Pharmaceutical compositions and methods useful for modulating angiogenesis and inhibiting metastasis and tumor fibrosis

Abstract

Methods and compositions suitable for modulating angiogenesis in a mammalian tissue are provided. Further provided are methods suitable for inhibiting metastasis and fibrosis in a mammalian tissue.

Description

FIELD AND BACKGROUND OF THE INVENTION

[0002] The present invention relates to pharmaceutical compositions and methods useful for modulating angiogenesis and for inhibiting metastasis and fibrosis in a mammalian tissue.

[0003] Angiogenesis

[0004] In an adult, formation of new blood vessels in normal or diseased tissues is regulated by two processes, recapitulated vasculogenesis (the transformation of pre-existing arterioles into small muscular arteries) and angiogenesis, the sprouting of existing blood vessels (which occurs both in the embryo and in the adult).

[0005] The process of angiogenesis is regulated by biomechanical and biochemical stimuli. Angiogenic factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are released by vascular cells, macrophages, and cell surrounding blood vessels. These angiogenic factors activate specific proteases that are involved in degradation of the basement membrane. As a result of this degradation, vascular cells migrate and proliferate thus leading to new blood vessel formation. Peri-endothelial cells, such as pericytes in the capillaries, smooth muscle cells in larger vessels and cardiac myocytes in the heart are recruited to provide maintenance and modulatory functions to the forming vessel.

[0006] The establishment and remodeling of blood vessels is controlled by paracrine signals, many of which are mediated by protein ligands which modulate the activity of transmembrane tyrosine kinase receptors. Among these molecules are vascular endothelial growth factor (VEGF) and its receptor families (VEGFR-1, VEGFR-2, neuropilin-1 and neuropilin-2), Angiopoietins 1-4 (Ang-1, Ang-2 etc.) and their respective receptors (Tie-1 and Tie-2), basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF), and transforming growth factor β (TGF-β).

[0007] The growth of solid tumors is limited by the availability of nutrients and oxygen. When cells within solid tumors start to produce angiogenic factors or when the levels of angiogenesis inhibitors decline, the balance between anti-angiogenic and angiogenic influences is perturbed, initiating the growth of new blood vessels from the existing vascular bed into the tumor. This event in tumor progression is known as the angiogenic switch (Folkman, 1990; Hanahan and Folkman 1996). It had been demonstrated that inhibitors of tumor angiogenesis are able to completely inhibit tumor growth in mice (Boehm et al., 1997; Bergers et al., 1999) and also inhibit tumor metastasis, a process that relies upon close contact between the vasculature and tumor cells (Zetter, 1998). It has also been demonstrated that angiogenesis plays an important role in the progression of breast cancer (Weidner, N. (1998; Degani et al., 1997; Guidi et al., 1997; Balsari et al., 1999).

[0008] Such findings have prompted the use of known anti-angiogenic factors in breast cancer therapy (Klauber et al., 1997; Harris et al., 1996; Weinstatsaslow et al., 1994) and a search for novel angiogenesis inhibitors.

[0009] During the past decade several novel inhibitors of angiogenesis have been isolated including inhibitors of VEGF signaling (Neufeld et al., 1999) and inhibitors of processes which lead to the maturation and stabilization of new blood vessels. Anti-integrin antibodies have been used as inhibitors of blood vessel maturation (Brooks et al., 1994; Brooks et al., 1998).

[0010] Although several anti-angiogenic drugs are now available commercially, the anti-angiogenic mechanisms of most of these drugs (e.g., angiostatin and endostatin) remain unclear (O'Reilly et al., 1997; Oreilly et al., 1996).

[0011] Since angiogenesis can be initiated by many (possibly compensatory) angiogenic factors it stands to reason that anti-angiogenic factors which target later processes in the angiogenic response such as vessel maturation or a combination of anti-angiogenic factors would be most effective in arresting vessel formation.

[0012] Platelet factor-4 (PF4) is an anti-angiogenic protein normally sequestered in platelets (Tanaka et al., 1997; Maione et al., 1990; Neufeld et al., 2000). PF4 inhibits angiogenesis using poorly defined mechanisms (Gengrinovitch et al., 1995; Brown, and Parish, 1994; Gupta, and Singh, 1994; Watson et al., 1994). It was previously speculated that PF4 binds to cell surface heparan-sulfate proteoglycans and in this manner inhibits the activity of angiogenic growth factors such as basic fibroblast growth factor (Watson et al., 1994).

[0013] While reducing the present invention to practice and while searching for alternative anti-angiogenic factors or targets, the present inventors have uncovered a novel PF4 binding protein which participates in modulating anglogenesis.

[0014] Tumor Metastasis

[0015] The transition from a localized tumor to an invasive and metastatic tumor represents a landmark in the development of malignant disease, since it is usually associated with a markedly worse prognosis. The understanding of the processes that govern this transition is therefore of prime importance.

[0016] In breast cancer, the transition from a localized to an invasive/metastatic tumor is associated in many cases with the formation of fibrotic foci and desmoplasia, which is the presence of unusually dense collagenous stroma, within the primary tumor (Colpaert et al., 2001; Hasebe et al., 2000). A similar correlation may exist in other types of cancers such as colon and pancreatic cancers (Nishimura et al., 1998; Ellenrieder et al., 2000). These observations represent apparent paradoxes at first glance, since invasiveness has long been associated with the destruction of extracellular matrix by extracellular matrix degrading enzymes like metalo-proteases (Stamenkovic, 2000; Duffy et al., 2000) and heparanase (Vlodavsky and Friedmann, 2001). However, it is possible that deposition of excess extracellular matrix may stimulate in turn expression of matrix degrading enzymes that will contribute under certain circumstances to tumor invasion. In fact, there is some evidence that an increase in extracellular matrix deposition can indeed influence the production of extracellular matrix degrading enzymes (Schuppan et al., 2001; Swada et al., 2001).

[0017] Several prior art studies have attempted to develop agents to treat breast cancer metastases (Sauer et al., 2002) including a study by Kim et al., (2000) that described apicidin [cyclo (N-O-methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl-L-2-amino-8-oxodecanoyl)], a fungal metabolite that was identified as an antiprotozoal agent known to inhibit parasite histone deacetylase (HDAC), that can inhibit the H-ras-induced invasive phenotype of MCF10A human breast epithelial cells. Another agent is the polymeric form of fibronectin that was shown to reduce tumor growth and to posses antimetastatic activity when administered systemically to tumor-bearing mice (Yi and Ruoslahti, 2001).

[0018] While reducing the present invention to practice, the present inventors have also discovered that the lysyl oxidase protein, LOR-1, participates in metastasis and as such can be used as a target for inhibiting metastasis and reducing tumor invasiveness.

SUMMARY OF THE INVENTION

[0019] According to one aspect of the present invention there is provided a method of modulating angiogenesis in a mammalian tissue, the method comprising administering into the mammalian tissue a molecule capable of modifying a tissue level and/or activity of at least one type of lysyl-oxidase to thereby modulate angiogenesis in the mammalian tissue.

[0020] According to another aspect of the present invention there is provided a method of modulating angiogenesis in a mammalian tissue, the method comprising administering into the mammalian tissue a nucleic acid construct being capable of expressing a polypeptide having lysyl-oxidase activity to thereby modulate angiogenesis within the mammalian tissue.

[0021] According to yet another aspect of the present invention there is provided a pharmaceutical composition useful for modulating angiogenesis in mammalian tissue comprising, as an active ingredient, a molecule capable of modifying a level and/or activity of at least one type of lysyl-oxidase of the mammalian tissue and a pharmaceutically effective carrier.

[0022] According to another aspect of the present invention there is provided a method of modulating angiogenesis in a mammalian tissue, the method comprising administering into the mammalian tissue a molecule capable of modifying a tissue level and/or activity of a polypeptide at least 75% homologous to the polypeptide set forth in SEQ ID: 2 or 9 to thereby modulate angiogenesis within the mammalian tissue. According to further features in preferred embodiments of the invention described below, the molecule is an antibody or an antibody fragment capable of binding with, and at least partially inhibiting the activity of, the at least one polypeptide.

[0023] According to still further features in the described preferred embodiments the antibody or the antibody fragment is directed against at least a portion of the polypeptide set forth in SEQ ID NO: 2, 3, 6, 8 or 9.

[0024] According to still further features in the described preferred embodiments the molecule is a polynucleotide capable of down regulating expression of the at least one type of lysyl-oxidase.

[0025] According to still further features in the described preferred embodiments the polynucleotide is at least partially complementary with the polynucleotide set forth in SEQ ID NO: 1, 4, 5 or 7.

[0026] According to still further features in the described preferred embodiments the molecule is a polypeptide having lysyl-oxidase activity.

[0027] According to still further features in the described preferred embodiments the polypeptide is as set forth in SEQ ID NO: 2, 3, 6, 8 or 9.

[0028] According to another aspect of the present invention there is provided a method of modulating angiogenesis in a mammalian tissue, the method comprising administering into the mammalian tissue a nucleic acid construct being capable of expressing a polypeptide having lysyl-oxidase activity to thereby modulate angiogenesis within the mammalian tissue.

[0029] According to still further features in the described preferred embodiments the polypeptide is at least 75% homologous to the polypeptide set forth in SEQ ID NO: 2, 3, 6, 8 or 9.

[0030] According to still another aspect of the present invention there is provided method of identifying molecules capable of modulating angiogenesis, the method comprising: (a) isolating molecules which exhibit specific reactivity with at least one type of lysyl-oxidase; and (b) testing the molecules within mammalian tissue so as to determine the angiogenesis modulation activity thereof.

[0031] According to still further features in the described preferred embodiments step (a) is effected by binding assays and/or lysyl-oxidase activity assays.

[0032] According to an additional aspect of the present invention there is provided method of determining the malignancy of cancerous tissue, the method comprising (a) determining a lysyl-oxidase expression level and/or activity of the cancerous tissue; and (b) comparing the lysyl-oxidase expression level and/or activity with that determined for control tissue to thereby determine the malignancy of the cancerous tissue.

[0033] According to another aspect of the present invention there is provided method of inhibiting metastasis and fibrosis in a mammalian tissue, the method comprising administering into the mammalian tissue a molecule capable of downregulating a tissue level and/or activity of at least one type of lysyl-oxidase to thereby inhibit metastasis in the mammalian tissue

[0034] According to still another aspect of the present invention there is provided a pharmaceutical composition useful for inhibiting metastasis and fibrosis in mammalian tissue comprising, as an active ingredient, a molecule capable of downregulating a level and/or activity of at least one type of lysyl-oxidase of the mammalian tissue and a pharmaceutically effective carrier.

[0035] According to further features in preferred embodiments of the invention described below, the molecule is an antibody or an antibody fragment capable of binding with, and at least partially inhibiting the activity of, the at least one polypeptide.

[0036] According to still further features in the described preferred embodiments the antibody or the antibody fragment is directed against at least a portion of the polypeptide set forth in SEQ ID NO: 2, 3, 6, 8 or 9.

