PHARMACEUTICAL COMPOSITIONS AND METHODS USEFUL FOR MODULATING ANGIOGENESIS

Abstract: A method of modulating angiogenesis in a mammalian tissue is provided. The method including 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.

Description: PHARMACEUTICAL COMPOSITIONS AND METHODS USEFUL FOR MODULATING ANGIOGENESIS FIELD AND BACKGROUND OF THE INVENTION The present invention relates to pharmaceutical compositions and methods useful for modulating angiogenesis. 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). 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 which 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. 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). 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 (1,2). It had been demonstrated that inhibitors of tumor angiogenesis are able to completely inhibit tumor growth in mice (3,4) and also inhibit tumor metastasis, a process that relies upon close contact between the vasculature and tumor cells (5). It has also been demonstrated that angiogenesis plays an important role in the progression of breast cancer (6-9). Such finding have prompted the use of known anti-angiogenic factors in breast cancer therapy (10-12) and a search for novel angiogenesis inhibitors. During the past decade several novel inhibitors of angiogenesis have been isolated including inhibitors of VEGF signaling (13) 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 (14,15). 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 (16,17). Since angiogenesis can be initiated by many (possibly compansatory) 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. Platelet factor-4 (PF4) is an anti-angiogenic protein normally sequestered in platelets (18-20). PF4 inhibits angiogenesis using poorly defined mechanisms (21-24). 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 (24). 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 angiogenesis. As demonstrated by the present study, this protein, which is termed herein LOR-1, is highly expressed in cultured endothelial cells as well as in other blood vessel cells. In addition, the expression levels of LOR-1 can be correlated to the metastatic properties of breast cancer derived cell lines, indicating that LOR-1 may play additional roles in tumor progression in addition to a role in angiogenesis. SUMMARY OF THE INVENTION 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. 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. According to yet another aspect of the present invention there is provided 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. 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. 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 NOs: 2, 3, 6, 8 or 9. 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. 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 NOs: 1, 4, 5 or 7. According to still further features in the described preferred embodiments the molecule is a polypeptide having lysyl-oxidase activity. According to still further features in the described preferred embodiments the polypeptide is as set forth in SEQ ID NOs: 2, 3, 6, 8 or 9. 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. 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 NOs: 2, 3, 6, 8 or 9. 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. According to still further features in the described preferred embodiments step (a) is effected by binding assays and/or lysyl-oxidase activity assays. According to an additional aspect of the present invention there is provided method of deteπnining 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. The present invention successfully addresses the shortcomings of the presently known configurations by providing pharmaceutical compositions and methods which can be used to treat disorders characterized by excessive or insufficient blood vessel formation. BRIEF DESCRIPTION OF THE DRAWINGS 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. In the drawings: 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 35 S-methionine. Extracts from the vector transfected cells (lane 2), from vector containing LOR-1 cDNA transfected cells (lane 4) or from 35 S-methionine labeled human umbilical vein endothelial cells (ΗUVEC) (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. 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. 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. 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 (10 7 /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. FIGs. 5a-b illustrate anti-factor-8 immunostaining of tumors generated by MCF-7 cells transfected with the expression vector alone (Figure 5 a) or with an expression vector containing the LOR-1 cDNA (Figure 5b). Counter staining was performed with hematoxelin-eosin (blue). Invasion of blood vessels into the tumor mass is more abundant in LOR-1 expressing tumors (Figure 5b) as compared to tumors generated by control cells which do not express LOR-1 (Figure 5a). FIGs. 6a-d illustrate liver sections of Wilson's disease patients (Figures 6c-d) and normal patients (Figures 6a-b) probed with a LOR-1 sense probe (Figures 6a, 6c) and antisense probe (Figures 6b, 6d). FIG. 7 illustrates results of a whole mount in-situ hybridization of a using a LOR-1 cDNA probe arid a 4 day old chick embryo. Strong expression of LOR-1 mRNA is observed in amnionic blood vessels (arrow). FIG. 8 illustrates sequence alignment of several lysyl oxidases including LOR-1. DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is of pharmaceutical compositions and methods which can be used to modulate angiogenesis. 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. The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions. Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings 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. As described in the Examples section which follows, the present inventors have uncovered a novel protein constituent of the angiogenic process. 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 (27,28,32,33), 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 Figure 8 which is further described in the Examples section which follows. 