated by non-catalytic membranal heparanase.

In a specifically preferred embodiment, the substance used by the methods of the invention may be identified by the screening method of the invention.

Both platelet activation and thrombin-mediated clot formation are essential to hemostasis. However, perturbations in either of these two hemostatic mechanisms may result in the formation of pathogenic thrombi (blood clots) which block blood flow to dependent tissues. This is the case in a variety of life-threatening vascular diseases, such as myocardial infarction, stroke, peripheral arterial occlusion and other blood system thromboses. Since various biochemical pathways contribute to vascular disease, treatment and prevention may focus on either inhibiting platelets, inhibiting thrombin or directly dissolving the blood clot.

Therefore, strategies to control platelet aggregation and release are desirable in the treatment of vascular disease. Furthermore, inhibition of platelet aggregation may also be desirable in the case of extracorporeal treatment of blood, such as in dialysis, cardiopulmonary bypass surgery, storage of platelets in platelet concentrates and following vascular surgery.

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.

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

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

Disclosed and described, it is to be understood that this invention is not limited to the particular examples, process steps, and materials disclosed herein as such process steps and materials 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.

Throughout this specification and the 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.

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.

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 Cells and cell culture Methylcholanthrene-induced non metastatic mouse Eb (L5 1 7SY) T- lymphoma cells were kindly provided by Dr. V. Schirrmacher (DKFZ, Heidelberg, Germany). The cells were grown in RPMI 1640 medium (Life Technologies, Grand Island, N. Y.) supplemented with (3-mercaptoethanol (5 x 10-5 M) and 10% FCS, as described [Goldshmidt (2002) ibid; Goldshmidt (2001) ibid.]. Cultures of bovine corneal endothelial cells were established from steer eyes and maintained in DMEM (lg glucose/liter) supplemented with 5% newborn calf serum, 10% FCS and Ing/ml bFGF, as described [Vlodavsky, I. In Current protocols in Cell Biology Vol. 1 pp. 10.14. 11- 10.14. 14 (1999 (b) )]. Confluent cells were dissociated with 0.05% trypsin and 0.02% EDTA and sub-cultured at a split ratio of 1: 10 [Vlodavsky (1999 (b)) ibid.]. Adult bovine aortic endothelial cells (ABAE) were established from bovine aorta and maintained in DMEM (1 g glucose/liter) supplemented 10% FCS and 1 ng/ml bFGF, as described [Vlodavsky (1999 (b)) ibid.].

Human umbilical endothelial cells (HUVEC) were kindly provided by Dr.

Neomi Lanir (Rambam Medical Center, Haifa, Israel) and were cultured in gelatin-coated dishes as described [Ilan et al. J. Cell Sci. Ill : 3621-3631 (1998) ]. Bovine aortic endothelial cells (BAEC) were kindly provided by Dr.

Nitzan Resnick (Technion, Haifa) [Shay-Salit et al. Proc. Natl. Acad. Sci.

USA 99: 9462-9467 (2002) ] and were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS and antibiotics. Mutant Chinese hamster ovary cells (pgsA-745), deficient in xylosyltransferase and unable to initiate glycosaminoglycan synthesis, were purchased from the ATCC and cultured in RPMI medium supplemented with 10% FCS and antibiotics. HT-29 colon carcinoma cells (also known as WiDr) were purchased from the ATCC and cultured in DMEM as above. These cells synthesize perlecan, a secreted proteoglycans, but no other proteoglycans [Fuki et al. J. Biol. Chem. 275,25742-25750 (2000) ] and are therefore a useful model system for HSPG-deficient cells. In vitro tube-like structure formation assay on Matrigel-coated plates were performed as described [Ilan et al. J. Cell Sci. 112: 3005-3014 (1999)]. HEK 293 cells, stably transfected with the human heparanase gene construct in the mammalian pSecTag vector (invitrogen), were kindly provided by lmClone Systems Inc. (New York, lKlY-). This plasmid vector provides the IgG K signal peptide to ensure efficient protein secretion, together with Myc and His tags at the protein C- terminus to enable easy detection and purification as described in detail [Zetser, et al. Cancer Research (2003) in press].

The U87-MG glioma cells were purchased from the American Type Culture Collection (ATCC). The cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with glutamine, pyruvate, antibiotics and 10% fetal calf serum in a humidified atmosphere containing 8% C02 at 37°C.

Plasmids The pcDNA3 plasmids (Invitrogen, NV Leek, Netherlands) containing the different hpa cDNA sequences encoding chicken-, human-, and chimeric- heparanase cDNAs (Chk-hpa, H-1Tpa and chimeric-hpa, respectively), or an empty pcDNA3 plasmid, all under the control of the CMV promoter, were prepared and subcloned as previously described [Goldshmidt, (2001) ibid.]. A construct encoding mutated chimeric heparanase (Mut-chimeric-hpa) was prepared as described [Goldshmidt, (2001) ibid.], except that a point mutation was first introduced in the H-hpa, replacing the proton donor Glu225 with alanin [Hulett (2000) ibid.].

Transfection Eb mouse lymphoma cells constitutively over-expressing the various heparanase constructs (chk-hpa, H-hpa, chimeric-hpa, Mut-chimeric-hpa), or the pcDNA3 vector alone, were generated as previously described [Goldshmidt, (2001) ibid..]. Briefly, Eb cells 0.5 x 106 cells/ml were incubated o (48-72 h, 37 C) with a total of 1-2 ig DNA and 6 Ill FuGene transfection reagent (Boehringer, Mannheim, Germany) in 94 Ill Optimem (GibcoBRL, Iiivitrogeii corporation) [Goldshmidt, (2001) ibid.]. Transfected cells were selected with 350 pg/ml G418 (GibcoBRL) and stable populations of heparanase expressing cells were obtained and maintained in growth medium containing 150 µg/ml G418 to avoid the overgrowth of non transfected cells. Expression of heparanase was evaluated by RT-PCR, activity measurements and immunostaining [Goldshmidt, (2001) ibid.].