[0037] According to still further features in the described preferred embodiments the molecule is a polynucleotide capable of downregulating expression of the at least one type of lysyl-oxidase.

[0038] According to still further features in the described preferred embodiments the polynucleotide is at least partially complementary with the polynucleotide set forth in SEQ ID NO: 1, 4, 5 or 7.

[0039] According to still another aspect of the present invention there is provided method of identifying molecules capable of inhibiting metastasis and fibrosis, the method comprising: (a) screening and identifying molecules which exhibit specific reactivity with at least one type of lysyl-oxidase; and (b) testing the metastasis and fibrosis inhibitory potential of the said molecules.

[0040] According to still further features in the described preferred embodiments step (a) is effected by binding assays and/or lysyl-oxidase activity assays.

[0041] According to yet another aspect of the present invention there is provided a method for inhibiting metastasis and fibrosis in a mammalian tissue, the method comprising administering to the mammalian tissue a molecule capable of downregulating a tissue level and/or activity of a polypeptide at least 75% homologous to the polypeptide set forth in ID NO: 2 or 9, to thereby inhibit metastasis and fibrosis in a mammalian tissue.

[0042] The present invention successfully addresses the shortcomings of the presently known configurations by providing pharmaceutical compositions and methods that can be used to treat metastatic cancer, formation of tumor fibrosis and other disorders characterized by excessive or insufficient blood vessel formation.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0045] In the Drawings:

[0046] FIG. 1 illustrates SDS-PAGE analysis of extracts of Pporcine aortic endothelial cells (PAE Cells) which were transfected with vector along (lane 1) or with vector containing the LOR-1 cDNA (lane 3) and metabolically labeled with 35S-methionine. Extracts from the vector transfected cells (lane 2), from vector containing LOR-1 cDNA transfected cells (lane 4) or from 35S-methionine labeled human umbilical vein endothelial cells (HUVEC) (lane 5) were purified on a PF4 affinity column. A Band corresponding in size to the original band observed in the HUVEC is evident (compare lanes 4 and 5); this band is absent in extracts of vector transfected cells.

[0047] FIG. 2 illustrates differential expression of LOR-1 in breast cancer derived cells of different metastatic potential. The metastatic potential of the cells increases from left to right, and is correlated to increased LOR-1 mRNA expression. The results correspond to northern blot analysis of LOR-1 mRNA expression. Data regarding the relative metastatic potential of the cell lines was derived from the literature.

[0048] FIG. 3 illustrates expression of recombinant LOR-1 in MCF-7 breast cancer cells (lane 1). Vector transfected MCF-7 cells (lane 2) and two clones of MCF-7 expressing recombinant LOR-1 (lane 3, clone 12, lane 4, clone 22) were grown for two days in serum free medium. The medium from an equal number of cells was collected, concentrated 30 fold using Centricon™, and 10 μl aliquots were electrophoresed using an SDS/PAGE gel. Proteins were blotted onto nitrocellulose, and LOR-1 protein was identified using an antibody directed against the C-terminal of LOR-1. A secondary alkaline-phosphatase coupled antibody and NBT-BICP staining were used to detect the primary bound antibody.

[0049] FIG. 4 illustrates tumor size as correlated to LOR-1 expression. Parental MCF-7 cells (par), MCF-7 cells transfected with pCDNA3 vector alone (vec) and two recombinant LOR-1 expressing MCF-7 cells (clones 12 and 24) were deposited under the skin of immune deficient mice (107/injection site) along with an estrogen slow release pellet. Six animals were used for each cell type implanted. The area of the tumors was measured every few days. Bars represent standard deviation from the mean.

[0050] FIGS. 5 a -b illustrate anti-factor-8 immunostaining of tumors generated by MCF-7 cells transfected with the expression vector alone (FIG. 5a) or with an expression vector containing the LOR-1 cDNA (FIG. 5b). Counter staining was performed with hematoxylin-eosin (blue). Invasion of blood vessels into the tumor mass is more abundant in LOR-1 expressing tumors (FIG. 5b) as compared to tumors generated by control cells which do not express LOR-1 (FIG. 5a).

[0051] FIGS. 6 a -d illustrate liver sections of Wilson's disease patients (FIGS. 6c-d) and normal patients (FIGS. 6a-b) probed with a LOR-1 sense probe (FIGS. 6a, 6c) and antisense probe (FIGS. 6b, 6d).

[0052] FIG. 7 illustrates results of a whole mount in-situ hybridization using a LOR-1 cDNA probe and a 4 day old chick embryo. Strong expression of LOR-1 mRNA is observed in amniotic blood vessels (arrow).

[0053] FIG. 8 illustrates sequence alignment of several lysyl oxidases including LOR-1.

[0054] FIGS. 9 a -b. illustrate the expression of LOR-1 and LOR-2 in human breast cancer derived cells: Total RNA was prepared from confluent MCF-7 cells (MCF-7), MDA-MB-231 cells (MDA-231) and MDA-MB-435 cells (MDA-435) and was subjected to Northern blot analysis using a LOR-1 (FIG. 9a) and LOR-2 (FIG. 9b) specific cDNA probes. LOR-1 specific hybridization signal is seen in tumors derived from both MDA-231 and MDA-435 cells but not in tumors derived from MCF-7 cells. LOR-2 specific hybridization signal is seen only in tumors derived from MDA-435 cells. LOR-1 and LOR-2 non-specific hybridization signals are seen in all tumors' RNA in a band that corresponds to the 28S rRNA.

[0055] FIGS. 9 c -f illustrate the expression pattern of LOR-1 in normal human breast (FIG. 9c), in in-situ non-invasive breast carcinoma (FIG. 9d), in grade-1 invasive ductal carcinoma (FIG. 9e) and in grade-3 invasive ductal carcinoma (FIG. 9f). A polyclonal affinity purified rabbit antibody directed against the C-terminal of LOR-1 was used to detect the expression of LOR-1. High level expression of LOR-1 protein is seen in the epithelium of the normal duct (FIG. 9c, open arrow); magnification×100. In in-situ non-invasive breast carcinoma (FIG. 9d) the cancer cells have filled the duct but are still confined to it. Many of the cells located at periphery of the tumor have lost their ability to express LOR-1, while at the center, the cells still express high levels of LOR-1 (FIG. 9d, empty arrow); magnification×200. In grade-1 invasive breast carcinoma the tumorigenic cells have formed pseudo-ducts (FIG. 9e, black arrows) but they do not express LOR-1 anymore. However, cells found in a nearby carcinoma in-situ express LOR-1 (FIG. 9e, empty arrow); magnification×200. In grade-3 invasive breast carcinoma the tumorigenic cells express large amounts of LOR-1 and the morphology is completely disorderly (FIG. 9f); magnification×200.

[0056] FIG. 10 a illustrates the expression of recombinant LOR-1 in MCF-7 cells transfected with the expression vector alone (vec) or with an expression vector containing the LOR-1 cDNA clone 12 or 24 (#12 or #24, respectively). LOR-1 proteins were deteceted by Western blot with an antibody directed against the C-terminal of human LOR-1.

[0057] FIG. 10 b illustrates post-translational processes of LOR-1. MCF-7/Tet-LOR1 cells grown in the presence of tetracycline were trypsinized and seeded into a 24-well dish (5×104 cells/well) in a serum free medium in the absence of tetracycline. Cell aliquots collected at the noted times following tetracycline removal and were analyzed for the presence of LOR-1 by Western blot as described in FIG. 10a.

[0058] FIG. 10 c illustrates the growth rate of tumors derived from control or LOR-1 expressing MCF-7 cells. MCF-7 cells transfected with the expression vector alone (vec) or with an expression vector containing the LOR-1 cDNA clone 12 or 24 were injected into the mammary fat pads of female athymic nude mice. Each cell type was implanted in 8 animals. Tumor area was measured at the indicated times. Error bars represent the standard error of the mean. The experiment was terminated and the mice sacrificed when the tumor reached a diameter of about 1 cm.

[0059] FIG. 10 d illustrates the relative size of tumor in mice 25 days following injection of MCF-7 cells. Shown are mice harboring tumors that developed from cells transfected with the expression vector alone (left) or with the expression vector containing LOR-1 cDNA clone 24 (center) or 12 (right).

[0060] FIGS. 11 a -h illustrate fibrotic foci and collagen deposits in tumors derived from MCF-7 cells expressing recombinant LOR-1. Hematoxilin-eosin staining demonstrate a few necrotic foci in a tumor derived from MCF-7 cells transfected with expression vector alone (FIG. 11a, arrow, magnification×20) and numerous necrotic foci in a tumor derived from MCF-7 cells transfected with expression vector containing LOR-1 cDNA clone 12 (FIG. 11b, arrows, magnification×20).

[0061] FIG. 11 c illustrates human keratin-7 immunostaining of tumors generated by MCF-7 cells transfected with expression vector containing clone 12. Counter staining was performed with hematoxylin-eosin (blue). Arrow points to nuclei of host cells concentrated in the fibrotic foci. No necrosis can be seen; magnification×40. FIGS. 11d-f illustrate collagen deposits in tumor cells as viewed by Masson's Trichrome stain. A few collagen deposits are seen in tumors generated from MCF-7 cells transfected with the expression vector alone (FIG. 11d, arrows, magnification×200). Thick collagen bundles are seen between tumor cells generated from MCF-7 cells transfected with expression vector containing clone 12 (FIG. 11e, magnification×200). The fibrotic area is full with collagen fibers and interspaced with host derived cells in tumors generated from MCF-7 cells transfected with expression vector containing clone 24 (FIG. 11f, magnification×200).

[0062] FIGS. 11 g -h illustrate blood vessels stained with Masson's Trichrome in tumors generated from MCF-7 cells transfected with the expression vector alone (FIG. 11g) or expression vector containing clone 12 (FIG. 11h); magnification×400.

[0063] FIGS. 12 a -d illustrate the deposition of collagen type-3 by reticulum stain in tumors generated from MCF-7 and C6 glioma cells transfected with expression vector alone (FIG. 12a, c) or vector expressing LOR-1 cDNA clone 12 (FIG. 12b,d); magnification×200.

[0064] FIGS. 13 a -i illustrate the invasiveness of tumors derived from MCF-7 cells expressing recombinant LOR-1. Shown are histological sections of tumors generated from MCF-7 cells transfected with an expression vector alone (FIGS. 13a, b) or a vector expressing LOR-1 (FIGS. 13c-h). Sections were labeled with either a monoclonal antibody specific to human keratin-7 (FIGS. 13 a-d and f-h, blue purple stain) or with an antibody directed against LOR-1 (FIG. 13e, red stain). Counterstain was with hematoxylin (light blue) in all sections. Black arrows designate cytokeratin-7 positive tumor cells invading the tumor pseudo-capsule (FIG. 13c), infiltrating between adjacent muscle bundles (FIG. 13d), or invading the vasculature (FIGS. 13f, g) and peri-neural space of nerves (FIG. 13h). White arrows designate LOR-1 positive tumor cells infiltrating muscles located next to the tumor (FIG. 13g). Blood vessels v; nerves n; muscle fibers m; magnification×100 for FIGS. 13a-c, f, h and ×200 for FIGS. 13d, e, g.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0065] The present invention is of pharmaceutical compositions and methods that can be used to modulate angiogenesis and to inhibit tumor invasiveness and tumor fibrosis. Specifically, the present invention can be used to suppress tumor growth and metastasis as well as to treat disorders such as, for example, arthritis, diabetic retinopathy, psoriasis and vasculitis.