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. It should be noted that prior art studies have shown that removal of copper from tumor tissues leads to inhibition of angiogenesis (30, 34). 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. Further support to the angiogenic activity of lysyl-oxidases is provided by the PF4-LOR-1 biding 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 Thus according to one aspect of the present invention there is provided a method of modulating angiogenesis. 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. 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. 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. The 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". Downregulators An antibody (polyclonal, monoclonal or monospecific) or an antibody portion (e.g., Fab fragment) directed 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. Numerous examples of antibody inhibitors are known in the art, including inhibitors of angiogenesis which target angiogenic factors (14, 15). As is described below, various anti-sense or ribozyme approaches can be used to reduce or abolish transcription or translation of a lysyl oxidase. 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 NOs:l, 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. 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. The ability of chemically synthesizing oligonucleotides and analogs thereof having a selected predetermined sequence offers means for downmodulating gene expression. Three types of gene expression modulation strategies may be considered. 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. 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 lriRNAs. 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 (58), growth (59), or entry into the S phase of the cell cycle (60) and to prevent receptor mediated responses (61). 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. 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 man milligram quantities. In addition, such oligonucleotides are poor cell membrane penetrants. Thus it is apparent that in order to meet all the above listed requirements, oligonucleotide analogs need to be devised in a suitable manner. 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. 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. 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 (62). 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 (-S02-). 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. Thus, antisense technology requires pairing of messenger RNA with an oligonucleotide to foπn 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 has 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. 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 2'0-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. 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. RNA oligonucleotides may also be designed so as to activate RNA interference mechanisms within the cell (RNAi). Oligonucleotides suitable for such purpose must be of a defined length and area of complementation (63). 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) . 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. 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. 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 ITLRNAS 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 (67). 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). The downregulators described hereinabove would be particularly useful for inhibiting angiogenesis in tumor tissue. It has been shown that PF4, a lysyl oxidase biding protein which inhibits angiogenesis in tumor tissue specifically accumulates in newly formed blood vessels of tumors (angiogenic vessels) but not in established blood vessels (31,35). 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) (13). Tumor associated blood vessels are therefore in a permanent state of hyperpermeability due to deregulated over-expression of VEGF (36,37) 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. Upregulators Several approaches can be utilized to increase the levels of lysyl oxidase and as such to enhance the formation of blood vessels. 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. 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. Suitable mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1 (+/-), pZeoSV2(+/-), P SecTag2, 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. Gene "knock-in" techniques can also be used to increase the expression levels of the lysyl oxidase. Enhancer elements can be "knocked-in" adjacent to endogenous lysyl oxidase coding sequences to thereby increase transcription therefrom. Further detail relating to the construction and use of knock-out and knock- in constructs is provided in references 64-66. It will be appreciated that direct administration of a polypeptide exhibiting lysyl oxidase activity can also be utilized for enhancing angiogenesis. 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. 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. (68). In order to modulate angiogenesis the upregulator/downregulator 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. 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. 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. 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. 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. 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. 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. 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. 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 (PNP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. Pharmaceutical compositions, which can be used orally, include push- fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. 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. 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. 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. 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. 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. 