For stable transfection, U87 cells were transfected with the chimeric-hepa gene construct (the human heparanase cDNA fused to the chicken heparanase signal peptide) and with control pcDNA3 vector, using the FuGene reagent according to the manufacturer instructions (Roche), selected with G418 (1000 ug/ml) for 3 weeks, expanded and pooled. This parental transfected cell population was further selected for high heparanase- expressing cells by generating pools of-50 colonies/100 mm culture dishes and evaluating heparanase expression by immunoblot analysis. The pool with the highest expression level was further expanded and designated as"Hi" throughout the manuscript, while the parental transfected cells are referred to as"Low".

Preparation of dishes coated with ECM Bovine corneal endothelial cells were plated into 35-mm tissue culture dishes at an initial density of 2 x 105 cells/ml and cultured as described above, except that 4% dextran T-40 was included in the growth medium [Vlodavsky (1999) ibid.]. On day 12, the subendothelial ECM was exposed by dissolving the cell layer with PBS containing 0.5% Triton X-100 and 20 mM NH40H, followed by four washes with PBS [Vlodavsky (1999 (b)) ibid.]. The ECM remained intact, free of cellular debris and firmly attached to the entire area <BR> <BR> <BR> <BR> <BR> 35<BR> <BR> of the tissue culture dish. To produ8ce sulfate labeled ECM, Na2 SO4 (25 Ci/ml) (Amersham, Buckinghamshire, UK) was added on days 2 and 5 after seeding and the cultures were incubated with the label without medium change and processed as described [Vlodavsky (1999 (b)) ibid.] Nearly 80% of <BR> <BR> <BR> <BR> <BR> <BR> the ivity was incorporated into HSPGs.

Heparanase activity of Eb transfected cells Hpa transfected Eb cells were incubated (24 h, 370C, pH 6.6) with 35S. labeled ECM. The incubation medium was centrifuged and the supernatant containing sulfate labeled degradation fragments was analyzed by gel filtration on a Sepharose CL-6B column (0.9 x 30 cm). Fractions (0.2 ml) were eluted with PBS and their radioactivity counted in a-scintillation counter. Degradation fragments of HS side chains were eluted from Sepharose 6B at 0.5 < Kav < 0.8 (peak II). Nearly intact HSPGs were eluted just after the Vo (Kav < 0.2, peak I) (23,31) [\71odavsky (1994) ibid. ; Vlodavsky (1999 (a)) ibid.]. The experiment was performed at least 3 times and the variation in elution positions (Kav values) did not exceed 15%.

Heparamase actiuity assay Preparation of ECM-coated dishes and determination of heparanase activity were performed as described in details elsewhere [Vlodavsky (1999 (b)) ibid. ; Goldshmidt (2002) ibid.]. Briefly, purified heparanase (1 p. g) was incubated (4 h, 370C, pH 6.6) with soluble 35S-labeled ECM (peak I). The incubation medium 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. For evaluating the enzymatic activity of immobilized heparanase, soluble sulfate-labeled ECM (peak I) was applied onto heparanase-coated dishes and following incubation (4 h, 37°C) was fractionated by gel filtration as above.

All experiments were repeated at least three times, with similar results. Akt activation is presented as mean + SE of at least five independent experiments quantified by densitometry analysis.

Cell l Ad hesion Hpa transfected Eb cells were grown (1 x lOG cells/ml, 48 h, 37°C) in complete medium in the presence of [3H]-thymidine (1 ACi/ml) (Amersham). The labeled cells were washed (x3) free of unincorporated thymidine and incubated (37°C, pH 7. 2) in complete medium for various time periods on either intact naturally produced ECM (15 min-8 h), or confluent vascular endothelial cell monolayers (2 h-24 h). Following incubation, cells were washed (x3) with serum free medium and the remaining firmly attached cells were solublized (2 h, 0.1 M NaOH, 37°C,) and counted in a ß-scintillation counter. In some experiments the ECM was pretreated with ECM degrading enzymes (heparanase, chodroitinase ABC, hyaluronidase). Cell adhesion was also preformed in the presence of 1 mg/ml RGD or RAD peptides (Calbiochem, La Jolla, CA).

Heparanase immobilization and adhesion studies Heparanase (1 pg/ml) was diluted with 50 mM carbonate/bicarbonate buffer, pH 9.6 and applied onto tissue culture dishes (16 h, 4°C). Dishes were subsequently washed and the remaining biding sites were blocked with 1% BSA for 1 h at 37°C. Control dishes were coated with BSA alone. HUVEC (2x105) were plated onto BSA-or heparanase-coated dishes (1 h, 37°C) and, following several washes, fixed with 4% PFA, photographed and counted. For immunoblot analysis, HUVEC were similarly plated onto heparanase-coated dishes or were left in suspension as control and cell lysates were prepared as below.

Immunoprecipitation and Western immunoblotting Eb cells were incubated on naturally produced ECM (2 x lOG cells, 20 and 60 min, RPMI complete medium, 37°C), rinsed with PBS and lysed with RIPA buffer (1% Triton X-100,1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 50 mM Tris, pH 8.0, 20 llg/ml aprotinin, 2 llg/ml leupeptin, 1 llg/ml pepstatin, 2 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate), followed by immunoprecipitation (IP) with anti-paxillin monoclonal antibodies (Transduction Laboratories, Lexington), and protein-G Sepharose beads (Sigma). The cell extracts were then subjected to 10 % SDS-PAGE and Western blot analysis, utilizing the anti-paxillin, or anti-phosphotyrosine (4G10, Upstate Biotechnologies, Lake Placid) monoclonal antibodies, followed by secondary peroxidase-conjugated goat anti-mouse antibodies.