[0066] The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.

[0067] 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 described in the Examples section. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

[0068] As described in the Examples section which follows, the present inventors have uncovered a novel protein constituent of the angiogenic process.

[0069] This protein, which is termed LOR-1 herein (SEQ ID NO: 2) belongs to the lysyl-oxidase family of enzymes which catalyze the formation of covalent crosslinks between lysine residues on adjacent collagen or elastin fibrils. The lysyl oxidase family includes four genes (Saito et al., 1997; Kim et al., 1995; Reiser et al., 1992; Jang et al., 1999), the protein sequences of which are presented in SEQ ID NOs: 3, 6, 8 and 9. A homology comparison between several lysyl oxidase family members is presented in FIG. 8 which is further described in the Examples section which follows.

[0070] Each member of the lysyl-oxidase family of enzymes includes a highly conserved lysyl-oxidase domain, the activity of which is highly dependent on the presence of copper.

[0071] It should be noted that prior art studies have shown that removal of copper from tumor tissues leads to inhibition of angiogenesis (Rabinovitz, 1999; Yoshida et al., 1995). This further substantiates the role of the lysyl-oxidase family of enzymes in angiogenesis since presumably, removal of copper leads to inhibition of lysyl-oxidases.

[0072] Further support to the angiogenic activity of lysyl-oxidases is provided by the PF4-LOR-1 binding assays presented herein. As mentioned hereinabove, PF4 is an inhibitor of angiogenesis. As such, the anti-angiogenic activity exhibited by PF4 may be effected through LOR-1 inhibition, which, as demonstrated in the Examples section which follows, is highly expressed in the endothelial cells lining blood vessels.

[0073] Thus according to one aspect of the present invention there is provided a method of modulating angiogenesis

[0074] The method is effected by administering into the mammalian tissue a molecule capable of modifying a tissue level and/or activity of at least one type of lysyl-oxidase to thereby modulate angiogenesis in the mammalian tissue.

[0075] As used herein, the phrase “tissue level” refers to the level of lysyl-oxidase protein present in active form in the tissue at a given time point. Protein levels are determined by factors such as, transcription and/or translation rates, RNA or protein turnover and/or protein localization within the cell. As such any molecule which effects any of these factors can modify the tissue level of the lysyl-oxidase.

[0076] As used herein the term “activity” refers to an enzymatic activity of the lysyl-oxidase. A molecule which can modify the enzymatic activity may directly or indirectly alter substrate specificity of the enzyme or activity of the catalytic site thereof.

[0077] There are numerous examples of molecules which can specifically modify the tissue level and/or activity of a lysyl-oxidase. Such molecules can be categorized into lysyl-oxidase “upregulators” or “downregulators”.

[0078] Downregulators

[0079] An antibody (polyclonal, monoclonal or monospecific) or an antibody portion (e.g., Fab fragment) directed against at least a portion of a lysyl-oxidase (e.g., region spanning the catalytic site) can be used to specifically inhibit lysyl-oxidase activity when introduced into the mammalian tissue. As such, an antibody or an antibody fragment directed at a lysyl-oxidase can be used to suppress or arrest the formation of blood vessels, and to inhibit tumor fibrosis and metastasis.

[0080] Numerous examples of antibody inhibitors are known in the art, including inhibitors of angiogenesis which target angiogenic factors (Brooks et al., 1994; Brooks et al., 1998).

[0081] As is described below, various approaches can be used to reduce or abolish transcription or translation of a lysyl oxidase. These include anti-sense or ribozyme approaches as well as RNA interference.

[0082] An antisense molecule which can be used with the present invention includes a polynucleotide or a polynucleotide analog of at least 10 bases, preferably between 10 and 15, more preferably between 50 and 20 bases, most preferably, at least 17, at least 18, at least 19, at least 20, at least 22, at least 25, at least 30 or at least 40 bases which is hybridizable in vivo, under physiological conditions, with a portion of a polynucleotide strand encoding a polypeptide at least 50% homologous to SEQ ID NO: 1, 4, 5 or 7 or at least 75% homologous to an N-terminal portion thereof as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap creation penalty equals 8 and gap extension penalty equals 2.

[0083] The antisense oligonucleotides used by the present invention can be expressed from nucleic acid construct administered into the tissue, in which case inducible promoters are preferably used such that antisense expression can be switched on and off, or alternatively such oligonucleotides can be chemically synthesized and administered directly into the tissue, as part of, for example, a pharmaceutical composition.

[0084] The ability of chemically synthesizing oligonucleotides and analogs thereof having a selected predetermined sequence offers means for downmodulating gene expression. Four types of gene expression modulation strategies may be considered.

[0085] At the transcription level, antisense or sense oligonucleotides or analogs that bind to the genomic DNA by strand displacement or the formation of a triple helix, may prevent transcription. At the transcript level, antisense oligonucleotides or analogs that bind target mRNA molecules lead to the enzymatic cleavage of the hybrid by intracellular RNase H. In this case, by hybridizing to the targeted mRNA, the oligonucleotides or oligonucleotide analogs provide a duplex hybrid recognized and destroyed by the RNase H enzyme. Alternatively, such hybrid formation may lead to interference with correct splicing. As a result, in both cases, the number of the target mRNA intact transcripts ready for translation is reduced or eliminated.

[0086] At the translation level, antisense oligonucleotides or analogs that bind target mRNA molecules prevent, by steric hindrance, binding of essential translation factors (ribosomes), to the target mRNA, a phenomenon known in the art as hybridization arrest, disabling the translation of such mRNAs.

[0087] Several prior art studies have shown that antisense oligonucleotides can be effective in vivo. For example, antisense molecules have been used to arrest hematopoietic cell proliferation (Szczylik et al., 1991), growth (Calabretta et al. 1991), or entry into the S phase of the cell cycle (Heikhila et al., 1987) and to prevent receptor mediated responses (Burch and Mahan, 1991).

[0088] Several considerations must be taken into account when designing antisense oligonucleotides. For efficient in vivo inhibition of gene expression using antisense oligonucleotides or analogs, the oligonucleotides or analogs must fulfill the following requirements (i) sufficient specificity in binding to the target sequence; (ii) solubility in water; (iii) stability against intra- and extracellular nucleases; (iv) capability of penetration through the cell membrane; and (v) when used to treat an organism, low toxicity. The binding affinity of an antisense oligonucleotide can be predicted (Walton et al., 1999, Biotechnol Bioeng 65: 1-9; Matveeva et al., 1998, Nature Biotechnology 16, 1374-1375.) Unmodified oligonucleotides are typically impractical for use as antisense sequences since they have short in vivo half-lives, during which they are degraded rapidly by nucleases. Furthermore, they are difficult to prepare in more than milligram quantities. In addition, such oligonucleotides are poor cell membrane penetrants.

[0089] Thus it is apparent that in order to meet all the above listed requirements, oligonucleotide analogs need to be devised in a suitable manner.

[0090] For example, problems arising in connection with double-stranded DNA (dsDNA) recognition through triple helix formation have been diminished by a clever “switch back” chemical linking, whereby a sequence of polypurine on one strand is recognized, and by “switching back”, a homopurine sequence on the other strand can be recognized. Also, good helix formation has been obtained by using artificial bases, thereby improving binding conditions with regard to ionic strength and pH.

[0091] In addition, in order to improve half-life as well as membrane penetration, a large number of variations in polynucleotide backbones have been done, nevertheless with little success.

[0092] Oligonucleotides can be modified either in the base, the sugar or the phosphate moiety. These modifications include, for example, the use of methylphosphonates, monothiophosphates, dithiophosphates, phosphoramidates, phosphate esters, bridged phosphorothioates, bridged phosphoramidates, bridged methylenephosphonates, dephospho internucleotide analogs with siloxane bridges, carbonate bridges, carboxymethyl ester bridges, carbonate bridges, carboxymethyl ester bridges, acetamide bridges, carbamate bridges, thioether bridges, sulfoxy bridges, sulfono bridges, various “plastic” DNAs, anomeric bridges and borane derivatives (Cook, 1991).

[0093] International patent application WO 89/12060 discloses various building blocks for synthesizing oligonucleotide analogs, as well as oligonucleotide analogs formed by joining such building blocks in a defined sequence. The building blocks may be either “rigid” (i.e., containing a ring structure) or “flexible” (i.e., lacking a ring structure). In both cases, the building blocks contain a hydroxy group and a mercapto group, through which the building blocks are said to join to form oligonucleotide analogs. The linking moiety in the oligonucleotide analogs is selected from the group consisting of sulfide (—S—), sulfoxide (—SO—), and sulfone (—SO2-).

[0094] International patent application WO 92/20702 describe an acyclic oligonucleotide which includes a peptide backbone on which any selected chemical nucleobases or analogs are stringed and serve as coding characters as they do in natural DNA or RNA. These new compounds, known as peptide nucleic acids (PNAs), are not only more stable in cells than their natural counterparts, but also bind natural DNA and RNA 50 to 100 times more tightly than the natural nucleic acids cling to each other. PNA oligomers can be synthesized from the four protected monomers containing thymine, cytosine, adenine and guanine by Merrifield solid-phase peptide synthesis. In order to increase solubility in water and to prevent aggregation, a lysine amide group is placed at the C-terminal region.

[0095] Thus, antisense technology requires pairing of messenger RNA with an oligonucleotide to form a double helix that inhibits translation. The concept of antisense-mediated gene therapy was already introduced in 1978 for cancer therapy. This approach was based on certain genes that are crucial in cell division and growth of cancer cells. Synthetic fragments of genetic substance DNA can achieve this goal. Such molecules bind to the targeted gene molecules in RNA of tumor cells, thereby inhibiting the translation of the genes and resulting in dysfunctional growth of these cells. Other mechanisms have also been proposed. These strategies have been used, with some success in treatment of cancers, as well as other illnesses, including viral and other infectious diseases.

[0096] Antisense oligonucleotides are typically synthesized in lengths of 13-30 nucleotides. The life span of oligonucleotide molecules in blood is rather short. Thus, they have to be chemically modified to prevent destruction by ubiquitous nucleases present in the body. Phosphorothioates are very widely used modification in antisense oligonucleotide ongoing clinical trials. A new generation of antisense molecules consist of hybrid antisense oligonucleotide with a central portion of synthetic DNA while four bases on each end have been modified with 2′O-methyl ribose to resemble RNA. In preclinical studies in laboratory animals, such compounds have demonstrated greater stability to metabolism in body tissues and an improved safety profile when compared with the first-generation unmodified phosphorothioate. Dozens of other nucleotide analogs have also been tested in antisense technology.