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. Thus, the present invention provides a method and pharmaceutical compositions useful modulating angiogenesis. Such modulation activity can be used to treat arthritis (38,39), diabetic retinopathy (40), psoriasis (41,42) and vasculitis (43,44). 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 (43,45-47). It is possible that some of these disease result from reduced or abolished lysyl oxidase activity which leads to the synthesis of a fragile extracellular matrix, and consequently, fragile blood vessels. As such, administration of lysyl oxidase encoding sequences or polypeptides can be used to correct some of the manifestations of these diseases. 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 (49) and aortic stenosis (50) are all potentially treatable by the method of the present invention. In addition, evidence presented in the Examples section which indicates that LOR-1 may be more highly expressed in metastatic cell lines than non-metastatic cell lines (Figure 3) suggests that levels of LOR-1 expression can be used as a diagnostic tool to determine the malignancy of cancer cells, and thus to determine what treatment regimen should be used. Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples. EXAMPLES Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion. 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, Maryland (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, CT (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, CA (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. EπXAMPLE l The role ofLORl in angiogenesis A study was conducted in efforts to further substantiate and characterize the role of LOR-1 in angiogenesis. Materials and Methods 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, FL, USA. Construction of LOR-1 expression vector, transfection into MCF-7 cells, and expression: The LOR-1 cDNA (SEQ ID NO:l) 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. 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 (51) as previously described for vascular endothelial growth factor (52). Serum free conditioned medium was collected from S-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). Experiments with nude mice: Modified or parental MCF-7 cells (10 7 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 (53). Tumors were measured periodically, following which, tumors at least 1 cm in 5 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. Ln-situ hybridization: Fragments encompassing nucleotides 922-1564 of LOR-1, nucleotides 976-1391 of LOL, nucleotides 400-950 of LO and l o 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 15 subsequent detection of hybridized probes was carried out essentially as previously described (54). 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 0 collected 10 days following each injection and an immunoglobulin fraction was purified using a protein A Sepharose affinity column (Pharmacia). Results LOR-1 purification: A PF4 affinity column was used to detect endothelial cell proteins which specifically interact with PF4. 5 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. Two liters of conditioned medium enabled a partial purification of one such binding protein which eluted from the column at a relatively high salt 0 concentrations (0.4-0.5 M NaCl). 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. 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 (28,29). 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. 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-al expression levels, as well as being induced by TGF-β and inhibited by phorbol esters and retinoic acid (27). The role of LOR-1 in tumor development: Recombinant LOR-1 expressed in PAE cells specifically bound with the PF4 affinity column (Figure 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. LOR-1 expression: ln-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. LOR-1 and cancer: As shown in Figure 2, a direct correlation between the expression levels of LOR-1 and the metastatic properties of breast cancer derived cell lines was demonstrated herein. 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. 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 (Figure 3). 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 (55). The rate of tumor development in the mice was continuously monitored (Figure 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. 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. 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 (Figure 5a and Figure 5b respectively). 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 (Figures 6a and 6c) and antisense probes (Figures 6b and 6d). Normal liver tissues expresses very low levels of LOR-1 (Figure 6b). However, fibrotic liver tissues such as those observed in Wilson's disease, exhibit a strong increase in hepatocyte expression of LOR-1 (Figures 6d). Expression of LOR-1 in chick embryos: Figure 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). 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. 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. 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Claims: WHAT IS CLAIMED IS: 1. A method of modulating angiogenesis in a mammalian tissue, the method comprising administering into the maimnalian 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 a portion of the polypeptide set forth in SEQ ID NOs: 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 NOs: 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 NOs: 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 NOs: 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 NOs: 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.

Inventors: AKIRI GAL, GENGROVITCH STELA, NEUFELD GERA, VADASZ ZAHAVA, AKIRI, GAL, GENGROVITCH, STELA, NEUFELD, GERA, VADASZ, ZAHAVA
Applicants: AKIRI GAL, GENGROVITCH STELA, NEUFELD GERA, TECHNION RES & DEV FOUNDATION, VADASZ ZAHAVA, AKIRI, GAL, GENGROVITCH, STELA, NEUFELD, GERA, TECHNION RESEARCH AND DEVELOPMENT FOUNDATION LTD., VADASZ, ZAHAVA
Application year: 2001
Main classification: A61K
IPC codes: A61K38, A61P35, C07K16, C12N9, C12N15