For the EC experiments, cell cultures were pre-treated with 1 mM orththovanadate for 10 min at 37°C, washed twice with ice cold PBS containing 1 mM orththovanadate and scraped into lysis buffer containing a cocktail of proteinase inhibitors (Roche, Indianapolis, IL), and lysate. samples were subjected to immunoblotting as described above.

Aittibodi. es a7id reage7ltS The following antibodies were purchased from Santa Cruz Biotechnology: anti ERK-2 (sc-154), anti phospho-ERK (sc-7383), anti Akt (sc-5298) and anti Myc epitope tag (sc-40). Anti phospho-Akt antibody was purchased from Cell Signaling Technology (Beverly, MA). Anti-heparanase antibody (&num 1453) was raised against the entire 65 kDa heparanase precursor isolated from the conditioned medium of heparanase-transfected 293 cells. This antibody was affinity purified on immobilized bacterially-expressed 50 kDa heparanase- GST fusion protein [Levy-Adam et al. Biochem. Biophy. Res. Comm. 308: 885- 891 (2003)]. Laminaran sulfate (LS) was purchased from Qingdao Third Pharmaceutical Compant (Qingdao, China) and was used at concentration of 10 ßg/ml [Miao et al. , Int. J. Cancer 83: 424-431 (1999) ]. The phosphatidylinositol 3 (PI 3) -kinase inhibitor LY 294002 was purchased from Sigma (Saint Louis, MO) and was dissolved in DMSO. Equivalent volume of the vehicle control was always run in parallel.

Heparamase upta. ke studies For uptake studies, the 65 kDa Myc-tagged heparanase precursor was added to confluent cell cultures at a concentration of 1 pIg/ml under serum-free conditions. At the indicated time points, the medium was aspirated, cells were washed twice with ice-cold PBS and total cell lysates were prepared as described below. Heparanase uptake and processing were analyzed by immunoblotting with anti c-Myc and anti heparanase (#1453) antibodies.

Matrigel invasion assay Eb cells were grown for 48 h in the presence of [3H]-thymidine (1 µCi/ml) (Amersham) and assayed (37°C, 5% C02 incubator, 6h) for invasion through Matrigel-coated filters using blind-well chemotaxis chambers and polycarbonate filters, as described [Goldshmidt (2002) ibid.]. Medium conditioned by 3T3 fibroblasts was applied as a chemoattractant and placed in the lower compartment of the Boyden chamber [Goldshmidt (2002) ibid.].

Following incubation for 5 and 24 lu at 37°C in 5% C02 incubator, the upper surface of the filter was wiped free of cells and migrated cells on the lower surface of the filter were fixed, stained with 0.5% crystal violet (Sigma) and counted by examination of at least five microscopic fields the filters counted in a (3-scintillation counter.

For EC, migration assay was similarly performed except that inserts were coated fibronectin.

Irzdirect Imnauofluorescertce Eb cells were permeabilized for 3 min in PBS containing 0.5% Triton X-100 and 4% formaldehyde, followed by fixation with 4% formaldehyde in PBS for 20 min. The cells were then incubated for 1 h with anti-paxillin monoclonal antibodies in PBS, washed, and further incubated (45 min, 24°C) with Cy3- conjugated goat anti-mouse antibodies and FITC-conjugated phalloidin. After extensive washes, coverslips were mounted in elvanol, and images of the double-stained cells were acquired using an Axioscope microscope (Zeiss, Oberkochen, Germany) equipped with a charged-coupled device (CCD) camera (1024x1024 pixels chip readout generating 12-bit digital data).

Heparanase staining was performed on either permeabilized (methanol,- 20°C, 5 min), or non-permeabilized (4% freshly prepared paraformaldehyde in PBS, 20 min, 4°C) cells. The cells were then incubated with anti-heparanase rabbit polyclonal antibodies (p9), kindly provided by Insight Ltd. (Rehovot, Israel), diluted to 2 µg/ml. These antibodies are directed against a specific peptide: PGKKVWLGETSSAYGGGAP (also denoted by SEQ ID NO.: 13) of the human heparanase enzyme. Cy2-conjugated goat anti-mouse antibodies (Jackson Laboratories, Bar Harbor, MA) diluted 1: 200, were used as secondary antibodies.

Example 1 Cell surface heparanase increases cell adhesion to ECM and endothelial cells Mouse Eb-lymphoma cells are anchorage independent, grow in suspension and exhibit no attachment or cell spreading when plated on regular, or ECM coated dishes. The inventors have noticed that Eb cells stably transfected with a cDNA encoding surface associated and secreted form of heparanase (chilueric-hpa) [Goldshmidt (2001) ibid.] became firmly attached to the tissue culture plastic, exhibiting an adherent phenotype and spreading, characteristic of anchorage-dependent cells (Fig. 1A). On the other hand, Eb cells over-expressing the human enzyme (H-hpa) and exhibiting the heparanase protein predominantly in perinuclear acidic vesicles [Goldshmidt (2001) ibid.], grew in suspension and remained floating (Fig. 1A), similar to mock transfected cells (not shown). To better elucidate the involvement of cell surface heparanase in cell adhesion and spreading, Eb cells transfected with surface associated (chk-hpa, chimeric-hpa)-or perinuclear (H-hpa)- heparanase were compared for their ability to adhere to a naturally produced subendothelial ECM and to a confluent vascular endothelial cell monolayer.