[0097] RNA oligonucleotides may also be used for antisense inhibition as they form a stable RNA-RNA duplex with the target, suggesting efficient inhibition. However, due to their low stability RNA oligonucleotides are typically expressed inside the cells using vectors designed for this purpose. This approach is favored when attempting to target a mRNA that encodes an abundant and long-lived protein.

[0098] Recent scientific publications have validated the efficacy of antisense compounds in animal models of hepatitis, cancers, coronary artery restenosis and other diseases. The first antisense drug was recently approved by the FDA. The drug, Fomivirsen, was developed by Isis, and is indicated for local treatment of cytomegalovirus in patients with AIDS who are intolerant of or have a contraindication to other treatments for CMV retinitis or who were insufficiently responsive to previous treatments for CMV retinitis (Pharmacotherapy News Network).

[0099] Several antisense compounds are now in clinical trials in the United States. These include locally administered antivirals, systemic cancer therapeutics. Antisense therapeutics has the potential to treat many life-threatening diseases with a number of advantages over traditional drugs. Traditional drugs intervene after a disease-causing protein is formed. Antisense therapeutics, however, block mRNA transcription/translation and intervene before a protein is formed, and since antisense therapeutics target only one specific mRNA, they should be more effective with fewer side effects than current protein-inhibiting therapy.

[0100] A second option for disrupting gene expression at the level of transcription uses synthetic oligonucleotides capable of hybridizing with double stranded DNA. A triple helix is formed. Such oligonucleotides may prevent binding of transcription factors to the gene's promoter and therefore inhibit transcription. Alternatively, they may prevent duplex unwinding and, therefore, transcription of genes within the triple helical structure.

[0101] Ribozymes may also be used as down regulators. Ribozymes are being increasingly used for the sequence-specific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest. The possibility of designing ribozymes to cleave any specific target RNA has rendered them valuable tools in both basic research and therapeutic applications. In the therapeutics area, ribozymes have been exploited to target viral RNAs in infectious diseases, dominant oncogenes in cancers and specific somatic mutations in genetic disorders. Most notably, several ribozyme gene therapy protocols for HIV patients are already in Phase 1 trials (Welch et al., 1998). More recently, ribozymes have been used for transgenic animal research, gene target validation and pathway elucidation. Several ribozymes are in various stages of clinical trials. ANGIOZYME was the first chemically synthesized ribozyme to be studied in human clinical trials. ANGIOZYME specifically inhibits formation of the VEGF-r (Vascular Endothelial Growth Factor receptor), a key component in the angiogenesis pathway. Ribozyme Pharmaceuticals, Inc., as well as other firms have demonstrated the importance of anti-angiogenesis therapeutics in animal models. HEPTAZYME, a ribozyme designed to selectively destroy Hepatitis C Virus (HCV) RNA, was found effective in decreasing Hepatitis C viral RNA in cell culture assays (Ribozyme Pharmaceuticals, Incorporated).

[0102] Another mechanism of down regulating enzymes at the transcript level is RNA interference (RNAi), an approach which utilizes small interfering dsRNA (siRNA) molecules that are homologous to the target mRNA and lead to its degradation (Carthew, 2001). RNAi is an evolutionarily conserved surveillance mechanism that responds to double-stranded RNA by sequence-specific silencing of homologous genes (Fire et al., 1998; Zamore et al., 2000). RNAi is initiated by the dsRNA-specific endonuclease Dicer, which promotes cleavage of long dsRNA into double-stranded fragments between 21 and 25 nucleotides long, termed small interfering RNA (siRNAs) (Zamore et al., 2000; Elbashir et al., 2001; Hammond et al., 2000; Bernstein et al., 2001). siRNA are incorporated into a protein complex that recognizes and cleaves target mRNAs (Nykanen et al., 2001).

[0103] RNAi has been increasingly used for the sequence-specific inhibition of gene expression. The possibility of interfering with any specific target RNA has rendered RNAi a valuable tool in both basic research and therapeutic applications. RNAi was first used for gene silencing in nematodes (Fire et al., 1991;Guo & Kemphues, 1995).

[0104] Recent scientific publications have validated the efficacy of such short double stranded RNA molecules in inhibiting target mRNA expression and thus have clearly demonstrated the therapeutic potential of such molecules. For example, RNAi has been utilized to inhibit expression of hepatitis C (McCaffrey et al., 2002), HIV-1 (Jacque et al., 2002), cervical cancer cells (Jiang and Milner 2002) and leukemic cells (Wilda et al., 2002).

[0105] Several considerations must be taken into account when designing RNAi for in vivo administration into mammalian cells. Since the introduction of dsRNA into mammalian cells does not result in efficient Dicer-mediated generation of siRNA (Caplen et al., 2000; Ui-Tei et al., 2000) short siRNA duplexes of typically 21 to 25-base pairs are utilized to initiate target cleavage.

[0106] Such siRNA molecules can be chemically synthesized as 21 to 25-nucleotide siRNA duplexes (Elbashir et al., 2001; McCaffrey et al., 2002). Synthetic siRNA oligonucleotide duplexes can be prepared with either ribonucleotide 3′ overhangs or with deoxyribonucleotide 3′ overhangs (Hohjoh 2002). They can also be prepared as a sense-stranded DNA/antisense-stranded RNA hybrids or vise versa.

[0107] The siRNA used by the present invention can be transcribed in vitro from plasmids and administered into the tissue. Transcripts that include two self-complementary siRNAs annealed to form a loop region can be further processed by single-stranded ribonucleases and/or other proteins into a functional duplex siRNA molecule (Leirdal and Sioud, 2002). siRNA can also be prepared from dsRNA by Escherichia coli RNase III cleavage into endoribonuclease-prepared siRNA (esiRNA).

[0108] Since approaches for introducing synthetic siRNA into cells by lipofection can result in low transfection efficiencies in some cell types and/or short-term persistence of silencing effects, vector mediated methods have been developed.

[0109] Thus, siRNA molecules utilized by the present invention are preferably delivered into cell using retroviruses. Delivery of siRNA using retroviruses provides several advantages over methods, such as lipofection, since retroviral delivery is more efficient, uniform and immediately selects for stable “knock-down” cells (Devroe and Silver, 2002).

[0110] Thus, siRNA molecules of the present invention are preferably transcribed from expression vectors which can facilitate stable expression of the siRNA transcripts once introduced into a host cell. These vectors are engineered to express small hairpin RNAs (shRNAs), which are processed in-vivo into siRNA molecules capable of carrying out gene-specific silencing (Brummelkamp, 2002; Paddison et al. 2002; Paul et al., 2002; Yu et al., 2002.

[0111] An example of a suitable expression vector is the pSUPER™, which includes the polymerase-III H1-RNA gene promoter with a well defined start of transcription and a termination signal consisting of five thymidines in a row (T5) (Brummelkamp, 2002). Most importantly, the cleavage of the transcript at the termination site is at a site following the second uridine, thus yielding a transcript which resembles the ends of synthetic siRNAs, which also contain nucleotide overhangs. siRNA is cloned such that it includes the sequence of interest, i.e., LOR-1 separated by a short spacer from the reverse complement of the same sequence. The resulting transcript folds back on itself to form a stem-loop structure, which mediates LOR-1 RNAi.

[0112] Another suitable siRNA expression vector encodes the sense and antisense siRNA under the regulation of separate polIII promoters (Miyagishi and Taira (2002) Nature Biotech. 20:497-500). The siRNA, generated by this vector also includes a 5 thymidine termination signal.

[0113] Oligoribonucleotide sequences of the present invention can also be placed under bi-directional promoters to produce both the sense and antisense transcripts from the same promoter construct, thus simplifying the construction of expression vectors and achieving an equal molar ratio of cellular sense and antisense sequences. Examples for bi-directional promoters are disclosed in U.S. pat. application No. 20020108142.

[0114] The downregulators described hereinabove would be particularly useful for inhibiting angiogenesis in tumor tissue. It has been shown that PF4, a lysyl oxidase binding protein which inhibits angiogenesis in tumor tissue specifically accumulates in newly formed blood vessels of tumors (angiogenic vessels) but not in established blood vessels (Hansell et al., 1995; Reiser et al., 1992).

[0115] Newly formed angiogenic blood vessels are more permeable to proteins than established blood vessels because the major inducer of angiogenesis in many angiogenic diseases is VEGF, a growth factor which also functions as a potent blood vessel permeabilizing factor (VPF) (Neufeld et al., 1999). Tumor associated blood vessels are therefore in a permanent state of hyperpermeability due to deregulated over-expression of VEGF (Shweiki et al., 1995; Rak et al., 1995) and as such, a downregulator molecule used by the method of the present invention would be able to extravasate efficiently from tumor blood vessels but much less efficiently from normal stabilized blood vessels.

[0116] Upregulators

[0117] Several approaches can be utilized to increase the levels of lysyl oxidase and as such to enhance the formation of blood vessels.

[0118] For example, a nucleic acid construct including a constitutive, inducible or tissue specific promoter positioned upstream of a polynucleotide encoding a polypeptide having lysyl oxidase activity, such as the polypeptide set forth in SEQ ID NO: 2, 3, 6, 8 or 9 can be administered into a mammalian tissue. The lysyl oxidase expressed from this construct would substantially increase the levels of lysyl oxidase within the cells of the tissue and as such enhance angiogenesis.

[0119] The polynucleotide segments encoding the lysyl oxidase can be ligated into a commercially available expression vector system suitable for transforming mammalian cells and for directing the expression of this enzyme within the transformed cells. It will be appreciated that such commercially available vector systems can easily be modified via commonly used recombinant techniques in order to replace, duplicate or mutate existing promoter or enhancer sequences and/or introduce any additional polynucleotide sequences.

[0120] Suitable mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(+/−), pZeoSV2(+/−), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, which are available from Invitrogen, pCI which is available from Promega, pBK-RSV and pBK-CMV which are available from Stratagene, pTRES which is available from Clontech, and their derivatives.

[0121] Gene “knock-in” techniques can also be used to increase the expression levels of the lysyl oxidase.

[0122] Enhancer elements can be “knocked-in” adjacent to endogenous lysyl oxidase coding sequences to thereby increase transcription therefrom.

[0123] Further detail relating to the construction and use of knock-out and knock-in constructs is provided elsewhere (Fukushige and Ikeda, 1996; Bedell et al., 1997; Bermingham et al., 1996).

[0124] It will be appreciated that direct administration of a polypeptide exhibiting lysyl oxidase activity can also be utilized for enhancing angiogenesis.

[0125] It will be appreciated that affinity binding assays and/or activity assays, the principles of which are well known in the art, can be used to screen for novel compounds (e.g., substrate analogs) which can specifically regulate activity of lysyl oxidase and as such can be used with the present invention.