The various heparanase constructs are schematically presented in Fig. 1B. As demonstrated in Fig. 1C, cells expressing surface associated heparanase (Chk-hpa, chimeric-hpa) adhered to ECM within minutes. Nearly 50% of the added cells attached to the ECM within 15 min of incubation, while 90-100% of the cells adhered to the ECM within 1 h, remaining firmly bound thereafter (Fig. 1C). Cell adhesion was followed by cell spreading (Fig. 1A, top-left). In contrast, cells transfected with the human heparanase (H-hpa), or mock transfected cells, exhibited very low or no adhesion to ECM even after a prolonged (i. e. , 8 h) incubation time (Fig. 1C). These experiments indicate that heparanase stimulates rapid and firm cell adhesion in non- adherent cells, provided that the enzyme is expressed on the cell surface and/or secreted.

The various hpa transfected cells were also tested for their ability to adhere to confluent, contact inhibited endothelial cell monolayers. Regardless of the expressed heparanase type, Eb cell adhesion to endothelial cell monolayers was relatively slow (about 15% of the added hpa-transfected cells adhered within 2 h) and reached a lower level (< 30 % cell adhesion within 24 h) as compared to cell adhesion to ECM. A three to five fold increased adhesion was noted with Chk-1zpa transfected cells over-expressing surface heparanase, vs. cells expressing the enzyme in perinuclear acidic vesicles (H- hpa), or mock transfected cells, respectively (Fig. 1D). Adhesion to endothelial cells was, however, much higher then cell adhesion to plastic, requiring at least 24 h of incubation and occurring exclusively with cells expressing the surface associated and secreted enzyme (not shown).

Since Eb murine lymphoma cells are non-adherent and grow in suspension, the'inventors have next examined whether heparanase can affect the adhesive properties of adherent cells, as well. It was found that human primary foreskin fibroblasts (HFF) that were first incubated with exogenously added 65 kDa latent heparanase attached much faster to the tissue culture plastic as compared with control untreated cells (Fig. 1F).

Again, heparanase-stimulated cell attachment was accompanied by an increased cell spreading (not shown).

Moreover, mock Eb cells (transfected with the control pCDNA3 plasmid) that were incubated in the presence or the absence of exogenously added 65 kDa heparanase, demonstrated significantly enhanced attachment to ECM (Fig.

1E), indicating that transfected heparanase as well as exogenously added heparanase significantly improves the adhesive properties of the cells. It is worthwhile mentioning that the inventors have previously demonstrated that recombinant latent heparanase (65 kDa) binds to the cell surface and is then processed into its highly active 50 kDa form [Nadav (2002) ibid.].

Example 2 RGD containing peptides inhibit heparanase-7nediated cell spreadiftg, but not adhesion Heparanase mediated cell adhesion is likely to involve interaction between the surface associated enzyme and the ECM HS. To investigate this possibility, ECM was treated with human recombinant heparanase prior to its incubation with chimeric-lzpa transfected Eb cells. As demonstrated in Fig. 2, exposure of ECM to heparanase had no effect on cell adhesion despite removal of-65% of the total sulfate labeled material, measured by release of labeled HS degradation products. Similarly, cell adhesion was not affected by a combined pre-treatment of the ECM with heparanase and chondroitinase ABC, removing 85-90% of the ECM sulfate labeled material (not shown). Cell attachment was also not inhibited in the presence of 10 ptg/ml heparin, again suggesting that cell adhesion mediated by cell surface heparanase dose not necessarily involve interaction with the ECM HS. The inventors have next examined the possible involvement of integrin receptors in heparanase- mediated cell adhesion. For this purpose, chimeric-hpa transfected Eb cells were incubated on ECM in the absence or presence of an RGD containing peptide (i. e., Gly-Arg-Gly-Asp-Ser-Pro). An RAD containing peptide (i. e. , Gly- Arg-Ala-Asp-Ser-Pro) was used as a control. As demonstrated in Fig. 2A, both peptides had no effect on the number of cell adhering to the ECM.

There was also no effect of the peptides on cell adhesion to ECM that was pretreated with active 50 kDa human recombinant heparanase (Fig. 2A). A marked difference was, however, noted upon microscopic examination of the attached cells. Whereas in the absence (Fig. 1A), or presence (Fig. 2B, bottom) of the RAD peptide the chimeric-hpa transfected cells were spread and exhibited cell processes, cells plated on ECM in the presence of the RGD peptide were firmly attached, but failed to spread and remained round (Fig.

2B, top). These results indicate that cell surface heparanase play a role in the initial cell attachment, while cell spreading involves the subsequent participation of integrin receptors.

Example 3 Heparana, seanediated cell attachm. ent is associated with paxillin recruitment and tyrosine phosphorylation Cell adhesion may result in generation of several types of cell-ECM attachment sites (e. g. , focal contacts, fibrillar adhesions) [Zamir, E. , and CTeiger, B. J. Cell Sci. 114: 3583-3590 (2001) ]. The inventors have next examined whether cell surface heparanase triggers the formation of characteristic cell-ECM adhesions. As shown in Fig. 3A, Chk-hpa transfected Eb cells adhere, spread and form elongated cellular processes when plated on ECM. The inventors observed that the cytoskeletal protein paxillin is organized in defined regions within these processes, however, these regions contained relatively low amounts of phosphotyrosine (not shown). Moreover, the actin cytoskeleton of the heparanase transfected Eb adherent cells was organized radially rather then in elongated stress fibers (Fig. 3A).

Integrin-mediated cell adhesion often results in the activation of various biochemical responses, including tyrosine phosphorylation of cytoskeletal molecules (e. g. , FAK, paxillin) [Turner (2000) ibid.]. These initial biochemical events may trigger the activation of signaling pathways (e. g. , ERK) that affect cellular responses (e. g. , cell proliferation). Therefore, the inventors examined whether cell adhesion stimulated by cell surface associated heparanase results in activation of signaling events. In both chimeric-hpa- and H-hpa-transfected Eb cells, paxillin was not phosphorylated on tyrosine when the cells were in suspension (Fig. 3B). However, when plated on ECM, a profound tyrosine phosphorylation of paxillin, lasting for at least 1 h, was observed in the adherent chimeric-hpa transfected Eb cells (Fig. 3B). These data suggest that heparanase may promote the formation of adhesion sites similar to the recently described 3-dimensional adhesions formed by cells plated on endothelial cell derived ECM [Cukierman (2001) ibid.]. In contrast to integrin mediated signaling activation, we did not detect ERK activation following heparanase-mediated cell adhesion (not shown).

Cell adhesion often results in activation of various biochemical responses, including tyrosine phosphorylation of cytoskeletal elements such as FAK and paxillin [Turner (2000) ibid.]. The inventors have therefore examined whether cell surface heparanase triggers the formation of characteristic cell- ECM adhesions. The inventors observed that lymphoma cell spreading on ECM was accompanied by organization of paxillin within cellular processes formed by the adhering cells. These regions, however, contained relatively low amounts of phosphotyrosine, exhibiting little or no co-localization with paxillin. Moreover, the actin cytoskeleton of the adherent Eb cells was organized radially rather then in elongated stress fibers. Altogether, these results suggest that heparanase may promote the formation of adhesion sites similar to the recently described 3D matrix adhesions, typical for cell adhering to 3-dimensional ECM, rather then focal contacts characteristic of cell attachment to 2-dimensional surfaces [Cukierman (2001) ibid.]. Unlike integrin induced cell signaling and activation, ERK activation in response to heparanase-mediated cell adhesion was not detected, nor was the heparanase-mediated early cell adhesion inhibited by RGD containing peptides. These peptides inhibited, however, spreading of the same 1zpa- transfected cells, indicating that a non-integrin primary attachment phase is first stimulated by cell surface heparanase, followed by integrin dependent cell spreading.

Example 4 Heparanase-nzediated cell adhesiot dose not require heparanase eltzmatic activity The above described cell adhesion mediated by heparanase expressed on the cell surface was detected in a physiological pH (-7. 4) in which heparanase binds to heparin or HS, but no enzymatic activity is detected [Toyoshima (1999) ibid.]. It is therefore conceivable that the heparanase mediated cell adhesion may be independent of its endoglycosidase activity. To investigate this possibility, the adhesive properties of heparanase transfected cells in the absence or presence of laminaran sulfate, a potent inhibitor of heparanase activity and experimental metastasis were examined [Miao, (1999) ibid.].

Laminaran sulfate is not a substrate for heparanase, but rather binds to the enzyme and inhibits its hydrolytic activity. As shown in Fig. 4A, laminaran sulfate failed to inhibit adhesion of Chk-hpa-and chimeric-hpa- (chim-hpa) transfected Eb cells to ECM, although it efficiently inhibited heparanase activity (not shown).

On the basis of sequence alignments of human and chicken heparanase with a number of glycosyl hydrolases from GH-A, Glu225 and Glu343 of human heparanase and Glu204 and Glu323 of chicken heparanase were identified as the likely proton donor and nucleophile residues, respectively [Goldshmidt (2001) ibid.]. The substitution of either or both of these residues with alanine and the subsequent expression of the mutant heparanases in C6-glioma and Eb-lymphoma cells (both lacking endogenous heparanase expression and/or activity) demonstrated that the HS-degrading capacity was abolished. In contrast, the alanine substitution of two other glutamic acid residues in the human heparanase (Glu378 and Glu396), both predicted to be outside the active site, did not affect heparanase activity [Hulett (2000) ibid.]. The highly conserved acidic residues, function as the putative proton donor at Glu225 and a nucleophile at Glu343 in human, mouse rat and chicken and are considered crucial for heparanase function, since site-directed mutagenesis of these residues, but not others predicted to be outside the active site, completely abolished heparanase activity. It should be appreciated that the location of Glu225 and Glu343 refers to the position of these residues on the amino acid sequence of human heparanase according to GenBank Accession No.

AF144325.

Thus, to further investigate the relation between heparanase activity and its involvement in cell adhesion, a mutated chimeric heparanase (Mut-chimeric- hpa, Fig. 1B), which lacks heparanase activity, was generated. For this purpose, the proton donor Glui25 in the active site of the human heparanase [Hulett (2000) ibid.] was point mutated and substituted with alanin in order to abolish heparanase activity. In addition, a Glu343 to Ala and a double mutant having Glu225 and Glu343 replaced by Ala, were constructed (not shown). Eb cells stable transfected with the mutated hpa construct (Glu225 to Ala) expressed the processed 50 kDa heparanase protein (Fig. 4B, inset), but failed to exhibit any heparanase activity (Fig. 4B). Cell surface biotinilation (not shown), as well as immunofluorescent staining (Fig. 4C, inset), using polyclonal anti-heparanase antibodies revealed intense cell surface expression of the mutated enzyme in non-permeablized cells, similar to that observed with chimeric-hpa transfected Eb cells over-expressing the active cell surface associated heparanase. Evaluation of cell adhesion revealed that cells expressing the Mut-chime ric-17, pa firmly attached to ECM (Fig. 4C) and endothelial cell monolayers (Fig. 4D) in a manner similar to chimeric-1 ? pa transfected cells. Mock transfected cells exhibited little or no adhesion.

Similar results were also demonstrated for the Glu343 and the double Gluis and Glu343 mutants (not shown). These results clearly indicate that heparanase-mediated cell adhesion is independent of its enzymatic activity, provided that the enzyme is expressed on the cell surface.