[0126] An assay suitable for use with this aspect of the present invention has been previously described in a study conducted by Bedell-Hogan et al., 1993.

[0127] As is clearly illustrated in the Examples section which follows, the present study also correlated expression levels of LOR-1 to the metastatic properties of breast cancer derived cell lines, indicating that LOR-1 may play additional roles in tumor invasiveness in addition to its role in angiogenesis.

[0128] Thus, the present invention also provides a method of inhibiting metastasis and/or fibrosis in a mammalian tissue. The method is effected by administering to the mammalian tissue a molecule capable of downregulating a tissue level and/or activity of at least one type of lysyl-oxidase.

[0129] The method of the present invention can be used to treat human patients that have been diagnosed with cancerous tumors, by administering any of the downregulating molecules described herein above, in order to reduce the tissue level and/or activity of at least one type of a lysyl oxidase.

[0130] As used herein, the phrase “cancerous tumor” refers to any malignant tumor within a human body including, but not limiting to, tumors with metastases. In addition, and without being bound to any particular type of cancerous tumor, the present invention is useful to treat breast cancer tumors, with or without metastases.

[0131] As used herein, the phrase “administering” refers to all modes of administration described hereinbelow with respect to the pharmaceutical compositions of the present invention.

[0132] These include, but not limit to, local administration at the tumor tissue, an organ where the cancerous tumor was diagnosed and/or related tissues that typically form metastases [Hortobagyi, 2002, Semin Oncol 29 (3 Suppl 11): 134-44; Morrow and Gradishar, 2002, 324: 410-4]. Examples of related tissue include lymph nodes adjacent to, for example, breast tissue and bones.

[0133] Administration can also be effected in a systemic manner in order to treat the affected tissue, i.e., the tissue where the cancerous tumor was formed and where metastases are present or likely to be formed with tumor progression.

[0134] Since any molecule capable of downregulating lysyl-oxidase activity can be utilized by the methods described hereinabove, the present invention also provide a method of identifying molecules capable of inhibiting metastasis and/or fibrosis.

[0135] This method is effected by screening and identifying molecules which exhibit specific reactivity with at least one type of lysyl-oxidase and testing a metastasis and/or fibrosis inhibitory potential of these molecules.

[0136] Numerous types of molecules can be screened for reactivity with at least one type of lysyl-oxidase, examples include, but are not limited to, molecules such as antisense oligonucleotides, siRNA and ribozymes that interact with a polynucleotide expressing a lysyl oxidase activity or molecules such as antibodies that interact with polypeptides having a lysyl oxidase activity. In addition, short peptides and other small molecules can also be screened by this method of the present invention.

[0137] Screening for cross reactivity can be effected by lysyl-oxidase enzymatic activity assays, by binding assays and the like. Examples of suitable assays are provided in Rodriguez et al., 2002, Arterioscler Thromb Vasc Biol 22: 1409-14; Wilson and Nock, 2002, Curr Opin Chem Biol 6: 81-5; Uetz, 2002, Curr Opin Chem Biol 6: 57-62; Stoll et al., 2002, Front Biosci 2002 7: c13-32).

[0138] Testing a metastatic phenotype of transformed tumor cells can be performed in-vitro since nearly all steps of the metastatic process, including attachment, matrix degradation and migration, can be modeled experimentally in-vitro by measuring invasion of a reconstituted basement membrane (RBM). Metastatic invasiveness of tumor cell can be modeled by migration of tumor cells into reconstituted basement membrane (RBM) in the presence and absence of a chemoattractant, such as fibroblast conditioned medium (FCM). The assay determines cells that have attached to the RBM, degraded the RBM enzymatically and, finally, cells that have penetrated the FCM side of the membrane.

[0139] Since in-vitro metastasis events correspond to steps observed in the metastatic spread of tumor cells through the basement membrane in-vivo, in-vitro invasiveness of cells can be assayed by the methods described in Albini et al., 1987 Cancer Research 47: 3239-3245, which is incorporated herein by reference in its entirety. Invasiveness assays and other methods for assessing metastatic affects, are described in Leyton et al., 1994 Cancer Research 54:3696-3699, which is incorporated by reference herein in its entirety. Reconstituted basement membrane preparations for use in accordance with the hereinabove described assays are readily available from numerous commercial suppliers. One suitable example membrane in this regard is “MATRIGEL” available from Collaborative Biomedical Products of Bedford, Mass.

[0140] In-vitro evaluation of tumor cell metastatic phenotype can also be effected by determining level and pattern of expression of one or more metastasis associated markers such protease markers, which are considered to be an integral part of tumor metastasis (see U.S. Pat. No.: 6,303,318). One example is the arachidonic acid, the release of which in cells can serve to indicate metastatic potential of a tumor (U.S. Pat. No: 6,316,416). In this regard, determining phospholipase A-2 (PLA2) activity, and the activity or abundance of factors that affect the activity of PLA2, such as uteroglobin protein (U.S. Pat. No.: 6,316,416) can serve as an indication of metastatic potential.

[0141] Determining pattern and level of expression of metastasis-associated markers can be effected by one of several methods known in the art.

[0142] The presence or level of proteins indicative of metastatic potential of tumors can be determined in cells by conventional methods well known to those of skill in the art. For instance, the techniques for making and using antibody and other immunological reagents and for detecting particular proteins in samples using such reagents are described in current protocols in immunology, Coligan et al., Eds., John Wiley & Sons, New York (1995), which is incorporated by reference herein in parts pertinent to making and using reagents useful for determining specific proteins in samples. As another example, immunohistochemical methods for determining proteins in cells in tissues are described in Volume 2, Chapter 14 of current protocols in molecular biology, Ausubel et al., Eds., John Wiley & Sons, Inc. (1994), which is incorporated by reference herein in part pertinent to carrying out such determinations. Finally, Linnoila et al., A.J.C.P. 97(2): 235-243 (1992) and Peri et al., J. Clin. Invest. 92: 2099-2109 (1992), incorporated herein as referred to above, describe techniques that may need, in part, in this aspect of the present invention.

[0143] Metastatic potential can also be determined in vivo at the mRNA level. The presence and/or level of mRNA transcripts can be determined by a variety of methods known to those of skill in the art. A given mRNA may be detected in cells by hybridization to a specific probe. Such probes may be cloned DNAs or fragments thereof, RNA, typically made by in-vitro transcription, or oligonucleotide probes, usually generated by solid phase synthesis. Methods for generating and using probes suitable for specific hybridization are well known and used in the art.

[0144] A variety of controls may be usefully employed to improve accuracy in mRNA detection assays. For instance, samples may be hybridized to an irrelevant probe and treated with RNAse A prior to hybridization, to assess false hybridization.

[0145] In order to modulate angiogenesis or inhibit metastasis or tumor fibrosis, the molecules used by the present invention can be administered to the individual per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.

[0146] As used herein a “pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration/targeting of a compound to a mammal.

[0147] As used herein the term “active ingredients”, refers to the preparation accountable for the biological effect, i.e. the upregulator/downregulator molecules used by the present invention to modulate angiogenesis and the downregulators molecules used by the present invention to inhibit metastasis and tumor fibrosis.

[0148] Hereinafter, the phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier” are interchangeably used to refer to a carrier, such as, for example, a liposome, a virus, a micelle, or a protein, or a diluent which do not cause significant irritation to the mammal and do not abrogate the biological activity and properties of the active ingredient. An adjuvant is included under these phrases.

[0149] Herein the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients, include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[0150] Techniques for formulation and administration of compositions may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.

[0151] Suitable routes of administration may, for example, include oral, rectal, transmucosal, transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, inrtaperitoneal, intranasal, or intraocular injections.

[0152] For injection, the active ingredients of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0153] For oral administration, the compounds can be formulated readily by combining the active ingredient with pharmaceutically acceptable carriers well known in the art. Such carriers enable the active ingredient of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0154] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0155] Pharmaceutical compositions, which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

[0156] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0157] The preparations described herein may be formulated for parenteral administration, e.g., by bolus injection or continuos infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

[0158] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

[0159] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.

[0160] The preparation of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

[0161] Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose.

[0162] The pharmaceutical composition may form a part of an article of manufacturing which also includes a packaging material for containing the pharmaceutical composition and a leaflet which provides indications of use for the pharmaceutical composition.

[0163] Thus, the present invention provides a method and pharmaceutical compositions useful modulating angiogenesis.

[0164] Such modulation activity can be used to treat arthritis (Koch, 1998; Paleolog and Fava, 1998), diabetic retinopathy (Miller et al., 1997), psoriasis (Detmar et al., 1994; Creamer et al., 1997) and vasculitis (Lie, 1992; Klipple and Riordan, 1989).

[0165] In addition, the present invention can also be used to treat disease characterized by fragile blood vessels, including Marfans syndrom, Kawasaki, Ehlers-Danlos, cutis-laxa, and takysu (Lie, 1992; Klipple and Riordan, 1989; Brahn et al., 1999; Cid et al., 1993; Hoffman et al., 1991).

[0166] It is possible that some of these diseases result from reduced or abolished lysyl oxidase activity which leads to the synthesis of a fragile extracellular matrix, and consequently, fragile blood vessels.

[0167] As such, administration of lysyl oxidase encoding sequences or polypeptides can be used to correct some of the manifestations of these diseases.

[0168] The present invention can also be used to treat diseases which are characterized by changes in the wall of blood vessels. For example, restenosis which is a common complication following balloon therapy, Fibromuscular dysplasia (Begelman, and Olin, 2000) and aortic stenosis (Palta et al., 2000) are all potentially treatable by the method of the present invention.

[0169] In addition, as is illustrated in the Examples section which follows, LOR-1 is more highly expressed in metastatic tumors and cell lines than in non-metastatic tumors and cell lines (FIGS. 3, 9, and 13). This suggests that levels of LOR-1 expression can be used as a diagnostic tool to determine the malignancy of cancer cells, as well as, to determine and implement suitable treatment regimens.

[0170] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

EXAMPLES

[0171] Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.

[0172] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., eds. (1985); “Transcription and Translation” Hames, B. D., and Higgins S. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

Example 1

The role of LOR1 in Angiogenesis

[0173] A study was conducted in efforts to further substantiate and characterize the role of LOR-1 in angiogenes is.

[0174] Materials and Methods

[0175] Human recombinant platelet factor-4 (PF4, GenBank Accession number M20901) which was produced in bacteria and subsequently refolded was supplied by Dr. Maione of Repligen Corp. (Boston, USA). Estrogen slow release pellets were obtained from Innovative Research of America, Sarasota, Fla., USA.

[0176] Construction of LOR-1 expression vector, transfection into MCF-7 cells, and expression: The LOR-1 cDNA (SEQ ID NO: 1) was cloned into a pCDNA3.1-hygro expression vector (Invitrogen Inc., USA) under the control of a CMV promoter. Clones of cells expressing LOR-1 were selected using hygromycine and assayed for LOR-1 expression using the polyclonal antisera described below.