Unlike the adhesion to ECM, Eb cells expressing the cell surface associated heparanase (Chk-hpa, chimeric-hpa) failed to attach to poly-L-lysine coated tissue culture plastic. In contrast, cells expressing the enzyme predominantly in perinuclear endosomal/lysosomal granules firmly adhered to poly-L-lysine (Fig. 4E), as compared to little or no adhesion to ECM (Fig.

1C & Fig. 4C). These results suggest that heparanase-mediated cell adhesion may in part be attributed to an effect on the net cell surface charge. Cell surface heparanase may interact with adjacent cell surface HS, reducing the net negative charge in this specific microenvironment, thereby accelerating interactions with the negatively charged ECM.

Example 5 Heparanase-mediated cell adhesion promotes cell invasion Previous studies revealed a correlation between heparanase expression and the metastatic potential of malignant cells [Parish (2001) ibid. ; Nakajima (1988) ibid. ; Goldshmidt (2002) ibid. ; Vlodavsky (1999 (a)) ibid.], as well as correlation between tumor metastasis and cell adhesion [Humphries, M.

Science 233: 467-470 (1986) ; Kohn, E. C. , and Liotta, L. A. Cancer Res. 55: 1856-1862 (1995); Borsig, L. et al. Proc. Natl. Acad. Sci. USA 98: 3352-3357 (2001); Hood; J. D. , and Cheresh, D. A. Nat. Rev. Cancer 2 : 91-100 (2002) ].

The inventors have recently demonstrated that cell surface expression and secretion of heparanase markedly promote cell invasion, both in Vit7'0 (matrigel invasion) and in vivo (metastatic dissemination) [Goldshmidt (2002) ibid.].

Since cell adhesion is a prerequisite for cell invasion, the invasive properties of Eb cells overexpressing the enzymatically inactive, surface associated heparanase (Mut-chimeric-17pa) were examined. For this purpose, Eb cells stable transfected with H-hpa, chimeric-hpa, or Mut-chimeric-hpa were compared for their ability to invade a reconstituted basement membrane (Matrigel). As demonstrated in Fig. 5, cells over-expressing the surface associated, active chimeric heparanase exhibited the highest degree of matrigel invasivon (4 fold higher than cells transfected with H-lzpa).

Interestingly, cells expressing the mutated, enzymatically inactive heparanase invaded the matrigel to an extent which was significantly higher (P = 0.002) than that exhibited by H-hpa transfected cells, albeit lower than the invasion capacity of chimeric-1lpa transfected cells overexpressing active heparanase (Fig. 5). The various 1V ? a-transfected cell types did not differ in their motility on. filters coated with collagen type IV, a process which does not involve enzymatic degradation, nor in their gelatinolytic activity, evaluated by zymography (not shown). These results indicate that cell surface heparanase facilitates cell invasion through its combined effect on cell adhesion and HS degradation.

The exact mechanism (s) by which heparanase promotes rapid cell adhesion is still unclear. The inventors found that cells expressing the various forms of cell surface associated heparanase, including the inactive enzyme, attached to HS-coated beads, while cells expressing intacellular heparanase, or mock transfected cells failed to attach to these beads (not shown). This result suggests that cell surface heparanase may bind to HS on the adhesion surface, whether ECM or endothelial cells, thereby forming a bridge between circulating cells and the blood vessel wall. It should be stated, however, that pre-incubation of the cells with excess of exogenous heparin did not inhibit their binding to ECM. Also, pre-treatment of the ECM with heparanase, hyaluronidase, chondroitinase ABC, or their combination did not inhibit the adhesion of cells expressing surface associated heparanase to ECM. It is therefore conceivable that the ECM counterpart on the adhering cells may not necessarily be a glycosaminoglycan (i. e. , heparan sulfate, hyaluronic acid, chondroitin sulfate, dermatan sulfate). On the other hand, cells expressing membrane associated heparanase attached poorly to surfaces coated with poly-L-lysine, while mock-or H-1wpa transfected cells attached quiet well to this positively charged substrate. Thus, heparanase may alter the cell surface charge, thereby modulating early cell adhesion. Moreover, while active heparanase cleaves the HS side chains, physiologically inactive (i. e. , pH >7. 2) cell surface heparanase can still bind to negatively charged residues and possibly mask them.

Cell surface HS and their core proteins may promote or inhibit cell adhesion and invasion {Wight (1992) ibid. ; David (1998) ibid. ; Sivaram, P. et al. J. Biol.

Chem. 270 : 29760-29765 (1995) ]. Inhibition occurs via HSPG binding and/or masking of inegrin receptors, or through a direct interaction of adhesion molecules with HS. In this case HS may affect integrin binding to the core protein via an RGD independent mode [Hayashi, Is., Madri, J. A. , and Yurchenco, P. D. J. Cell Biol. 119: 945-959 (1992) ]. In view of this possibility, and without being bound by the theory, the inventors propose that binding of cell surface heparanase to HS may liberate the inhibitory effect of HS, enabling integrin binding and subsequent cell adhesion.

An effect of heparanase on the surface properties of cells may trigger the observed re-organization of cytoskeletal molecules (e. g. , F-actin, paxillin) and tyrosine phosphorylation of paxillin. Actin re-organization and tyrosine phosphorylation are associated with cell adhesion and motility [West, K. et al. J. Cell Biol. 154: 161-176 (2001)]. The inventors found that heparanase- mediated cell adhesion also promoted cell invasion through a reconstituted BM (Matrigel). Inactive heparanase was found to stimulate cell invasion, possibly due to its effect on cell adhesion and cytoskeletal organization, both critically involved in cell invasion [Kohn (1995) ibid.].

Example 6 <BR> <BR> <BR> <BR> Heparanase induces activatio7t of the PI3'K/Alzt sig7zal transductio7z pathway in endothelial cells Heparanase is subjected to processing at the C-terminus end Under certain physiological conditions vascular endothelial cells (EC) are likely to be exposed locally to elevated heparanase levels. In order to study the effect of heparanase on EC, human (HUVEC) and bovine (BAEC) EC were incubated with exogenously added Myc-tagged latent 65 kDa heparanase precursor and heparanase uptake was followed by means of immunoblotting (Fig. 6A). As was noted previously for primary fibroblasts [Nadav (2001) ibid.] heparanase rapidly interact with primary EC in culture.

Immunoblot analysis with the anti-Myc epitope antibody revealed a strong signal already 15 min (HUVEC, upper panel, left) or 30 min (BAEC, upper panel, right) following heparanase addition. Interestingly, the anti Myc antibody only recognized a single protein band that corresponded to the added 65 kDa heparanase form, while a lower protein band was not detected even at later time points when heparanase processing was evident (second panel, 2 h, 4 h). Reactivity with the anti Myc antibody declined rapidly and precedes heparanase processing that became detectable by 60 min and was mainly apparent by 2 and 4 hours (Hepa, second panel). This may suggest that in addition to the processing at Glu109-Ser110 and Glnl57-Lysl58 that removes the linker domain and ultimately generate the 8 kDa and 50 kDa heparanase subunits, heparanase is also subjected to processing at the C- terminus, resulting in the loss of the tag sequences.

Heparanase induces A1et phosplTorylation The inventors have next examined the possible involvement of heparanase in inducing the PI3'K/Akt pathway, which is known to be involved in adhesion andmigration. As shown by Fig. 6, the addition of the latent 65 kDa heparanase protein resulted in similar Akt phosphorylation in EC. Akt activation appeared maximal 30 min after heparanase addition and was subsequently declined to basal levels (Fig. 6A, third panel). Moreover, heparanase-induced Akt phosphorylation exhibit dose-dependency (Fig. 6B), reaching maximal effect at 1 g/ml and about 5-fold increase as judged by densitometry analysis (Fig. 6C). This Akt activation was considered specific as the phosphorylation level of other signaling molecules such as MAPK was not induced or even reduced (Fig. 6A, fifth panel).

HSPG are riot reqzcired for hepara7aase-irzdzcced Akt actiuatiorz The rapid and efficient uptake of heparanase by various cell types is thought to be mediated, at least in part, by membranous HSPG [Nadav (2001) ibid.].

In order to evaluate the possible involvement of HSPG in Akt activation, heparanase was added to CHO 745 cells that exhibit minimal HS as well as chondroitin sulfate synthesis [Esko, J. D. Curr. Op. Cell Biol. 3: 805-816 (1991) ]. Heparanase addition to these HS-deficient cells resulted in Akt activation, similar in magnitude and kinetics to the one observed in EC (Fig.

7A, left, upper panel). This apperant HSPG-independent heparanase function was further confirmed by an additional cell type, HT-29. This human colon tumor-derived cell line synthesizes perlecan, a secreted HSPG mainly found in the ECM, but no other HSPG [Fuki (9000) ibid.]. The addition of heparanase to HT-29 cells resulted in time-dependent Akt activation (Fig.

7A, right, upper panel), further supporting the notion that heparanase- induced Akt activation does not relay upon membranous HS. As was noted for EC (Fig. 6A), Akt activation appeared specific and MAPK phosphorylation was not affected by heparanase addition (Fig. 7A, third panel). Densitometry analysis revealed a comparable, approximately 4 to 5 fold increase of Akt phosphorylation upon heparanase addition to EC and to HS-deficient cells (Fig. 7B). Furthermore, Akt activation was also evident upon heparanase addition to heparitinase-treated EC (data not shown). These findings suggest that cell surface molecules other than HSPGs are involved in heparanase- mediated Akt activation.

Example 7 <BR> <BR> <BR> <BR> Heparanase-i7tduced A/et activation is indepe7ldent of its enzymatic activity In terms of kinetics, maximal Akt activation appeared 30 minutes following heparanase addition and precedes its processing into the 50 kDa active form that was first detected 60 minutes after its addition (Fig. 6A), suggesting that Akt activation is independent of heparanase enzymatic activity. In order to further confirm that Akt activation is enzymatic activity-independent, CHO 745 cells were incubated with heparanase in the presence or absence of the heparanase inhibitor laminaran sulfate (LS, Fig. 8, left panel). The present inventors have previously reported that the chemically sulfated polysaccharide LS inhibit heparanase activity and exhibit anti metastatic effects [Miao (1999) ibid.].

The addition of heparanase to CHO 745 cells resulted in a 4-fold increase of Akt activation, in agreement with our previous results (Fig. 7A, left panel).

Interestingly, the addition of heparanase to CHO 745 cells together with LS at 10 Lg/Ml, concentration that completely abolish heparanase enzymatic activity [Miao (1999) ibid.], did not prevent Akt phosphorylation (Fig. 8A, B).

On the contrary, the addition of heparanase combined with LS resulted in further nearly two-fold induction of Akt phosphorylation (Fig. 8A, B). It is now well documented that several HS-bound growth factors prototyped by FGF family members, requires HS fragments or heparin in order to establish stable complex with their receptor and exert maximal biological effect [Ornitz, D. M. BioEssay 22: 108-112 (2000); Powers et al. Endocrine-Related Cancer 7: 16-197 (2000); Pellegrini, L. Curr. Op. Structural Biol. 11: 629-634 (2001) ]. Being a linear sulfated polysaccharide, the inventors have questioned whether the augmentation of Akt activation by LS could be reproduced by commercial heparin. To this end, HUVEC cells were left untreated or incubated with heparanase without or with heparin. As shown in Fig. 8C, Akt activation by heparanase was augmented by heparin in a manner and in magnitude comparable to LS (Fig. 8C-D), while heparin by itself had no effect (not shown). These findings support the concept that Akt activation is mediated by yet unknown heparanase receptor and that heparin may be required for maximal receptor activity.