[0177] Construction of platelet factor-4 affinity columns and purification of LOR-1 on such columns: PF4 was coupled to sepharose using a modification of the method of Miron and Wilchek (Wilchek and Miron, 1982) as previously described for vascular endothelial growth factor (Soker et al., 1998). Serum free conditioned medium was collected from 35S-methionine labeled MCF-7 cells which over-expressed LOR-1. The conditioned medium was passed through the column twice. The column was washed with phosphate buffered saline (300 mM NaCl, pH-7.2) and eluted with PBS (containing 2M NaCl).

[0178] Experiments with nude mice: Modified or parental MCF-7 cells (107 cells per animal) were implanted under the skin of nude mice. A pellet of slow release estrogen was implanted 1 cm away as previously described (Zhang et al., 1995). Tumors were measured periodically, following which, tumors at least 1 cm in size were removed and immuno-histologicaly analyzed using a commercial antibody directed against a factor 8 like antigen which served as a specific marker for endothelial cells.

[0179] In-situ hybridization: Fragments encompassing nucleotides 922-1564 of LOR-1, nucleotides 976-1391 of LOL, nucleotides 400-950 of LO and nucleotides 1061-1590 of LOR-2 (as numbered from the ATG codon of these sequences) where each independently subcloned into the Bluescript SK and KS (Stratagene) vectors. A DIG cRNA labeling kit of Boehringer-Mannheim was used to transcribe sense (s) and antisense (as) digoxigenin-labeled cRNA probes from the T7 promoter of the Bluescript constructs. Hybridization and subsequent detection of hybridized probes was carried out essentially as previously described (Cohen et al., 2001).

[0180] Anti-LOR-1 polyclonal antisera: Antisera was generated by injecting a recombinant peptide containing the C-terminal 200 amino-acids of LOR-1 (amino acids 540-744 of SEQ ID NO: 2) into female rabbits. Serum was collected 10 days following each injection and an immunoglobulin fraction was purified using a protein A Sepharose affinity column (Pharmacia).

[0181] Results

[0182] LOR-1 purification: A PF4 affinity column was used to detect endothelial cell proteins which specifically interact with PF4.

[0183] Two PF4 binding proteins were detected in conditioned medium of human umbilical vein endothelial cells (HUVEC), whereas PF4 binding proteins were not detected in detergent extracts of endothelial cells.

[0184] Two liters of conditioned medium enabled a partial purification of one such binding protein which eluted from the column at a relatively high salt concentrations (0.4-0.5 M NaCl).

[0185] Further purification of this protein was performed using reverse phase high pressure liquid chromatography and SDS/PAGE chromatography. The PF4 binding protein did not bind to heparin nor was it a heparan-sulfate proteoglycan since heparinase digestion failed to change its mobility in SDS/PAGE experiments.

[0186] Partial sequencing and database comparison revealed that the PF4 binding protein of the present invention (LOR-1) belongs to a family of proteins containing a lysyl-oxidase like domain (Kim et al., 1995; Kim et al., 1999). Lysyl-oxidases are copper dependent enzymes that participate in the synthesis of the extracellular matrix by catalyzing the formation of covalent bonds between lysines of adjacent collagen or elastin fibers.

[0187] The full length amino acid sequence of LOR-1 (as deduced from the isolated cDNA sequence) displayed a high degree of identity to WS9-14, a protein over expressed in senescent fibroblasts, in several types of adherent cells (but not in non-adherent cells) and in fibroblasts, in which it was correlated to pro-collagen I-a1 expression levels, as well as being induced by TGF-β and inhibited by phorbol esters and retinoic acid (Saito et al., 1997).

[0188] The role of LOR-1 in tumor development: Recombinant LOR-1 expressed in PAE cells specifically bound with the PF4 affinity column (FIG. 1). Since LOR-1 is a member of the LO family it was hypothesized that it participates in ECM formation during angiogenesis. Furthermore it was also hypothesized that PF4 suppresses or inhibits the pro-angiogenic activity of LOR-1 thus inhibiting the later stages of blood vessel formation and as a result limiting tumor growth.

[0189] LOR-1 expression: In-situ hybridization demonstrated that LOR-1 is expressed in a wide variety of tissues and cell types including fibroblasts, adipocytes, nerve cells, endothelial cells and a variety of epithelial cells. Several cell types, such as liver hepatocytes, did not express LOR-1; of the 4 LO family members examined (all except for LoxC) LOR-1 was the only one expressed in endothelial cells of blood vessels.

[0190] LOR-1 and cancer: as shown in FIG. 2, a direct correlation between the expression levels of LOR-1 and the metastatic properties of breast cancer derived cell lines was demonstrated herein.

[0191] Since the epithelial cells which line the milk ducts of normal breast tissue (from which most breast tumors arise) express large amounts of LOR-1 it is possible that the less metastatic lines lost LOR-1 expression rather than gained it.

[0192] To substantiate its role in metastasis, LOR-1 cDNA was expressed in non-metastatic breast cancer derived MCF-7 cell lines which do not normally express LOR-1. The expression of LOR-1 was examined using rabbit polyclonal antibodies generated as described above (FIG. 3).

[0193] A control cell line which was transfected with an empty expression vector, and an MCF-7 cell line expressing LOR-1 were implanted under the skin of immune deficient mice along with an estrogen slow release pellet as described above. Estrogen was added since the development of tumors from this non-metastatic cell line is estrogen dependent (Mcleskey et al., 1993).

[0194] The rate of tumor development in the mice was continuously monitored (FIG. 4); tumors 1 cm in size were excised and subjected to histological analysis as described above. Interestingly, the rate of tumor development varied between the two cell lines, with some tumors exhibiting slower growth in the LOR-1 expressing MCF-7 and yet other exhibiting slower growth in the control cells.

[0195] In order to overcome expression level problems, LOR-1 cDNA was placed under the control of a tetracycline induced promoter (The TET-off system). Such a construct will enable to determine conclusively whether the reduced rate of tumor growth observed in LOR-1 expressing cells is indeed caused by LOR-1.

[0196] Tumors expressing large amounts of LOR-1 were sectioned and stained with an antibody directed against factor 8 like antigen, a specific marker of endothelial cells. Control tumor tissue predominantly stained at the capsule around the tumor while in the LOR-1 expressing tumors pronounced staining was observed in inner regions of the tumor tissue (FIG. 5a and FIG. 5b respectively).

[0197] The role of LOR-1 in Wilson's disease and in other chronic liver diseases: Normal and diseased liver tissue were probed with LOR-1 sense (FIGS. 6a and 6c) and antisense probes (FIGS. 6b and 6d). Normal liver tissues expresses very low levels of LOR-1 (FIG. 6b). However, fibrotic liver tissues such as those observed in Wilson's disease, exhibit a strong increase in hepatocyte expression of LOR-1 (FIG. 6d).

[0198] Expression of LOR-1 in chick embryos: FIG. 7 illustrates LOR-1 expression of LOR-1 mRNA in blood vessels of a developing chick embryo. Whole-mount in-situ hybridization of 4 day old chick embryos revealed LOR-1 mRNA expression in blood vessels located in the amnion (arrow).

[0199] The Lysyl oxidase family: A homology comparison between five members of the lysyl oxidase family which includes the LO and LOL subfamily and the LOR-1 and LOR-2 subfamily revealed a strong homology at the C-terminal portion which includes the conserved lysyl oxidase motif. LOR-1 and LOR-2 are characterized by long N-terminal stretches which are not found in LO and LOL.

Example 2

MCF-7 Breast Cancer Cells Expressing Recombinant Lysyl Oxidase Related Protein-1 (LOR-1) form Invasive Tumors Characterized by Extensive Fibrosis

[0200] A study was conducted in efforts to further substantiate and characterize the role of LOR-1 in inhibiting metastasis and tumor fibrosis.

[0201] Materials and Methods

[0202] Estrogen pellets (17β-estradiol, 0.72 mg/pellet, 60-days release) were from Innovative Research of America, FL, USA. Masson Trichrome stain kit was purchased from Bio-Optica (Milano, Italy), Reverse Transcriptase, G418 were from GIBCO BRL (U.K.), Hygromycin B, Tetracycline hydrochloride, Reticulum stain kit fast green and Sirius red (direct red 80) were from Sigma (USA) , Restriction enzymes, T4 ligase were from New England Biolabs (USA). The bacterial expression vector pQE-30 and the nickel affinity column were obtained from Qiagen (Germany) , 32p-dATP was purchased from NEN (USA). Monoclonal anti cytokeratin-7 (CAM 5.2) coupled to FITC was acquired from Becton Dickinson (USA), and monoclonal mouse anti vimentin (clone V9) was purchased from DAKO Denmark). Anti FITC alkaline phosphates-conjugated was purchased from ROCHE (USA), CAS block, citrate and EDTA antigen retrieval buffers were from Zymed (USA).

[0203] Cell culture: MCF-7 breast cancer cells were kindly provided by Dr. Hadasa Degani (Weizmann Institute, Israel). The MDA-MB-435 breast cancer cell line was kindly provided by Dr. Israel Vlodavsky (Technion, Israel). The MDA-MB-23 1 cells were kindly provided by Dr. Michael Klagsbrun (Harvard University, USA). These cell lines were routinely cultured in Dulbecco's modified eagle medium supplemented with gentamicin, amphotericin, glutamine and 10% Fetal Calf Serum (FCS). Human umbilical vein derived endothelial cells were isolated and cultured as described (Neufeld and Gospodarowics, 1988). Tissue culture media, sera, and cell culture supplements were from Beth-Haemek Biological Industries, Israel, or from Gibco-BRL. MCF-7 TetOff cells (Clontech, USA) containing the tetracycline trans-activator (tTA) were grown in DMEM medium containing 10% Tet system approved fetal calf serum (Clontech), in the presence of 100 μg/ml G418, 150 μg/ml Hygromycin B and 1 μg/ml Tetracyclin.

[0204] Cloning of the LOR1 and LOR2 cDNA: Total RNA (4 μg) from HUVEC cells (for LOR1) or melanoma cells (for LOR2) was reversed transcribed using MMLV reverse transcriptase (GIBCO BRL) as described (Chomczynski and Sacchi, 1987). The LOR-1 and LOR-2 cDNAs were amplified using the Expand Long High Fidelity PCR system (ROCHE) and the following pairs of amplification primers: For LOR-1 SEQ ID NOs: 10 and 11, and for LOR-2 SEQ ID NOs: 12 and 13. The 2.3 kb cDNA of LOR-1 (SEQ ID NO: 1) and the 2.26 KB of LOR-2 (SEQ ID NO: 4) were subcloned into the pGEM-T Easy vector (Promega) by T-A cloning.

[0205] Generation of polyclonal antibodies against human LOR1: A cDNA fragment containing nucleotides 1641-2253 of LOR1 (SEQ ID NO: 14) was amplified using the Expand High Fidelity PCR kit and a pair of amplification primers (SEQ ID NOs: 15 and 16). The PCR product was subcloned into the pGEM-T Easy vector (Promega) by T-A cloning.

[0206] A 613 bp LOR1 cDNA fragment (SEQ ID NO: 14) was digested with the Sph-I and Hind III restriction enzymes and ligated into the bacterial expression vector pQE-30, which added an in-frame sequence encoding a six histidine (6×His) tag to the 5′ end of the insert. The resulting plasmid was used to produce a recombinant, 6×His tagged 23 kDa peptide (SEQ ID NO: 17). The peptide was purified from bacterial cell extracts using nickel affinity chromatography, and further purified using SDS-PAGE. The gel was electroblotted onto nitrocellulose and the band containing the peptide was cut out from the blot, solubilized in DMSO, and used to immunize rabbits. Antiserum was affinity purified on protein-A sepharose followed by affinity purification on a column to which the recombinant peptide was coupled using a previously described method (Wilchek and Miron, 1982). The antibody was eluted from the column using 0.1 M glycine at pH 3.

[0207] Transfections: To constitutively express LOR1, the full length LOR1 cDNA (SEQ ID NO: 1), was digested out of the pGEM-T easy vector (promega) with Hind III and XbaI (which were incorporated into the primers used for the cloning of the LOR1 cDNA) and ligated into the mammalian expression vector pcDNA3.1 Hygro (Invitrogen, USA) to generate the expression vector pcDNA-LOR1. Empty pCDNA3. 1 Hygro plasmid or pcDNA-LOR1 plasmid (10-20 μg) were stably transfected into MCF-7 cells using electroporation with a BioRad gene pulser (960 μF, 0.28 V). Stable transfectants were selected using 300 μg/ml hygromycin B. Clones expressing recombinant LOR1 were obtained in two consecutive stable transfections and screened for LOR1 expression using our anti-LOR-1 polyclonal antibodies. Conditioned medium was collected after 48 hours from transfected cells and LOR-1 expression was monitored using western blot is analysis (FIG. 10A).

[0208] To inducively express LOR1, full length LOR1 cDNA (SEQ ID NO: 1) was cloned intQ the pTET-Splice vector (Clontech), which enables an inducible expression under the control of tetracycline (Tet off system). The pTET-Splice plasmid DNA was digested with Hind III and SpeI and ligated to the 2.3 kb HLOR1 cDNA fragment which was rescued out of the pCDNA-LOR1 plasmid using Hind III and XbaI. The resultant plasmid, which was designated as pTET-LOR1, was co-transfected into MCF-7 TetOff cells along with pTK-Hygro at a ratio of 20:1 respectively. LOR-1 expressing cells were selected in medium containing 100 μg/ml G418, 150 μg/ml hygromycin B and 1 μg/ml tetracycline. Stable transfectants were screened for inducible expression of LOR1 using western blot analysis 48 hours following removal of the tetracycline from the growth media. The clone having the highest induction levels in the absence of tetracycline and lowest basal expression levels in the presence of tetracycline was selected and designated MCF-7/Tet-LOR1.

[0209] C6 glioma cells were transfected and screened for LOR1 expression as described above.

[0210] Northern blot Analysis: Total RNA was extracted from cultured cells using Tri-Reagent (MRC, Cincinnati) according to the manufacture's instructions. Total RNA (15 μg) was loaded on a 1.2% agarose gel and Northern blot analysis was carried out as previously described (Cohen et al., 2001). LOR-1 and LOR-2 32P labeled cDNA fragments; nucleotides 1-660 of SEQ ID NO: 18 and 1061-1590 of SEQ ID NO: 19 (respectively) were used as probes.

[0211] Protein blot analysis: Serum free conditioned media (40 μl) was separated on a 8% SDS-PAGE gel and the proteins were electroblotted onto a nitrocellulose filter using semi-dry electroblotting. The filter was blocked for 1 hour at room temperature with TBST buffer containing 10 mM Tris-HCl (pH-7.0), 0.15 M NaCl, and 0.3% Tween-20 supplemented with 10% low fat milk. The filter was incubated over night at 4° C. with affinity purified rabbit anti-LOR1 polyclonal antibody in TBST (1:2500). The blot was subsequently washed 3 times in TBST and incubated with goat anti-rabbit IgG peroxidase-conjugated secondary antibodies for 1 hour at room temperature. Bound antibody was visualized using the ECL detection system (Biological Industries, Israel).

[0212] Nude Mice Experiments: Slow release pellets containing 17β-estradiol (0.72 mg/pellet, 60 day release, Innovative Research) were pre-implanted subcutaneously in 6-8 weeks old female athymic nude mice (CD1). MCF-7 cells (107 cells/mouse) were injected into the mammary fat pads. Tumor size was measured with a caliper once or twice a week, and tumor volume was determined using the following formula: volume=width2×length×0.52 (O'Reilly et al., 1999). Mice were sacrificed 4 weeks following injection of the MCF-7 cells. In other experiments, the tumors were excised when reaching a diameter of 0.8 cm. The primary tumor, the liver and the lungs were removed, weighted, fixed in 10% buffered formalin and embedded in paraffin.

[0213] The development of tumors from C6 glioma cells expressing recombinant LOR-1 was also studied. Cells (2×105 cells/animal) transfected with a control expression vector or with an expression vector containing LOR1 cDNA were injected subcutaneously in the hind limb. Mice were sacrificed 3 weeks after the injection of the cells. The primary tumors were removed, fixed in 10% buffered formalin, and embedded in paraffin for analysis.

[0214] Histology and Immunohistochemistry: Formalin-fixed, paraffin-embedded tissues were cut into serial sections of 5 μm each and used for immunohistochemistry. Sections were deparaffinized by heating to 60° C. for 1 hour, washed twice with xylen for 5 min. and rehydrated by consecutive washes in 100%, 95%, and 70% ethanol followed by a wash in water. Endogenous peroxidase activity was inhibited by a 15 minute incubation with 3% hydrogen peroxide in methanol, followed by washes with water and PBS. The sections were then antigen retrieved by heating twice for 10 minutes in a microwave oven to 90° C. in citrate buffer at pH-6.2 (for cytokeratin and vimentin antibodies) or in 1 mM EDTA buffer (for LOR1 antibody); blocking was performed using CAS block (zymed). Following blocking, the sections were incubated for 1.5 hr at room temperature with the following antibodies, all diluted with in antibody diluent reagent solution (Zymed): affinity purified anti-LOR1 antibody (1:30-1:50), monoclonal anti human cytokeratin-7 antibodies-FITC conjugated (1:50), or with monoclonal antibodies directed against vimentin (1:50) . The sections were then washed 3 times with TBST, and secondary detection was applied using anti FITC alkaline phosphates-conjugate (Roche) at a 1:200 dilution (for anti cytokeratin) or DAKO Envision detection system (anti rabbit or anti mouse—HRP). Sections were developed in 3-amino-9-ethylcarbazole (AEC, DAKO) solution, counterstained by Hematoxylin, and photographed under a microscope. In control experiments the primary antibodies were omitted. Masson Trichrome and reticulum stains were according to manufacture's protocol. For Sirius red stain sections were incubated for 5 min. with 0.03% (w/v) fast green solution, rinsed twice with 1% acetic acid, incubated for 15 min. in 0.1% Sirius red solution, rinsed as above, dehydrated and examined under polarizing light microscope.

[0215] Results

[0216] LOR-1 is expressed in highly metastatic breast cancer derived cell types but not in non-metastatic MCF-7 cells: Desmoplasia and formation of fibrotic foci in breast cancer tumors is associated with the transition from a localized, relatively benign tumor to an invasive/metastatic tumor (Colpaert et al., 2001; Hasebe et al., 2000). Lysyl-oxidases contribute to the deposition of collagen by covalently cross-linking collagen monomers (Smith-Mungo and Kagan, 1998). To find out whether expression of lysyl-oxidases is associated with the invasive/metastatic phenotype several human breast cancer derived cell types have been screened for the expression of lysyl-oxidases. Northern blot analysis revealed that the LOR-1 gene is expressed in the highly malignant, hormone independent MDA-MB-231 and MDA-MB-435 cells (Price et al., 1990) but not in hormone dependent non-metastatic MCF-7 cells (FIG. 9a). LOR-2 on the other hand was expressed only in MDA-MB-435 cells but not in the MDA-MB-231 or in MCF-7 cells (FIG. 9b).

[0217] Highly malignant grade 1 breast carcinoma cells do not express LOR-1 while highly malignant cells of grade 3 carcinomas express LOR-1: LOR-1 expression was confined to the milk ducts in both normal breast (FIG. 9c) and in in-situ ductal carcinoma (FIG. 9d). However, in grade 1 well-differentiated ductal breast carcinoma, wherein the tumor cells migrate out of the ducts to form pseudo ducts, the cells do not express LOR-1 (FIG. 9e, black arrows). On the other hand, the cells of grade 3 ductal breast carcinoma tumors, a highly malignant tumor characterized by desmoplasia, express high levels of LOR-1 (FIG. 9f).

[0218] Tumors generated in mice from LOR-1 MCF-7 transfected cells have a slower growth rate. In order to determine whether LOR-1 expression contributes to the progression of breast tumors and to the invasive/metastatic phenotype, non-invasive MCF-7 cells have been transfected with an expression plasmid containing LOR-1 cDNA (SEQ ID NO: 1). Several transfectant clones have been isolated and selected for their LOR-1 expression. The conditioned medium of two LOR-1 expressing clones (clone 12 and 24) and of a clone transfected with an expression vector alone (vec) was assayed with LOR-1 antibodies directed against the C-terminal portion of LOR-1 (SEQ ID NO: 17). Western blot analysis revealed higher levels of LOR-1 expression in clone 12 cells as compared with clone 24 cells, and no expression in cells transfected with the expression vector alone (FIG. 10a).

[0219] In addition, both LOR-1 expressing cells displayed extra protein bands of about 70 kDa (FIGS. 10a, b) suggesting that LOR-1 may undergo proteolytic processing like other members of the lysyl-oxidase family (Borel et al., 2001; Panchenko et al., 1996). To verify that the low molecular weight forms are produced as a result of post-translational processing, LOR-1 cDNA has been expressed in MCF-7 cells under the control of a Tetracycline inducible promoter (Shockett and Schatz, 1996). It can be seen that once the tetracycline inhibition is removed, the cells start to produce full length LOR-1 which is then converted into a shorter, 70 kDa C-terminal containing form (FIG. 10b). It is not yet clear whether all of these forms are enzymatically active.

[0220] To substantiate its role in tumor growth and metastasis LOR-1 producing cells and cells transfected with expression vector alone were pre-implanted subcutaneously in nude mice (described above). Interestingly, the growth rate of tumors containing LOR-1 expressing cells was retarded as compared with that of tumors developed from parental cells (not shown) or cells transfected with the expression vector alone (FIGS. 10c, d).

[0221] To determine if the decreased tumor growth rate was a result of slower proliferation, the proliferation rate of empty vector transfected MCF-7 with that of clone 12 and clone 24 cells were also compared; significant differences in their rates of proliferation was not detected (data not shown).