Although the 50 kDa active heparanase enzyme was not detected in the heparanase preparation used herein by means of immunoblotting (not shown), heparanase activity became evident applying the inventor's sulfate- labeled ECM assay (Fig. 9A). It is therefore possible that Akt activation resulted in from these very low levels of the active heparanase form. In order to further role out the necessity of heparanase activity for Akt activation, heparanase was adhere to tissue culture dishes under high pH conditions (carbonate/bicarbonate buffer, pH 9.6). As shown by Fig. 9A, exposure of active heparanase to high pH conditions irreversibly inactivated the enzyme.

In addition, and in contrast with the soluble protein, the immobilized inactive heparanase protein is not subjected to uptake and processing, creating an experimental system in which inactive heparanase can only function from outside the cell. The immobilized heparanase protein facilitated 8-fold increase of EC adhesion compared with control BSA-coated dishes (Fig. 9B), supporting the notion that under certain physiological conditions heparanase may mediate cell'adhesion [Goldshmidt (2003) ibid. ; Gilat (1995) ibid.].

HUVEC adhesion to the immobilized inactive heparanase resulted in a marked 10-fold induction of Akt phosphorylation (Fig. 9C, upper panel; Fig.

9D), implying that Akt activation is heparanase activity-independent.

Interestingly, under these experimental conditions MAPK was also activated (Fig. 9D, third panel ; Fig. 9E), suggesting that additional adhesion-related pathways were engaged.

Example 8 Heparanase izduces Akt-depe7zdent EC migrsatio71 In order to evaluate the possible effect of exogenously added heparanase on EC behavior, HUVEC were plated on top of Matrigel-coated dishes. Under these conditions, EC rearrange themselves into lumen-containing tube-like structures [Ilan (1999) ibid.] in a process that resemble several features of angiogenesis. Control cultures exhibited only limited network formation (Con. Fig. 10A). In contrast, the addition of heparanase resulted in a well- organized EC structures composed from interconnected, elongated EC (Hepa, Fig. 10A). EC network formation on Matrigel-coated dishes was rapid and completed within 24 h. Thus, EC proliferation is not likely to play a major role in this assay, but rather cell migration.

In order to evaluate the effect of heparanase on EC migration, confluent HUVEC cultures were scraped with the wide end of 1 ml pipette tip and cell migration into the wounded area was followed over 7 days in culture without or with heparanase addition. As shown in Fig. 10B, heparanase addition stimulated a significant migratory response compared with non-treated cultures. Enhanced migration rates upon heparanase addition were further confirmed and quantified by migration assay on fibronectin-coated inserts.

Under these conditions, the addition of heparanase resulted in 2. 5-fold increase in HUVEC migration rates (Fig. 10C, D). Importantly, the addition of heparanase together with the PI 3-kinase inhibitor LY 294002 abolished the pro-migratory effect of heparanase (Figs. 10C, 10D), while the heparanase inhibitor LS had no effect on EC migration (Figs. 10C, 10E), in agreement with the present invention's finding that heparanase enzymatic activity is not required for Akt activation (Fig. SA, 8B). Moreover, heparanase stimulated a 5-fold increase of EC invasion through Matrigel-coated inserts and this effect was similarly prevented by LY 294002 (Fig. 10F, 10G). These findings clearly indicate that heparanase directly stimulates EC motility.

This effect is PI 3-kinase dependent, is likely mediated by heparanase- induced Akt activation and involved a putative heparanase receptor.

Example 9 Heparanase expression enhances U87 cell spreading and integrin a, cti. uatioit In order to rule out the possibility that activation of Akt by heparanase may be specific for endothelial cells, the inventors have next examined the adhesive properties and activation of adhesion related signaling pathways in glioma cells transfected with heparanase (U87 low and high expressing clones).

Careful examination yielded no differences between control and heparanase- transfected cell attachment rates to fibronectin-coated or uncoated surfaces (data not shown). Interestingly however, significant differences in cell spreading were noted. One hour after plating, control Vo cells (control) appeared rounded or slightly spread (Fig. 11A, left panel). In contrast, heparanase transfected U87 Low and Hi cells appeared to be significantly better spread. This increase in cell spreading was confirmed by staining for several characteristic components of focal adhesion complexes, namely phospho tyrosine (second panel), phospho-FAK (third panel), paxillin (fourth panel) and filamentous actin (fifth panel). The staining pattern of all the above markers strongly argues for an increased cell spreading and focal contact formation in heparanase-transfected cells. Moreover, a marked increase in FAK (Fig. 11B, upper panel) and Akt (second panel) phosphorylation levels was observed in heparanase transfected Low and Hi cell extracts prepared 1 h after plating, while other signaling components such as phospho-p38 (third panel) and phospho-JNK (fourth panel) were unchanged or even reduced (phospho-ERK, fifth panel) upon heparanase over expression, at the time point examined. In addition, an increase in Rac activation was noted in the heparanase transfectants (Fig. 11B, bottom panel), while Rho activation was not detected (data not shown). Furthermore, heparanase-transfected Low and Hi cells exhibited an increase in cell migration (Fig. 11C), further supporting an elevation of cell motility upon heparanase over-expression mediated, possibly, by Akt activation.