[0222] Tumors that develop from LOR-1 producing MCF-7 cells contain many necrotic and fibrotic foci rich in collagen deposits: Hematoxilin-eosin staining of tumor sections revealed major necrosis in tumors generated from LOR-1 expressing MCF-7 cells (FIG. 11b) and only a few necrotic areas in tumors generated from parental (not shown) or MCF-7 cells transfected with expression vector alone (FIG. 11a).

[0223] LOR-1 expressing tumors also contained extensive fibrotic areas mainly composed of host derived cells such as mouse fibroblasts rather than MCF-7 cells. These cells are easily distinguishable from the host cells since they do not react with an antibody against human keratin 7 (FIG. 11c).

[0224] Since LOR-1 was shown to oxidize lysine residues on collagen-I (Vadasz et al., 2002) it was anticipated that it may induce collagen cross-linking and deposition. However, this activity is probably depended on the existence of collagen since LOR-1 expressing MCF-7 cells grown in culture do not produce more collagen than parental MCF-7 cells (data not shown). To further understand the involvement of LOR-1 in tumor progression tumors derived from LOR-1 expressing MCF-7 cells have been stained with Mason's Trichrome, a reagent that reacts primarily with collagen-I and produces an azure color (Pinder et al., 1994). (1) Tumors that developed from parental or MCF-7 cells transfected by an expression vector alone contained limited amounts of collagen that was scattered between the tumor cells (FIG. 11d, arrows). In-contrast, tumors that developed from clone 12 or clone 24 LOR-1 expressing MCF-7 cells contained denser deposits of collagen between the tumor cells (FIG. 11e). Furthermore, the fibrotic and necrotic areas in these tumors appeared choke full with collagen fibers (FIG. 11f). In addition, while the blood vessels within tumors that developed from vector-transfected MCF-7 cells remained almost unstained (FIG. 11g, arrows), the blood vessels in tumors derived from LOR-1 expressing MCF-7 cells were sheathed by a thick layer of collagen (FIG. 11h, arrows).

[0225] These experiments indicate that LOR-1 affects both collagen-I production and deposition in MCF-7 derived breast cancer tumors. To substantiate the involvement of LOR-1 in collagen deposition C6 glioma cells were also subcutaneously injected into mice. While tumors generated from C6-glioma cells did not contain large amounts of collagen, tumors generated from LOR-1 expressing C6-glioma cells were rich with collagen (data not shown). These results indicate a more general effect for LOR-1 on collagen-I deposits in tumors. In addition, Reticulum staining of collagen-III revealed that tumors generated from LOR-1 expressing MCF-7 or C6-glioma cells contained thicker and higher concentrations of collagen-Ill fibers (FIG. 12b, 12d) as compared with tumors generated from cells transfected with the expression vector alone (FIGS. 12a, c).

[0226] Expression of LOR-1 in MCF-7 cells transforms the cells into invasive cells in-vivo: It was reported that the appearance of fibrotic foci in breast cancer tumors correlates with their degree of invasiveness (Hasebe et al., 2000). To substantiate the involvement of LOR-1 in tumor invasiveness human keratin-7 staining was employed. Tumors generated from MCF-7 cells transfected by the expression vector alone were surrounded by thick capsules with sharp borders. No staining of human keratin-7 was observed within the capsule (FIG. 13a) or in between blood vessels, nerves and muscles located adjacent to the capsules (FIG. 13b, arrows). In contrast, in tumors generated from LOR-1 expressing MCF-7 cells, human keratin-7 positive cells were observed within the capsule (FIG. 13c). Furthermore, in many areas the tumor cells migrated on-mass through the capsule and invaded muscles (FIG. 13d), nerves (FIG. 13e) and blood vessels (FIG. 13f). The invading cells were identified as the transfected LOR-1 expressing MCF-7 cells using an anti-LOR-1 antibody (FIG. 13g). These observations provide a strong evidence that the production of LOR-1 by breast cancer tumor cells contribute to the transition from localized non-invasive tumors to invasive tumors. Furthermore, aggregates of tumor cells were also detected inside lymph vessels adjacent to the tumors indicating that the LOR-1 expressing MCF-7 cells are metastatic (Luna, 1968).

[0227] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents, patent applications and sequences identified by their accession numbers mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent, patent application or sequence identified by their accession number was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

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Claims

1. A method of modulating angiogenesis in a mammalian tissue, the method comprising administering into the mammalian tissue a molecule capable of modifying a tissue level and/or activity of at least one type of lysyl-oxidase to thereby modulate angiogenesis in the mammalian tissue.

2. The method of claim 1, wherein said molecule is an antibody or an antibody fragment capable of binding with, and at least partially inhibiting said activity of, said at least one polypeptide.

3. The method of claim 2, wherein said antibody or said antibody fragment is directed against at least an antigenic portion of the polypeptide set forth in SEQ ID NO: 2, 3, 6, 8 or 9.

4. The method of claim 1, wherein said molecule is a polynucleotide capable of downregulating expression of said at least one type of lysyl-oxidase.

5. The method of claim 4, wherein said polynucleotide is at least partially complementary with the polynucleotide set forth in SEQ ID NO: 1, 4, 5 or 7.

6. The method of claim 1, wherein said molecule is a polypeptide having lysyl-oxidase activity.

7. The method of claim 6, wherein said polypeptide is as set forth in SEQ ID NO: 2, 3, 6, 8 or 9.

8. A method of modulating angiogenesis in a mammalian tissue, the method comprising administering into the mammalian tissue a nucleic acid construct being capable of expressing a polypeptide having lysyl-oxidase activity to thereby modulate angiogenesis within the mammalian tissue.

9. The method of claim 8, wherein said polypeptide is at least 75% homologous to the polypeptide set forth in SEQ ID NO: 2, 3, 6, 8 or 9.

10. A method of identifying molecules capable of modulating angiogenesis, the method comprising: (a) isolating molecules which exhibit specific reactivity with at least one type of lysyl-oxidase; and (b) testing said molecules within mammalian tissue so as to determine the angiogenesis modulation activity thereof.

11. The method of claim 10, wherein step (a) is effected by binding assays and/or lysyl-oxidase activity assays.

12. A pharmaceutical composition useful for modulating angiogenesis in mammalian tissue, the composition-of matter comprising, as an active ingredient, a molecule capable of modifying a level and/or activity of at least one type of lysyl-oxidase of the mammalian tissue and a pharmaceutically effective carrier.

13. The pharmaceutical composition of claim 12, wherein said molecule is an antibody or an antibody fragment capable of binding with, and at least partially inhibiting said activity of, said at least one type of lysyl-oxidase.

14. The pharmaceutical composition of claim 13, wherein said antibody or said antibody fragment is directed against at least a portion of the polypeptide set forth in SEQ ID NO: 2, 3, 6, 8 or 9.

15. The pharmaceutical composition of claim 12, wherein said molecule is a polynucleotide capable of downregulating expression of said at least one type of lysyl-oxidase.

16. The pharmaceutical composition of claim 15, wherein said polynucleotide is at least partially complementary with the polynucleotide set forth in SEQ ID NO: 1, 4, 5 or 7.

17. The pharmaceutical composition of claim 12, wherein said molecule is a polypeptide having lysyl-oxidase activity.

18. The pharmaceutical composition of claim 12, wherein said polypeptide is as set forth SEQ ID NO: 2, 3, 6, 8 or 9.

19. A method of modulating angiogenesis in a mammalian tissue, the method comprising administering into the mammalian tissue a molecule capable of modifying a tissue level and/or a lysyl-oxidase activity of a polypeptide at least 75% homologous to the polypeptide set forth in SEQ ID NO: 2 to thereby modulate angiogenesis in the mammalian tissue.

20. A method of modulating angiogenesis in a mammalian tissue, the method comprising administering into the mammalian tissue a nucleic acid construct being capable of expressing a polypeptide at least 75% homologous to the polypeptide set forth in SEQ ID NO: 2 to thereby modulate angiogenesis within the mammalian tissue.

21. A method of determining the malignancy of cancerous tissue, the method comprising (a) determining a lysyl-oxidase expression level and/or activity of the cancerous tissue; and (b) comparing said lysyl-oxidase expression level and/or activity with that determined for control tissue to thereby determine the malignancy of the cancerous tissue.

22. A method of inhibiting metastasis and/or fibrosis in a mammalian tissue, the method comprising administering to the mammalian tissue a molecule capable of downregulating a tissue level and/or activity of at least one type of lysyl-oxidase to thereby inhibit metastasis in the mammalian tissue.

23. The method of claim 22, wherein said molecule is an antibody or an antibody fragment capable of specifically binding with said at least one type of lysyl-oxidase.

24. The method of claim 22, wherein said antibody or said antibody fragment is directed against at least an antigenic portion of the polypeptide set forth in SEQ ID NO: 2, 3, 6, 8 or 9.

25. The method of claim 22, wherein said molecule is a polynucleotide capable of downregulating expression of said at least one type of lysyl-oxidase.

26. The method of claim 25, wherein said polynucleotide is at least partially complementary with the polynucleotide set forth in SEQ ID NO: 1, 4, 5 or 7.

27. A method of identifying molecules capable of inhibiting metastasis and/or fibrosis, the method comprising: (a) screening and identifying molecules which exhibit specific reactivity with at least one type of lysyl-oxidase; and (b) testing a metastasis and/or fibrosis inhibitory potential of said molecules.

28. The method of claim 27, wherein step (a) is effected by binding assays and/or lysyl-oxidase activity assays.

29. The method of claim 27, wherein step (b) is effected in-vivo or in-vitro.

30. A pharmaceutical composition useful for inhibiting metastasis and/or fibrosis in mammalian tissue, comprising, as an active ingredient, a molecule capable of downregulating a level and/or activity of at least one type of lysyl-oxidase present within the mammalian tissue and a pharmaceutically effective carrier.

31. The pharmaceutical composition of claim 30, wherein said molecule is an antibody or an antibody fragment capable of specifically binding with said at least one type of lysyl-oxidase.

32. The pharmaceutical composition of claim 31, wherein said antibody or said antibody fragment is directed against at least an antigenic portion of the polypeptide set forth in SEQ ID NO: 2, 3, 6, 8 or 9.

33. The pharmaceutical composition of claim 30, wherein said molecule is a polynucleotide capable of downregulating expression of said at least one type of lysyl-oxidase.

34. The pharmaceutical composition of claim 33, wherein said polynucleotide is at least partially complementary with the polynucleotide set forth in SEQ ID NO: 1, 4, 5 or 7.

35. A method of inhibiting metastasis and fibrosis in a mammalian tissue, the method comprising administering to the mammalian tissue a molecule capable of downregulating a tissue level and/or a lysyl-oxidase activity of a polypeptide at least 75% homologous to the polypeptide set forth in ID NO: 2 or 9, to thereby inhibit metastasis and fibrosis in the mammalian tissue.